Archive for the ‘Ubiquitinylation’ Category

Lesson 9 Cell Signaling:  Curations and Articles of reference as supplemental information for lecture section on WNTs: #TUBiol3373

Stephen J. Wiilliams, Ph.D: Curator

The following contain curations of scientific articles from the site  intended as additional reference material  to supplement material presented in the lecture.

Wnts are a family of lipid-modified secreted glycoproteins which are involved in:

Normal physiological processes including

A. Development:

– Osteogenesis and adipogenesis (Loss of wnt/β‐catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes)

  – embryogenesis including body axis patterning, cell fate specification, cell proliferation and cell migration

B. tissue regeneration in adult tissue

read: Wnt signaling in the intestinal epithelium: from endoderm to cancer

And in pathologic processes such as oncogenesis (refer to Wnt/β-catenin Signaling [7.10]) and to your Powerpoint presentation


The curation Wnt/β-catenin Signaling is a comprehensive review of canonical and noncanonical Wnt signaling pathways


To review:












Activating the canonical Wnt pathway frees B-catenin from the degradation complex, resulting in B-catenin translocating to the nucleus and resultant transcription of B-catenin/TCF/LEF target genes.

Fig. 1 Canonical Wnt/FZD signaling pathway. (A) In the absence of Wnt signaling, soluble β-catenin is phosphorylated by a degradation complex consisting of the kinases GSK3β and CK1α and the scaffolding proteins APC and Axin1. Phosphorylated β-catenin is targeted for proteasomal degradation after ubiquitination by the SCF protein complex. In the nucleus and in the absence of β-catenin, TCF/LEF transcription factor activity is repressed by TLE-1; (B) activation of the canonical Wnt/FZD signaling leads to phosphorylation of Dvl/Dsh, which in turn recruits Axin1 and GSK3β adjacent to the plasma membrane, thus preventing the formation of the degradation complex. As a result, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it promotes the expression of target genes via interaction with TCF/LEF transcription factors and other proteins such as CBP, Bcl9, and Pygo.

NOTE: In the canonical signaling, the Wnt signal is transmitted via the Frizzled/LRP5/6 activated receptor to INACTIVATE the degradation complex thus allowing free B-catenin to act as the ultimate transducer of the signal.

Remember, as we discussed, the most frequent cancer-related mutations of WNT pathway constituents is in APC.

This shows how important the degradation complex is in controlling canonical WNT signaling.

Other cell signaling systems are controlled by protein degradation:

A.  The Forkhead family of transcription factors

Read: Regulation of FoxO protein stability via ubiquitination and proteasome degradation

B. Tumor necrosis factor α/NF κB signaling

Read: NF-κB, the first quarter-century: remarkable progress and outstanding questions

1.            Question: In cell involving G-proteins, the signal can be terminated by desensitization mechanisms.  How is both the canonical and noncanonical Wnt signal eventually terminated/desensitized?

We also discussed the noncanonical Wnt signaling pathway (independent of B-catenin induced transcriptional activity).  Note that the canonical and noncanonical involve different transducers of the signal.

Noncanonical WNT Signaling

Note: In noncanonical signaling the transducer is a G-protein and second messenger system is IP3/DAG/Ca++ and/or kinases such as MAPK, JNK.

Depending on the different combinations of WNT ligands and the receptors, WNT signaling activates several different intracellular pathways  (i.e. canonical versus noncanonical)


In addition different Wnt ligands are expressed at different times (temporally) and different cell types in development and in the process of oncogenesis. 

The following paper on Wnt signaling in ovarian oncogenesis shows how certain Wnt ligands are expressed in normal epithelial cells but the Wnt expression pattern changes upon transformation and ovarian oncogenesis. In addition, differential expression of canonical versus noncanonical WNT ligands occur during the process of oncogenesis (for example below the authors describe the noncanonical WNT5a is expressed in normal ovarian  epithelia yet WNT5a expression in ovarian cancer is lower than the underlying normal epithelium. However the canonical WNT10a, overexpressed in ovarian cancer cells, serves as an oncogene, promoting oncogenesis and tumor growth.

Wnt5a Suppresses Epithelial Ovarian Cancer by Promoting Cellular Senescence

Benjamin G. Bitler,1 Jasmine P. Nicodemus,1 Hua Li,1 Qi Cai,2 Hong Wu,3 Xiang Hua,4 Tianyu Li,5 Michael J. Birrer,6Andrew K. Godwin,7 Paul Cairns,8 and Rugang Zhang1,*

A.           Abstract

Epithelial ovarian cancer (EOC) remains the most lethal gynecological malignancy in the US. Thus, there is an urgent need to develop novel therapeutics for this disease. Cellular senescence is an important tumor suppression mechanism that has recently been suggested as a novel mechanism to target for developing cancer therapeutics. Wnt5a is a non-canonical Wnt ligand that plays a context-dependent role in human cancers. Here, we investigate the role of Wnt5a in regulating senescence of EOC cells. We demonstrate that Wnt5a is expressed at significantly lower levels in human EOC cell lines and in primary human EOCs (n = 130) compared with either normal ovarian surface epithelium (n = 31; p = 0.039) or fallopian tube epithelium (n = 28; p < 0.001). Notably, a lower level of Wnt5a expression correlates with tumor stage (p = 0.003) and predicts shorter overall survival in EOC patients (p = 0.003). Significantly, restoration of Wnt5a expression inhibits the proliferation of human EOC cells both in vitro and in vivo in an orthotopic EOC mouse model. Mechanistically, Wnt5a antagonizes canonical Wnt/β-catenin signaling and induces cellular senescence by activating the histone repressor A (HIRA)/promyelocytic leukemia (PML) senescence pathway. In summary, we show that loss of Wnt5a predicts poor outcome in EOC patients and Wnt5a suppresses the growth of EOC cells by triggering cellular senescence. We suggest that strategies to drive senescence in EOC cells by reconstituting Wnt5a signaling may offer an effective new strategy for EOC therapy.

Oncol Lett. 2017 Dec;14(6):6611-6617. doi: 10.3892/ol.2017.7062. Epub 2017 Sep 26.

Clinical significance and biological role of Wnt10a in ovarian cancer. 

Li P1Liu W1Xu Q1Wang C1.

Ovarian cancer is one of the five most malignant types of cancer in females, and the only currently effective therapy is surgical resection combined with chemotherapy. Wnt family member 10A (Wnt10a) has previously been identified to serve an oncogenic function in several tumor types, and was revealed to have clinical significance in renal cell carcinoma; however, there is still only limited information regarding the function of Wnt10a in the carcinogenesis of ovarian cancer. The present study identified increased expression levels of Wnt10a in two cell lines, SKOV3 and A2780, using reverse transcription-polymerase chain reaction. Functional analysis indicated that the viability rate and migratory ability of SKOV3 cells was significantly inhibited following Wnt10a knockdown using short interfering RNA (siRNA) technology. The viability rate of SKOV3 cells decreased by ~60% compared with the control and the migratory ability was only ~30% of that in the control. Furthermore, the expression levels of β-catenin, transcription factor 4, lymphoid enhancer binding factor 1 and cyclin D1 were significantly downregulated in SKOV3 cells treated with Wnt10a-siRNA3 or LGK-974, a specific inhibitor of the canonical Wnt signaling pathway. However, there were no synergistic effects observed between Wnt10a siRNA3 and LGK-974, which indicated that Wnt10a activated the Wnt/β-catenin signaling pathway in SKOV3 cells. In addition, using quantitative PCR, Wnt10a was overexpressed in the tumor tissue samples obtained from 86 patients with ovarian cancer when compared with matching paratumoral tissues. Clinicopathological association analysis revealed that Wnt10a was significantly associated with high-grade (grade III, P=0.031) and late-stage (T4, P=0.008) ovarian cancer. Furthermore, the estimated 5-year survival rate was 18.4% for patients with low Wnt10a expression levels (n=38), whereas for patients with high Wnt10a expression (n=48) the rate was 6.3%. The results of the present study suggested that Wnt10a serves an oncogenic role during the carcinogenesis and progression of ovarian cancer via the Wnt/β-catenin signaling pathway.

Targeting the Wnt Pathway includes curations of articles related to the clinical development of Wnt signaling inhibitors as a therapeutic target in various cancers including hepatocellular carcinoma, colon, breast and potentially ovarian cancer.


2.         Question: Given that different Wnt ligands and receptors activate different signaling pathways, AND  WNT ligands  can be deferentially and temporally expressed  in various tumor types and the process of oncogenesis, how would you approach a personalized therapy targeting the WNT signaling pathway?

3.         Question: What are the potential mechanisms of either intrinsic or acquired resistance to Wnt ligand antagonists being developed?


Other related articles published in this Open Access Online Scientific Journal include the following:

Targeting the Wnt Pathway [7.11]

Wnt/β-catenin Signaling [7.10]

Cancer Signaling Pathways and Tumor Progression: Images of Biological Processes in the Voice of a Pathologist Cancer Expert

e-Scientific Publishing: The Competitive Advantage of a Powerhouse for Curation of Scientific Findings and Methodology Development for e-Scientific Publishing – LPBI Group, A Case in Point 

Electronic Scientific AGORA: Comment Exchanges by Global Scientists on Articles published in the Open Access Journal – Four Case Studies



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A Reconstructed View of Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP


There has always been Personalized Medicine if you consider the time a physician spends with a patient, which has dwindled. But the current recognition of personalized medicine refers to breakthrough advances in technological innovation in diagnostics and treatment that differentiates subclasses within diagnoses that are amenable to relapse eluding therapies.  There are just a few highlights to consider:

  1. We live in a world with other living beings that are adapting to a changing environmental stresses.
  2. Nutritional resources that have been available and made plentiful over generations are not abundant in some climates.
  3. Despite the huge impact that genomics has had on biological progress over the last century, there is a huge contribution not to be overlooked in epigenetics, metabolomics, and pathways analysis.

A Reconstructed View of Personalized Medicine

There has been much interest in ‘junk DNA’, non-coding areas of our DNA are far from being without function. DNA has two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), and the pyrimidines (cytosine [C], thymine [T], and  no uracil [U]),  while RNA contains only A, G, C, and U (no T).  The Watson-Crick proposal set the path of molecular biology for decades into the 21st century, culminating in the Human Genome Project.

There is no uncertainty about the importance of “Junk DNA”.  It is both an evolutionary remnant, and it has a role in cell regulation.  Further, the role of histones in their relationship the oligonucleotide sequences is not understood.  We now have a large output of research on noncoding RNA, including siRNA, miRNA, and others with roles other than transcription. This requires major revision of our model of cell regulatory processes.  The classic model is solely transcriptional.

  • DNA-> RNA-> Amino Acid in a protein.

Redrawn we have

  • DNA-> RNA-> DNA and
  • DNA->RNA-> protein-> DNA.

Neverthess, there were unrelated discoveries that took on huge importance.  For example, since the 1920s, the work of Warburg and Meyerhoff, followed by that of Krebs, Kaplan, Chance, and others built a solid foundation in the knowledge of enzymes, coenzymes, adenine and pyridine nucleotides, and metabolic pathways, not to mention the importance of Fe3+, Cu2+, Zn2+, and other metal cofactors.  Of huge importance was the work of Jacob, Monod and Changeux, and the effects of cooperativity in allosteric systems and of repulsion in tertiary structure of proteins related to hydrophobic and hydrophilic interactions, which involves the effect of one ligand on the binding or catalysis of another,  demonstrated by the end-product inhibition of the enzyme, L-threonine deaminase (Changeux 1961), L-isoleucine, which differs sterically from the reactant, L-threonine whereby the former could inhibit the enzyme without competing with the latter. The current view based on a variety of measurements (e.g., NMR, FRET, and single molecule studies) is a ‘‘dynamic’’ proposal by Cooper and Dryden (1984) that the distribution around the average structure changes in allostery affects the subsequent (binding) affinity at a distant site.

What else do we have to consider?  The measurement of free radicals has increased awareness of radical-induced impairment of the oxidative/antioxidative balance, essential for an understanding of disease progression.  Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Various studies have confirmed that metals activate signaling pathways and the carcinogenic effect of metals has been related to activation of mainly redox sensitive transcription factors, involving NF-kappaB, AP-1 and p53.

I have provided mechanisms explanatory for regulation of the cell that go beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, such as, the cell death pathways, expressed in apoptosis and repair.  Nevertheless, there is still a missing part of this discussion that considers the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment.

There is heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics.  There is also heterogeneity in the immediate interstices between cancer cells.  Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. In the case of breast cancer, there is interaction with estrogen , and we refer to an androgen-unresponsive prostate cancer.

Finally,  the interaction between enzyme and substrates may be conditionally unidirectional in defining the activity within the cell.  The activity of the cell is dynamically interacting and at high rates of activity.  In a study of the pyruvate kinase (PK) reaction the catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Experiments found that in there were differences in the active form of pyruvate kinase that were clearly related to the environmental condition of the assay – glycolitic or glyconeogenic. The conformational changes indicated by differential regulatory response were used to present a dynamic conformational model functioning at the active site of the enzyme. In the model, the interaction of the enzyme active site with its substrates is described concluding that induced increase in the vibrational energy levels of the active site decreases the energetic barrier for substrate induced changes at the site. Another example is the inhibition of H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate. An investigation of the inhibition revealed that a covalent bond was formed between the nicotinamide ring of the NAD+ and the enol form of pyruvate.  The isoenzymes of isocitrate dehydrogenase, IDH1 and IDH2 mutations occur in gliomas and in acute myeloid leukemias with normal karyotype. IDH1 and IDH2 mutations are remarkably specific to codons that encode conserved functionally important arginines in the active site of each enzyme. In this case, there is steric hindrance by Asp279 where the isocitrate substrate normally forms hydrogen bonds with Ser94.

Personalized medicine has been largely viewed from a lens of genomics.  But genomics is only the reading frame.  The living activities of cell processes are dynamic and occur at rapid rates.  We have to keep in mind that personalized in reference to genotype is not complete without reconciliation of phenotype, which is the reference to expressed differences in outcomes.


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Ubiquitin researchers win Nobel

Larry H. Bernstein, MD, FCAP, Curator


Ciechanover, Hershko, and Rose awarded for discovery of ubiquitin-mediated proteolysis

Nature Cell Biology 2, E171 (2000)

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2004 “for the discovery of ubiquitin-mediated protein degradation” jointly to

Aaron Ciechanover
Technion – Israel Institute of Technology, Haifa, Israel,

Avram Hershko
Technion – Israel Institute of Technology, Haifa, Israel and

Irwin Rose
University of California, Irvine, USA


Proteins labelled for destruction

Proteins build up all living things: plants, animals and therefore us humans. In the past few decades biochemistry has come a long way towards explaining how the cell produces all its various proteins. But as to thebreaking down of proteins, not so many researchers were interested. Aaron Ciechanover, Avram Hershko and Irwin Rose went against the stream and at the beginning of the 1980s discovered one of the cell’s most important cyclical processes, regulated protein degradation. For this, they are being rewarded with this year’s Nobel Prize in Chemistry.

Aaron Ciechanover, Avram Hershko and Irwin Rose have brought us to realise that the cell functions as a highly-efficient checking station where proteins are built up and broken down at a furious rate. The degradation is not indiscriminate but takes place through a process that is controlled in detail so that the proteins to be broken down at any given moment are given a molecular label, a ‘kiss of death’, to be dramatic. The labelled proteins are then fed into the cells’ “waste disposers”, the so called proteasomes, where they are chopped into small pieces and destroyed.


Avram Hershko is an Israeli biochemist and winner of the 2004 Nobel Prize for Chemistry.

Hershko (born December 31, 1937) was born as Hersko Ferenc in Karcag, Hungary. In 1950, Hershko and his family emigrated from Hungary to Israel, where he adopted the name Avram. Hershko received his M.D. and Ph.D. from the Hadassah Medical School of the Hebrew University. In 1965-67, Hershko worked as a physician in the Israel Defense Forces.

In 1969-72, Hershko was a postdoctoral fellow with the late Dr. Gordon Tomkins at the University of California, San Francisco.

In 1987, Hershko was awarded the Weizmann Prize for Sciences, an honor given to top Israeli scientists. In 1994, he won the Israeli Prize for his contributions to Israeli society through biochemistry and medicine.

In 2004, Hershko was awarded the Nobel Prize in Chemistry “for the discovery of ubiquitin-mediated protein degradation.”

Ciechanover was born in Haifa, a year before the establishment of Israel. He is the son of Bluma (Lubashevsky), a teacher of English, and Yitzhak Ciechanover, an office worker.[1] His family were Jewish immigrants from Poland before World War II.

He earned a master’s degree in science in 1971 and graduated from Hadassah Medical School in Jerusalem in 1974. He received his doctorate in biochemistry in 1981 from the Technion – Israel Institute of Technology in Haifa before conducting postdoctoral research in the laboratory of Harvey Lodish at the Whitehead Instituteat MIT from 1981-1984. He is currently a Technion Distinguished Research Professor in the Ruth and Bruce Rappaport Faculty of Medicine and Research Institute at the Technion.

Ciechanover is a member of the Israel Academy of Sciences and Humanities, the Pontifical Academy of Sciences, and is a foreign associate of the United States National Academy of Sciences.

As one of Israel’s first Nobel Laureates in Science, he is honored in playing a central role in the history of Israel and in the history of the Technion – Israel Institute of Technology


  • Ciechanover, A., Hod, Y. and Hershko, A. (1978). A Heat-stable Polypeptide Component of an ATP-dependent Proteolytic System from Reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105.
  • Ciechanover, A., Heller, H., Elias, S., Haas, A.L. and Hershko, A. (1980). ATP-dependent Conjugation of Reticulocyte Proteins with the Polypeptide Required for Protein Degradation. Proc. Natl. Acad. Sci. USA 77, 1365–1368.
  • Hershko, A. and Ciechanover, A. (1982). Mechanisms of intracellular protein breakdown. Annu. Rev. Biochem. 51, 335–364.

Interview Transcript

Transcript from an interview with the 2004 Nobel Laureates in Chemistry Aaron Ciechanover, Avram Hershko and Irwin Rose, on 9 December 2004. Interviewer is Joanna Rose, science writer.

Aaron Ciechanover, Avram Hershko and Irwin Rose during the interview

Dr Ciechanover, Dr Hershko and Dr Rose, my congratulations to the Nobel Prize and welcome to this interview. I know that you two started as medical doctors but you are in science now, and you get the prize for scientific research. How come you left medicine?

Avram Hershko: Well, I started out as a medical student, I wanted to be a doctor. And during my medical studies I studied biochemistry. That was one of the subjects that every medical student studies, so I liked it very much. I liked, you know, the whole concept of biochemistry, of looking for chemical processes in cells, so we had, we could take off one year from the studies to spend in research in the lab. I also found a very good teacher, Jacob Mager, and I wanted to spend it with him, so I did. That’s how I got involved in biochemistry. Afterwards, I finished my medical studies but already, I, after that one year, I knew that I will go to biochemistry and not to practical medicine. That’s how I started. So, it’s, it’s, like all things in life, it starts by some kind of accident or so, that was the accident, I met a subject during my studies that I liked.

And a good teacher.

Avram Hershko: And a very good teacher.

Was it also a topic, an issue that you were interested in?

Avram Hershko: No, no, not yet, not yet. Mager was interested in many subjects so that was … Actually, I continued with him after my army service as a doctor, and during the course of a couple of years I evolved in four completely different subjects, protein, synthesis, purine metabolism, and a certain disease called glucose-6-phosphate dehydrogenase deficiency, because he was interested in many things, so that gave me a very good background, a very, very, you know, very good basic background.

What about you, Dr Ciechanover?

I fell in love with biochemistry …

Aaron Ciechanover: Surely you can repeat the story verbatim. The same very story, I started in the same medical school, and after four years I decided to try and taste, I fell in love with biochemistry, too.

Like ten years later.

Aaron Ciechanover: Exactly ten years later, and I also decided to taste it, and at that time at medical school they let students take one year off for medical studies, try some research, so I went into biochemistry, same very story, different mentor. And, a wonderful mentor, and I studied lipids.


Aaron Ciechanover: Not proteins at all, and then exactly, made a decision, that that’s it. But I had, because of obligations to serve in Israel in the military as a physician. I completed my medical studies, went to serve in the army, but meanwhile, in between, I was looking already for a future mentor, in biochemistry, and Avram was at the time abroad, in the University of California in San Francisco, and I got rave recommendation, that he is a great teacher and a great biochemist, and I wrote him, and he was ready to accept me, and there started this story. More or less.

So did you go to the States?

Aaron Ciechanover: No, no, he came here. He returned to /- – -/ fellow, he started a new department in Haifa, which was a new medical school, I joined him, not initially on this project, on a different one because I still had to serve in the army. It’s a little bit complicated date-wise, but basically it’s the same very story, mentorship, the same footstep, without knowing where I am going.

You will never know.

Aaron Ciechanover: I never know, but it’s basically, ten years later the same very footsteps.

Oh, that’s funny. What about you, Dr Rose? How did you get …

Irwin Rose: I have an anomalist’s story. It doesn’t, there is no precedent for this. We moved from the east coast to the town of Spokane, Washington, when I was about 13 years old, and I did not adapt very well to the, to the style of the place, and I spent most of my time in the public library. And I enjoyed the company of the Journal of Biological Chemistry, because it was the book shaped thing, in those days, you know, it was the small journal …

Avram Hershko: At the age of 13?

Irwin Rose: No, you know, like a couple of years, you know, I was very unpopular with the other students, and so I read the Journal of Biological … the small, the small Journal of Biological Chemistry, and I found an article I thought I understood. And I read it and I thought I understood it to the point where I could make some suggestions as to how it would be, the experiment might work, and then I was very satisfied with that, and then I … I didn’t spend much time in science at that point. Went into the navy, got out of the navy, tried to go to the University of California at Berkeley, but due to the failure to find the bulletin board announcing the laboratory time of organic chemistry, I couldn’t do my organic chemistry there.

So I said OK, I’ll be a biochemist …

So I went back to the State College of Washington and there I was influenced, I would say, by the embryology teacher, who was a very strong personality in terms of academic research, he tried to encourage his students. Then I went to the University of Chicago and there was a big shock to learn all the new kinds of things that they were teaching there, in organic chemistry and that sort of stuff, and very attractive concepts, and things began to come together in my mind as to how chemistry worked and how I might be able to exploit some of the early kinds of techniques that were being used in organic chemistry into biochemistry, which was something I was attracted to, due to my reading of the Journal of Biological Chemistry. So at that point I signed up, there was a big gymnasium, and people were signing people up for which major you were going to go into. So I said OK, I’ll be a biochemist.

So I entered into the department of biochemistry, never saw the chairman of biochemistry because he was the appointed ambassador to Britain for the United States. So I floated around in the department of biochemistry and learned some interesting things, and then I began to … I never wanted to work with a mentor, because I always wanted to have my own reputation and be free to do what I wanted to do. So I worked with the weakest people in the department. Don’t make that public. No, I don’t mention the names, but … so I did that sort of thing and that way I came to learn some more independence, and once in a while I did a good experiment, and so I had more confidence that I could do research, and so that’s how it got started.

Avram Hershko: Can I mention the story that you did your PhD or eight counts per minute or …

Irwin Rose: Oh yes, well, in those days people weren’t counting, people counted on planchettes. And you …

Avram Hershko: Puckered.

Irwin Rose: Well, it could be, depends on they were flat.

Avram Hershko: You dried them, didn’t you?

Irwin Rose: Yes, you dried them out, depending … yes, that’s right. You had to dry them out, it depends on what the compound was, but if it was trillium you had to get an infinitely thin layer so that you wouldn’t get self-absorption.

Avram Hershko: It’s common, self-absorption on a planchette.

Irwin Rose: Did you guys do that, too?

Aaron Ciechanover: Yeah, yeah, yeah.

Avram Hershko: We had a counter with only three /- – -/ so we moved it like that …

Irwin Rose: Oh yeah, yeah.

Avram Hershko: … it was a big excitement.

Irwin Rose: So I wasn’t that primitive. You were doing these things in Israel, an advanced state.

Aaron Ciechanover: You came to our country.

Irwin Rose: I did. I came to Israel. But anyway, yes, so we did those things. And even if you had eight counts above background, if there were eight, there were eight. That’s right. So you could do some experiments. That’s how it worked out.

So how did you meet together?

Avram Hershko: Well, that’s another story. I got interested in protein degradation during my post-doc fellowship in San Francisco, and when I came back to Israel I continued with that, and at that time it was a very obscure field, you know. People, there were all kinds of, not too many people were interested in it. Those that were interested were not very good. So I looked for somebody, and so my first time I think I came up and I looked for somebody to spend a sabbatical with. I couldn’t find anybody that attracted me. So then I met Ernie at a meeting in 1976, one year before, before my sabbatical was due. And do you remember, we met in the breakfast, so I said can I, just began to talk …

Irwin Rose: It’s alright, I forgot.

… it turned out that he was interested in protein degradation. And that was a secret …

Avram Hershko: … breakfast table, so I knew who he was, he was very well known for his work on enzyme mechanism. That I knew, but then I asked him what are you interested in, in other things? So it turned out that he was interested in protein degradation. And that was a secret, it was a secret because he never published anything on it, and I asked him how come you never published anything, and so he said there is nothing worth publishing on protein degradation. So that’s what he said.

Irwin Rose: Yeah, that was my opinion. Well, because I hadn’t done anything, you don’t say it right.

Avram Hershko: OK. Well, that’s how I remember it. And anyhow, I liked that attitude very much, and asked, I asked him can I spend my sabbatical with you? And he said yes, so that’s how it started, and then Aaron, the same year he started his PhD with me, and after my sabbatical the following, the summer after my sabbatical, Aaron joined us, and then he joined us for a couple of summers afterwards, so that’s how, that’s how the whole connection started.

But how come you pick up an obscure field in science, to work on?

Irwin Rose: Well, I’ll tell you, because when I first worked at Yale, the guy who had a lab next to me had made the original observation that there was a protein, there was an energy dependent on protein breakdown. Now, nobody believed him, but he had made some pretty strong observations that if you …

Avram Hershko: Here, we could mention names.

Irwin Rose: Yes, Melvin Simpson. He made these important observations.

Aaron Ciechanover: He hardly believed himself, because when you go into discussion on the paper, you kind of come to a convoluted argument whether it’s a direct requirement or indirect. We can do the conclusion that it’s indirect.

When was it?

Avram Hershko: 1953, so …

Irwin Rose: So I didn’t read the paper, but I had this man in the laboratory next to me and he said, he made this observation and I got very interested in it. And worked on it for, on sabbatical, and when I went to England and when I went to Israel I got mice from Mager, it turned out the same guy, but he wasn’t there at the time, and … but I never found an energy dependence on the protein breakdown. And it turns out later on that a fellow named Art Haas who had been a post doc with me, made the observation that if you’re not careful when you break cells, there’s a lysosomal enzyme that degrades the ubiquitin. So I never would have found it, you know. Somebody else had to make the observation that you could make a self-resistent that … that would show an ATP dependence on protein breakdown. It was not for me, but I did work on it earlier, and that’s the, that’s why I told you that I’d never made any important observations.

But you three work together. How does it work, to do things together?

Irwin Rose: I don’t do anything.

You do nothing? Who is the worker?

Avram Hershko: Well, that’s, first of all, that’s not true. I remember that you made some ubiquitin preparation …

Irwin Rose: I did.

Avram Hershko: Yes, and it fell on the floor, and then you collected it up from the floor … yeah, yeah. That first step is to boil the extra, because ubiquitin is heat stable, so you boiled it but then it fell on the floor, but you picked it up and it was good, yeah.

Irwin Rose: It was good, nothing could destroy it.

Irwin Rose: It was a licence only enzyme.

Aaron Ciechanover: The /- – -/ can take it, but not the floor.

Avram Hershko: But, yeah, but when I came to his lab we already had his first step, which was the fractionation, well, the reticulocyte cell-free system system was actually established in the laboratory of somebody else, Alfred Goldberg in Harvard, but they didn’t …

Aaron Ciechanover: /Inaudible./

Avram Hershko: No, no, but, yeah, but he made it first, he made it first.

Aaron Ciechanover: The first publication was from Harvard, no doubt.

Avram Hershko: But then he didn’t progress, but then he didn’t do what he should have done, which is fractionation. It’s hard to purify right away, but ATP dependent enzyme, he never found it. And what we did was fractionation and constitution, so we already had this first step of separating it into two, two fractions, fraction one and fraction two.

… we didn’t really understand that it’s binding …

So during these two years between the beginning of ’77 when I write to your lab and December of ’79, when we made the breakthrough in your lab, we purified the component from fraction one, we found it a heat stable protein, and then you had a part in that, you also boiled ubiquitin, and then in Haifa we found that it gets … when we labelled it with iodine and we found it gets bound to proteins and ATP dependent reaction, but we didn’t really understand that it’s binding, its co-herent binding the substate until that summer in 1971 in the laboratory of Rose where you invited me, together with Aaron who was then my graduate student in /- – -/ who was there. 1979, 1979. So that is when, when the discovery that ubiquitin …

Irwin Rose: Shall I tell the story about the ubiquitin?

Avram Hershko: Yes. I think I have finished. So then, that’s how I remember it, and how …

Irwin Rose: OK, well, here they had a heat stable factor that was required, and they made the observation that the ubiquitin went on to proteins. And so one of my post docs went to a post doc of another student, of another faculty member at the Fox Chase Cancer Centre, and said, there was a conversation, and do you know of any examples of a protein covalently linked to a protein? And this post doctoral fellow said yes, there is in the nucleus, a protein called ubiquitin that’s covalently linked to histone. And so they rushed to look at the amino acid composition of that so-called ubiquitin, and they compared it to the amino acid composition which you had published, I guess …

Aaron Ciechanover: No, not yet.

Irwin Rose: Not yet published.

Aaron Ciechanover: But in the end it was published back to back with JBC.

Irwin Rose: No, no, no. But how did they know the conversation …

Aaron Ciechanover: No, because they knew, the end story is that the Wilkinson paper came back to back with ours on the /- – -/.

Avram Hershko: OK. Let’s not go into the detail.

Irwin Rose: Well, for some reason or other, they found confidence…

Avram Hershko: They knew that I published that.

Irwin Rose: Really, and I was not a leak.

Avram Hershko: No, no, you were not.

Aaron Ciechanover: No, he was in the lab, he was free and did this. We didn’t hide anything.

Irwin Rose: OK, you’re getting the inside story here. Now, wait a second.

I have a statement from your colleague. “At first nobody cared about your work, and those that knew something about it, they didn’t believe it.” Was it so …?

Irwin Rose: Who said that?

Avram Hershko: That was, that was Fred Goldberg, yeah.

Aaron Ciechanover: Let’s not mention names.

Avram Hershko: Oh! No, we didn’t mention names.

That citation is right.

Aaron Ciechanover: I’ll tell you, I’ll tell you a funny story. I left the lab in ’81, basically after my PhD was completed I submitted it and I went to Harvard, I went to MIT to do a post doc fellow, and Harvard carried out weekly seminars. And in this weekly seminar, one of the founders in the field of proteolysis, one of the originally, not the founder, but it doesn’t matter. A famous scientist in the field presented the weekly seminar at Harvard. I knew of him because he was our competitor for many years, and I went to hear the seminar, so I crossed the river by the bus, I took the shuttle bus that goes /- – -/ and I was sitting in the very back bench. And this was probably about two weeks before you came to visit, it was the very beginning of my, do you remember when I met you, I came to the airport to pick you up.

Avram Hershko: Yeah, yeah.

Aaron Ciechanover: And then, near me, was sitting a very famous scientist that I only later realised that his name is Arthur Dee, a very famous scientist, and after this presentation of the professor, this was only ’81 when we had like eight or nine papers already in the literature with a huge amount of information there. And he was a protein researcher and he raised his hand, I remember very well, and the other guy, when we were both  /- – -/ he said, you know, I have a question to ask you. There is a fellow in Haifa by the name of Hershko, and another one with a very complicated Polish name that I cannot even pronounce, that published a series of papers on a small protein that is attached to other proteins and marks them for degradation, can you comment on it? And he basically dismissed it as an artefact.

… it adds to our benefit, because they left us alone for seven successive years …

And I don’t, I don’t criticise him, all I’m telling you it was symbolic for me enough for after eight papers in the literature, this was the spirit in the field from people who worked in the field, and there were very few. As a matter of fact, it adds to our benefit, because they left us alone for seven successive years, even after I left the lab to work out basically the entire system. The next scientist to join the field was a scientist at MIT, Alex Varshavsky, who joined in ’84, ’83, but published in ’84, and given I was there and collaborated, so for seven successive years they let us lay the entire stone down in the literature so I don’t criticise him, actually I appreciate him tremendously for letting us do it. You know, in retrospect.

But I wonder, how do you survive as a scientist when nobody believes you somehow? Nobody’s interested. You become kind of non-visible.

Irwin Rose: You’re making observations, and the observations get published, so the observations are true. Whether anybody will say that belongs to a big story like it turns out to be is not predictable, but so you don’t make claims like that. You say that this is very interesting and so on and so on and so on, and you keep following it up, and it doesn’t necessarily become the centre of attention yet, until you build a big enough story. I think that’s the way it works.

We all survive because funding for research was generous in those days, you know. It’s been less generous now, and we have a peer review system which is more critical and so I think you have to, you have to add successively to the picture you’re trying to portray. It’s not sufficient to just provide data. So I think that’s part of it. But I agree that it’s important to be left alone for a sufficient amount of time in order to be able to do it, and not feel that you’re in the middle of a big activity already, so you know, you need to do that sort of thing.

So do you think you would get support today for such work, which was kind of apart?

Avram Hershko: Well, I hope the fund /- – -/ look up your website and will hear these things. Because it’s … yeah, Joe Goldstein, you know, a Nobel Laureate and a good one, wrote a nice article about this year’s Lasker Award, in which he compared science to a sculpture by this British sculptor who had his stone, it was a huge stone of two and a half ton, on which another stone, and another stone, and another stone, and at the end is a little stone, so he said that in science there are big stones and small stones. The important science is the opposite. When you have a little stone, and on top of it you put a bigger stone and then a bigger stone. If you throw out a big stone at the beginning so there’s a lot of publicity sometimes nothing comes out of it, and the scientist, to find his little stone, on which the other stones can be built. So I recommend to read his article.

Now you find the small stones, Dr Rose, in your kitchen, as I understand it. You have a small laboratory there?

Irwin Rose: You want to talk about my kitchen?

Yeah. Your laboratory, I would say.

Irwin Rose: Well, when I retired from Fox Chase I took my spectrophotometer and a lot of my chemicals, based on a sort of suggestion of Dr … his recommendation. So I took all my chemicals and my spectrophotometer and my constant temperature bath and so forth with me to Irvine, and when the person whose laboratory I was sitting decided to retire, I had to do something with the spectrophotometer and so I found a place in my kitchen for it. And this was very convenient because it saved me a lot of time. I didn’t have to go to work every day and if I had a little experiment to do I could do it in my kitchen. So that was very good, although I’ve got a lot of chemicals that I have no use for and I’d like to take them back.

Aaron Ciechanover: Send them over, send them over.

Irwin Rose: I’ll send them over. I’ll get a box.

Avram Hershko: But I worry that you don’t have an ice machine. You need an ice machine.

Irwin Rose: No, I don’t have an ice machine. But I have a freezer and I can make ice cubes and I can break them up.

It’s kind of worrying, in science. So you can work when everything’s /- – -/ ?

Irwin Rose: Yeah, that’s right, exactly.

So are you the kind of scientists that work all day and all night long, kind of nerd scientists?

So that’s my recommendation, do not retire. Do not retire fellas. …

Irwin Rose: I think we all work all day and all night long. I do. I don’t have any hobbies, you know, I’m very embarrassed when people ask me what are my hobbies, I don’t have any hobbies. I mean, it’s just enough to keep up with the things I’m trying to solve. You know, I used to work on little puzzles and so on and so forth. Each puzzle requires attention and, so you get an idea. You get your ideas at different times. Sometimes your wife makes a statement and you say: aha, maybe you’re right. And so you go off to your kitchen, and do a little experiment, so you try to, that’s the way you make progress, if you continue these things. So that’s my recommendation, do not retire. Do not retire fellas.

Avram Hershko: I won’t.

Aaron Ciechanover: I’m never going to.

You worked together in the beginning, you were the graduate student of Dr Hershko, how was it to separate from each other?

Aaron Ciechanover: Well, it’s the nature of science, I think, because you know, you graduate, you go your post doctorate fellowship, and Avram was gracious enough to bring me back, but now is independent and that’s the entire idea, if you bring a young scientist back, you give him a bench, start up funds, and then you tell him now in five years, come back in five years, and show the committees that you worked for something. So actually, you know, it would be unnatural if we would have continued to work together. So, each of us is independent. Now we’re in the same institute and that’s the whole idea of children that grow up, students that become their own, scientists on their own, I think that’s the way.

Do you compete with each other?

Avram Hershko: No, there is enough to do in the ubiquitin field, we don’t feel that we had to compete. There are different aspects of the ubiquitin field. I am working on cell cycle and he works on …

Aaron Ciechanover: /- – -/. Completely different.

How is it to live in a small country with big problems and to get funds for science?

Avram Hershko: It is not easy, it is not easy. You have to know the daily tension which is of course distractive. The funds are small, some funds for science are small. Graduate students have to go to serve in the army and things like that, so it’s more difficult than elsewhere, but it’s possible, it’s possible.

And now everybody’s happy. About the Nobel Prize. So thank you very much for sharing your thoughts with us, and being with us.


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MLA style: “Transcript from an interview with the 2004 Nobel Laureates in Chemistry Aaron Ciechanover, Avram Hershko and Irwin Rose, on 9 December 2004”. Nobel Media AB 2014. Web. 5 Sep 2015. <>


Other related articles published in this Open Access Online Scientific Journal include the following:


Innovations in Israel – Nobel Prize in Chemistry 2004, 2011

Reporter: Aviva Lev-Ari, PhD, RN


Ubiquitin Pathway Involved in Neurodegenerative Diseases

Larry H Bernstein, MD,  FCAP


Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator: Larry H Bernstein, MD, FCAP


Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD


Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 






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Protein-binding, Protein-Protein interactions & Therapeutic Implications

Writer and Curator: Larry H. Bernstein, MD, FCAP 

7.3  Protein-binding, Protein-Protein interactions & Therapeutic Implications

7.3.1 Action at a Distance. Allostery_Delabarre_allostery review

7.3.2 Chemical proteomics approaches to examine novel histone modifications

7.3.3 Misfolded Proteins – from Little Villains to Little Helpers… Against Cancer

7.3.4 Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer

7.3.5 Putting together structures of epidermal growth factor receptors

7.3.6 Complex Relationship between Ligand Binding and Dimerization in the Epidermal Growth Factor Receptor

7.3.7 IGFBP-2.PTEN- A critical interaction for tumors and for general physiology

7.3.8 Emerging-roles-for-the-Ph-sensing-G-protein-coupled-receptor

7.3.9 Protein amino-terminal modifications and proteomic approaches for N-terminal profiling

7.3.10 Protein homeostasis networks in physiology and disease

7.3.11 Proteome sequencing goes deep

7.3.1 Action at a Distance. Allostery_Delabarre_allostery review

DeLaBarre B1Hurov J1Cianchetta G1Murray S1Dang L2.
Chem Biol. 2014 Sep 18; 21(9):1143-61

Cancer cells must carefully regulate their metabolism to maintain growth and division under varying nutrient and oxygen levels. Compelling data support the investigation of numerous enzymes as therapeutic targets to exploit metabolic vulnerabilities common to several cancer types. We discuss the rationale for developing such drugs and review three targets with central roles in metabolic pathways crucial for cancer cell growth: pyruvate kinase muscle isozyme splice variant 2 (PKM2) in glycolysis, glutaminase in glutaminolysis, and mutations in isocitrate dehydrogenase 1 and 2 isozymes (IDH1/2) in the tricarboxylic acid cycle. These targets exemplify the drugging approach to cancer metabolism, with allosteric modulation being the common theme. The first glutaminase and mutant IDH1/2 inhibitors have entered clinical testing, and early data are promising. Cancer metabolism provides a wealth of novel targets, and targeting allosteric sites promises to yield selective drugs with the potential to transform clinical outcomes across many cancer types.

Based on knowledge acquired to date, there is no doubt that cancer metabolism provides a wealth of novel therapeutic targets and multiple innovative ways in which to exploit metabolic vulnerabilities for therapeutic benefit. More comprehensive reviews cover the breadth of metabolic targets that are currently under investigation (Stine and Dang, 2013; Vander Heiden, 2011). The following sections of this review focus on PKM2, glutaminase, and mutated IDH1/2 as exemplary metabolism targets under investigation for development of cancer therapies.
Drugging Glycolysis: Targeting Pyruvate Kinase Muscle Isozyme Alternative Splice Variant 2 PK catalyzes the last step of glycolysis, converting phosphoenolpyruvate (PEP) to pyruvate, while producing one molecule of ATP. The reaction encompasses two chemical steps: the first involves a phosphoryl transfer from PEP to ADP, forming an enolate intermediate and ATP, and the second involves protonation of the enolate intermediate, forming pyruvate (Robinson and Rose, 1972). PKM2 is one of four PK isoforms in humans. PKM1 and PKM2 result from the alternative splicing of exons 9 and 10 of the PKM gene, which encode a stretch of amino acids that differ at 23 positions between PKM1 and PKM2. PKM1 is constitutively active in skeletal muscle and brain tissue, but is not allosterically regulated. PKM2 is expressed in fetal and proliferating tissues, has low basal activity compared with PKM1, and is allosterically regulated. R-type pyruvate kinase (PKR) and L-type pyruvate kinase (PKL) are transcribed via different promoters from the PKLR gene. PKR is expressed in erythrocytes and PKL in the liver. PKR, PKL, and PKM1 exist as stable tetramers,whereas PKM2 forms tetramers (high activity form), dimers (low activity form), and monomers (Mazurek, 2011).

Figure 1. Central Metabolic Pathways Utilized by Cancer Cells *denotes mutated isoenzyme.

Pyruvate Kinase Muscle Isozyme Alternative Splice Variant 2 in Cancer Cell Metabolism Cancer cells predominantly express PKM2, which can be downregulated by tyrosine kinase growth factor signaling pathways, allowing metabolic flexibility. Phosphotyrosine peptides have been shown to suppress PKM2 activity by binding tightly to PKM2, thereby catalyzing the release of fructose 1,6-bisphosphate (FBP), resulting in a switch to the low activity dimer state (Christofk et al., 2008b; Hitosugi et al., 2009). This downregulation is thought to support tumor growth and proliferation by allowing for the shunting of glycolytic intermediates toward other biosynthetic pathways (i.e., pentose phosphate and serine pathways). In keeping with this model, the activation of PKM2 in cancer cells using small molecule agonists resulted in serine auxotrophy (Kung et al., 2012). Consistent with the hypothesis that PKM2 is a critical metabolic switch, there is growing evidence that, depending on the cellular stress environment, PKM2activity canberegulated byposttranslational modification such as acetylation (Lv et al., 2011), phosphorylation (Hitosugi et al., 2009), cysteine oxidation (Anastasiou et al., 2011), and proline hydroxylation (Luo et al., 2011). The utility of PKM2 activators in the clinic has yet to be determined, but recent work with tumor xenografts with a PKM2 activator suggests that this may be a viable approach (Parnell et al., 2013). As PKM2 tetramers show greater than 50-fold higher activity than PKM2 monomers (Anastasiou et al., 2012), one consideration when designing drugs to activate PKM2 for therapeutic means would be the need for small-molecule ligands capable of driving the enzyme toward its optimally active tetrameric form, thus forcing cancer cells into a less flexible metabolic state.

Structure of Pyruvate Kinase Muscle Isozyme Alternative Splice Variant 2 The structure of the PKM2 tetramer is summarized in Figure 2A. PKM2 is allosterically activated in a ‘‘feedforward’’ manner by the upstream glycolytic metabolite, FBP, which induces a shift to the active tetrameric conformation (Christofk et al., 2008b; Dombrauckas et al., 2005). PKM2 can be independently allosterically activated by serine (Chaneton et al., 2012), which binds in a distinct pocket that can also accommodate the inhibitor phenylalanine (Protein Data Bank [PDB] ID: 4FXJ). The binding of phenylalanine results in a tetrameric form distinct from the active conformer (Morgan et al., 2013). It is not clear how the change from serine to phenylalanine elicits such a dramatic change in protein behavior, or whether there is any biological interaction between serine activation and phenylalanine inhibition of PKM2 in cancer cells. Of note, PKM1 and PKL/R are not activated by serine, despite the conservation of the serine binding site in all PK isoforms.
Figure 2. Three Different Metabolic Enzymes and Their Allosteric Inhibitors Protomers are depicted as cartoon ribbons in blue, green, yellow, and cyan. Synthetic allostery is depicted in stick format with red highlight. (A) Structure of tetrameric PKM2:AGI-980 (4:2 complex) from PDB 4G1N. AGI-980 is shown in stick rendering near the center of tetramer. Each PK monomer consists of four domains, usually designated A, B, C, and N (Dombrauckas et al., 2005). The tetramer is a dimer-of-dimers with approximate D2 symmetry. The dimer is formed between the A domains of each monomer, while the tetramer is formed via dimerization along the C subunit interfaces of each dimer. The active site of PKM2 lies within a cleft between the A and B domain, illustrated by a PEP analog (red spheres). The FBP binding pocket is located entirely within the C domain (pink spheres). The natural allosteric site of serine is also shown (black spheres). (B)Tetrameric GAC:BPTES (4:2 complex) from PDB 3UO9. Glutamate molecules are shown as spheres. (C) Dimeric IDH2R140Q:AGI-6780 (2:1 complex) from PDB 4JA8 (Wang et al., 2013). NADP molecules are shown as spheres.
Discovery of Allosteric Activators of Pyruvate Kinase Muscle Isozyme Alternative Splice Variant 2 A number of small molecules that potently activate PKM2 have been discovered by various groups (Table 1). Interestingly, all seven X-rayco-complexescurrentlyavailableshowcompoundsbound at a novel binding pocket distinct from the FBP and serine binding sites, which would otherwise allow cells to overcome negative regulation by phosphotyrosines (Kung et al., 2012). The compounds found in structures 3GQY, 3GR4 (Boxer et al., 2010), 3H6O (Jiang et al., 2010), 3ME3, and 3U2Z (Anastasiou et al., 2012) were identified by screening the NIH Small Molecule Repository, and can be classified into two distinct chemical series, both of which establish very similar interactions with PKM2 (Table 1). Analogues in these two classes selectively activated PKM2 allosterically with good selectivity against PKM1, PKL, and PKR (Anastasiou et al., 2012; Boxer et al., 2010; Jiang et al., 2010). The molecule found in the structure 4JPG (Guo et al., 2013) is similar to the two series described above, where the pyrimidone ring is found between the two Phe26 residues (Table 1). Interestingly, the activator found in the 4G1N structure (Kung et al., 2012) sits in the same pocket as the NIH compounds. However, the interactions are quite different, with the side chains of the two Phe26 that line the pocket assuming distinct conformations. This activator wraps around the two aromatic residues, which pushes it closer to the walls of the pocket, allowing for a richer series of interactions with PKM2 (Table 1). There are two additional series of PKM2 activators that have been reported for which no structural information is available (Table 1)(Parnell et al., 2013; Xu et al., 2014; Yacovan et al., 2012). Members of this series were shown to have an activation level comparable to that of FBP, with selectivity for PKM2 over PKL, PKR, and PKM1. PKM2 offers a very interesting example of an allosterically regulated enzyme. Different allosteric sites have so far been identified for three different types of activator (FBP, serine, and small-molecule ligands) and all activate PKM2 by stabilizing the tetrameric form. It is remarkable that molecules as small as serine can dramatically alter this protein’s conformational landscape and aggregation state and lead to an active enzyme. This unusual allosteric site revealed by the small-molecule ligands is of particular curiosity, largely because neither its function nor its native ligands are known. All of the drug-like activators described above bind at the dimer–dimer interface and seem to act by displacing water from the mainly apolar pocket, thus contributing to the stabilization of the tetramer. While these PKM2 activators show promising preclinical data, none have yet entered clinical development.

Table 1. Biochemical Properties of Small Molecule PKM2 Inhibitors Series PDB ID Ligand Reference Binding Characteristics

Substituted N,N’diarylsulfonamide 3GQY (Boxer et al., 2010)

  •  All completely buried within A-A’ interface, 35A ˚ from FBP pocket
  •  Binding pocket lined with residues equivalent to those of PKM2 molecules forming A-A’ interface
  •  All sandwiched between phenyl rings of the two Phe26 from different monomers
  •  All additionally interact with side chain of Phe26 through slightly distorted T-shaped p-p interactions (two such interactions for substituted N,N0diarylsulfonamides and one for thieno[3,2-b]pyrrole[3,2-] pyridazinones)
  1. 3GR4 (Boxer et al., 2010) 3ME3 (Anastasiou et al., 2012)
  2. Thieno[3,2-b]pyrrole [3,2-d]pyridazinone 3H6O (Jiang et al., 2010)
  3. 3U2Z (Anastasiou et al., 2012)
  4. 2-((1H-benzo[d]imidazol1-yl)methyl)-4H-pyrido [1,2-a]pyrimidin-4-ones
  5. 4JPG (Guo et al., 2013)
  • Pyrimidone ring found between the two Phe26 residues forming p-p interactions with the aromatic rings
  • Carbonyl interacts with a bridging water molecule
  • Benzimidazole reaches a region of the activator pocket that is not occupied in any of the published crystal structures
  • One of the imidazole nitrogens forms an H-bond with Lys311, which is normally part of a salt bridge to Asp354

Quinolone sulfonamides 4G1N (Kung et al., 2012)

  •  Quinoline moiety sits on a flat, mainly apolar surface defined by Phe26, Leu27 and Met30 from chain A and Phe26, Tyr390 and Leu394 from chain A’
  •  One of the two oxygen atoms of the sulfonamide accepts an H bond from the backbone oxygen of Tyr390, the other interacts with a water molecule
  •  The oxygen of the amide moiety forms an H-bond with side-chain nitrogen of Lys311
  •  Terminal aromatic ring sits in the other copy of the quinoline pocket d Aromatic rings of the side chains of the two Phe26 lining the pocket almost perpendicular (not parallel); activator wrapped around the two aromatic residues

3-(trifluoromethyl)-1Hpyrazole-5-carboxamide (Parnell et al., 2013; Xu et al., 2014)

  • Cocrystal structure of one compound bound to tetrameric PKM2 obtained but file not available for download from PDB: described as bound to the allosteric site at the dimer–dimer interface

5-((2,3-dihydrobenzo[b] [1,4]dioxin-6-yl)sulfonyl)-2methyl-1-(methylsulfonyl) indoline scaffold (Yacovan et al., 2012)

  • Cocrystal structure of one compound bound to PKM2 obtained but not available for download from the PDB: described as bound to dimer interface
  • Interactions very similar to those established by thieno [3,2-b]pyrrole[3,2-d]pyridazinone series above

Drugging Glutaminolysis: Targeting the Glutaminase C Variant Glutaminase catalyzes the conversion of glutamine to glutamate and ammonia. Glutamate can be oxidized to a-ketoglutarate (aKG), which then anaplerotically feeds into the TCA cycle as a means of providing proliferating cells with biosynthetic intermediates and ATP (Figure 1); glutamate is also used as a substrate for the generation of glutathione, which provides protection from redox stress (Hensley et al., 2013; Shanware et al., 2011). The ammonia produced during the reaction can be used in certain tissues like the kidney to provide pH homeostasis, and nitrogen derived from glutamine is utilized in nucleotide biosynthetic and glycosylation pathways.

Table 2. Characteristics of Small Molecule Glutaminase Inhibitors

BPTES N-(5–[1,3,4]thiadiazol-2yl)-2-phenylacetamide 6 (Shukla et al., 2012)

  • Similar potency but better water solubility vs. BPTES d Attenuated growth of P493 human lymphoma B cells in vitro d Diminished tumor growth in P493 tumor xenograft SCID mice with no apparent toxicity

CB-839 (Calithera) (Gross et al., 2014)

  • Orally bioavailable d Binds at allosteric sites of GLS1 KGA and GAC d Potent, selective, time-dependent reversible inhibition with slow recovery time
  • Anti-proliferative activity (double-digit nM potency) in cellular proliferation assays in wide range of tumors
  • Currently in Phase I trials of locally-advanced/metastatic refractory solid tumors (triple negative breast cancer, NSCLC, RCC, mesothelioma) and hematological cancers [ NCT02071927, NCT02071862, NCT02071888]

Dibenzophenanthridines Compound 968 (Katt et al., 2012; Wang et al., 2010)

  • Modest potency in the low mM concentrations d Loses all inhibitory activity against glutaminase activated by preincubation with inorganic phosphate (phosphate does not affect BPTES potency)
  • Anti-proliferative activity in breast cancer cell line at 10 mmol/L concentration

There are three isoforms of IDH. IDH1 is located in both the peroxisome and the cytosol, whereas IDH2 and IDH3 are located in mitochondria. It is unclear what the relative contributions of the IDH2 and IDH3 isoforms are to overall mitochondrial TCA function. IDH1 and IDH2 are both obligatory homodimeric proteins and use NADP+ as a cofactor, whereas IDH3 uses NAD+ as a cofactor and is a heterotrimeric protein comprising alpha, beta, and gamma subunits. All three isozymes require either Mg2+ or Mn2+ asdivalent metal cofactors for catalysis.The dimeric structure of IDH2 is shown in Figure 2C.

Mutant Isocitrate Dehydrogenase in Cancer Cell Metabolism The role of IDH mutations in cancer metabolism was recognized following the observation of frequent and recurrent mutations of IDH1 and IDH2 in patients with glioma and AML, initially identified by genomic deep sequencing and subsequent comparative genetic analyses (Parsons et al., 2008; Yan et al., 2009). These mutations were originally characterized as loss of function (Mardis etal.,2009; Parsonsetal.,2008; Yanet al.,2009), suggesting that mutated IDH acts as a tumor suppressor due to the loss of catalytic conversion of isocitrate to aKG (Zhaoetal., 2009). However, with the exception of cases of haploinsufficiency, the heterozygous mutation pattern of IDH is more consistent with an oncogene role. Subsequently, IDH mutations were shown to possess the neomorphic activity to generate the oncometabolite, 2-hydroxyglutarate (2HG) (Dang et al., 2009; Gross et al., 2010; Ward et al., 2010). With a single codon substitution, the kinetic properties of the mutant IDH isozyme are significantly altered, resulting in an obligatory sequential ordered reaction in the reverse direction (Rendina et al., 2013). Indeed, the critical kinetic observation of mutant IDH was not only the loss of affinity for isocitrate, but also a dramatic increase in NADPH affinity by three orders of magnitude (Dang et al.,2009), suggesting a substantial change in protein dynamics imparted by the mutation. The only known homeostatic 2HG clearance mechanism is the relatively inefficient reconversion of 2HG back to aKG by D-2hydroxyglutarate dehydrogenase. Therefore, 2HG accumulates when over-produced by mutant IDH. 2HG itself has been shown to be sufficient to drive the malignant phenotype (Rakheja et al., 2013). Abnormally high 2HG levels impair aKG-dependent dioxygenases through competitive inhibition, including those that modify DNA and histones (i.e., Jumonji domain-containing histone demethylases and the ten-eleven translocation (TET) family of 50-methylcytosine hydroxylases) (Chowdhury et al., 2011; Figueroa et al., 2010), as well as EglN prolyl hydroxylase in regulating hypoxia-inducible factor (Losman et al., 2013). This results in altered epigenetic status that blocks cell differentiation. These findings, combined with the inhibitory effects of fumarate and succinate on the same families of aKG-dependent enzymes, highlight a critical and fascinatingnetwork that ties together central metabolic pathways and epigenetic control. Remarkably, mutations in TET2 are mutually exclusive with IDH mutations in AML, strongly suggesting that, in this context, the tumorigenic effects of 2HG are at least in part driven by inhibition of TET2. The precise targets of IDH mutations with associated 2HG production (and TET2 mutations) that promote tumorigenesis are currentlyunknown;however,itisclearthatIDH1/2andTET2mutations lead to a block in hematopoietic cell differentiation (Figueroa et al., 2010; Lu et al., 2012; Moran-Crusio et al., 2011; Wang et al., 2013). To date, no IDH3 mutation associated with cancer has been reported (Krell et al., 2011; Reitman and Yan, 2010), suggesting that the role of IDH1/2 has a greater impact on tumorigenesis. Targeting mutated isoforms of IDH1/2 therefore presents a logical approach to cancer therapy. A consideration in designing suchdrugsistheheterozygoussomaticnatureoftheIDH1/2mutation, which likely yields a mixture of homo- and heterodimers; statistically, heterodimers should be the major species in vivo. Mutant homodimers and wild-type-mutant heterodimers both efficiently catalyze the production of 2HG from aKG (Dang et al., 2009; Rendina et al., 2013). However, the heterodimer is potentially more oncogenic, as it is more efficient at producing 2HG than homodimeric mutants (Pietrak et al., 2011) due to an increased local concentration of substrate while conserving NADPH. The heterodimer as a molecular target therefore becomes an important consideration in this scenario.

Structure of Isocitrate Dehydrogenase Structurally, both IDH1 and IDH2 comprise three main domains: the large domain, the small domain, and the clasp region (Yang et al., 2010). A simplified description of protein motion is provided in Figure 3 (Rendina et al., 2013; Xu et al., 2004). The dynamic of motion may differ slightly between IDH1 and IDH2 mutants. IDH1 mutants appear to open wider than IDH2 mutants to the point of unwinding a helix termed ‘‘seg2’’ (Yang et al., 2010). In contrast, the open form of IDH2 does not involve the melting of any secondary structure, and as a consequence has a much narrower range of motion (Taylor et al., 2008; Wang et al., 2013). This differential in protein dynamics could possibly explain the differential responses of IDH1 and IDH2 to inhibitors. X-ray structures of IDH3 have not yet been reported, but it appears to be distinct from IDH1 and IDH2 in terms of primary sequence and predicted quaternary organization (Kim et al., 1995; Ramachandran and Colman, 1980). There are three arginine residues in the enzyme active site that are predicted to play a central role in electrostatic stabilization and proper geometric orientation of isocitrate via its acidic moieties as the substrate binds in the active site. With the exception of the novel G97D or G97N codon mutation (Ward et al., 2012), virtually all confirmed IDH mutations that generate high levels of 2HG occur in one of these arginines (i.e., IDH1-R132 and IDH2-R172/R140) (Losman and Kaelin, 2013) and have in common a substitution of one of the diffuse positive charges of the respective arginine’s guanidinium moiety.
Discovery of Inhibitors against Mutated Isocitrate Dehydrogenase Several inhibitors of mutant IDH isoforms that block 2HG production in vitro and in vivo have been recently described. The first potent and specific IDH1 inhibitors reported were the phenylglycine series, specifically AGI-5198 (Popovici-Muller et al., 2012; Rohle et al., 2013) and subsequently ML309 (Davis et al., 2014)(Table 3), which were shown to be rapid-equilibrium inhibitors specific for IDH1-R132-codon mutations. These compounds inhibited IDH1-R132H competitively with respect to aKG and uncompetitively with respect to NADPH, suggesting that they preferably bind to the enzyme-NADPH ternary complex. Notably, they do not appreciably cross-react against the IDH2-R140Q mutant isozyme, suggesting a unique binding mode in IDH1-R132 that does not favorably exist in IDH2R140. Because no X-ray co-complex has been reported for this series, the exact mode of binding cannot be ascertained at this time. Preclinical data indicated 2HG inhibition and antitumor effects in vitro and in vivo (Table 3). These phenylglycine compounds appear to be excellent chemical tools for tumor biology investigation, but optimization of their properties is likely required for further therapeutic development. Co-complexes of IDH1-R132H with two different 1-hydroxypyridin-2-one inhibitors have been reported (Zheng et al., 2013), but the quality of the crystal structure data supporting the mechanism of inhibition is poor. AG-120, a selective, potent inhibitor of mutated IDH1, is currently in clinical development for the treatment of cancers with IDH1 mutations (Table 3), but there is currently no published information on this inhibitor. Another inhibitor of mutated IDH1 has been reported recently (Table 3) (Deng et al., 2014). Co-complex X-ray studies revealed that Compound1 binds mutated IDH1 allosterically at the dimer interface resulting in an asymmetric open conformation. Distinctively, Compound 1 displaces the conserved catalytic Tyr139 and further disrupts the Mg2+ binding network, consistent with kinetic results of competitive inhibition with respect to Mg2+, but not with aKG substrate. Others have reported modeling of inhibitors into the active site of IDH1, but experimental evidence is lacking (Chaturvedi et al., 2013; Davis et al., 2014). The first reported potent and selective IDH2 inhibitor was the urea-sulfonamide series, AGI-6780 (Wang et al., 2013), a timedependent slow-tight binder to IDH2-R140Q exhibiting noncompetitive inhibition with respect to substrate and uncompetitive inhibition with respect to NADPH, and nanomolar potency for 2HG inhibition (Table 3). This compound showed good inhibitory selectivity for IDH2-R140Q, with no effect on the closely related IDH1 and IDH1-R132H isozymes. At doses that effectively blocked 2HG to basal levels, AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human AML cells in vitro, suggesting potential to reverse leukemic phenotype in AML tumors harboring the IDH2 mutation. Unlike the case of IDH1 above, the published structure of AGI-6780 co-complexed with IDH2-R140Q allows for detailed analysis of its inhibitory mechanism (Wang et al., 2013). In the X-ray structure, a single molecule
of AGI-6780 binds at the interface of two protomers (Figure 2C). The allosteric inhibition appears to arise from the ability of AGI6780 to keep the IDH2-R140Q mutant enzyme in an open orientation, thereby preventing the NADPH cofactor and substrate aKG from coming close to the catalytic Mg2+ binding site (see Figure 3). The highly symmetric AGI-6780 binding pocket extends deep into the protein interface and is closed over by loops composed of residues 152–167, which also fold over the binding pocket, providing anexplanation for the time-dependent inhibition kinetics. AGI-6780 makes several direct H-bond interactions from its urea group and amide nitrogen to Gln316, but a significant amount of binding energy arises from van der Waals contacts between the protein and hydrophobic surfaces of AGI-6780. The in vivo potential for this compound is not known, since its pharmacokinetic properties were not reported. Nevertheless, this effective mode of inhibition serves as an important molecular model for the design of bioisosteric compounds. OtherIDH2inhibitorsareunderdevelopment,notablyAG-221, a first-in-class, orally available inhibitor (Table 3) which demonstrated a survival advantage in a preclinical study of a primary human IDH2 mutant AML xenograft mouse model (Yen et al., 2013). Early phase I clinical trial data for AG-221 show promise, with meaningful clinical responses in evaluable AML patients harboring IDH2 mutations (Stein et al., 2014). To date, there is no published example of a molecule that inhibits both IDH1 and IDH2 mutant isoforms with equipotency.

Table 3.Characteristics of Small Molecule Inhibitors of Mutant IDH

PhenylglycineAGI-5198 (Popovici-Mulleretal., 2012; Rohleetal.,2013)
N-cyclohexyl-2-(N-(3-fluorophenyl)-2(2-methyl-1H-imidazol-1-yl)acetamido)2-(o-tolyl)acetamide IDH1-R132H

  • Good potency against enzyme and in U87cell line overexpressing R132H mutation (IC50= 70nM)
  • Good oral exposure in rodents at high doses (>300mg/kg), which were likely at levels saturating hepatic clearance mechanisms
  • Plasma 2HG inhibition > 90% (BID dosing) in xenograft model of U87-R132H tumors
  • Promoted differentiation of glioma cells via induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation at near-complete 2HG inhibition
  • inhibited plasma 2HG and delayed growth of IDH1-mutant but not wild-type glioma xenografts in mice

ML309 (Davis et al.,2014)
2-(2-(1H-benzo[d]imidazol-1-yl)-N-(3fluorophenyl)acetamido)-N-cyclopentyl2-o-tolylacetamide IDH1-R132H IDH1-R132C dIC50=68nM(R132H)

  • Inhibited 2HG production in glioblastoma cell line (IC50 = 250 nM) with minimal cytotoxicity
  • 1-hydroxypyridin2-one Compounds2and3 (Zhengetal.,2013)
    6-substituted1-hydroxypyridin-2-oneIDH1-R132H IDH1-R132C
  • K i= 190 and 280 nM (forR132H)
  • Inhibited production of 2HG in IDH1 mutated cells

AG-120 (Agios)

  • Orally available, selective, potent inhibitor
  • PhaseI studies ongoing in advanced solid tumors (NCT02073994; NCT02074839)

Allostery as an Approach to Drugging Metabolic Enzymes Is Important in Cancer All enzymes discussed in this article are allosterically targeted by small molecule modulators. With the exception of the enzymes of lipid metabolism, it is striking that there are very few examples of the regulation of metabolic enzymes by drug-like molecules at the catalytic site. We believe that this observation will hold true for the wider set of metabolic enzymes. Metabolic pathways are typically regulated by upstream and downstream metabolites through feedforward and feedback mechanisms. This regulation occurs typically through binding at allosteric sites, which have distinctly different properties relative to active sites. Therefore regulation can come from effectors that may have very different properties to the substrate. This review describes the potential therapeutic impact of specific allosteric regulators of PKM2, glutaminase, and IDH. Additionally, preclinical studies of tool compounds demonstrated that allosteric regulators of other enzymes involved in cancer cell metabolism could provide more therapeutic opportunities (Table 4). Substrates and products of metabolic enzymes tend to be small and very polar, and often include crucial metal ions and their ligands, so it is likely that targeting their catalytic pockets will yield molecules with similar properties. From a drug-discovery point of view, targeting allosteric sites is appealing as hydrophilic substrate-binding sites are generally not hospitable to strong interactions with small molecule drugs, which gain potency to a large extent through hydrophobic interactions. In addition, as activity of most metabolic enzymes is regulated by multimerization, the formation of multimers provides opportunity for binding sites to form at protein–protein interfaces.

Table 4. Examples of Allostery in Cancer Cell Metabolism

TH           Tyrosine hydroxylase         Haloperidol                                           Activator             Catecholamine metabolism               (Casu and Gale, 1981)
PDK1      Pyruvate dehydrogenase
kinase isozyme1                  3,5-diphenylpent-2-enoicacids                         Activator             TCAcycle                                                (Stroba et al., 2009)
BCKDK  Branched chain keto acid
dehydrogenase kinase   (S)-a-chloro-phenylpropionicacid[(S)-CPP]     Inhibitor              Branch-chain amino acid                   (Tso et al., 2013)
ACACA   Acetyl-CoA carboxylase
alpha                                 5-tetradecyloxy-2-furoicacid (TOFA)                  Inhibitor              Fatty acid  synthesis                            (Wang et al.,2009)

FBP1     Fructose-1,6
bisphosphatase1               Benzoxazole benzene sulfonamide1                    Inhibitor              Glycolysis                                        (von Geldern et al., 2006)
ALADA minolevulinate
dehydratase                     wALAD in1 benzimidazoles                                     Inhibitor              Haem synthesis                                    (Lentz et al., 2014)
TYR       Tyrosinase         2,3-dithiopropanol                                                   Inhibitor              Melanin metabolism                    (Wood and Schallreuter, 1991)
DBHD  opamine beta
hydroxylase-2H-phthalazinehydrazone (hydralazine;HYD)
2-1H-pyridinonehydrazone (2-hydrazinopyridine;HP)
2-quinoline-carboxylicacid (QCA)
1H-imidazole-4-aceticacid (imidazole-4-aceticacid;IAA)                             Inhibitor         Neurotransmitter synthesis                    (Townes et al.,1990)
deaminase        5-iodo-2’-deoxyuridine5’-triphosphate                                 Inhibitor          Nucleotide metabolism                      (Prusoff and Chang, 1968)
TYMP  Thymidine
phosphorylase     5’-O-tritylinosine (KIN59)                                                    Inhibitor          Nucleotide metabolism                         (Casanova et al.,2006)
TYMS Thymidylate
synthase         1,3-propanediphosphonicacid (PDPA)                                     Inhibitor          Nucleotide   metabolism                        (Lovelace et al.,2007)

Figure 3. Simplified Description of IDH Protein Motion The large domain (residues 1–103 and 286–414) forms nearly all of the NADPH cofactor binding residues and roughly half of the substrate binding residues.The small domain(residues 104–136 and 186–285) contains the remaining substrate binding residues and the metal binding residues. The interface between the two protomers is formed by both the small domain and the clasp region (residues 137–185). The large domain moves away from the small domain to facilitate NADPH cofactor exchange and substrate binding. The large domain then closes up against the small domain, thereby completing the substrate binding pocket and bringing the cofactor, substrate, and metal into close contact with each other and with the key catalytic residues to facilitate hydride transfer between substrate and cofactor and enzyme-assisted carboxylation/decarboxylation. Subsequent opening of the large domain from the small domain would enable product release and cofactor exchange to complete the catalytic cycle (Rendina et al., 2013; Xu et al., 2004).

7.3.2 Chemical proteomics approaches to examine novel histone modifications

Xin LiXiang David Li
Current Opinion in Chemical Biology Feb 2015; 24:80–90


  • A variety of novel histone PTMs have been identified by MS-based methods.
  • Regulatory mechanisms and cellular functions of most novel histone PTMs remain unknown, due to lack of knowledge about their readers, erasers and writers.
  • Chemical proteomics approaches provide valuable tools to characterize novel histone PTMs.
  • The application of photoaffinity probes helps the profiling of histone PTMs’ readers, erasers and writers.

Histone posttranslational modifications (PTMs) play key roles in the regulation of many fundamental cellular processes, such as gene transcription, DNA damage repair and chromosome segregation. Significant progress has been made on the detection of a large variety of PTMs on histones. However, the identification of these PTMs’ regulating enzymes (i.e. ‘writers’ and ‘erasers’) and functional binding partners (i.e. ‘readers’) have been a relatively slow-paced process. As a result, cellular functions and regulatory mechanisms of many histone PTMs, particularly the newly identified ones, remain poorly understood. This review focuses on the recent progress in developing chemical proteomics approaches to profile readers, erasers and writers of histone PTMs. One of such efforts involves the development of the Cross-Linking-Assisted and SILAC-based Protein Identification (CLASPI) approach to examine PTM-mediated protein–protein interactions.

Table 1    Novel histone PTMs                      functions
1             Lysine formylation             Arising from oxidative damage of DNA modification sites overlap with lysine acetylation and methylation, potentially interfere with normal regulation of these PTMs

2      Lysine propionylation  p300,c CREB-binding protein,c Sirt1,c Sirt2,c Sirt3c
Structurally similar with lysine acetylation, regulated by same set of enzymes, H3K23pr may be regulatory for cell metabolism
3    Lysine butyrylation       p300,c CREB-binding protein,c Sirt1,c Sirt2,c Sirt3c
Structurally similar with lysine acetylation, regulated by same set of enzymes
4    Lysine malonylation    Sirt5c
Changing the positively charged lysine to negatively charged residue, likely to affect the chromatin structure
5   Lysine succinylation    Sirt5c
A  mutation to mimic crotonyl lysine that changes lysine to glutamic acid of histone H4K31, reduces cell viability
6  Lysine crotonylation   Sirt1,c Sirt2,c Sirt3
Enriched at active gene promoters potential enhancers in mammalian genomes, male germ cell differentiation
7 Lysine 2-hydroxyiso
butyrylation                     HDAC1-3c
Associated with gene transcription
8  Lysine 4-oxononoylation    Modified by 4-oxo-2-nonenal, generated under oxidative stress, prevents nucleosome assembly in vitro
9 Lysine 5-hydroxylation   JMJD6
suppress lysine acetylation and methylation
10 Glutamine methylation   Nop1  (yeast), fibrillarin (huma)
human histone H2AQ105
11 Serine and
threonine GlcNAcylation  O-GlcNAc transferase
H2BS112 GlcNAcylation promotes K120 monoubiquitination, H3S10 GlcNAcylation suppresses phosphorylation of site
12 Serine and threonine acetylation
13 Serine palmitoylation   Lpcat1
catalyzed H4S47 palmitoylation, Ca2+-dependent, regulates global RNA synthesis
14  Cysteine glutathionylation
H3.2 and H3.3
conserved cysteine, but not H3.1, destabilize the nucleosomal structure
15 Cysteine fatty-acylation
H3.2 C110
16 Tyrosine hydroxylation

Fig. 1. Schematic description of a MS-based method for the identification of novel histone PTMs.

Fig. 2. Chemical proteomics approaches to profile readers and erasers of histone PTMs.
(a) Photo-cross-linking strategy to capture proteins recognizing histone PTMs.
(b) Chemical structure of photoaffinity peptide probes.
Modifications of interest were labeled in green; photo-cross-linkers were labeled in red; chemical handles (alkyne) were labeled in blue; the sequence of probe C and probes 1–5 were derived from the
histone H3 1–15 amino acids residues, the sequence of probe 6 was derived from the histone H4 1–19 amino acids residues.
(c) Schematic for the CLASPI strategy to profile proteins that bind certain histone mark in whole-cell proteomes

Consistent with our findings, Tate and coworkers [57] recently reported the development of a photoaffinity probe based on a succinylated glutamate dehydrogenase (GDH) peptide for capturing Sirt5
as the corresponding desuccinylase. In addition to the application of photo-cross-linking strategy for examining the histone PTMs with known erasers, we recently used CLASPI with a photoaffinity
probe (probe 5, Figure 2b) to profile proteins that recognize a novel histone mark, crotonylation at histone H3K4 (H3K4cr, Table 1, Entry 6) [25], whose erasers were unknown. This study revealed,
for the first time, that Sirt3 can recognize the H3K4cr mark and efficiently catalyze the removal of histone crotonylation marks. More importantly, Sirt3 was found to regulate histone Kcr level in
cells and may potentially modulate gene transcription through its decrotonylase activity [58]. By converting bisubstrate inhibitors of HATs (histone peptides with certain lysine residues covalently
attached to Ac-CoA) to clickable photoaffinity probes (for example, probe 6, Figure 2b), they carried out the first systematic profiling of HATs in whole-cell proteomes [59].  We  anticipate  that  similar methods can be used to search for writers of novel histone PTMs such as Kmal, Ksucc, Kcr and Khib (Table 1) since the corresponding acyl-CoAs are presumed to be the acyl donors.

We have shown, in this review, the applications and recent advances of chemical tools, in combination with MS-based proteomics approaches, for the detection and characterization of histone
PTMs and their readers, erasers and writers.

This article belongs to a special issue

Omics Edited By Benjamin F Cravatt and Thomas Kodadek

Editorial overview: Omics: Methods to monitor and manipulate biological systems: recent advances in ‘omics’

Benjamin F Cravatt, Thomas Kodadek
Current Opinion in Chemical Biology Feb 2015; 24:v–vii

7.3.3 Misfolded Proteins – from Little Villains to Little Helpers… Against Cancer

Ansgar Brüning1,* and Julia Jückstock
Front Oncol. 2015; 5: 47

The application of cytostatic drugs targeting the high proliferation rates of cancer cells is currently the most commonly used treatment option in cancer chemotherapy. However, severe side effects and resistance mechanisms may occur as a result of such treatment, possibly limiting the therapeutic efficacy of these agents. In recent years, several therapeutic strategies have been developed that aim at targeting not the genomic integrity and replication machinery of cancer cells but instead their protein homeostasis. During malignant transformation, the cancer cell proteome develops vast aberrations in the expression of mutated proteins, oncoproteins, drug- and apoptosis-resistance proteins, etc. A complex network of protein quality-control mechanisms, including chaperoning by heat shock proteins (HSPs), not only is essential for maintaining the extravagant proteomic lifestyle of cancer cells but also represents an ideal cancer-specific target to be tackled. Furthermore, the high rate of protein synthesis and turnover in certain types of cancer cells can be specifically directed by interfering with the proteasomal and autophagosomal protein recycling and degradation machinery, as evidenced by the clinical application of proteasome inhibitors. Since proteins with loss of their native conformation are prone to unspecific aggregations and have proved to be detrimental to normal cellular function, specific induction of misfolded proteins by HSP inhibitors, proteasome inhibitors, hyperthermia, or inducers of endoplasmic reticulum stress represents a new method of cancer cell killing exploitable for therapeutic purposes. This review describes drugs – approved, repurposed, or under investigation – that can be used to accumulate misfolded proteins in cancer cells, and particularly focuses on the molecular aspects that lead to the cytotoxicity of misfolded proteins in cancer cells.


How Do Proteins Fold and What Makes Misfolded Proteins Dangerous?

For an understanding of misfolded proteins, it is necessary to understand how cellular proteins attain and then further maintain their native conformation and how mature proteins and unfolded proteins are generated and converted into each other.

The principles and mechanisms of protein folding were one of the major research topics and achievements of biochemical research in the last century. For decades, Anfinsen’s model, which explained protein structure by thermodynamic principles applying to the polypeptide’s inherent amino acid sequence (1), was to be found in the introductory sections of all textbooks in protein biochemistry. According to Anfinsen’s thermodynamic hypothesis, the structure with the lowest conformational Gibbs free energy was finally taken by each single polypeptide due to a thermodynamic and stereochemical selection for side chain relations that form most stable and effective enzymes or structural proteins (1). Beyond this individual selection for the energetically most optimized conformation, evolution also selected for amino acid sequences that energetically allowed the smoothest and most “frustration-free” folding processes via a thermodynamic “folding funnel” (1–3).

Whereas Anfinsen’s model preferred the side chain elements as preferential organizing structures, recent hypotheses have inversely proposed the backbone hydrogen bonds as the driving force behind protein folding (4). According to the former theory, the finally folded protein was assumed to attain a single defined structure and shape (1, 4), and the unfolded conditions were described as being represented by a structureless statistical coil with nearly indefinite conformations – a so-called “featureless energy landscape” (4). The latter model assumes that a protein selects during its folding process from a limited repertoire of stable scaffolds of backbone hydrogen bond-satisfied α-helices and β-strands (4). This also implies that unfolded proteins are not structureless, shoelace-like linear amino acid alignments as often depicted in cartoons for graphical reasons, but actually, at least in part, retain discrete and stable scaffolds.

Once the protein has attained its final conformation, the problem of stabilizing this structure arises. Hydrophobic interactions that press non-polar side chains into the center of the protein are assumed to be a major force in protein stabilization (5, 6). At the protein surface, polar interactions, mainly by hydrogen bonds of polar side chains and backbone structure, are assumed to be of similar importance (6). Salt bridges and covalent disulfide bonds were identified as further forces supporting the stability of proteins (6). Accordingly, all conditions that interfere with these stabilizing forces, including extreme temperature, salt concentrations, and redox conditions, may lead to protein misfolding.

Another aspect that must be taken into account when studying protein folding relates to the very different conditions found in viable cells when compared to test tube conditions. Considering the life-cycle of a protein, each protein begins as a growing polypeptide chain protruding from the ribosomal exit tunnel and with several of its future interacting amino acid binding partners not even yet attached to the growing chain of the nascent polymer. In these ribosomal exit tunnels, first molecular interactions and helical structures are formed, and evidence exists to support the notion that the speed of translation is regulated by slow translating codon sequences just to optimize these first folding processes (7). After leaving the ribosomal tunnel, nascent polypeptides are also directly welcomed by chaperoning protein complexes, which facilitate and further guide the folding process of newly synthesized proteins (8). It is believed that a high percentage of nascent proteins are subject to immediate degradation due to early folding errors (9). Since many nascent proteins are synthesized in parallel at polysomes, the temporal and spatial proximity of unfolded peptides brings the additional risk of protein aggregation (10). Moreover, as mentioned above, even incomplete folding intermediates and partially folded states may form energetically but not physiologically active metastable structures (11, 12). An immediate, perinatal guidance and chaperoning of newborn proteins is therefore essential to creating functional, integrative proteins and to avoiding misfolded, function-less polypeptides with potentially cytotoxic features.

Since protein structure and function are coupled, misfolded proteins are, at first, loss-of-function proteins that might reduce cell viability, in particular when generated in larger quantities. A more dangerous feature of misfolded proteins, however, lies in their strong tendency toward abnormal protein–protein interactions or aggregations, which is reflected by the involvement of misfolded proteins and their aggregates in several amyloidotic diseases, including neurodegenerative syndromes such as Alzheimer’s disease and Parkinson’s disease (13, 14). The fact that several of these intracellular and extracellular protein aggregates contain β-sheet-like structures and form filamentous structures also supports the notion that misfolded proteins are not necessarily structureless protein coils or unspecific aggregates, at least when they are formed by homogenous proteins as in the case of several neurodegenerative diseases (13). Paradoxically, these larger aggregates appear to reflect a cell protective mechanism so as to sequester or segregate smaller, but highly reactive, nucleation cores of condensing protein aggregates (13).

Unspecific hydrophobic interactions, in particular, have been held responsible for protein aggregations that form when terminally folded proteins lose their native conformation and expose buried hydrophobic side chains on their surface (15, 16). These hydrophobic interactions are also believed to be the most problematic issues with newly synthesized polypeptides on single ribosomes or polysomes (12). Once exposed to the surface, the hydrophobic structures will quickly find possible interaction partners. The intracellular milieu can be regarded as a “crowded environment” (17), fully packed with proteins in close contact and near to their solubility limit (8, 12). Thus, misfolded proteins not only aggregate among each other but may also attach to normal native proteins and inhibit their function and activity. Since such misfolding effects and interactions can also include nuclear DNA replication and repair enzymes (18), misfolded proteins may not only exert proteotoxic but also genotoxic effects, thereby endangering the entire cellular “interactome” (19) by interfering both with the integrity of the proteome (proteostasis) and the genome. Therefore, a misfolded protein is not simply a loss-of-function protein but also a promiscuous little villain that might act like a free radical, exerting uncontrolled danger to the cell.

The way in which cells deal with misfolded proteins strongly depends on the nature, strength, length, and location of the damage induced by the various insults. Management of misfolded proteins can be achieved by heat shock protein (HSP)-mediated protein renaturation (repair); proteasomal, lysosomal, or autophagosomal degradation (recycling); intracellular disposal (aggregation); or – in its last consequence if overwhelmed – by programed cell death (despair). In the following paragraphs, the cellular management of misfolded proteins is described and therapeutic options to induce misfolded proteins in cancer cells are presented.

Hsp90 and Hsp90 Inhibitors

The best-known and evolutionarily most-conserved mechanism to protect against protein misfolding is the binding and refolding process mediated by so-called heat shock proteins (HSPs). HSPs recognize unfolded or misfolded proteins and facilitate their restructuring in either an ATP-dependent (large HSPs) or energy-independent manner (low weight HSPs). HSP of 90 kDa (hsp90) is a constitutively expressed HSP and is regarded as the most common and abundantly expressed HSP in eukaryotic cells (20, 21). Although commonly referred to as hsp90, it consists of a variety of isoforms that are encoding for cytosolic (hsp90α1, α2, β), mitochondrial (TRAP1), or endoplasmic reticulum (ER)-resident (GRP94) forms. Its primary function is less that of a stress response protein and more to bind to a certain group of client proteins unable to maintain a stable configuration without being assisted by hsp90 (20, 22, 23). Steroid hormone receptors (estrogen receptor, glucocorticoid receptor), cell cycle regulatory proteins (CDK4, cyclin D, polo-like kinase), and growth factor receptors and their downstream targets (epidermal growth factor receptor 1, HER2, AKT) are among the best-studied client proteins of hsp90 (20–22). Also, several cancer-specific mutations generating otherwise instable oncoproteins, such as mutant p53 or bcr-abl, rely on hsp90 chaperoning to keep them in a soluble form, thereby facilitating the extravagant but vulnerable “malignant lifestyle” of hsp90-addicted cancer cells (21, 24). Accordingly, hsp90 has been assumed to be a prominent target, in particular for hormone-responsive and growth factor receptor amplification-dependent cancer types.

The microbial antibiotics geldanamycin and radicicol are the prototypes of hsp90 inhibitors. Based on intolerable toxicity, these molecules had to be chemically modified for application in humans, and most of the ongoing clinical studies with hsp90 inhibitors are aimed at identifying semi-synthetic derivatives of these lead compounds with an acceptable risk profile. Unfortunately, most recent studies using geldanamycin derivatives have provided disappointing results because of toxicities and insufficient efficacy (22, 25–27). Studies with radicicol (resorcinol) derivatives, in particular with ganetespib, appear to be more promising because of fewer adverse effects (22, 25–27). Liver and ocular (retinal) toxicities have been described as main adverse effects of hsp90 inhibition, and appeared to be experienced less with ganetespib than with most of the first generation hsp90 inhibitors (28).

Since both geldanamycin and radicicol target the highly conserved and unique ATP-binding domain of hsp90, new synthetic inhibitors have also been generated by rational drug design (22, 25–27). However, none of the various natural or synthetic hsp90 inhibitors under investigation have yet provided convincing clinical data, and future studies will show whether hsp90 can eventually be added to the list of effective cancer targets.

Hsp70, Hsp40, Hsp27, and HSF1

Hsp90 is assisted by several other HSPs and non-chaperoning co-factors, finally forming a large protein complex that recruits and releases client proteins in an energy-dependent manner (21, 22, 29). Client proteins for hsp90 are first bound to hsp70, which transfers the prospective client to hsp90 through the mediating help of an hsp70–hsp90 organizing protein (HOP). Binding of potential hsp90 client proteins to hsp70 is facilitated by its co-chaperone hsp40 (23, 30). Exposed hydrophobic amino acids, the typical feature of misfolded proteins, have been described as the main recognition signal for hsp70 proteins (15, 16, 31). Hsp70 proteins are not only supporter proteins for hsp90 but also represent a large chaperone family capable of acting independently of hsp90 and that can be found in all cellular compartments, including cytosol and nucleus (hsp70, hsp72, hsc70), mitochondria (GRP75 = mortalin), and the ER (GRP78 = BiP). Hsp70 chaperones may act on misfolded or nascent proteins either as “holders” or “folders” (31), which means that they prevent protein aggregation either by sheltering these aggregation-prone protein intermediates or by allowing these proteins to fold/refold into their native form in an assisted mechanism within a protected environment (31). Hsc70 (HSPA8) is a constitutively expressed major hsp70 isoform that is an essential factor for normal protein homeostasis even in unstressed cells (16). Misfolded proteins can also be destined by hsp70 proteins for their ultimate degradation. Proteins that expose KFERQ amino acid motifs on their surface during their unfolding process are preferentially bound by hsc70 and can be directed to lysosomes in a process called chaperone-mediated autophagy (CMA) (32, 33). In another mechanism of targeted protein degradation, interaction of hsc70 with the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) leads to ubiquitination of misfolded proteins and thus their destination of the ubiquitin-proteasome protein degradation pathway (34, 35). Since hsc70 is essential for normal protein homeostasis and its knock-out is lethal in mice (16, 36), hsc70 inhibition might not be an optimal target for cancer-specific induction of misfolded proteins. This contrasts with the inducible forms of hsp70 such as hsp72 (HSPA1), which are upregulated in a cell stress-specific manner and are often found to be constitutively overexpressed in cancer tissues (16, 36). Transcriptional activation of these inducible HSPs is mediated by the heat shock factor 1 (HSF1), which also regulates expression of hsp40 and the small HSP hsp27 by sharing a common promoter consensus sequence (heat shock response element) for HSF1 binding (37). HSF1 was also found to be constitutively activated in cancer tissues, modulating several cell cycle- and apoptosis-related pathways via its target genes (38–40). HSF1 itself is kept inactive in the cytosol by binding to hsp90, and the recruitment of hsp90 to misfolded proteins is considered a main activation mechanism to release monomeric HSF1 for its subsequent trimerization, post-translational activation, and nuclear translocation (24, 41). Also, since hsp90 inhibition causes hsp70 induction by HSF1 activation as a compensatory feed-back mechanism (24), combined inhibition of hsp90 and hsp70, or of hsp90 and HSF1 might be a more effective therapeutic approach for cancer treatment than single HSP targeting alone.

Indeed, several small-molecule inhibitors and aptamers for hsp70, hsp40, and hsp27 have been designed (16, 42–44), but most of them remain in pre-clinical development, or are either not applicable in humans or associated with intolerable side effects (16, 42–44). Notably, the natural bioflavonoid quercetin was shown to inhibit phosphorylation and transcriptional activity of the heat shock transcription factor HSF1, thus reducing HSP expression at its most basal level (45–48). This HSP and HSF1 inhibition may also contribute to the observed cancer-preventing effects of a flavonoid-rich diet, which includes fruits and vegetables. However, due to their low bioavailability, the concentrations of flavonoids needed to induce direct cytotoxic effects in cancer cells for (chemo-)therapeutic reasons are obviously not achievable in humans, even when applied as nutritional supplements (49). More effective and clinically more easily applicable inhibitors of HSF1 are therefore urgently sought. Promising HSF1 targeting strategies are currently under development, although are apparently not yet suited for clinical applications (24, 50, 51).

SP Williams Comment:

There is a new hsp90- inhibitor, ganetespib, which is active against ovarian cancer in vitro and in vivo. Clinical trials are looking at this in cisplatin refractory cases. This was identified by a network analysis from a previous siRNA screen on ovarian cancer cells for pathways related to growth inhibition in an effort to find possible targets against CP resistance. The reference is

Protein Ubiquitination and Proteasomal Degradation

Ubiquitin is a 76 amino acid polypeptide that can covalently be attached via its carboxy-terminus to free (lysyl) amino groups of proteins. Ubiquitination of proteins generates a cellular recognition motif that is involved in various functions ranging from transcription factor and protein kinase activation to DNA repair and protein degradation – depending on the extent and exact location of this post-translational modification (52, 53). Monoubiquitination of peptides of more than 20 amino acids was found to be a minimal requirement for protein degradation, but the canonical fourfold (poly-)ubiquitination with three further lysine (K48) side chain-linked ubiquitins appears to be most apt for an effective and rapid substrate recognition by the proteasome (54). This canonical polyubiquitin structure, as well as several other mixed polyubiquitin structures, can be recognized by the external 19S subunits of the 26S proteasome complex (54, 55). Prior to degradation of ubiquitinated proteins by the proteasomal 20S core subunit, the attached ubiquitin chains are released by the external 19S subunits for recycling, although they can also be co-degraded by the proteasome (56). After first passing the 19S subunit, the proteasomal target proteins are then unfolded in an energy-dependent manner and introduced into the narrow enzymatic cavity of proteasome for degradation. The barrel-shaped 20S proteasomal core complex contains three different proteolytic activities in duplicate (β1: caspase-like-, β2: tryptic-, and β5: chymotryptic activity), which initiate an efficient cleavage of the proteasomal target proteins into smaller peptides (57).

It is important to note that specific ubiquitination and ensuing proteasomal degradation is not an exclusive degradation mechanism of misfolded proteins but is also used to regulate the expression level of several native cell cycle regulatory proteins [cyclins, proliferating cell nuclear antigen (PCNA), p53], signaling pathway molecules (β-catenin, IκB), and survival factors (mcl-1) during the course of normal protein homeostasis and cell cycle progression (53, 55, 57, 58). Moreover, proteasomes are involved in protein maturation, including the processing and maturation of the NF-κB transcription factor subunit p50 and the drug-resistant protein MDR1 (57). Therefore, targeting proteasomal activity has not only been of interest for the generation of misfolded, cytotoxic proteins but also for interfering with the expression of proteins involved in several hallmarks of cancer, including cell cycle progression, signal transduction, and apoptosis.

Proteasome Inhibitors

Bortezomib (PS-341, Velcade ™) has long been known as a paragon of a clinically applicable proteasome inhibitor. Bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma (55, 59, 60). The great expectations of transferring the success of bortezomib to non-hematological solid cancer types have unfortunately not yet been fulfilled. It has been suggested that the high antibody-producing capacity of myeloma cells and thus the need for an efficient proteasomal degradation system to cope with the recycling process of misfolded ER-generated antibodies [ER-associated degradation process (ERAD); see below] might contribute to the high sensitivity of myeloma cells to bortezomib (9, 60, 61). Originally, bortezomib was developed to inhibit the proteasomal degradation of the NF-κB inhibitor IκB, thus targeting the pro-inflammatory, but also cancer-promoting, effect of the NF-κB transcription factor (55, 60, 62). Recent insights indicate that the anti-tumoral effect of bortezomib is not only mediated by its NF-κB inhibitory activity but also by its ability to induce accumulation of misfolded proteins in the cytosol and the ER (60, 62–65). However, the use of bortezomib, even for highly sensitive multiple myeloma, is limited by its strong tendency to induce a proteasome inhibition-independent peripheral neuropathy by acting on neuronal mitochondria (61). Since neurodegenerative diseases are associated with protein misfolding and aggregation, the neuropathological effects of bortezomib might also be assumed to be mediated by the possible proteotoxic effects of bortezomib in neuronal cells. However, although proteasome inhibitor-induced neurodegeneration and inclusion body formation have been described in animal models, similarities between proteasome inhibitor-induced neurodegeneration and Parkinson’s disease-like histopathological features could not be established (66).

Table 1 Drugs described in this review and their mechanism of action (MOA), status of approval, and main adverse effects.

Aggresome Formation and Re-Solubilization: Role of HDAC6

As depicted above, proteasome and HSP inhibition will eventually lead to the accumulation of misfolded and polyubiquitinated proteins. Based on their inherent cohesive properties mediated by their exposed hydrophobic surfaces, both ubiquitinated and non-ubiquitinated misfolded proteins tend to adhere as small aggregates (Figure ​(Figure1).1). Individual ubiquitinated proteins and small ubiquitinated aggregates can be recognized by specific ubiquitin-binding proteins such as HDAC6 via its zinc finger ubiquitin-binding domain. HDAC6 is an unusual histone deacetylase located in the cytosol that regulates microtubule acetylation and is also able to bind ubiquitinated proteins. Based on HDAC6’s additional ability to bind to microtubule motor protein dynein, these aggregates are actively transported along the microtubular system into perinuclear aggregates around the microtubule organizing center (MTOC) (108384). Recognition of small, scattered ubiquitinated aggregates by HDAC6 has been described as being mediated by unanchored ubiquitin chains, which are generated by aggregate-attached ubiquitin ligase ataxin-3 (85). Whereas proteasomal target proteins are primarily tagged by K-48 (lysine-48) linked ubiquitins; K-63 linked ubiquitin chains appear to be a preferential modification for aggresomal targeting by HDAC6 and were assumed to mediate a redirection from proteasomal degradation to aggresome formation in the case of proteasomal inhibition or overload (86). Accordingly, aggresome formation is not an unspecific protein aggregation but a specific, ubiquitin-controlled sorting process. Furthermore, these aggresomes consist not only of misfolded and deposited proteins but have also been shown to contain a large amount of associated HSPs and ubiquitin-binding proteins, including HDAC6 [Figure ​[Figure1;1; (108384)]. Aggresomes contain, and are also surrounded by, large numbers of proteasomes (108384), which help to resolubilize these aggregates not only through their intrinsic proteasomal digestion but also by generating unanchored K63-branched polyubiquitin chains, which then stimulate HDAC6-mediated autophagy, another cellular disposal mechanism in involving HDAC6 (87). Notably, HDAC6 has also been shown to control further maturation of autophagic vesicles by stimulating autophagosome–lysosome fusion (Figure ​(Figure1)1) in a manner different from the normal autophagosome–lysosome fusion process (88).

Figure 1

Drugs that inhibit folding or disposal of misfolded proteins. Native mature proteins, nascent proteins, or misfolded proteins can be prevented from folding or refolding by small and large heat shock protein inhibitors, of which the hsp90 inhibitors based 

The HDAC6 multitalent also exerts its deacetylase activity on hsp90 and modifies hsp90 client binding by facilitating its chaperoning of steroid hormone receptors and HSF1 (8991). Recruitment of HDAC6 to ubiquitinated proteins leads to the dissociation of the repressive HDAC6/hsp90/HSF1 complex (91) and allows the release of transcriptionally active HSF1 to the nucleus. The engagement of HDAC6 at the aggresome–autophagy pathway hence also indirectly facilitates HSF1 activity. p97/VCP (valosin-containing protein), another binding partner of HDAC6 and itself a multi-interactive, ATP-dependent chaperone (9294), is assumed to be involved not only in the specific separation of hsp90 and HSF1 by its “segregase” activity but also in the binding and remodeling of polyubiquitinated proteins before their delivery to the proteasome (9395). Additionally, p97/VCP dissociates polyubiquitinated proteins bound to HDAC6 (91). Accumulation of polyubiquitinated proteins thus leads to HDAC6-dependent HSF1 activation and HSP induction, p97/VCP-dependent recruitment and “preparation” of polyubiquitinated proteins to proteasomes, and, in the case of pharmacological proteasome inhibition or physiological overload, to an HDAC6-dependent detoxification of polyubiquitinated proteins by the aggresome/autophagy pathway.

Pharmacological Inhibition of Aggresome Formation: HDAC6 Inhibitors

The central involvement of HDAC6 in aggresome formation and clearance makes HDAC6 one of the most interesting druggable targets for the induction of proteotoxicity in cancer cells. Also, HDAC6 has been found to be overexpressed in various cancer tissues, associated with advanced cancer stages and increased neoplastic transformation (96). Several pan-histone deacetylase inhibitors have been developed and tested in clinical studies for a variety of diseases, including different types of cancer (9798). Although hematological malignancies responded best to most of the already clinically tested pan-histone deacetylase inhibitors, the efficacy on solid cancer types was disappointingly poor and also associated with intolerable side effects (98). The unforeseeable pleiotropic epigenetic mechanism caused by non-specific (nuclear) histone deacetylase inhibitors may also limit their application for use in cancer treatment or HDAC6 inhibition, and has led to the search for selective HDAC6 inhibitors with no inhibitory effects on transcription modifying histone deacetylases. Through screening of small molecules under the rationale of selecting for tubulin deacetylase inhibitors with no cross-reactive histone deacetylase activity, the HDAC6 inhibitor tubacin was identified, and suggested for use in the treatment of neurodegenerative diseases or to reduce cancer cell migration and angiogenesis (99). Hideshima et al. then proved the hypothesis that the combined use of bortezomib with tubacin leads to an accumulation of non-disposed cytotoxic proteins and aggregates in cancer cells (100). Indeed, a synergistic effect of these two drugs against multiple myeloma cells could be observed with no detectable toxic effect on peripheral blood mononuclear cells (100). This and follow-up studies also revealed the efficacy of tubacin as a single agent against leukemia cells (100101) and a chemo-sensitizing effect on cytotoxic drugs in breast- and prostate-cancer cells (102).

Endoplasmic Reticulum Stress

Besides the cytosol, the ER is a major site for protein synthesis, in particular for those proteins destined for extracellular secretion, the cell membrane, or their retention within the endomembrane system. At the rough ER, nascent proteins are co-translationally transported across the ER membrane into the ER lumen (107), where they immediately encounter ER-resident chaperones, most prominently represented by hsp70 family member BiP/GRP78 and hsp90 family member GRP94 to help proper protein folding (15108). Most of these proteins also undergo post-translational modifications, including N- or O-linked glycosylation or protein disulfide bridge-building (109110), thereby adding further mechanisms of protein stabilization but also challenges for proper protein folding.

Similar to the situation in cytosolic protein biosynthesis, a large proportion of nascent proteins in the ER are assumed to misfold and to go “off-pathway” even under normal physiological conditions. Furthermore, the ER lumen, narrowly sandwiched between two phospholipid membranes, has been described as an even more densely crowded environment than the cytosol, additionally facilitating unspecific protein attachments and aggregations (15). Since, with the exception of bulk reticulophagy, the lumen of the ER contains no endogenous protein degradation system, and the anterograde transport of ER proteins to the Golgi, lysosomes, endosomes, or the extracellular environment requires properly folded proteins, a retrograde transport of ER proteins into the cytosol remains the only possible mechanism of preventing misfolded protein accumulation within the ER. In this ERAD, misfolded proteins are re-exported across the ER membrane by a specific multi protein complex, ubiquitinated by ER membrane-integrated ubiquitin ligases, and finally become degraded by cytosolic proteasomes (111112). Notably, association of the cytosolic p97/VCP protein, an important interacting partner with HDAC6, has also been described as being an essential factor for driving the luminal proteins through the ER membrane pore complex into the cytosol (92,112).

Accordingly, all agents and conditions that interfere with these folding, maturation, and retranslocation processes can lead to protein misfolding and aggregation within this sensitive organelle. Chemicals that act as glycosylation inhibitors (tunicamycin), calcium ionophore inhibitors (A23187, thapsigargin), heavy metal ions (cadmium, lead), reducing agents (dithiothreitol), as well as conditions like hypoxia or oxidative stress, all lead to a phenomenon called ER stress (113116). In the ER-stress response, a triad of ER membrane-resident signaling receptors and transducers, IRE1, ATF6, and PERK1, become activated and lead to the transcriptional activation of cytosolic and ER-resident chaperones to cope with the increasing number of misfolded proteins. Induction of autophagy (reticulophagy; ER-phagy) may also occur and supports the removal of damaged regions of the ER (117). Under very intensive or even unmanageable ER-stress conditions, a variety of pro-apoptotic pathways ensue, including CHOP induction, c-JUN-kinase activation, and caspase cleavage (118120), which eventually prevails over the cytoprotective arm of the ER-stress response and may lead to apoptosis. Targeting of protein folding within the ER is therefore a very promising strategy to induce apoptosis in cancer cells, in particular in those cancer cells characterized by an unphysiologically high protein secretion rate, such as, for example, multiple myeloma cells. Whereas the above-mentioned drugs such as tunicamycin or thapsigargin are valuable tools for cell biology studies, they display unacceptable toxicities in humans and are not suited for therapeutic applications. Interestingly, several already established drugs used for non-cancerous diseases have been described as inducing ER stress at pharmacologically relevant concentrations in humans as an off-target effect (113116). The non-steroidal anti-inflammatory COX-2 inhibitor celecoxib is an approved drug to treat various forms of arthritis and pain, but has also been described as exerting ER stress by functioning as a SERCA (sarco/ER Ca2+ ATPase) inhibitor (113116). However, although well tolerated in humans, the ER-stress-inducing ability of celecoxib seems to be weaker than that of direct SERCA inhibitors such as thapsigargin, and the usefulness of celecoxib against advanced cancer has been questioned (116). Various HIV protease inhibitors have been described as inducing ER stress in human tissue cells as a side effect (121123). In particular the HIV drugs lopinavir, saquinavir, and nelfinavir appear to be potent inducers of the ER-stress reaction, leading to a focused interest in these drugs for the induction of ER stress and apoptosis in cancer cells (116124128). In fact, with currently over 27 clinical studies in cancer patients2, nelfinavir, either used as a single agent or in combination therapy, is on the list of the most promising prospective candidates to induce selective proteotoxicity in cancer cells at pharmacologically relevant concentrations. Although the exact mechanism by which nelfinavir induces ER stress is not yet clear, it was shown that nelfinavir causes the upregulation of cytosolic and ER-resident HSPs, and induces apoptosis in cancer cells associated with caspase activation and induction of the pro-apoptotic transcription factor CHOP (125126). Nelfinavir was also shown to be combinable with bortezomib to enhance its activity on cancer cells (129). Since the retrograde transport of misfolded ER proteins is inhibited by the p97/VCP inhibitor eeyarestatin (130131), we recently tested the combination of eeyarestatin with nelfinavir but found no synergistic effect between these two agents in cervical cancer cells (132). In contrast, eeyarestatin markedly sensitized cervical cancer cells to bortezomib treatment (132), which was also observed in preceding studies in which eeyarestatin was used to augment the ER-stress-inducing ability of bortezomib in leukemia cells (131).

Induction of proteotoxicity through the accumulation of misfolded proteins has evolved as a new treatment modality in the fight against cancer. Clinically approved drugs such as bortezomib and carfilzomib provide evidence of the functionality of this approach. Newly developed agents like the HDAC6 inhibitor ACY-1215 or repurposed drugs like nelfinavir or disulfiram are currently being tested in clinical trials with cancer patients and will hopefully further broaden our arsenal of anti-cancer drugs. Notably, most proteotoxic agents that have been approved or are in clinical trials target the ubiquitin-proteasome-system (UPS) and are mainly effective in multiple myeloma cells, which rely on a functional ER/ERAD/UPS for excessive and proper antibody production. Similarly, it can be assumed that other cancer cell types with a marked secretory phenotype may also be affected by ER/ERAD/UPS inhibitors. In accordance with this notion, a recent dose-escalating Phase Ia study with nelfinavir as a single agent, that covered a large variety of solid cancer entities, revealed response rates primarily in patients with neuroendocrine tumors (140). In most other solid cancer types, however, the chemo-sensitizing or combination effects of proteotoxic drugs may prevail, and have become the focus of an increasing number of very promising clinical and pre-clinical studies.

7.3.4 Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer

Friend or Foe: Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer

Chen S1Zhang D2

FEBS Open Bio. 2015 Jan 30; 5:91-8

The endoplasmic reticulum (ER) protein 29 (ERp29) is a molecular chaperone that plays a critical role in protein secretion from the ER in eukaryotic cells. Recent studies have also shown that ERp29 plays a role in cancer. It has been demonstrated that ERp29 is inversely associated with primary tumor development and functions as a tumor suppressor by inducing cell growth arrest in breast cancer. However, ERp29 has also been reported to promote epithelial cell morphogenesis, cell survival against genotoxic stress and distant metastasis. In this review, we summarize the current understanding on the biological and pathological functions of ERp29 in cancer and discuss the pivotal aspects of ERp29 as “friend or foe” in epithelial cancer.

The endoplasmic reticulum (ER) is found in all eukaryotic cells and is complex membrane system constituting of an extensively interlinked network of membranous tubules, sacs and cisternae. It is the main subcellular organelle that transports different molecules to their subcellular destinations or to the cell surface [10,85].

The ER contains a number of molecular chaperones involved in protein synthesis and maturation. Of the ER chaperones, protein disulfide isomerase (PDI)-like proteins are characterized by the presence of a thioredoxin domain and function as oxido-reductases, isomerases and chaperones [33]. ERp29 lacks the active-site double-cysteine (CxxC) motif and does not belong to the redox-active PDIs [5,47]. ERp29 is recognized as a characterized resident of the cellular ER, and it is expressed ubiquitously and abundantly in mammalian tissues [50]. Protein structural analysis showed that ERp29 consists of N-terminal and C-terminal domains [5]: N-terminal domain involves dimerization whereas the C-terminal domain is essential for substrate binding and secretion [78]. The biological function of ERp29 in protein secretion has been well established in cells [8,63,67].

ERp29 is proposed to be involved in the unfolded protein response (UPR) as a factor facilitating transport of synthesized secretory proteins from the ER to Golgi [83]. The expression of ERp29 was demonstrated to be increased in cells exposed to radiation [108], sperm cells undergoing maturation [42,107], and in certain cell types both under the pharmacologically induced UPR and under the physiological conditions (e.g., lactation, differentiation of thyroid cells) [66,82]. Under ER stress, ERp29 translocates the precursor protein p90ATF6 from the ER to Golgi where it is cleaved to be a mature and active form p50ATF by protease (S1P and S2P) [48]. In most cases, ERp29 interacts with BiP/GRP78 to exert its function under ER stress [65].

ERp29 is considered to be a key player in both viral unfolding and secretion [63,67,77,78] Recent studies have also demonstrated that ERp29 is involved in intercellular communication by stabilizing the monomeric gap junction protein connexin43 [27] and trafficking of cystic fibrosis transmembrane conductance regulator to the plasma membrane in cystic fibrosis and non-cystic fibrosis epithelial cells [90]. It was recently reported that ERp29 directs epithelial Na(+) channel (ENaC) toward the Golgi, where it undergoes cleavage during its biogenesis and trafficking to the apical membrane [40]. ERp29 expression protects axotomized neurons from apoptosis and promotes neuronal regeneration [111]. These studies indicate a broad biological function of ERp29 in cells.

Recent studies demonstrated a tumor suppressive function of ERp29 in cancer. It was found that ERp29 expression inhibited tumor formation in mice [4,87] and the level of ERp29 in primary tumors is inversely associated with tumor development in breast, lung and gallbladder cancer [4,29].

However, its expression is also responsible for cancer cell survival against genotoxic stress induced by doxorubicin and radiation [34,76,109]. The most recent studies demonstrate other important roles of ERp29 in cancer cells such as the induction of mesenchymal–epithelial transition (MET) and epithelial morphogenesis [3,4]. MET is considered as an important process of transdifferentiation and restoration of epithelial phenotype during distant metastasis [23,52]. These findings implicate ERp29 in promoting the survival of cancer cells and also metastasis. Hence, the current review focuses on the novel functions of ERp29 and discusses its pathological importance as a “friend or foe” in epithelial cancer.

2. ERp29 regulates mesenchymal–epithelial transition

2.1. Epithelial–mesenchymal transition (EMT) and MET

The EMT is an essential process during embryogenesis [6] and tumor development [43,96]. The pathological conditions such as inflammation, organ fibrosis and cancer progression facilitate EMT [16]. The epithelial cells after undergoing EMT show typical features characterized as: (1) loss of adherens junctions (AJs) and tight junctions (TJs) and apical–basal polarity; (2) cytoskeletal reorganization and distribution; and (3) gain of aggressive phenotype of migration and invasion [98]. Therefore, EMT has been considered to be an important process in cancer progression and its pathological activation during tumor development induces primary tumor cells to metastasize [95]. However, recent studies showed that the EMT status was not unanimously correlated with poorer survival in cancer patients examined [92].

In addition to EMT in epithelial cells, mesenchymal-like cells have capability to regain a fully differentiated epithelial phenotype via the MET [6,35]. The key feature of MET is defined as a process of transdifferentiation of mesenchymal-like cells to polarized epithelial-like cells [23,52] and mediates the establishment of distant metastatic tumors at secondary sites [22]. Recent studies demonstrated that distant metastases in breast cancer expressed an equal or stronger E-cadherin signal than the respective primary tumors and the re-expression of E-cadherin was independent of the E-cadherin status of the primary tumors [58]. Similarly, it was found that E-cadherin is re-expressed in bone metastasis or distant metastatic tumors arising from E-cadherin-negative poorly differentiated primary breast carcinoma [81], or from E-cadherin-low primary tumors [25]. In prostate and bladder cancer cells, the nonmetastatic mesenchymal-like cells were interacted with metastatic epithelial-like cells to accelerate their metastatic colonization [20]. It is, therefore, suggested that the EMT/MET work co-operatively in driving metastasis.

2.2. Molecular regulation of EMT/MET

E-cadherin is considered to be a key molecule that provides the physical structure for both cell–cell attachment and recruitment of signaling complexes [75]. Loss of E-cadherin is a hallmark of EMT [53]. Therefore, characterizing transcriptional regulators of E-cadherin expression during EMT/MET has provided important insights into the molecular mechanisms underlying the loss of cell–cell adhesion and the acquisition of migratory properties during carcinoma progression [73].

Several known signaling pathways, such as those involving transforming growth factor-β (TGF-β), Notch, fibroblast growth factor and Wnt signaling pathways, have been shown to trigger epithelial dedifferentiation and EMT [28,97,110]. These signals repress transcription of epithelial genes, such as those encoding E-cadherin and cytokeratins, or activate transcription programs that facilitate fibroblast-like motility and invasion [73,97].

The involvement of microRNAs (miRNAs) in controlling EMT has been emphasized [11,12,18]. MiRNAs are small non-coding RNAs (∼23 nt) that silence gene expression by pairing to the 3′UTR of target mRNAs to cause their posttranscriptional repression [7]. MiRNAs can be characterized as “mesenchymal miRNA” and “epithelial miRNA” [68]. The “mesenchymal miRNA” plays an oncogenic role by promoting EMT in cancer cells. For instance, the well-known miR-21, miR-103/107 are EMT inducer by repressing Dicer and PTEN [44].

The miR-200 family has been shown to be major “epithelial miRNA” that regulate MET through silencing the EMT-transcriptional inducers ZEB1 and ZEB2 [13,17]. MiRNAs from this family are considered to be predisposing factors for cancer cell metastasis. For instance, the elevated levels of the epithelial miR-200 family in primary breast tumors associate with poorer outcomes and metastasis [57]. These findings support a potential role of “epithelial miRNAs” in MET to promote metastatic colonization [15].

2.3. ERp29 promotes MET in breast cancer

The role of ERp29 in regulating MET has been established in basal-like MDA-MB-231 breast cancer cells. It is known that myosin light chain (MLC) phosphorylation initiates to myosin-driven contraction, leading to reorganization of the actin cytoskeleton and formation of stress fibers [55,56]. ERp29 expression in this type of cells markedly reduced the level of phosphorylated MLC [3]. These results indicate that ERp29 regulates cortical actin formation through a mechanism involved in MLC phosphorylation (Fig. 1). In addition to the phenotypic change, ERp29 expression leads to: expression and membranous localization of epithelial cell marker E-cadherin; expression of epithelial differentiation marker cytokeratin 19; and loss of the mesenchymal cell marker vimentin and fibronectin [3] (Fig. 1). In contrast, knockdown of ERp29 in epithelial MCF-7 cells promotes acquisition of EMT traits including fibroblast-like phenotype, enhanced cell spreading, decreased expression of E-cadherin and increased expression of vimentin [3,4]. These findings further substantiate a role of ERp29 in modulating MET in breast cancer cells.

Fig. 1  ERp29 triggers mesenchymal–epithelial transition. Exogenous expression of ERp29 in mesenchymal MDA-MB-231 breast cancer cells inhibits stress fiber formation by suppressing MLC phosphorylation. In addition, the overexpressed ERp29 decreases the

2.4. ERp29 targets E-cadherin transcription repressors

The transcription repressors such as Snai1, Slug, ZEB1/2 and Twist have been considered to be the main regulators for E-cadherin expression [19,26,32]. Mechanistic studies revealed that ERp29 expression significantly down-regulated transcription of these repressors, leading to their reduced nuclear expression in MDA-MB-231 cells [3,4] (Fig. 2). Consistent with this, the extracellular signal-regulated kinase (ERK) pathway which is an important up-stream regulator of Slug and Ets1 was highly inhibited [4]. Apparently, ERp29 up-regulates the expressions of E-cadherin transcription repressors through repressing ERK pathway. Interestingly, ERp29 over-expression in basal-like BT549 cells resulted in incomplete MET and did not significantly affect the mRNA or protein expression of Snai1, ZEB2 and Twist, but increased the protein expression of Slug [3]. The differential regulation of these transcriptional repressors of E-cadherin by ERp29 in these two cell-types may occur in a cell-context-dependent manner.

Fig. 2  ERp29 decreases the expression of EMT inducers to promote MET. Exogenous expression of ERp29 in mesenchymal MDA-MB-231 breast cancer cells suppresses transcription and protein expression of E-cadherin transcription repressors (e.g., ZEB2, SNAI1 and Twist), ..

2.5. ERp29 antagonizes Wnt/ β-catenin signaling

Wnt proteins are a family of highly conserved secreted cysteine-rich glycoproteins. The Wnt pathway is activated via a binding of a family member to a frizzled receptor (Fzd) and the LDL-Receptor-related protein co-receptor (LRP5/6). There are three different cascades that are activated by Wnt proteins: namely canonical/β-catenin-dependent pathway and two non-canonical/β-catenin-independent pathways that include Wnt/Ca2+ and planar cell polarity [84]. Of note, the Wnt/β-catenin pathway has been extensively studied, due to its important role in cancer initiation and progression [79]. The presence of Wnt promotes formation of a Wnt–Fzd–LRP complex, recruitment of the cytoplasmic protein Disheveled (Dvl) to Fzd and the LRP phosphorylation-dependent recruitment of Axin to the membrane, thereby leading to release of β-catenin from membrane and accumulation in cytoplasm and nuclei. Nuclear β-catenin replaces TLE/Groucho co-repressors and recruits co-activators to activate expression of Wnt target genes. The most important genes regulated are those related to proliferation, such as Cyclin D1 and c-Myc [46,94], which are over-expressed in most β-catenin-dependent tumors. When β-catenin is absent in nucleus, the transcription factors T-cell factor/lymphoid enhancer factors (TCF/LEF) recruits co-repressors of the TLE/Groucho family and function as transcriptional repressors.

β-catenin is highly expressed in the nucleus of mesenchymal MDA-MB-231 cells. ERp29 over-expression in this type of cells led to translocation of nuclear β-catenin to membrane where it forms complex with E-cadherin [3] (Fig. 3). This causes a disruption of β-catenin/TCF/LEF complex and abolishes its transcription activity. Indeed, ERp29 significantly decreased the expression of cyclin D1/D2 [36], one of the downstream targets of activated Wnt/β-catenin signaling [94], indicating an inhibitory effect of ERp29 on this pathway. Meanwhile, expression of ERp29 in this cell type increased the nuclear expression of TCF3, a transcription factor regulating cancer cell differentiation while inhibiting self-renewal of cancer stem cells [102,106]. Hence, ERp29 may play dual functions in mesenchymal MDA-MB-231 breast cancer cells by: (1) suppressing activated Wnt/β-catenin signaling via β-catenin translocation; and (2) promoting cell differentiation via activating TCF3 (Fig. 3). Because β-catenin serves as a signaling hub for the Wnt pathway, it is particularly important to focus on β-catenin as the target of choice in Wnt-driven cancers. Though the mechanism by which ERp29 expression promotes the disassociation of β-catenin/TCF/LEF complex in MDA-MB-231 cells remains elusive, activating ERp29 expression may exert an inhibitory effect on the poorly differentiated, Wnt-driven tumors.

Fig. 3  ERp29 over-expression “turns-off” activated Wnt/β-catenin signaling. In mesenchymal MDA-MB-231 cells, high expression of nuclear β-catenin activates its downstream signaling involved in cell cycles and cancer stem cell

3. ERp29 regulates epithelial cell integrity

3.1. Cell adherens and tight junctions

Adherens junctions (AJs) and tight junctions (TJs) are composed of transmembrane proteins that adhere to similar proteins in the adjacent cell [69]. The transmembrane region of the TJs is composed mainly of claudins, tetraspan proteins with two extracellular loops [1]. AJs are mediated by Ca2+-dependent homophilic interactions of cadherins [71] which interact with cytoplasmic catenins that link the cadherin/catenin complex to the actin cytoskeleton [74].

The cytoplasmic domain of claudins in TJs interacts with occludin and several zona occludens proteins (ZO1-3) to form the plaque that associates with the cytoskeleton [99]. The AJs form and maintain intercellular adhesion, whereas the TJs serve as a diffusion barrier for solutes and define the boundary between apical and basolateral membrane domains [21]. The AJs and TJs are required for integrity of the epithelial phenotype, as well as for epithelial cells to function as a tissue [75].

The TJs are closely linked to the proper polarization of cells for the establishment of epithelial architecture[86]. During cancer development, epithelial cells lose the capability to form TJs and correct apico–basal polarity [59]. This subsequently causes the loss of contact inhibition of cell growth [91]. In addition, reduction of ZO-1 and occludin were found to be correlated with poorly defined differentiation, higher metastatic frequency and lower survival rates [49,64]. Hence, TJs proteins have a tumor suppressive function in cancer formation and progression.

3.2. Apical–basal cell polarity

The apical–basal polarity of epithelial cells in an epithelium is characterized by the presence of two specialized plasma membrane domains: namely, the apical surface and basolateral surface [30]. In general, the epithelial cell polarity is determined by three core complexes. These protein complexes include: (1) the partitioning-defective (PAR) complex; (2) the Crumbs (CRB) complex; and (3) the Scribble complex[2,30,45,51]. PAR complex is composed of two scaffold proteins (PAR6 and PAR3) and an atypical protein kinase C (aPKC) and is localized to the apical junction domain for the assembly of TJs [31,39]. The Crumbs complex is formed by the transmembrane protein Crumbs and the cytoplasmic scaffolding proteins such as the homologue of Drosophila Stardust (Pals1) and Pals-associated tight junction protein (Patj) and localizes to the apical [38]. The Scribble complex is comprised of three proteins, Scribble, Disc large (Dlg) and Lethal giant larvae (Lgl) and is localized in the basolateral domain of epithelial cells [100].

Fig. 4  ERp29 regulates epithelial cell morphogenesis. Over-expression of ERp29 in breast cancer cells induces the transition from a mesenchymal-like to epithelial-like phenotype and the restoration of tight junctions and cell polarity. Up-regulation and membrane

The current data from breast cancer cells supports the idea that ERp29 can function as a tumor suppressive protein, in terms of suppression of cell growth and primary tumor formation and inhibition of signaling pathways that facilitate EMT. Nevertheless, the significant role of ERp29 in cell survival against drugs, induction of cell differentiation and potential promotion of MET-related metastasis may lead us to re-assess its function in cancer progression, particularly in distant metastasis. Hence, it is important to explore in detail the ERp29’s role in cancer as a “friend or foe” and to elucidate its clinical significance in breast cancer and other epithelial cancers. Targeting ERp29 and/or its downstream molecules might be an alternative molecular therapeutic approach for chemo/radio-resistant metastatic cancer treatment

7.3.5 Putting together structures of epidermal growth factor receptors

Bessman NJ, Freed DM, Lemmon MA
Curr Opin Struct Biol. 2014 Dec; 29:95-101


  • Several studies suggest flexible linkage between extracellular and intracellular regions. • Others imply more rigid connections, required for allosteric regulation of dimers. • Interactions with membrane lipids play important roles in EGFR regulation. • Cellular studies suggest half-of-the-sites negative cooperativity for human EGFR.

Numerous crystal structures have been reported for the isolated extracellular region and tyrosine kinase domain of the epidermal growth factor receptor (EGFR) and its relatives, in different states of activation and bound to a variety of inhibitors used in cancer therapy. The next challenge is to put these structures together accurately in functional models of the intact receptor in its membrane environment. The intact EGFR has been studied using electron microscopy, chemical biology methods, biochemically, and computationally. The distinct approaches yield different impressions about the structural modes of communication between extracellular and intracellular regions. They highlight possible differences between ligands, and also underline the need to understand how the receptor interacts with the membrane itself.

Growth factor receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) have been the subjects of intense study for many years [1,2]. There are 58 RTKs in the deduced human
proteome, and all play key roles in regulating cellular processes such as proliferation, differentiation, cell survival and metabolism, cell migration, and cell cycle control [3].  Importantly, aberrant activation
of RTK signaling by mutation, gene amplification, gene translocation or other mechanisms has been causally linked to cancers, diabetes, inflammation, and other diseases. These observations have prompted
the development of many targeted therapies that inhibit RTKs such as EGFR [4], Kit, VEGFR, or their ligands — typically employing therapeutic antibodies [5] or small molecule tyrosine kinase inhibitors [6].
Following the initial discoveries for EGFR [7] and the platelet-derived growth factor receptor (PDGFR) [8] that ligand-stabilized dimers are essential for RTK signaling, structural studies over the past decade
or so have guided development of quite sophisticated mechanistic views[1]. Each RTK has a ligand-binding extracellular region (ECR) that is linked by a single transmembrane a-helix to an intracellular
tyrosine kinase domain (TKD). Structures of the isolated ECRs and TKDs from several RTKs point to surprising mechanistic diversity across the larger family [1]. Unliganded RTKs exist as an equilibrium
mixture of inactive monomers, inactive dimers and active dimers (Figure 1), except for the extreme case of the insulin receptor (IR), which is covalently dimerized [9]. Extracellular ligand can bind to monomers,
to inactive dimers, or to active dimers — in each case pushing the equilibria shown in Figure 1 towards the central ligand-bound active dimer. Thus, ligand binding can drive receptor dimerization (Figure 1,
upper), or can promote inactive-to-active conformational transitions in dimers (Figure 1, lower). Regardless of pathway, the intracellular TKD of the ligand-stabilized dimer becomes activated either through
trans-autophosphorylation or through induced allosteric changes [1,10]. Roles for other parts of the receptor in RTK activation, including the juxtamembrane (JM) and transmembrane (TM) segments, have
also become clearer. The key current challenge for the field is to assemble data from many studies of isolated RTK parts into coherent views of how the intact receptors are regulated in their native membranes.
We will focus here on recent efforts to do this for the EGFR (or ErbB receptor) family. The missing links in intact RTKs: flexible or rigid? A central goal in extrapolating to the intact RTKs from studies of
isolated soluble domains is to understand how the individual parts of the receptor communicate with one another. The methods that have been used to produce and study the isolated domains inevitably
yield the impression that inter-domain linkers are flexible and disordered. For example, extracellular juxtamembrane regions have typically only been observed as C-terminal extensions of  the soluble ECR.
Similarly, intracellular juxtamembrane regions have been encountered predominantly as N-terminal extensions of TKD constructs, or as short peptides. In each of these contexts, the JM regions are incomplete,
and may appear disordered and flexible simply because key structural restraints have been removed. Nonetheless, this possible artifact has strongly influenced thinking about linkages between the extracellular
and intracellular regions [11], and in turn about mechanisms of RTK signaling. Highly flexible linkages between extracellular and intracellular regions of RTKs are fully consistent with simpler ligand-induced
dimerization models for transmembrane signaling by RTKs. It is more difficult, however, to understand how subtle allosteric communication across the membrane could be achieved if the linkages are truly
flexible. For example, since flexible linkage implies structural independence of the extracellular and intracellular regions, it is difficult to envision how a transition from inactive to active dimer in Figure 1
could be controlled precisely by ligand without more rigid (or restricted) connections.

Recent experimental studies with intact — or nearly intact — EGFR differ in the impressions they provide about how flexibly or rigidly the extracellular and intracellular regions are linked. Springer’s laboratory used cysteine crosslinking and mutagenesis approaches to investigate this issue for EGFR expressed in Ba/F3 cells [12]. They were unable to identify any specific JM or TM region interfaces
that were required for EGFR signaling, leading them to argue that the linkage across the membrane is too flexible to transmit a specific orientation between the extracellular and intracellular regions.
Consistent with this, negative-stain electron microscopy studies of (nearly) full-length EGFR in dodecylmaltoside micelles showed that a given extracellular dimer can be linked to several different
arrangements of the intracellular kinase domain [13,14]. Similarly, dimers driven by inhibitor binding to the intracellular TKD could couple to multiple different ECR conformations [13]. Biochemical
studies are also consistent with such structural independence of the extracellular and intracellular  regions [15,16]. Contrasting with these observations, however, Schepartz and colleagues have
reported that different precise conformations within the EGFR intracellular region can be induced by distinct activating ligands [17]. They used a method called bipartite tetracysteine display that
reports on formation of a chemically detectable tetracysteine motif when two cysteine pairs come together at  the dimer  interface. EGF activation of the receptor led to formation of a  tetracysteine
motif that requires the intracellular JM helix  [18] shown in Figure 2a to form antiparallel coiled-coil dimers  (Figure 2b/c) as proposed by Kuriyan and colleagues [19,20]. Surprisingly, transforming
growth factor-a (TGFa),which also activates EGFR, did not bring these two cysteine pairs together in the same way — arguing that TGFa does not induce formation of the same intracellular antiparallel
coiled-coil. Instead, activation of EGFR with TGFa (but not EGF) stabilized an alternative tetracysteine motif, consistent with a different intracellular JM structure. Evidence for ‘inside-out’ signaling
in EGFR has also been reported, where alterations in the intracellular JM region directly influence allosteric EGF binding to the ECR of the intact receptor analyzed in CHO cells [21–23]. The contradictory
views of flexibility versus rigidity  in linkages between the domains leave the path to understanding the intact receptor unclear, although it seems  reasonable doubt that  the inactive dimers known to
form in the absence of ligand [24–26] could be regulated by extracellular ligand if all linkages are always highly flexible.
Does the membrane hold the key?
All of the studies that support direct conformational communication between the extracellular and intracellular regions of EGFR were performed in cells [17,21,22]. By contrast, most of those that
explicitly suggest otherwise were performed in detergent micelles [13,14,15] — where the potentially important influences of specific membrane lipids (or membrane geometry) are absent. Studies of intact  EGFR in liposomes with defined lipid compositions [27] have shown that the ganglioside GM3 inhibits ligand-independent activation (and dimerization) of the receptor, apparently through interactions with a  site in its extracellular JM region. McLaughlin and colleagues [28,29] also proposed a model in which interaction of the intracellular JM region (and TKD) with anionic phospholipids in the inner leaflet of  the plasma membrane (notably PtdIns(4,5)P2) exerts an inhibitory effect that must be overcome in order for EGFR to signal. Association of the JM and TM regions with specific membrane lipids is likely to  define specific structures in the linkages between the EGFR extracellular and intracellular regions that are more well-defined (and potentially rigid) than is typically appreciated. Recent studies have begun to  shed some structural light on how membrane interactions with the intracellular JM region of EGFR might influence the signaling mechanism. Endres et al. [20] found that simply tethering the complete  intracellular region of EGFR to the inner leaflet of the plasma membrane maintains the TKD in a largely monomeric state and inhibits its kinase activity. Parallel computational studies [30] suggest that this  results from the previously proposed [29] inhibitory interaction of the JM and TKD regions of EGFR with the negatively charged membrane surface. The data of Endres et al. [20] further indicated that TM-mediated dimerization reverses this inhibitory effect. Moreover, NMR studies of a 60-residue peptide containing the TM and part of  the JM region solubilized in lipid bicelles led them to conclude that specific  TM dimerization through an N terminal GxxxG motif stabilizes formation of an antiparallel coiled-coil between the two JM fragments in the dimer — the same JM coiled-coil shown in Figure 2b/c that was  investigated in the bipartite tetracysteine display studies of  intact EGF-bound EGFR described above [17,19]. Independent solid-state NMR studies of a similar TM-JM peptide from the EGFR relative
ErbB2 in vesicles containing acidic phospholipids [31] further suggested that an activating mutation in the TM domain leads to release of  the JM region from the anionic membrane surface. Collectively,
these data suggest that ligand-induced dimerization of the receptor (or reorientation of receptors within a dimer) may engage the TM domain in a specific dimer that promotes both the formation of activating
interactions in the JM region and the disruption of inhibitory interactions between the JM region (and possibly TKD) and the membrane surface.

Negative cooperativity 
A key characteristic of ligand binding at the cell surface to EGFR [36], IR [37], and other receptors [38] is negative cooperativity — which is lost when soluble forms of the ECR from human EGFR [39]
or IR [40] are studied in isolation. Several studies have shown that intracellular and/or transmembrane regions are required for this negative cooperativity to be manifest [21,22,40,41], implying that
these parts of the receptor contribute to breaking the symmetry of the dimer — as required for the two sites to have distinct binding properties [42]. Such propagation of dimer asymmetry across the
membrane would surely require defined structures in the regions that connect extracellular and intracellular regions, and is difficult to reconcile with highly flexible JM linkers.
In brief, binding of one ligand stabilizes a singly-liganded asymmetric dimer in which the unoccupied ligand-binding site is compromised [43]. The binding affinity of the second ligand is thus reduced,
constituting a half-of-the-sites mode of negative cooperativity [44]. Leahy’s group has provided important evidence consistent with a similar mechanism in the cases of human EGFR and ErbB4 [16].
By comparing human ErbB receptor ECR dimer crystal structures with different bound ligands, Leahy and colleagues went on to identify two types of dimer interface [16], a ‘flush’ interface that resembles
the asymmetric (singly-liganded) dimer seen for the Drosophila EGFR [43] and a ‘staggered’ interface seen in the ECRs from EGFR (with bound EGF [12]) and ErbB4 (with bound neuregulin1b[16]).
These observations suggest that the ‘flush’ interface drives the most  stable dimers, which are singly liganded (Figure 2b). Binding of the second ligand is weaker, and also forces the dimer interface
into the less stable ‘staggered’ conformation (Figure 2c). Taken together, these findings suggest both a structural basis for negative cooperativity and a possible structural distinction between singly-liganded
and doubly-liganded ErbB receptor dimers.

A model for EGFR activation
The model shown in Figure 2 summarizes key proposed steps in the activation of human EGFR. In the absence of ligand, the ECR exists in a tethered conformation with the domain II ‘dimerization
arm’ engaged in an intramolecular interaction with domain IV that occludes the dimer interface [49]. The TKDs and the N-terminal portions of each intracellular JM region are thought to be engaged
in autoinhibitory interactions with the membrane surface [20,28,29,30].

Figure 2. More detailed view of EGF-induced activation of EGFR, as described in the text.
In the absence of ligand (a), the ECR adopts a tethered conformation, with an autoinhibitory tether interaction between domains II and IV. The TKD and JM regions lie against the membrane, making what
are believed to be additional autoinhibitory interactions. Domains I and III of the ECR are colored red, and domains II and IV are green. The JM helix is shown as a short cylinder and labeled in magenta.
The N-lobes and C-lobes of the kinase are also labeled, and both helix aC (blue) and the short helix in the activation loop (green) that interacts with aC to inhibit the TKD [50] are shown. The C-tail is
also depicted as a curve bearing 5 tyrosines. As described in the text, binding of a single ligand (b) induces formation of a singly-liganded dimer with a ‘flush’ (presumed asymmetric) ECR dimer interface.
The JM region forms an anti-parallel helix, as labeled in magenta, and the TKDs form an asymmetric dimer in which the activator (grey) allosterically activates the receiver (shown with an amber N-lobe).
It is not clear how the extracellular and intracellular asymmetry is structurally related, if at all. Finally, a second ligand binds to yield a more symmetric dimer with the ‘staggered’ ECR interface (c) described
in the text.

Conclusions Our mechanistic understanding of EGFR and its relatives has advanced dramatically in recent years, and the past year or two has seen substantial progress in putting the results of studies
with isolated domains together into initial views of how the intact receptor works. New insights into the origin of allosteric regulation of EGFR have been gained through a combination of innovative
structural, biochemical, cellular, and computational studies. A self-consistent picture is beginning to emerge. Two key issues remain unclear, however, and represent the current frontiers in studies of EGFR.
The first — for which we describe progress in this review — centers on the influence of specific interactions of the receptor with membrane lipids, which seem likely to define the structural ‘connections’
between extracellular and intracellular regions of the receptor. The second centers on the role of the carboxy-terminal 230 amino acids, which is believed to play a regulatory role for which little detail has
so far been defined [55].

7.3.6 Complex Relationship between Ligand Binding and Dimerization in the Epidermal Growth Factor Receptor

Bessman NJ1Bagchi A2Ferguson KM2Lemmon MA3.
Cell Rep. 2014 Nov 20; 9(4):1306-17.


  • Preformed extracellular dimers of human EGFR are structurally heterogeneous • EGFR dimerization does not stabilize ligand binding
    • Extracellular mutations found in glioblastoma do not stabilize EGFR dimerization • Glioblastoma mutations in EGFR increase ligand-binding affinity

The epidermal growth factor receptor (EGFR) plays pivotal roles in development and is mutated or overexpressed in several cancers. Despite recent advances, the complex allosteric regulation of EGFR remains incompletely understood. Through efforts to understand why the negative cooperativity observed for intact EGFR is lost in studies of its isolated extracellular region (ECR), we uncovered unexpected relationships between ligand binding and receptor dimerization. The two processes appear to compete. Surprisingly, dimerization does not enhance ligand binding (although ligand binding promotes dimerization). We further show that simply forcing EGFR ECRs into preformed dimers without ligand yields ill-defined, heterogeneous structures. Finally, we demonstrate that extracellular EGFR-activating mutations in glioblastoma enhance ligand-binding affinity without directly promoting EGFR dimerization, suggesting that these oncogenic mutations alter the allosteric linkage between dimerization and ligand binding. Our findings have important implications for understanding how EGFR and its relatives are activated by specific ligands and pathological mutations.

X-ray crystal structures from 2002 and 2003 (Burgess et al., 2003) yielded the scheme for ligand-induced epidermal growth factor receptor (EGFR) dimerization shown in Figure 1. Binding of a single ligand to domains I and III within the same extracellular region (ECR) stabilizes an “extended” conformation and exposes a dimerization interface in domain II, promoting self-association with a KD in the micromolar range (Burgess et al., 2003, Dawson et al., 2005, Dawson et al., 2007). Although this model satisfyingly explains ligand-induced EGFR dimerization, it fails to capture the complex ligand-binding characteristics seen for cell-surface EGFR, with concave-up Scatchard plots indicating either negative cooperativity (De Meyts, 2008, Macdonald and Pike, 2008) or distinct affinity classes of EGF-binding site with high-affinity sites responsible for EGFR signaling (Defize et al., 1989). This cooperativity or heterogeneity is lost when the ECR from EGFR is studied in isolation, as also described for the insulin receptor (De Meyts, 2008).

Figure 1

Structural View of Ligand-Induced Dimerization of the hEGFR ECR

(A) Surface representation of tethered, unliganded, sEGFR from Protein Data Bank entry 1NQL (Ferguson et al., 2003). Ligand-binding domains I and III are green and cysteine-rich domains II and IV are cyan. The intramolecular domain II/IV tether is circled in red.

(B) Hypothetical model for an extended EGF-bound sEGFR monomer based on SAXS studies of an EGF-bound dimerization-defective sEGFR variant (Dawson et al., 2007) from PDB entry 3NJP (Lu et al., 2012). EGF is blue, and the red boundary represents the primary dimerization interface.

(C) 2:2 (EGF/sEGFR) dimer, from PDB entry 3NJP (Lu et al., 2012), colored as in (B). Dimerization arm contacts are circled in red.

Here, we describe studies of an artificially dimerized ECR from hEGFR that yield useful insight into the heterogeneous nature of preformed ECR dimers and into the origins of negative cooperativity. Our data also argue that extracellular structures induced by ligand binding are not “optimized” for dimerization and conversely that dimerization does not optimize the ligand-binding sites. We also analyzed the effects of oncogenic mutations found in glioblastoma patients (Lee et al., 2006), revealing that they affect allosteric linkage between ligand binding and dimerization rather than simply promoting EGFR dimerization. These studies have important implications for understanding extracellular activating mutations found in EGFR/ErbB family receptors in glioblastoma and other cancers and also for understanding specificity of ligand-induced ErbB receptor heterodimerization

Predimerizing the EGFR ECR Has Modest Effects on EGF Binding

To access preformed dimers of the hEGFR ECR (sEGFR) experimentally, we C-terminally fused (to residue 621 of the mature protein) either a dimerizing Fc domain (creating sEGFR-Fc) or the dimeric leucine zipper from S. cerevisiae GCN4 (creating sEGFR-Zip). Size exclusion chromatography (SEC) and/or sedimentation equilibrium analytical ultracentrifugation (AUC) confirmed that the resulting purified sEGFR fusion proteins are dimeric (Figure S1). To measure KD values for ligand binding to sEGFR-Fc and sEGFR-Zip, we labeled EGF with Alexa-488 and monitored binding in fluorescence anisotropy (FA) assays. As shown in Figure 2A, EGF binds approximately 10-fold more tightly to the dimeric sEGFR-Fc or sEGFR-Zip proteins than to monomeric sEGFR (Table 1). The curves obtained for EGF binding to sEGFR-Fc and sEGFR-Zip showed no signs of negative cooperativity, with sEGFR-Zip actually requiring a Hill coefficient (nH) greater than 1 for a good fit (nH = 1 for both sEGFRWT and sEGFR-Fc). Thus, our initial studies argued that simply dimerizing human sEGFR fails to restore the negatively cooperative ligand binding seen for the intact receptor in cells.

One surprise from these data was that forced sEGFR dimerization has only a modest (≤10-fold) effect on EGF-binding affinity. Under the conditions of the FA experiments, isolated sEGFR (without zipper or Fc fusion) remains monomeric; the FA assay contains just 60 nM EGF, so the maximum concentration of EGF-bound sEGFR is also limited to 60 nM, which is over 20-fold lower than the KD for dimerization of the EGF/sEGFR complex (Dawson et al., 2005, Lemmon et al., 1997). This ≤10-fold difference in affinity for dimeric and monomeric sEGFR seems small in light of the strict dependence of sEGFR dimerization on ligand binding (Dawson et al., 2005,Lax et al., 1991, Lemmon et al., 1997). Unliganded sEGFR does not dimerize detectably even at millimolar concentrations, whereas liganded sEGFR dimerizes with KD ∼1 μM, suggesting that ligand enhances dimerization by at least 104– to 106-fold. Straightforward linkage of dimerization and binding equilibria should stabilize EGF binding to dimeric sEGFR similarly (by 5.5–8.0 kcal/mol). The modest difference in EGF-binding affinity for dimeric and monomeric sEGFR is also significantly smaller than the 40- to 100-fold difference typically reported between high-affinity and low-affinity EGF binding on the cell surface when data are fit to two affinity classes of binding site (Burgess et al., 2003, Magun et al., 1980).

Mutations that Prevent sEGFR Dimerization Do Not Significantly Reduce Ligand-Binding Affinity

The fact that predimerizing sEGFR only modestly increased ligand-binding affinity led us to question the extent to which domain II-mediated sEGFR dimerization is linked to ligand binding. It is typically assumed that the domain II conformation stabilized upon forming the sEGFR dimer in Figure 1C optimizes the domain I and III positions for EGF binding. To test this hypothesis, we introduced a well-characterized pair of domain II mutations into sEGFRs that block dimerization: one at the tip of the dimerization arm (Y251A) and one at its “docking site” on the adjacent molecule in a dimer (R285S). The resulting (Y251A/R285S) mutation abolishes sEGFR dimerization and EGFR signaling (Dawson et al., 2005, Ogiso et al., 2002). Importantly, we chose isothermal titration calorimetry (ITC) for these studies, where all interacting components are free in solution. Previous surface plasmon resonance (SPR) studies have indicated that dimerization-defective sEGFR variants bind immobilized EGF with reduced affinity (Dawson et al., 2005), and we were concerned that this reflects avidity artifacts, where dimeric sEGFR binds more avidly than monomeric sEGFR to sensor chip-immobilized EGF.

Surprisingly, our ITC studies showed that the Y251A/R285S mutation has no significant effect on ligand-binding affinity for sEGFR in solution (Table 1). These experiments employed sEGFR (with no Fc fusion) at 10 μM—ten times higher than KD for dimerization of ligand-saturated WT sEGFR (sEGFRWT) (KD ∼1 μM). Dimerization of sEGFRWT should therefore be complete under these conditions, whereas the Y251A/R285S-mutated variant (sEGFRY251A/R285S) does not dimerize at all (Dawson et al., 2005). The KD value for EGF binding to dimeric sEGFRWT was essentially the same (within 2-fold) as that for sEGFRY251A/R285S (Figures 2B and 2C; Table 1), arguing that the favorable Gibbs free energy (ΔG) of liganded sEGFR dimerization (−5.5 to −8 kcal/mol) does not contribute significantly (<0.4 kcal/mol) to enhanced ligand binding. …

Thermodynamics of EGF Binding to sEGFR-Fc

If there is no discernible positive linkage between sEGFR dimerization and EGF binding, why do sEGFR-Fc and sEGFR-Zip bind EGF ∼10-fold more strongly than wild-type sEGFR? To investigate this, we used ITC to compare EGF binding to sEGFR-Fc and sEGFR-Zip (Figures 3A and 3B ) with binding to isolated (nonfusion) sEGFRWT. As shown in Table 1, the positive (unfavorable) ΔH for EGF binding is further elevated in predimerized sEGFR compared with sEGFRWT, suggesting that enforced dimerization may actually impair ligand/receptor interactions such as hydrogen bonds and salt bridges. The increased ΔH is more than compensated for, however, by a favorable increase in TΔS. This favorable entropic effect may reflect an “ordering” imposed on unliganded sEGFR when it is predimerized, such that it exhibits fewer degrees of freedom compared with monomeric sEGFR. In particular, since EGF binding does induce sEGFR dimerization, it is clear that predimerization will reduce the entropic cost of bringing two sEGFR molecules into a dimer upon ligand binding, possibly underlying this effect.

Possible Heterogeneity of Binding Sites in sEGFR-Fc

Close inspection of EGF/sEGFR-Fc titrations such as that in Figure 3A suggested some heterogeneity of sites, as evidenced by the slope in the early part of the experiment. To investigate this possibility further, we repeated titrations over a range of temperatures. We reasoned that if there are two different types of EGF-binding sites in an sEGFR-Fc dimer, they might have different values for heat capacity change (ΔCp), with differences that might become more evident at higher (or lower) temperatures. Indeed, ΔCp values correlate with the nonpolar surface area buried upon binding (Livingstone et al., 1991), and we know that this differs for the two Spitz-binding sites in the asymmetric Drosophila EGFR dimer (Alvarado et al., 2010). As shown in Figure 3C, the heterogeneity was indeed clearer at higher temperatures for sEGFR-Fc—especially at 25°C and 30°C—suggesting the possible presence of distinct classes of binding sites in the sEGFR-Fc dimer. We were not able to fit the two KD values (or ΔH values) uniquely with any precision because the experiment has insufficient information for unique fitting to a model with four variables. Whereas binding to sEGFRWT could be fit confidently with a single-site binding model throughout the temperature range, enforced sEGFR dimerization (by Fc fusion) creates apparent heterogeneity in binding sites, which may reflect negative cooperativity of the sort seen with dEGFR. …

Ligand Binding Is Required for Well-Defined Dimerization of the EGFR ECR

To investigate the structural nature of the preformed sEGFR-Fc dimer, we used negative stain electron microscopy (EM). We hypothesized that enforced dimerization might cause the unliganded ECR to form the same type of loose domain II-mediated dimer seen in crystals of unliganded Drosophila sEGFR (Alvarado et al., 2009). When bound to ligand (Figure 4A), the Fc-fused ECR clearly formed the characteristic heart-shape dimer seen by crystallography and EM (Lu et al., 2010, Mi et al., 2011). Figure 4B presents a structural model of an Fc-fused liganded sEGFR dimer, and Figure 4C shows a calculated 12 Å resolution projection of this model. The class averages for sEGFR-Fc plus EGF (Figure 4A) closely resemble this model, yielding clear densities for all four receptor domains, arranged as expected for the EGF-induced domain II-mediated back-to-back extracellular dimer shown in Figure 1 (Garrett et al., 2002, Lu et al., 2010). In a subset of classes, the Fc domain also appeared well resolved, indicating that these particular arrangements of the Fc domain relative to the ECR represent highly populated states, with the Fc domains occupying similar positions to those of the kinase domain in detergent-solubilized intact receptors (Mi et al., 2011). …

Our results and those of Lu et al. (2012)) argue that preformed extracellular dimers of hEGFR do not contain a well-defined domain II-mediated interface. Rather, the ECRs in these dimers likely sample a broad range of positions (and possibly conformations). This conclusion argues against recent suggestions that stable unliganded extracellular dimers “disfavor activation in preformed dimers by assuming conformations inconsistent with” productive dimerization of the rest of the receptor (Arkhipov et al., 2013). The ligand-free inactive dimeric ECR species modeled by Arkhipov et al. (2013) in their computational studies of the intact receptor do not appear to be stable. The isolated ECR from EGFR has a very low propensity for self-association without ligand, with KD in the millimolar range (or higher). Moreover, sEGFR does not form a defined structure even when forced to dimerize by Fc fusion. It is therefore difficult to envision how it might assume any particular autoinhibitory dimeric conformation in preformed dimers. …

Extracellular Oncogenic Mutations Observed in Glioblastoma May Alter Linkage between Ligand Binding and sEGFR Dimerization

Missense mutations in the hEGFR ECR were discovered in several human glioblastoma multiforme samples or cell lines and occur in 10%–15% of glioblastoma cases (Brennan et al., 2013, Lee et al., 2006). Several elevate basal receptor phosphorylation and cause EGFR to transform NIH 3T3 cells in the absence of EGF (Lee et al., 2006). Thus, these are constitutively activating oncogenic mutations, although the mutated receptors can be activated further by ligand (Lee et al., 2006, Vivanco et al., 2012). Two of the most commonly mutated sites in glioblastoma, R84 and A265 (R108 and A289 in pro-EGFR), are in domains I and II of the ECR, respectively, and contribute directly in inactive sEGFR to intramolecular interactions between these domains that are thought to be autoinhibitory (Figure 5). Domains I and II become separated from one another in this region upon ligand binding to EGFR (Alvarado et al., 2009), as illustrated in the lower part of Figure 5. Interestingly, analogous mutations in the EGFR relative ErbB3 were also found in colon and gastric cancers (Jaiswal et al., 2013).

We hypothesized that domain I/II interface mutations might activate EGFR by disrupting autoinhibitory interactions between these two domains, possibly promoting a domain II conformation that drives dimerization even in the absence of ligand. In contrast, however, sedimentation equilibrium AUC showed that sEGFR variants harboring R84K, A265D, or A265V mutations all remained completely monomeric in the absence of ligand (Figure 6A) at a concentration of 10 μM, which is similar to that experienced at the cell surface (Lemmon et al., 1997). As with WT sEGFR, however, addition of ligand promoted dimerization of each mutated sEGFR variant, with KD values that were indistinguishable from those of WT. Thus, extracellular EGFR mutations seen in glioblastoma do not simply promote ligand-independent ECR dimerization, consistent with our finding that even dimerized sEGFR-Fc requires ligand binding in order to form the characteristic heart-shaped dimer. …

We suggest that domain I is normally restrained by domain I/II interactions so that its orientation with respect to the ligand is compromised. When the domain I/II interface is weakened with mutations, this effect is mitigated. If this results simply in increased ligand-binding affinity of the monomeric receptor, the biological consequence might be to sensitize cells to lower concentrations of EGF or TGF-α (or other agonists). However, cellular studies of EGFR with glioblastoma-derived mutations (Lee et al., 2006, Vivanco et al., 2012) clearly show ligand-independent activation, arguing that this is not the key mechanism. The domain I/II interface mutations may also reduce restraints on domain II so as to permit dimerization of a small proportion of intact receptor, driven by the documented interactions that promote self-association of the transmembrane, juxtamembrane, and intracellular regions of EGFR (Endres et al., 2013, Lemmon et al., 2014, Red Brewer et al., 2009).

Setting out to test the hypothesis that simply dimerizing the EGFR ECR is sufficient to recover the negative cooperativity lost when it is removed from the intact receptor, we were led to revisit several central assumptions about this receptor. Our findings suggest three main conclusions. First, we find that enforcing dimerization of the hEGFR ECR does not drive formation of a well-defined domain II-mediated dimer that resembles ligand-bound ECRs or the unliganded ECR from Drosophila EGFR. Our EM and SAXS data show that ligand binding is necessary for formation of well-defined heart-shaped domain II-mediated dimers. This result argues that the unliganded extracellular dimers modeled by Arkhipov et al. (2013)) are not stable and that it is improbable that stable conformations of preformed extracellular dimers disfavor receptor activation by assuming conformations that counter activating dimerization of the rest of the receptor. Recent work from the Springer laboratory employing kinase inhibitors to drive dimerization of hEGFR (Lu et al., 2012) also showed that EGF binding is required to form heart-shaped ECR dimers. These findings leave open the question of the nature of the ECR in preformed EGFR dimers but certainly argue that it is unlikely to resemble the crystallographic dimer seen for unligandedDrosophila EGFR (Alvarado et al., 2009) or that suggested by computational studies (Arkhipov et al., 2013).

This result argues that ligand binding is required to permit dimerization but that domain II-mediated dimerization may compromise, rather than enhance, ligand binding. Assuming flexibility in domain II, we suggest that this domain serves to link dimerization and ligand binding allosterically. Optimal ligand binding may stabilize one conformation of domain II in the scheme shown in Figure 1 that is then distorted upon dimerization of the ECR, in turn reducing the strength of interactions with the ligand. Such a mechanism would give the appearance of a lack of positive linkage between ligand binding and ECR dimerization, and a good test of this model would be to determine the high-resolution structure of a liganded sEGFR monomer (which we expect to differ from a half dimer). This model also suggests a mechanism for selective heterodimerization over homodimerization of certain ErbB receptors. If a ligand-bound EGFR monomer has a domain II conformation that heterodimerizes with ErbB2 in preference to forming EGFR homodimers, this could explain several important observations. It could explain reports that ErbB2 is a preferred heterodimerization partner of EGFR (Graus-Porta et al., 1997) and might also explain why EGF binds more tightly to EGFR in cells where it can form heterodimers with ErbB2 than in cells lacking ErbB2, where only EGFR homodimers can form (Li et al., 2012).

7.3.7 IGFBP-2/PTEN: A critical interaction for tumours and for general physiology?

The insulin-like growth factor family of proteins, together with insulin, form an evolutionarily conserved system that helps to coordinate the metabolic status and activity of organisms with their nutritional environment. When food is abundant, the IGF/insulin signalling pathway is switched on and cell proliferation and other activities are enhanced; while when food is limited, such activities are suppressed to conserve energy and resources [1,2]. The IGF axis consists of two ligands IGF-I and -II, a series of heterotetrameric tyrosine kinase receptors and six high affinity binding proteins IGFBP-1 to-6. These IGFBPs not only regulate the reservoir, availability and functions of IGFs but also have direct actions upon cell behaviour that are independent of IGF-binding [3]. The six IGFBPs are conserved in all placental mammals having evolved from serial duplication of genes that were present throughout vertebrate evolution [4]. Each of the six IGFBPs has evolved unique functions that presumably have conferred some evolutionary advantage and hence have been conserved across mammalian evolution. After IGFBP-3, IGFBP-2 is the second most abundant binding protein in the circulation throughout adult life in humans. While circulating IGFBP-3 levels peak during puberty and decrease thereafter, IGFBP-2 levels are highest in infancy and old age. Together with the other five IGFBPs, IGFBP-2 regulates IGF availability and actions and has pleiotropic effects on normal and neoplastic tissues [3]. One of the clear distinctive structural features of IGFBP-2 is that it contains an Arg-Gly-Asp (RGD) sequence that enables functional interactions with integrin receptors [4]. This structural element is only present in one of the other IGFBPs, IGFBP-1. Although the RGD sequence was only acquired in IGFBP-1 during mammalian evolution it was present within IGFBP-2 from early vertebrate evolution indicating that it has been a long retained functional characteristic of IGFBP-2 [4]. The integrin receptors are critical for the anchorage of cells to the extracellular matrix (ECM) within tissues and hence for maintaining tissue architecture [5,6]. In solid tissue an important safeguard is imposed by linking normal cell functions and proliferation to appropriate cues from the ECM that are mediated by signals from attachment receptors such as the integrin receptors. Anchoragedependent growth is a common feature of normal cells and loss of attachment results in a form of apoptosis called anoikis. The integrin receptors interact with growth factor receptors in an ancillary and permissive manner to ensure that the signals for growth and survival occur in the appropriate setting and not inappropriately in detached cells. It has also become clear that integrin receptors serve many other roles in regulating cell functions and integrating cues from the surrounding ECM [5,6]. Over the last few decades, as the role of IGFBPs as extracellular modulators of IGF-availability and actions has emerged, there has also been a gradual characterization of the intracellular counter-regulatory components that modulate the signals initiated by IGF-receptor activation. There has been considerable progress in charting the signalling cascades initiated from these receptors but it is evident that the reason needs to be mechanisms for inactivating the pathways in intervening periods in preparation for subsequent activation. Throughout the canonical kinase cascades, activated by receptor ligation, at each node there is a corresponding phosphatase that returns the pathway to the inactive state and modulates the signal. The extracellular regulators of these phosphatases have however received much less attention than the activating kinases. That the extracellular counter-regulators may act in synchrony and be linked to the intracellular counter-regulators has only recently started to be revealed. Transgenic over-expression of IGFBP-2 at supra-physiological levels in mice results in reduced somatic growth [7] and this growth deficit is more pronounced when these mice were crossed with mice with raised growth hormone/IGF-I [8] implying that the growth inhibitory effect was due to sequestration of IGF-I. As with most of the IGFBP-family [3], there are also however multiple lines of evidence that IGFBP-2 has cellular actions that are independent of its ability to bind IGFs. There is evidence that IGFBP-2 initiates intrinsic cellular signalling through specific binding of its RGD-motif to integrin receptors, particularly the α5β1 integrin.In addition IGFBP-2 appears to modulate IGF and epidermal growth factor signalling through interactions with α5β3 integrins [9]. A heparin binding domain also exists in IGFBP-2 and it has been shown to bind to glycosaminoglycans [10], heparin [11], and other proteoglycans such as the receptor protein tyrosine phosphatase-β (RPTPβ) [12,13]. In addition,IGFBP-2has been reported to be localized on the cell surface, in the cytoplasm and on the nuclear membrane[14]. Several groups have now reported nuclear localization of IGFBP-2 [15–17]. A functional nuclear localization sequence in the central domain of IGFBP-2 has been reported that appears to interact with importin-α [18]. In the nucleus IGFBP-2 has been reported to regulate the expression of vascular endothelial growth factor [19].
IGFBP-2 and metabolic regulation
Epidemiological studies of human populations have indicated that IGFBP-2 levels are reduced in obesity, metabolic syndrome and type 2 diabetes and are inversely correlated with insulin sensitivity [20]. That these associations were due to a metabolic role for IGFBP-2, rather thanitjustbeingamarkerofdisturbance,hasbeenconfirmedinanumber of animal models. Using a transgenic IGFBP-2 over-expressing mouse model, Wheatcroft and coworkers found that IGFBP-2 was able to protect mice from high-fat/high-energy induced obesity and insulin resistance, and also protected the mice from the age-related development of glucose intolerance and hypertension [21]. Over-expression of IGFBP-2 induced by Leptin in wild type or obese mice similarly resulted in reduced plasma glucose and insulin levels [22]. All these data indicate a metabolic role for IGFBP-2 in glucose homeostasis.
IGFBP-2 and cancer
As indicated above, the early reports had implied that IGFBP-2 was generally a negative regulator of IGF-activity; the systemic growth restriction observed in transgenic mice over-expressing IGFBP-2 was followed by observations that chemically induced colorectal cancers were inhibited in this model [23]. Despite this there has been an accumulation of evidence indicating that IGFBP-2 is positively associated with the malignant progression of a wide range of cancers, as has been reviewed previously [24]. Raised serum levels of IGFBP-2 have been reported and positive associations between tumor IGFBP-2 expression and malignancy or metastasis have been observed for a variety of cancers, including glioma [25], breast [26], prostate [27], lung [28], colon [29] and lymphoid tumor [30]. Subsequent work has generally been consistent with this association between IGFBP-2 and cancer progression. In view of the majority of evidence, indicating that IGFBP-2 interacting with IGFs generally inhibited cell growth, it was suggested thatIGF-independentactionswereprobablyresponsibleforpositiveassociations between IGFBP-2 and tumourgrowth and progression [24]. The explanation for the increased expression of IGFBP-2 that has beenreportedformanydifferentcancersappearstocomefromthefactorsthat have been shown to regulate IGFBP-2 expression.The tumor suppressor gene p53, which is the most mutated gene in many human cancers, has been reported to transcriptionally regulate IGFBP-2 [31].

There also appears to again be reciprocal feedback as p53 mRNA in the breast cancer cell line Hs578T increased significantly after treatment with human recombinant IGFBP-2, suggesting a close interaction between IGFBP-2 and p53 [14]. A number of hormonal regulators of IGFBP-2 expression have been described including hCG, FSH, TGF-β, IL1, estradiol, glucocorticoids, EGF, IGF-I, IGF-II and insulin [24]. The stimulation of IGFBP-2 expression by EGF, IGF-I, IGF-II and insulin has been shown to be via the PI3K/AKT/mTOR pathway in breast cancer cells [32] and in adipocytes [33]. This is one of the most well characterisedsignallingpathwaysactivatedbyinsulinandIGFs.Inaddition the critical counter-regulatory phosphatase that deactivates this pathway the phosphatase and tensin homologue PTEN has been shown to downregulate the expression of IGFBP-2 [34]. This suggests another autoregulatory loop in which activation of the PI3K/AKT/mTOR pathway by IGFs induces the expression of IGFBP-2 that then sequesters the IGFs and modulates the signal. As activating mutations in the PI3K pathway or loss of PTEN are very common across a variety of human cancers, this plus the effect of p53, probably accounts for the common dysregulation of IGFBP-2 observed across many cancers. Using prostate cancer cell lines it has also been shown that local IGFBP-2 expression is metabolically regulated; IGFBP-2 expression was increased in hyperglycemic conditions through acetylation of histones H3 and H4 associated with the IGFBP-2 promoter, furthermore this up-regulation of IGFBP-2 mediated hyperglycemia-induced chemo-resistance [35].

The signaling kinase PI3K plays a fundamental role that has been maintained throughout most of evolution. The ability to control growth and development according to the availability of nutrients provides a survival advantage and therefore has been selectively retained throughout evolution. Evidence has accumulated to indicate that the PI3K pathway provides this control in all species from yeast to mammals.Various forms of the PI3K enzyme exist that are classified into three groups (classes I, II, and III). Only one of these forms is present in yeast and is equivalent to mammalian class III PI3K: this acts as a nutrient sensor and is directly activated by the availability of amino acids and then itself activates mTOR/S6K1 to regulate cell growth and development [36]. In mammals class 1API3K has evolved: this form is not directly activated by nutrients but consists of heterodimers comprising a catalytic p110 subunit and a regulatory p85 subunit that enables the enzyme to be controlled by receptor tyrosine kinases, classically the insulin and insulin-like growth factor receptors (IR and IGF-IR) [37]. This enables the regulation of PI3K by social nutritionally dependent signals rather than by nutrients directly. It is not by chance that the insulin/IGF/PI3K pathway plays a fundamental role in regulating both metabolism and growth as it clearly is an advantage to synchronize the set processes and this synchronized control has been maintained throughout evolution.

Phosphatase and tensin homolog (PTEN)
Of all the intracellular counter-regulators of the IGF-pathway the one that has received the most attention in relation to cancer is PTEN. PTEN is a lipid tyrosine phosphatase that negatively regulates the Akt/ PKB signaling pathway by specifically dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate and thereby reduces AKT activation to reduce signals for cell metabolism, proliferation and survival [37]. PTEN is the second most often mutated tumor suppressor in human cancers, after p53[38]. Aberrant PTEN activity occurs due to mutation, homozygous deletion, loss of heterozygosity, or epigenetic silencing. Lost or reduced activity of PTEN has been observed in a great variety of cancers, including breast [39], prostate [40,41], colorectal [42], lung[43], glioblastoma [44], endometrial [45], etc. It has been demonstrated that deregulation of PTEN is involved in tumorigenesis, tumor progression and also the predisposition of many cancers [46]. AsPI3K/Akt signaling is critical for the metabolic effects of insulin. It is clear that PTEN will also play a role in modulating the metabolic actions of insulin. Consistent with this mice genetically modified to have haploinsufficiency of PTEN were observed to be hypersensitive to insulin [47]. Similarly humans with haplo-insufficiency due to mutations in PTEN were found to have enhanced insulin sensitivity [48]. Recently an increase in insulin sensitivity due to suppression of PTEN has been described in grizzly bears in preparation for hibernation, indicating that this is a mechanism for physiological adaptation [49]. Although the genetic defects resulting in PTEN loss in cancers and the intrinsic mechanisms for regulation of PTEN have been well characterised; there have been relatively few reports of external cell regulators. Of interest one of the few extrinsic regulators that has been described is IGF-II [50]. IGF-II is the most abundant growth factor present in most human tissues and activates the PI3K/AKT/mTOR pathway. Just as the induction of IGFBP-2 by activation of the PI3K pathway suggests an autoregulatory feedback loop extrinsic to the cell;the induction of PTEN by IGF-II via PI3K suggests an additional feedback loop that is intrinsic within the cell (Fig. 1). Activation of the pathway by IGF-II induces expression of PTEN that then attenuates the signal; conversely when the pathway is not activated then PTEN expression is reduced making the cell more responsive for when an activation signal is next received.One of the mechanisms that has emerged,to explain this feedback loop, indicates that the signaling output of the PI3K/PTEN pathway is balanced by asynchronous regulation of the activity of both PI3K and PTEN. The p85α regulatory subunit of PI3K that binds to and represses the activity of the p110 catalytic subunit also binds directly to PTEN at a regulatory site within PTEN where serine/threonine phosphorylation occurs to inactivatePTEN.The p85α subunit binds to unphosphorylated PTEN at this site and enhances its lipid phosphatase activity 3-fold [51]. The nature of this feedback loop has been further extended by reports that PTEN can suppress the expression of IGF-II [52,53]. As IGF-II induces PTEN, the ability of PTEN to subsequently reduce IGF-II expression may enable the cell to protect itself from over-stimulation. In contrast loss of PTEN may increase the expression of IGF-II resulting inactivation of the PI3K/AKT/mTOR pathway that is then unopposed.

PTEN/IGFBP-2 interactions
In view of the recognized importance of loss of PTEN for a variety of cancers there has been considerable interest in identifying biomarkers that could be used clinically to indicate loss of PTEN within tumors. An unbiased screen of human prostate cancer xenografts and human glioblastoma samples using microarray-based expression profiling found that the most significant gene was IGFBP-2 and it was confirmed in experimental models that IGFBP-2 was inversely regulated by PTEN [54]. This was consistent with all of the subsequent studies indicating that the expression of IGFBP-2 was regulated by the PI3K/AKT/mTOR pathway. An increase in tumor IGFBP-2 has also been associated with loss of PTEN in human breast cancer samples[55]. In the same year that a screen revealed IGFBP-2 as the best marker for loss of PTEN; the nature of the interaction between these two proteins was extended by the demonstration that at the cellular level IGFBP-2 can inversely regulate PTEN. With human breast cancer cells it was confirmed that IGF-II stimulated PTEN expression and that this was modulated by the binding of IGF-II to IGFBP-2, but when IGFBP-2 was not bound to IGF-II it was able to suppress PTEN via an interaction with cell surface integrin receptors (Fig. 1) [56]. Subsequent work with human prostate cancer cells indicated that the interaction of IGFBP-2 with integrin receptors could also result in PTEN inactivation via increasing its phosphorylation [57].

Fig.1. A proposed autoregulatory feedback loop of IGFBP-2/PTEN interaction. Binding of IGF-II to the IGF-IR activates the PI3K pathway. Induction of PI3K activates Akt and mTOR signaling and leads to cell proliferation and cell survival. The regulatory subunit of PI3K,p85, also binds and activates PTEN through dephosphorylation. This increased PTEN subsequently blocks IGFII production to avoid overstimulation. On the other hand, activated PI3K pathway induces IGFBP-2 expression, which when unbound to IGF-II, suppresses PTEN via an interaction with integrin receptors and/or the receptor protein tyrosine phosphatase β(RPTPβ). Thus the negative control of PTEN on PI3K signaling is counteracted. These feedback loops enable the extrinsic balance between IGF-II and IGFBP-2 to be tightly integrated to the intrinsic balance between PI3K and PTEN.

The ability of IGFBP-2 to regulate PTEN, originally observed in human cancer cell lines has subsequently been confirmed in a variety of normal cell types from different tissues. In IGFBP-2 knock-out mice a decrease in hematopoietic stem cell survival and cycling has been associated with an increase in PTEN and this appeared to be mediated by the heparin binding domain (HBD) within IGFBP-2 as the administration of a peptide analogue could restore the deficit [58]. Similarly a decrease in bone mass in the IGFBP-2 knock-out mice has been attributed to an increase in PTEN and again the use of a peptide analogue appeared to implicate the IGFBP-2HBD [59]. It was subsequently reported that the IGFBP-2HBD mediated an interaction with the RPTPβ resulting in dimerization and consequent inactivation of RPTPβ and that this reduction in phosphatase activity cooperated with IGF-I activation of the IGF-IR to enhance the phosphorylation and inactivation of PTEN [12]. Recently IGFBP-2 has been reported to also suppress PTEN in human skeletal muscle cells [60] and human visceral adipocytes [61] by interacting with integrin receptors. A similar association between IGFBP-2 and PTEN has been implicated as playing a role in murine skeletal muscle cell differentiation, although the functional regulation was not directly investigated in that study [62].

Evidence from a variety of different sources have indicated a close regulatory feedback loop between IGFBP-2 and PTEN. Work using a variety of different cell types from different tissues and different species has indicated that IGFBP-2 inversely regulates PTEN. There are reports that this is mediated via the IGFBP-2 RGD domain interacting with integrin receptors and by the IGFBP-2 HBD interacting with proteoglycans; the relative involvement of each of these domains and their functional interactions will require further work to elucidate. These studies however suggest a general mechanism that plays a role in a variety of normal physiological processes in addition to having important implications for the progression of many different cancers. The phosphatase PTEN has an important role in determining insulin sensitivity and the extent that IGFBP-2 exerts a metabolic role in regulating PTEN to determine insulin-sensitivity is yet to be examined. The extracellular balance between IGF-II and IGFBP-2 seems tightly linked with the intracellular balance between PI3K and PTEN (Fig. 1). When driving, in order to move forward there is a synchronous application of the accelerator and a removal of the brake. It appears that the cell also synchronizes activation of an essential regulatory pathway with the removal of the tightly linked inactivation pathway.

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[9] G.K.Wang,etal., Aninteraction betweeninsulin-likegrowthfactor-bindingprotein 2 (IGFBP2) and integrin alpha5 is essential for IGFBP2-induced cell mobility, J. Biol. Chem. 281 (20) (2006) 14085–14091. [10] T.Arai,W.BusbyJr.,D.R.Clemmons,Bindingofinsulin-likegrowthfactor(IGF)IorII to IGF-binding protein-2 enables it to bind to heparin and extracellular matrix, Endocrinology 137 (11) (1996) 4571–4575. [11] J. Lund, et al., Heparin-binding mechanism of the IGF2/IGF-binding protein 2 complex, J. Mol. Endocrinol. 52 (3) (2014) 345–355.
[12] X. Shen, et al., Insulin-like growth factor (IGF) binding protein 2 functions coordinately with receptor protein tyrosinephosphatase βandtheIGF-Ireceptorto regulate IGF-I-stimulated signaling, Mol. Cell. Biol. 32 (20) (2012) 4116–4130.
[13] V.C.Russo, etal.,Insulin-like growth factor binding protein-2 bindingto extracellularmatrixplaysacriticalroleinneuroblastomacellproliferation,migration,andinvasion, Endocrinology 146 (10) (2005) 4445–4455.
[14] K.W. Frommer, etal., IGF-independent effects of IGFBP-2 on the human breast cancer cell line Hs578T, J. Mol. Endocrinol. 37 (1) (2006) 13–23.
[15] K. Miyako, et al., PAPA-1 Is a nuclear binding partner of IGFBP-2 and modulates its growth-promoting actions, Mol. Endocrinol. 23 (2) (2009) 169–175.
[16] X.Terrien,etal.,IntracellularcolocalizationandinteractionofIGF-bindingprotein-2 with the cyclin-dependent kinase inhibitor p21CIP1/WAF1 during growth inhibition, Biochem. J. 392 (Pt 3) (2005) 457–465.
[17] R.M. Villani, et al., Patched1 inhibits epidermal progenitor cell expansion and basal cell carcinoma formation by limiting Igfbp2 activity, Cancer Prev. Res. (Phila.) 3 (10) (2010) 1222–1234.
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[19] W.J.Azar,etal.,IGFBP-2enhancesVEGFgenepromoteractivityandconsequentpromotion of angiogenesis by neuroblastoma cells, Endocrinology 152 (9) (2011) 3332–3342.
[20] S.B. Wheatcroft, M.T. Kearney, IGF-dependent and IGF-independent actions of IGFbinding protein-1 and -2: implications for metabolic homeostasis, Trends Endocrinol. Metab. 20 (4) (2009) 153–162. [21] S.B. Wheatcroft, et al., IGF-binding protein-2 protects against the development of obesity and insulin resistance, Diabetes 56 (2) (2007) 285–294.

7.3.8 Emerging roles for the pH-sensing G protein-coupled receptors in response to acidotic stress

Edward J Sanderlin, Calvin R Justus, Elizabeth A Krewson, Li V Yang
Cell Health & Cytoskel Mar 2015; 2015(7): 99—109

Protons (hydrogen ions) are the simplest form of ions universally produced by cellular metabolism including aerobic respiration and glycolysis. Export of protons out of cells by a number of acid transporters is essential to maintain a stable intracellular pH that is critical for normal cell function. Acid products in the tissue interstitium are removed by blood perfusion and excreted from the body through the respiratory and renal systems. However, the pH homeostasis in tissues is frequently disrupted in many pathophysiologic conditions such as in ischemic tissues and tumors where protons are overproduced and blood perfusion is compromised. Consequently, accumulation of protons causes acidosis in the affected tissue. Although acidosis has profound effects on cell function and disease progression, little is known about the molecular mechanisms by which cells sense and respond to acidotic stress. Recently a family of pH-sensing G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), and GPR68 (OGR1), has been identified and characterized. These GPCRs can be activated by extracellular acidic pH through the protonation of histidine residues of the receptors. Upon activation by acidosis the pH-sensing GPCRs can transduce several downstream G protein pathways such as the Gs, Gq/11, and G12/13 pathways to regulate cell behavior. Studies have revealed the biological roles of the pH-sensing GPCRs in the immune, cardiovascular, respiratory, renal, skeletal, endocrine, and nervous systems, as well as the involvement of these receptors in a variety of pathological conditions such as cancer, inflammation, pain, and cardiovascular disease. As GPCRs are important drug targets, small molecule modulators of the pH-sensing GPCRs are being developed and evaluated for potential therapeutic applications in disease treatment.

Cellular metabolism produces acid as a byproduct. Metabolism of each glucose molecule by glycolysis generates two pyruvate molecules. Under anaerobic conditions the metabolism of pyruvate results in the production of the glycolytic end product lactic acid, which has a pKa of 3.9. Lactic acid is deprotonated at the carboxyl group and results in one lactate ion and one proton at the physiological pH. Under aerobic conditions pyruvate is converted into acetyl-CoA and CO2 in the mitochondria. CO2in water forms a chemical equilibrium of carbonic acid and bicarbonate, an important physiological pH buffering system. The body must maintain suitable pH for proper physiological functions. Some regulatory mechanisms to control systemic pH are respiration, renal excretion, bone buffering, and metabolism.14 The respiratory system can buffer the blood by excreting carbonic acid as CO2 while the kidney responds to decreased circulatory pH by excreting protons and electrolytes to stabilize the physiological pH. Bone buffering helps maintain systemic pH by Ca2+ reabsorption and mineral dissolution. Collectively, it is clear that several biological systems require tight regulation to maintain pH for normal physiological functions. Cells utilize vast varieties of acid-base transporters for proper pH homeostasis within each biological context.58 Some such transporters are H+-ATPase, Na+/H+exchanger, Na+-dependent HCO3/C1 exchanger, Na+-independent anion exchanger, and monocarboxylate transporters. Cells can also maintain short-term pH homeostasis of the intracellular pH by rapid H+ consuming mechanisms. Some such mechanisms utilize metabolic conversions that move acids from the cytosol into organelles. Despite these cellular mechanisms that tightly maintain proper pH homeostasis, there are many diseases whereby pH homeostasis is disrupted. These pathological conditions are characterized by either local or systemic acidosis. Systemic acidosis can occur from respiratory, renal, and metabolic diseases and septic shock.14,9 Additionally, local acidosis is characterized in ischemic tissues, tumors, and chronically inflamed conditions such as in asthma and arthritis caused by deregulated metabolism and hypoxia.1015

Acidosis is a stress for the cell. The ability of the cell to sense and modulate activity for adaptation to the stressful environment is critical. There are several mechanisms whereby cells sense acidosis and modulate cellular functions to facilitate adaptation. Cells can detect extracellular pH changes by acid sensing ion channels (ASICs) and transient receptor potential (TRP) channels.16 Apart from ASIC and TRP channels, extracellular acidic pH was shown to stimulate inositol polyphosphate formation and calcium efflux.17,18 This suggested the presence of an unknown cell surface receptor that may be activated by a certain functional group, namely the imidazole of a histidine residue. The identity of the acid-activated receptor was later unmasked by Ludwig et al as a family of proton-sensing G protein-coupled receptors (GPCRs). This group identified human ovarian cancer GPCR 1 (OGR1) which upon activation will produce inositol phosphate and calcium efflux through the Gq pathway.19 These pH-sensing GPCR family members, including GPR4, GPR65 (TDAG8), and GPR68 (OGR1), will be discussed in this review (Figure 1). The proton-sensing GPCRs sense extracellular pH by protonation of several histidine residues on their extracellular domain. The activation of these proton-sensing GPCRs facilitates the downstream signaling through the Gq/11, Gs, and G12/13 pathways. Their expression varies in different cell types and play critical roles in sensing extracellular acidity and modulating cellular functions in several biological systems.

Figure 1 Biological roles and G protein coupling of the pH-sensing GPCRs.
Abbreviation: GPCRs, G protein-coupled receptors.

Role for the pH-sensing GPCRs in the immune system and inflammation

Acidic pH is a main characteristic of the inflammatory loci.14,20,21 The acidic microenvironment in inflamed tissue is predominately due to the increased metabolic demand from infiltrating immune cells, such as the neutrophil. These immune cells increase oxygen consumption and glucose uptake for glycolysis and oxidative phosphorylation. When oxygen availability is limited, cells often undergo anaerobic glycolysis. This process generates increasing amounts of lactic acid, thereby creating a local acidic microenvironment within the inflammatory loci.22 This presents a role for the pH-sensing GPCR GPR65 (TDAG8) in inflammation and immune cell function.23 TDAG8 was originally identified by cloning as an orphan GPCR which was observed to be upregulated during thymocyte apoptosis.24,25GPR65 (TDAG8) is predominately expressed in lymphoid tissues such as the spleen, lymph nodes, thymus, and leukocytes.2426 It was demonstrated that GPR65 inhibited pro-inflammatory cytokine secretion, which includes IL-6 and TNF-α, in mouse peritoneal macrophages upon activation by extracellular acidification. This cytokine inhibition was shown to occur through the Gs-cAMP-protein kinase A (PKA) signaling pathway.23,27 Treatment with dexamethasone, a potent glucocorticoid, increased GPR65 expression in peritoneal macrophages. Following dexamethasone treatment, there was an inhibition of TNF-α secretion in a manner dependent on increased expression of GPR65.28Another report provides an anti-inflammatory role for GPR65 in arthritis.29 Type II collagen-induced arthritis was increased in GPR65-null mice in comparison to wild-type mice. These studies taken together suggest GPR65 serves as a negative regulator in inflammation.30 However, one study provided a function for GPR65 as a positive modulator in inflammation.31 GPR65 was reported to increase eosinophil viability in the acidic microenvironment by reducing apoptosis through the cAMP pathway. As eosinophils are central in asthmatic inflammation and allergic airway disease, GPR65 may play a role in increasing asthmatic inflammation.31 On the other hand, GPR65 has shown little involvement in immune cell development. One report indicates that GPR65 knockout mice had normal immune development and function.26 Modulation of inflammation by GPR65 is complex and must be examined within each specific pathology.23

In addition to GPR65, GPR4 is also involved in the inflammatory response. Endothelial cells compose blood vessels that often penetrate acidic tissue microenvironments such as the inflammatory loci. Among the pH-sensing GPCR family, GPR4 has the highest expression in endothelial cells. Response to inflammation by vascular endothelial cells facilitates the induction of inflammatory cytokines that are involved in the recruitment of leukocytes for adherence and transmigration into inflamed tissues. Activation of GPR4 by acidosis in human umbilical vein endothelial cells, among other endothelial cell types, increased the expression of a broad range of pro-inflammatory genes including chemokines, cytokines, PTGS2, NF-κB pathway genes, and adhesion molecules.32 Moreover, human umbilical vein endothelial cells, when treated with acidic pH, increased GPR4-mediated endothelial adhesion to leukocytes.32,33 Altogether, GPR65 and GPR4 provide differential regulation of the inflammatory response through their acid sensing capabilities. GPR65 predominately demonstrates function in the inhibition of the inflammatory response whereas GPR4 activation exacerbates inflammation.

Role for the pH-sensing GPCRs in the cardiovascular system

Taken together, both GPR4 and GPR68 play roles in regulating the function of the cardiovascular system. GPR4 regulates blood vessel stability and endothelial cell function and GPR68 increases cardiomyogenic and pro-survival gene expression while also mediating aortic smooth muscle cell gene expression.

Role for the pH-sensing GPCRs in the renal system

GPR4 is expressed in the kidney cortex, isolated kidney collecting ducts, inner and outer medulla, and in cultured inner and outer medullary collecting duct cells.59 In mice deficient for GPR4, renal acid excretion and the ability to respond to metabolic acidosis was reduced.59 In response to acidosis, inner and outer medullary collecting duct cells produced cAMP, a second messenger for the Gs G-protein pathway, through the GPR4 receptor.59 In renal HEK293 epithelial cells GPR4 overexpression was found to increase the activity of PKA.60 In addition, the protein expression of H+-K+-ATPase α-subunit (HKα2) was increased following GPR4 overexpression dependent on increased PKA activity.60

GPR68 has also been reported to alter proton export of HEK293 cells by stimulating the Na+/H+exchanger and H+-ATPase.58 The activation of GPR68 by acidosis was found to stimulate this effect through a cluster of extracellular histidine residues and the Gq/PKC signaling pathway.58 In GPR68-null mice the expression of the pH-sensitive kinase Pyk2 in the kidney proximal tubules was upregulated which might compensate for GPR68 deficiency.58 Taken together, GPR4 and GPR68 may both be necessary for successful systemic pH buffering by controlling renal acid excretion.

Role for the pH-sensing GPCRs in the respiratory system

Aoki et al demonstrated that GPR68-deficient mice were resistant to asthma along with inhibiting Th2 cytokine and immunoglobulin E production.68 This study concludes that GPR68 in dendritic cells is crucial for the onset of asthmatic responses.68 Moreover, GPR65 has been implicated as having a role in respiratory disorders as it is highly expressed in eosinophils, hallmark cells for asthmatic inflammation.69 Kottyan et al showed that GPR65 increased the viability of eosinophils within an acidic environment through the cAMP pathway in murine asthma models.31 In summary, GPR68 and GPR65 play important roles in the respiratory system and asthma. GPR68 regulates gene expression in airway epithelial, smooth muscle and immune cells while GPR65 enhances the survival of airway eosinophils in response to acidosis.

Role for the pH-sensing GPCRs in the skeletal system

GPR65 has also been reported as a pH sensor in bone. GPR65 is expressed in osteoclasts and its activity may inhibit Ca2+ resorption.81 Disruption of GPR65 gene exacerbated osteoclastic bone resorption in ovariectomized mice.81 The relative bone density of GPR65-null mice was less than control mice.81 In cultured osteoclast cells from mice deficient for GPR65, the normal inhibition of osteoclast formation in response to acidosis was abrogated.81 Taken together, this data suggest that the activation of GPR65 may enhance bone density, thus the GPR65 signaling may be important for disease processes such as osteoporosis and other bone density disorders.

Role for the pH-sensing GPCRs in the endocrine system

GPR68 has also been found to modify insulin production and secretion. In GPR68 knockout mice insulin secretion in response to glucose administration was reduced when compared to wild-type mice although blood glucose was not significantly altered.84 GPR68 deficiency in this respect may reduce insulin secretion but at the same time increase insulin sensitivity. In addition, stimulation of GPR68 in islet cells by acidosis increased the secretion of insulin through the Gq/11 G-protein signaling.84

Role for the pH-sensing GPCRs in the nervous system and nociception

Acidosis causes pain by exciting nociceptors located in sensory neurons. Several types of ion channels and receptors, such as ASICs, TRPV1, and proton-sensing GPCRs, have been identified as nociceptors in response to acidosis. ASICs and TRPV act as proton-gated membrane-bound channels, which are activated by acidic pH and mediate multimodal sensory perception including nociception.8688  GPR65 activation sensitized the response of TRPV1 to capsaicin. The results suggest high accumulation of protons post inflammation may not only stimulate nociceptive ion channels such as TRPV1 to trigger pain, but also activate proton-sensing GPCRs to regulate heightened sensitivity to pain.89 Furthermore, Hang et al demonstrated GPR65 activation elicited cancer-related bone pain through the PKA and phosphorylated CREB (pCREB) signaling pathway in the rat model.90 Collectively, GPR4, GPR65, and GPR68 are all expressed in the dorsal root ganglia; GPR65 is a functional receptor involved in nociception and the nervous system by sensitizing inflammatory pain and the evocation of cancer-related bone pain.

Role for the pH-sensing GPCRs in tumor biology

The tumor microenvironment is highly heterogeneous. Hypoxia, acidosis, inflammation, defective vasculature, poor blood perfusion, and deregulated cancer cell metabolism are hallmarks of the tumor microenvironment.9193 The acidity in the tumor microenvironment is owing to the altered cancer cell metabolism termed the “Warburg Effect”. This metabolic phenotype allows the cancer cells to preferentially utilize glycolysis over oxidative phosphorylation as a primary means of energy production.94 This process occurs even in normoxic tissue environments where sufficient oxygen is available. Due to this phenomenon, the Warburg Effect is often termed “aerobic glycolysis”. This unique metabolic phenotype produces vast quantities of lactic acid, which serve as a proton source for acidification. Upon disassociation of lactic acid to one lactate molecule and one proton, the monocarboxylate transporter and proton transporters export lactate and protons into the extracellular tumor microenvironment.95 The proton-sensing GPCRs are activated by acidic pH and facilitate tumor cell modulation in response to extracellular acidification. GPR4, GPR65, and GPR68 play roles in tumor cell apoptosis, proliferation, metastasis, angiogenesis, and immune cell function.19,27,32,33,44,45,96,97

GPR4 has had conflicting reports in terms of tumor suppressing or promoting activities. One study demonstrated that GPR4 could act as a tumor metastasis suppressor, when overexpressed and activated by acidic pH in B16F10 melanoma cells, by impeding migration and invasion of tumor cells.45 GPR4 overexpression also significantly inhibited the lung metastasis of B16F10 melanoma cells in mice.45 Another study utilizing the B16F10 melanoma cell line which overexpressed GPR4 showed an increase in mitochondrial surface area and a significant reduction in membrane protrusions by quantification of 3D morphology.98 These data point to a decrease in cancer cell migration when GPR4 is overexpressed and provides another example of GPR4 as exhibiting tumor metastasis suppressor function.98 However, in another report GPR4 malignantly transformed immortalized NIH3T3 fibroblasts.99 This presents GPR4 with tumor-promoting capabilities. The conflicting reports seem to indicate the functional ability of GPR4 to act as a tumor promoter and a tumor suppressor depending on the context of certain cell types and biological systems.

Reports with GPR65 involvement in cancer cells provide evidence in favor for cancer cell survival; however, opposing evidences suggest GPR65 functions as a tumor suppressor. In the same report suggesting GPR4 is oncogenic due to GPR4 transforming immortalized NIH3T3 fibroblasts, GPR65 overexpression was able to transform the mouse NMuMG mammary epithelial cell line.99 Another group demonstrated in NCI-H460 human non-small cell lung cancer cells that GPR65 promotes cancer cell survival in an acidic microenvironment.100 Conversely, a recent study showed that GPR65 inhibited c-Myc oncogene expression in human lymphoma cells.101 Furthermore, GPR65 messenger ribonucleic acid expression was reduced by more than 50% in a variety of human lymphoma samples when compared to normal lymphoid tissues, therefore implying GPR65 has a tumor suppressor function in lymphoma.101 GPR65 has also been shown to increase glucocorticoid-induced apoptosis in murine lymphoma cells.102 These reports highlight cell type dependency and biological context for GPR65 activity as a tumor suppressor or promoter.

GPR68 also has roles in tumor biology as a potential tumor suppressor or a tumor promoter. Reports have shown that GPR68 can inhibit cancer metastasis, reduce cancer cell proliferation, and inhibit migration. One study showed that when GPR68 was overexpressed in prostate cancer cells, metastasis to the lungs, diaphragm, and spleen was inhibited.97 When GPR68 was overexpressed in ovarian cancer (HEY) cells, cellular proliferation and migration were significantly reduced, and cell adhesion to the extracellular matrix was increased.96 Another study reported GPR68 expression was critical for the tumor cell induced immunosuppression in myeloid-derived cells. This study proposed that GPR68 promotes M2 macrophage development and inhibits T-cell infiltration, and thereby facilitates tumor development.103 In summary, the biological roles of GPR4, GPR65, and GPR68 in tumor biology are complex and both tumor-suppressing and tumor-promoting functions have been reported, primarily dependent on cell type and biological milieu.

Development of small molecule modulators of the pH-sensing GPCRs

GPCRs are critical receptors for the regulation of many physiological operations. It is of little surprise that GPCRs have become a central focus of pharmaceutical development. In fact, 30%–50% of therapeutics focuses on modulating GPCR activity.104,105 In view of the diverse roles of the pH-sensing GPCRs in the context of multiple biological systems, targeting these receptors with small molecules and other modulators could serve as potential therapeutics for diseases associated with deregulated pH homeostasis. There have been recent developments in the characterization of GPR4 antagonists along with agonists for GPR65 and GPR68.29,32,50,106 The GPR4 antagonist demonstrated effectiveness in vitro to reduce the GPR4-mediated inflammatory response to acidosis in endothelial cells.32 The GPR65 agonist, BTB09089, showed in vitro effects in GPR65 activation of immune cells to inhibit inflammatory response; however, the activity of BTB09089 was not strong enough for the use in animal models in vivo.29 The GPR68 agonist, lsx, exhibited pro-neurogenic activity and induced hippocampal neurogenesis in young mice.107 It was also demonstrated that lsx suppressed the proliferation of malignant astrocytes.108 To date, however, much advancement needs to be done in development of efficacious agonists and antagonists of the pH-sensing GPCRs coupled with a capacity to target specific tissue dysfunction in the midst of systemic drug administration to optimize therapeutic effects and minimize potential adverse effects.

Concluding remarks

Cells encounter acidotic stress in many pathophysiologic conditions such as inflammation, cancer, and ischemia. Intricate molecular mechanisms, including a large array of acid/base transporters and acid sensors, have evolved for cells to sense and respond to acidotic stress. Emerging evidence has demonstrated that a family of the pH-sensing GPCRs can be activated by extracellular acidotic stress and regulate the function of multiple physiological systems (Table 1). The pH-sensing GPCRs also play important roles in various pathological disorders. Agonists, antagonists and other modulators of the pH-sensing GPCRs are being actively developed and evaluated as potential novel treatment for acidosis-related diseases.

Table 1 The main biological functions of the pH-sensing GPCRs

7.3.9 Protein amino-terminal modifications and proteomic approaches for N-terminal profiling

Lai ZW1, Petrera A2, Schilling O3.
Curr Opin Chem Biol. 2015 Feb; 24:71-9

Amino-/N-terminal processing is a crucial post-translational modification affecting almost all proteins. In addition to altering the chemical properties of the N-terminus, these modifications affect protein activation, conversion, and degradation, which subsequently lead to diversified biological functions. The study of N-terminal modifications is of increasing interest; especially since modifications such as proteolytic truncation or pyroglutamate formation have been linked to disease processes. During the past decade, mass spectrometry has played an important role in facilitating the investigation of N-terminal modifications. Continuous progress is being made in the development and application of robust methods for the dedicated analysis of native and modified protein N-termini in a proteome-wide manner. Here we highlight recent progress in our understanding of protein N-terminal biology as well as outlining present enrichment strategies for mass spectrometry-based studies of protein N-termini.


    • N-terminal acetylation, pyroglutamate formation, N-degrons and proteolysis are reviewed.• N-terminomics provide comprehensive profiling of modification at protein N-termini in a proteome-wide manner.• We outline a number of established methodologies for the enrichment of protein N-termini through positive and negative selection strategies.• Peptidomics-based approach is beneficial for the study of post-translational processing of protein N-termini.

 Introduction The life of every protein begins at the amino-terminus, also known as the N-terminus. During the initiation of mRNA translation into proteins or polypeptides, newly synthesized amino
acid chains form the N-termini and are the first to exit the ribosomes into the cytosol or the endoplasmic reticulum. The N-termini of these proteins or protein precursors often contain a signaling peptide
sequence proximal to the N-terminus, which may function as a ‘zip-code’ to direct the delivery of a protein to a cellular compartment as well as orchestrating protein maturation via different post-translational
modifications (PTMs) such as acetylation or proteolysis. These modifications often determine protein activity or stability; thus being crucial for the tight regulation of cellular homeostasis (Figure 1).
Mass spectrometry (MS) based analyses of protein N-termini, termed N-terminomics, is a promising tool to tackle these problems. In the past decade, we have witnessed significant progress in the
area of mass spectrometric investigation of post-translational modifications such as phosphorylation or glycosylation [1].  Similarly, MS-based studies of protein N-termini are gaining momentum.
Recent progress in positional proteomics using advanced MS platforms combined with a number of effective enrichment strategies has reinforced significant interest in N-terminomics.
Here we outline some of the most current highlights on proteomics-based studies on N-terminal modifications, including N-acetylation, pyroglutamate formation, proteolysis, and N-terminal degrons
(Figure 2). We also present a number of recent N-terminomic methodologies for the study of protein N-termini.

Acetylation of protein N-termini represents an abundant post-translational modification in eukaryotes, affecting nearly all cytoplasmic proteins. This  modification is catalyzed by the N-terminal
acetyltransferase (Nat) enzyme complex, which transfers an acetyl group to the N-termini of newly synthesized proteins during translation (Figure 2). Initial findings highlighted that N-terminal
acetylation protects proteins from degradation [2–4]. Recent studies however yield a more diverse picture. N-terminal acetylation may also play a role in protein delivery and localization [5–7],
protein complex formation and generation of specific degradation signals in cellular proteins via the N-degron pathway [9,10]. Loss of N-terminal acetylation through inactive acetyltransferases leads to
smaller aggregates of prion proteins [11]. In addition, N-terminal acetyltransferases have been described to also function as N-terminal proprionyltransferases [12].  Genetic mutation in the Naa10 gene,
encoding the NatA catalytic subunit, is known to cause N-terminal acetyltransferase deficient phenotypes. This genetic mutation has also been linked to X-linked disorder of infancy, causing lethality in
male infants[13]. The multifunctional roles of N-acetyltransferases as well as the importance of  N-terminal acetylation have been previously reviewed in [14]. Few MS-based studies have emerged that
specifically investigate acetylated N-termini in a proteome wide manner. The structural and functional integrity of actomyosin fibers depends on active NatB. A novel methodology determines the
extent of N-terminal acetylation in vivo through chemical, stable-isotope coded acetylation of proteins before their mass spectrometric analysis [16].

Pyroglutamate conversion of N-terminal glutamate and glutamine Many proteins and biologically active peptides exhibit an N-terminal pyroglutamic acid (pGlu) residue. This post
translational modification originates from the conversion of N-terminal glutamate and glutamine into pyroglutamic acid by glutaminyl cyclase or isoglutaminyl cyclase (Figure 2). N-terminal
pGlu influences structural stability as well as biological activity of peptides and proteins [17]. pGlu protects proteins from degradation by aminopeptidases [18] as well as regulating the
biological activity of peptide hormones, neuropeptides or chemokines [19]. Examples include thyrotropin releasing hormone (TRH), gonadotropin-releasing hormone, and the human
chemokines MCP-1 and 2. The presence of N-terminal pGlu in some amyloidogenic peptides, such as amyloid-b peptides, increases their hydrophobicity, resulting in an accelerated
aggregation [20]. Modulating the extent of N-terminal pGlu formation through pharmaceutical inhibition of glutaminyl cyclase is considered a promising strategy, for example, to
increase the degradation of inflammatory and neurotoxic peptides. Inhibition of glutaminyl cyclase has alleviated liver inflammation by destabilizing the chemokine MCP1 (CCL2) [21].
Proteolytic degradation of this promigratory chemokine by inhibiting glutaminyl cyclase was also proposed as an attractive novel strategy in preventing thyroid cancer metastasis [22].
Given the functional relevance of N-terminal pGlu in pathological conditions, an MS-based approach to profile this modification may be particularly useful.

N-terminal degrons N-terminal residues have a strong impact on protein stability and half-life. Firstly described in 1986 by Varshavsky and colleagues [25], the N-end rule pathway
has been identified in a broad range of species, from mammals to bacteria, and from yeast to plants [26]. This control of protein degradation in eukaryotes and bacteria is governed
by the formation and recognition of specific sequences at protein N-termini, called N-degrons. The main determinant of an N-degron is an N-terminal destabilizing residue. In eukaryotes,
two N-end rule pathways are being distinguished: the Ac/N-end rule pathway targets proteins through their N-terminally acetylated residues while the Arg/N-rule pathway targets
unacetylated N-terminal residues and involves N-terminal arginylation [26]. Proteolytic processing leading to new protein N-termini is increasingly recognized to play an important
role in the formation of N-degrons. In eukaryotes, N-degron mediated protein degradation occurs through the  ubiquitin–proteasome system. N degrons are recognized by E3
ubiquitin ligases called N-recognins, which induce protein ubiquitylation. Recent studies showed that the N-end rule pathway can be regulated by various mechanisms [26].
Hemin, the ferric (Fe3+) counterpart of heme, and short peptides can bind to components of the N-end rule pathway and impede their functionality [26]. Although the N-end rule
pathway has been molecularly dissected in great detail, numbers of identified physiological substrates undergoing N-end rule degradation have remained limited. A recent study
has expanded the range of substrates targeted by the Arg/N-end rule. Kim and colleagues have shown that N terminal Met followed by a hydrophobic residue functions as an N-degron
[27]. N-terminal Met followed by a small residue is typically removed by aminopeptidases in a cotranslational manner (Figure 2). However, approximately 15% of the genes in mammals
or yeast encode for an N-terminal Met followed by a larger hydrophobic residue. This specific N-degron is targeted by the Ac/N-end rule pathway when the N-terminal Met is acetylated.
The Arg/N-end rule acts instead on the non-acetylated N-terminal Met. As previously mentioned, novel N-degrons can be generated by preceding proteolysis. Piatkov and colleagues
investigated this concept for proteolytic cleavage products that occur during apoptosis [28]. They find that numerous proapoptotic fragments are short lived substrates of Arg/N-end
rule pathway, attributing to this pathway an anti-apoptotic role. Notably, the corresponding N-degron sequences are evolutionary conserved.

Figure 1 Protein N-termini are susceptible to various post-translational modification.
For a more comprehensive overview of all possible N terminal modification, see [60].

Figure 2 Examples of N-terminal mofications: acetylation, pyroglutamate conversion, proteolysis and N-degron processing via deamidation and amino acid conjugation.

Proteolytic processing of N-termini Proteolysis has long been regarded a degradation process. It is now increasingly recognized as an important posttranslational modification
with an array of proteases mediating cellular signaling via the precise processing of bioactive proteins and peptides. The study of cleavage events using N-terminomics is particularly
useful for the identification of proteolytic substrates. Proteolytic cleavage of proteins and polypeptides results in the generation of cleavage fragments with new N-termini and
C-termini. Numerous recent proteomic studies highlighted differential regulation of proteases in different disease settings. MALDI-TOF in combination with enzymatic assays
established reduced levels of dipeptidyl-peptidase (DPP)4 in the serum of patients suffering from metastatic prostate cancer [31]. Another proteomic based study,  using isotope
coded affinity tag (ICAT) labeling showed bacterial leucine aminopeptidase from Plasmodium chabaudi to be significantly upregulated in periodontal disease [32]. Mass spectrometry
was also used for the functional characterization of proteases.

7.3.10 Protein homeostasis networks in physiology and disease

Although most text books of biochemistry describe the process of protein folding to a three dimensional native state as an intrinsic property of the primary sequence, it is becoming increasingly clear that this process can go wrong in an almost infinite number of ways. In fact, many different diseases are caused by the misfolding and aggregation of certain proteins without genetic mutations in the primary sequence. An integrative view of the mechanisms that maintain protein folding homeostasis is emerging, which could be thought as a balanced and dynamic network of interconnected processes tightly regulated by a series of quality control mechanisms. This protein homeostasis network involves families of folding catalysts, co-factors under specific environmental and metabolic conditions. Maintaining protein homeostasis is particularly challenging in specialized secretory cells where the high demand for protein synthesis generates a constant source of stress that could lead to proteotoxicity.

Protein folding is assisted and monitored by diverse interconnected processes that follow a sequential pattern over time. The calnexin/calreticulin cycle ensures the proper folding of glycosylated proteins through the secretory pathway, which establishes the final pattern of disulfide bond formation through interactions with the disulfide isomerase ERp57. Coupled to this cycle is the ER-associated degradation (ERAD) pathway, which translocates terminally misfolded proteins to the cytosol for degradation by proteasomes. In addition, macroautophagy is becoming a relevant mechanism for the clearance of damaged proteins and abnormal protein aggregates through lysosomal hydrolysis, a process also referred to as ERAD-II. The folding status at the ER is constantly monitored by the Unfolded Protein Response (UPR), a specialized signaling pathway initiated by the activation of three types of stress sensors. The process underlying the surveillance of protein folding stress by the UPR is not fully understood, but it may require coupling to key folding mediators such as BiP or the direct recognition of the misfolded peptides by stress sensors. The UPR regulates genes and processs related to almost every folding step in the secretory pathway to reduce the load of misfolded proteins, including protein translation into the ER, translocation, folding, quality control, ERAD, the redox status, and many other related functions. Protein folding stress is observed in many disease conditions such as cancer, diabetes, and neurodegeneration. For example, abnormal protein aggregation and the accumulation of protein inclusions is associated with Parkinson’s and Alzheimer’s Disease, and amyotrophic lateral sclerosis. In those diseases and many others, neuronal dysfunction and disease progression correlates with the presence of a strong ER stress response; however, the direct in vivo role of the UPR in the disease process has been experimentally defined in only a few cases. Therapeutic strategies are currently being developed to increase protein folding and clearance of misfolded proteins, with the goal of alleviating ER stress.

In this issue of Current Opinion in Cell Biology we present a series of focused reviews from recognized experts in the field, that provide an overview of mechanisms underlying protein folding and quality control, and how balance of protein homeostasis is maintained in physiology and deregulated in diseases. Daniela Roth and William Balch integrate the concept of protein homeostasis networks into an interesting model termed FoldFx, showing how the interconnection between different pathways in the context of the cellular proteome determines the energetic barrier required to generate a functional folded peptide. The authors have previously proposed the term Proteostasis to refer to the set of interacting activities that maintain the health of the proteome and the organism (protein homeostasis). The ER is a central subcellular compartment for protein synthesis and quality control in the secretory pathway. Yukio Kimata and Kenji Kohno give an overview of the signaling pathways that control adaptation to ER stress and maintenance of protein folding homeostasis. The authors summarize the models proposed so far for the activation of UPR stress sensors, and discuss how this directly or indirectly relates to the accumulation of unfolded proteins in the ER lumen. Chronic or irreversible ER stress triggers cell death by apoptosis. Gordon Shore, Feroz Papa, and Scott Oakes summarize the complex signaling pathways initiating apoptosis by ER stress, where cross talk between the ER and the mitochondria play a central role. The authors focus on addressing the role of the BCL-2 protein family on the activation of intrinsic mitochondrial apoptosis pathways, highlighting different cytosolic and transcriptional events that determine the transition between adaptive responses to apoptosis programmed by the UPR to eliminate irreversibly injured cells.

Although diverse families of chaperones, foldases and co-factors are expressed at the ER, only a few protein folding networks have been well defined. However, molecular explanations for specific substrate recognition and quality control mechanisms are poorly defined. Here we present a series of reviews covering different aspects of protein maturation. Amy Lee summarizes what is known about the biology of the key ER folding chaperone BiP/Grp78, and its emerging role in diverse pathological conditions including cancer. In two reviews, David B. Williams and Linda M. Hendershot describe the best characterized mechanism of protein quality control at the ER, the calnexin cycle. In addition, they give an overview of the function of a family of ER foldases, the protein disulfide isomerases (PDIs), in folding, quality control and degradation of abnormally folded proteins. PDIs are also becoming key factors in establishing the redox tone of the ER. Riccardo Bernasconi and Maurizio Molinari overview the ERAD process and how this pathway affects the efficiency of the protein folding process at the ER and its relation to pathological conditions.

Lysosomal-mediated degradation is becoming a fundamental process for the control of the haft-life of proteins and the degradation of misfolded, aggregate prone proteins. Ana Maria Cuervo reviews the relevance of Chaperone-mediated autophagy in the selective degradation of soluble cytosolic proteins in lysosomes, and also points out a key role for Chaperone-mediated autophagy in the cellular defense against proteotoxicity. David Rubinsztein and Guido Kroemer present two reviews highlighting the emerging relevance of macroautophagy in maintaining the homeostasis of the nervous system. They also discuss the actual impact of macroautophagy in the clearance of protein aggregates related to neurodegenerative diseases, including Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease among others. In addition, recent evidence suggesting an actual impairment of macroautophagy as a causative factor in aging-related disorders is also discussed.

Alterations in protein homeostasis underlie the etiology of many diseases affecting the nervous system, in addition to cancer and diabetes. Fumiko Urano summarizes the impact of ER stress in β cell dysfunction and death during the progression of type 1 and type 2 diabetes, as well as in genetic forms of diabetes such as Wolfram syndrome. The occurrence of basal ER stress is observed in specialized secretory cells and organs, including plasma B cells. Roberto Sitia covers several aspects of how proteotoxic stresses physiologically contribute to regulate the biogenesis, function and lifespan of B cells, and speculates about the possible impact of ER stress in the treatment of multiple myeloma. Claudio Soto describes the specific role of calcineurin, a key phosphatase in the brain, in the occurrence of synaptic dysfunction and neuronal death in prion-related disorders. We also present provide a review summarizing the emerging role of ER stress and the UPR in most neurodegenerative diseases related to protein misfolding. We also discuss the particular mechanisms currently proposed to be involved in the generation of protein folding stress at the ER in these pathologies, and speculate about possible therapeutic interventions to treat neurodegenerative diseases.

Strategies to increase the efficiency of quality control mechanisms, to reduce protein aggregation and to enhance folding are suggested to be beneficial in the setting of diseases associated with the disruption of protein homeostasis. Finally, Jeffery Kelly overviews recent chemical and biological therapeutic strategies to restore protein homeostasis, which could be achieved by enhancing the biological capacity of the proteostasis network or through small molecule to stabilize misfolding-prone proteins. In summary, this volume ofCurrent Opinion in Cell Biology compiles the most recent advances in understanding the impact of protein folding stress in physiology and disease, and integrates a variety of complex mechanisms that evolved to maintain protein homeostasis in a dynamic way in the context of a changing environment. The biomedical applications of developing strategies to cope with protein folding stress have profound implications for the treatment of the most prevalent diseases in the human population.

7.3.11 Proteome sequencing goes deep
Advances in mass spectrometry (MS) have transformed the scope and impact of protein characterization efforts. Identifying hundreds of proteins from rather simple biological matrices, such as yeast, was a daunting task just a few decades ago. Now, expression of more than half of the estimated ∼20,000 human protein coding genes can be confirmed in record time and from minute sample quantities. Access to proteomic information at such unprecedented depths has been fueled by strides in every stage of the shotgun proteomics workflow-from sample processing to data analysis-and promises to revolutionize our understanding of the causes and consequences of proteome variation.
    • Recent MS advances have transformed the depth of coverage of the human proteome.• Expression of half the estimated human protein coding genes can be verified by MS.• MS sample preparation, instrumentation, and data analysis techniques are highlighted.

Mammalian proteomes  are complex [3]. The human proteome contains ~20,300 protein-coding genes; however, non-synonymous single nucleotide polymorphisms (nsSNPs), alternative
splicing events, and post-translational modifications (PTMs) all occur and exponentially increase the number of distinct proteoforms [4–6]. Detection of 5000 proteins in a proteomic
experiment was a considerable achievement just a few years ago [7–9]. More recently, two groups identified over 10,000 protein groups in a single experiment. Through extensive protein
and peptide fractionation (72 fractions) and digestion with multiple enzymes, Nagaraj et al. identified 10,255 protein groups from HeLa cells over 288 hours of instrument analysis [10].
A comparison with paired RNA-Seq data revealed nearly complete overlap between the detected proteins and the expressed transcripts. In that same year, a similar strategy enabled
the identification of 10,006 proteins from the U2OS cell line [11]. Kim and co-workers analyzed 30 human tissues and primary cells over 2000 LC–MS/MS experiments, resulting
in the detection of 293,000 peptides with unique amino acid sequences and evidence for 17,294 gene products [16]. Wilhelm et al. amassed a total of 16,857 LC–MS/MS experiments
from human cell lines, tissues, and body fluids. These experiments produced 946,000 unique peptides, which map to 18,097 protein coding genes [17]. Together, these two studies
provide direct evidence for protein translation of over 90% of  human genes (Figure 2). New developments in mass spectrometer technology have increased the rate at which proteomes
can be analyzed. We describe developments in sample preparation, MS instrumentation, and bioinformatics that have been key to obtaining comprehensive proteomic coverage.
Further, we consider how access to such proteomic detail will impact genomic  research.

Aurelian Udristioiu


Aurelian Udristioiu

Lab Director at Emergency County Hospital Targu Jiu

Mg²+ is critical for maintaining the positional integrity of closely clustered phosphate groups. These clusters appear in numerous and distinct parts of the cell nucleus and cytoplasm. The Mg²+ ion maintains the integrity of nucleic acids, ribosomes and proteins. In addition, this ion acts as an oligo-element with role in energy catalysis. Biological cell membranes and cell walls exhibit poly-anionic charges on the surface. This finding has important implications for the transport of ions, particularly because different membranes preferentially bind different ions. Both Mg²+ and Ca²+ regularly stabilize membranes by cross-linking the carboxylated and phosphorylated head groups of lipids.

Notable document –

Theor Biol Med Model. 2010 Jun 9;7:19.
Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations.
Matveev VV1.
Cell physiologist at Institute of Cytology, Russian Academy of Sciences

According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen’s dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity.”One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity.”Josiah Willard Gibbs (1839-1903).

To date, numerous mechanisms, signal pathways, and different factors have been found in the cell. Researchers are naturally eager to find commonalities in the mechanisms of cellular regulation. I would like to propose a substantial approach to problems of cell physiology – the structural ground that produces signals and underlies the diversity of cellular mechanisms.

The methodological basis for the proposed hypothesis results from studies by the scientific schools of Dmitrii Nasonov [1] and Gilbert Ling [26], which have gained new appreciation over the last 20-30 years owing to advances in protein physics [7] in the study of properties of globular proteins, their unfolding and folding, as well as the discovery of novel states of the protein molecule: the natively unfolded and the molten globule. The key statement for the rationale of the present paper is that the specificity of interactions of polypeptide chains with each other (at the intra- and inter-molecular levels) can be provided only by their secondary structures, primarily α-helices and β-sheets.

Nasonov’s school discovered and studied a fundamental phenomenon — the nonspecific reaction of the cell to external actions [1], while works by Ling [5] and his followers allow the mechanisms of this phenomenon to be understood.

The above-mentioned cell reaction has been called nonspecific because diverse physical and chemical factors produce the same complex of structural changes in the cell: an increase in the turbidity and macroscopic viscosity of the cytoplasm and in the adsorption of hydrophobic substances by cytoplasmic proteins. It is of primary importance that the same changes also occur in the cell during its transition into the active state: muscle contraction, action potential, enhancement of secretory activity (for details, see [8]). Hence, from the point of view of structural changes, there is no fundamental difference between the result of action on the cell of hydrostatic pressure and, for instance, muscle contraction. In both cases, proteins are aggregated.

Nasonov called the cause of these changes the stages of cell protein denaturation, as the changes of properties of isolated proteins during denaturation are very similar to the changes in the cytoplasm during the nonspecific reaction. As a result, the denaturational theory of cell excitation and damage was created [1]. The structural changes of protein denaturation were unclear in Nasonov’s time. Nowadays, it is assumed that the denaturation is the destruction of the tertiary and secondary structure of a protein. Below I give two definitions, for the denaturation of natively folded (globular) proteins and for natively unfolded proteins.

A key notion in physiology is the resting state of the cell. This is implicit in the concept of the threshold character of the action of stimuli on the cell, which has played a historical role in the development of physiological science. It is the threshold that is the boundary between two states — rest and activity. But in effect, all our knowledge about cells concerns active cells, not cells in the resting state. It is in the active cell that variable changes occur that can be recorded. Nothing happens in the resting cell, so there is nothing to be recorded in it. Nevertheless, it is obvious that the resting state is the initial cell state, the starting point for all changes occurring in the cell.

What characterizes the structural aspect of the cell in the state of rest? It is only in Ling’s work [5] that I have found a clear answer to this question. The answer can be interpreted as follows: if all resting cell proteins were arranged in one line, it would turn out that most of the peptide bonds in this superpolypeptide would be accessible to solvent (water), while only a few would be included in secondary structures. When the cell is activated, the ratio between the unfolded and folded areas is changed sharply to the opposite: the proportion of peptide bonds accessible to solvent decreases markedly, whereas the proportion included in secondary structures rises significantly. These two extreme states of cell proteins, suggested by Ling, provide a basis for further consideration.

If Ling’s approach is combined with Nasonov’s theory, we obtain several interesting consequences. First of all, it is clear that proteins with maximally unfolded structures form the structural basis of resting cells because they are inactive, i.e., do not interact with other proteins or other macromolecules. The situation changes when an action on the cell exceeds the threshold: completely or partially unfolded key proteins begin to fold when new secondary protein structures are formed. Owing to these new secondary structures, the proteins become capable of reacting, i.e., intramolecular aggregation (folding of individual polypeptides into globules) and intermolecular aggregation (interaction of some proteins with others) begin. A distinguishing feature of these aggregational processes is their absolutely specific character, which is ensured by the amino acid composition, shape, and size of the secondary structures. The structures appearing have physiological meaning, so such aggregation is native and the secondary structures causing it are centers of native aggregation. Another source of secondary structures necessary for native aggregation is the molten globule.

The ability of cells to return to the initial state, the state of rest, means that native aggregation is completely reversible, and the structures appearing in the course of native aggregation are temporary and are disassembled as soon as they cease to be necessary. Native aggregation can involve both the whole cell and individual organelles, compartments, and structures, and activation of proteins is of a threshold rather than a spontaneous character.

The meaning of the proposed hypothesis of native aggregation is that the primary cause of any functional changes in cell is the appearance, as a result of native aggregation, of temporary structures, continually appearing and disintegrating during the life of the cell. Since native aggregation is initiated by external stimuli or regulatory processes and the structures appearing have a temporary character, these structures can be called signal structures.

Signal structures can have different properties: (i) they can be centers of binding of ions, molecules (solutes), and proteins; (ii) they can have enzymatic activity; (iii) they can form channels and intercellular contacts; (iv) they can serve as matrices organizing the interactions of molecules in synthetic and transport processes; (iv) they can serve as receptors for signal molecules; (v) they can serve as the basis for constructing even more complex supramolecular structures. These structures “flash” in the cell space like signal lights, perform their role, and disappear, to appear in another place and at another time. The meaning of the existence of the structural “flashes” is that during transition into the active state the cell needs new resources, functions, mechanisms, regulators, and signals. As soon as the cell changes to the resting state, the need for these structures disappears, and they are disassembled. Extreme examples of native aggregation are muscle contraction, condensation of chromosomes, the appearance of the division spindle, and interactions of ligands with receptors.

Thus, the present paper will consider the meaning and significance of native aggregation as the universal structural basis of the active cell. The basis of pathological states is the inability of the cell to return to the resting state and errors in the formation of signal structures. The presentation of native aggregation is based on three pillars: (i) reversible protein aggregation is a structural basis of cell activity (Nasonov’s School); (ii) the operation of the living cell or its individual structures can be regarded as a repetitive sequence of transitions between two states (active and resting), a key role in which belongs to natively unfolded proteins (Ling’s approach); (iii) the specificity of interactions of separate parts of a single polypeptide chain with each other (folding) or the interaction of separate polypeptide chains among themselves (self-assembly, aggregation) can be provided only by protein secondary structures.

The goal of this paper is the enunciation of principles, rather than a review of facts corresponding to these principles.

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Novel Approaches to Cancer Therapy

Writer sand Curator: Larry H. Bernstein, MD, FCAP

11.1       Novel Approaches to Cancer Therapy

11.1.1 Electrically-driven modulation of surface-grafted RGD peptides for .. cell adhesion

11.1.2 The metabolic state of cancer stem cells—a target for cancer therapy

11.1.3 Regulation of tissue morphogenesis by endothelial cell-derived signals

11.1.4 Novel approach to bis(indolyl)methanes. De novo synthesis of 1-hydroxyimino-methyl derivatives with anti-cancer properties

11.1.5 Synthesis and Biological Evaluation of New 1,3-Thiazolidine-4-one Derivatives of 2-(4-Isobutylphenyl)propionic Acid molecules

11.1.6 Targeting pyruvate kinase M2 contributes to radiosensitivity of NSCLC cells

11.1.7 The tyrosine kinase inhibitor nilotinib has antineoplastic activity in prostate cancer cells but up-regulates the ERK survival signal—Implications for targeted therapies

11.1.8 PAF and EZH2 Induce Wnt.β-Catenin Signaling Hyperactivation

11.1.9 PAF Makes It EZ(H2) for β-Catenin Transactivation

11.1.10 PI3K.AKT.mTOR pathway as a therapeutic target in ovarian cancer

11.1.11 Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

11.1.12 Curcumin-could-reduce-the-monomer-of-ttr-with-tyr114cys-mutation via autophagy in cell model of familial amyloid polyneuropathy.

11.1.1 Electrically-driven modulation of surface-grafted RGD peptides for .. cell adhesion

Lashkor M1Rawson FJStephenson-Brown APreece JAMendes PM.
Chem Commun (Camb). 2014 Dec 21; 50(98):15589-92

Reported herein is a switchable surface that relies on electrically-induced conformational changes within surface-grafted arginine–glycine–aspartate (RGD) oligopeptides as the means of modulating cell adhesion

Stimuli-responsive surfaces that are capable of modulating their biological properties in response to an external stimuli, including temperature,1,2 light,3 magnetic field4 and electrical potential,59 are of growing interest for a variety of biological and medical applications.10,11 Switchable surfaces that can be controlled on-demand are playing an increasingly important part in the development of highly sensitive biosensors,1215novel drug delivery systems1618 and functional microfluidic, bioanalysis, and bioseparation systems.1922Additionally, dynamic, synthetic surfaces that can control the presentation of regulatory signals to a cell are expected to have a significant impact in the field of tissue engineering and regenerative medicine, and to provide unprecedented opportunities in fundamental studies of cell biology.23,24 The availability of sophisticated and functional switchable surfaces is expected to emulate more complex in vivo like extracellular environments, and provide a powerful means to probe and control the dynamic interactions between the cell and its external environments.

The majority of studies on stimuli-responsive surfaces reported to date either rely2529 on controlling non-specific interactions (i.e., hydrophobic/hydrophilic and electrostatic) of the biomolecules with the active surface, or have focused3032 on demonstrating modulation of specific biomolecular interactions using relatively simple biological systems (e.g. biotin–streptavidin) and conditions (i.e. water or buffer solutions). For example, Zareie et al. 30 fabricated a mixed self-assembled monolayer (SAM) on gold comprising oligo(ethylene glycol) (OEG) thiol molecules and shorter disulfides carrying biotin end-groups that regulated the interaction between biotin and streptavidin in water. The OEG thiols were able to switch in response to a change in temperature below and above their lower critical solution temperature (LCST = 37 °C). At 23 °C the structure of the OEG molecules was fully extended hindering the shorter biotin disulfide components. On the contrary, at 45 °C the OEG backbone collapsed, thus allowing the specific interaction between the biotin molecule on the surface and the protein streptavidin in solution. In our previous work,79 electrically controlled switching has been applied to regulate the conformational changes of modified positively charged oligolysine peptides tethered to a gold surface, such that biotin moieties incorporated into the oligolysines could be reversibly exposed or concealed on demand, as a function of surface potential. Switchable SAMs used to control biomolecular interactions via an electrical stimulus are particularly appealing because of their fast response times, ease of creating multiple individually addressable switchable regions on the same surface, as well as low-drive voltage and electric fields, which are compatible with biological systems.33 Our previous reported electrically switchable surface was able to control directly the biomolecular interactions between biotin and neutravidin in phosphate buffer saline (PBS) solution.

However, switchable surfaces have been scarcely used, thus far, to control biomolecular interactions on more complex systems such as those involving modulation of cell responsiveness.3437 Jonkheijm and co-workers35 have reported a cucurbit[8]uril-based SAM system to electrochemically control the release of cells. Charged end groups on SAM surfaces have been exploited to electrically control the early stages of bacterial cell adhesion37 and form patterned surfaces with two independent dynamic functions for inducing cell migration.36 In spite of these efforts, given cellular complexity and diversity, such studies are very limited in number, as are the opportunities to further understand and control the complex interplay of events and interactions occurring within living cells.

Herein, we report on a stimuli-responsive surface that relies on electrically-induced conformational changes within surface-grafted arginine–glycine–aspartate (RGD) oligopeptides as the means of modulating cell adhesion. RGD, which is present in most of the adhesive ECM proteins (e.g. fibronectin, vitronectin, laminin and collagen), is specific for integrin-mediated cell adhesion.38 The RGD modified electrode is used here to dynamically regulate the adhesion of immune macrophage cells. The stimuli-responsive surface is fabricated on a gold surface and comprises a mixed SAM consisting of two components (Fig. 1): (i) an oligopeptide containing a terminal cysteine for attachment to the gold surface, three lysine residues as the main switching unit, and a glycine–arginine–glycine–aspartate–serine (GRGDS) as the recognition motif for cell adhesion –C3K-GRGDS, and (ii) an ethylene glycol-terminated thiol (C11TEG) to space out the oligopeptides. Since the charged backbone of the oligopeptide can be potentially harnessed79 to induce its folding on the surface upon an application of an electrical potential, we reasoned that such conformational changes can be employed to selectively expose under open circuit (OC) conditions (bio-active state) or conceal under negative potential (bio-inactive state) the RGD to the cell and dynamically regulate cell adhesion.



RDG oligopeptide SAM utilised for controlling specific cellular interactions

Fig. 1 Schematic of the dynamic RDG oligopeptide SAM utilised for controlling specific cellular interactions. The electrically switchable SAM exposes the RGD peptide and supports cell adhesion under open circuit (OC) conditions (no applied potential), while …

Mixed SAMs of C3K-GRGDS : C11TEG were formed from a solution ratio of 1 : 40 and characterised by X-ray photoelectron spectroscopy (XPS) (Fig. S2, ESI). XPS analysis confirmed the formation of the C3K-GRGDS:C11TEG mixed monolayer and displayed signals from S, N, C and O. The chemical state of the sulphur atom was probed using the XPS spectra of the S 2p emission (Fig. S2, ESI). The S 2p spectrum (Fig. S2a, ESI) consists of two doublet peaks, with one doublet peak at 162.0 eV (S 2p3/2) and 163.2 eV (S 2p1/2), indicating that the sulphur is chemisorbed on the gold surface.39 A second small doublet peak can be observed at 163.8 eV and 165.0 eV, which can be attributed to the S–H bond, indicating a small presence of unbound sulphur. No sulphur peaks above 166 eV were observed, indicating that no oxidised sulphur is present at the surface. The N 1s spectrum (Fig. S2b, ESI) can be de-convoluted into two peaks, which support the presence of the peptide on the surface. The first peak centred at 400.5 eV is attributed to amino (NH2) and amide (CONH) moieties. The second peak centred at 402.8 eV is ascribed to protonated amino groups.40 Note that no nitrogen peak was observed for pure C11TEG SAMs. The C 1s spectrum (Fig. S2c, ESI) can be de-convoluted into three peaks, which are attributed to five different binding environments. The peak at 285.0 eV is attributed to C–C bonds,41 while the peak at 286.7 eV corresponds to C 1s of the three binding environments of C–S, C–N and C–O.41 The third and smaller peak (288.6 eV) is assigned to the C 1s photoelectron of the carbonyl moiety, C O.41 The O 1s spectrum (Fig. S2d, ESI) is de-convoluted into two different peaks, corresponding to two different binding environments, arising from the C–O (533.3 eV) and C O (532.0 eV) bonds.41 From integrating the area of the S 2 p and N 1s peaks and taking into consideration that the C3K-GRGDS oligopeptide consists of 15 N atoms and 1 S atom and C11TEG has no N and 1 S atom only, it was possible to infer that the ratio of C3K-GRGDS:C11TEG on the surface is 1 : 10 ± 2. The presence of C11TEG was utilised not only to ensure sufficient spatial freedom for molecular reorientation of the surface bound oligopeptide, but also to stop non-specific binding to the surface.

The C3K-GRGDS:C11TEG mixed SAMs were shown to support adhesion of immune macrophage cells as determined by cell counting42,43 (Fig. 2). When RAW 264.7 mouse macrophages were cultured on theC3K-GRGDS:C11TEG mixed SAM in supplemented Dulbecco’s Modified Eagle Medium (DMEM), the number of cells adhered to the surface increased with incubation time, reaching 1792 ± 157 cells per mm2after 24 hours. This is in contrast with the weak cell adhesion observed in two control surfaces, pureC11TEG SAMs and clean gold, in which the number of cells that adhere was 60% and 50% lower, respectively, after 24 hours (Fig. 2).



Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAM, pure C11TEG SAM and bare gold surfaces

Fig. 2 Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAM, pure C11TEG SAM and bare gold surfaces that were normalized against the density of cells adherent onto the C3K-GRGDS:C11TEG mixed SAM. The surfaces were cultured in RAW 264.7 mouse macrophage cells under OC conditions for 24 hours.

In order to demonstrate that the C3K-GRGDS:C11TEG mixed SAMs can support or resist cell adhesion on demand, the macrophage cells were cultured on the C3K-GRGDS:C11TEG mixed SAM in DMEM medium under OC conditions and applied negative potential (–0.4 V) for a period of 1 h. Note that DMEM contains a mixture of inorganic salts, amino acids, glucose and vitamins. On application of the applied potential of –0.4 V the number of adherent cells was 70% less compared to the C3K-GRGDS:C11TEGmixed SAMs under OC conditions, Fig. 3. Similar switching efficiencies have been observed in another oligopeptide system using different DMEM solutions.44 These findings suggest that the negative potential induces the conformational changes in the C3K moiety of C3K-GRGDS in the SAM which in turn leads to the RGD moiety being concealed and hence reducing the binding of the cells.



Density of adhered cells on C3K-GRGDS:C11TEG, C11TEG, C6EG-GRGDS:C11TEG mixed SAMs

Fig. 3 Density of adhered cells on C3K-GRGDS:C11TEG, C11TEG, C6EG-GRGDS:C11TEG mixed SAMs that were normalized against the density of cells adherent onto the C3K-GRGDS:C11TEG mixed SAM. The surfaces were cultured in RAW 264.7 for 1 h under OC conditions or while applying –0.4 V.

Previous studies have shown that small conformational and orientational changes in proteins and peptides modulate the availability and potency of the active sites for cell surface receptors.4547 Thus, in a similar manner, small changes in the conformation/orientation of the RGD peptide on the surface induced by application of an electrical potential are able to affect the binding activity of the peptide. Recently, we have conducted detailed theoretical8 and experimental9 studies aimed at understanding the switching mechanism of oligopeptide-based switchable surfaces, that similarly as in the case of the C3K-GRGDS:C11TEG mixed SAMs, use lysine residues to act as the switching unit. These previous studies unraveled that the surface-appended oligolysines undergo conformational changes between fully extended, partially extended and collapsed conformer structures in response to an applied positive potential, open circuit conditions and negative electrical potential, respectively. Thus, these previous findings allow us to propose that when a negative potential is applied to the GRGDS:C11TEG mixed SAM surface, the oligopeptide chain adopts a collapsed conformation on the surface and the RGD binding motif is partially embedded on the C11TEGmatrix, thus showing no bioactivity (“OFF” state).

In order to verify that the changes in adhesion upon application of a negative surface potential occur due to changes in the conformational orientation of the RGD instead of cell repulsion or cell damage due to the presence of an electrical potential, control mixed SAMs were also prepared using C11TEG and a peptide where the 3 lysine residues as the switching unit were replaced by 6 non-switchable ethylene glycol units –C6EG-GRGDS (Fig. S1, ESI). Fig. 3 demonstrates that cells adhered in similar numbers to the C11TEGand C6EG-GRGDS:C11TEG mixed SAMs under OC conditions and an applied negative potential. These results provide strong evidence that control over cell adhesion using the C3K-GRGDS:C11TEG mixed SAM is due to a conformational behaviour of the lysine-containing oligopeptide that can either expose or conceal the RGD moiety.

Cell viability was checked following application of –0.4 V for 1 h by performing a trypan blue assay. Cells that were dead were stained blue due to a break down in membrane integrity. Incubation of the cells under a negative potential had negligible effect on cell viability, which was greater than 98%. Cyclic voltammetric studies (outlined in detail in the Fig. S3, ESI) were also performed to demonstrate that no significant faradaic process occur over the potential range studied, and thus ions are not participating in redox reactions and consequently redox chemistry is not being significantly affected by application of the potential used. In agreement with other studies,35,36,48 we conclude that the electrical modulation of the surface neither affected cell viability nor induced any redox process in the medium that could have had an effect on cells.

We then addressed the question of whether the C3K-GRGDS:C11TEG surfaces could be switched between different cell adhesive states (cell-resistant and cell-adhesive states). To begin with, we investigated the switching from a cell-adhesive state to a cell-resistant state, and the possibility to detach the cells from the substrate upon the application of a negative potential. Cells were incubated in the C3K-GRGDS:C11TEGmixed SAMs for 1 h under OC conditions, thereby exposing the RGD moiety and allowing for cell attachment. This step was followed by the application of a potential of –0.4 V for 1 h in order to detach the cells from the surface, by concealing the RGD moieties. Cell counts showed no significant differences between the pre and post application of the –0.4 V, suggesting that the electrostatic force generated by the applied negative electrical potential might not be sufficient to disrupt the RGD–integrin interaction. These results were to a certain extent expected since adherent cells are able to withstand strong detachment forces due to the adhesion being mediated by multiple RGD–integrin bonds in parallel.49

In contrast, a reversal of the switching sequence demonstrated that our surfaces can be dynamically switched from a non-adhesive to cell-adhesive state. Cells were incubated in the C3K-GRGDS:C11TEG mixed SAMs for 1 h while holding the potential at –0.4 V for 1 h making the RGD peptide inaccessible for recognition by the corresponding integrin. As above, the number of adherent cells when a negative potential of –0.4 V was applied was 70% of the number that adhered to the C3K-GRGDS:C11TEG mixed SAMs under OC conditions, Fig. 4. The potential was then shifted to open circuit conditions for 1 h on those exposed to a potential of –0.4 V, which resulted in a significant increase in the number of cells as a result of the exposure of the RGD moiety to the cells (Fig. 4). These values were similar to those obtained for the samples that were only incubated for 1 hour under OC conditions (Fig. 4), indicating that the surfaces were highly effective at switching from a non-adhesive to cell-adhesive state.



Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAMs

Fig. 4  Microscopic images and density of adhered cells on C3K-GRGDS:C11TEG mixed SAMs that were incubated with cells for 1 h while applying –0.4 V and subsequently in OC conditions for 1 h. The density was normalized against the density of cells adherent onto C3K-GRGDS:C11TEG mixed SAMs that were incubated with cells in OC conditions for 1 h.

In summary, an electrically switchable surface has been devised and fabricated that is capable of efficiently exposing and concealing the RGD cell adhesion motif and dynamically regulate the adhesion of immune macrophage cells. This study will no doubt be useful in developing more realistic dynamic extracellular matrix models and is certainly applicable in a wide variety of biological and medical applications. For instance, macrophage cell adhesion to surfaces plays a key role in mediating immune response to foreign materials.50 Thus, development of such dynamic in vitro model systems that can control macrophage cell adhesion on demand are likely to provide new opportunities to understand adhesion signaling in macrophages51 and develop effective approaches for prolonging the life-span of implantable medical devices and other biomaterials.52

11.1.2 The metabolic state of cancer stem cells—a target for cancer therapy

Vlashi E1Pajonk F2.
Free Radic Biol Med. 2015 Feb; 79:264-8


  • Bulk tumor cell populations rely on aerobic glycolysis.
  • Cancer stem cells are in a specific metabolic state.
  • Cancer stem cells in breast cancer, glioblastoma, and leukemia rely on oxidative phosphorylation of glucose.

In the 1920s Otto Warburg first described high glucose uptake, aerobic glycolysis, and high lactate production in tumors. Since then high glucose uptake has been utilized in the development of PET imaging for cancer. However, despite a deepened understanding of the molecular underpinnings of glucose metabolism in cancer, this fundamental difference between normal and malignant tissue has yet to be employed in targeted cancer therapy in the clinic. In this review, we highlight attempts in the recent literature to target cancer cell metabolism and elaborate on the challenges and controversies of these strategies in general and in the context of tumor cell heterogeneity in cancer.



11.1.3 Regulation of tissue morphogenesis by endothelial cell-derived signals

Saravana K. RamasamyAnjali P. KusumbeRalf H. Adams
Trends Cell Biol  Mar 2015; 25(3):148–157


  • Endothelial cells lining blood vessels induce organ formation and other morphogenetic processes in the embryo.
  • Blood vessels are also an important source of paracrine (angiocrine) signals acting on other cell types in organ regeneration.
  • Vascular niches and endothelial cell-derived signals generate microenvironments for stem and progenitor cells.

Endothelial cells (ECs) form an extensive network of blood vessels that has numerous essential functions in the vertebrate body. In addition to their well-established role as a versatile transport network, blood vessels can induce organ formation or direct growth and differentiation processes by providing signals in a paracrine (angiocrine) fashion. Tissue repair also requires the local restoration of vasculature. ECs are emerging as important signaling centers that coordinate regeneration and help to prevent deregulated, disease-promoting processes. Vascular cells are also part of stem cell niches and have key roles in hematopoiesis, bone formation, and neurogenesis. Here, we review these newly identified roles of ECs in the regulation of organ morphogenesis, maintenance, and regeneration.

Figure 1. Role of endothelial cells (ECs) during organogenesis

Figure 2. Endothelial cells (ECs) in lung regeneration

Figure 3. Liver endothelium in regeneration and fibrosis.

Vascular cells have key roles in morphogenesis and regeneration

Vascular cells have key roles in morphogenesis and regeneration

Figure 4. Functional roles of the bone vasculature

Figure 5. Vascular niche for neurogenesis.

Concluding remarks

The examples provided in this review highlight the important roles of ECs in tissue development, patterning, homeostasis, and regeneration. The endothelium often takes a central position in these processes and there are many reasons why ECs are ideally positioned as the source of important instructive, angiocrine signals. The vascular transport network extends into every organ system and needs to be embedded in those tissues in a certain spacing or pattern, which places ECs in central and, therefore, strategic positions for the regulation of morphogenesis and organ homeostasis.

Given that ECs and other cell types frequently form functional units, such as kidney glomeruli, liver lobules, or lung alveoli, the assembly, differentiation, and function of the different cellular components needs to be tightly coordinated. In addition, because circulating blood cells extensively rely on the vascular conduit system and frequently interact with the endothelium, it is perhaps not surprising that ECs contribute to niche microenvironments. During tissue repair, proliferative cell expansion processes are sometimes temporally separated from cell differentiation and tissue patterning events. The latter has to involve the restoration of a fully functional vascular network so that ECs appear ideally suited as the source of molecular signals that can trigger or suppress processes in the surrounding tissue.


11.1.4 Novel approach to bis(indolyl)methanes. De novo synthesis of 1-hydroxyimino-methyl derivatives with anti-cancer properties

Grasso C, et al.
Eur J Medicinal Chem 01/2015; 93:9-15.

A versatile and broad range approach to previously unknown bis(indolyl)methane oximes based on two consecutive hetero Diels-Alder cycloaddition reactions of electrophilic conjugated nitrosoalkenes with indoles is disclosed. The cytotoxic properties and selectivity of some adducts against several human cancer cell lines pointing to a promising role in the development of anti-tumoural drugs, in particular for leukemia and lymphoma.

Novel approach to bis(indolyl)methanes: De novo synthesis of 1-hydroxyiminomethyl derivatives with anti-cancer properties. Available from:

_Novel_approach_to_bis-28indolyl-29methanes_De_novo_synthesis_of_1-hydroxyiminomethyl_ derivatives_with_anti-cancer_properties [accessed Apr 11, 2015].

The one-pot synthetic strategy to bis(indolyl)methanes is outlined in Scheme 3. The starting a,a 0-dihalogenooximes 3 were efficiently prepared from the respective ketones by known procedures [58,61]. These compounds, in the presence of base, were converted, in situ, into the corresponding transient and reactive nitrosoalkenes 4, which were intercepted bya first molecule of the appropriate indole 5 originating the intermediate indole oximes 6. The initially formed tetrahydroxazines undergo ring-opening to the corresponding oximes, under the driving force of the energy gain on rearomatisation. Subsequent dehydro-halogenation of 6 produces nitrosoalkenes 7 which reacted with a second molecule of indole, producing the target bis(indolyl)methanes 8. The results obtained are summarised in Table 1.

The reaction yields may be considered generally good, taking into account that the synthetic process involves a sequence of reactions. On the other hand, no other products could be obtained, which indicates that the reactions were regioselective. The results have shown also that both alkyl and aryl oximes can be used in the synthesis of bis(indolyl)methanes. Starting from aryl oximes 3aef the expected (E) oximes 9 were obtained as single or major products (Entries 1e11) whereas alkyl oxime 3g reacted with indole to give the (Z)-oxime 10g as the major product (Entries 12e13). The stereochemistry assignment of oximes 9 and 10 was confirmed by analysis of the NOESY spectra of 9d, 9g, 10d and 10g. In the spectra of 10d and 10g, connectivity was observed between the hydroxyl proton and the phenyl protons and the methyl protons, respectively, whereas in the case of 9d and 9g no connectivity was observed. Moreover, oximes 9 and 10 are also characterized by 1H NMR spectra with different features. The chemical shift of the methylenic proton appears at higher value for (E)-oximes 9 (9b: δ  6.81 ppm; 9d: δ  = 6.82 ppm; 9g: δ = 6.39 ppm) than for the corresponding (Z) oximes 10 (10b: δ = 5.74 ppm; 10d: δ = 5.77 ppm; 10g: δ = 5.41 ppm).

The synthesis of two isomeric oximes from the reaction of arylnitrosoethylenes with pyrrole and dipyrromethanes has been previously observed [62]. The process was rationalized considering the conjugate addition of the heterocycle to the nitrosoalkene, at the s-cis or s-trans conformation, followed by rearomatization of the pyrrole unit leading to (E)- and (Z)-oxime, respectively. Thus, the synthesis of the BIM oximes via 1,4-conjugate addition of indole to the nitrosoelkene cannot be ruled out.

The use of water as solvent in Diels- Alder reactions has been shown to be advantageous, not only in environmental terms but also inducing critical improvements in reaction times, yields and selectivity [51,63]. We observed that carrying out the synthesis of bis(indolyl)methanes in water using dichloromethane as co-solvent is a valuable alternative to the use of dichloromethane as the only solvent. Generally the yields were better or comparable to those obtained in dichloromethane and reaction time significantly shorter (the reaction time was reduced from 36 h to 3 h). Clearly the efficiency of the reaction, using H2O/CH2Cl2 system, amongst the nitrosoalkenes bearing halogenated aryl substituents increases in the order F > Cl > Br > H the order of electron withdrawing ability and consequently the order of the expected effectiveness for an inverse electron demand Diels-Alder reaction (entries 2, 5, 7 and 9). However, the isolated yields from the reaction carried out in CH2Cl2 do not reflect the expected reactivity, which can be explained considering differences in the efficiency of the purification process.

The cytotoxicity of compounds 9a, 9e and 9d was evaluated in different tumorl cell lines, namely HepG2 (hepatocellular carcinoma), MDA-MB-468 (human breast carcinoma), RAW 264.7 (murine leukemic monocyte macrophages), THP1 (human acute monocytic leukaemia), U937 (human leukaemic monocytic lymphoma) and EL4 cells (murine T-lymphoma). The compounds’ selectivity towards tumoural cells was assessed determining their cytotoxicity with respect to two non-tumoural derived cell lines S17 (murine bone marrow) and N9 cells (murine microglial). Results of the half maximal concentrations (IC50) are shown in Table 2 together with the toxicity of etoposide, a known antitumoural drug. Compound 9e was considerably less cytotoxic on tumor cell lines than the other two compounds, with IC50 values ranging from 35.7 (HepG2) to 124 mM (THP1) and was not selective. Compounds 9a and 9d, however, were considerably cytotoxic to all cells tested, with IC50 values ranging from 1.62 (THP1) to 23.9 mM (RAW) and from 10.7 (MDA) to 34.1 mM (U937), respectively. Compound 9a was particularly active against non-adherent cell lines with IC50 values ranging from 1.62 in THP1 to 1.65 mM in EL4.

Some conclusions regarding structure activity relationships can be redrawn based on the biological evaluation of these bis(indolyl)methanes. There is a dramatic difference in anticancer activitybetweenN-unsubstituted bis(indolyl)methanes 9a and the Nmethyl substituted derivative 9e, the latter characterized by high IC50 values. On the other hand, the significantly lower IC50 values observed for 9a for non-adherent cell lines in comparisonwith the ones obtained for 9d demonstrates that the presence of the bromo substituent leads to higher cytotoxic activity.

The observed high cytotoxicity of compound 9a against THP1, EL4 and U937 cell lines led us to extend the study to BIMs 9c, 9g and 10g (Table 3). Compound 9c, bearing a 4-fluorophenyl substituent, showed moderate anti-cancer activity which reinforces the observation that the 4-bromophenyl group is crucial to ensure low IC50 values. On the other hand, alkyl oximes 9g and 10g were even less cytotoxic against THP1, EL4 and U937 cell lines. None of these compounds were selective towards the tumor cell lines (selectivity index calculated for non-tumour cell line S17). In addition to having displayed higher toxicity towards the nontumor cell lines than all the studied compounds, compound 9a demonstrated the highest selectivity indexes: 9.86-14.2. Further studies using 9a as scaffold in the development of anti-tumoural drugs for leukaemia and lymphoma is worth pursuing since it presents lower IC50 and higher selectivity than etoposide.


The reliable preparation of a variety of unknown BIMs bearing different oxime substituents at the methylene bridge was presented. This strategy, supported on the robust and proved methodology of Diels-Alder cyclo addition reactions of electrophilic nitrosoalkenes with electron rich indoles, may pave the way for the synthesis of a vast library of new compounds.

Table 1 Preparation of bis(indolyl)methane oxime

Scheme 1. Selected biological active bis(indolyl)methanes.

Scheme 2. Common methods for BIMs’ preparation [27e44].

Scheme 3. Synthetic strategy towards BIM oximes.

Synthesis of a new bis(indolyl)methane that inhibits growth and induces apoptosis in human prostate cancer cells

Marrelli M., et al.
Natural product research 08/2013; 27(21).

The synthesis and the antiproliferative activity against the human breast MCF-7, SkBr3 and the prostate LNCaP cancer cell lines of a series of bis(indolyl)methane derivatives are reported. The synthesis of new compounds was first accomplished by the reaction of different indoles with trimethoxyacetophenone in the presence of catalytic amounts of hydrochloric acid. A second procedure involving the use of oxalic acid dihydrate [(CO2H)2·2H2O] and N-cetyl-N,N,N-trimethylammonium bromide in water was carried out and led to better yields. Compound 5b significantly reduced LNCaP prostate cancer cell viability in a dose-dependent manner, with an IC50 of 0.64 ± 0.09 μM. To determine whether the growth inhibition was associated with the induction of apoptosis, treated cells were stained using DAPI. LNCaP cells treated with 1 μM of 5b showed the morphological changes characteristic of apoptosis after 24 h of incubation.

11.1.5 Synthesis and Biological Evaluation of New 1,3-Thiazolidine-4-one Derivatives of 2-(4-Isobutylphenyl)propionic Acid molecules

Vasincu IM1Apotrosoaei M2Panzariu AT3Buron F4Routier S5Profire L6
Molecules. 2014 Sep 18; 19(9):15005-25

New thiazolidine-4-one derivatives of 2-(4-isobutylphenyl)propionic acid (ibuprofen) have been synthesized as potential anti-inflammatory drugs. The structure of the new compounds was proved using spectral methods (FR-IR, 1H-NMR, 13C-NMR, MS). The in vitro antioxidant potential of the synthesized compounds was evaluated according to the total antioxidant activity, the DPPH and ABTS radical scavenging assays. Reactive oxygen species (ROS) and free radicals are considered to be involved in many pathological events like diabetes mellitus, neurodegenerative diseases, cancer, infections and more recently, in inflammation. It is known that overproduction of free radicals may initiate and amplify the inflammatory process via upregulation of genes involved in the production of proinflammatory cytokines and adhesion molecules. The chemical modulation of acyl hydrazones of ibuprofen 3a–l through cyclization to the corresponding thiazolidine-4-ones 4a–n led to increased antioxidant potential, as all thiazolidine-4-ones were more active than their parent acyl hydrazones and also ibuprofen. The most active compounds are the thiazolidine-4-ones 4e, m, which showed the highest DPPH radical scavenging ability, their activity being comparable with vitamin E.

In order to improve the anti-inflammatory effect and safety profile of representative NSAIDs, one research strategy is derivatization of the carboxylic acid group with various heterocyclic systems (oxazole, izoxazole, pyrazole, oxadiazole, thiazole, thiadiazole, triazole, etc.) [9,10]. In the past two decades there has been considerable interest in the role of reactive oxygen species (ROS) in inflammation [11]. ROS mediate the oxidative degradation of cellular components and alteration of protease/antiprotease balance with damage to the corresponding tissue. In the early stages of the inflammatory process, ROS exert their actions through activation of nuclear factors, such as NFkB or AP-1, that induce the synthesis of cytokines. In later stages, endothelial cells are activated due to the synergy between free radicals and cytokines, promoting the synthesis of inflammatory mediators and adhesion of molecules. In the last step free radicals react with different cellular components (trypsin, collagen, LDL, DNA, lipids) inducing the death of cells [12,13].

The thiazolidine-4-one moiety is a heterocycle that has received more attention in the last years due its important biological properties [14]. Many effects have been found, including anti-inflammatory and analgesic [15], antitubercular [16], antimicrobial and antifungal [17], antiviral, especially as anti-HIV agents [18], anticancer, antioxidants [19], anticonvulsants [20] and antidiabetic activity [21]. In the present study, some new derivatives of ibuprofen that contain thiazolidine-4-one scaffolds were synthesized in order to obtain compounds with double effect—antioxidant and anti-inflammatory properties. The structures of the compounds were assigned based on their spectral data (FT-IR, 1H-NMR, 13C-NMR, MS) and the compounds were screened for their in vitro antioxidant potential.

The 1,3-thiazolidine-4-one derivatives 4am were synthesized in several steps using the method summarized in Scheme 1 and Table 1. First 2-(4-isobutylphenyl)propionic acid (ibuprofen, 1) was reacted with thionyl chloride, followed by treatment with dry ethanol to get 2-(4-isobutylphenyl)propionic acid ethyl ester, which was turned in 2-(4-isobutylphenyl)propionic acid hydrazide (2) by reaction with 66% hydrazine hydrate [22]. The condensation of compound 2 with various aromatic aldehydes allowed the preparation of the corresponding hydrazone derivatives 3al in satisfactory yields. Finally, the hydrazone derivatives of ibuprofen upon reaction with mercaptoacetic acid led to the thiazolidine-4-one derivatives 4al in moderate to good yields. By reduction of compound 4g in presence of tin chloride and few drops of acetic acid in ethanol, the thiazolidine-4-one 4m was obtained in 90% yield. Acetylation of 4m with acetyl chloride gave thiazolidine-4-one 4n in moderate yield.

In the acyl hydrazone series most of the the tested compounds showed a radical scavenging ability comparable with ibuprofen (Table 4). The most active compounds were 3e and 3f which are about three times and two times more active than their parent compound, respectively. The scavenging ability of the acyl hydrazones was improved by cyclization to the corresponding thiazolidine-4-one derivatives, these compounds all being more active than ibuprofen, except for compound 4j which contains a CF3 group in the metaposition of phenyl ring (Table 5). The most active compounds were 4e and 4m which contain NO2 and NH2 groups in ortho and paraposition of the phenyl ring, respectively. For these compounds the radical scavenging ability (%) was 94.42 ± 0.43 and 94.88 ± 0.57, which means that the compounds are about 23 times more active than ibuprofen (4.15 ± 0.22). The activity of these compounds is comparable with that of vitamin E used as positive control. Important radical scavenging ability was also shown by compound 4b(81.31 ± 0.55), which contains a Cl group in the para position of the phenyl ring, the compound being 20 times more active than ibuprofen.

The acyl hydrazone derivatives showed an antioxidant activity comparable with ibuprofen. The most active compound in this series was 3h, with radical scavenging activity of 13.31 ± 0.81, which means that this compound is three times more active than ibuprofen (4.42 ± 0.18). In the thiazolidine-4-one series the most active compounds were 4b4e and 4k, which contain Cl(4), NO2(2) and CN(4), respectively, as substituents on the phenyl ring. These compounds, which showed a scavenging ability of around 50%, are 12 times more active than ibuprofen. In comparison with the corresponding acyl hydrazones 3b3e and 3k the thiazolidine-4-ones were 10 times (4b), seven times (4e) and 13 times (3k) more active. The improved antiradical activity of acyl hydrazones by cyclization to form thiazolidine-4-ones was also observed for compounds 3d3f and 3g. The most favorable influence was observed for acyl hydrazone 4g, which contains a NO2 in the para position of the phenyl ring. The corresponding thiazolidine-4-one (4g, 37.14 ± 1.10) is 22 times more active than 3g (1.67 ± 0.35). These data strongly support the favorable influence of the thiazolidine-4-one ring on the antioxidant potential of these compounds. The tested compounds were less active than vitamin E.

In this study new heterocyclic compounds that combine the thiazolidine-4-one structure with the arylpropionic acid one have been synthesized. The structure of the new compounds was proved using spectral methods (IR, 1H-NMR, 13C-NMR, MS). The compounds were evaluated for their antioxidant effects using in vitro assays: total antioxidant activity, DPPH and ABTS radical scavenging ability. All thiazolidin-4-one derivatives 4an showed improved antioxidant effects in comparison with the corresponding acyl hydrazones 3al and ibuprofen, the parent compound. The encouraging preliminary results illustrate the antioxidant potential of the synthesized compounds and motivate our next research focused on their anti-inflammatory effects in chronic and acute inflammation models.

11.1.6 Targeting pyruvate kinase M2 contributes to radiosensitivity of NSCLC cells

Meng MB1Wang HH2Guo WH3Wu ZQ2Zeng XL2Zaorsky NG4, et al.
Cancer Lett. 2015 Jan 28; 356(2 Pt B):985-93

Aerobic glycolysis, a metabolic hallmark of cancer, is associated with radioresistance in non-small cell lung cancer (NSCLC). Pyruvate kinase M2 isoform (PKM2), a key regulator of glycolysis, is expressed exclusively in cancers. However, the impact of PKM2 silencing on the radiosensitivity of NSCLC has not been explored. Here, we show a plasmid of shRNA-PKM2 for expressing a short hairpin RNA targeting PKM2 (pshRNA-PKM2) and demonstrate that treatment with pshRNA-PKM2 effectively inhibits PKM2 expression in NSCLC cell lines and xenografts. Silencing of PKM2 expression enhanced ionizing radiation (IR)-induced apoptosis and autophagy in vitro and in vivo, accompanied by inhibiting AKT and PDK1 phosphorylation, but enhanced ERK and GSK3β phosphorylation. These results demonstrated that knockdown of PKM2 expression enhances the radiosensitivity of NSCLC cell lines and xenografts as well as may aid in the design of new therapies for the treatment of NSCLC.

Knockdown of PKM2 expression increases the sensitivity of NSCLC cells to radiotherapy in vitro

To examine PKM2 expressions levels in the normal lung epithelial cell and the NSCLC cell lines, we evaluated the expression levels of PKM2 in normal lung bronchial epithelial cell BEAS-2B and five NSCLC cell lines including A549, H460, H1299, H292, and H520 by Western blotting assays, and our results demonstrated that PKM2 expression was elevated in almost five NSCLC cell lines examined compared to autologous normal lung bronchial epithelial cell, although the expression levels fluctuated slightly depending on the different cell lines (Fig.1A). To test the role of PKM2 in the sensitivity of NSCLC to radiotherapy, we generated plasmids of pshRNA-PKM2 and control pshRNA-Con by inserting the DNA fragment for a pshRNA specifically targeting the PKM2 or control into the pGenesil2 vector. After demonstrating the authenticity, A549 and H460 cells were transfected with the plasmid for 48h and the levels of PKM2 expression were tested by Western blot assays. Obviously, transfection with control plasmid did not significantly modulate PKM2 expression; while transfection with pshRNA-PKM2 reduced the levels of PKM2 expression (Fig.1B and Appendix: Supplementary Fig.S1A). Quantitative analysis revealed that transfection with pshRNA-PKM2 significantly reduced PKM2 expressions as compared with that in the mock-treated and control pshRNA-Con plasmid-transfected cells, respectively (p<0.05, Fig.1C). Mock-treated and pshRNA-PKM2-trasnfected A549 and H460 cells were subjected to IR (4Gy), and 12 and 24h after IR, these cells, together with un-irradiated mock-treated, pshRNA-Con-transfected, and pshRNA-PKM2-trasnfected cells, were tested for cell viability by trypan blue staining. Knockdown of PKM2 reduced the percentage of A549 viable cells by 12.6–20% and IR treatment decreased the frequency of viable cells by 17.1–28.2%. However, the percentages of viable cells in the PKM2-silencing and irradiated cells were reduced by 27.7–48.7%, which were significantly lower than that in other groups (Fig.1D, p<0.05). Furthermore, it was consistent with the above results of A549 cells that knockdown of PKM2 significantly reduced the percentage of H460 viable cells (Appendix: Supplementary Fig.S1B). In addition, to further validate PKM2 silencing on their radiosensitivity,unirradiated control, mock-treated, and pshRNA-PKM2 transfected A549 cells were subjected to IR (0, 2, 4, 6, and 8Gy), and two weeks after IR, these cells were tested for the capacity for colony formation. The results showed that the numbers of colonies formed by pshRNA-PKM2 cells were significantly decreased compared with that of mock-treated and control cells; however, there was no significant change in mock-treated cells compared with control cells. These results suggested that pshRNA-PKM2 cells were more sensitive to IR than mock-treated and control cells (Fig.1E and F). Given that IR usually causes DNA double-strand breaks [28], we characterized the frequency of γ-H2AX nuclear foci positive cells by immunofluorescent assays. While IR treatment dramatically increased the frequency of γ-H2AX+ cells, the same dose of IR further significantly increased the percentages of γ-H2AX+ cells when combined with PKM2 silencing at 12 and 24h after IR, and there was a significant difference in γ-H2AX+ cells between these two groups at 12 and 24 h after IR (Fig. 1G and H, p < 0.05).

Fig. 1. The PKM2 expression levels in the normal lung epithelial cell and the NSCLC cell lines and knockdown of PKM2 expression enhance the radiosensitivity of A549 cells in vitro. The expression levels of PKM2 in normal lung bronchial epithelial cell BEAS-2B and five NSCLC cell lines including A549, H460, H1299, H292, and H520 were determined by Western blotting assay (A). A549 cells were transfected with pshRNA-PKM2 or pshRNA-Con plasmid for 48h, and the levels of PKM2 expression were determined by Western blot assays using a PKM2-specific antibody and β-actin as an internal control (B and C). Data are representative images or expressed as mean±SD of the relative levels of PKM2 to control β-actin in individual groups of cells from three separate experiments. # p

Knockdown of PKM2 enhances IR-induced apoptosis in NSCLC cells

Next, we tested the impact of PKM2-silencing on IR-induced cell death types. One day after IR, the apoptotic cells in the irradiatedmock-treated,pshRNA-PKM2-trasnfected cells, and one group of cells that had been pre-treated with 30μM Z-VAD for 1h prior to IR, together with mock-treated, unirradiated pshRNA-Contransfected, and pshRNA-PKM2-trasnfected groups of cells were characterized by TUNEL assays and/or FACS analysis (Fig.2A and C). In comparison with that in mock-treated and control plasmid transfected cells, the frequency of apoptotic cells in the PKM2 silencing or IR-treated cells increased moderately, while the percentages of apoptotic cells in the cells receiving combined treatment with IR and PKM2-silencing were significantly greater. However, the frequency of apoptotic cells in the Z-VAD-pretreated cells was partially reduced. Apparently, knockdown of PKM2 and IR induced apoptosis in NSCLC cells in vitro (Fig. 2B and D, and Appendix: Supplementary Fig.S1C).

Fig. 2. Knockdown of PKM2 expression enhances IR-induced apoptosis in A549 cells. A549 cells were transfected with, or without, pshRNA-Con or pshRNA-PKM2 for 48h and treated with, or without, Z-VAD for 1h. Subsequently, the cells were subjected to IR, and 24h later, the frequency of apoptotic cells was determined by TUNEL assays and FACS. (A and C) TUNEL and FACS analyses of apoptotic cells. (B and D) Quantitative analysis of the percentage of apoptotic cells. Data are representative images or expressed as mean%±SD of individual groups of cells from three independent experiments. * p

Knockdown of PKM2 enhances IR-induced autophagy in NSCLC cells

The cell autophagy is characterized by the formation of numerous autophagic vacuoles, autophagosome, in the cytoplasm and elevated levels of the microtubule-associated protein 1 light chain 3 (LC3)-II [29]. To test the impact of PKM2 silencing on IR-induced autophagy, the presence of autophagosome in mock-treated, pshRNACon-transfected, pshRNA-PKM2-transfected, IR-treated alone, IR + pshRNA-PKM2-transfected, and 1 mM 3-MA-pretreated IR + pshRNA-PKM2-transfected cells was characterized by electronic microphotography (EM). Intriguingly and importantly, numerous autophagosomes were detected in the IR + pshRNAPKM2-transfected cells, and only a few were detected in the sensitivity of the NSCLC cells to radiotherapy in vitro. It was noted that pshRNA-Con had almost no effect on A549 cells, therefore, some subsequently experiments did not set this group.

Fig. 3. Knockdown of PKM2 and IR induce A549 cell autophagy. A549 cells were transfected with, or without, pshRNA-Con or pshRNA-PKM2 for 48h and treated with, or without, 3-MA for 1h. Subsequently, the cells were subjected to IR, and 2h later, the presence of autophagic vacuoles and autolysosomes in A549 cells was determined by EM and the relative levels of LC3-I, LC3-II, AKT, ERK1/2, and control β-actin expression and AKT, ERK1/2, GSK3β, PDK1 phosphorylation were determined by Western blot assays using specific antibodies. Data are representative images and expressed as mean values of the relative levels of target protein to control in individual groups of cells from three separate experiments. The relative levels of target protein to control in mock-treated cells were designated as 1. (A) EM analysis of autophagic vacuoles and autophagosomes. Black arrows point to autophagic vacuoles and autophagosomes in the cytoplasma of A549 cells. (B) Western blot analysis of LC3-I and LC3-II expression. The values indicate the ratios of the relative levels of LC3-II to LC3-I in individual groups. (C) Western blotting analysis of individual signal events. The values indicate the relative levels of target protein to control β-actin in individual groups of cell

Fig. 4. The impact of 3-MA or/and V-ZAD on cell viability, colony formation, apoptosis and autophagy in A549 cells. A549 cells were transfected with, or without, pshRNACon or pshRNA-PKM2 for 48h and pre-treated with, or without, 3-MA or V-ZAD for 1h, respectively. Subsequently, the cells were subjected to IR. Twenty-four hours later and two weeks, the viability, apoptosis, and colony formation were determined. Two hours after treatment, autophagy and the relative levels of LC3-I and LC3-II expression in different groups of cells were determined. Data are representative images and expressed as mean%±SD of individual groups of cells from three separate experiments. (A) The percentages of viable cells. (B) The capacity of cell colony formation. (C) Quantitative analysis of apoptotic cells. (D) Western blot analysis of LC3-I and LC3-II expression. The values indicate the ratios of LC3-II to LC3-I in individual groups of cells. * p

Fig. 5. Treatment with pshRNA-PKM2 enhances the IR-inhibited growth of implanted tumors in mice. The nude mice were inoculated with A549 cells and when the tumor grew at 50mm3 in one dimension, the mice were randomized and treated with vehicle (PS), plasmid of pshRNA-Con or pshRNA-PKM2 alone or IR (4Gy×7f) alone or in combination with pshRNA-PKM2 and IR, respectively. The body weights and tumor growths of individual mice were monitored longitudinally. At the end of the in vivo experiment, the tumor tissues were dissected out and the frequency of apoptotic cells, the presence of autophagosomes and the expression of PKM2 were determined by TUNEL, EM and immunohistochemistry, respectively. Data are representative images or expressed as mean±SD of individual groups of mice (n=6 per group). (A) The body weights of mice. (B and C) The tumor growth curve of implanted tumors and the log-transformed tumor growth curve of implanted tumors in mice. (D) Quantitative analysis of the frequency of apoptotic cells.(E) EM analysis of autophagy. (F)The expression of PKM2.(G) Quantitative analysis of PKM2 expression.The cells with brown cytoplasma were considered as positive anti-PKM2 staining and the percentage of PKM2-positive cells was obtained by dividing the numbers of the PKM2-positive cells by the total number of cancer cells in the same field.

11.1.7 The tyrosine kinase inhibitor nilotinib has antineoplastic activity in prostate cancer cells but up-regulates the ERK survival signal—Implications for targeted therapies

Schneider M1Korzeniewski N2Merkle K2Schüler J, et al.
Urol Oncol. 2015 Feb; 33(2):72.e1-7

Background: Novel therapeutic options beyond hormone ablation and chemotherapy are urgently needed for patients with advanced prostate cancer. Tyrosine kinase inhibitors (TKIs) are an attractive option as advanced prostate cancers show a highly altered phosphotyrosine proteome. However, despite favorable initial clinical results, the combination of the TKI dasatinib with docetaxel did not result in improved patient survival for reasons that are not known in detail. Methods: The National Cancer Institute-Approved Oncology Drug Set II was used in a phenotypic drug screen to identify novel compounds with antineoplastic activity in prostate cancer cells. Validation experiments were carried out in vitro and in vivo. Results: We identified the TKI nilotinib as a novel compound with antineoplastic activity in hormone-refractory prostate cancer cells. However, further analyses revealed that treatment with nilotinib was associated with a significant up-regulation of the phospho-extracellular-signal-regulated kinases (ERK) survival signal. ERK blockade alone led to a significant antitumoral effect and enhanced the cytotoxicity of nilotinib when used in combination. Conclusions: Our findings underscore that TKIs, such as nilotinib, have antitumoral activity in prostate cancer cells but that survival signals, such as ERK up-regulation, may mitigate their effectiveness. ERK blockade alone or in combination with TKIs may represent a promising therapeutic strategy in advanced prostate cancer.

Identification of nilotinib as a novel antineoplastic compound in prostate cancer cells

Using the NCI-Approved Oncology Drug Panel II for a phenotypic drug screen of normal prostate epithelial cells and prostate cancer cell lines (Fig. 1) [7], we identified the TKI nilotinib as a positive hit in hormone-refractory DU-145 prostate cancer cells.

Fig. 1. Discovery of nilotinib as a novel antineoplastic agent in prostate cancer cells using a phenotypic drug screen. Overview of the drug screen procedure (see text for details).

Results were confirmed using annexin V staining, which showed a significant induction of apoptosis beginning at 24 hours (Fig. 2A). The IC50 of nilotinib against DU-145 cells was determined at 10 μM using an MTT cell viability assay (Fig. 2B). Immunoblot experiments confirmed an induction of apoptosis using PARP cleavage in DU-145 cells and in hormonerefractory PC-3 prostate cancer cells at this drug concentration (Fig. 2C). An onset of apoptosis at 24 hours was likewise confirmed using PARP cleavage at a nilotinib concentration of 10 μM(Fig. 2D). PWR-1E prostate epithelial cells and hormone-sensitive prostate LNCaP prostate cancer cells were not found to undergo enhanced apoptosis when treated with nilotinib (not shown).

Fig. 2. Antitumoral effects of nilotinib in prostate cancer cells: (A) flow cytometric analysis of DU-145 prostate cancer cells for annexin V to detect apoptotic cells after treatment with 10 μM of nilotinib for the indicated intervals; (B) cell viability (MTT) assay to determine the IC50 of nilotinib in DU-145 cells (24-h treatment); (C and D) immunoblot analysis of DU-145 and PC-3 prostate cancer cells for PARP cleavage (arrow) at nilotinib concentrations and time intervals as indicated. GAPDH is shown for protein loading; and (E) colony growth assay of DU-145 cells after drug treatment and washout as shown. Cells grown in 60-mm dishes were stained with crystal violet to visualize viable cells at the time points indicated. (Color version of figure is available online.

To further confirm the effect of nilotinib on prostate cancer cell growth, we performed a colony growth assay in which DU-145 cells were treated with nilotinib for 72 hours followed by a washout of the drug and continued culture for additional 9 days (Fig. 2E). We found that nilotinib induced significant cytotoxicity after 72 hours and that a minor regrowth of cancer cells did not occur until 6 to 9 days after the washout, which is comparable to other TKIs [8]. Next, we sought to identify the targets of nilotinib in DU-145 prostate cancer cells. Overall, 5 well-established targets, including ABL1, KIT, PDGFRA, DDR1, and NQO2, were analyzed for their role in the drug response. We found that protein expression of 3 of these targets (ABL1, KIT, and PDGFRA) was not detectable in DU-145 cells and that small interfering RNA–mediated knockdown of the remaining 2 targets, DDR1 and NQO2, did not result in apoptosis (not shown). Collectively, these results show a significant antitumoral activity of nilotinib in prostate cancer cells. However, this effect was associated with a relatively high IC50 and was independent of known nilotinib targets.

Nilotinib up-regulates the ERK survival signal in prostate cancer cells

To further investigate why relatively high concentrations of nilotinib were required to induce cytotoxicity, we analyzed 40,6-diamidino-2-phenylindole–stained DU-145 cells treated with 10 μM of nilotinib for 24 hours using fluorescence microscopy (Fig. 3A).

Fig. 3. Nilotinib up-regulates the ERK survival signal in prostate cancer cells. (A) Fluorescence microscopic analysis of DAPI-stained DU-145 cells. (B and C) Immunoblot analyses of DU-145 cells (B) or DU-145 cells in comparison with LNCaP and PC-3 cells (C) treated with nilotinib for the expression of phospho-ERK1/2 T202/Y204 and total ERK. Immunoblot for GAPDH is shown as a loading control. (D) Immunohistochemical staining of xenografted DU-145 cells after 21 days of treatment with 75 mg/kg/d of nilotinib for phospho-ERK1/2 T202/Y204 expression. It can be noted that tumors explanted from vehicle-treated mice showed mostly positivity at the tumor periphery, whereas tumors explanted from nilotinib-treated mice showed a more evenly distributed phospho-ERK immunostaining (left panels). Quantification of phospho-ERK–positive DU-145 xenografts explanted after 21 days of treatment. Mean and standard errors of positive cells per high-power field (HPF; [1]40) from at least 3 tumors are given (right panel). (E) Immunoblot analysis of DU-145 cells treated with U0126 alone or in combination with nilotinib shows abrogation of phospho-ERK1/2 T202/Y204 expression by U0126. (F) Quantification of viable cells compared with that of controls using the MTT assay after treatment with U0126 (10 μM) or nilotinib (10 μM) or both and after either pretreatment (24 h) or simultaneous treatment (72 h). DAPI ¼ 40,6-diamidino-2-phenylindole. (Color version of figure is available online.)

We found that, despite the presence of apoptotic cells, there was also a population of actively dividing tumor cells in the presence of nilotinib as well as a population of viable but multinucleated cells (Fig. 3A). We interpreted these results as evidence that a subset of tumor cells has the ability to resist TKI treatment. To reconcile these results, we analyzed the activation of ERK1/2, which is known to function as a prosurvival signal in TKI-treated tumor cells [9,10]. We detected a robust overexpression of phospho-ERK1/2 T202/Y204 in nilotinib-treated DU-145 cells (Fig. 3B). An up-regulation of phospho-ERK1/2 T202/Y204 was also detectable in nilotinib-treated LNCaP cells, albeit at a lower level, and was not found in PC-3 cells (Fig. 3C). To further corroborate the evidence of phospho-ERK upregulation in vivo, we analyzed explanted DU-145 xenografts from a representative experiment in which nilotinib was used at a 75-mg/kg/d concentration. This initial dosage was based on published animal experiments [11] but yielded no or incomplete tumor control in our experiment (data not shown).

In vivo antitumoral activity of nilotinib and ERK blockade

Our results raised 2 important questions First, can a higher dose of nilotinib induce improved tumor control, and second, is a combination of nilotinib with the MEK inhibitor U0126 to block ERK activity superior to nilotinib alone?

Fig. 4. In vivo antitumoral activity of nilotinib and ERK blockade in prostate cancer cells: (A) tumor growth curves of DU-145 xenografts in NMRI-nude mice and (B) analysis of tumor volumes on day 21. Asterisks indicate statistical significance (**P r 0.01 and ***P r 0.001). (Color version of figure is available online.)

11.1.8 PAF and EZH2 Induce Wnt.β-Catenin Signaling Hyperactivation

Jung HY1Jun SLee MKim HCWang XJi HMcCrea PDPark JI
Mol Cell. 2013 Oct 24; 52(2):193-205

Fine-control of Wnt signaling is essential for various cellular and developmental decision making processes. However, deregulation of Wnt signaling leads to pathological consequences including cancer. Here, we identify a novel function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells, and required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing secondary axis with β-catenin target gene upregulation. Upon Wnt signaling activation, PAF is dissociated from PCNA, and directly binds to β-catenin. Then, PAF recruits EZH2 to β-catenin transcriptional complex, and specifically enhances Wnt target gene transactivation, independently of EZH2’s methyltransferase activity. In mouse, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling, and propose a novel regulatory mechanism of Wnt signaling during tumorigenesis. Fine-control of Wnt signaling is essential for various cellular and developmental decision making processes. However, deregulation of Wnt signaling leads to pathological consequences including cancer. Here, we identify a novel function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells, and required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing secondary axis with β-catenin target gene upregulation. Upon Wnt signaling activation, PAF is dissociated from PCNA, and directly binds to β-catenin. Then, PAF recruits EZH2 to β-catenin transcriptional complex, and specifically enhances Wnt target gene transactivation, independently of EZH2’s methyltransferase activity. In mouse, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling, and propose a novel regulatory mechanism of Wnt signaling during tumorigenesis.

Keywords: Wnt, β-catenin, PAF, KIAA0101, EZH2

Strict regulation of stem cell proliferation and differentiation is required for mammalian tissue homeostasis, and its repair in the setting of tissue damage. These processes are precisely orchestrated by various developmental signaling pathways, with dysregulation contributing to disease and genetic disorders, including cancer (Beachy et al., 2004). Cancer is initiated by the inactivation of tumor suppressor genes and activation of oncogenes. For instance, colon cancer cells harbor genetic mutations in Wnt/β-catenin pathway constituents such as adenomatous polyposis coli (APC), Axin, and β-catenin (Polakis, 2007). In mouse models, inactivation of APC or activation of β-catenin results in the development of intestinal hyperplasia and adenocarcinoma (Moser et al., 1990), indicating that hyperactivation of Wnt signaling promotes intestinal tumorigenesis.

In canonical Wnt signaling, Wnt ligand induces stabilization of β-catenin protein via inhibition of the protein destruction complex (glycogen synthase kinase 3, APC, casein kinase I, and Axin). Then, activated β-catenin is translocated into the nucleus and binds to its nuclear interacting partners, TCF/LEF. Finally, β-catenin-TCF/LEF transactivates the expression of its target genes (Clevers and Nusse, 2012).

Although various Wnt/β-catenin modulators have been identified (Wnt homepage;, the pathological relevance of these modulators to tumorigenesis remains elusive. Also, many reports have suggested that mutation-driven Wnt signaling activation can be enhanced further (Goentoro and Kirschner, 2009He et al., 2005Suzuki et al., 2004Vermeulen et al., 2010), which implies the presence of an additional layer of Wnt-signaling regulation in cancer beyond genetic mutations in APC or β-catenin. Here, we unraveled a novel function of the DNA repair gene, PAF (PCNA-associated factor) /KIAA0101). PAF was shown to be involved in translesion DNA synthesis (TLS), an error-prone DNA repair process that permits DNA replication machinery to replicate DNA lesions with specialized translesion DNA polymerase (Emanuele et al., 2011Povlsen et al., 2012Sale et al., 2012). Our comprehensive approaches uncover that cancer-specifically expressed PAF hyperactivates Wnt/β-catenin signaling and induces intestinal tumorigenesis.

Mitogenic role of PAF via Wnt signaling

To identify colon cancer-specific Wnt signaling regulators, we analyzed multiple sets of human colon cancer tissue samples using the publicly available database (, and selected genes that are highly expressed in colon cancer cells (fold change > 2; P < 0.0001; top 10% ranked). Among several genes, we investigated the biological role of PAF, based on its significant overexpression in human colon adenocarcinoma with correlated expression of Axin2, a well-established specific target gene of β-catenin (Jho et al., 2002Lustig et al., 2002)(Figure 1A). To validate our in silico analysis, we performed immunostaining of colon cancer tissue microarray, and confirmed that PAF was highly expressed in colon cancer cells, whereas its expression was barely detectable in normal intestine (Figure 1B). Consistently, PAF was strongly expressed and mainly localized in the nucleus of colon cancer cell lines (Figure 1C). Additionally, we found that PAF was not expressed in non-transformed cells such as NIH3T3, mouse embryonic fibroblasts, and mammary epithelial cells (data not shown). Next, to assess the relevance of PAF upregulation in colon cancer cell proliferation, we depleted endogenous PAF using short hairpin RNAs (shRNAs) in these cell lines. Intriguingly, PAF knockdown (sh-PAF) inhibited colon cancer cell proliferation (Figures 1D and 1E). Given that PAF was shown to interact with PCNA via PIP box (Yu et al., 2001), we also examined whether PAF-PCNA interaction is required for mitogenic effects of PAF. In reconstitution experiments, sh-PAF-induced cell growth inhibition was rescued by ectopic expression of both shRNA non-targetable wild-type PAF (nt-PAF) and PIP mutant PAF (mutPIP-PAF) (Figure 1F), indicating that the PAF-PCNA interaction is not necessary for PAF-mediated colon cancer cell proliferation. Interestingly, PAF knockdown downregulated cell proliferation–related genes (Cyclin D1 and c-Myc) (Figure 1G). Given that Cyclin D1 and c-Myc are β-catenin direct target genes (He et al., 1998Tetsu and McCormick, 1999), PAF likely participates in regulating Wnt/β-catenin signaling. Interestingly, PAF depletion-induced downregulation of Cyclin D1 andc-Myc was only observed in SW620 colon cancer cells, but not in Panc-1 and MDA-MB-231 cells (Figure 1G), indicating the specific effects of PAF on Cyclin D1 and c-Myc expression in colon cancer cells. We also assessed the effects of PAF knockdown on Axin2. Indeed, PAF knockdown suppressed Axin2transcription in colon cancer cells (Figure 1H). Moreover, as nt-PAF did, β-catenin ectopic expression reverted sh-PAF–induced cell growth arrest (Figure 1I), implying that PAF might be functionally associated with Wnt/β-catenin. We also examined whether other mitogenic signaling pathways mediate PAF’s mitogenic role. Of note, except HT29, other colon cancer cell lines (SW620, HCT116, HCC2998, and HCT15) harbor oncogenic mutations in K-Ras gene. Nonetheless, PAF depletion induced the suppression of cell growth on all five colon cancer cells (Figure 1D), indicating that PAF’s mitogenic function is independent of Ras/MAPK signaling activation. Additionally, overexpression of wild-type Akt or constitutively active form of Akt (myristoylated form of Akt [Myr-Akt]) did not rescue sh-PAF-induced inhibition of cell proliferation (Figure 1I). Moreover, β-catenin activation did not revert cell proliferation suppression resulted from MAPK or PI3K inhibition (Figure 1J), indicating that β-catenin-mediated mitogenic function is independent of MAPK and PI3K signaling pathways. These results suggest that PAF contributes to colon cancer cell proliferation, possibly via Wnt/β-catenin signaling.

Figure 1 Mitogenic role of PAF in colon cancer cells

PAF positively modulates Wnt signaling

Given that many cancers develop as a result of deregulation of developmental signalings (Beachy et al., 2004), analyzing PAF expression during development may provide insights into the mechanisms of PAF-mediated signaling regulation. Whole mount immunostaining of mouse embryos, showed that PAF was specifically enriched in the apical ectodermal ridge (AER) of the limb bud, midbrain, hindbrain, and somites (Figure 2A and data not shown). During limb development, AER induction is specifically coordinated by active Wnt signaling (Figure 2B)(Kengaku et al., 1998). Using, Axin2-LacZ, a β-catenin reporter (Lustig et al., 2002), mouse embryos, we confirmed the specific activation of Wnt signaling in AER (Figure 2C). Intriguingly, Wnt signaling activity as exhibited in the AER, overlapped with the pattern of PAF expression (Figures 2A and 2C). Given that (1) Wnt signaling is deregulated in most colon cancer, (2) PAF is highly overexpressed in colon cancer cells, (3) PAF is required for colon cancer cell proliferation (Figure 1D), and (4) PAF is enriched in AER where Wnt signaling is active (Figure 2A), we hypothesized that PAF modulates the Wnt signaling pathway. To test this, we first examined the impact of PAF on β-catenin transcriptional activity using TOPFLASH reporter assays. In HeLa cells, PAF knockdown decreased β-catenin reporter activation by 6-bromoindirubin-3′-oxime, a GSK3 inhibitor (Figure 2D). Similarly, Wnt3A-induced transcriptional activation of Axin2 was also inhibited by PAF depletion (Figure 2E). These data suggest that PAF might be required for Wnt target gene expression.

Figure 2 Activation of Wnt signaling by PAF

To gain better insight of PAF’s role in Wnt signaling regulation, we utilized Xenopus laevis embryos for axis duplication assays (Funayama et al., 1995), as previously performed (Park et al., 2009). Because of Wnt signaling’s pivotal role in vertebrate anterior-posterior axis development, the effects of Xenopus PAF (xPAF) on Wnt signaling can be monitored and quantified on the basis of secondary axis formation following injection of in vitro transcribed mRNAs. xβ-catenin mRNA, titrated to a subphenotypic level when expressed in isolation, was co-injected with xPAF mRNA into ventrovegetal blastomeres. Unlike the controls (β-catenin and β-galactosidase mRNA), the experimental group (β-catenin and xPAF mRNA) displayed axis-duplications (Figures 2F-H). Of note, the ventrovegetal injection of xPAF mRNA alone failed to induce secondary axes (data not shown), showing that PAF hyperactivates Wnt/β-catenin signaling only in the presence of active β-catenin. Consistent with the results of axis duplication assays, qRT-PCR assays showed that xPAF expression upregulated expression of Siamois and Xnr3, β-catenin targets in frogs (Figure 2I). Furthermore, we examined the specificity of PAF on Wnt/β-catenin signaling activity, using various luciferase assays. Ectopic expression of PAF hyperactivates Wnt3A or LiCl, a GSK3 inhibitor, -induced activation of β-catenin target gene reporter activity (MegaTOPFLASH, Siamoisc-Myc, and Cyclin D1). Of note, BMP/Smad pathway also plays an essential role in the developing limb AER (Ahn et al., 2001). However, PAF knockdown or overexpression did not affect BMP/Smad or FoxO signalings, respectively, (Figure 2J) indicating the specificity of PAF in regulating Wnt signaling. These results suggest that PAF positively modulates Wnt/β-catenin signaling in vitro and in vivo.

PAF-EZH2-β-catenin transcriptional complex formation

Next, we investigated the molecular mechanism underlying PAF hyperactivation of Wnt signaling. Given that stabilization of β-catenin protein is a key process in transducing Wnt signaling, we asked whether PAF affects β-catenin protein level. However, we found that the level of β-catenin protein was not altered by PAF knockdown or overexpression (Figures 2E and ​and3A),3A), leading us to test whether PAF controls the β-catenin/TCF transcriptional complex activity. Owing to the nuclear specific localization of PAF in colon cancer cells (Figure 1C), we tested whether PAF interacts with β-catenin transcriptional complex. Using a glutathione S-transferase (GST) pull-down assay, we found that PAF bound to β-catenin and TCF proteins (Figure 3B). Also, endogenous PAF interacted with β-catenin and TCF3 in SW620 cells that display constitutive hyperactivation of Wnt signaling by APC mutation (Figure 3C). Moreover, binding domain mapping assays showed that the Armadillo repeat domain of β-catenin was essential for its interaction with PAF (Figure 3D). Although PAF is a cell cycle-regulated anaphase-promoting complex substrate (Emanuele et al., 2011), PAF-β-catenin interaction was not affected (Figure S1). These data suggest that PAF directly binds to β-catenin transcriptional complex and this interaction is independent of cell cycle. Next, due to interaction of PAF with β-catenin and TCF, we tested whether PAF is also associated with Wnt/β-catenin target genes. First, we analyzed the subnuclear localization of PAF by chromatin fractionation. We found that PAF was only detected in the chromatin fraction of HCT116 cells (Figure 3E). Additionally, chromatin immunoprecipitation (ChIP) assays showed that both ectopically expressed and endogenous PAF occupied the TCF-binding element (TBE)-containing proximal promoter of the β-catenin targets (c-Myc and Cyclin D1) in HCT116 cells (Figures 3F and 3G). These data show that PAF is specifically associated with the promoters of Wnt/β-catenin target genes.

Figure 3 PAF-EZH2-β-catenin transcriptional complex at target gene promoters

In intestine, Wnt/β-catenin signaling constitutively activates intestinal stem cells (ISCs) to give rise to progenitor cells, which replenishes intestinal epithelium (Figure 3H). Given the involvement of PAF on Wnt/β-catenin signaling regulation (Figure 2), we analyzed the spatial expression of PAF in intestinal epithelium. Immunostaining showed that PAF was specifically expressed in B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) positive intestinal stem cells (ISCs)(Figures 3I and 3J). Bmi1 and its associated components in Polycomb-repressive complex 1 (PRC1) and 2 (PRC2) are shown to epigenetically regulate gene expression (Sparmann and van Lohuizen, 2006). Due to (1) specific association of PAF with TBEs of β-catenin target promoters (Figures 3F and 3G) and (2) co-localization with Bmi1 positive ISCs (Figure 3J), we asked whether PAF is associated with components of PRC1 and PRC2, using co-immunoprecipitation (co-IP) assays. Intriguingly, PAF interacted with both Bmi1 and enhancer of zeste homolog 2 (EZH2) in SW620 cells (Figure 3K), which led us to test whether either Bmi1 or EZH2 is functionally associated with PAF-mediated Wnt signaling hyperactivation. To do this, we assessed the effects of Bmi1 and EZH2 on β-catenin transcriptional activity, using β-catenin reporter assays. We observed that ectopic expression of EZH2 upregulated β-catenin transcriptional activity, but Bmi1 overexpression did not (data not shown), implying that EZH2 might be associated with Wnt signaling activation. Binding domain mapping analysis showed that EZH2 bound to PAF via the middle region of EZH2 including the CXC cysteine-rich domain (Figure 3L). In conjunction with the Bmi1-containing PRC1, EZH2-containing PRC2 catalyzes histone H3 lysine 27 trimethylation (H3K27me3) via histone methyltransferase domain. Despite the crucial role of EZH2 in H3K27me3-meidated gene regulation, we found that other core components of PRC2, EED, and Suz12 were not associated with PAF (Figure 3K). Moreover, although EZH2 overexpression in cancer induces PRC4 formation in association with the NAD+-dependent histone deacetylase Sirt1 (Kuzmichev et al., 2005), the PAF-EZH2 complex did not contain Sirt1 (Figure 3K). These data indicate that PAF-EZH2 complex is distinct from the conventional PRCs in cancer cells. Also, we questioned whether PCNA is required for PAF’s interaction with either PAF or β-catenin. Interestingly, β-catenin-PAF and EZH2-PAF complexes existed only in PCNA-free fractions (Figure 3M, compare lanes 1 and 2), which is consistent with PCNA-independent mitogenic role of PAF in colon cancer cell proliferation (Figure 1I). Due to exclusive interaction of PAF with either PCNA or β-catenin, we asked whether Wnt signaling activation affects either PAF-β-catenin or PAF-PCNA interaction. Co-IP assays showed that, in HeLa cells, PAF-β-catenin interaction was only detected upon LiCl treatment, while PAF-EZH2 interaction remained constant. Moreover, PAF-PCNA association was decreased by LiCl or Wnt3A treatment (Figures 3N and 3O, compare lanes 3 and 4). These data suggest that Wnt signaling activation is required for PAF-β-catenin interaction. Due to absence of endogenous Wnt signaling activity in HeLa cells, we also assessed the effects of active Wnt/β-catenin signaling on PAF-PCNA binding in colon cancer cell lines that exhibit hyperactivation of Wnt signaling by genetic mutations in APC or β-catenin alleles. Surprisingly, PAF-PCNA interaction was barely detectable in colon cancer cell lines, whereas 293T and HeLa cells displayed strong PAF-PCNA association (Figure 3P), implying that active β-catenin may sequester PAF from PCNA. In binding domain mapping analysis, we also found that N-terminal and PIP regions are required for β-catenin interaction (Figure S2), suggesting that β-catenin competes with PCNA for PAF interaction. These results suggest that, upon Wnt signaling activation, PAF is conditionally associated with β-catenin transcriptional complex.

PAF activates β-catenin target genes by recruiting EZH2 to promoters

Previous studies showed that EZH2 interacts with β-catenin (Li et al., 2009Shi et al., 2007). Also, we found that PAF is physically associated with EZH2, independently of PRC2 complex (Figure 3). These evidences prompted us to ask whether EZH2 mediates PAF-induced Wnt signaling hyperactivation. Given PAF-EZH2-β-catenin complex formation, we tested whether EZH2 is also associated with the promoters of β-catenin target genes. Intriguingly, PAF, EZH2, and β-catenin steadily co-occupied the promoters of c-Myc,Cyclin D1, and Axin2 in HCT116 cells carrying β-catenin mutation, whereas PCNA, EED, and Suz12 did not (Figure 4A), which recapitulates PRC2 complex-independent association of EZH2 with PAF (see Figures 3K and 3N). Next, we asked whether PAF, EZH2, and β-catenin are recruited to β-catenin target gene promoter upon Wnt signaling activation, as PAF-β-catenin interaction was dependent of Wnt signaling activation (Figure 3N). In HeLa cells, we found that PAF, EZH2, and β-catenin conditionally bound to TBEs in the c-Myc and Axin2 promoters, only upon LiCl treatment (Figure 4B), indicating that Wnt signaling activation is a prerequisite for PAF-β-catenin-EZH2’s promoter association. To further confirm the specificity of PAF-EZH2-β-catenin’s recruitment to β-catenin target promoters, we performed ChIP promoter scanning of 10 kb of the c-Myc promoter, and found that PAF, EZH2, and β-catenin specifically co-occupied the proximal promoter containing TBEs of the c-Myc gene (at -1037 and -459 bp) (He et al., 1998) in HCT116 cells (Figure 4C). Also, the analysis of EZH2 ChIP-sequencing data from mouse embryonic stem cells showed that EZH2 was specifically enriched in the proximal promoters of β-catenin targets (Lef1Lgr5c-Myc, and Axin2) (Figure 4D).

Figure 4 PAF promotes EZH2-β-catenin interaction

Next, we asked whether EZH2 promoter recruitment is necessary for activation of β-catenin target gene transcription. Previously, depletion of EZH2 was shown to inhibit c-Myc expression in DLD-1 colon cancer cells (Fussbroich et al., 2011). Consistently, EZH2 knockdown downregulated β-catenin target genes, Axin2and Cyclin D1 in HCT116 cells (Figure 4E), and decreased LiCl-induced β-catenin reporter activation (Figure 4F), suggesting that EZH2 is required for PAF-mediated Wnt target gene hyperactivation. These results are also supported by previous finding that EZH2 enhances β-catenin transcriptional activity by connecting β-catenin with the Med1/RNA polymerase II (Pol II) complex (Shi et al., 2007). Indeed, Med1/TRAAP220 and Pol II conditionally binds to c-Myc and Axin2 promoters in LiCl-treated HeLa cells (Figure 4G). Given that PRC2-indepednent interaction between EZH2 and PAF (Figures 3K and 3N), we asked whether EZH2’s histone methyltransferase activity is dispensable in β-catenin regulation. We utilized an EZH2 point mutant (F681I) that disrupts the contact between the EZH2 hydrophobic pocket and histone lysine residue H3K27 (Joshi et al., 2008). Ectopic expression of either EZH2 or EZH2-F681I enhanced β-catenin reporter activity (Figure 4H). Also, PAF knockdown did not change the H3K27 methylation status (H3K27me3) of proximal promoters of c-MycAxin2Cyclin D1, and DCC in HCT116 cells (Figure 4I). These results indicate a methyltransferase-independent role of EZH2 in transactivating β-catenin targets.

Due to PAF’s (1) small size (111 amino acids, one α-helix), (2) lack of a specific catalytic domain, and (3) binding to both β-catenin and EZH2, PAF may facilitate the interaction between EZH2 and β-catenin through recruiting EZH2 to the promoter. We tested this using ChIP assays for EZH2 in the setting of PAF depletion. Indeed, PAF-depleted HCT116 cells displayed decreased EZH2-association at the c-Myc promoter (Figure 4J), suggesting that PAF assists or is needed to recruit EZH2 to β-catenin transcriptional complex. Also, β-catenin knockdown decreased recruitment of PAF and EZH2 to promoters (Figure 4K), showing that PAF and EZH2 occupy target promoters via β-catenin. We then asked whether PAF promotes β-catenin-EZH2 binding. In vitro binding assays showed that the addition of GST-PAF protein increased EZH2-β-catenin association (Figure 4L). Moreover, ectopic expression of PAF promoted the EZH2-β-catenin interaction in HeLa cells treated with LiCl (Figure 4M). Additionally, we tested whether Wnt signaling-induced post-translational modification of either β-catenin or PAF is required for EZH2 interaction. However, in GST pull-down assays, we found that bacterially expressed either GST-β-catenin or –PAF bound to EZH2 (Figure S3). Due to the lack of post-translational modification in GST protein expression system, these data indicate that post-translation modification of either β-catenin or PAF is not necessary for EZH2 interaction. Together, these results suggest that PAF acts as a molecular adaptor to facilitate EZH2-β-catenin complex, and subsequently enhances the transcriptional activity of the β-catenin transcriptional complex at Wnt target promoters (Figure 4N).

Intestinal tumorigenesis following PAF conditional expression

Having determined that PAF is overexpressed in colon cancer cells and hyperactivates Wnt/β-catenin signaling, we aimed to determine whether mimicking PAF overexpression drives intestinal tumorigenesis, using genetically engineered mouse models. To conditionally express PAF, we generated doxycycline (doxy)-inducible PAF transgenic mice (TetO-PAF-IRES-emGFP [iPAF]). For intestine-specific expression of PAF, we used iPAF:Villin-Cre:Rosa26-LSL-rtTA mouse strains. Villin-Cre is specifically expressed in intestinal epithelial cells (IECs), including ISCs and progenitor cells. Cre removes a floxed stop cassette (loxP-STOP-loxP [LSL]) from the Rosa26 allele and induces rtTA expression. Upon doxy treatment, rtTA drives the transcriptional activation of the tetracycline-responsive element promoter, resulting in conditional transactivation of PAF selectively in IECs. We also utilized the Rosa26-rtTA strain for ubiquitous expression of PAF (Figure 5A and Figure S4). First, we examined the effects of PAF induction on IEC proliferation using a crypt organoid culture system (Figure S5A). Intriguingly, PAF conditional expression (2 weeks) induced expansion of the crypt organoids (Figures 5B and 5C), which recapitulates the mitogenic function of PAF (Figure 1). In mouse, IEC-specific PAF expression (iPAF:Villin-Cre:Rosa26-LSL-rtTA; 2 months) developed adenoma in both small intestine and colon (Figure 5D). Also, microscopic analysis using hematoxylin and eosin (H&E) staining showed aberrant IEC growth and crypt foci formation (Figures 5E and 5F), with disorganized epithelial cell arrangements (Figure S5B). Consistently, PAF-induced IEC hyperproliferation was manifested by increased Ki67 expression, a mitotic marker (Figure 5G). Importantly, these lesions exhibited the upregulation of CD44, a β-catenin target gene, whereas CD44 was expressed strictly in the crypts of normal intestine (Figure 5H). Next, we examined whether PAF directly hyperactivates Wnt/β-catenin in vivo using BAT-gal, a β-catenin reporter transgenic mouse carrying multiple TBEs followed by a LacZ reporter. To quantify the early effects of PAF on β-catenin activity, we treated mice with doxy for 1 week, and found that short-term induction of PAF increased β-catenin transcriptional activity as represented by enhanced X-gal staining (Figure 5I). Moreover, conditional PAF expression upregulated the β-catenin target genes, Axin2Lgr5CD44Cyclin D1, and c-Myc in crypt organoids (Figure 5J). Additionally, mice ubiquitously expressing PAF exhibited intestinal hypertrophy (Figure S5C), which is similar to that induced by R-Spondin1, a secreted Wnt agonist (Kim et al., 2005). These data strongly suggest that PAF expression is sufficient to initiate intestinal tumorigenesis via Wnt signaling hyperactivation.

Figure 5 Induction of intestinal neoplasia by PAF expression

Herein we reveal the unexpected role of PAF in modulating Wnt/β-catenin signaling. PAF enhances the transcription of Wnt targets by recruiting EZH2 to the β-catenin transcriptional complex. This is similar to the mechanism by which Lgl/BCL9 binds to β-catenin and thereby recruits the PHD-finger protein Pygopus, to bridge the β-catenin/TCF complex to Med12 and Med13 (Carrera et al., 2008). Importantly, due to specific overexpression of PAF in cancer cells, our studies identified an additional layer of the regulatory mechanism of β-catenin target gene transactivation.

In cancer cells, the upregulation of EZH2 contributes to tumorigenesis through the epigenetic repression of various genes including tumor suppressor genes, Wnt antagonists, and DNA repair genes (Chang et al., 2011Cheng et al., 2011Kondo et al., 2008). Our results propose a noncanonical function of EZH2 in activating β-catenin target genes in conjunction with PAF. Consistently, recent study also suggests methyltransferase activity-independent function of EZH2 in gene activation (Xu et al., 2012). Moreover, this non-canonical role of EZH2 is supported by several lines of evidence: (a) EZH2 transactivates β-catenin target genes (Li et al., 2009Shi et al., 2007) (Figures 4E and 4F); (b) EZH2 overexpression in murine mammary epithelium induces ductal hyperplasia (Li et al., 2009), which phenocopies that in a ∆Nβ-catenin (constitutively active form of β-catenin) mouse model (Imbert et al., 2001); (c) EZH2 occupies β-catenin target promoters (Figures 4A-D); and (d) EZH2’s methyltransferase activity is dispensable for β-catenin target activation (Figures 4H and 4I). Moreover, similar to PAF expression in the AER (Figure 2A), EZH2 is also specifically expressed there to maintain of Hox cluster gene transcription (Wyngaarden et al., 2011). Thus, it is plausible that EZH2 and PAF cooperatively control Hox gene activation in the developing limb. Interestingly, despite the presence of a physical and functional connection between Bmi1 and EZH2 in H3K27me3-mediated gene repression, EZH2 is expressed only in crypt IECs including ISCs (Figure S6), whereas Bmi1 is expressed in ISCs at position 4 (Figure 3J), implying a Bmi1-independent role for EZH2 in gene regulation. These results demonstrate the novel function of EZH2 in β-catenin target gene activation, independent of the histone methyltransferase activity of EZH2.

Previously, we found that TERT, a catalytic subunit of telomerase, positively modulates Wnt signaling (Park et al., 2009), elucidating a non-telomeric function of telomerase in development and cancer. Here our results propose that one component of DNA damage bypass process also functions in regulating Wnt signaling, dependent of context. In cancer, PAF overexpression may play a dual role in inducing (a) cell hyperproliferation (via Wnt signaling hyperactivation) and (b) the accumulation of mutations arising from DNA lesion bypass (by PAF-mediated TLS) (Povlsen et al., 2012). Importantly, PAF is only expressed in cancer cells, but not in normal epithelial cells. Thus, upon DNA damage, instead of cell growth arrest to permit high-fidelity DNA repair, the PAF overexpression in cancer cells is likely to induce DNA lesion bypass by facilitating TLS. However, in the setting of Wnt signaling deregulation, nuclear β-catenin sequesters PAF from PCNA and utilize PAF as a co-factor of transcriptional complex, which induces Wnt signaling hyperactivation and possibly lead to increased mutagenesis.

We observed that PAF marked the stemness of ISCs and mouse embryonic stem cells (Figure S7), implicating its roles in stem cell regulation under physiological conditions. In a previous study, a PAFgermline knockout mouse model displayed defects in hematopoietic stem cell self-renewal (Amrani et al., 2011), suggesting a crucial role of PAF in stem cell maintenance and activation. In the intestine, β-catenin activation in Lgr5-positive or Bmi1-positive cells is sufficient to develop intestinal adenoma (Barker et al., 2009Sangiorgi and Capecchi, 2008), suggesting an essential role of tissue stem cells in tumor initiation. Considering PAF expression in Bmi1-positive ISCs, PAF upregulation in ISCs likely hyperactivates the Wnt/β-catenin signaling and contributes to intestinal tumor initiation.

Despite the critical role of Wnt signaling in early vertebrate, development PAF germline knockout mice are viable (Amrani et al., 2011). It is noteworthy that, whereas deletion of any core component in the Wnt signaling pathway causes embryonic lethality, mice with germline knockout of Wnt signaling modulators, including Nkd1/2Pygo1/2, and BCL9/9-2, exhibit no lethal phenotypes (Deka et al., 2010Schwab et al., 2007Zhang et al., 2007). This may result from the robustness of Wnt signaling during embryogenesis because of functional compensation not only via the presence of multiple Wnt signaling regulators per se but also via other types of signaling crosstalk. Therefore, as described previously in pRb studies (Sage et al., 2003), acute deletion of PAF in a conditional knockout mouse model may disrupt the developmental balance or tissue homeostasis, and then reveal the full spectrum of the physiological and pathological roles of PAF in tumorigenesis. Taken together, our findings reveal unexpected function of PAF and EZH2 in modulating Wnt signaling, and highlight the impacts of PAF-induced Wnt signaling deregulation on tumorigenesis.

11.1.9 PAF Makes It EZ(H2) for β-Catenin Transactivation

Xinjun Zhang1 and Xi He1
Mol Cell. 2013 Oct 24; 52(2)

In this issue of Molecular Cell, Park and colleagues (Jung et al., 2013) show that PAF (PCNA-associatedfactor) binds to and hyperactivates transcriptional function of β-catenin in colon cancer cells by recruiting EZH2 to the coactivator complex. PAF-β-catenin and PAF-PCNA interactions are competitive, raising the question of whether β-catenin might regulate PCNA-dependent DNA replication and repair.

Wnt signaling through stabilization of transcription co-activator β-catenin plays critical roles in animal development and tissue homeostasis, and its deregulation is involved in myriad human diseases including cancer (Clevers and Nusse, 2012). Notably, most colorectal cancers (CRCs) have elevated β-catenin signaling caused by mutations of Wnt pathway components such as the tumor suppressor APC (Adenomatosis polyposis coli) and β-catenin itself (Clevers and Nusse, 2012). Much effort has focused on studying β-catenin-dependent transactivation in CRCs, including the current study by Park and colleagues that identifies PAF as an unexpected β-catenin co-activator (Jung et al., 2013).

PAF, for PCNA (proliferating cell nuclear antigen)-associated factor (also known as KIAA0101 or p15PAF), is an interacting partner of PCNA (Yu et al., 2001). PCNA has a key role in DNA replication and repair by assembling various DNA polymerase and repair complexes at the replication fork (Mailand et al., 2013). Dynamic regulation of PAF abundance and/or interaction with PCNA appears to be important for engaging DNA damage repair and bypass pathways (Emanuele et al., 2011Povlsen et al., 2012). PAF is overexpressed in many types of cancers and required for cell proliferation (e.g., Yu et al., 2001).

In the current study (Jung et al., 2013), Jung et al. show that PAF is overexpressed in CRCs in a manner that parallels expression of Axin2, an established Wnt/β-catenin target gene. PAF knockdown inhibits CRC proliferation, and this effect is independent of PAF-PCNA interaction and can be rescued by a PAF mutant that does not binds to PCNA or by β-catenin overexpression. PAF knockdown downregulates the expression of Wnt/β-catenin target genes Cyclin D1c-Myc, and Axin2 in a CRC line, leading the authors to hypothesize that PAF participates in Wnt/β-catenin signaling. Indeed PAF knockdown reduces, and its overexpression augments, Wnt/β-catenin responsive TOPFLASH reporter and target gene expression induced by Wnt3a or by pharmacological agents that stabilize β-catenin. In Xenopus embryos, PAF synergizes with β-catenin to induce Wnt target gene expression and axis duplication (a hallmark of Wnt/β-catenin activation). In mouse embryos, PAF is highly expressed in regions known for Wnt/β-catenin signaling such as the apical ectodermal ridge of the limb bud. Therefore PAF appears to be a positive regulator of Wnt/β-catenin signaling in CRCs and vertebrate embryos.

PAF does not affect β-catenin protein levels and is localized in the nucleus. Protein binding assays show that PAF interacts, directly or indirectly, with β-catenin (via the Armadillo-repeat domain) and its DNA-bound partner TCF (T Cell factor). Indeed PAF is associated with promoters of Wnt/β-catenin target genes in chromatin in CRC cells. Interestingly in the mouse intestine, the PAF protein is enriched in Bmi (B lymphoma Mo-MLV insertion region 1 homolog)-positive stem cells (at the “+4” position) (Sangiorgi and Capecchi, 2008). Bmi1 is a component of Polycomb Repressive Complex 1 (PRC1), which, together with the PRC2 complex that modifies Histone H3, has critical functions in transcriptional epigenetic silencing. Previous studies have suggested that a core PRC2 component, EZH2 (enhancer of zeste homolog 2), is a partner and paradoxically a co-activator of β-catenin, acting in a manner that is independent of EZH2’s methyltransferase activity (Li et al., 2009Shi et al., 2007). Jung et al. found that PAF indeed interacts with both Bmi1 and EZH2, but not other PRC2 components, and EZH2 overexpression augments β-catenin transcriptional activity. PAF, EZH2, and β-catenin are found to co-occupy promoters of several Wnt/β-catenin target genes in CRC and mouse ES cells, and PAF depletion decreases EZH2 association with the c-Myc promoter, and β-catenin depletion decreases the association of both PAF and EZH2 with the promoter. Thus the β-catenin-PAF-EZH2 complex appears to constitute a chain of co-activators (Figure 1), and indeed PAF, which binds to both β-catenin and EZH2, enhances β-catenin-EZH2 co-immunoprecipitation. Together with an earlier study (Shi et al., 2007), these results suggest a model that PAF brings EZH2 and the associated RNA polymerase II Mediator complex to β-catenin target genes for transactivation in CRCs (Figure 1). Consistent with this model, transgenic overexpression of PAF in the mouse intestine induces β-catenin-dependent target and reporter gene expression, intestinal overgrowth, and adenoma formation in vivo and crypt organoid expansion in vitro, resembling Wnt/β-catenin signaling activation in the gastrointestinal tract.



β-catenin transactivation

Figure 1 β-catenin transactivation mediated by PAF and EZH2 in the G1 phase and a speculative role of β-catenin in modulating PAF-PCNA-dependent DNA replication and repair/bypass pathways in the S phase.

PAF and EZH2 represent newer additions to β-catenin’s plethora of co-activators (Mosimann et al., 2009), which offer multiple routes to engage the basal transcription apparatus. These co-activators may have partially redundant and/or context-dependent functions for numerous Wnt/β-catenin-dependent gene programs. Mouse mutants that lack an individual β-catenin co-activator are often viable (MacDonald et al., 2009Mosimann et al., 2009). Paf−/− mice are viable but exhibit defects in hematopoietic stem cell properties (Amrani et al., 2011). PAF is also expressed in self-renewing mouse ES cells but the expression is downregulated upon ES cell differentiation (Jung et al., 2013). Whether PAF has a general role in self-renewal of embryonic and adult stem cells through its role in β-catenin signaling or DNA replication and repair pathways remains to be investigated.

PAF-β-catenin interaction is observed under Wnt stimulation, likely as a consequence of β-catenin accumulation (Jung et al., 2013). In some cell types PAF is ubiquitinated and degraded by the anaphase promoting complex and thus exhibits the lowest level in the G1 phase of the cell cycle (Emanuele et al., 2011). In these cells PAF may have a limited role as a co-activator for β-catenin-dependent transcription, which primarily occurs in G1. But in CRC and other cancers where PAF is overexpressed, PAF may have a prominent role as a β-catenin co-activator.

PAF-PCNA interaction is well documented (e.g., Yu et al., 2001). Surprisingly however, in CRCs with high levels of β-catenin, PAF-PCNA interaction is barely detectable (Jung et al., 2013). Conversely, in cells where the basal level of Wnt/β-catenin signaling is low, PAF-PCNA interaction is detected but is diminished by Wnt3a or pharmacological agents that stabilize β-catenin (Jung et al., 2013). PAF seems to interact with β-catenin and PCNA via an overlapping domain (although this remains to be better defined), offering a possible explanation why PAF-β-catenin and PAF-PCNA complexes appear to be mutually exclusive (Jung et al., 2013). This may simply reflect the fact that PAF-β-catenin (for RNA transcription) and PAF-PCNA (for DNA replication/repair) complexes act in G1 and S, respectively (Figure 1). However, when β-catenin levels are high in Wnt-stimulated cells or in CRCs, one may speculate that β-catenin accumulation could inhibit PAF-PCNA complex formation in the S phase, thereby enabling Wnt/β-catenin signaling to modulate PAF-PCNA-dependent DNA replication and repair/bypass pathways (Figure 1). This would constitute an unsuspected role for Wnt/β-catenin signaling in genomic stability beyond its established transcriptional function and could have implications to tumorigenesis.

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11.1.10 PI3K.AKT.mTOR pathway as a therapeutic target in ovarian cancer

Li H1Zeng JShen K.
Arch Gynecol Obstet. 2014 Dec; 290(6):1067-78

Background: Ovarian cancer is one of the major causes of death in women worldwide. Despite improvements in conventional treatment approaches, such as surgery and chemotherapy, a majority of patients with advanced ovarian cancer experience relapse and eventually succumb to the disease; the outcome of patients remains poor. Hence, new therapeutic strategies are urgently required. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) is activated in approximately 70 % of ovarian cancers, resulting in hyperactive signaling cascades that relate to cellular growth, proliferation, survival, metabolism, and angiogenesis. Consistent with this, a number of clinical studies are focusing on PI3K pathway as an attractive target in the treatment of ovarian cancer. In this review, we present an overview of PI3K pathway as well as its pathological aberrations reported in ovarian cancer. We also discuss inhibitors of PI3K pathway that are currently under clinical investigations and the challenges these inhibitors face in future clinical utility.Methods: PubMed was searched for articles of relevance to ovarian cancer and the PI3K pathway. In addition, the was also scanned for data on novel therapeutic inhibitors targeting the PI3K pathway. Results: Genetic aberrations at different levels of PI3K pathway are frequently observed in ovarian cancer, resulting in hyperactivation of this pathway. The alterations of this pathway make the PI3K pathway an attractive therapeutic target in ovarian cancer. Currently, several inhibitors of PI3K pathway, such as PI3K/AKT inhibitors, rapamycin analogs for mTOR inhibition, and dual PI3K/mTOR inhibitors are in clinical testing in patients with ovarian cancer. Conclusions: PI3K pathway inhibitors have shown great promise in the treatment of ovarian cancer. However, further researches on selection patients that respond to PI3K inhibitors and exploration of effective combinatorial therapies are required to improve the management of ovarian cancer.

Fig.1. Inputs from receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCR) to class I PI3K.

Fig. 2. Schematic representation of the PI3K/AKT/mTOR signaling pathway.

Fig.3. PI3K/AKT/mTOR inhibitors.

AKT inhibitors

AKT inhibitors can be grouped into three classes including lipid based phosphatidylinositol (PI) analogs, ATP-competitive inhibitors, and allosteric inhibitors. Perifosine, which is the most clinically studied AKT inhibitor, is a lipid-based PIanalog that targets the pleckstrin homology domain of AKT, preventing its translocation to the cell membrane. Amongthe three classes of AKT inhibitors, allosteric AKT inhibitors display highly specific selectivity for AKT isoforms. Considering the genetic background of ovarian cancer, allosteric AKT inhibitors such as MK2206 that can target both AKT1 and AKT2 might be the best agents for treating ovarian cancer.In clinical trials, AKT inhibitors have shown similar toxicities to those caused by PI3K inhibitors, such as hyperglycemia, rashes, stomatitis, and gastrointestinal side effects [25].

mTOR inhibitors

Rapamycin and its analogs Rapamycin (sirolimus), a potent inhibitor of mTORC1, was first isolated in 1975 from the bacterium Streptomyces hygroscopicus. Rapamycin inhibits mTORC1 by first binding to the intracellular protein FK506 binding protein 12 (FKBP12). The resultant rapamycin–FKBP12 complex then binds to the FKBP12–rapamycin-binding domain (FRB) of mTORC1 and inhibits the serine/threonine kinase activity of mTORC1 via an allosteric mechanism. In contrast to mTORC1, the rapamycin–FKBP12 complex cannot interact with the FRB domain of mTORC2, and thus,mTORC2 is generally resistant to rapamycin treatment [12]. As rapamycin displays very poor water solubility, which limits its clinical use, several soluble ester analogs of rapamycin (rapalogs) have been developed [12]. Currently, these analogs include temsirolimus, everolimus, and ridaforolimus. Temsirolimus and everolimus are formulated for intravenous and oral administration, respectively. Ridaforolimus was initially developed as an intravenous formulation, but an oral formulation was subsequently produced [12,28]. Clinically, rapalogs are generally well tolerated, with the most common side effects including stomatitis, rashes, fatigue, hyperglycemia, hyperlipidemia, hypercholesterolemia, and myelosuppression [3,12,25].

ATP-competitive inhibitors

Different from rapalogs, ATP-competitive inhibitors do not require co-factors such as FKBP12 to bind to mTOR. By competingwith ATP for theATP-binding sites of mTOR, this class of mTOR inhibitors can inhibit the kinase activity of both mTORC1 and mTORC2. Although there is a concern that the simultaneous inhibition of mTORC1 and mTORC2 might result in greater toxicities in normal tissues, ATP-competitive mTOR inhibitors have been shown to display stronger anti-proliferative activity than rapalogs across a broad range of cancers includingovarian cancer [12,15].


Metformin,the most commonly prescribed oral anti-diabetic agent, has been shown to reduce the incidence of malignancies in patients with diabetes. The activation of 5′ adenosine monophosphateactivated protein kinase (AMPK) by metformin plays an important role in mediating the drug’s effects. AMPK activation results in the phosphorylation and activation of TSC2, which exerts inhibitory effects on mTORC1. Metformin-induced AMPK activation also reduces AKT activity by inhibiting insulin receptor substrate 1 (IRS-1). Ultimately, AMPK activation results in the inhibition of the PI3K/AKT/mTOR signaling pathway, making metformin an effective treatment for cancer [28].

mTORC1 inhibitors              mTORC1                      Dual PI3K/mTOR inhibitors

PI3K inhibitors                     Class I PI3K                   mTORC2

AKT inhibitors                        AKT                              mTORC ½  inhibitors

PI3K inhibitors

Pan-class I PI3K inhibitors Pan-class IPI3K inhibitors can inhibit the kinase activity ofall 4 isoforms of classI PI3K.The main advantage of pan-class IPI3K inhibitors is that most cancer cells express multiple PI3K isoforms with redundant oncogenic signaling functions. Early clinical trials have suggested that the most common toxicitiesof pan-class IPI3K inhibitors are hyperglycemia, skin toxicities, stomatitis, and gastrointestinal side effects. Of these, hyperglycemia is likely to be a mechanism-based toxicity given the well described role of PI3K in insulin receptor signaling [3,25].

Isoform-selective PI3K inhibitors

This class of agents target the specific PI3K p110 isoforms involved in particular types of cancer. The p110α isoform (which is encoded by the PIK3CA gene) is a frequent genetic driver (PIK3CA mutations) of ovarian cancer, whereas p110β activity is known to be essential in cancer cells lacking PTEN. As for the p110δ isoform, it plays a fundamental role in the survival of normal B cells and is implicated in malignancies affecting this lineage. Thus, the main theoretical advantage of these inhibitors is that they have the potential to completely block the relevant target whilst causing limited toxicities compared with pan-PI3K inhibitors. Consistent withthese findings, preclinical studies have detected significant activities of PI3Kα inhibitor in tumors exhibiting PIK3CA mutations, PI3Kβ inhibitors in tumors with PTEN loss, and PI3Kδ inhibitors in hematologic malignancies. In addition, PI3Kδ inhibitors have already shown very promising activity in patients with chronic lymphocytic leukemia [26].

Dual PI3K/mTOR inhibitors

Structural similarities between the ATP-binding domain of p110 and the catalytic domain of mTOR have led to the development of a class of agents that inhibit both class I PI3K and mTORC1/2. Theoretically, dual mTOR/PI3K inhibitors should lead to more complete suppression of the PI3K/AKT/mTOR pathway than targeting either component independently.In agreement with this, in preclinical studies of ovarian cancer dual PI3K/mTOR inhibitors were found to exhibit greater in vitro and in vivo anti-tumor activity than mTOR inhibitors alone [27]. The safety profile of these inhibitors is similar to that of pan-PI3K inhibitors, with common adverse events including nausea, diarrhea, fatigue, and vomiting [3,25]. 


11.1.11 Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway

Mira JP1Benard VGroffen JSanders LCKnaus UG.
Proc Natl Acad Sci U S A. 2000 Jan 4; 97(1):185-9.

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3-Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

Uncontrolled cell proliferation is a major feature of cancer. Experimental cellular models have implicated some members of the Rho GTPase family in this process. However, direct evidence for active Rho GTPases in tumors or cancer cell lines has never been provided. In this paper, we show that endogenous, hyperactive Rac3 is present in highly proliferative human breast cancer-derived cell lines and tumor tissues. Rac3 activity results from both its distinct subcellular localization at the membrane and altered regulatory factors affecting the guanine nucleotide state of Rac3. Associated with active Rac3 was deregulated, persistent kinase activity of two isoforms of the Rac effector p21-activated kinase (Pak) and of c-Jun N-terminal kinase (JNK). Introducing dominant-negative Rac3 and Pak1 fragments into a breast cancer cell line revealed that active Rac3 drives Pak and JNK kinase activities by two separate pathways. Only the Rac3–Pak pathway was critical for DNA synthesis, independently of JNK. These findings identify Rac3 as a consistently active Rho GTPase in human cancer cells and suggest an important role for Rac3 and Pak in tumor growth.

Rac proteins are members of the Rho GTPase family and act as molecular switches in regulating a variety of biological response pathways, including cell motility, gene transcription, cell transformation, and cell-cycle progression (1). The Rac family includes Rac1, the myeloid-lineage-specific Rac2, and the recently cloned Rac3 proteins (2). Rac3 differs from Rac1 and Rac2 in two domains, the insert region and the C terminus, which influence transformation (34), interaction with guanine nucleotide exchange factors (GEFs) (56), and subcellular localization (78). Small GTPases, including Rac, cycle between an inactive GDP-bound state and an active GTP-bound state. Two classes of regulatory factors, GTPase-activating proteins (GAPs) and GEFs, determine by their opposing effects the ratio of GDP versus GTP, which is bound to the GTPase (1). GAP proteins increase the intrinsic rate of GTP hydrolysis, rendering the GTPase inactive, whereas GEFs enhance the exchange of bound GDP for GTP, thereby activating the protein. Active Rac regulates distinct downstream signaling pathways by interacting with specific effector proteins, including a family of serine-threonine protein kinases termed Paks (p21-activated kinases) (911).

Apart from its well documented role in cytoskeletal rearrangements in growth factor-stimulated cells (12), Rac1 is required for Ras-induced malignant transformation and is involved in transcription and growth control (11314). Recently, the importance of the Rac effector Pak in cell transformation has been highlighted by inhibiting RasV12- and Rac1V12-induced transformation of Rat-1 fibroblasts with a catalytically inactive form of Pak (1516). The involvement of Rac1 in driving cell-cycle progression through the G1 phase and stimulating DNA synthesis has been shown by introducing dominant-active and -negative Rac1 mutants into fibroblasts (1718). However, the signaling pathways used by Rac to control mitogenesis and proliferation still remain poorly understood. Overexpression of constitutively active Rac-effector-domain mutants in fibroblasts indicated that although Rac1 mediated cyclin D1 transcription by Pak in NIH 3T3 cells (19), Pak was not involved in the DNA synthesis of Swiss 3T3 cells (20). Accumulating evidence, however, suggests higher complexity where Pak-binding proteins, such as the GEF Pix, contribute to the Rac–Pak interaction in vivo and influence subsequent cellular functions (2123).

All biological functions listed above have been attributed to Rac1 in experimental cell systems using overexpression or microinjection of mutant forms. Endogenously active Rho GTPases, including Rac, have not yet been observed. In this paper, we describe a consistently active Rac3 GTPase leading to hyperactivity of its effector protein kinase, Pak, in human breast cancer-derived epithelial cell lines. Analysis of growth properties and DNA synthesis revealed that both proteins are required to convey the highly proliferative phenotype displayed by these cells.

Highly Proliferating Cancer Cells Contain Hyperactive Rac3.

Comparison of growth rates among several breast cancer cell lines showed that three lines (MDA-MB 435, T47D, and MCF 7) grew faster under normal and low-serum conditions (Fig. ​(Fig.1).1). Interestingly, in contrast to MDA-MD 231 and Hs578T cells, these three highly proliferative cell lines do not possess mutated Ras (2829). To assess whether Rho GTPases drive this cellular phenotype, we determined whether these cell lines contained active GTP-bound Rac or Cdc42. We used a recently described assay, the PBD-pulldown assay (24), which is based on the specific binding of the GTP-bound forms of Rac and Cdc42 to the PBD of Pak (10). Neither active Rac1 (Fig. ​(Fig.22A) nor active Cdc42 (data not shown) could be detected in any of the cell lysates obtained from serum-starved cells. However, both proteins were detected if the PBD-pulldown assay was performed with in vitro guanosine 5′-[γ-thio]triphosphate (GTP[γS])-loaded cell lysates, confirming that Rac1 and Cdc42 were present in their inactive GDP-bound forms in these cells (Fig. ​(Fig.22A for Rac1). Next we wanted to determine whether active Rac3 was present in breast cancer cell lines. Because Rac3 effectors have not yet been characterized, we demonstrated by overlay binding and kinase assays that Rac3 bound to and activated Pak as efficiently as Rac1 (data not shown). We verified that the PBD-pulldown assay specifically detected the active GTP-bound form of Rac3 (GTP[γS]-loaded Rac3wt or Rac3V12, Fig. ​Fig.22B) and not the inactive form. To probe for Rac3 protein in breast cell lysates, a Rac3-specific antibody was used. GST-PBD-pulldown experiments from cell lysates revealed the presence of hyperactive Rac3 in highly proliferative cell lines (MDA-MB 435, T47D, and MCF 7), but not in normal breast cell lines or in less proliferative breast cancer cells (Fig. ​(Fig.22C). Additionally, as indicated by the virtual absence of Rac3 in the supernatant of the PBD pulldown, all the Rac3 protein present in these cell lines was active (Fig. ​(Fig.22C). To demonstrate that consistent Rac3 activation is not limited to cell lines, we performed an initial screening of human metastatic breast cancer tissues and found active Rac3 in one of three samples, underlining the potential clinical relevance of the cellular findings (Fig. ​(Fig.22D).

Differential growth rates of human breast cell lines.  pq0104939001

Differential growth rates of human breast cell lines. pq0104939001

Differential growth rates of human breast cell lines.

Figure 1 Differential growth rates of human breast cell lines. Human breast cell lines, including HMEC 184 (○), MDA-MB 231 (▵), Hs578T (□), MDA-MB 435 (●), T47D (▴), and MCF 7 (♦), were grown in 10% serum (A) or 0.5% serum (B) conditions. The cells were split in duplicate over 6-well plates at 5 × 105 cells per well and counted daily with a hemocytometer for 4 days. Data shown in A and B are representative of three independent experiments.

Figure 2 Active Rac3 is present in highly proliferative cell lines and in human breast cancer tissue. (A and C) Cell lysates from serum-starved breast cancer cell lines without (A and C) or after (+) GTP[γS] loading (A) were incubated with 10 μg of GST-PBD. Active Rac proteins (PBD pulldown) were detected by immunoblot with anti-Rac1 (A) or anti-Rac3 antibodies (C). Blotting of PBD supernatants revealed the GDP-bound form of Rac3 in lysates. Equal amounts of Rac3 protein were detected by immunoblot (IB) in all cell lines. (B) A PBD-pulldown assay of extracts from HeLa cells expressing Myc-Rac3wt or -Rac3 mutants, followed by an anti-Myc immunoblot, detected only active Rac3 (GTP[γS] loading or Rac3V12). (D) PBD pulldown of lysates obtained from three different human metastatic breast cancer tissues, followed by anti-Rac1 and anti-Rac3 immunoblots, revealed active Rac3 in tissue 1. (E) PBD pulldown of lysates derived from MDA-MB 435 and MDA-MB 231 cells expressing LacZ control or Myc-Rac3wt without or after in vitro GTP[γS] loading. Consistent activation of Myc-Rac3wt occurred only in MDA-MB 435 cells. (F) Subcellular localization of Rac1 and Rac3. Cytosol (c) and membranes (m) were obtained after nitrogen cavitation and fractionation of breast cancer cell lines and immunoblotted with anti-Rac1 and anti-Rac3 antibodies. All blots are representative of at least three experiments.

Subcellular Localization and GTPase-Regulatory Factors Influence Rac3 Activity.

Constitutive activation of Ras proteins in cancer cells is often caused by activating point mutations at the switch I or II regions (29). cDNA cloning and complete sequence analysis of full-length Rac3 did not reveal any mutations in the breast cell lines studied and did not explain the observed Rac3 activation. GTPase-regulatory proteins such as GEFs and GAPs, which are usually regulated by upstream stimuli, control cycling between the active and inactive forms of Rac. To confirm the presence of an altered regulatory mechanism involved in Rac3 activation, we used the PBD-pulldown assay to analyze the activation state of Myc-tagged Rac3wt transfected into either MDA-MB 231, a cell line harboring only GDP-Rac3, or MDA-MB 435, a cell line that contains endogenous, active GTP-Rac3. Fig. ​Fig.22E shows that activated Myc-Rac3 was detected only in the MDA-MB 435 cell line, confirming that the regulation of the GDP/GTP state of Rac3 was altered in these cells. We then investigated several upstream stimuli that have been shown to affect GTPase-regulatory proteins (283032). We excluded the possibility of an autocrine growth-stimulatory loop by culturing MDA-MB 231 cells with the conditioned medium from MDA-MB 435, which did not affect the Rac3 activation state (data not shown). Treatment of cell cultures with phosphatidylinositol 3-kinase or tyrosine kinase inhibitors, including wortmannin, LY294002, and genistein, did not decrease Rac3 activation (data not shown). At this point, we speculated that an oncogenic, Rac3-specific GEF is present in certain breast cancer cells. GEFs possess a pleckstrin homology domain that is essential for membrane localization and for their oncogenic properties (533). Analysis of the subcellular localization of the Rac family members revealed that Rac3 is located in the membranes of breast epithelial cell lines, independently of its activation state (Fig. ​(Fig.22F). In contrast, endogenous Rac1 in its inactive GDP-bound state was essentially cytosolic (Fig. ​(Fig.22F). Thus, the distinct localization of Rac3 and Rac1 may contribute to their different activation states in certain breast cancer cell lines. It is conceivable that the highly proliferative cell lines (Fig. ​(Fig.1)1) express a constitutively active, membrane-bound Rho GEF that activates adjacent Rac3 protein. This hypothesis was further supported by using an hydroxymethylglutaryl-CoA reductase inhibitor, lovastatin, that interferes with isoprenoid synthesis and thereby with posttranslational processing of GTPases. Unprocessed Rac3 from lovastatin-treated MDA-MB 435 cells was predominantly cytosolic and inactive (GDP-Rac3) (data not shown). The requirement of membrane localization for consistent Rac3 activity was further supported by using a Rac3S189 mutant. Replacing cysteine-189 of the CAAX box with serine abolishes isoprenoid incorporation, rendering the GTPase cytosolic. This Rac3 mutant remained in its inactive GDP-bound state when transfected into MDA-MB 435 cells (data not shown).

Several Rho GTPase-regulating GEFs have been identified (5), including the Rac1-specific GEF Tiam-1, which has been linked to tumors such as invasive T-lymphomas (34). Although Tiam-1 is expressed in virtually all tissues, no evidence of oncogenic truncations or alternative splicing of Tiam-1 transcripts has been found (35). A variation of Tiam-1 transcript levels in certain cancer cell lines might lead to overexpression and possibly activation of Tiam-1 protein. However, the activation state of Rac3 protein in the cell lines used in this study does not seem to correlate with Tiam-1 expression levels as reported by Habets et al. (35). Hyperactivity of Rac3 in cancer cells could also result from an absent or dysfunctional Rac3-specific GAP protein. By accelerating the intrinsic GTP hydrolysis rate, GAPs render the GTPase inactive and act as tumor suppressors. Deletion or mutations in the RasGAP gene NF1 and the RhoGAP homologs bcr and DLC-1 have been reported in cancer cells (3637).

Active Rac3 Drives Epithelial Cell Proliferation.

To study whether active Rac3 could account for the high proliferation rate of certain breast cancer cells, we expressed a constitutively active Rac3 mutant (Rac3V12) in normal mammary epithelial cells (HMEC 184) that contain only GDP-Rac3 (Fig. ​(Fig.22C). Rac3V12 expression significantly increased the incorporation of BrdUrd into nascent DNA (Fig. ​(Fig.3),3), emphasizing that transfection of active Rac3 drives epithelial cell proliferation.

Rac3V12 induces DNA synthesis in human mammary epithelial cells pq0104939003

Rac3V12 induces DNA synthesis in human mammary epithelial cells pq0104939003

Rac3V12 induces DNA synthesis in human mammary epithelial cells

Figure 3 Rac3V12 induces DNA synthesis in human mammary epithelial cells. HMEC 184 cells, infected with recombinant LacZ or Rac3V12 Semliki Forest virus, were allowed to express protein for 14 h in serum-free medium containing 10 μM BrdUrd. Cells were fixed and stained with anti-Myc antibody for Myc-Rac3V12 expression level (Upper) or with FITC-conjugated anti-BrdUrd antibody for BrdUrd incorporation (Lower). The presence of bright fluorescent nuclei indicates BrdUrd-positive cells. The percentage was calculated after counting 400 cells in each of three independent experiments.

Hyperactive Pak and c-Jun Kinases in Cancer Cells.

The signaling cascade utilized by Rac proteins to control cell proliferation still remains to be identified (19), but might involve Paks. We analyzed Pak activity in cell lysates derived from serum-starved breast cancer cell lines by using in-gel kinase assays and by usingin vitro kinase assays after immunoprecipitation with Pak-specific antibodies. Pak activity was increased 4- to 6-fold in the three cell lines containing active Rac3 (Fig. ​(Fig.44A). This increased kinase activity was mainly associated with the Pak2 isoform, which can phosphorylate and positively regulate Raf-1 activity, another key component in cell proliferation (3840).

Figure 4 Rac3 activates Pak and JNK by two different pathways. (A) Breast cancer cell lysates from serum-starved cells were analyzed for Pak and JNK activities. Pak activities in cell lysates were analyzed by in-gel kinase assays. JNK activity was determined by 

Intracellular Rac-regulated signaling pathways impinge on distinct mitogen-activated protein kinase cascades. Constitutively active Rac has been shown to positively regulate the activity of the stress-activated kinases JNK and p38 (1). Moreover, ERK activity can be indirectly stimulated by Rac or mediated by crosstalk between the distinct mitogen-activated protein kinase cascades (141). Determination of distinct mitogen-activated protein and stress-activated protein kinase activities in the breast cell lines studied here showed that consistent Rac3 and Pak kinase activities were associated with enhanced JNK activity (Fig. ​(Fig.44A). In contrast, no correlation existed between p38 or ERK kinase activities and active Rac3 or Pak (data not shown).

Rac3 Triggers Pak and JNK Activities by Separate Pathways.

To determine whether the highly proliferative phenotype of breast cancer cells depends directly on a consistently active Rac3-Pak-JNK cascade, we used virus-mediated protein expression in MDA-MB 435 cells to examine the ability of Rac3 and Paks to control JNK activation and cellular proliferation. The importance of Pak as an effector protein in Rac-mediated activation of JNK is still controversial and seems to be cell-type-dependent (42). Expression of the PBD domain, which controls the activity of both Rac and Pak (21), completely inhibited Pak and JNK stimulation (Fig. ​(Fig.44B). The mutation of leucine to phenylalanine at position 107 of the PBD domain suppresses the autoinhibitory function of the PBD (21). Thus, PBD F107 will act only to sequester active Rac3 and blocks its ability to bind and activate endogenous effectors. Expression of either dominant-negative Rac3N17 or PBD F107 almost completely blocked Pak and JNK activities, demonstrating that Rac3 is upstream of these proteins (Fig. ​(Fig.44B). Moreover, Pak kinase activity can be inhibited independently of Rac3 by overexpressing the kinase autoinhibitory domain, PID, which does not interact with Rac (2143). Transfection of PID into MDA-MB 435 cells dramatically inhibited Pak activity as expected, but did not decrease JNK activation (Fig. ​(Fig.44B). Our results indicate that in MDA-MB 435 cells, consistent stimulation of JNK by Rac3 is independent of PAK activity and that Rac3 initiates two different pathways involving Pak and JNK, respectively.

Rac3 and Pak Are Both Required for Breast Cancer Cell Proliferation.

We subsequently determined which of these two Rac3 pathways promoted the increased cell proliferation in breast cancer cell lines with hyperactive Rac3. We studied the consequence of expressing inhibitory Rac mutants or Pak fragments on DNA synthesis. LacZ-expressing MDA-MB 435 cells still proliferated in low-serum conditions and 35% incorporated BrdUrd (Fig. ​(Fig.5).5). This percentage increased to 50% when Rac3wt, which will be partially activated in these cells (Fig. ​(Fig.22E), is expressed (Fig. ​(Fig.55 Bottom Right). In contrast, expression of inhibitory proteins, including Rac3N17 or the PBD that suppressed Pak and JNK activation (Fig. ​(Fig.44B), almost completely blocked S-phase entry, as indicated by the absence of BrdUrd incorporation (Fig. ​(Fig.5).5). Expression of the PID that inhibited Pak kinase activity without affecting JNK stimulation (Fig. ​(Fig.44B) also arrested proliferation in MDA-MB 435 cells (Fig. ​(Fig.5).5). These experiments emphasize the crucial role of active Rac3 for DNA synthesis in breast cancer cell lines and demonstrate that Pak kinase activity is necessary for Rac3-induced proliferation.

Rac3 mediates proliferation in MDA-MB 435 cells  pq0104939005

Rac3 mediates proliferation in MDA-MB 435 cells pq0104939005

Rac3 mediates proliferation in MDA-MB 435 cells

Figure 5 Rac3 mediates proliferation in MDA-MB 435 cells by a Pak-dependent pathway. MDA-MB 435 cells growing in 0.5% FBS were infected with Semliki Forest virus encoding for LacZ, Rac3N17, Pak1-PBD, Pak1-PBD F107, Pak1-PID, or Rac3wt. After 12 to14 h of protein expression in serum-free medium, 20 μM BrdUrd was added for 20 min before the cells were fixed and stained with anti-Myc antibody and phalloidin for expression (Top) or with FITC-conjugated anti-BrdUrd antibody for BrdUrd incorporation (Lower five micrographs). The presence of bright fluorescent nuclei indicates BrdUrd-positive cells. The percentage was calculated after counting 400 cells in each of four independent experiments.

Our results establish the persistent activation of a small Rho GTPase, Rac3, and the effector kinase Pak in human breast cancer cells. In contrast to Rac1, endogenous Rac3 is localized at the plasma membrane in both guanine nucleotide states. It seems likely that a Rac3 regulatory protein is altered or deleted in highly proliferating cancer cells, and that its specificity toward Rac3 results from the adjacent location of both proteins at the membrane and/or from discrete Rac3 domains, which convey a specific interaction. The cytoskeletal phenotypes of serum-starved breast cancer cells, such as ruffles or lamellipodia typical of Rac1 protein activation, did not seem to correlate with the GDP versus GTP state of endogenous Rac3. This may suggest that Rac family members are specialized in certain cellular functions, as already reported for Rac2 in leukocyte phagocytosis (44) and now demonstrated by us for Rac3 in cancer cell proliferation. Our studies establish further that endogenous, active Rac3 is essential for breast cancer cell proliferation via a Pak-dependent pathway. Paks have been shown to directly phosphorylate Raf kinase, which binds to retinoblastoma protein and regulates its function (45), and to interact with cyclin-dependent kinases to up-regulate cyclin D1 expression (46). Initial screening of various human cancer-derived cell lines revealed the presence of hyperactive Rac3 and Pak kinase in other types of highly proliferating tumors (data not shown). Further investigations, primarily in animal models and clinical settings, will be necessary to assess whether loss of Rac3 and Pak regulation correlates with certain breast tumor stages and is accompanied by specific alterations in cell-cycle regulators. Approaches to inhibit Rac3 or Pak activity would then open a new avenue for cancer therapeutics.

11.1.12 Curcumin-could-reduce-the-monomer-of-ttr-with-tyr114cys-mutation via autophagy in cell model of familial amyloid polyneuropathy.

Li H1Zhang Y1Cao L1Xiong R1Zhang B1Wu L1Zhao Z1Chen SD2
Drug Des Devel Ther. 2014 Oct 31; 8:2121-8

Transthyretin (TTR) familial amyloid polyneuropathy (FAP) is an autosomal dominant inherited neurodegenerative disorder caused by various mutations in the transthyretin gene. We aimed to identify the mechanisms underlying TTR FAP with Tyr114Cys (Y114C) mutation. Our study showed that TTR Y114C mutation led to an increase in monomeric TTR and impaired autophagy. Treatment with curcumin resulted in a significant decrease of monomeric TTR by recovering autophagy. Our research suggests that impairment of autophagy might be involved in the pathogenesis of TTR FAP with Y114C mutation, and curcumin might be a potential therapeutic approach for TTR FAP.

Transthyretin (TTR) familial amyloid polyneuropathy (FAP) is an autosomal dominant inherited disease, characterized clinically by progressive sensory, motor, and autonomic impairment, which typically lead to death around a decade after diagnosis.1 Since the first identification of TTR with Val30Met mutation (TTR V30M), the most common gene mutation in FAP patients, more than 100 TTR mutations have been found to cause FAP.2 However, the detailed pathogenesis underlying TTR FAP remains undefined. Previous studies of the TTR V30M mutant have shown that misfolding and self-aggregation of TTR are implicated in the pathogenesis of TTR FAP involving abnormal endoplasmic reticulum (ER) stress.3

Corresponding to the various TTR gene mutations and a wide range of geographical distributions, FAP presents diverse characteristics in genotype-phenotype in different regions. We have recently published the first report of a TTR Tyr114Cys (TTR Y114C) mutation in a Chinese family with TTR FAP.4 Compared with TTR V30M, the TTR Y114C mutation showed different clinical manifestations, and was also observed in a Japanese family.5,6 This suggests that the pathogenesis of the TTR Y114C and TTR V30M mutations might be different. Studies focused on monomer generation and tetramer depolymerization have been performed.1,2 However, the mechanisms underlying the clearing of the abnormally increased monomer are unknown.

Autophagy is the major lysosomal pathway via which cells degrade intracytoplasmic protein. It is widely accepted that autophagy plays a key role in the process of amyloid deposition in certain neurodegenerative diseases, including alpha-synuclein, beta peptides, tau oligomers, and misfolded prion protein.7 Therefore, autophagy may be involved in degradation of the TTR monomer in TTR FAP.

Curcumin and its analogs have demonstrated a protective effect in many diseases involving antimicrobial, antitubercular,8 and anticancer mechanisms,9 and they can also modulate innate immunity.10 Of note, curcumin has been shown to promote autophagy.11 Therefore, we hypothesized that autophagy might be involved in the pathogenetic mechanism of the TTR Y114C mutation in TTR FAP and curcumin might have potential therapeutic role in this disease. In this study, we aimed to identify the role of autophagy in the pathogenetic mechanism of TTR FAP and to assess the therapeutic effect of curcumin in the disease.

TTR Y114C mutation led to increased monomeric TTR and impaired autophagy in vitro

To investigate the alteration of monomeric TTR with different mutations, we generated HEK293T cell lines with wild-type TTR, TTR Y114C, and stable overexpression of TTR V30M. Wild-type TTR represented the normal control and TTR V30M represented the positive control. Western blotting analysis of the TTR level in the cells when cultured for 24 hours showed that the monomer of TTR Y114C and TTR V30M was increased by approximately 2.3 times and 2.78 times, respectively, compared with wild-type TTR (Figure 1A and B). Mutation of TTR Y114C was related to the increase in monomeric TTR, as well as the mutation of TTR V30M.

Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C dddt-8-2121Fig1

Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C dddt-8-2121Fig1

Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C

Figure 1 Changes in autophagy and endoplasmic reticulum stress related to wild-type TTR, TTR V30M, and TTR Y114C.

Next we investigated the activation of several markers associated with ER stress, including ER-resident chaperone BiP and p-eIF2α. Our results showed the levels of BiP and p-eIF2α is higher in TTR V30M than those in wild-type TTR. In contrast, BiP and p-eIF2α levels in TTR Y114C were similar to those in wild-type TTR (Figure 1A and C), indicating ER stress might not be the main pathogenetic mechanism for the TTR Y114C mutation. We then investigated whether autophagy plays a role in the mechanism of TTR Y114C mutation. LC3-II is well known to be a robust marker of autophagosomes, and immunofluorescent staining of LC3-II can be used to assay for autophagosome formation. A high ratio of LC3-II to LC3-I would indicate induction of autophagy. Our results revealed that the ratio of LC3-II/I was markedly decreased for TTR Y114C, but less suppressed for TTR V30M (Figure 1A and D). Likewise, a significant decrease in LC3-II immunoreactivity was detected in TTR Y114C (Figure 1E). The results of Western blotting and immunofluorescence indicated that autophagy in TTR Y114C was significantly downregulated. Therefore, impaired autophagy might be responsible for the pathogenesis of TTR Y114C mutation.

Curcumin decreased monomeric TTR by promoting autophagy

The effects of curcumin were investigated in TTR Y114C and wild-type TTR stable overexpressed HEK293T cells. Curcumin did not show toxic effects in the stable overexpressed cell lines at curcumin concentrations below 10 µM (Figure 2A and B). We chose 5 µM as the experimental concentration, because it is the minimal effective concentration of curcumin in these cell lines. Further, we wanted to determine whether curcumin could decrease monomeric TTR by promoting autophagy at the minimal effective concentration. Therefore, we used curcumin (2.5 µM and 5 µM) as a protective agent to assess whether it could decrease monomeric TTR with mutation by promoting autophagy. Quantification of LC3-II and LC3-I indicated markedly higher activation of LC3 in TTR Y114C treated with curcumin 5 µM for 24 hours (Figure 2D). In contrast, treatment with curcumin at different concentrations could not activate LC3 in wild-type TTR (Figure 2C, E). We next examined the ratio of monomers to tetramers in TTR Y114C, which was significantly decreased after 24 hours of treatment with 5 µM curcumin compared with no treatment with curcumin (Figure 2D and F). However, for wild-type TTR, the ratio of monomers to tetramers was unchanged after treatment with curcumin (Figure 2C and E). These results indicate that treatment with curcumin 5 µM for 24 hours was able to decrease the monomer in the TTR Y114C mutation by promoting autophagy.

Curcumin decreased monomeric TTR by promoting autophagy dddt-8-2121Fig2

Curcumin decreased monomeric TTR by promoting autophagy dddt-8-2121Fig2

Curcumin decreased monomeric TTR by promoting autophagy

Figure 2 Curcumin decreased monomeric TTR by promoting autophagy.

Protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA

To further validate whether the decrease in monomer by curcumin in our experiments was mediated by autophagy, 3-MA, an inhibitor of autophagosome formation, was implied to negatively regulate autophagy. 3-MA (1 mM) was added to the cell culture medium 2 hours before curcumin and incubated for 24 hours. Analysis of LC3, tetrameric TTR, and monomeric TTR from TTR Y114C revealed that 3-MA partly reversed the LC3 II activation induced by curcumin and increased the monomer of TTR Y114C (Figure 3). These results confirm that curcumin induced the decrease in the TTR Y114C monomer by promoting the autophagy pathway.

Figure 3 Protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA.


TTR FAP is a severe autosomal dominant inherited disease, for which the treatment options are limited. Liver transplantation performed early in the course of the disease is the only therapeutic strategy known to stabilize this neuropathy.1,13 More recently, tafamidis meglumine, a potent inhibitor of misfolding and deposition of mutated TTR, has completed an 18-month, placebo-controlled Phase II/III clinical trial for the treatment of FAP.14 However, in June 2012, the US Food and Drug Administration Peripheral and Central Nervous System Drugs Advisory Committee rejected this drug, stating a lack of convincing data supporting its efficacy.15 Hence, it is important to identify the pathogenetic mechanism of FAP to find an alternative effective treatment strategy.

Accumulating studies focused on the TTR mutation gene and protein have provided insights into the pathogenesis of TTR FAP, including decreased stability of TTR tetramers, conformational change in the crystal structure of variant TTR, altered kinetics of denaturation, and disturbing endoplasmic ER quality control system.1,1618 Previous studies have demonstrated that increased levels of ER stress are correlated with extracellular TTR deposition. Two ER stress markers, BiP and p-eIF2α, have been observed to be present and upregulated in the salivary gland tissue of FAP patients.3 However, the precise molecular mechanisms underlying TTR FAP and its phenotypic heterogeneity are not yet fully understood.

Our current study investigated whether the two mutations, TTR Y114C and TTR V30M, share the same pathogenesis and evaluated the effect of pathogenic mutations on the clearance of the monomer. Our results show that the ratio of LC3-II/I was markedly decreased, while BiP and p-eIF2α levels remained constant in TTR Y114C when compared with wild-type TTR and TTR 30M. The results of our research indicate the impaired autophagy contributed to the TTR Y114C mutation, but not ER stress. This observation indicates that abnormal accumulation of TTR caused by a different mutation might be cleared by different pathways, and more studies are necessary to confirm whether this difference applies to other TTR mutations.

Curcumin is known to have neuroprotective properties through a variety of mechanisms.811 Our research indicates that curcumin decreased the monomeric TTR by promoting autophagy, and without toxic effects. Moreover, this protective effect of curcumin on TTR Y114C could be partially blocked by 3-MA. Pullakhandam et al showed that curcumin binds to wild-type TTR and prevents urea-induced perturbations in the tertiary structure of TTR in vitro.19 Recently, Ferreira et al reported that dietary curcumin modulated TTR amyloidogenicity.20 Therefore, curcumin might be an effective therapy for FAP involving multiple molecular pathways.

Overall, our findings show that abnormal accumulation of TTR caused by different mutations might be cleared in different ways, and curcumin might be an effective therapy for FAP by promoting autophagy. Further studies are necessary to determine whether this phenomenon exists in other TTR mutations.

Stephen Williams, PhD

For PI3K and related inhibitors of PI3K/AKT/mTOR i would refer you to two people who should be in the discussion of this signaling pathway and PI3K/AKT inhibitors used for chemotherapy. The first is Dr. Mien-Chie Hung and the second is Dr. Gordon Mills. They both had been at MD Anderson and developed some of the first inhibitors as well as the earliest discoveries of overactivity of PI3K/AKT in ovarian cancer.
Next the field had never progressed any inhibitors past Stage II as there has been some serious toxicities seen in preclinical phases (most long term tox studies are done after patients are enrolled in phase I).

I would refer to three papers

Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin

A new mutational AKTivation in the PI3K pathway

These will show how inhibitors of certain isoforms of PI3K (namely delta) had to be developed to circumvent some of the severe toxicity seen with the earliest inhibitors (wortmanin and LY294002.

Take your PIK: phosphatidylinositol 3-kinase inhibitors race through the clinic and toward cancer therapy

Targeting the phosphoinositide 3-kinase (PI3K) pathway in cancer

Development of PI3K Inhibitors in Breast Cancer by Aggerwal nice review

Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics will explain about some of the toxicities and describes the one PI3K that has made it to phase II

Most of them have failed and I believe now are being thought as an adjuvant not front line therapy

Aurelian Udristioiu


Aurelian Udristioiu

Lab Director at Emergency County Hospital Targu Jiu

In experimental models, disrupting the MDM2–p53
interaction restored p53 function and sensitized tumors to
chemotherapy or radiotherapy. (Kojima et al., 2005). This
strategy could be particularly beneficial in treating
cancers that do not harbor TP53 mutations. For example
in hematologic malignancies, such as multiple myeloma,
chronic lymphocytic leukemia (CLL), acute lymphoblastic
leukemia (ALL), acute myeloid leukemia (AML), and
Hodgkin’s disease, the induction of p53 – using a small
MDM2-inhibitor molecule, nutlin-3 – can induce the
apoptosis of malignant cells. Nutlins are a group of cisimidazoline
analogs, first identified by Vassilev et al.
(2004), which have a high binding potency and selectivity
for MDM2. Crystallization data have shown that nutlin-3
mimics the three residues of the helical region of the
trans-activation domain of p53 (Phe19, Trp23 and
Leu26), which are conserved across species and critical
for binding to MDM2 (Wade et al., 2010). Nutlin-3
displaces p53 by competing for MDM2 binding. It has
also been found that nutlin-3 potently induces apoptosis
in cell lines derived from hematologic malignancies,
including AML, myeloma, ALL, and B-cell CLL (Secchiero
et al., 2010).

Stephen J Williams, PhD

Now as far as PKM2 you would want to look at a company called Synta Pharmaceuticals and their inhibitor Elesclomal. elesclomol binds copper ions causing a change in conformation that enables its uptake through membranes and into cells. Elesclomol binds copper in an oxidative, positively charged state called Cu(II). Once inside mitochondria, the elesclomol-Cu(II) complex interacts with the energy production mechanism of the cell, or the electron transport chain. This interaction reduces the copper from Cu(II) to Cu(I), resulting in a cascade of reduction-oxidation, or redox, reactions, that causes a rapid increase of oxidative stress, disruption of mitochondrial energy production, and ultimately, triggering of the mitochondrial apoptosis pathway.

The important part is that it seemed, to prefer tumors which had lower LDH activity, meaning that these tumor cells actually did have a more active electron transport chain than tumors with high LDH (Warburg) and therefore in clinical trials the tumors with lower LDH activity responded more favorably. for press release and study results

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Warburg Effect Revisited – 2

Writer and Curator: Larry H. Bernstein, MD, FCAP

Finding Dysregulation in the Cancer Cell

2.1.         Warburg Effect Revisited

One of the great observations of the 20th century was the behavior of cancer cells to proliferate and rely on anaerobic glycolysis for the source of energy.  This was a restatement of the Pasteur effect, described 60 years earlier by the great French scientist in yeast experiments.  The experiments with yeast were again reperformed by Jose EDS Roselino, a Brazilian biochemist, who established an explanation for it 50 years after Warburg.  It is quite amazing the mitochondria were not yet discovered at the time that Warburg carried out the single-cell thickness measurements in his respiratory apparatus. He concluded from the observation that the cancer cells grew in a media that became acidic from producing lactic acid, that the cells were dysfunctional in the utilization of oxygen, as nonmalignant cells efficiently utilized oxygen. He also related the metabolic events to observations made by Meyerhof.  The mitochondria and the citric acid cycle at this time had not yet been discovered, and the latter was, worked out by Hans Krebs and Albert Szent-Gyorgi, both of whom worked with him on mitochondrial metabolism.  The normal cell utilizes glucose efficiently and lipids as well, generating energy through oxidative phosphorylation, with the production of ATP in a manner previously described in these posts.  Greater clarity was achieved with the discovery of Coenzyme A, and finally the electron transport chain (ETC).  This requires that the pyruvate be directed into the tricarboxylic acid cycle and to go through a series of reactions producing succinate and finally malate.

The following great achievements were made with regard to elucidating these processes:

1922 Archibald Vivian Hill United Kingdom “for his discovery relating to the production of heat in the muscle[26]
Otto Fritz Meyerhof Germany “for his discovery of the fixed relationship between the consumption of oxygen and the metabolism of lactic acid in the muscle”[26]
1931 Otto Heinrich Warburg Germany “for his discovery of the nature and mode of action of the respiratory enzyme[34]
1937 Albert Szent-Györgyi von Nagyrapolt Hungary “for his discoveries in connection with the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid[40]
1953 Sir Hans Adolf Krebs United Kingdom “for his discovery of the citric acid cycle[53]
Fritz Albert Lipmann United States “for his discovery of co-enzyme A and its importance for intermediary metabolism”[53]
1955 Axel Hugo Theodor Theorell Sweden “for his discoveries concerning the nature and mode of action of oxidation enzymes”[55]
1978 Peter D. Mitchell United Kingdom “for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory[77]
1997 Paul D. Boyer United States “for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)”[96]
John E. Walker United Kingdom


 1967  Manfred Eigen   and the other half jointly to:

Ronald George Wreyford Norrish and Lord George Porter for their studies of extremely fast chemical reactions, effected by disturbing the equlibrium by means of very short pulses of energy.

1965   FRANÇOIS JACOB , ANDRÉ LWOFF And JACQUES MONOD for their discoveries concerning genetic control of enzyme and virus synthesis.

1964 KONRAD BLOCH And FEODOR LYNEN for their discoveries concerning the mechanism and regulation of the cholesterol and fatty acid metabolism.

If there is a more immediate need for energy (as in stressed muscular activity) with net oxygen insufficiency, the pyruvate is converted to lactic acid, with acidemia, and with much less ATP production, but the lactic academia and the energy deficit is subsequently compensated for.    The observation made by Jose EDS Rosalino was that yeast grown in a soil deficient in oxygen don’t put down roots.

^I. Topisirovic and N. Sonenberg

Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXVI ”A prominent feature of cancer cells is the use of aerobic glycolysis under conditions in which oxygen levels are sufficient to support energy production in the mitochondria (Jones and Thompson 2009; Cairns et al. 2010). This phenomenon, named the “Warburg effect,” after its discoverer Otto Warburg, is thought to fuel the biosynthetic requirements of the neoplastic growth (Warburg 1956; Koppenol et al. 2011) and has recently been acknowledged as one of the hallmarks of cancer (Hanahan and Weinberg 2011). mRNA translation is the most energy-demanding process in the cell (Buttgereit and Brand 1995).

Again, the use of aerobic glycolysis expression has been twisted.”

To understand my critical observation consider this: Aerobic glycolysis is the carbon flow that goes from Glucose to CO2 and water (includes Krens cycle and respiratory chain for the restoration of NAD, FAD etc.

Anerobic glyclysis is the carbon flow that goes from glucose to lactate. It uses conversion of pyruvate to lactate to regenerate NAD.

“Pasteur effect” is an expression coined by Warburg, which refers to the reduction in the carbon flow from glucose when oxygen is offered to yeasts. The major reason for that is in general terms, derived from the fact that carbon flow is regulated by several cell requirements but mainly by the ATP needs of the cell. Therefore, as ATP is generated 10 more efficiently in aerobiosis than under anaerobiosis, less carbon flow is required under aerobiosis than under anaerobiosis to maintain ATP levels. Warburg, after searching for the same regulatory mechanism in normal and cancer cells for comparison found that transformed cell continued their large flow of glucose carbons to lactate despite the presence of oxygen.

So, it is wrong to describe that aerobic glycolysis continues in the presence of oxygen. It is what it is expected to occur. The wrong thing is that anaerobic glycolysis continues under aerobiosis.
^Aurelian Udristioiu (comment)
In cells, the immediate energy sources involve glucose oxidation. In anaerobic metabolism, the donor of the phosphate group is adenosine triphosphate (ATP), and the reaction is catalyzed via the hexokinase or glucokinase: Glucose +ATP-Mg²+ = Glucose-6-phosphate (ΔGo = – 3.4 kcal/mol with hexokinase as the co-enzyme for the reaction.).

In the following step, the conversion of G-6-phosphate into F-1-6-bisphosphate is mediated by the enzyme phosphofructokinase with the co-factor ATP-Mg²+. This reaction has a large negative free energy difference and is irreversible under normal cellular conditions. In the second step of glycolysis, phosphoenolpyruvic acid in the presence of Mg²+ and K+ is transformed into pyruvic acid. In cancer cells or in the absence of oxygen, the transformation of pyruvic acid into lactic acid alters the process of glycolysis.

The energetic sum of anaerobic glycolysis is ΔGo = -34.64 kcal/mol. However a glucose molecule contains 686kcal/mol and, the energy difference (654.51 kcal) allows the potential for un-controlled reactions during carcinogenesis. The transfer of electrons from NADPH in each place of the conserved unit of energy transmits conformational exchanges in the mitochondrial ATPase. The reaction ADP³+ P²¯ + H²à ATP + H2O is reversible. The terminal oxygen from ADP binds the P2¯ by forming an intermediate pentacovalent complex, resulting in the formation of ATP and H2O. This reaction requires Mg²+ and an ATP-synthetase, which is known as the H+-ATPase or the Fo-F1-ATPase complex. Intracellular calcium induces mitochondrial swelling and aging. [12].

The known marker of monitoring of treatment in cancer diseases, lactate dehydrogenase (LDH) is an enzyme that is localized to the cytosol of human cells and catalyzes the reversible reduction of pyruvate to lactate via using hydrogenated nicotinamide deaminase (NADH) as co-enzyme.

The causes of high LDH and high Mg levels in the serum include neoplastic states that promote the high production of intracellular LDH and the increased use of Mg²+ during molecular synthesis in processes pf carcinogenesis (Pyruvate acid>> LDH/NADH >>Lactate acid + NAD), [13].

The material we shall discuss explores in more detail the dysmetabolism that occurs in cancer cells.

Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?

Warburg Effect Revisited

AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo

AKT Signaling Variable Effects

Otto Warburg, A Giant of Modern Cellular Biology

The Metabolic View of Epigenetic Expression

Metabolomics Summary and Perspective

2.1.1       Cancer Metabolism  Oncometabolites: linking altered  metabolism with cancer

Ming Yang, Tomoyoshi Soga, and Patrick J. Pollard
J Clin Invest Sep 2013; 123(9):3652–3658

The discovery of cancer-associated mutations in genes encoding key metabolic enzymes has provided a direct link between altered metabolism and cancer. Advances in mass spectrometry and nuclear magnetic resonance technologies have facilitated high-resolution metabolite profiling of cells and tumors and identified the accumulation of metabolites associated with specific gene defects. Here we review the potential roles of such “oncometabolites” in tumor evolution and as clinical biomarkers for the detection of cancers characterized by metabolic dysregulation.

The emerging interest in metabolites whose abnormal accumulation causes both metabolic and nonmetabolic dysregulation and potential transformation to malignancy (herein termed “oncometabolites”) has been fueled by the identification of cancerassociated mutations in genes encoding enzymes with significant roles in cellular metabolism (1–5). Loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDH) cause the accumulation of fumarate and succinate, respectively (6), whereas gain-offunction isocitrate dehydrogenase (IDH) mutations increase levels of D–2-hydroxyglutarate (D-2HG) (7, 8). These metabolites have been implicated in the dysregulation of cellular processes including the competitive inhibition of α-ketoglutarate–dependent (α-KG–dependent) dioxygenase enzymes (also known as 2-oxoglutarate–dependent dioxgenases) and posttranslational modification of proteins (1, 4, 9–11). To date, several lines of biochemical and genetic evidence support roles for fumarate, succinate, and D-2HG in cellular transformation and oncogenesis (3, 12).

The Journal of Clinical Investigation   Volume 123   Number 9   September 2013

ventional gene sequencing methods may lead to false positives due to genetic polymorphism and sequencing artifacts (98). In comparison, screening for elevated 2HG levels is a sensitive and specific approach to detect IDH mutations in tumors. Whereas patient sera/plasma can be assessed in the case of AML (7, 8, 21, 99), exciting advances with proton magnetic resonance spectroscopy (MRS) have been made in the noninvasive detection of 2HG in patients with gliomas (100–103). Using MRS sequence optimization and spectral fitting techniques, Maher and colleagues examined 30 patients with glioma and showed that the detection of 2HG correlated 100% with the presence of IDH1 or IDH2 mutations (102). Andronesi et al. further demonstrated that two-dimensional correlation spectroscopy could effectively distinguish 2HG from chemically similar metabolites present in the brain (103). Negative IHC staining for SDHB correlates with the presence of SDH mutations, whether in SDHB, SDHC, or SDHD (104). This finding is most likely explained by the fact that mutations in any of the four subunits of SDH can destabilize the entire enzyme complex. PGLs/PCCs associated with an SDHA mutation show negative staining for SDHA as well as SDHB (105). Therefore, IHC staining for SDHB is a useful diagnostic tool to triage patients for genetic testing of any SDH mutation, and subsequent staining for the other subunits may further narrow the selection of genes to be tested. In contrast, detection of FH protein is often evident in HLRCC tumors due to retention of the nonfunctional mutant allele (106). However, staining of cysts and tumors for 2SC immunoreactivity reveals a striking correlation between FH inactivation and the presence of 2SC-modified protein (2SCP), which is absent in non-HLRCC tumors and normal tissue controls (106). IHC staining for 2SCP thus provides a robust diagnostic biomarker for FH deficiency (107).

Therapeutic targeting Because D-2HG is a product of neomorphic enzyme activities, curtailing the D-2HG supply by specifically inhibiting the mutant IDH enzymes provides an elegant approach to target IDH-mutant cancers. Indeed, recent reports of small-molecule inhibitors against mutant forms of IDH1 and IDH2 demonstrated the feasibility of this method. An inhibitor against IDH2 R140Q was shown to reduce both intracellular and extracellular levels of D-2HG, suppress cell growth, and increase differentiation of primary human AML cells (108). Similarly, small-molecule inhibition of IDH1 R132H suppressed colony formation and increased tumor cell differentiation in a xenograft model for IDH1 R132H glioma (58). The inhibitors exhibited a cytostatic rather than cytotoxic effect, and therefore their therapeutic efficacy over longer time periods may need further assessment (109). Letouzé et al. showed that the DNA methytransferase inhibitor decitabine could repress the migration capacities of SDHB-mutant cells (40). However, for SDH- and FH-associated cancers, a synthetic lethality approach is worth exploring because of the pleiotrophic effects associated with succinate and fumarate accumulation.

Outlook The application of next-generation sequencing technologies in the field of cancer genomics has substantially increased our understanding of cancer biology. Detection of germline and somatic mutations in specific tumor types not only expands the current repertoire of driver mutations and downstream effectors in tumorigenesis, but also sheds light on how oncometabolites may exert their oncogenic roles. For example, the identification of mutually exclusive mutations in IDH1 and TET2 in AML led to the characterization of TET2 as a major pathological target of D-2HG (34, 110). Additionally, the discovery of somatic CUL3, SIRT1, and NRF2 mutations in sporadic PRCC2 converges with FH mutation in HLRCC, in which NRF2 activation is a consequence of fumarate-mediated succination of KEAP1, indicating the functional prominence of the NRF2 pathway in PRCC2 (73). In light of this, the identification of somatic mutations in genes encoding the chromatin-modifying enzymes histone H3K36 methyltransferase (SETD2), histone H3K4 demethylase JARID1C (KDM5C), histone H3K27 demethylase UTX (KDM6A), and the SWI/SNF chromatin remodelling complex gene PBRM1 in clear cell renal cell carcinoma (111–113) highlights the importance of epigenetic modulation in human cancer and raises the potential for systematic testing in other types of tumors such as those associated with FH mutations. Technological advances such as those in gas and liquidchromatography mass spectrometry (114, 115) and nuclear magnetic resonance imaging (102) have greatly improved the ability to measure low-molecular-weight metabolites in tumor samples with high resolution (116). Combined with metabolic flux analyses employing isotope tracers and mathematical modeling, modern-era metabolomic approaches can provide direct pathophysiological insights into tumor metabolism and serve as an excellent tool for biomarker discovery. Using a data-driven approach, Jain and colleagues constructed the metabolic profiles of 60 cancer cell lines and discovered glycine consumption as a key metabolic event in rapidly proliferating cancer cells (117), thus demonstrating the power of metabolomic analyses and the relevance to future cancer research and therapeutics.

Acknowledgments The Cancer Biology and Metabolism Group is funded by Cancer Research UK and the European Research Council under the European Community’s Seventh Framework Programme (FP7/20072013)/ERC grant agreement no. 310837 to Dr. Pollard. Professor Soga receives funding from a Grant-in-Aid for scientific research on Innovative Areas, Japan (no. 22134007), and the Yamagata Prefectural Government and City of Tsuruoka.

Address correspondence to: Patrick J. Pollard, Cancer Biology and Metabolism Group, Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom. Phone: 44.0.1865287780; Fax: 44.0.1865287787; E-mail:

  1. Yang M, Soga T, Pollard PJ, Adam J. The emerging role of fumarate as an oncometabolite. Front Oncol. 2012;2:85. 2. Ward PS, Thompson CB. Metabolic reprogramming: a cancer hallmark even warburg did not anticipate. Cancer Cell. 2012;21(3):297–308. 3. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009; 324(5930):1029–1033. 4. Thompson CB. Metabolic enzymes as oncogenes or tumor suppressors. N Engl J Med. 2009; 360(8):813–815. 5. Schulze A, Harris AL. How cancer metabolism is tuned for proliferation and vulnerable to disruption. Nature. 2012;491(7424):364–373.
  1. Pollard PJ, et al. Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations. Hum Mol Genet. 2005; 14(15):2231–2239. 7. Ward PS, et al. The common feature of leukemiaassociated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell. 2010; 17(3):225–234.

Because D-2HG is a product of neomorphic enzyme activities, curtailing the D-2HG supply by specifically inhibiting the mutant IDH enzymes provides an elegant approach to target IDH-mutant cancers. Indeed, recent reports of small-molecule inhibitors against mutant forms of IDH1 and IDH2 demonstrated the feasibility of this method. An inhibitor against IDH2 R140Q was shown to reduce both intracellular and extracellular levels of D-2HG, suppress cell growth, and increase differentiation of primary human AML cells (108). Similarly, small-molecule inhibition of IDH1 R132H suppressed colony formation and increased tumor cell differentiation in a xenograft model for IDH1 R132H glioma (58). The inhibitors exhibited a cytostatic rather than cytotoxic effect, and therefore their therapeutic efficacy over longer time periods may need further assessment (109). Letouzé et al. showed that the DNA methytransferase inhibitor decitabine could repress the migration capacities of SDHB-mutant cells (40). However, for SDH- and FH-associated cancers, a synthetic lethality approach is worth exploring because of the pleiotrophic effects associated with succinate and fumarate accumulation.

Technological advances such as those in gas and liquid chromatography mass spectrometry (114, 115) and nuclear magnetic resonance imaging (102) have greatly improved the ability to measure low-molecular-weight metabolites in tumor samples with high resolution (116). Combined with metabolic flux analyses employing isotope tracers and mathematical modeling, modern-era metabolomic approaches can provide direct pathophysiological insights into tumor metabolism and serve as an excellent tool for biomarker discovery. Using a data-driven approach, Jain and colleagues constructed the metabolic profiles of 60 cancer cell lines and discovered glycine consumption as a key metabolic event in rapidly proliferating cancer cells (117), thus demonstrating the power of metabolomic analyses and the relevance to future cancer research and therapeutics.

Figure 1 D-2HG produced by mutant IDH1/2 affects metabolism and epigenetics by modulating activities of α-KG–dependent oxygenases. Wild-type IDH1 and IDH2 catalyze the NADP+-dependent reversible conversion of isocitrate to α-KG, whereas cancer-associated gain-of-function mutations enable mutant IDH1/2 (mIDH1/2) to catalyze the oxidation of α-KG to D-2HG, using NADPH as a cofactor. Because D-2HG is structurally similar to α-KG, its accumulation can modulate the activities of α-KG–utilizing dioxygenases. Inhibition of 5mC hydroxylase TET2 and the KDMs results in increased CpG island methylation and increased histone methylation marks, respectively, thus blocking lineage-specific cell differentiation. Inhibition of collagen prolyl and lysyl hydroxylases (C-P4Hs and PLODs, respectively) leads to impaired collagen maturation and disrupted basement membrane formation. D-2HG can also stimulate the activities of HIF PHDs, leading to enhanced HIF degradation and a diminished HIF response, which are associated with increased soft agar growth of human astrocytes and growth factor independence of leukemic cells. Together these processes exert pleiotrophic effects on cell signaling and gene expression that probably contribute to the malignancy of IDH1/2-mutant cells.
Figure 2 Candidate oncogenic mechanisms of succinate and fumarate accumulation. SDH and FH are Krebs cycle enzymes and tumor suppressors. Loss-of-function mutations in SDH and FH result in abnormal accumulation of Krebs cycle metabolites succinate (Succ) and fumarate (Fum), respectively, both of which can inhibit the activities of α-KG–dependent oxygenases. Inhibition of HIF PHDs leads to activation of HIF-mediated pseudohypoxic response, whereas inhibition of KDMs and TET family of 5mC hydroxylases causes epigenetic alterations. Fumarate is electrophilic and can also irreversibly modify cysteine residues in proteins by succination. Succination of KEAP1 in FH deficiency results in the constitutive activation of the antioxidant defense pathway mediated by NRF2, conferring a reductive milieu that promotes cell proliferation. Succination of the Krebs cycle enzyme Aco2 impairs aconitase activity in Fh1-deficient MEFs. Fumarate accumulation may also affect cytosolic pathways by inhibiting the reactions involved in the biosynthesis of arginine and purine. AcCoA, acetyl CoA; Mal, malate; OAA, oxaloacetate; Succ-CA, succinyl CoA. Emerging concepts: linking hypoxic signaling and cancer metabolism.

Lyssiotis CA, Vander-Heiden MG, Muñoz-Pinedo C, Emerling BM.
Cell Death Dis. 2012 May 3; 3:e303

The Joint Keystone Symposia on Cancer and Metabolism and Advances in Hypoxic Signaling: From Bench to Bedside were held in Banff, Alberta, Canada from 12 to 17 February 2012. Drs. Reuben Shaw and David Sabatini organized the Cancer and Metabolism section, and Drs. Volker Haase, Cormac Taylor, Johanna Myllyharju and Paul Schumacker organized the Advances in Hypoxic Signaling section. Accumulating data illustrate that both hypoxia and rewired metabolism influence cancer biology. Indeed, these phenomena are tightly coupled, and a joint meeting was held to foster interdisciplinary interactions and enhance our understanding of these two processes in neoplastic disease. In this report, we highlight the major themes of the conference paying particular attention to areas of intersection between hypoxia and metabolism in cancer.

One opening keynote address was delivered by Craig Thompson (Memorial Sloan-Kettering, USA), in which he provided a comprehensive perspective on the current thinking around how altered metabolism supports cancer cell growth and survival, and discussed areas likely to be important for future discovery. In particular, Thompson highlighted the essential roles of glucose and glutamine in cell growth, how glucose- and glutamine-consuming processes are rewired in cancer and how this rewiring facilitates anabolic metabolism. These topics were at the core of many of the metabolism presentations that described in detail how some metabolic alterations contribute to the properties of transformed cells.

The other keynote address was delivered by Peter Ratcliffe (University of Oxford, UK), in which he provided a historical perspective on the progress of how signaling events sense oxygen. Mammals have evolved multiple acute and long-term adaptive responses to low oxygen levels (hypoxia). This response prevents a disparity in ATP utilization and production that would otherwise result in a bioenergetic collapse when oxygen level is low. Multiple effectors have been proposed to mediate the response to hypoxia including prolyl hydroxylases, AMPK, NADPH oxidases and the mitochondrial complex III. Currently, however, the precise mechanism by which oxygen is sensed in various physiological contexts remains unknown. Indeed, this was an active point of debate, with Peter Ratcliffe favoring the prolyl hydroxylase PHD2 as the primary cellular oxygen sensor.

Anabolic glucose metabolism and the Warburg effect

Nearly a century ago, Warburg noted that cancer tissues take up glucose in excess than most normal tissues and secrete much of the carbon as lactate. Recently, headway has been made toward determining how the enhanced glucose conversion to lactate occurs and contributes to cell proliferation and survival. Heather Christofk (University of California, Los Angeles, USA) and John Cleveland (the Scripps Research Institute, USA) described a role for the lactate/pyruvate transporter MCT-1 in carbon secretion, and suggested that blocking lactate or pyruvate transport may be a strategy to target glucose metabolism in cancer cells. Kun-Liang Guan (University of California, San Diego, USA) described a novel feedback loop to control glucose metabolism in highly glycolytic cells. Specifically, he discussed how glucose-derived acetyl-CoA can be used as a substrate to modify two enzymes involved in glucose metabolism, pyruvate kinase M2 (PKM2) and phosphoenolpyruvate carboxylase (PEPCK). In both cases, acetylation leads to protein degradation and decreased glycolysis and gluconeogenesis, respectively. Data presented from Matthew Vander Heiden’s laboratory (Koch Institute/MIT, USA) illustrated that loss of pyruvate kinase activity can accelerate tumor growth, suggesting that the regulation of glycolysis may be more complex than previously appreciated. Almut Schulze (London Research Institute, UK) discussed a novel regulatory role for phosphofructokinase in controlling glucose metabolism and Jeffrey Rathmell (Duke University, USA) discussed parallels between glucose metabolism in cancer cells and lymphocytes that suggest many of these phenotypes could be a feature of rapidly dividing cells.

Glutamine addiction

Cancer cells also consume glutamine to support proliferation and survival. Alfredo Csibi (Harvard Medical School, USA) described how mTORC1 promotes glutamine utilization by indirectly regulating the activity of glutamate dehydrogenase. This work united two major themes at the meeting, mTOR signaling and glutamine metabolism, highlighting the interconnectedness of signal transduction and metabolic regulation. Richard Cerione (Cornell University, USA) described a small molecule inhibitor of glutaminase that can be used to target glutamine-addicted cancer cells. Christian Metallo (University of California, San Diego, USA), Andrew Mullen (University of Texas Southwestern Medical School, USA) and Patrick Ward (Memorial Sloan-Kettering, USA) presented data demonstrating that the carbon skeleton of glutamine can be incorporated into newly synthesized lipids. This contribution of glutamine to lipid synthesis was most pronounced in hypoxia or when the mitochondrial electron transport chain was compromised.

Signal transduction and metabolism

The protein kinases AMPK and mTOR can function as sensors of metabolic impairment, whose activation by energy stress controls multiple cellular functions. Grahame Hardie (University of Dundee, UK) and Reuben Shaw (Salk Institute, USA) highlighted novel roles for AMPK, including inhibition of viral replication, and the control of histone acetylation via phosphorylation of class IIa HDACs, respectively. Brandon Faubert (McGill University, USA) reported on an AMPK-dependent effect on glucose metabolism in unstressed cells. Brendan Manning (Harvard Medical School, USA) found that chronic activation of mTOR in the mouse liver, due to genetic ablation of this complex, promotes the development of liver cancer. Kevin Williams (University of California, Los Angeles, USA) discussed how growth signaling can control both lipid and glucose metabolism by impinging on SREBP-1, a transcription factor downstream of mTOR. AMPK-independent control of mTOR was addressed by John Blenis (Harvard Medical School, USA), who discussed the possible role of mTOR stabilizing proteins as mediators of mTOR inactivation upon energetic stress. David Sabatini (Whitehead Institute/MIT, USA) discussed several aspects of amino-acid sensing by Rag GTPases and showed that constitutive activation of the Rag GTPases leads to metabolic defects in mice.

One of the outcomes of AMPK activation and mTOR inhibition is autophagy, which can provide amino acids and fatty acids to nutrient-deprived cells. Ana Maria Cuervo (Albert Einstein College of Medicine, USA) and Eileen White (Rutgers University, USA) illuminated the role of chaperone-mediated autophagy (CMA) and macroautophagy, respectively, in tumor survival. White described a role for macroautophagy in the regulation of mitochondrial fitness, maintenance of TCA cycle and tumorigenesis induced by oncogenic Ras. Cuervo described how CMA is consistently elevated in tumor cells, and how its inactivation leads to metabolic impairment via p53-mediated downregulation of glycolytic enzymes.

Oncogene-specific changes to metabolism

Lewis Cantley (Harvard Medical School, USA) described a metabolic role for oncogenic Kras in the rewiring of glucose metabolism in pancreatic cancer. Specifically, Myc-mediated transcription (downstream of MEK-ERK signaling) both enhances glucose uptake and diverts glucose carbon into the nonoxidative pentose phosphate pathway to facilitate nucleotide biosynthesis. Alejandro Sweet-Cordero (Stanford University, USA) described how oncogenic Kras increases glycolysis and represses mitochondrial respiration (via decreased pyruvate dehydrogenase phosphatase 1 (PDP1) expression) in colon cancer. While these studies indicate that hyperstimulation of the Erk pathway suppresses PDH flux through suppression of PDP1, Joan Brugge (Harvard Medical School, USA) described studies showing that reduction of Erk signaling in normal epithelial cells also causes suppression of PDH flux, in this case through loss of repression of PDK4. The seemingly contradictory nature of these results highlighted an important theme emphasized throughout the week-long conference—that cellular context has an important role in shaping how oncogenic mutations or pathway activation rewires metabolism.

Targeting cancer metabolism

There was extensive discussion around targeting metabolism for cancer therapy. Metformin and phenformin, which act in part by mitochondrial complex I inhibition, can activate AMPK and influence cancer cell metabolism. Kevin Struhl (Harvard Medical School, USA) described how metformin can selectively target cancer stem cells, whereas Jessica Howell (Harvard Medical School, USA) described how the therapeutic activity of metformin relies on both AMPK and mTOR signaling to mediate its effect. Similarly, David Shackelford (University of California, Los Angeles, USA) demonstrated efficacy for phenformin in LKB1-deficient mouse models.

Several presentations, including those by Taru Muranen (Harvard Medical School, USA), Karen Vousden and Eyal Gottlieb (both from the Beatson Institute for Cancer Research, UK), provided insight into genetic control mechanisms that cancer cells use to promote survival under conditions of increased biosynthesis. As an example, Vousden illustrated how p53 loss can make cancer cells more dependent on exogenous serine. Several additional presentations, including those by Gottlieb, Richard Possemato (Whitehead Institute/MIT, USA), Michael Pollak (McGill University, USA) and Kevin Marks (Agios Pharmaceuticals, USA), also included data highlighting the important role of serine biosynthesis and metabolism in cancer growth. Collectively, these data highlight a metabolic addiction that may be therapeutically exploitable. Similarly, Cristina Muñoz-Pinedo (Institut d’Investigació Biomèdica, Spain) described how mimicking glucose deprivation with 2-deoxyglucose can cause programmed cell death and may be an effective cancer treatment.

Regulation of hypoxic responses

Peter Carmeliet (University of Leuven, Belgium) highlighted the mechanisms of resistance against VEGF-targeted therapies. Roland Wenger (University of Zurich, Switzerland) discussed the oxygen-responsive transcriptional networks and, in particular, the difference between the transcription factors HIF-1α and HIF-2α. Importantly, he demonstrated a rapid role for HIF-1α, and a later and more persistent response for HIF-2α. These results were central to a recurrent theme calling for the distinction of HIF-1α and HIF-2α target genes and how these responses mediate divergent hypoxic adaptations.

Advances in hypoxic signaling

Brooke Emerling (Harvard Medical School, USA) introduced CUB domain-containing protein 1 (CDCP1) and showed persuasive data on CDCP1 being a HIF-2α target gene involved in cell migration and metastasis, and suggested CDCP1 regulation as an attractive therapeutic target. Johannes Schodel (University of Oxford, UK) described an elegant HIF-ChIP-Seq methodology to define direct transcriptional targets of HIF in renal cancer.

Randall Johnson (University of Cambridge, UK) emphasized that loss of HIF-1α results in decreased lung metastasis. Lorenz Poellinger (Karolinska Institutet, Sweden) focused on how hypoxia can alter the epigenetic landscape of cells, and furthermore, how the disruption of the histone demethylase JMJD1A and/or the H3K9 methyltransferase G9a has opposing effects on tumor growth and HIF target gene expression.

Paul Schumacker (Northwestern University, USA) further emphasized the importance of mitochondrial ROS signaling under hypoxic conditions showing that ROS could be detected in the inter-membrane space of the mitochondria before activating signaling cascades in the cytosol. He also presented evidence for mitochondria as a site of oxygen sensing in diverse cell types. Similarly, Margaret Ashcroft (University College London, UK) argued for a critical role of mitochondria in hypoxic signaling. She presented on a family of mitochondrial proteins (CHCHD4) that influence hypoxic signaling and tumorigenesis and suggested that CHCHD4 is important for HIF and tumor progression.  Glutaminolysis: supplying carbon or nitrogen or both for cancer cells?

Dang CV
Cell Cycle. 2010 Oct 1; 9(19):3884-6

A cancer cell comprising largely of carbon, hydrogen, oxygen, phosphorus, nitrogen and sulfur requires not only glucose, which is avidly transported and converted to lactate by aerobic glycolysis or the Warburg effect, but also glutamine as a major substrate. Glutamine and essential amino acids, such as methionine, provide energy through the TCA cycle as well as nitrogen, sulfur and carbon skeletons for growing and proliferating cancer cells. The interplay between utilization of glutamine and glucose is likely to depend on the genetic make-up of a cancer cell. While the MYC oncogene induces both aerobic glycolysis and glutaminolysis, activated β-catenin induces glutamine synthesis in hepatocellular carcinoma. Cancer cells that have elevated glutamine synthetase can use glutamate and ammonia to synthesize glutamine and are hence not addicted to glutamine. As such, cancer cells have many degrees of freedom for re-programming cell metabolism, which with better understanding will result in novel therapeutic approaches.

Figure 1. Glutamine, glucose and glutamate are imported into the cytoplasm of a cell. Glucose is depicted to be converted primarily (large powder blue arrow) to lactate via aerobic glycolysis or the Warburg effect or channeled into the mitochondrion as pyruvate and converted to acetyl-CoA for oxidation. Glutamine is shown imported and used for different processes including glutaminolysis, which involves the conversion of glutamine to glutamate and ammonia by glutaminase (GLS). Glutamate is further oxidized via the TCA cycle to produce ATP and contribute anabolic carbon skeletons. Some cells can import glutamate and use ammonia to generate glutamine through glutamine synthetase (GLUL); glutamine could then be used for different purposes including glutathione synthesis (not shown).

The liver is organized into lobules, which have zones of cells around the perivenous region enriched with glutamine synthetase, which detoxifies ammonia by converting it to glutamine through the amination of glutamate (Fig. 1). As such, liver cancers vary in the degree of glutamine synthetase expression depending on the extent of anaplasia or de-differentiation. Highly undifferentiated liver cancers tend to be more glycolytic than those that retain some of the differentiated characteristics of liver cells. Furthermore, glutamine synthetase (considered as a direct target of activated β-catenin, which also induces ornithine aminotransferase and glutamate transporters) expression in liver cancers has been directly linked to β-catenin activation or mutations.  Hence, the work by Meng et al. illustrates, first and foremost, the metabolic heterogeneity amongst cancer cell lines, such that the ability to utilize ammonia instead of glutamine by Hep3B cells depends on the expression of glutamine synthetase. The Hep3B cells are capable of producing glutamine from glutamate and ammonia, as suggested by the observation that a glutamine-independent derivative of Hep3B has high expression of glutamine synthetase. In this regard, Hep3B could utilize glutamate directly for the production of α-ketoglutarate or to generate glutamine for protein synthesis or other metabolic processes, such as to import essential amino acids.  In contrast to Hep3B, other cell lines in the Meng et al. study were not demonstrated to be glutamine independent and thus become ammonia auxotrophs. Hence, the mode of glutamine or glucose utilization is dependent on the metabolic profile of cancer cells.
The roles of glutamine in different cancer cell lines are likely to be different depending on their genetic and epigenetic composition. In fact, well-documented isotopic labeling studies have demonstrated a role for glutamine to provide anapleurotic carbons in certain cancer and mammalian cell types. But these roles of glutaminolysis, whether providing nitrogen or anabolic carbons, should not be generalized as mutually exclusive features of all cancer cells. From these considerations, it is surmised that the expression of glutamine synthetase in different cancers will determine the extent by which these cancers are addicted to exogenous glutamine.  The Warburg effect and mitochondrial stability in cancer cells

Gogvadze V, Zhivotovsky B, Orrenius S.
Mol Aspects Med. 2010 Feb; 31(1):60-74

The last decade has witnessed a renaissance of Otto Warburg’s fundamental hypothesis, which he put forward more than 80 years ago, that mitochondrial malfunction and subsequent stimulation of cellular glucose utilization lead to the development of cancer. Since most tumor cells demonstrate a remarkable resistance to drugs that kill non-malignant cells, the question has arisen whether such resistance might be a consequence of the abnormalities in tumor mitochondria predicted by Warburg. The present review discusses potential mechanisms underlying the upregulation of glycolysis and silencing of mitochondrial activity in cancer cells, and how pharmaceutical intervention in cellular energy metabolism might make tumor cells more susceptible to anti-cancer treatment.

mitochondrial stabilization gr1

mitochondrial stabilization gr1

Fig. 1. (1) Oligomerization of Bax is mediated by the truncated form of the BH3-only, pro-apoptotic protein Bid (tBid); (2) Bcl-2, Bcl-XL, Mcl-1, and Bcl-w, interact with the pro-apoptotic proteins, Bax and Bak, to prevent their oligomerization; (3) The anti-apoptotic protein Bcl-XL prevents tBid-induced closure of VDAC and apoptosis by maintaining VDAC in open configuration allowing ADT/ATP exchange and normal mitochondrial functioning; (4) MPT pore is a multimeric complex, composed of VDAC located in the OMM, ANT, an integral protein of the IMM, and a matrix protein, CyPD; (5) Interaction with VDAC allows hexokinase to use exclusively intramitochondrial ATP to phosphorylate glucose, thereby maintaining high rate of glycolysis.

mitochodrial stabilization gr2

mitochodrial stabilization gr2

Fig. 2. Different sites of therapeutic intervention in cancer cell metabolism. (1) The non-metabolizable analog of glucose, 2-deoxyglucose, decreases ATP level in the cell; (2) 3-bromopyruvate suppresses the activity of hexokinase, and respiration in isolated mitochondria; (3) Phloretin a glucose transporter inhibitor, decreases ATP level in the cell and markedly enhances the anti-cancer effect of daunorubicin; (4) Dichloroacetate (DCA) shifts metabolism from glycolysistoglucoseoxidation;(5)Apoptolidin,aninhibitorofmitochondrialATPsynthase,inducescelldeathindifferentmalignantcelllineswhenapplied together with the LDH inhibitor oxamate (6).

Warburg Symposium Oxidative phosphorylation in cancer cells

Giancarlo Solaini Gianluca SgarbiAlessandra Baracca

BB Acta – Bioenergetics 2011 Jun; 1807(6): 534–542

Research Highlights

►Mitochondrial hallmarks of tumor cells.►Complex I of the respiratory chain is reduced in many cancer cells.►Oligomers of F1F0ATPase are reduced in cancer cells.►Mitochondrial membranes are critical to the life or death of cancer cells.

Evidence suggests that mitochondrial metabolism may play a key role in controlling cancer cells life and proliferation. Recent evidence also indicates how the altered contribution of these organelles to metabolism and the resistance of cancer mitochondria against apoptosis-associated permeabilization are closely related. The hallmarks of cancer growth, increased glycolysis and lactate production in tumours, have raised attention due to recent observations suggesting a wide spectrum of oxidative phosphorylation deficit and decreased availability of ATP associated with malignancies and tumour cell expansion. More specifically, alteration in signal transduction pathways directly affects mitochondrial proteins playing critical roles in controlling the membrane potential as UCP2 and components of both MPTP and oxphos complexes, or in controlling cells life and death as the Bcl-2 proteins family. Moreover, since mitochondrial bioenergetics and dynamics, are also involved in processes of cells life and death, proper regulation of these mitochondrial functions is crucial for tumours to grow. Therefore a better understanding of the key pathophysiological differences between mitochondria in cancer cells and in their non-cancer surrounding tissue is crucial to the finding of tools interfering with these peculiar tumour mitochondrial functions and will disclose novel approaches for the prevention and treatment of malignant diseases. Here, we review the peculiarity of tumour mitochondrial bioenergetics and the mode it is linked to the cell metabolism, providing a short overview of the evidence accumulated so far, but highlighting the more recent advances. This article is part of a Special Issue entitled: Bioenergetics of Cancer.

Mitochondria are essential organelles and key integrators of metabolism, but they also play vital roles in cell death and cell signaling pathways critically influencing cell fate decisions [1][2] and [3]. Mammalian mitochondria contain their own DNA (mtDNA), which encodes 13 polypeptides of oxidative phosphorylation complexes, 12S and 16S rRNAs, and 22 tRNAs required for mitochondrial function [4]. In order to synthesize ATP through oxidative phosphorylation (oxphos), mitochondria consume most of the cellular oxygen and produce the majority of reactive oxygen species (ROS) as by-products [5]. ROS have been implicated in the etiology of carcinogenesis via oxidative damage to cell macromolecules and through modulation of mitogenic signaling pathways [6][7] and [8]. In addition, a number of mitochondrial dysfunctions of genetic origin are implicated in a range of age-related diseases, including tumours [9]. How mitochondrial functions are associated with cancer is a crucial and complex issue in biomedicine that is still unravelled [10] and [11], but it warrants an extraordinary importance since mitochondria play a major role not only as energy suppliers and ROS “regulators”, but also because of their control on cellular life and death. This is of particular relevance since tumour cells can acquire resistance to apoptosis by a number of mechanisms, including mitochondrial dysfunction, the expression of anti-apoptotic proteins or by the down-regulation or mutation of pro-apoptotic proteins [12].

Cancer cells must adapt their metabolism to produce all molecules and energy required to promote tumor growth and to possibly modify their environment to survive. These metabolic peculiarities of cancer cells are recognized to be the outcome of mutations in oncogenes and tumor suppressor genes which regulate cellular metabolism. Mutations in genes including P53, RAS, c-MYC, phosphoinosine 3-phosphate kinase (PI3K), and mTOR can directly or through signaling pathways affect metabolic pathways in cancer cells as discussed in several recent reviews [13][14][15][16] and [17]. Cancer cells harboring the genetic mutations are also able to thrive in adverse environments such as hypoxia inducing adaptive metabolic alterations which include glycolysis up-regulation and angiogenesis factor release [18] and [19]. In response to hypoxia, hypoxia-induced factor 1 (HIF-1) [20], a transcription factor, is up-regulated, which enhances expression of glycolytic enzymes and concurrently it down regulates mitochondrial respiration through up-regulation of pyruvate dehydrogenase kinase 1 (PDK1) (see recent reviews [21] and [22]). However, several tumours have been reported to display high HIF-1 activity even in normoxic condition, now referred to as pseudohypoxia [23][24] and [25]. In addition, not only solid tumours present a changed metabolism with respect to matched normal tissues, hematological cell malignancies also are characterized by peculiar metabolisms, in which changes of mitochondrial functions are significant [26],[27] and [28], therefore indicating a pivotal role of mitochondria in tumours independently from oxygen availability.

Collectively, actual data show a great heterogeneity of metabolism changes in cancer cells, therefore comprehensive cellular and molecular basis for the association of mitochondrial bioenergetics with tumours is still undefined, despite the numerous studies carried out. This review briefly revisits the data which are accumulating to account for this association and highlights the more recent advances, particularly focusing on the metabolic and structural changes of mitochondria.

Mitochondria-related metabolic changes of cancer cells

Accumulating evidence indicate that many cancer cells have an higher glucose consumption under normoxic conditions with respect to normal differentiated cells, the so-called “aerobic glycolysis” (Warburg effect), a phenomenon that is currently exploited to detect and diagnose staging of solid and even hematological malignancies [27]. Since the initial publication by Otto Warburg over half a century ago [29], an enormous amount of studies on many different tumours have been carried out to explain the molecular basis of the Warburg effect. Although the regulatory mechanisms underlying aerobic and glycolytic pathways of energy production are complex, making the prediction of specific cellular responses rather difficult, the actual data seem to support the view that in order to favour the production of biomass, proliferating cells are commonly prone to satisfy the energy requirement utilizing substrates other than the complete oxidation of glucose (to CO2 and H2O). More precisely, only part (40 to 75%, according to [30]) of the cells need of ATP is obtained through the scarcely efficient catabolism of glucose to pyruvate/lactate in the cytoplasm and the rest of the ATP need is synthesized in the mitochondria through both the tricarboxylic acid (TCA) cycle (one ATP produced each acetyl moiety oxidized) and the associated oxidative phosphorylation that regenerates nicotinamide- and flavin-dinucleotides in their oxidized state(NAD+ and FAD). This might be due to the substrate availability as it was shown in HeLa cells, where replacing glucose with galactose/glutamine in the culture medium induced increased expression of oxphos proteins, suggesting an enhanced energy production from glutamine [31]. As a conclusion the authors proposed that energy substrate can modulate mitochondrial oxidative capacity in cancer cells. A direct evidence of this phenomenon was provided a few years later in glioblastoma cells, in which it was demonstrated that the TCA cycle flux is significantly sustained by anaplerotic alfa-ketoglutarate produced from glutamine and by acetyl moieties derived from the pyruvate dehydrogenase reaction where pyruvate may have an origin other than glucose [32]. The above changes are the result of genetic alteration and environmental conditions that induce many cancer cells to change their metabolism in order to synthesize molecules necessary to survive, grow and proliferate, including ribose and NADPH to synthesize nucleotides, and glycerol-3 phosphate to produce phospholipids. The synthesis of the latter molecules requires major amount of acetyl moieties that are derived from beta-oxidation of fatty acids and/or from cytosolic citrate (citrate lyase reaction) and/or from the pyruvate dehydrogenase reaction. Given the important requirement for NADPH in macromolecular synthesis and redox control, NADPH production in cancer cells besides being produced through the phosphate pentose shunt, may be significantly sustained by cytosolic isocitrate dehydrogenases and by the malic enzyme (see Ref. [33] for a recent review). Therefore, many cancer cells tend to have reduced oxphos in the mitochondria due to either or both reduced flux within the tricarboxylic acid cycle and/or respiration (Fig. 1). The latter being also caused by reduced oxygen availability, a typical condition of solid tumors, that will be discussed below.

Schematic illustration of mitochondrial metabolism and metabolic reprogramming in tumours gr1

Schematic illustration of mitochondrial metabolism and metabolic reprogramming in tumours gr1

Fig. 1. Schematic illustration of mitochondrial metabolism and metabolic reprogramming in tumours. In normal cells (A), glucose is phosphorylated by HK-I, then the major part is degraded via glycolysis to pyruvate, which prevalently enters the mitochondria, it is decarboxylated and oxidized by PDH to acetyl-coenzyme A, which enters the TCA cycle where the two carbons are completely oxidized to CO2 whereas hydrogen atoms reduce NAD+ and FAD, which feed the respiratory chain (turquoise). Minor part of glycolytic G-6P is diverted to produce ribose 5-phosphate (R-5P) and NADPH, that will be used to synthesize nucleotides, whereas triose phosphates in minimal part will be used to synthesize lipids and phospholipids with the contribution of NADPH and acetyl-coenzyme A. Amino acids, including glutamine (Gln) will follow the physiological turnover of the proteins, in minimal part will be used to synthesize the nucleotides bases, and the excess after deamination will be used to produce energy. In the mitochondria inner membranes are located the respiratory chain complexes and the ATP synthase (turquoise), which phosphorylates ADP releasing ATP, that in turn is carried to the cytosol by ANT (green) in exchange for ADP. About 1–2% O2 uptaken by the mitochondria is reduced to superoxide anion radical and ROS. In cancer cells (B), where anabolism is enhanced, glucose is mostly phosphorylated by HK-II (red), which is up-regulated and has an easy access to ATP being more strictly bound to the mitochondria. Its product, G-6P, is only in part oxidized to pyruvate. This, in turn, is mostly reduced to lactate being both LDH and PDH kinase up-regulated. A significant part of G-6P is used to synthesize nucleotides that also require amino acids and glutamine. Citrate in part is diverted from the TCA cycle to the cytosol, where it is a substrate of citrate lyase, which supplies acetyl-coenzyme A for lipid and phospholipid synthesis that also requires NADPH. As indicated, ROS levels in many cancer cells increase.

Of particular relevance in the study of the metabolic changes occurring in cancer cells, is the role of hexokinase II. This enzyme is greatly up-regulated in many tumours being its gene promoter sensitive to typical tumour markers such as HIF-1 and P53 [30]. It plays a pivotal role in both the bioenergetic metabolism and the biosynthesis of required molecules for cancer cells proliferation. Hexokinase II phosphorylates glucose using ATP synthesized by the mitochondrial oxphos and it releases the product ADP in close proximity of the adenine nucleotide translocator (ANT) to favour ATP re-synthesis within the matrix (Fig. 1). Obviously, the expression level, the location, the substrate affinity, and the kinetics of the enzyme are crucial to the balancing of the glucose fate, to either allowing intermediates of the glucose oxidation pathway towards required metabolites for tumour growth or coupling cytoplasmic glycolysis with further oxidation of pyruvate through the TCA cycle, that is strictly linked to oxphos. This might be possible if the mitochondrial-bound hexokinase activity is reduced and/or if it limits ADP availability to the mitochondrial matrix, to inhibit the TCA cycle and oxphos. However, the mechanism is still elusive, although it has been shown that elevated oncogene kinase signaling favours the binding of the enzyme to the voltage-dependent anion channel (VDAC) by AKT-dependent phosphorylation [34] (Fig. 2). VDAC is a protein complex of the outer mitochondrial membrane which is in close proximity of ANT that exchanges ADP for ATP through the inner mitochondrial membrane [35]. However, the enzyme may also be detached from the mitochondrial membrane, to be redistributed to the cytosol, through the catalytic action of sirtuin-3 that deacylates cyclophilin D, a protein of the inner mitochondrial membrane required for binding hexokinase II to VDAC (Fig. 2[36]. Removing hexokinase from the mitochondrial membrane has also another important consequence in cancer cells: whatever mechanism its removal activates, apoptosis is induced [37] and [38]. These observations indicate hexokinase II as an important tool used by cancer cells to survive and proliferate under even adverse conditions, including hypoxia, but it may result an interesting target to hit in order to induce cells cytotoxicity. Indeed, a stable RNA interference of hexokinase II gene showed enhanced apoptosis indices and inhibited growth of human colon cancer cells; in accordance in vivo experiments indicated a decreased tumour growth [39].

Schematic illustration of the main mitochondrial changes frequently occurring in cancer cells gr2

Schematic illustration of the main mitochondrial changes frequently occurring in cancer cells gr2

Fig. 2. Schematic illustration of the main mitochondrial changes frequently occurring in cancer cells. The reprogramming of mitochondrial metabolism in many cancer cells comprises reduced pyruvate oxidation by PDH followed by the TCA cycle, increased anaplerotic feeding of the same cycle, mostly from Gln, whose entry in the mitochondrial matrix is facilitated by UCP2 up-regulation. This increases also the free fatty acids uptake by mitochondria, therefore β-oxidation is pushed to produce acetyl-coenzyme A, whose oxidation contributes to ATP production. In cancer cells many signals can converge on the mitochondrion to regulate the mitochondrial membrane permeability, which may respond by elevating the MPTP (PTP) threshold, with consequent enhancement of apoptosis resistance. ROS belong to this class of molecules since it can enhance Bcl2 and may induce DNA mutations. Dotted lines indicate regulation; solid lines indicate reaction(s).

Respiratory chain complexes and ATP synthase

Beyond transcriptional control of metabolic enzyme expression by oncogenes and tumour suppressors, it is becoming evident that environmental conditions affect the mitochondrial energy metabolism, and many studies in the last decade indicate that mitochondrial dysfunction is one of the more recurrent features of cancer cells, as reported at microscopic, molecular, biochemical, and genetic level [7], [40] and [41]. Although cancer cells under several conditions, including hypoxia, oncogene activation, and mDNA mutation, may substantially differ in their ability to use oxygen, only few reports have been able to identify a strict association between metabolic changes and mitochondrial complexes composition and activity. In renal oncocytomas [42] and in lung epidermoid carcinoma [43], the NADH dehydrogenase activity and protein content of Complex I were found to be strongly depressed; subsequently, in a thyroid oncocytoma cell line [44] a similar decrease of Complex I activity was ascribed to a specific mutation in the ND1 gene of mitochondrial DNA. However, among the respiratory chain complexes, significant decrease of the only Complex I content and activity was found in K-ras transformed cells in our laboratory [45], and could not be ascribed to mtDNA mutations, but rather, based on microarray analysis of oxphos genes, we proposed that a combination of genetic (low transcription of some genes) and biochemical events (assembly factors deficiency, disorganization of structured supercomplexes, and ROS-induced structural damage) might cause the Complex I defects.

In some hereditary tumours (renal cell carcinomas) a correlation has been identified between mitochondrial dysfunctions and content of oxphos complexes [46]. For instance, the low content of ATP synthase, often observed in clear cell type renal cell carcinomas and in chromophilic tumours, seems to indicate that the mitochondria are in an inefficient structural and functional state [46]. However, it cannot be excluded that, in some cases, the structural alteration of ATP synthase may offer a functional advantage to cells exhibiting a deficient respiratory chain for instance to preserve the transmembrane electrical potential (Δψm) [47]. It is likely that low levels of ATP synthases may play a significant role in cancer cell metabolism since it has been reported that in tumours from many different tissues, carcinogenesis specifically affects the expression of F1-ATPase β subunit, suggesting alterations in the mechanisms that control mitochondrial differentiation (see for a detailed review [48]). What it seems intriguing is the overexpression of the inhibitor protein, IF1, reported in hepatocellular carcinomas [49] and [50] and in Yoshida sarcoma [51]. Normally, this protein binds to the F1 domain of the ATP synthase inhibiting its activity [52], and it is believed to limit the ATP hydrolysis occurring in the mitochondria of hypoxic cells, avoiding ATP depletion and maintaining Δψm to a level capable to avoid the induction of cell death [5]. But why is its expression in cancer cells enhanced in front of a reduced F1-ATPase β subunit?

The first possibility is that IF1 has a function similar to that in normal cells, simply avoiding excessive ATP hydrolysis therefore limiting Δψm enhancement, but in cancer cells this is unlikely due to both the reduced level of ATP synthase [46] and the high affinity of IF1 for the enzyme. A second possibility might be that cancer cells need strongly reduced oxphos to adapt their metabolism and acquire a selective growth advantage under adverse environmental conditions such as hypoxia, as it has been experimentally shown [53]. Finally, IF1 might contribute to the saving of the inner mitochondrial membrane structure since it has been reported its capability to stabilize oligomers of ATP synthase, which in turn can determine cristae shapes [54]. In this regard, recent experimental evidence has shed some light on a critical role of mitochondrial morphology in the control of important mitochondrial functions including apoptosis [55] and oxidative phosphorylation [56]. In particular, dysregulated mitochondrial fusion and fission events can now be regarded as playing a role in cancer onset and progression [57]. Accordingly, mitochondria-shaping proteins seem to be an appealing target to modulate the mitochondrial phase of apoptosis in cancer cells. In fact, several cancer tissues: breast, head-and-neck, liver, ovarian, pancreatic, prostate, renal, skin, and testis, showed a pattern suggestive of enlarged mitochondria resulting from atypical fusion [58].

Mitochondrial membrane potential in cancer cells

Critical mitochondrial functions, including ATP synthesis, ion homeostasis, metabolites transport, ROS production, and cell death are highly dependent on the electrochemical transmembrane potential, a physico-chemical parameter consisting of two components, the major of which being the transmembrane electrical potential (Δψm) (see for a recent review [59]). In normal cells, under normoxic conditions, Δψm is build up by the respiratory chain and is mainly used to drive ATP synthesis, whereas in anoxia or severe hypoxia it is generated by the hydrolytic activity of the ATP synthase complex and by the electrogenic transport of ATP in exchange for ADP from the cytosol to the matrix, operated by the adenine nucleotide translocator [17]. Dissipation of the mitochondrial membrane potential (proton leak) causes uncoupling of the respiratory chain electron transport from ADP phosphorylation by the ATP synthase complex. Proton leak functions as a regulator of mitochondrial ROS production and its modulation by uncoupling proteins may be involved in pathophysiology, including tumours. In addition, Δψm plays a role in the control of the mitochondrial permeability transition pore (MPTP), that might be critical in determining reduced sensitivity to stress stimuli that were described in neoplastic transformation [60], implying that dysregulation of pore opening might be a strategy used by tumour cells to escape death. Indeed, it has recently been reported that ERK is constitutively activated in the mitochondria of several cancer cell types, where it inhibits glycogen synthase kinase-3-dependent phosphorylation of CyP-D and renders these cells more refractory to pore opening and to the ensuing cell death [61].

It is worth mentioning a second protein of the inner mitochondrial membrane, the uncoupling protein, UCP2 (Fig. 2), which contributes to regulate Δψm. Indeed, recent observations evidenced its overexpression in various chemoresistent cancer cell lines and in primary human colon cancer. This overexpression was associated with an increased apoptotic threshold [62]. Moreover, UCP2 has been reported to be involved in metabolic reprogramming of cells, and appeared necessary for efficient oxidation of glutamine [63]. On the whole, these results led to hypothesize an important role of the uncoupling protein in the molecular mechanism at the basis of the Warburg effect, that suppose a reduced Δψm-dependent entry of pyruvate into the mitochondria accompanied by enhanced fatty acid oxidation and high oxygen consumption (see for a review [64]). However, in breast cancer Sastre-Serra et al. [65] suggested that estrogens by down-regulating UCPs, increase mitochondrial Δψm, that in turn enhances ROS production, therefore increasing tumorigenicity. While the two above points of view concur to support increased tumorigenicity, the mechanisms at the basis of the phenomenon appear on the opposite of the other. Therefore, although promising for the multiplicity of metabolic effects in which UCPs play a role (see for a recent review [66]), at present it seems that much more work is needed to clarify how UCPs are related to cancer.

A novel intriguing hypothesis has recently been put forward regarding effectors of mitochondrial function in tumours. Wegrzyn J et al. [67] demonstrated the location of the transcription factor STAT3 within the mitochondria and its capability to modulate respiration by regulating the activity of Complexes I and II, and Gough et al. [68] reported that human ras oncoproteins depend on mitochondrial STAT3 for full transforming potential, and that cancer cells expressing STAT3 have increased both Δψm and lactate dehydrogenase level, typical hallmarks of malignant transformation (Fig. 2). A similar increase of Δψm was recently demonstrated in K-ras transformed fibroblasts [45]. In this study, the increased Δψm was somehow unexpected since the cells had shown a substantial decrease of NADH-linked substrate respiration rate due to a compatible reduced Complex I activity with respect to normal fibroblasts. The authors associated the reduced activity of the enzyme to its peculiar low level in the extract of the cells that was confirmed by oxphos nuclear gene expression analysis. This significant and peculiar reduction of Complex I activity relative to other respiratory chain complexes, is recurrent in a number of cancer cells of different origin [42][44][45] and [69]. Significantly, all those studies evidenced an overproduction of ROS in cancer cells, which was consistent with the mechanisms proposed by Lenaz et al. [70] who suggested that whatever factor (i.e. genetic or environmental) initiate the pathway, if Complex I is altered, it does not associate with Complex III in supercomplexes, consequently it does not channel correctly electrons from NADH through coenzyme Q to Complex III redox centres, determining ROS overproduction. This, in turn, enhances respiratory chain complexes alteration resulting in further ROS production, thus establishing a vicious cycle of oxidative stress and energy depletion, which can contribute to further damaging cells pathways and structures with consequent tumour progression and metastasis [69].

Hypoxia and oxidative phosphorylation in cancer cells

Tumour cells experience an extensive heterogeneity of oxygen levels, from normoxia (around 2–4% oxygen tension), through hypoxia, to anoxia (< 0.1% oxygen tension). The growth of tumours beyond a critical mass > 1–2 mm3 is dependent on adequate blood supply to receive nutrients and oxygen by diffusion [88]. Cells adjacent to capillaries were found to exhibit a mean oxygen concentration of 2%, therefore, beyond this distance, hypoxia occurs: indeed, cells located at 200 μm displayed a mean oxygen concentration of 0.2%, which is a condition of severe hypoxia [89]. Oxygen shortage results in hypoxia-dependent inhibition of mitochondrial activity, mostly mediated by the hypoxia-inducible factor 1 (HIF-1)[90] and [91]. More precisely, hypoxia affects structure, dynamics, and function of the mitochondria, and in particular it has a significant inhibitory effect on the oxidative phosphorylation machinery, which is the main energy supplier of cells (see Ref. [22] for a recent review). The activation of HIF-1 occurs in the cytoplasmic region of the cell, but the contribution of mitochondria is critical being both cells oxygen sensors and suppliers of effectors of HIF-1α prolyl hydroxylase like α-ketoglutarate and probably ROS, that inhibit HIF-1α removal [92]. As reported above, mitochondria can also promote HIF-1α stabilization if the TCA flux is severely inhibited with release of intermediate molecules like succinate and fumarate into the cytosol. On the other hand, HIF-1 can modulate mitochondrial functions through different mechanisms, that besides metabolic reprogramming [7][22][93] and [94], include alteration of mitochondrial structure and dynamics[58], induction of microRNA-210 that decreases the cytochrome c oxidase (COX) activity by inhibiting the gene expression of the assembly protein COX10 [95], that also increases ROS generation. Moreover, these stress conditions could induce the anti-apoptotic protein Bcl-2, which has also been reported to regulate COX activity and mitochondrial respiration [96] conferring resistance to cells death in tumours (Fig. 2). This effect might be further enhanced upon severe hypoxia conditions, since COX is also inhibited by NO, the product of activated nitric oxide synthases [97].

The reduced respiration rate occurring in hypoxia favours the release of ROS also by Complex III, which contribute to HIF stabilization and induction of Bcl-2 [98]. In addition, hypoxia reduces oxphos by inhibiting the ATP synthase complex through its natural protein inhibitor IF1 (discussed in a previous section), which contributes to the enhancement of the “aerobic glycolysis”, all signatures of cancer transformation.

The observations reported to date indicate that cancer cells exhibit large varieties of metabolic changes which are associated with alterations in the mitochondrial structure, dynamics and function, and with tumour growth and survival. On one hand, mitochondria can regulate tumour growth through modulation of the TCA cycle and oxidative phosphorylation. The altered TCA cycle provides intermediates for both macromolecular biosynthesis and regulation of transcription factors such as HIF, and it allows cytosolic reductive power enhancement. Oxphos provides significant amounts of ATP which varies among tumour types. On the other hand, mitochondria are crucial in controlling redox homeostasis in the cell, inducing them to be either resistant or sensitive to apoptosis. All these reasons locate mitochondria at central stage to understanding the molecular basis of tumour growth and to seeking for novel therapeutical approaches.

Due to the complexity and variability of mitochondrial roles in cancer, careful evaluation of mitochondrial function in each cancer type is crucial. Deeper and more integrated knowledge of mitochondrial mechanisms and cancer-specific mitochondrial modulating means are expected for reducing tumorigenicity and/or improving anticancer drugs efficacy at the mitochondrial level. Although the great variability of biochemical changes found in tumour mitochondria, some highlighted peculiarities such as reduced TCA cycle flux, reduced oxphos rate, and reduced Complex I activity with respect to tissue specific normal counterparts are more frequent. In addition, deeper examination of supramolecular organization of the complexes in the inner mitochondrial membrane has to be considered in relation to oxphos dysfunction.  Oxidation–reduction states of NADH in vivo: From animals to clinical use

Mayevsky A, Chance B.
Mitochondrion. 2007 Sep; 7(5):330-9

Mitochondrial dysfunction is part of many pathological states in patients, such as sepsis or stroke. Presently, the monitoring of mitochondrial function in patients is extremely rare, even though NADH redox state is routinely measured in experimental animals. In this article, we describe the scientific backgrounds and practical use of mitochondrial NADH fluorescence measurement that was applied to patients in the past few years. In addition to NADH, we optically measured the microcirculatory blood flow and volume, as well as HbO(2) oxygenation, from the same tissue area. The four detected parameters provide real time data on tissue viability, which is critical for patients monitoring.

(very important article)  Mitochondria in cancer. Not just innocent bystanders

Frezza C, and Gottlieb E
Sem Cancer Biol 2009; 19: 4-11

The first half of the 20th century produced substantial breakthroughs in bioenergetics and mitochondria research. During that time, Otto Warburg observed abnormally high glycolysis and lactate production in oxygenated cancer cells, leading him to suggest that defects in mitochondrial functions are at the heart of malignant cell transformation. Warburg’s hypothesis profoundly influenced the present perception of cancer metabolism, positioning what is termed aerobic glycolysis in the mainstream of clinical oncology. While some of his ideas stood the test of time, they also frequently generated misconceptions regarding the biochemical mechanisms of cell transformation. This review examines experimental evidence which supports or refutes the Warburg effect and discusses the possible advantages conferred on cancer cells by ‘metabolic transformation’.

Fig.1. Mitochondria as a crossroad for catabolic and anabolic pathways in normal and cancer cells. Glucose and glutamine are important carbon sources which are metabolized in cells for the generation of energy and anabolic precursors. The pathways discussed in the text are illustrated and colour coded: red, glycolysis; white, TCA cycle; pink, non-essential amino acids synthesis; orange, pentose phosphate pathway and nucleotide synthesis; green, fatty acid and lipid synthesis; blue, pyruvate oxidation in the mitochondria; brown, glutaminolysis; black, malic enzyme reaction. Solid arrows indicate a single step reaction;dashed-dotted arrows indicate transport across membranes and dotted arrows indicate multi-step reactions. Abbreviations: HK, hexokinase; AcCoA, acetyl co-enzyme A; OAA, oxaloacetate; αKG, α-ketoglutarate.

Fig. 2. Mitochondria as a target for multiple metabolic transformation events. Principal metabolic perturbations of cancer cells are induced by genetic reprogramming and environmental changes. The activation of Akt and MYC oncogenes and the loss of p53 tumor suppressor gene are among the most frequent events in cancer. Furthermore, all solid tumors are exposed to oxidative stress and hypoxia hence to HIF activation.These frequent changes in cancer cells trigger a dramatic metabolic shift from oxidative phosphorylation to glycolysis. In addition, direct genetic lesions of mtDNA or of nuclear encoded mitochondrial enzyme (SDH or FH) can directly abrogate oxidative phosphorylation in cancer. 3- D structures of the respiratory complexes in the scheme were retrieved from Protein DataBank ( except for complex I which was retrieved from [87]. PDB codes are as follow: SDH (II), 1 LOV; complex III (III), 1BGY; COX (IV), 1OCC; ATP synthase (V), 1QO1.

Fig. 3. The physiological roles of SDH in the TCA cycle and the ETC and its potential roles in cancer. (A) Ribbon diagram of SDH structure (PBD code: 1LOV). The catalytic subunits: the flavoprotein (SDHA) and the iron-sulphur protein (SDHB) are depicted in red and yellow, respectively, and the membrane anchors and ubiquinone binding proteins SDHC and SDHD are depicted in cyan and green, respectively. (B) Other than being a TCA enzyme, SDH is an additional entry point to the ETC (most electrons are donated from NADH to complex I—not shown in this diagram). The electron flow in and out of complex II and III is depicted by the yellow arrows. During succinate oxidation to fumarate by SDHA, a two-electron reduction of FAD to FADH2 occurs. Electrons are transferred through their on–Sulphur centres on SDHB to ubiquinone (Q) bound to SDHC and SDHD in the inner mitochondrial membrane (IMM), reducing it to ubiquinol (QH2). Ubiquinol transfers its electrons through complex III, in a mechanism named the Q cycle, to cytochrome c (PDB: 1CXA). Electrons then flow from cytochrome c to COX where the final four-electron reduction of molecular oxygen to water occurs (not shown in this diagram). Complex III is the best characterized site of ROS production in the ETC, where a single electron reduction of oxygen to superoxide can occur (red arrow). It was proposed that obstructing electron flow within complex II might support a single electron reduction of oxygen at the FAD site (red arrow). Superoxide is dismutated to hydrogen peroxide which can then leave the mitochondria and inhibit PHD in the cytosol, leading to HIF[1] stabilization. Succinate or fumarate, which accumulate in SDH- or FH-deficient tumors, can also leave the mitochondria and inhibit PHD activity in the cytosol. The red dotted line represents the outer mitochondrial membrane (OMM).  Mitochondria in cancer cells: what is so special about them?

Gogvadze V, Orrenius S, Zhivotovsky B.
Trends Cell Biol. 2008 Apr; 18(4):165-73

The past decade has revealed a new role for the mitochondria in cell metabolism–regulation of cell death pathways. Considering that most tumor cells are resistant to apoptosis, one might question whether such resistance is related to the particular properties of mitochondria in cancer cells that are distinct from those of mitochondria in non-malignant cells. This scenario was originally suggested by Otto Warburg, who put forward the hypothesis that a decrease in mitochondrial energy metabolism might lead to development of cancer. This review is devoted to the analysis of mitochondrial function in cancer cells, including the mechanisms underlying the upregulation of glycolysis, and how intervention with cellular bioenergetic pathways might make tumor cells more susceptible to anticancer treatment and induction of apoptosis.

Glucose utilization pathway

Glucose utilization pathway

Figure 1. Glucose utilization pathway. When glucose enters the cell, it is phosphorylated by hexokinase to glucose-6-phosphate, which is further metabolized by glycolysis to pyruvate. Under aerobic conditions, most of the pyruvate in non-malignant cells enters the mitochondria, with only a small amount being metabolized to lactic acid. In mitochondria, pyruvate dehydrogenase (PDH) converts pyruvate into acetyl-CoA, which feeds into the Krebs cycle. Oxidation of Krebs cycle substrates by the mitochondrial respiratory chain builds up the mitochondrial membrane potential (Dc) – the driving force for ATP synthesis. By contrast, in tumor cells, the oxidative (mitochondrial) pathway of glucose utilization is suppressed, and most of the pyruvate is converted into lactate. Thus, the fate of pyruvate is determined by the relative activities of two key enzymes – lactate dehydrogenase and pyruvate dehydrogenase.

Mechanisms of mitochondrial silencing in tumors

Mechanisms of mitochondrial silencing in tumors

Figure 2. Mechanisms of mitochondrial silencing in tumors. The activity of PDH is regulated by pyruvate dehydrogenase kinase 1 (PDK1), the enzyme that phosphorylates and inactivates pyruvate dehydrogenase. HIF-1 inactivates PDH through PDK1 induction, resulting in suppression of the Krebs cycle and mitochondrial respiration. In addition, HIF-1 stimulates expression of the lactate dehydrogenase A gene, facilitating conversion of pyruvate into lactate by lactate dehydrogenase (LDH). Mutation of p53 can suppress the mitochondrial respiratory activity through downregulation of the Synthesis of Cytochrome c Oxidase 2 (SCO2) gene, the product of which is required for the assembly of cytochrome c oxidase (COX) of the mitochondrial respiratory chain. Thus, mutation of p53 can suppress mitochondrial respiration and shift cellular energy metabolism towards glycolysis.

Production of ROS by mitochondria

In any cell, the majority of ROS are by-products of mitochondrial respiration. Approximately 2% of the molecular oxygen consumed during respiration is converted into the superoxide anion radical, the precursor of most ROS. Normally, a four-electron reduction of O2, resulting in the production of two molecules of water, is catalyzed by complex IV (COX) of the mitochondrial respiratory chain. However, the electron transport chain contains several redox centers (e.g. in complex I and III) that can leak electrons to molecular oxygen, serving as the primary source of superoxide production in most tissues. The one-electron reduction of oxygen is thermodynamically favorable for most mitochondrial oxidoreductases. Superoxide-producing sites and enzymes were recently analyzed in detail in a comprehensive review [87]. ROS, if not detoxified, oxidize cellular proteins, lipids, and nucleic acids and, by doing so, cause cell dysfunction or death. A cascade of water and lipid soluble antioxidants and antioxidant enzymes suppresses the harmful ROS activity. An imbalance that favors the production of ROS over antioxidant defenses, defined as oxidative stress, is implicated in a wide variety of pathologies, including malignant diseases. It should be mentioned that mitochondria are not only a major source of ROS but also a sensitive target for the damaging effects of oxygen radicals. ROS produced by mitochondria can oxidize proteins and induce lipid peroxidation, compromising the barrier properties of biological membranes. One of the targets of ROS is mitochondrial DNA (mtDNA), which encodes several proteins essential for the function of the mitochondrial respiratory chain and, hence, for ATP synthesis by oxidative phosphorylation. mtDNA, therefore, represents a crucial cellular target for oxidative damage, which might lead to lethal cell injury through the loss of electron transport and ATP generation. mtDNA is especially susceptible to attack by ROS, owing to its close proximity to the electron transport chain, the major locus for free-radical production, and the lack of protective histones. For example, mitochondrially generated ROS can trigger the formation of 8-hydroxydeoxyguanosine as a result of oxidative DNA damage; the level of oxidatively modified bases in mtDNA is 10- to 20-fold higher than that in nuclear DNA. Oxidative damage induced by ROS is probably a major source of mitochondrial genomic instability leading to respiratory dysfunction.

Figure 3. Stabilization of mitochondria against OMM permeabilization in tumor cells. OMM permeabilization is a key event in apoptotic cell death. (a) During apoptosis, tBid-mediated oligomerization of Bax causes OMM permeabilization and release of cytochrome c (red circles). (b) Bcl-2 protein binds Bax and prevents its oligomerization. A shift in the balance between pro- apoptotic and antiapoptotic proteins in cancer cells, in favor of the latter, reduces the availability of Bax and prevents OMM permeabilization. (c) Upregulation of hexokinase in tumors and its binding to VDAC in the OMM not only facilitates glucose phosphorylation using mitochondrially generated ATP but keeps VDAC in the open state, preventing its interaction with tBid (de).

Figure 4. Shifting metabolism from glycolysis to glucose oxidation. Utilization of pyruvate is controlled by the relative activities of two enzymes, PDH and LDH. In cancer cells, PDH activity is suppressed by PDH kinase-mediated phosphorylation, and, therefore, instead of entering the Krebs cycle, pyruvate is converted into lactate. Several attempts have been made to redirect pyruvate towards oxidation in the mitochondria. Thus, inhibition of PDK1 by dichloroacetate might stimulate the activity of PDH and, hence, direct pyruvate to the mitochondria. A similar effect can be achieved by inhibition of LDH by oxamate. Overall, suppression of PDK1 and LDH activities will stimulate mitochondrial ATP production and might be lethal to tumor cells, even if these inhibitors are used at non-toxic doses. In addition, stimulation of mitochondrial function, for example though overexpression of mitochondrial frataxin, a protein associated with Friedreich ataxia, was shown to stimulate oxidative metabolism and inhibited growth in several cancer cell lines [86].  Glucose avidity of carcinomas

Ortega AD1, Sánchez-Aragó M, Giner-Sánchez D, Sánchez-Cenizo L, et al.
Cancer Letters 276 (2009) 125–135

The cancer cell phenotype has been summarized in six hallmarks [D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (1) (2000) 57-70]. Following the conceptual trait established in that review towards the comprehension of cancer, herein we summarize the basis of an underlying principle that is fulfilled by cancer cells and tumors: its avidity for glucose. Our purpose is to push forward that the metabolic reprogramming that operates in the cancer cell represents a seventh hallmark of the phenotype that offers a vast array of possibilities for the future treatment of the disease. We summarize the metabolic pathways that extract matter and energy from glucose, paying special attention to the concerted regulation of these pathways by the ATP mass-action ratio. The molecular and functional evidences that support the high glucose uptake and the “abnormal” aerobic glycolysis of the carcinomas are detailed discussing also the role that some oncogenes and tumor suppressors have in these pathways. We overview past and present evidences that sustain that mitochondria of the cancer cell are impaired, supporting the original Warburg’s formulation that ascribed the high glucose uptake of cancer cells to a defective mitochondria. A simple proteomic approach designed to assess the metabolic phenotype of cancer, i.e., its bioenergetic signature, molecularly and functionally supports Warburg’s hypothesis. Furthermore, we discuss the clinical utility that the bioenergetic signature might provide. Glycolysis is presented as the “selfish” pathway used for cellular proliferation, providing both the metabolic precursors and the energy required for biosynthetic purposes, in the context of a plethora of substrates. The glucose avidity of carcinomas is thus presented as the result of both the installment of glycolysis for cellular proliferation and of the impairment of mitochondrial activity in the cancer cell. At the end, the repression of mitochondrial activity affords the cancer cell with a cell-death resistant phenotype making them prone to malignant growth.

Fig. 1. Pathways of glucose metabolism. The model shows some of the relevant aspects of the metabolism of glucose. After entering the cell by specific transporters, glucose can be (i) catabolized by the pentose phosphate pathway (PPP) to obtain reducing power in the form of NADPH, (ii) used for the synthesis of carbohydrates or (iii) utilized by glycolysis to generate pyruvate and other metabolic intermediates that could be used in different anabolic processes (blue rectangles). In the cytoplasm, the generated pyruvate can be reduced to lactate and further exported from the cell or oxidized in the mitochondria by pyruvate dehydrogenase to generate acetyl-CoA, which is condensed with oxaloacetate in the tricarboxylic acid cycle (TCA cycle). The operation of the TCA cycle completes the oxidation of mitochondrial pyruvate. Different pathways that drain intermediates of the TCA cycle (oxaloacetate, succinyl-CoA, a-ketoglutarate and citrate) for biosynthetic purposes (blue rectangles) are represented. The transfer of electrons obtained in biological oxidations (NADH/FADH2) to molecular oxygen by respiratory complexes of the inner mitochondrial membrane (in green) is depicted by yellow lines. The utilization of the proton gradient generated by respiration for the synthesis of ATP by the H+-ATP synthase (in orange) in oxidative phosphorylation (OXPHOS) is also indicated. The incorporation of glutamine carbon skeletons into the TCA cycle is shown. The utilization of NADPH in anabolic pathways is also indicated.

Fig. 3. Fluxes of matter and energy in differentiated, proliferating and cancer cells. In differentiated cells, the flux of glycolysis is low because the requirement for precursors for anabolic purposes is low and there is a high energy yield by the oxidation of pyruvate in mitochondrial oxidative phosphorylation (OXPHOS). In this situation, mitochondrial activity produces large amounts of ROS that are normally quenched by the cellular antioxidant defense. In proliferating and cancer cells, there is a high demand of glucose to provide metabolic precursors for the biosynthesis of the macromolecules of daughter cells and because most of the energy required for anabolic purposes derives from non-efficient non-respiratory modes (glycolysis, pentose phosphate pathway) of energy generation. Limiting mitochondrial activity in these situations ensures less ROS production and their further downstream consequences. In addition, cancer cells have less overall mitochondrial complement or activity than normal cells by repressing the biogenesis of mitochondria.

Fig. 2. Genetic alterations underlying the glycolytic phenotype of cancer cells. The diagram represents the impact of gain-of-function mutations in oncogenes (ovals) and loss-of-function mutations in tumor suppressors (rectangles) in glycolysis and in the mitochondrial utilization of pyruvate in cancer cells. Hypoxia (low O2) induces the stabilization of HIF-1, which promotes transcriptional activation of the glucose transporter, glycolytic genes and PDK1. The expression of PDK1 results in the inactivation of pyruvate dehydrogenase and thus in a decreased oxidation of pyruvate in the TCA cycle concurrent to its enhanced cytoplasmic reduction to lactate by lactate dehydrogenase (LDHA). In addition, HIF1a reciprocally regulates the expression of two isoforms of the cytochrome c oxidase complex. The oncogen myc also supports an enhanced glycolytic pathway by transcriptional activation of glycolytic genes. High levels of c-myc could also promote the production of reactive oxygen species (ROS) that could damage nuclear (nDNA) and mitochondrial (mtDNA). The loss-of-function of the tumor suppressor p53 promotes an enhanced glycolytic phenotype by the repression of TIGAR expression. Likewise, loss-of-function of p53 diminished the expression of SCO2, a gene required for the appropriate assembly of cytochrome c oxidase, and thus limits the activity of mitochondria in the cancer cell.

Jose E S Roselino

  1. Warburg Effect revisited
    It is very interesting the series of commentaries following Warburg Effect revisited. However, it comes as no surprise that almost all of them have small or greater emphasis in the molecular biology (changes in gene expression) events of the metabolic regulation involved.
    I would like to comment on some aspects: 1- Warburg did the initial experiments following Pasteur line of reasoning that aimed at carbon flow through the cell (yeast in his case) instead of describing anything inside the cell. It is worth to recall that for the sake of his study, Pasteur considered anything inside the cell under the domain of divine forces. He, at least in defence of his work, entirely made outside the cell, considered that inside the cells was beyond human capability of understanding – He has followed vitalism as his line of reasoning in defence of his work – Interestingly, the same scientist that has ruled out spontaneous generation when Pasteurization was started. Therefore, Pasteur measured everything outside the cell (mainly sugar, ethanol – the equivalent of our lactic acid end product of anaerobic metabolism) and found that as soon as yeasts were placed in the presence of oxygen, sugar was consumed at low speed in comparison with the speed measured in anaerobiosis and ethanol was also produced at reduced speed. This is an indication of a fast biological regulatory mechanism that obviously, do not require changes in gene expression. As previously said, Warburg work translated for republishing in the Journal Biological Chemistry mentioned “grana” for mitochondria calling attention on an “inside-the-cell” component. It seems that, there is not a unique, single site of metabolism, where the Pasteur Effect – Warburg Effect seems to be elicited by the shift from anaerobiosis to aerobiosis or vice versa.
    In order to find a core for the mechanism the best approach seems to take into account one of the most important contributions of one of the greatest north-American biochemists, Briton Chance. He has made it with his polarographic method of following continuously the oxygen consumption of the cell´s mitochondria.
    Mitochondria burn organic carbon molecules under a very stringent control mechanism of oxidative-phosphorylation ATP production. Measured in the form of changes in the speed of oxygen consumption over time as Respiratory Control Ratio (RCR). When no ATP is required by the cell, oxygen consumption goes at low speed (basal or state II or IV). When ADP is offered to the mitochondria as an indication that ATP synthesis is necessary, oxygen consumption is activated in state III respiration. Low respiration means low burning activity of organic (carbon) molecules what in this case, means indirectly low glucose consumption. While high respiration is the converse – greater glucose consumption.
    Aerobic metabolism of glucose to carbonic acid and water provides a change in free energy enough for 38 molecules of ATP (the real production is +/- 32 ATP in aerobic condition) while glucose to lactic acid metabolism in anaerobiosis leads to 2 ATP production after discounting the other 2 required at initial stages of glucose metabolism.
    The low ATP yield in anaerobiosis explains the fast glucose metabolism in anaerobiosis while the control by RCR in mitochondria explains the reduction in glucose metabolism under aerobiosis as long as the ATP requirements of the cell remains the same – This is what it is assumed to happen in quiescent cells. Not necessarily in fast growing cells as cancer cells are. However, this will not be discussed here. In my first experiments in the early seventies, with M. Rouxii a dimorphic mold-yeast biological system the environmental change (aerobic – anaerobic) led to morphogenetic change presented as morphogenetic expression of the Pasteur Effect. In this case, the enzyme that replaces mitochondria in ATP production (Pyruvate Kinase) converting phosphoenolpyruvate into pyruvate together with ADP into ATP, shows changes that can be interpreted as change in gene expression together with new self-assembly of enzyme subunits. (Dimer AA – yeast in anaerobic growth or sporangiospores- converted into dimer AB in aerobic mold). In Leloir opinions at that time, PK I (AA) was only highly glycosylated, while PK II (AB) was less glycosylated without changes in gene expression.

    In case you read comments posted, you will see that the reference to aerobic glycolysis, continues to be made together with, new deranged forms of reasoning as is indicated by referring to: Mitochondrial role in ion homeostasis…
    Homeostasis is a regulation of something, ions, molecules, pH etc. that is kept outside the cell, therefore any role for mitochondria on it is only made indirectly, by its ATP production.
    However, mitochondria has a role together with other cell components in the regulation of for instance, intracellular Ca levels (Something that is not a homeostatic regulation). This is a very important point for the following reason: Homeostasis is maintained as a composite result of several differentiated cellular, tissue and organ functions. Differentiated function is something clearly missing in cancer cells. The best form to refer to the mitochondrial function regarding ions is to indicate a mitochondrial role in ion fluxes.
    In short, to indicate how an environmental event or better saying condition could favour genetic changes instead of being caused by genetic changes is to follow the same line of reasoning that is followed in understanding the role of cardioplegia. To stop heart beating is adequate for heart surgery it is also adequate for heart cells by sparing the ATP use during surgery and therefore, offering better recovery condition to the heart afterwards.
    In the case, here considered, even assuming that the genome is not made more unstable during hypoxic condition it is quite possible to understand that sharing ATP with both differentiated cell function and replication may led quality control of DNA in short supply of much needed ATP and this led to maintenance of mutations as well as less organized genome.

    • Thank you. I enjoy reading your comments. They are very instructive. I don’t really think that I comprehend the use of the term “epigenetics” and longer. In fact, it was never clear to me when I first heard it used some years ago.

      The term may have been closely wedded to the classic hypothesis of a unidirectional DNA–> RNA–> protein model that really has lost explanatory validity for the regulated cell in its environment. The chromatin has an influence, and protein-protein interactions are everywhere. As you point out, these are adjusting to a fast changing substrate milieu, and the genome is not involved. But in addition, the proteins may well have a role in suppression or activation of signaling pathways, and thereby, may well have an effect on gene expression. I don’t have any idea about how it would work, but mutations would appear to follow the metabolic condition of the cell over time. It would appear to be – genomic modification.

  2. In aerobic glucose metabolism, the oxidation of citric acid requires ADP and Mg²+, which will increase the speed of the reaction: Iso-citric acid + NADP (NAD) — isocitrate dehydrogenase (IDH) = alpha-ketoglutaric acid. In the Krebs cycle (the citric cycle), IDH1 and IDH2 are NADP+-dependent enzymes that normally catalyze the inter-conversion of D-isocitrate and alpha-ketoglutarate (α-KG). The IDH1 and IDH2 genes are mutated in > 75% of different malignant diseases. Two distinct alterations are caused by tumor-derived mutations in IDH1 or IDH2: the loss of normal catalytic activity in the production of α-ketoglutarate (α-KG) and the gain of catalytic activity to produce 2-hydroxyglutarate (2-HG), [22].
    This product is a competitive inhibitor of multiple α-KG-dependent dioxygenases, including demethylases, prolyl-4-hydroxylase and the TET enzymes family (Ten-Eleven Translocation-2), resulting in genome-wide alternations in histones and DNA methylation. [23]
    IDH1 and IDH2 mutations have been observed in myeloid malignancies, including de novo and secondary AML (15%–30%), and in pre-leukemic clone malignancies, including myelodysplastic syndrome and myeloproliferative neoplasm (85% of the chronic phase and 20% of transformed cases in acute leukemia), [24].
    Normally, cells in the body communicate via intra-cytoplasmic channels and maintain the energetic potential across cell membranes, which is 1-2.5 µmol of ATP in the form of ATP-ADP/ATP-ADP-IMP. These normal energetic values occur during normal cell division. If the intra-cellular and extra-cellular levels of Mg2+ are high, the extra-cellular charges of the cells will not be uniformly distributed.
    This change in distribution induces a high net positive charge for the cell and induces a loss of contact inhibition via the electromagnetic induction of oscillation [28, 29, 30]. Thereafter, malignant cells become invasive and metastasize.
    -22. Hartmann C, Meyer J, Balss J. Capper D, et al. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol 2009; 118: 464-474.

    23. Raymakers R.A, Langemeijer S.M., Kuiper R.P, Berends M, et al. Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat. Genet 2009; 41; 838–849.

    24 Wagner K, Damm F, Gohring G., Gorlich K et al. Impact of IDH1 R132 mutations and an IDH1 single nucleotide polymorphism in cytogenetically normal acute myeloid leukemia: SNP rs11554137 is an adverse prognostic factor. J. Clin. Oncol.2010; 28: 2356–2364.
    Plant Molecular Biology 1989; 1: 271–303.

    29. Chien MM, Zahradka CE, Newel MC, Fred JW. Fas induced in B cells apoptosis require an increase in free cytosolic magnesium as in early event. J Biol Chem.1999; 274: 7059-7066.

    30. Milionis H J, Bourantas C L, Siamopoulos C K, Elisaf MS. Acid bases and electrolytes abnormalities in Acute Leukemia. Am J Hematol 1999; (62): 201-207.

    31. Thomas N Seyfried; Laura M Shelton.Cancer as a Metabolic Disease. Nutr Metab 2010; 7: 7

    – Aurelian Udristioiu, M.D,
    – Lab Director, EuSpLM,
    – City Targu Jiu, Romania
    AACC, National Academy of Biochemical Chemistry (NACB) Member, Washington D.C, USA.









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