Posts Tagged ‘cancer therapeutics’

37th Annual J.P. Morgan HEALTHCARE CONFERENCE: News at #JPM2019 for Jan. 8, 2019: Deals and Announcements

Reporter: Stephen J. Williams, Ph.D.


JP Morgan Healthcare Conference Update: FDA, bluebird, Moderna and the Price of Coffee

Researcher holding test tube up behind circle of animated research icons

Tuesday, January 8, was another busy day in San Francisco for the JP Morgan Healthcare Conference. One interesting sideline was the idea that the current government shutdown could complicate some deals. Kent Thiry, chief executive officer of dialysis provider DaVita, who is working on the sale of its medical group to UnitedHealth Group this quarter, said, “We couldn’t guarantee that even if the government wasn’t shut down, but we and the buyer are both working toward that goal with the same intensity if not more.”

And in a slightly amusing bit of synchrony, U.S.Food and Drug Administration (FDA)Commissioner Scott Gottlieb’s keynote address that was delivered by way of video conference from Washington, D.C., had his audio cut out in the middle of the presentation. Gottlieb was talking about teen nicotine use and continued talking, unaware that his audio had shut off for 30 seconds. When it reconnected, the sound quality was reportedly poor.

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bluebird bio’s chief executive officer, Nick Leschlygave an update of his company’s pipeline, with a particular emphasis on a proposed payment model for its upcoming LentiGlobin, a gene therapy being evaluated for transfusion-dependent ß-thalassemia (TDT). The gene therapy is expected to be approved in Europe this year and in the U.S. in 2020. Although the price hasn’t been set, figures up to $2.1 million per treatment have been floated. Bluebird is proposing a five-year payment program, a pledge to not raise prices above CPI, and no costs after the payment period.

Eli Lilly’s chief executive officer David Ricks, just days after acquiring Loxo Oncologyoffered up projections for this year, noting that 45 percent of its revenue will be created by drugs launched in 2015. Those include Trulicity, Taltz and Verzenio. The company also expects to launch two new molecular entities this year—nasal glucagons, a rescue medicine for high blood sugar (hyperglycemia), and Lasmiditan, a rescue drug for migraine headaches.

CNBC’s Jim Cramer interviewed Allergan chief executive officer Brent Saunders, in particular discussing the fact the company’s shares traded in 2015 for $331.15 but were now trading for $145.60. Cramer noted that the company’s internal fundamentals were strong, with multiple pipeline assets and a strong leadership team. Some of the stock problems are related to what Saunders said were “unforced errors,” including intellectual property rights to Restasis, its dry-eye drug, and Allergan’s dubious scheme to protect those patents by transferring the rights to the Saint Regis Mohawk Tribe in New York. On the positive side, the company’s medical aesthetics portfolio, dominated by Botox, is very strong and the overall market is expected to double.

One of the big areas of conversation is so-called “flyover tech.” Biopharma startups are dominant in Boston and in San Francisco, but suddenly venture capital investors have realized there’s a lot going on in between. New York City-based Radian Capital, for example, invests exclusively in markets outside major U.S. cities.

“At Radian, we partner with entrepreneurs who have built their businesses with a focus on strong economics rather than growth at all costs,” Aly Lovett, partner at Radian, told The Observer. “Historically, given the amount of money required to stand up a product, the software knowledge base, and coastal access to capital, health start-ups were concentrated in a handful of cities. As those dynamics have inverted and as the quality of living becomes a more important factor in attracting talent, we’re not seeing a significant increase in the number of amazing companies being built outside of the Bay Area.”

“Flyover companies” mentioned include Bind in Minneapolis, Minnesota; Solera Health in Phoenix, Arizona; ClearDATA in Austin, Texas; Healthe, in Eden Prairie, Minnesota; HistoSonics in Ann Arbor, Michigan; and many others.

Only a month after its record-breaking IPO, Moderna Therapeutics’ chief executive officer Stephane Bancelspent time both updating the company’s clinical pipeline and justifying the company’s value despite the stock dropping off 26 percent since the IPO. Although one clinical program, a Zika vaccine, mRNA-1325, has been abandoned, the company has three new drugs coming into the clinic: mRNA-2752 for solid tumors or lymphoma; mRNA-4157, a Personalized Cancer Vaccine with Merck; and mRNA-5671, a KRAS cancer vaccine. The company also submitted an IND amendment to the FDA to add an ovarian cancer cohort to its mRNA-2416 program.

One interesting bit of trivia, supplied on Twitter by Rasu Shrestha, chief innovation officer for the University of Pittsburgh Medical Center, this year at the conference, 33 female chief executive officers were presenting corporate updates … compared to 19 men named Michael. Well, it’s a start.

And for another bit of trivia, Elisabeth Bik, of Microbiome Digest, tweeted, “San Francisco prices are so out of control that one hotel is charging the equivalent of $21.25 for a cup of coffee during a JPMorgan conference.”

Other posts on the JP Morgan 2019 Healthcare Conference on this Open Access Journal include:

#JPM19 Conference: Lilly Announces Agreement To Acquire Loxo Oncology

36th Annual J.P. Morgan HEALTHCARE CONFERENCE January 8 – 11, 2018

37th Annual J.P. Morgan HEALTHCARE CONFERENCE: #JPM2019 for Jan. 8, 2019; Opening Videos, Novartis expands Cell Therapies, January 7 – 10, 2019, Westin St. Francis Hotel | San Francisco, California


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AACR2016 – Cancer immunotherapy

Curator: Larry H. Bernstein, MD, FCAP



AACR 2016: Cancer Immunotherapy and Beyond

At this year’s meeting there was a palpable buzz around subjects ranging from microbiomics to the tumor microenvironment and cancer vaccines, big data to in vitro and in vivo modeling and drug delivery (to name just a few).

By all accounts, this year’s American Association of Cancer Research’s 2016 annual meeting (AACR) was dominated by immunotherapy writ large. Which is not to say the meeting was devoid of other topics – there was certainly palpable buzz around subjects ranging from microbiomics to the tumor microenvironment and cancer vaccines, big data to in vitro and in vivo modeling and drug delivery (to name just a few), that kept the meeting’s 19,500 attendees rapt in New Orleans April 16-20. Yet in the 12-13 years that David R. Soto-Pantoja, Ph.D., has been attending AACR, the Wake Forest University School of Medicine assistant professor of Cancer Biology has “never seen anything take over so much like immunotherapy.”

That being said, it’s not always easy to put cancer research into neat little boxes. Researchers interested in cell signaling, and oncologists concerned with genomics, may have found themselves in sessions dedicated to finding and exploiting neo-antigens (one of immunotherapy’s buckets).

Immunotherapy Buckets

For years there have been certain pillars used to target cancer: chemotherapy, surgery, radiation therapy, and more recently targeted therapies. “And now immunotherapy,” says Wafik El-Deiry, M.D., Ph.D., Deputy Director for Translational Research at the Fox Chase Cancer Center. “This is an emerging and expanding field that is going to be very intensely explored in every direction you can imagine. I wouldn’t even try to pretend that we know what all the buckets are at this point.”

One known bucket that  isfast being filled is the area of checkpoint inhibitors (checkpoint blockades). Over the past few years several therapeutics have been developed that had to do with getting the immune system to either better recognize, better target, or better fight, a tumor, with some success. Recall that last year former president Jimmy Carter, earlier diagnosed with metastatic melanoma, was declared “cancer free” following treatment with radiation and an antibody targeted against the immune programmed cell death – 1 (PD-1) antigen. PD-1 keeps the immune cells in check; blockading PD-1 de-inhibits, or “releases the brakes” of these cells and allow them free rein to attack the cancer. Encouraging results from several trials and follow-ups of PD-1 and other checkpoint inhibitors, such as anti-PDL-1 and anti-CTLA-4, were presented. These are in some cases being combined for even greater efficacy.

In addition to trials, “a lot of people, whether in posters or in other smaller talks, delve into the scientific mechanism at the cellular and immunological level of how those worked,” remarked Emil Lou, M.D., Ph.D., assistant professor of Medicine in the Division of Hematology, Oncology and Transplantation at the University of Minnesota.

Another bucket  receiving many contributions is the area of CAR-T cells: T cells imbued with a chimeric antigen receptor. T cells that have been harvested from a patient are given a receptor that will recognize a new protein – CD-19, found on B cells, for example – expanded, and re-infused (adoptively transferred) back into the patient to attack the cancer — B cell lymphomas, in the present example. There were many hurdles and pitfalls to be overcome – from finding the right antigen and designing the CAR, to controlling the CAR-T cell’s response – yet the field “has gone at such a rapid pace that it’s very clinically relevant,” says Dr. Lou.

Much familiar (and not-so-familiar) technology, such as high content (phenotypic) screening platforms like the IntelliCyt, is being leveraged to help with array testing, selection, and even manufacturing of CAR-T cells, says Janette Phi, the company’s CBO.

Miltenyi Biotec touted their CliniMACS Prodigy, a benchtop-sized automated cell processing and separation platform, for manufacturing CAR-T cells. It can take a patient’s cells from apheresis and selection on beads, through viral transduction to final product “in 8-10 days,” says clinical instrument specialist Kevin Longin. “It’s a GMP lab without a GMP lab.”

But making bespoke CAR-Ts is a “very expensive approach,” notes Dr. Soto-Pantoja. There were discussions at the meeting about generating off-the-shelf CAR-T cells.

Gene Editing

One way to do this is to use gene editing techniques such as CRISPR/Cas9 to knock out the proteins that could cause rejection of these foreign cells by the patient’s own immune system, or graft-versus-host disease (in which the introduced cells treat the host as foreign).

CRISPR has really become a hot buzz word, says Dr. Lou. “More from the basic science side, and slowly making its way into the clinical talks – it’s not ready for prime time.” While gene editing spins off a different conversation about ethics – people are hesitant about its capacity to create designer babies — he notes that “in cancer it’s helpful to be able to study and reproduce the genes that are driving cancer, in the lab.”

CRISPR is not just for knocking genes in or out, either. In his plenary talk, MIT’s W. M. Keck Career Development Professor of Biomedical Engineering Feng Zhang, Ph.D., discussed a host of other uses to which his lab and others have put the CRISPR/Cas9 system and its relatives. They can be used to create selective transcriptional activation or repression, for example, to recruit epigenetic writers, erasers, and readers, to edit RNA transcripts, and even to identify non-coding regions of the genome.

Neo-antigens and Other Biomarkers

Gene therapy and gene editing are not the only ways in which the complement of expressed proteins is altered. Among the hallmarks of cancer is genetic mutation of oncogenes, tumor suppressor genes, and others – whether as point mutations, duplications, insertions and deletions (indels), or fusions. Because they’re mutated and encode other amino acids, they are “foreign” and may be recognized by the immune system as such, Dr. El-Deiry points out. “One needs to analyze these neo-antigens — to figure out what they are – as well as to analyze the various immune cell subsets that may react with those neo-antigens.” There are many tools that researchers are using to do just that, and there was no shortage of vendors from instrument and assay manufacturers and software developers to service providers – in areas like next-generation genomic sequencing (for both genomics and transcriptomics) and its associated preparatory and analytics, flow cytometry, and antibody development, to name just a few – vying for the attention of AACR conventioneers.

Neo-antigens are, of course, only one mark of a cancerous cell or tissue. In fact, most biomarkers used to diagnose or track cancers in the lab or the clinic rely on “signatures” — collections of multiple markers, each of which in and of themselves may be considered within the normal range but taken together (at the levels expressed) correlate with disease, prognosis, or likely response to treatment. Many panels, available on different platforms, are currently approved for clinical testing and others are working toward that goal.

It’s important to realize that cancers are often not static – they evolve, often as the result of treatment, sometimes selecting for a resistant population. “We’re trying to overcome the mistake of treating a patient’s cancer, after their tumor has grown despite different types of chemotherapies, based on what the information was at the time of diagnosis, when in reality the tumor has potentially transformed into something different,” relates Dr. Lou. Serial biopsies under the auspices of clinical trials, mostly in lung cancer, have revealed the evolution of cancer genomics and an understanding of how to better target therapy to the patient’s tumor at the time of recurrence and progression.

But taking a biopsy is an invasive and sometimes risky procedure.

Liquid Biopsy

It has been known for many years that cells, nucleic acids, and even vesicles derived from tumors can be found in blood and other fluids. “The idea that we can biopsy less invasively by using blood-based biomarkers is really coming to maturity just in the last two years or so,” says Dr. Lou. There is currently a lot of excitement about the possibility of using these as liquid biopsies to “provide a window into the cancer,” says Shane Booth, D. Phil., CTO of Angle LLC. They can look for the presence or evolution of a tumor, for example to monitor the effect of therapy.

Only a single circulating tumor cell (CTC) platform, CELLSEARCH, has thus far been approved for clinical use. Dr. Booth estimates that there are currently perhaps 20-30 different companies with technologies to isolate CTCs, most (like CELLSEARCH) based on affinity capture or (like Angle’s Parsortix platform) on “very sophisticated, bleeding-edge filtration techniques”. These differ from each other in a variety of ways including whether the platform performs analysis, whether it is specific or agnostic to the type of cancer, whether cells can be recovered (and whether they can be recovered alive) for downstream use, the purity of CTCs, and the sample preparation required.

Several companies are offering platforms or services to look at cell free DNA (cfDNA, aka circulating tumor DNA, ctDNA). Trovagene, for example, has assays to examine DNA in blood or urine for common BRAF, KRAS, and EGFR mutations. Meanwhile Nanostring Technologies uses digital molecular barcoding to multiplex hundreds of assays from single molecules without amplification.

Caris Life Sciences’ ADAPT Biotargeting System can profile the different proteins, miRNAs, and DNA found in exosomes. “It’s still early times, but these kinds of test will in the future be used to try and make some predictions about prognosis or response to therapy,” says Dr. El-Deiry.

Personalized Medicine

If there was a theme (implicitly) pervading AACR 2016 more than that of immunotherapy it was that of personalized medicine (aka precision medicine, individualized medicine). From genomic sequencing to determine whether a patient will benefit from (or be harmed by) a given therapy, to examining the microenvironment in which a tumor is found, one size no longer fits all.

The challenge, notes Dr. Soto-Pantoja, is to take the results seen in cases such as checkpoint inhibitor therapy, in which about one third of patients are seen to benefit, and figure out how to extend that to other patients and apply that to other types of cancer.



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Advances in Cancer Immunotherapy

Larry H. Bernstein, MD, FCAP, Curator



Dramatic remissions in blood cancer in immunotherapy treatment trial

“We are at the precipice of a revolution in cancer treatment based on using immunotherapy.” — Stanley Riddell, MD

Recent advances in an immune-cell cancer treatment — a type of immunotherapy* using engineered immune cells to target specific molecules on cancer cells — are producing dramatic results for people with cancer, according to Stanley Riddell, MD, an immunotherapy researcher and oncologist at Seattle’s Fred Hutchinson Cancer Research Center.**

Riddell and his colleagues have refined new methods of engineering a patient’s own immune cells to better target and kill cancer cells while decreasing side effects. In laboratory and clinical trials, the researchers are seeing “dramatic responses” in patients with tumors that are resistant to conventional high-dose chemotherapy, “providing new hope for patients with many different kinds of malignancies,” Riddell said.

Twenty-seven out of 29 patients with an advanced blood cancer who received experimental, “living” immunotherapy as part of a clinical trial experienced sustained remissions, in preliminary results of an ongoing study at Fred Hutchinson Cancer Research Center.

Boosting natural immune response

Adoptive T-cell transfer aims to boost a patient’s immune cells’ ability to recognize and attack cancer cells. (1) T cells are extracted from the patient’s blood, (2) genetically engineered to produce a molecule that recognizes cancer cells and grown in the laboratory, and (3) infused back into the patient to (4) improve immune response. (credit: LUNGevity Foundation)

The immune system produces two major types of immune reaction to protect the body: one uses antibodies secreted by B cells; the other uses T cells.

Riddell’s team takes T cells from the patient’s body, re-engineers them, and infuses them back into the patient to create an army of cancer-fighting immune cells. (credit: Fred Hutchinson Cancer Research Center)

T cells are white blood cells that detect foreign or abnormal cells — including cancerous or infected cells — and initiate a process that targets those cells for attack. But the natural immune response to a tumor is often neither potent nor persistent enough, so Riddell and associates pioneered a new way to boost this immune response using a method known as “adoptive T-cell transfer.”

With adoptive T-cell transfer, immune cells are engineered to recognize and attack the patient’s cancer cells. Researchers extract T cells from a patient’s blood and then introduce genes into those T cells so they synthesize highly potent receptors (called chimeric antigen receptors, or CARs) that can recognize and target the cancer cell.

A single treatment of a relatively small number of the re-engineered T cells only takes about 30 minutes, and within weeks, the patient goes into a complete remission. (credit: Fred Hutchinson Cancer Research Center)

They grow the T cells in a laboratory for about two weeks and then infuse the engineered cells back into the patient, where they can home in on the tumor site and destroy the cancer cells.

Sustained remission of B cell cancers

Riddell’s team has recently developed a refined version of this process that increases the effectiveness of the immune response while reducing negative side effects, such as neurological symptoms, fevers, and large decreases in blood pressure.

In a study published in the journal Nature Biotechnology, Riddell and his team describe tagging the potent T-cell receptor (with amino acid sequences called Strep-tag), and the resulting effect on human cancer cells in the laboratory and on a mouse model of lymphoma.

Those results, using the latest version of this experimental immunotherapy, suggest sustained remission in cases of B cell cancers that previously relapsed and had become resistant to treatment.***

“The results are simply astounding,” Riddell said. We are treating patients with advanced leukemia and lymphoma that have failed every conventional therapy and radiation therapy, including transplants … in a single treatment. Within weeks, the patient goes into remission.”

“In my years as a oncologist and as a research scientist, I have never seen a treatment that has that spectacular response rate in its initial testing in patients,” Riddell said. His team is initiating trials in lung, breast, sarcoma, melanoma, and soon in pancreatic cancer. The opportunities for this technology are “incredible” and the approach has the potential to also treat common cancers such as kidney and colon cancer, he said.

“We are at the precipice of a revolution in cancer treatment based on using immunotherapy.”

Funding for Riddell’s research was provided by Juno Therapeutics.

* For approximately 100 years, the main tools to treat cancer were surgery, chemotherapy, and radiation therapy. But since around 2000, doctors have had access to a type of immunotherapy based on engineered antibodies that can target specific molecules on cancer cells. For example, trastuzumab (Herceptin) can be used for some types of breast cancer and stomach cancer. The new treatment approach used by Riddell’s team is based on a new type of immunotherapy using engineered immune cells to kill cancer, rather than antibodies.

** Stanley Riddell. Engineering T cells for safe and effective cancer immunotherapy. 2016 Annual Meeting of the American Association for the Advancement of Science, Washington, D.C., February 2016.

*** Such as acute lymphoblastic leukemia, Non-Hodgkin lymphoma, and chronic lymphocytic leukemia.

Abstract of Acquisition of a CD19 negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T cell therapy

Administration of lymphodepletion chemotherapy followed by CD19-specific chimeric antigen receptor (CAR)-modified T cells is a remarkably effective approach to treat patients with relapsed and refractory CD19+ B cell malignancies. We treated 7 patients with B-cell acute lymphoblastic leukemia (B-ALL) harboring rearrangement of the mixed lineage leukemia (MLL) gene with CD19 CAR-T cells. All patients achieved complete remission in the bone marrow by flow cytometry after CD19 CAR-T cell therapy; however, within one month of CAR-T cell infusion two of the patients developed acute myeloid leukemia that was clonally related to their B-ALL, a novel mechanism of CD19-negative immune escape. These reports have implications for the management of patients with relapsed and refractory MLL-B-ALL who receive CD19 CAR-T cell therapy.

Abstract of Inclusion of Strep-tag II in design of antigen receptors for T-cell immunotherapy

Adoptive immunotherapy with genetically engineered T cells has the potential to treat cancer and other diseases. The introduction of Strep-tag II sequences into specific sites in synthetic chimeric antigen receptors or natural T-cell receptors of diverse specificities provides engineered T cells with a marker for identification and rapid purification, a method for tailoring spacer length of chimeric receptors for optimal function, and a functional element for selective antibody-coated, microbead-driven, large-scale expansion. These receptor designs facilitate cGMP manufacturing of pure populations of engineered T cells for adoptive T-cell therapies and enable in vivo tracking and retrieval of transferred cells for downstream research applications.


It is great that immunotherapy is being highlighted! However the approach they are using is misguided. Cancer occurs from constant chemical attack by free radicals and other types of chemical or forms of damage like radiation. The objective is prevention and secret is in the diet. If you already have it you have to eliminate all the bad stuff and start consuming nutrients that will enhance your immune system so it takes care of the cancer with the T cells. Watch this video and go to minute 38 where the Doc starts explaining this.


Having survived terminal cancer with a dietary approach, what you say is too simplistic.

Cancer is anything that interferes with any of the many growth inhibition pathways the prevent individual cells within the cooperative society of cells that is an animal body from growing in a fashion that puts the whole cooperative system at risk.

Certainly diet, largely via its effect on our immune system, and certainly in some degrees by other mechanisms also, can play a huge role in that. The particular regime I am on is strictly vegan, largely raw, and high dose vitamin c and supplementation of other vitamin/mineral complexes in very low doses.

The work in this article looks very promising, and in most people it would be unnecessary if they changed their diet and bought the contribution from animal products (meat, dairy, fish and foul etc) to below 10% of total calories. Going to zero seems to slightly reduce the risk even further, but not hugely. Along with that one needs to reduce stress (which seems to be not directly about external factors, but more accurately how we contextualise and respond to them).


Immunotherapy historically has involved all arms of the immune system in experimental treatments. That includes not only trained white blood cells, but B-cell antibodies and T-cell antibodies. In some experiments they attached poisons such as ricin to kill the cancer cells.Indeed most anti-cancer drugs can theoretically be attached to antibodies to kill of cancer cells specifically.Most approaches have had miraculous cures and remissions of hopelessly ill cancer patients who were dying.They are not offered to people who have no other hope except as small treatment studies.Why? Oncol;ogy is a big medical business, to cure it outright would put Oncologists out of work.The giant pharmaceutical companies that sell super expensive drugs would lose great gobs of money.They have some of the biggest lobbies in congress to maintain their business.
Often Immunotherapy of whatever form will have dangerous side effects.Some people do die from the treatments.It is unetihcal to refuse to give people who have a few weeks or minths to live a shot at these miracle treatments. In the case of enhanced T-cell therapy such as this one it can be difficult to control how extreme the body attacks. Today they have the means to put in genetic switches which will simply turn off the T-cells or any other cell line, by turning off the genes responsible for the action.One such switch is being produced by the company Intrexon using the insect molting hormone ecdysone to stop and start the genes of any organism.There almost certainly could be analogous techniques to biochemically create similar results if we understand how this one works.— I will be dead and gone a thousand years before any of this is cheaply available to the general population.


Despite the fact that immunotherapy has attracted considerable interest in recent years because of major progress in the identification of human tumor antigens (TA) suitable for clinical use, considerable obstacles to the development of clinically effective immunotherapy still exists including inability to:

induce expansion of large pools of antigen specific CD8+ T cells

maintain durable anti-tumor immunity > 5 years

overcome inherent tolerogenic mechanisms, such as CD4+CD25+ regulatory T cells (Tregs)

Unfortunately understanding the effectiveness of this new protocol with respect to resolving these obstacles takes time and future studies with larger cohorts.



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PI3K delta isoform selective inhibitor

Larry H. Bernstein, MD, FCAP, Curator





1 Vote


N-((1S)-1-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine, WO2008118468


 CAS 1608125-21-8

Chemical Formula: C21H16FN7
Exact Mass: 385.14512

Phosphoinositide-3 kinase delta inhibitor


PI3K delta isoform selective inhibitor is being investigated in human clinical trials for the treatment of PI3K-mediated conditions or disorders, such as cancers and/or proliferative diseases

Useful for treating PI3K-mediated disorders such as acute myeloid leukemia, myelo-dysplastic syndrome, myelo-proliferative diseases, chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, non-Hodgkins lymphoma, B-cell lymphoma, or breast cancer.

Amgen is developing AMG-319, a small molecule PI3K-δ inhibitor, for treating lymphoid malignancies and solid tumors including, head and neck squamous cell carcinoma.

AMG-319 is a highly selective, potent, and orally bioavailable small molecule inhibitor of the delta isoform of the 110 kDa catalytic subunit of class IA phosphoinositide-3 kinases (PI3K) with potential immunomodulating and antineoplastic activities. PI3K-delta inhibitor AMG 319 prevents the activation of the PI3K signaling pathway through inhibition of the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), thus decreasing proliferation and inducing cell death. Unlike other isoforms of PI3K, PI3K-delta is expressed primarily in hematopoietic lineages. The targeted inhibition of PI3K-delta is designed to preserve PI3K signaling in normal, non-neoplastic cells.

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Angiogenesis Inhibitors [9.5]

Writer and Curator: Larry H Bernstein, MD, FCAP

This article has the following structure:

9.5.1 Motesanib (AMG 706)

9.5.2 Drugs that block cancer blood vessel growth (anti angiogenics)

9.5.3 Recent Advances in Anti-Angiogenic Therapy of Cancer

9.5.4 Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

9.5.5 LUCITANIB a VEGFR/FGFR dual kinase inhibitor in Phase 2 trials

9.5.1 Motesanib (AMG 706)


Motesanib (AMG 706) is an experimental drug candidate originally developed by Amgen[1] but is now being investigated by theTakeda Pharmaceutical Company. It is an orally administered small molecule belonging to angiokinase inhibitor class which acts as an antagonist of VEGF receptorsplatelet-derived growth factor receptors, and stem cell factor receptors.[2] It is used as the phosphate salt motesanib diphosphate.

Motesanib, also known as AMG-706, is an orally administered multikinase inhibitor that selectively targets VEGF receptors, platelet-derived growth factor receptors, and Kit receptors.




9.5.2 Drugs that block cancer blood vessel growth (anti angiogenics)

When it has reached 1 to 2mm across, a tumor needs to grow its own blood vessels in order to continue to get bigger. Some cancer cells make a protein called vascular endothelial growth factor (VEGF). The VEGF protein attaches to receptors on cells that line the walls of blood vessels within the tumour.

Drugs that block blood vessel growth factor

Some drugs block vascular endothelial growth factor (VEGF) from attaching to the receptors on the cells that line the blood vessels. This stops the blood vessels from growing.

A drug that blocks VEGF is bevacizumab (Avastin). It is also a monoclonal antibody.

Drugs that block signalling within the cell

Some drugs stop the VEGF receptors from sending growth signals into the blood vessel cells. These treatments are also called cancer growth blockers or tyrosine kinase inhibitors (TKIs).

Sunitinib (Sutent) is a type of TKI that blocks the growth signals inside blood vessel cells. It is used to treat kidney cancer and a rare type of stomach cancer called gastrointestinal stromal tumour (GIST).

Drugs that affect signals between cells

Some drugs act on the chemicals that cells use to signal to each other to grow. This can block the formation of blood vessels. Drugs that works in this way include thalidomide and lenalidomide (Revlimid).

Each drug has different side effects. You can look up the name of your drug in our cancer drug section to find out about the side effects you may have.

To find trials using anti angiogenesis treatment go to our clinical trials database and type ‘angiogenesis’ into the search box.

Tumors can cause their blood supply to form by giving off chemical signals that stimulate angiogenesis. Tumors can also stimulate nearby normal cells to produce angiogenesis signaling molecules. The resulting new blood vessels “feed” growing tumors with oxygen and nutrients, allowing the cancer cells to invade nearby tissue, to move throughout the body, and to form colonies of cancer cells, called metastases. Because tumors cannot grow beyond a certain size or spread without a blood supply, scientists are trying to find ways to block tumor angiogenesis.

Angiogenesis requires the binding of signaling molecules, such as vascular endothelial growth factor (VEGF), to receptors on the surface of normal endothelial cells. When VEGF and other endothelial growth factors bind to their receptors on endothelial cells, signals within these cells are initiated that promote the growth and survival of new blood vessels.

Angiogenesis inhibitors interfere with various steps in this process. For example, bevacizumab (Avastin®) is a monoclonal antibody that specifically recognizes and binds to VEGF (1). When VEGF is attached to bevacizumab, it is unable to activate the VEGF receptor. Other angiogenesis inhibitors, including sorafenib and sunitinib, bind to receptors on the surface of endothelial cells or to other proteins in the downstream signaling pathways, blocking their activities (2).

The U.S. Food and Drug Administration (FDA) has approved bevacizumab to be used alone forglioblastoma that has not improved with other treatments and to be used in combination with other drugs to treat metastatic colorectal cancer, some non-small cell lung cancers, and metastatic renal cell cancer. Bevacizumab was the first angiogenesis inhibitor that was shown to slow tumor growth and, more important, to extend the lives of patients with some cancers.

The FDA has approved other drugs that have antiangiogenic activity, including sorafenib (Nexavar®), sunitinib(Sutent®), pazopanib (Votrient®), and everolimus (Afinitor®). Sorafenib is approved for hepatocellular carcinoma and kidney cancer, sunitinib and everolimus for both kidney cancer and neuroendocrine tumors, and pazopanib for kidney cancer.

Angiogenesis inhibitors are unique cancer-fighting agents because they tend to inhibit the growth of blood vessels rather than tumor cells. In some cancers, angiogenesis inhibitors are most effective when combined with additional therapies, especially chemotherapy. It has been hypothesized that these drugs help normalize the blood vessels that supply the tumor, facilitating the delivery of other anticancer agents, but this possibility is still being investigated.

Angiogenesis inhibitor therapy does not necessarily kill tumors but instead may prevent tumors from growing. Therefore, this type of therapy may need to be administered over a long period.

Initially, it was thought that angiogenesis inhibitors would have mild side effects, but more recent studies have revealed the potential for complications that reflect the importance of angiogenesis in many normal body processes, such as wound healing, heart and kidney function, fetal development, and reproduction. Side effects of treatment with angiogenesis inhibitors can include problems with bleeding, clots in the arteries (with resultant stroke or heart attack), hypertension, and protein in the urine (35). Gastrointestinal perforation and fistulas also appear to be rare side effects of some angiogenesis inhibitors.

In addition to the angiogenesis inhibitors that have already been approved by the FDA, others that target VEGF or other angiogenesis pathways are currently being tested in clinical trials (research studies involving patients). If these angiogenesis inhibitors prove to be both safe and effective in treating human cancer, they may be approved by the FDA and made available for widespread use.

In addition, phase I and II clinical trials are testing the possibility of combining angiogenesis inhibitor therapy with other treatments that target blood vessels, such as tumor-vascular disrupting agents, which damage existing tumor blood vessels (6).

9.5.3 Recent Advances in Anti-Angiogenic Therapy of Cancer

Rajeev S. Samant and Lalita A. Shevde
Oncotarget. 2011 Mar; 2(3): 122–134.

More than forty anti-angiogenic drugs are being tested in clinical trials all over the world. This review discusses agents that have approved by the FDA and are currently in use for treating patients either as single-agents or in combination with other chemotherapeutic agents.

Tumor angiogenesis is generation of a network of blood vessels within the cancerous growth. This process can occur two ways: The more accepted model involves the release of signaling molecules by the tumor cells; these molecules activate the surrounding tissue to promote growth of new blood vessels. This stimulates vascular endothelial cells to divide rapidly [910]. The other model proposes the generation of new vasculature by vasculogenic mimicry. This model argues that the tumor cells trans-differentiate in endothelial-like cells and create structures from inside of the tumor tapping into a nearby blood vessel [4].

Escape of the tumor cell from the confines of the primary tumor to distant body parts is the pre-requisite for hematogenous metastasis. This escape route is provided by the tumor vasculature. Thus, it was envisioned that inhibition of angiogenesis will also lead to inhibition of metastasis. This phenomenon was demonstrated by very elegant mouse model studies using angiostatin [1112]. Angiostatin was also demonstrated to be secreted by some primary tumors leading to restricted growth of the metastasis leading to “dormancy” of the metastasis. Mice deficient in angiogenesis (Id1 & Id3 deficient) showed significantly less tumor take rates [13]. Independent studies showed absence of metastasis in angiogenesis deficient mice [1415]. Defective angiogenesis was attributed to impaired VEGF-dependent recruitment of precursor endothelial cells from the bone marrow to the newly developing tumor vasculature [16].

Metastasis of malignant tumors to regional lymph nodes is one of the early signs of cancer spread in patients, and it occurs at least as frequently as hematogenous metastasis [17]. Particularly, in cancers, such as breast cancer, lymphatic metastasis is a predominant route for tumor spread. The contribution of lymphatic system to the tumor growth is an area that is relatively less studied. However, lymphatic vessels are speculated to contribute to tumor growth and metastasis in a variety of ways. The VEGF, FGF2 and PDGF produced by vascular endothelial cells are proposed to be involved in the activation of lymphatic endothelial cells, which in turn produce matrix metalloproteases and urokinase plasminogen activator (uPA) that can promote malignant tumor growth. Thus, there exists a synergistic crosstalk between the tumor and the lymphatic vessels and blood vessels.

Angiogenesis is a complex and intricately regulated process. Like all other regulated biological phenomena, angiogenesis has activators or pro-angiogenic factors and inhibitors or anti-angiogenic factors [9].

The Activators

Tumor cells activate signaling pathways that promote uncontrolled proliferation and survival. These include the PI3K/AKT/mTOR pathway, Hedgehog pathway and, Wnt pathway [1824] that produce pro-angiogenic signaling intermediates [2526]. Among the several reported activators of angiogenesis present in cells two proteins appear to be the most important for sustaining tumor growth: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF and bFGF are secreted by the tumor into the surrounding tissue. They bind to their cognate receptors on endothelial cells. This activates a signaling cascade that transmits a nuclear signal prompting target genes to activate endothelial cell growth. Activated endothelial cells also produce matrix metalloproteinases (MMPs). These MMPs break down the extracellular matrix and allow the migration of endothelial cells. The division and migration of the endothelial cells leads to formation of new blood vessels [2728].

The Inhibitors

If angiogenesis is so critical for the tumor growth, then agents that inhibit angiogenesis would have great therapeutic value. With the discovery of endostatin, the concept of anti-angiogenic therapy was launched and popularized by Dr. Folkman [29]. Angiogenesis inhibitors have been discovered from a variety of sources. Some are naturally present in the human body e.g. specific fragments of structural proteins such as collagen or plasminogen (angiostatin, endostatin, tumstatin) [30]. Others are natural products in green tea, soy beans, fungi, mushrooms, tree bark, shark tissues, snake venom etc. [31]. A plethora of synthetic compounds are also characterized to have anti-angiogenic properties [32].


Since angiogenesis is an event critical to primary tumor growth as well as metastasis, anti-angiogenic treatment of tumors is a highly promising therapeutic avenue [33]. Thus, for over last couple of decades, there has been a robust activity aimed towards the discovery of angiogenesis inhibitors [3435]. More than forty anti-angiogenic drugs are being tested in human cancer patients in clinical trials all over the world. From the several anti-angiogenic agents reported, we have focused this review on discussing those agents that have received FDA approval in the United States and are currently in use for treating patients either as a single-agent or in combination with other chemotherapeutic agents (Figure ​(Figure1).1). Based on functionality, the anti-angiogenic drugs can be sub-divided into three main groups:

angiogenesis inhibitors oncotarget-02-122-g001

angiogenesis inhibitors oncotarget-02-122-g001

Figure 1

Targets of FDA-approved angiogenesis inhibitors: Angiogenesis inhibitors impact both, the tumor as well as the endothelial cells resulting in the disruption of the effects of the microenvironment in promoting tumor growth and angiogenesis

Drugs that inhibit growth of endothelial cells

e.g. Endostatin and combretastatin A4, cause apoptosis of the endothelial cells [36]. Thalidomide is also a potent inhibitor of endothelial cell growth [37].

Drugs that block angiogenesis signaling

e.g. anti-VEGF antibodies (Avastin, FDA approved for colorectal cancer), Interferon-alpha (inhibits the production of bFGF and VEGF) [36].

Drugs that block extracellular matrix breakdown

e.g. inhibitors of MMPs [38].


Conventional chemotherapy is usually a systemic therapy that tries to capture a narrow therapeutic window offered by rapid proliferation of tumor cells compared to the normal cells. Chemotherapy has significant side effects such as hair loss, diarrhea, mouth ulcer, infection, and low blood counts. Anti-angiogenic therapy has several advantages over chemotherapy as it is mostly not directed towards directly killing cells but stopping the blood vessel formation, an event that is rare in tissues other than growing tumor. Hence it is well tolerated by the patients and has fewer side effects [29]. There are currently seven approved anti-cancer therapies in two primary categories:

  1. Monoclonal antibodies directed against specific pro-angiogenic growth factors and/or their receptors
  2. Small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors.

Besides these, inhibitors of mTOR (mammalian target of rapamycin), proteasome inhibitors and thalidomide have also been reported to indirectly inhibit angiogenesis through mechanisms that are not completely understood.


Four monoclonal antibody therapies are approved to treat several tumor types:

Bevacizumab (Avastin®)

The first FDA approved angiogenesis inhibitor, Avastin is a humanized monoclonal antibody that binds biologically active forms of vascular endothelial growth factor (VEGF) and prevents its interaction with VEGF receptors (VEGFR-1 and VEGFR-2), thereby inhibiting endothelial cell proliferation and angiogenesis. Bevacizumab has been tested in phase I studies in combination with chemotherapy with a good safety profile [39]. This treatment is approved for metastatic colorectal cancer and non-small cell lung cancer [4043]. Bevacizumab has also evolved as a first line of treatment in combination with paclitaxel in breast cancer patients by virtue of its ability to double median progression-free survival (PFS) [44]. In combination with chemoendocrine therapy (including capecitabine and vinorelbine, and letrozole) bevacizumab treatment significantly decreased the percentage of viable circulating endothelial cells and prevented the chemotherapy-induced mobilization of circulating progenitors [45]. In combination with irinotecan, bevacizumab significantly increased PFS in glioma patients [4647]. VEGF has emerged as a compelling therapeutic target for leukemias. Inhibition of angiogenesis in hematological malignancies interdicts the angiogenesis within the bone marrow ecosystem comprised of multiple cell types, including fibroblasts, endothelial progenitor cells, endothelial cells, dendritic cells and, malignant cells, blocking the availability of nutrients to cancer cells and disrupting crosstalk between the various cell types to curtail the malignant phenotype [48].

Cetuximab (Erbitux®)

This is a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), preventing ligand binding and activation of the receptor resulting in internalization and degradation of the receptor culminating in inhibition of cell proliferation and angiogenesis. Cetuximab downregulated VEGF expression in a dose-dependent manner in a human colorectal carcinoma (CRC) cell line and in human CRC mouse xenografts [49]. The xenografts also showed a significant reduction in blood vessel counts following several rounds of cetuximab treatment [49], indicating that the tumor-promoting effects of EGFR overexpression may be mediated through VEGF stimulation and tumor angiogenesis. This treatment is approved for metastatic CRC and head and neck cancer [50] in patients who are refractory to irinotecan-based chemotherapy. In combination with irinotecan (an inhibitor of topoisomerase I), cetuximab is the first monoclonal antibody that has been approved by the FDA as second-line treatment for metastatic colorectal cancer [5152]. In Phase I and Phase III trials [5354] cetuximab significantly improved the effects of radiotherapy in patients with unresectable (cannot be removed by surgery) squamous cell carcinoma of the head and neck (SCCHN). Cetuximab has also been shown to sensitize cells to radiation and chemotherapy, potentially through blocking EGFR nuclear import and the associated activation of DNA protein kinase enzymes necessary for repairing radiation- and chemotherapy-induced DNA damage [55]. Compared to radiation alone, cetuximab plus radiation therapy can nearly double the median survival in patients with a certain kind of head and neck cancer that has not spread to other parts of the body [54] making cetuximab the only drug achieving interesting response rate in second line treatment of advanced SCCHN [56]. Cetuximab was also found to be tolerated well in combination with cisplatin, or carboplatin, and fluorouracil [5758].

Panitumumab (Vectibix™)

It is a fully humanized anti-EGFR monoclonal antibody that binds specifically to the human EGFR. Panitumumab is a recombinant human monoclonal antibody [59]; therefore, the risk of an infusion reaction is minimized. Vectibix® is indicated as a single agent for the treatment of EGFR-expressing, metastatic colorectal carcinoma with disease progression on or following fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens [6062]. The effectiveness of Vectibix® as a single agent for the treatment of EGFR-expressing, metastatic CRC is based on progression-free survival [6364]. Panitumumab is used in patients who are not responding to regimens containing fluorouracil, oxaliplatin, and irinotecan [60]. Patients often receive panitumumab after receiving bevacizumab or cetuximab. Panitumumab can be given with FOLFOX (oxaliplatin, leucovorin, and fluorouracil) or FOLFIRI (irinotecan, leucovorin, and fluorouracil) regimens, or as a single agent. Currently no data are available that demonstrate an improvement in disease-related symptoms or increased survival with Vectibix® in colon cancer [65]. This drug is also being tested for aerodigestive track and head and neck cancer [6667].

Trastuzumab (Herceptin®)

Is a humanized monoclonal antibody that binds the extracellular domain of HER-2, which is overexpressed in 25-30% of invasive breast cancer tumors [68]. HER2-positive breast cancer is highly aggressive disease with high recurrence rate, poorer prognosis with decreased survival compared with HER2-negative breast cancer [69]. Herceptin® is designed to target and block the function of HER2 protein overexpression. This is the first humanized antibody is approved for Breast cancer [70]. Herceptin® is approved by the FDA to treat HER2 positive breast cancer that has metastasized after treatment with other anticancer drugs [71]. It is also approved to be used with other drugs to treat HER2-positive breast cancer that has spread to the lymph nodes to be used after surgery. The FDA first approved Herceptin in September 1998 [7173]. In November 2006, the FDA approved Herceptin as part of a treatment regimen containing doxorubicin, cyclophosphamide and paclitaxel, for the adjuvant treatment of patients with HER2-positive, node-positive breast cancer ( In January 2008, the FDA approved Herceptin as a single agent for the adjuvant treatment of HER2-overexpressing node-negative (ER/PR-negative or with one high-risk feature) or node-positive breast cancer, following multi-modality anthracycline-based therapy ( Trastuzumab is also being studied in the treatment of other types of cancers such as pancreatic [74], endometrial [75], lung [76], cervical [77] and ovarian cancer [78]


Protein tyrosine kinases have emerged as crucial targets for therapeutic intervention in cancer especially because they play an important role in the modulation of growth factor signaling. As per (, there are 43 ongoing studies on tyrosine kinase inhibitors in angiogenesis. Since discussing all of them is beyond the scope of this article, we have focused our discussion on the three TKIs that are currently approved as anti-cancer therapies:

Erlotinib (Tarceva®)

Erlotinib hydrochloride (originally coded as OSI-774) is an orally available, potent, reversible, and selective inhibitor of the EGFR (ErbB1) tyrosine kinase activity. Erlotinib hydrochloride has been approved by FDA for treatment of patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen [7980]. Interesting recent studies have demonstrated that since Erlotinib and Bevacizumab act on two different pathways critical to tumor growth and dissemination, administering these drugs concomitantly may confer additional clinical benefits to cancer patients with advanced disease. This combination therapy may prove to be a viable second-line alternative to chemotherapy in patients with NSCLC [81]. Also, for patients with locally advanced, unresectable or metastatic pancreatic carcinoma, Erlotinib has received FDA approval for the treatment in combination with gemcitabine [8283]. Erlotinib is also being studied in the treatment of other types of cancers. For example combination of Erlotinib with Bevacizumab has been evaluated in metastatic breast cancer [84], hepatocellular carcinoma [85] and in metastatic renal cancer [86] as phase II trials. Outcomes for prostate, cervical and colorectal cancers treated with Erlotinib are cautiously optimistic [8789].

Sorafenib (Nexavar®)

Sorafenib is an orally active inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, and Raf-1 tyrosine kinase activity [90]. It has received the approval of FDA for the treatment of patients with unresectable hepatocellular carcinoma [91] and advanced renal cell carcinoma [92]. However, not all advanced hepatocellular carcinoma patients were able to tolerate sorafenib and some patients experienced tumor progression [91]. Sorafenib has shown improvements in PFS in patients with renal cell carcinoma [93]. It is one of the aggressively studied drugs. According to the NCI clinical trials search results, there are about 168 active clinical trials involving sorafenib in a variety of cancers.

Sunitinib (Sutent®)

Sunitinib targets activity of multiple tyrosine kinases such as VEGFR-1, VEGFR-2, VEGFR-3, PDGFR- β, and RET [94]. It is approved by FDA as Sunitinib malate for treating advanced (metastatic) renal cell carcinoma [95]. It is also approved by FDA for gastrointestinal stromal tumor (GIST) in patients whose disease has progressed or who are unable to tolerate treatment with imatinib (Gleevec), the current treatment for GIST patients [9596]. Sunitinib has shown early evidence of anti-tumor activity in Phase II trials in US, European and Asian patients with locally advanced, unresectable and metastatic hepatocellular carcinoma. A Phase III trial of sunitinib in hepatocellular carcinoma is ongoing [97]. According to the NCI clinical trials search results, Sunitinib is currently evaluated in about 150 active clinical trials. It is evaluated for ovarian [98], breast [99] and non small cell lung cancer [100] among others [101].

Inhibitors of mTOR

mTOR plays a part in the PI3 kinase/AKT pathway involved in tumor cell proliferation and angiogenesis [102]. Rapamycin and related mTOR inhibitors inhibit endothelial cell VEGF expression, as well as VEGF-induced endothelial cell proliferation [103]. Inhibitors of mTOR are an important class of anti-angiogenic agents. These include: deforolimus, everolimus, rapamycin (sirolimus), and temsirolimus [104105]. Temsirolimus (Toricel™) is a small molecule inhibitor of mTOR, approved for treating advanced renal cell carcinoma [106]. It is a type of rapamycin analog and a type of serine/threonine kinase inhibitor, it is also called CCI-779. In pre-clinical models combination therapy for treating breast cancer using anti-estrogen, ERA-923, and temsirolimus has been successfully tested [107]. It is found to be highly effective against human melanoma when tested in combination with cisplatin and DTIC (in independent studies) in a SCID mouse xenotranplantation model [108109]. There are over 41 active studies of Temsirolimus for a variety of solid tumors [110]. mTOR inhibition has also been strongly advocated in as a putative cancer therapeutic strategy for urologic malignancies [111]. In a pilot study (6 patients) with imatinib-resistant CML, rapamycin induced major and minor leukocyte responses, with an observed decrease in the mRNA levels of VEGFA in circulating leukaemic cells [112]. Combination treatments for breast cancer with aromatase inhibitor [113] and letrozol [114] are also being evaluated. Rapamycin treatment brought partial responses (>50% reduction in the absolute number of blood blasts) and stable disease in adult refractory/relapsed AML [115]. In a recent report, Deforolimus was studied in a Phase 2 trial in pretreated patients with various hematological malignancies, including ALL, AML, CLL, CML, MDS, agnogenic myeloid metaplasia, mantle cell lymphoma and T-cell leukemia/lymphoma [116]. Overall, 40% of deforolimus-treated patients experienced hematological improvement or stable disease.


Bortezomib (Velcade®)

Is a proteasome inhibitor that disrupts signaling of cancer cells, leading to cell death and tumor regression. It is the first compound in its class to be used in clinical practice. It has indirect anti-angiogenic properties [117]. While its exact mechanism is not understood, it induces the pro-apoptotic BH3-only family member NOXA in a p53 independent fashion triggering of a caspase cascade culminating in apoptosis in melanoma and myeloma cells [118]. It is FDA-approved for the treatment of myeloma that has relapsed after two prior treatments (or where resistance has developed following the last treatment). It was also found to induce high quality responses as third line salvage therapy with acceptable toxicity in a significant proportion of homogeneously pre-treated myeloma patients with progressive disease after autologous transplantation and thalidomide. [119]. In a Phase 3 trial involving 669 myeloma patients treated with at least one prior therapy, bortezomib increased median, improved overall survival, and increased response rate, compared with high-dose dexamethasone [120]. In combination with doxorubicin and gemcitabine, bortezomib was also found to be effective in heavily pretreated, advanced Cutaneous T cell Lymphomas (CTCL) [121]. Bortezomib was also reported to be active as a single agent for patients with relapsed/refractory CTCL and Peripheral T Cell Lymphoma (PTCL) with skin involvement [122]. On the contrary, the use of bortezomib was discouraged after a phase II study revealed that found in combination with dexamethasone, bortezomib is not active in heavily pre-treated patients with relapsed Hodgkin’s lymphoma [123124].

Thalidomide (Thalomid®)

Possesses immunomodulatory, anti-inflammatory, and anti-angiogenic properties, although the precise mechanisms of action are not fully understood. Thalidomide was the first angiogenesis inhibitor to demonstrate clinical efficacy in multiple myeloma [37125]. Specifically in myeloma, thalidomide down-regulated VEGF secretion from bone marrow endothelial cells obtained from patients with active disease. In a landmark Phase 2 clinical trial, 169 previously treated patients with refractory myeloma received thalidomide monotherapy [126]. Partial response, was achieved in 30% of patients, and 14% achieved a complete or nearly complete remission. The survival rate at 2 years was 48%. These results led to many subsequent clinical studies of thalidomide in myeloma, leading ultimately to FDA approval of the drug in 2006, for the treatment of newly diagnosed multiple myeloma, in combination with dexamethasone. In the pivotal Phase 3 trial, the response rate in patients receiving thalidomide plus dexamethasone was 63% compared to 41% with dexamethasone alone [127]. Long-term outcome measures, including time-to-progression (TTP) and PFS, were recently reported for a 470 patient randomized, placebo-controlled Phase 3 clinical trial of a similar protocol in newly diagnosed multiple myeloma, with comparable overall response rates [128]. Significant increases resulted in both median TTP and median PFS for the thalidomide plus dexamethasone group versus dexamethasone alone.

Thalidomide was found to be moderately tolerated and minimally effective in patients with histologically proven advanced hepatocellular carcinoma [129]. Thalidomide provided no survival benefit for patients with multiple, large, or midbrain metastases when combined with WBRT (whole-brain radiation therapy) [130]. On the contrary, thalidomide did not significantly add to the efficacy of the fludarabine, carboplatin, and topotecan (FCT) regimen in poor prognosis AML patients [131] and was also ineffective in improving prognosis or decreasing plasma VEGF levels in patients with persistent or recurrent leiomyosarcoma of the uterus [132].


While conventional anti-angiogenic therapy is based on Maximum Tolerated Doses (MTD), the cells involved in angiogenesis may regenerate during the three- to four-week interval between cycles of the chemotherapy. Taking advantage of the fact that endothelial cells are about 10–100 times more susceptible to chemotherapeutic agents than cancer cells, therapy based on daily, oral, low-dose chemotherapeutic drugs was designed. Metronomic chemotherapy refers to the close, rhythmic administration of low doses of cytotoxic drugs, with minimal or no drug-free breaks, over prolonged periods. Metronomic therapy appears promising mainly due to the fact that its anti-angiogenic and anti-tumorigenic effects are accompanied by low toxicity, limited side effects, no need for hospitalization and allowing for feasible combinations with selective inhibitors of angiogenesis. There are several foreseeable advantages and opportunities for metronomic chemotherapy: activity against the parenchymal and stromal components, pro-apoptotic activity, reduction of the likelihood of emergence of acquired resistance, feasibility of long term administration and acceptable systemic side effects [133]. In a pilot phase II study conducted by Correale et al [134] to investigate the toxicity and activity of the novel metronomic regimen of weekly cisplatin and oral etoposide in high-risk patients with NSCLC, the objective response rate was 45.2%, disease control was 58.1%, meantime to progression and survival were 9 and 13 months, respectively. Pharmacokinetic analysis showed that this regimen allowed a greater median monthly area under the curve of the drugs than conventional schedules. In a Phase I trial of metronomic dosing of docetaxel and thalidomide, of the 26 patients with advanced tumors enrolled, prolonged freedom from disease progression was observed in 44.4% of the evaluable patients [135].

Circulating endothelial progenitor cells (EPCs) also participate in tumor angiogenesis. In a study comparing the effects of metronomic chemotherapy over conventional dose-dense chemotherapy, it was found that the numbers of circulating EPCs and the plasma levels of VEGF increased sharply, doubling pre-therapeutic levels at day 21 after conventional chemotherapy, whereas under low-dose metronomic chemotherapy, the numbers of circulating EPCs decreased significantly and VEGF plasma concentrations remained unchanged. These observations provide evidence that conventional dose-dense chemotherapy leads to rebound EPC mobilization even when given with adjuvant intention, while low-dose metronomic scheduling of cytotoxic substances such as trofosfamide may sharply reduce EPC release into the circulation. [136].

Combined bevacizumab and metronomic oral cyclophosphamide was also discovered to be a safe and effective regimen for heavily pre-treated ovarian cancer patients [137]. Treatment with metronomic capecitabine and cyclophosphamide in combination with bevacizumab was shown to be effective in advanced breast cancer and additionally was minimally toxic [138]. Metronomic treatment with carboplatin and vincristine associated with fluvastatin and thalidomide significantly increased survival of pediatric brain stem tumor patients. Tumor volume showed a significant reduction accompanied by increased quality of life [139]. Thus, given the fact that the most evident effect of selective anti-angiogenic agents (i.e. bevacizumab) is the significant prolonging of the duration of response obtainable by chemotherapy alone, with minimal possible side effects of cytotoxic agents given in association metronomic chemotherapy should be considered both as novel up-front or maintenance treatment in patients with biologically poorly aggressive advanced cancer diseases [140].

Overall, metronomic chemotherapy was able to induce tumor stabilization and prolong the duration of clinical benefit, without much associated toxicity. Emerging evidence suggests that metronomic chemotherapy could also activate the host immune system and potentially induce tumor dormancy [141143].


While angiogenesis as a hallmark of tumor development and metastasis is now a validated target for cancer treatment, the overall benefits of anti-angiogenic drugs from the perspective of impacting survival have left much to desire, endorsing a need for developing more effective therapeutic regimens e.g., combining anti-angiogenic drugs with established chemotherapeutic drugs [144145]. There are now several agents that target the tumor vasculature through different pathways, either by inhibiting formation of the tumor neovasculature or by directly targeting the mature tumor vessels. The main body of evolving evidence suggests that their effects are compounded by their synergistic use with conventional chemotherapy rather than individual agents. Anti-angiogenic drugs such as bevacizumab can bring about a transient functional normalization of the tumor vasculature. This can have an additive effect when co-administered with chemo/radiotherapy. But long term inhibition of angiogenesis reduces tumor uptake of co-administered chemotherapeutic agents. This underscores the need for discovering new targets for anti-angiogenic therapy in order to effectively prohibit angiogenesis and circumvent mechanisms that contribute to resistance mechanisms that emerge with long term use of anti-angiogenic therapies. It also warrants a need to define reliable surrogate indicators of effectiveness of the anti-angiogenic therapy as well as dependable markers for identifying the patients who are most likely to benefit from the combination of anti-angiogenic therapy and conventional chemotherapy.

Several new frontiers are emerging. New advances in understanding endothelial cells, which constitute the tumor vasculature, towards developing antiangiogenic strategies are one of the important ones [146147]. Novel cellular targets such as integrins and microRNAs and novel treatment options such as possible use of pharmaconutrients to modulate angiogenic pathways need careful testing and evaluation [148151]. Finally, the administration of these drugs in a metronomic schedule is likely to improve the overall response to anti-angiogenic drugs making it feasible to administer them with conventionally toxic chemotherapeutic drugs, thus increasing the armamentarium of drug combinations that can be employed for treatment.

9.5.4 Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

El-Kenawi AE1, El-Remessy AB.
Br J Pharmacol. 2013 Oct; 170(4):712-29.

Angiogenesis, a process of new blood vessel formation, is a prerequisite for tumor growth to supply the proliferating tumor with oxygen and nutrients. The angiogenic process may contribute to tumour progression, invasion and metastasis, and is generally accepted as an indicator of tumor prognosis. Therefore, targeting tumor angiogenesis has become of high clinical relevance. The current review aimed to highlight mechanistic details of anti-angiogenic therapies and how they relate to classification and treatment rationales. Angiogenesis inhibitors are classified into either direct inhibitors that target endothelial cells in the growing vasculature or indirect inhibitors that prevent the expression or block the activity of angiogenesis inducers. The latter class extends to include targeted therapy against oncogenes, conventional chemotherapeutic agents and drugs targeting other cells of the tumor micro-environment. Angiogenesis inhibitors may be used as either monotherapy or in combination with other anticancer drugs. In this context, many preclinical and clinical studies revealed higher therapeutic effectiveness of the combined treatments compared with individual treatments. The proper understanding of synergistic treatment modalities of angiogenesis inhibitors as well as their wide range of cellular targets could provide effective tools for future therapies of many types of cancer.

Two major processes of blood vessel formation are implicated in the development of vascular system: vasculogenesis and angiogenesis. Vasculogenesis prevails in the embryo and refers to the formation ofde novo blood vessels by in situ differentiation of the mesoderm-derived angioblasts and endothelial precursors. Angiogenesis is the formation of new capillaries from pre-existing vessels and circulating endothelial precursors (Polverini, 2002; Chung et al., 2010; Ribatti and Djonov, 2012). Angiogenesis is a tightly controlled dynamic process that can occur physiologically in those tissues that undergo active remodeling in response to stress and hypoxia (Carmeliet, 2003; Folkman, 2007). However, it can be aberrantly activated during many pathological conditions such as cancer, diabetic retinopathy as well as numerous ischemic, inflammatory, infectious and immune disorders (Carmeliet, 2003; Ali and El-Remessy, 2009; Willis et al., 2011). Although the concept of proposing angiogenesis inhibitors as anticancer drugs received considerable skepticism when first presented by Dr. Folkman in the early 1970s (Folkman, 1971), active research in the field and subsequent clinical trials eventually resulted in US Food and Drug Administration (FDA) approval of bevacizumab for colorectal cancer in 2004 (Cohen et al., 2007). Since then, several angiogenic inhibitors have been identified. This review will provide an overview of the key mechanisms involved in tumor angiogenesis, classification of angiogenesis inhibitors as well as treatment rationales from the mechanistic point of view.

Sustained angiogenesis as a hallmark of cancer

Proliferating tumours tend to activate an angiogenic phenotype to fulfil their increased demand of oxygen and nutrients (Hanahan and Folkman, 1996; Carmeliet, 2005). Additionally, paracrine release of anti-apoptotic factors from activated endothelial cells in the newly formed vasculature supplies tumour cells with a survival privilege (Folkman, 2003). Consequently, in order to progress, tumors tend to activate an event called ‘angiogenic switch’ by shifting the balance of endogenous angiogenesis inducers and inhibitors towards a pro-angiogenic outcome. As a result, dormant lesion progresses into outgrowing vascularized tumor and eventually into a malignant phenotype (Hanahan and Folkman, 1996; Baeriswyl and Christofori, 2009). Hypoxia drives such imbalance through up-regulation of the transcription factor hypoxia inducible factor-1α (HIF-1α), which in turn increases the expression of many angiogenesis inducers as well as suppresses the expression of endogenous angiogenesis inhibitors (Pugh and Ratcliffe, 2003). In spite of that, accumulating evidence indicates that angiogenic cascade can be also driven by alternative HIF-1-independent pathways (Mizukami et al., 2007; Arany et al., 2008; Lee, 2013).

As summarized in Table 1, the angiogenesis inducers are a wide range of mediators that include many growth factors, a plethora of cytokines, bioactive lipids, matrix-degrading enzymes and a number of small molecules (Folkman, 1995; Folkman, 2003; Lopez-Lopez et al., 2004; Bouis et al., 2006; El-Remessy et al., 2007; Bid et al., 2011; MacLauchlan et al., 2011; Murakami, 2011; Fagiani and Christofori, 2013; Qin et al., 2013). Pro-angiogenic growth factors mostly activate a series of surface receptors in a series of paracrine and autocrine loops with the VEGF-A signaling representing the critical rate-limiting step, physiologically and pathologically. VEGF-A (traditionally known as VEGF) is the most potent VEGF isoform that acts mainly on VEGF receptor 2 (VEGFR2) to mediate vascular permeability, endothelial proliferation, migration and survival (Takahashi and Shibuya, 2005; Bouis et al., 2006). In spite of the well-established master roles of VEGF signaling in literature, those processes are probably accomplished through a highly regulated interplay between VEGF and other pro-angiogenic factors. In this context, basic fibroblast growth factor (bFGF) activation of the endothelium is required for maintenance of VEGFR2 expression and the ability to respond to VEGF stimulation (Murakami et al., 2011). Similarly, sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid that can directly contribute to tumor angiogenesis (reviewed in Sabbadini, 2011), is needed for VEGF-induced blood vessel formation, indicating the cooperation between S1P and VEGF in tumor angiogenesis (Visentin et al., 2006). As a net result, the pro-angiogenic interplay of those ligands and others dominates over the activities of two dozen endogenous angiogenesis inhibitors that can be either matrix-derived inhibitors or non–matrix-derived inhibitors (Nyberg et al., 2005).

Table 1. Pro-angiogenic mediators implicated in tumor angiogenesis

Category Examples References
Growth factors VEGFs Bouis et al., 2006
FGFs Ibid
TGFs Ibid
PDGFs Ibid
Insulin-like growth factors Lopez-Lopez et al., 2004; Bid et al., 2011
ANGs Fagiani and Christofori, 2013
Cytokines IL-8 Strieter et al., 2004
CSF-1 Lin et al., 2006
Bioactive lipids PGE2 Wang and Dubois, 2010
S1P Murakami, 2011
Matrix-degrading enzymes MMPs Bourboulia and Stetler-Stevenson, 2010
Heparanases Vlodavsky and Friedmann, 2001
Small mediators NO MacLauchlan et al., 2011
Peroxynitrite El-Remessy et al., 2007
Serotonin Qin et al., 2013
Histamine Qin et al., 2013

The multistep angiogenic process starts with vasodilation and increased permeability of existing vessels in response to tumor cell-secreted VEGF. This is accompanied by loosening of pericytes covering mediated by angiopoietin-2 (ANG2), a ligand of tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (TIE2) receptor (Bergers and Benjamin, 2003; Jain, 2003; Fagiani and Christofori, 2013). Meanwhile, many secreted matrix-degrading enzymes, such as MMPs and heparanases, function in concert to dissolve the basement membrane and to remodel the extracellular matrix (ECM) as well as to liberate more pro-angiogenic growth factors (bFGF and VEGF) from matrix heparan sulfate proteoglycans (HSPGs) respectively (Houck et al., 1992; Whitelock et al., 1996; Vlodavsky and Friedmann, 2001; Tang et al., 2005; van Hinsbergh and Koolwijk, 2008). The overall chemotactic angiogenic stimuli guide endothelial cells to migrate, to align into tube-like structures and to eventually form new blood vessels. However, such blood vessels are characterized by being disorganized, chaotic, hemorrhagic and poorly functioning (Bergers and Benjamin, 2003).

The angiogenic phenotype in tumor micro-environment can further be sustained and extravagated by the recruitment of other types of stromal cells. Stromal cells such as fibroblasts, mesenchymal stem cells and various bone marrow-derived myeloid cells including macrophages, TIE2-expressing monocytes, neutrophils and mast cells contribute to tumor angiogenesis through their production of growth factors, cytokines and proteases (Murdoch et al., 2008; Joyce and Pollard, 2009; Cirri and Chiarugi, 2011). For example, in response to cancer cell-derived TGF-β, PDGF or bFGF, fibroblasts are transformed to an activated phenotype with a higher proliferative activity and myofibroblastic characteristics (Kalluri and Zeisberg, 2006; Cirri and Chiarugi, 2011). Such carcinoma-associated fibroblasts (CAFs) were shown to promote angiogenesis and metastasis by secreting large amounts of MMP-2 and MMP-9 as well as by expressing many cytokines and chemokines that resulted in immune cell infiltration (Gerber et al., 2009; Giannoni et al., 2010). Furthermore, it has been shown that PDGF-C produced by CAFs is able to elicit VEGF production from tumor cells, thereby sustaining the angiogenic shift (Crawford et al., 2009). Similarly, tumor-associated macrophages (TAMs), one of the bone marrow myeloid-derived cells, are induced to develop into polarized type II (alternatively activated or M2 macrophages), upon exposure to tumor hypoxia and tumor cell-derived cytokines (Leek et al., 2002; Rogers and Holen, 2011). M2 macrophages tend to produce many pro-angiogenic growth factors, cytokines and matrix-degrading enzymes such as VEGF, PDGF, bFGF, TNF-α, COX-2, MMP-9, MMP-7 and MMP-12 (Lewis and Pollard, 2006).

From another perspective, angiogenesis may be dispensable for progression of some malignancies. For example, some tumours may co-opt pre-existent vessels as an alternative way to obtain blood supply. Vessel co-option was first described in the brain, one of the most densely vascularized organs, in which tumours may develop in earlier stages without the activation of angiogenic response (Holashet al., 1999; Leenders et al., 2002; Bergers and Benjamin, 2003; Hillen and Griffioen, 2007). In another example, hypovascularized tumors such as pancreatic ductal adenocarcinoma may involve certain adaptation to flourish in the absence of prominent angiogenesis (Bergers and Hanahan, 2008). Obviously, in both cases, tumors may be intrinsically indifferent to angiogenesis inhibitors. However, in most other cases, therapy directed towards the vasculature of solid tumors is being considered as an important direction in cancer treatment.

Classification of angiogenesis inhibitors

Growth of newly formed vessels in tumor micro-environment can be inhibited directly by targeting endothelial cells in the growing vasculature or indirectly by targeting either tumor cells or the other tumor-associated stromal cells. Therefore, angiogenesis inhibitors can be classified into direct and indirect inhibitors (Kerbel and Folkman, 2002; Folkman, 2007).

Direct endogenous inhibitors of angiogenesis

Direct endogenous inhibitors of angiogenesis, such as angiostatin, endostatin, arrestin, canstatin, tumstatin and others, are fragments released on proteolysis of distinct ECM molecules. Endogenous inhibitors prevent vascular endothelial cells from proliferating, migrating in response to a spectrum of angiogenesis inducers, including VEGF, bFGF, IL-8 and PDGF (Kerbel and Folkman, 2002; Abdollahi et al., 2004; Mundel and Kalluri, 2007; Ribatti, 2009). This direct anti-angiogenic effect may be mediated by interference with endothelial integrins along with several intracellular signaling pathways (Mundel and Kalluri, 2007). For example, the ability of tumstatin-derived active peptide to inhibit angiogenesis and tumour growth is associated with the expression of the adhesion receptor, αvβ3 integrin, on tumor endothelial cells (Eikesdal et al., 2008). Through binding αvβ3 integrin, full tumstatin was found to inhibit endothelial cell activation of focal adhesion kinase, PI3K, Akt, mammalian target of rapamycin (mTOR) and others (Maeshima et al., 2002). Direct targeting of those signaling pathways by endogenous inhibitors was thought to be the least likely to induce acquired drug resistance because they target endothelial cells with assumed genetic stability rather than unstable mutating tumour cells (Kerbel and Folkman, 2002). However, endostatin has not yet led to any documented benefit to patients in randomized phase III trials, or even modest activity in phase II trials (Ellis and Hicklin, 2008).

Indirect inhibitors of angiogenesis

Indirect inhibitors of angiogenesis classically prevent the expression or block the activity of pro-angiogenic proteins (Folkman, 2007). For example, Iressa, an EGF receptor (EGFR) TK inhibitor (TKI), blocks tumour expression of many pro-angiogenic factors; bevacizumab, a monoclonal antibody, neutralizes VEGF after its secretion from tumour cells whereas sunitinib, a multiple receptor TKI, blocks the endothelial cell receptors (VEGFR1, VEGFR2 and VEGFR3), preventing their response to the secreted VEGF (Folkman, 2007; Roskoski, 2007). In addition, this class extends to include conventional chemotherapeutic agents, targeted therapy against oncogenes and drugs targeting other cells of the tumor micro-environment (Kerbel et al., 2000; Ferrara and Kerbel, 2005).

Conventional chemotherapeutic agents

Conventional chemotherapeutic agents have been shown to have anti-angiogenic properties in addition to the ability to induce direct cancer cell death. Such chemotherapeutic agents can affect the endothelial cell population in the tumour bed during treatment cycles because they have significantly higher proliferation rates than resting endothelium outside a tumor, making them more susceptible to cytotoxic effect (Kerbel et al., 2000; Folkman, 2003). However, the cyclic treatment rationale of cytotoxic drugs allows the potential damage to the tumour vasculature to be repaired during the long breaks. Thus, continuous low doses of chemotherapeutic agents were suggested as a way to reduce side effects and drug resistance (Drevs et al., 2004). This modality is termed metronomic therapy, and clinically, it refers to the daily administration of 5–10% of the phase II-recommended dose of the chemotherapeutic agent (Penel et al., 2012). The extended use of such low doses of cytotoxic agents elicits an anti-angiogenic activity through induction of endothelial cell apoptosis and decreasing the level of circulating endothelial precursors (Hamano et al., 2004; Shahrzad et al., 2008). In clinical investigations, metronomic dosing of cyclophosphamide and others showed promising efficacy in patients with advanced, multiple metastasized and/or multiple pretreated solid tumours (Lord et al., 2007; Fontana et al., 2010; Nelius et al., 2011; Gebbia et al., 2012; Briasoulis et al., 2013; Navid et al., 2013).

VEGF-targeted therapy

VEGF-targeted therapy includes neutralizing antibodies to VEGF (e.g. bevacizumab) or VEGFRs (e.g. ramucirumab), soluble VEGFR/VEGFR hybrids (e.g. VEGF-Trap) and TKIs with selectivity for VEGFRs (e.g. sunitinib and sorafenib; Baka et al., 2006; Ellis and Hicklin, 2008; Hsu and Wakelee, 2009). Bevacizumab, a humanized monoclonal antibody against all isoforms of VEGF-A, has been approved for the treatment of colorectal, lung, glioblastoma and renal cell carcinoma (Hsu and Wakelee, 2009). Many other clinical trials with promising efficacy were also conducted in other cancers such as head and neck cancer, hepatocellular carcinoma, ovarian cancer, metastatic melanoma and gastric cancer (Argiris et al., 2011; 2013; Burger et al., 2011; Ohtsu et al., 2011; Fang et al., 2012; Minor, 2012; Schuster et al., 2012; Van Cutsem et al., 2012). However, for metastatic breast cancer, bevacizumab had been initially granted an accelerated FDA approval, which was later withdrawn due to lack of improvement evidence in disease-related symptoms or overall survival (Burstein, 2011; Montero et al., 2012). Similarly, clinical trials showed that the addition of bevacizumab to the treatment regimens of advanced pancreatic cancer did not extend overall survival (Chiu and Yau, 2012). The neutralization of VEGF-A can also be achieved by soluble receptor construct (VEGF-Trap) that monomerically ‘traps’ the different isoforms of VEGF-A, in addition to VEGF-B and placental growth factor (Rudge et al., 2007). VEGF-Trap showed clinical benefit in a phase III trial of oxaliplatin pretreated metastatic patients with colorectal cancer, and is currently being investigated in a prostate cancer phase III trial (Gaya and Tse, 2012). TKIs are small molecules with different chemical structures that have the ability to interact physically with the highly conserved kinase domain shared by different VEGFRs as well as PDGF receptors (PDGFRs), FGF receptors (FGFRs), EGFR, Raf kinases and c-Kit (a receptor of the pluripotent cell growth factor, stem cell factor). Such interaction directly inhibits tyrosine phosphorylation and the subsequent many downstream pro-angiogenic signaling networks (Baka et al., 2006; Ivy et al., 2009). Those multi-targeted TKIs demonstrated efficacy against various solid malignancies in different clinical trials, some of which have lead eventually to FDA approval of sunitinib and sorafenib. Sunitinib, known to inhibit several receptor TKs (RTKs) including VEGFR1–3, PDGFR-α, PDGFR-β, c-Kit, colony-stimulating factor-1 receptor (CSF-1R) and Flt-3, was approved for the treatment of renal cell carcinoma and gastrointestinal stromal cell tumours. Sorafenib that acts also by inhibiting VEGFR1–3 and PDGFR-β in addition to the serine–threonine kinases Raf-1, B-Raf, was approved for hepatocellular carcinoma in addition to renal cell carcinoma (Llovet et al., 2008; Ivy et al., 2009; Huang et al., 2010).

FGF-targeted therapies

FGF-targeted therapies were recently reconsidered as promising anti-angiogenic and anti-tumor agents after a long period of little attention for drug development, partly due to redundancy (Bono et al., 2013). The FGFR superfamily with its 18 ligands and four receptors has been involved in endothelial cell migration, proliferation and differentiation (Presta et al., 2005). Therapeutic targeting of FGF/FGFR signalling was accomplished by either monoclonal antibodies that inhibit FGFs binding, small molecules that inhibit FGFR TK activity or allosteric modulators that bind the extracellular FGFR domain. Monoclonal antibodies against bFGF displayed potent anti-tumor and anti-angiogenic effects in different preclinical cancer models, which warrant further clinical evaluation (Zhao et al., 2010; Wang et al., 2012). Pan inhibitors of the FGFR TKs such as AZD4547 (blocks the activity of FGFR1–3) and ponatinib (blocks all the FGFR isoforms) elicited potent anti-tumor activities in preclinical investigations so they are currently being evaluated in clinical trials. Those inhibitors displayed the greatest potency in FGFR-driven cancer models, which may be attributed to the interference with the oncogenic functions of either amplified or constitutively active FGFR (Dutt et al., 2011; Zhao et al., 2011; Gavine et al., 2012; Gozgit et al., 2012). Accordingly, further studies are needed to evaluate the relative contribution of angiogenic versus oncogenic inhibitory mechanisms towards the overall anti-tumor activity. The allosteric antagonist of the FGFR, SSR128129E, showed a strong anti-angiogenic activity in addition to tumour growth and metastasis inhibitory effects in animal models of arthritis and cancer respectively. Because allosteric modulators leave a residual level of baseline signalling, they have the ability to fine-tune target biological responses. As a result, allosteric multi-FGFR inhibitors may have an improved benefit/risk ratio that is not attainable with the other TKIs (Bonoet al., 2013; Herbert et al., 2013). However, preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional pro-angiogenic RTKs that may also reduce the risk of drug resistance (Hilberg et al., 2008). For example, dual inhibition of VEGFRs and FGFRs using brivanib produced enduring tumour stasis and angiogenic blockade following the failure of VEGF-targeted therapies (Allen et al., 2011). Furthermore, triple inhibition of FGFRs, VEGFRs and PDGFR(s) using dovitinib (TKI258) or nintedanib (BIBF 1120) displayed broad-spectrum anti-tumour activities in several tumour xenograft models as well as promising data in clinical trials. Combined inhibition of FGFR/VEGFR/PDGFR targets not only tumour cells, but also endothelial cells, pericytes and smooth muscle cells, resulting in an effective inhibition of tumour growth, angiogenesis and metastasis even in advanced tumour stages (Hilberg et al., 2008; Ledermann et al., 2011; Taeger et al., 2011; Chenet al., 2012; Angevin et al., 2013).

Oncogene-targeted therapy

Oncogenes, genes that cause the transformation of normal cells into cancerous cells, are thought to up-regulate many pro-angiogenic proteins. Therefore, anticancer drugs that were developed for their capacity to block an oncogene also have an indirect anti-angiogenic activity (Kerbel et al., 2000; Bergers and Benjamin, 2003; Folkman, 2003). For example, dasatinib and other inhibitors of sarcoma (Src), an aberrantly activated non-RTK associated with many human malignancies, showed potent anti-angiogenic effects through the down-regulation of VEGF and IL-8 (Summy et al., 2005; Han et al., 2006; Haura et al., 2010). Another example is to target the oncogenic Ras using farnesyl transferase (FT) inhibitors, which inhibit post-translational farnesylation of Ras that governs the latter’s activity (Awada et al., 2002). FT inhibitors were found to inhibit tumor VEGF expression and block FTase-dependent Ras activation, which is critically involved in VEGF-elicited angiogenic signal transduction and angiogenesis (Han et al., 2005; Izbicka et al., 2005; Kim et al., 2010). In addition to classical oncogenes inhibition, interference with other tumor-deregulated signaling pathways would offer another approach in targeting angiogenesis. For example, inhibitors of heat shock protein 90 (HSP90), a chaperone molecule known to protect oncoproteins from misfolding and degradation in the protein-rich intracellular environment, were found to prevent VEGF production and to disrupt multiple pro-angiogenic signalling pathways in numerous cancer cells. They were also shown to inhibit tumour growth and vascularity of different human tumor xenografts (Sanderson et al., 2006; Langet al., 2007; Eccles et al., 2008; Trepel et al., 2010; Moser et al., 2012). Proteasome inhibitors, such as bortezomib (PS-341) or MG-132, were also shown to reduce tumour growth and vascularity of squamous cell carcinoma and pancreatic cancer xenograft probably through inhibition of NF–κB-dependent release of pro-angiogenic gene products, VEGF and IL-8 (Sunwoo et al., 2001; Nawrocki et al., 2002; Matsuo et al., 2009). Similarly, inhibition of B-cell lymphoma 2 (Bcl-2), a prosurvival protein that regulates apoptosis by preventing the mitochondrial release of pro-apoptogenic factors, was shown to prevent NF-κB-mediated release of the pro-angiogenic factors IL-8 and CXC chemokine ligand 1 (CXCL1) as well as VEGF in tumor-associated endothelial cells and pancreatic cell lines respectively (Karl et al., 2005; Wang et al., 2008). Moreover, (−)-gossypol, a natural BH3 mimetic that inhibits BH3 domain of Bcl-2 as well as related prosurvival proteins (Bcl-xL and Mcl-1), was shown to remarkably decrease microvessel density in human prostate tumour PC-3 xenografts through decrease of VEGF and IL-8 release as well as blocking multiple steps in VEGF-activated biological events (Karaca et al., 2008; Pang et al., 2011).

Matrix degrading and remodelling-targeted therapy

Matrix degrading and remodelling are activated by tumors to modify local micro-environment, which in turn promote their angiogenic potential (Bergers et al., 2000; Vlodavsky and Friedmann, 2001). Up-regulation of expression and activity of several endogenous MMPs including MMP-2, MMP-9 as well as MMP-3 and MMP-7 have been identified in invasive tumors (for a review, see Bourboulia and Stetler-Stevenson, 2010). Consequently, inhibitors of MMPs were extensively pursued as a therapeutic strategy for treating cancer. Unfortunately, MMPs intervention strategies had met with limited clinical success because of severe toxicities and associated metastasis-promoting effect (Coussens et al., 2002; Devy et al., 2009). Furthermore, the paradoxical roles of tissue inhibitors of metalloproteinases (TIMPs) may contribute to such failure depending on the net balance of TIMPs and MMPs in tumour stroma (Jiang et al., 2002). As a result, efforts were directed at therapies exploiting endogenous MMP inhibitors, TIMPs or monoclonal antibodies against individual MMPs (Martens et al., 2007; Jarvelainen et al., 2009). For example, DX-2400, a highly selective fully human MMP-14 inhibitory antibody, was found to block pro-MMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions (Devy et al., 2009). Alternatively, in order to reduce toxicity and enhance drug delivery, polymeric nanoparticulate delivery systems could be used to target individual components of ECM. For example, targeted delivery of antisense inhibitors of laminin-8, a vascular basement membrane component, by conjugation to the natural drug carrier β-poly(L-malic acid) significantly reduced tumour microvessel density and increased animal survival in an experimental model of glioblastoma (Fujita et al., 2006). Similarly, a nano delivery system that incorporate peptides against proteolytically processed type IV collagen significantly accumulated in tumors and blocked angiogenesis in experimental models (Mueller et al., 2009). However, the highly sulfated oligosaccharides, Heparan (HS) mimetics highly sulfated oligosaccharides, were shown to have a heparanase-inhibiting effect sequestering, in turn, many heparan sulfate proteoglycan (HSPG)-binding factors (Johnstone et al., 2010; Dredge et al., 2011). In preclinical studies, HS mimetics have effectively targeted multiple HSPG-dependent functions and have resulted in decreased in vivo tumor growth, tumor invasion, tumor metastasis and angiogenesis (Johnstone et al., 2010; Dredge et al., 2011; Zhou et al., 2011). Clinically, the heparanase inhibitor PI-88 showed preliminary efficacy as an adjunct therapy for post-operative hepatocellular carcinoma (Liu et al., 2009).

Tumour-associated stromal cell-targeted therapy

Tumour-associated stromal cells crosstalk is a perquisite for the formation of a tumour vasculature, an essential step for tumour progression (Lorusso and Ruegg, 2008). Interference with those crosstalk circuits through intervention of cellular adhesion (highlighted in next paragraph) or tumor-induced recruitment of different stromal cells may be considered as an indirect way of anti-angiogenic therapy (Ferrara and Kerbel, 2005). The latter can be supported by studies in which inhibition of macrophage infiltration, for example, by either genetic ablation of the macrophage CSF-1 or liposomal clodronate-induced macrophage depletion, was shown to delay the angiogenic switch and malignant transition (Giraudo et al., 2004; Lin et al., 2006). Furthermore, CSF-1R kinase inhibitors were found to reduce tumor-associated vascularity in two different tumor mouse models (Kubota et al., 2009; Mantheyet al., 2009). In addition, clodronate and other related bisphosphonates, originally used to treat skeletal complications in patients with tumour-induced osteolysis, were shown to exert potent anti-tumour and anti-angiogenic effects in many other studies (Fournier et al., 2002; Santini et al., 2003; Stathopoulos et al., 2008). Zoledronic acid, a third-generation bisphosphonate, was also found to reduce a number of tumour-associated macrophages and shift their phenotype from M2 to M1, resulting in a reduction in TAM-associated production of VEGF in murine models of spontaneous mammary carcinogenesis and mesothelioma (Coscia et al., 2010; Veltman et al., 2010). Clinically, repeated low-dose therapy with zoledronic acid, which maintains active drug plasma concentration, was able to induce an early remarkable and long-lasting decrease of VEGF levels in patients with cancer (Santini et al., 2007). In another example, inhibition of mobilization of neutrophils, from bone marrow and their infiltration into tumour, using neutralizing anti–prokineticin-2, an antibody against a secreted protein known also as BV8, was shown to impair the initial angiogenic switch in a multistage pancreatic beta cell tumorigenesis model (Shojaei et al., 2008). Furthermore, the neutralizing anti-BV8 was found to prevent myeloid cell-dependent tumour angiogenesis in several xenograft models (Shojaei et al., 2007). Cancer-associated fibroblasts (CAF) can also be targeted with thapsigargin analogue coupled with peptides specific for fibroblast activation protein (FAP), a CAF membrane-bound protease whose catalytic site has access to the peritumoural fluid of the tumor micro-environment. This extracellular activation results in the death of CAFs as well as pericytes and endothelial cells within milieu of different human tumor xenografts (Brennen et al., 2012).

Cell adhesion molecules (CAMs)-targeted therapy

CAMs are cell surface proteins known to be involved in binding with other counter-receptors on adjacent cells or surrounding ECM macromolecules (Aplin et al., 1998). Many CAMs, such as αv-integrins, E-selectin, N-cadherin and VE-cadherin, have been implicated in tumour angiogenesis (Bischoff, 1997; Tei et al., 2002; Nakashima et al., 2003; Weis and Cheresh, 2011). For example, αv-integrins are expressed on surface of endothelial cells and can determine whether cells can adhere to and survive in a particular micro-environment. A number of matrix-derived fragments have the ability to act as endogenous angiogenesis inhibitors through binding to integrins on endothelial cells, disrupting physical connections and suppressing signalling events associated with cell survival, migration and proliferation (Nyberg et al., 2005). Consequently, integrins antagonism using peptidomimetics (e.g. cilengitide), monoclonal antibodies (e.g. volociximab) or oral small-molecule compounds have been investigated in a wide range of malignancies (Huveneers et al., 2007). Cilengitide is a cyclized pentapeptide peptidomimetic designed to compete for the arginine-glycine-aspartic acid (RGD) peptide sequence, thereby blocking the ligation of the αvβ3 and αvβ5 integrins to matrix proteins (Hariharan et al., 2007). Cilengitide is mainly under clinical development for glioblastoma; however, clinical trials of other malignancies such as head and neck cancer as well as lung cancer were also initiated (Reardon and Cheresh, 2011; Vermorken et al., 2012; Manegold et al., 2013). Alternatively, cyclic peptides containing RGD motif could guide nanoparticulate delivery system, which incorporates anti-angiogenic cytotoxic agents such as doxorubicin, paclitaxel or combretastatin A4, to accumulate specifically in tumor vasculature with no overt systemic toxicity (Murphy et al., 2008; Ruoslahti et al., 2010; Wang et al., 2011). Volociximab, a chimeric humanized monoclonal antibody that selectively inhibits the αvβ1 integrin interaction with fibronectin, has been evaluated also in clinical trials for solid tumours such as renal cell carcinoma, recurrent ovarian cancer, advanced non–small-cell lung cancer and metastatic pancreatic cancer (Figlin et al., 2006; Evans et al., 2007; Jarvelainen et al., 2009; Vergote et al., 2009; Besse et al., 2013). Cadherins constitute a superfamily of molecules that mediate calcium-dependent cell–cell adhesions. The intracellular domains of cadherins directly bind to β-catenin and link with cytoskeletal components, providing the molecular basis for stable cell–cell adhesion (Zhang et al., 2010). Targeting cadherin signalling may also represent another way for tumor angiogenesis intervention. For example, ADH-1, a cyclic pentapeptide containing the cell adhesion recognition site (His-Ala-Val) required for N-cadherin adhesion, was shown to possess anti-angiogenic and anti-tumour activity (Blaschuk et al., 2005; Blaschuk, 2012). Similarly, monoclonal antibody directed against specific region of VE-cadherin was able to inhibit tumor angiogenesis and growth with no side effects on normal vasculature (Corada et al., 2002; May et al., 2005).

Inflammatory angiogenesis-targeted therapy

Targeting inflammatory angiogenesis, responsible for a substantial part of tumour vascularization initiated by infiltrating leukocytes, may be considered as another indirect anti-angiogenic strategy (Albini et al., 2005). Moreover, as mentioned before, tumour-infiltrating leukocytes contribute into malignant progression through production of many pro-inflammatory cytokines, chemokines and enzymes that can mostly induce angiogenic cascade (Balkwill et al., 2005). Such vital roles have been supported by the early observation that nonsteroidal anti-inflammatory drugs can inhibit tumour angiogenesis and, in turn, tumor progression (Albini et al., 2005). For example, ibuprofen was found to decrease tumor growth and metastatic potential in mice models through modulation of angiogenesis (Yao et al., 2005). Moreover, selective inhibitors of COX-2, an inducible enzyme that catalyses the production of prostanoids from arachidonic acid, were also shown to inhibit angiogenesis (Tsujii et al., 1998; Wei et al., 2004). The anti-angiogenic effect of COX-2 inhibitors may be contributed, in part, by decreasing the COX-2 metabolic product PGE2, the predominant PG in solid tumors known to stimulate cancer cells to produce pro-angiogenic factors such as VEGF and bFGF as well as many other factors belonging to CXC chemokines family (Strieter et al., 2004; Wang et al., 2006; Wang and Dubois, 2010). Members of the CXC chemokine family are heparin-binding proteins that possess disparate regulative roles in angiogenesis. For example, the ELR+ CXC chemokines, characterized by highly conserved three amino acid motifs (Glu-Leu-Arg; ‘ELR’ motif), are potent promoters of angiogenesis, whereas the IFN-inducible (ELR−) CXC chemokines are inhibitors of angiogenesis (Strieter et al., 2004). The use of repertaxin, originally designed to target the ELR+ CXC chemokine receptors CXCR1 and CXCR2 on neutrophils to prevent their migration to sites of inflammation, was found to inhibit tumor angiogenesis, thereby suppressing tumour progression in a genetic model of pancreatic ductal adenocarcinoma (Ijichi et al., 2011). It would be beneficial to explore other small-molecule CXCR2 antagonists that have already been developed for the treatment of inflammatory diseases in different preclinical models of cancer, especially inflammation-associated cancers (refer to Chapman et al., 2009 for a list of newly developed CXCR2 antagonists used in the treatment of inflammatory diseases of the lung).

Mechanisms of enhanced therapeutic efficacy

  • Dual targeting of tumor vasculature
  • Targeting different cell types of tumor micro-environment
  • Normalization of tumor vasculature
  • Chemosensitization of tumor cells
  • Interference with the repair of cytotoxic drug-induced damage and resistance mechanisms

Consequences of anti-angiogenic therapy with other anticancer therapy

  • Contrary to initial expectations, treatment with angiogenesis inhibitors was associated with unexpected toxicities. The toxicity profiles of those inhibitors reflect the systemic disturbance of growth factor signalling pathways that mediate their anti-angiogenic activity (Elice and Rodeghiero, 20102012). In this context, disturbance of the tight endothelial cell-platelet interaction that maintains vascular integrity results in bleeding complications, gastrointestinal perforations, and disturbed wound and ulcer healing (Verheul and Pinedo, 2007). In general, the incidence of those adverse effects increases when anti-angiogenic agent is combined with chemotherapy. For example, bleeding complications have been observed in patients with colorectal cancer treated with chemotherapy in combination with bevacizumab (Kabbinavar et al., 2003; Giantonio et al., 2006). In non–small-cell lung cancer, some patients treated with bevacizumab in combination with carboplatin and paclitaxel experienced severe or fatal pulmonary haemorrhage (Johnson et al., 2004). Furthermore, a higher incidence of gastrointestinal perforation was observed in patients with colorectal cancer given bevacizumab in combination with chemotherapy compared with chemotherapy alone (Hurwitz et al., 2004). Similarly, thrombotic events have been observed in patients treated with angiogenesis inhibitors, especially when these agents are given in combination with chemotherapy (Verheul and Pinedo, 2007). Treatment of patients with cancer with angiogenesis inhibitors is frequently associated with hypertension, which may require the addition of regular anti-hypertensive agent (Izzedine et al., 2009).

Summary and future directions

  • Angiogenesis is a critical process that occurs pathologically in many malignancies due to changing balance of endogenous angiogenesis inducers and inhibitors, leading to the activation of nearby endothelial cells to form new vasculature. Consequently, angiogenesis can be targeted to restrict initiation, growth and progression of most of angiogenesis-dependent malignancies. Numerous angiogenic inhibitors have been identified, some of which are currently being investigated in clinical trials and some others were even approved for cancer therapies. These angiogenesis inhibitors were classified based on their target into two main classes: direct and indirect inhibitors. Indirect angiogenesis inhibitors can be further subclassified based on their interference mechanisms with the angiogenic cascade. A list of major categories and molecular targets for angiogenesis inhibitors is shown in Table 2.
  • Most angiogenesis inhibitors conferred clinical benefits mainly when combined with other chemotherapeutic/targeted therapies rather than being used as monotherapy. Unfortunately, many anti-angiogenic agents were shown to be associated with overt systemic toxicity as well as resistance emergence and disease recurrence. Drug resistance in anti-angiogenic therapy may result from a plethora of pro-angiogenic factors released by inappropriately functioning host cells in the tumor micro-environment as a compensatory mechanism. Therefore, the strategy of targeting endothelial cells alone may not be enough as explained in the previous texts, requiring the proposal of different rationales in which other cellular compartments of tumor micro-environment are targeted to attain proper anti-angiogenic and anti-tumor response. That highlights the importance of considering tumor micro-environment as a dynamic system, as depicted in Figure 1 in which interference with any of its components may be an approach to interfere with cancer hallmarks, including angiogenesis.

9.5.5 LUCITANIB a VEGFR/FGFR dual kinase inhibitor in Phase 2 trials

Dr.  Anthony Melvin Crasto


6-(7-((l-aminocyclopropyl)methoxy)-6-methoxyquinolin-4-yloxy)- N-methyl- 1 -naphthamide
1058137-23-7 (E-3810 free base); 1058137-84-0  (E-3810 HCl salt)
E-3810, E-3810 amine, UNII-PP449XA4BH, E3810, Lucitanib [INN], AL3810
Molecular Formula:C26H25N3O4
Molecular Weight:443.4944 g/mol
Spiro Substituted Compounds As Angiogenesis Inhibitors [US8163923] 2008-09-18 2012-04-24
A 4-(3-methoxypropoxy)-3-methylpyridinyl derivative of timoprazole that is used in the therapy of STOMACH ULCERS and ZOLLINGER-ELLISON SYNDROME. The drug inhibits H(+)-K(+)-EXCHANGING ATPASE which is found in GASTRIC PARIETAL CELLS.
For in advanced solid tumors.
Lucitanib (E-3810): Lucitanib, also known as E-3810,  is a novel dual inhibitor targeting human vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs) with antiangiogenic activity. VEGFR/FGFR dual kinase inhibitor E-3810 inhibits VEGFR-1, -2, -3 and FGFR-1, -2 kinases in the nM range, which may result in the inhibition of tumor angiogenesis and tumor cell proliferation, and the induction of tumor cell death. Both VEGFRs and FGFRs belong to the family of receptor tyrosine kinases that may be upregulated in various tumor cell type
Lucitanib (E-3810) Structure


Lucitanib is an oral, potent inhibitor of the tyrosine kinase activity of fibroblast growth factor receptors 1 through 3 (FGFR1-3), vascular endothelial growth factor receptors 1 through 3 (VEGFR1-3) and platelet-derived growth factor receptors alpha and beta (PDGFR α-ß). We own exclusive development and commercial rights to lucitanib on a global basis, excluding China. Lucitanib rights to markets outside of the U.S. and Japan have been sublicensed to Les Laboratoires Servier (Servier). We are collaborating with Servier on the global clinical development of lucitanib.

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Nonhematological cancers [4.2]

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

Characteristics and Types

Tumors are considered to be cell growths that are either benign or malignant proliferations. Those that are benign may be reactive, but they do not have the characteristics of a malignancy.  The main features we are concerned with are:

  1. Cancer cells utilize glucose by the anaerobic glycolysis energy pathway as the primary energy source, and this is despite a sufficient supply of oxygen. This characteristic is referred to as the Warburg Effect, as it was originally described by Otto Warburg in the 1920s, and he had derived a parallel to the observations of yeast cells by Louis Pasteur 60 years earlier.
  2. Cancer cells proliferate and take on features common to cancer cells and less characteristic in expression than the cells of origin.
  3. Cancer cells loose the properties of cell-cell attachment, and this is associated with metastasis to proximate or to distant sites.
  4. As a result of Warburg’s original studies, he concluded that cancer cells have impaired respiration (mitochondria were not yet described).
  5. It would be known much later that there is an impairment of the balance between cell repair and cell death.
  6. Malignant tumors are divided into solid tumors and hematological malignancies. This discussion is focused only on malignant solid tumors.
  7. The types of cancer can be classified according to the tissue of origin: mesenchymal (sarcoma) and epithelial (carcinoma), and by source of origin:

Brain Cancer
Breast Cancer
Kidney Cancer
Lung Cancer
Ovarian Cancer
Pancreatic Cancer
Prostate Cancer
Stomach Cancer

There are many studies showing positive associations between solid tumors and pesticide exposure. In particular, the large well-designed cohort studies consistently show statistically significant positive associations. The relationships are most consistent for high exposure levels such as those found in occupational settings. The results frequently show dose response relationships, and quality of studies was generally good. Overall, these findings strongly support a reduction of pesticide use, particularly for those individuals with occupational exposure (agriculture, pesticide applicators) at high doses.
Otto Warburg called attention to this in the 1950’s. However, we also know that viruses can have a role in the causation of cancers. Moreover, cancers may occur in sites of chronic inflammation.  I have not said anything about the association of specific mutations with types of cancer, and associations in some cases with specific populations at a greater risk than the broader population.

Profiling Solid Tumor Heterogeneity by LCM and Biological MS of Fresh-Frozen Tissue Sections

Donald J. Johann, Sumana Mukherjee, DaRue A. Prieto, Timothy D. Veenstra, Josip Blonder
Laser Capture Microdissection
Methods in Molecular Biology 2011; 755, pp 95-106

The heterogeneous nature of solid tumors represents a common problem in mass spectrometry (MS)-based analysis of fresh-frozen tissue specimens. Here, we describe a method that relies on synergy between laser capture microdissection (LCM) and MS for enhanced molecular profiling of solid tumors. This method involves dissection of homogeneous histologic cell types from thin fresh-frozen tissue sections via LCM, coupled with liquid chromatography (LC)-MS analysis. Such an approach enables an in-depth molecular profiling of captured cells. This is a bottom-up proteomic approach, where proteins are identified through peptide sequencing and matching against a specific proteomic database. Sample losses are minimized, since lysis, solubilization, and digestion are carried out directly on LCM caps in buffered methanol using a single tube, thus reducing sample loss between these steps. The rationale for the LCM-MS coupling is that once the optimal method parameters are established for a solid tumor of interest, homogeneous histologic tumor/tissue cells (i.e., tumor proper, stroma, etc.) can be effectively studied for potential biomarkers, drug targets, pathway analysis, as well as enhanced understanding of the pathological process under study.

A microRNA expression signature of human solid tumors defines cancer gene targets

Stefano Volinia*†‡, George A. Calin*‡, Chang-Gong Liu*, et al.
PNAS  Feb 14, 2006; 103(7): 2257–2261

Small noncoding microRNAs (miRNAs) can contribute to cancer development and progression and are differentially expressed in normal tissues and cancers. From a large-scale miRnome analysis on 540 samples including lung, breast, stomach, prostate, colon, and pancreatic tumors, we identified a solid cancer miRNA signature composed by a large portion of overexpressed miRNAs. Among these miRNAs are some with well characterized cancer association, such as miR-17-5p, miR-20a, miR-21, miR-92, miR-106a, and miR-155. The predicted targets for the differentially expressed miRNAs are significantly enriched for protein-coding tumor suppressors and oncogenes (P < 0.0001). A number of the predicted targets, including the tumor suppressors RB1 (Retinoblastoma 1) and TGFBR2 (transforming growth factor, beta receptor II) genes were confirmed experimentally. Our results indicate that miRNAs are extensively involved in cancer pathogenesis of solid tumors and support their function as either dominant or recessive cancer genes.

Diagnosis of and therapy for solid tumors with radiolabeled antibodies and immune fragments

Carrasquillo JA, Krohn KA, Beaumier P, McGuffin RW, et al.
Cancer Treatment Reports [1984, 68(1):317-328]

Antibodies which are directed against human tumor-associated antigens can potentially be used as carriers of radioactivity for in vivo diagnosis (radioimmunodetection) or treatment (radioimmunotherapy) of solid tumors, including colon, hepatomacholangiocarcinoma,  and melanoma.  Murine  monoclonal antibodies (MOAB), produced by the hybridoma technique of Kohler and Milstein, are replacing conventional heterosera as sources of antibodies, because MOAB can be produced in large quantities as reproducible reagents with homogeneous binding properties. We have studied  human melanoma using MOAB IgG and Fab fragments that recognize the human melanoma-associated antigens p97and “high-molecular-weight antigen.” Both antigens are found in the membrane of melanomas at much larger concentrations than in normal adult tissues. We have performed radioimmunodetection studies with whole immunoglobulin and have detected 88% of lesions greater than 1.5 cm. We have used Fab fragments for radioimmunotherapy and have found that large doses of radiolabeled antibodies (up to 342 mCi) can be repetitively given to patients without excessive end-organ toxicity. Two of three patients treated with high-dose radiolabeled antimelanoma Fab showed an effect from the treatment. Although both technical and biologic problems remain, the use of radiolabeled antibodies that are directed against tumor-associated antigens holds future promise as a new therapeutic approach to solid tumors that are resistant to conventional therapy.

Solid tumors include cancers of the brain, ovary, breast, colon and other tissues. Many people believe that one quality solid tumors share is a reliance on cancer stem cells. These cancer stem cells are thought to divide to produce the bulk of the cells that make up the tumor.

The hypothesis suggests that unlike most cells of a tumor, the cancer stem cells divide very slowly and are less likely to be destroyed by chemotherapies that kill the fast-growing tumor cells. The thought is that cancers might recur because the chemotherapy kills the bulk of the tumor, but leaves behind the cancer stem cells that can, over time, form a new tumor.

Stem cell scientists are studying cancer stem cells from solid tumors in the lab to find ways of destroying them. If these cancer stem cells share characteristics that allow them to be destroyed by the same drug, then a single new drug could significantly improve cancer treatment for a range of different cancer types.

Resminostat – by 4SC

Despite decades of concentrated effort, medicine has yet to achieve a decisive breakthrough for many types of cancer. 4SC is focusing on fields of research with an especially high academic interest and future potential – such as epigenetics, cancer stem cells, cancer immunotherapy and other key molecular signal transduction pathways that contribute to the development and persistence of cancer diseases.

Resminostat is 4SC’s lead oncology compound. Resminostat is an oral histone-deacetylase (HDAC) inhibitor with an innovative epigenetic mechanism of action that potentially enables the compound to be deployed as a novel, targeted tumour therapy for a broad spectrum of oncological indications, both as a monotherapy and, in particular, in combination with other cancer drugs.

Epigenetic mode of action

HDAC inhibitors modify the three-dimensional chromatin DNA structure of tumour cells and can trigger cell differentiation, which can ultimately result in programmed cell death (apoptosis). HDAC inhibitors therefore offer a mechanism of action that has the potential to halt tumour progression and induce tumour regression. Furthermore, resminostat – due to its epigenetic mode of action – can develop an additional synergetic effect in combined treatments with other traditional cancer therapies and also fight the development of resistance to other cancer medications.

An example: In preclinical studies, resminostat has been shown to effectively inhibit epithelial-mesenchymal transition (EMT). EMT, which may be promoted through the administration of certain conventional cancer therapies, leads to the formation of particularly aggressive tumour cells, which ultimately may result in greater proliferation of cancer cells in patients and the patients’ death.

On the whole, a reinforcing positive therapeutic effect is expected to be achieved through well-tolerated parallel administration of an epigenetic compound such as resminostat and a traditional cancer drug. Combination therapy thus aims to improve the success of the treatment as a whole.

Resminostat – by 4SC in Europe and its Japanese development partner Yakult Honsha in Asia – has been investigated to date in a broad clinical Phase I/II program in the four indications of liver cancer (hepatocellular carcinoma, HCC), Hodgkin Lymphoma (HL), colorectal cancer (CRC), and non-small-cell lung cancer (NSCLC).

Notably, in both tumor indications, HCC and HL, gene expression levels of the new biomarker ZFP64 measured prior to study treatment start in blood cells of patients, were identified to be indicative of survival outcome upon treatment with resminostat. Hereby, the set of patients with a high level of ZFP64 gene expression at baseline showed a statistically significant increase of median overall survival compared with patients with low ZFP64 expression levels.

4SC is prioritizing the further development of resminostat in the liver cancer indication. 4SC’s goal is to progress resminostat in combination with sorafenib as a first-line therapy for HCC until market approval. 4SC’s main focus is the use of the resminostat/sorafenib combination as a first-line treatment for HCC patients, while the use as a second-line therapy remains an attractive additional option.

4SC Discovery is a drug discovery company based in Planegg-Martinsried near Munich. It was founded in December 2011 as a wholly owned subsidiary of 4SC AG.

Response Evaluation Criteria in Solid Tumors

Response Evaluation Criteria In Solid Tumors (RECIST) is a set of published rules that define when tumors in cancer patients improve (“respond”), stay the same (“stabilize”), or worsen (“progress”) during treatment. The criteria were published in February 2000 by an international collaboration including the European Organisation for Research and Treatment of Cancer (EORTC), National Cancer Institute of the United States, and the National Cancer Institute of Canada Clinical Trials Group. Today, the majority of clinical trials evaluating cancer treatments for objective response in solid tumors use RECIST.

These criteria were developed and published in February 2000, and subsequently updated in 2009. They are specifically NOT meant to determine whether patients have improved or not, as these are tumor-centric, not patient centric criteria. This distinction must be made by both the treating physicians and the cancer patients themselves. Many oncologists in their daily clinical practice follow their patient’s malignant disease by means of repeated imaging studies and make decisions about continuing therapy on the basis of both objective and symptomatic criteria. It is not intended that these RECIST guidelines play a role in that decision making, except if determined appropriate by the treating oncologist.

  • CT and MRI are the best currently available and reproducible methods to measure target lesions selected for response assessment. Conventional CT and MRI should be performed with cuts of 10 mm or less in slice thickness contiguously. Spiral CT should be performed using a 5 mm contiguous reconstruction algorithm. This applies to tumors of the chest, abdomen and pelvis. Head and neck tumors and those of extremities usually require specific protocols.
  • Lesions on chest X-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable.
  • When the primary endpoint of the study is objective response evaluation, ultrasound (US) should not be used to measure tumor lesions. It is, however, a possible alternative to clinical measurements of superficial palpable lymph nodes, subcutaneous lesions and thyroid nodules. US might also be useful to confirm the complete disappearance of superficial lesions usually assessed by clinical examination.
  • The utilization of endoscopy and laparoscopy for objective tumor evaluation has not yet been fully and widely validated. Their uses in this specific context require sophisticated equipment and a high level of expertise that may only be available in some centers. Therefore, the utilization of such techniques for objective tumor response should be restricted to validation purposes in specialized centers. However, such techniques can be useful in confirming complete pathological response when biopsies are obtained.
  • Tumor markers alone cannot be used to assess response. If markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response when all lesions have disappeared.
  • Cytology and histology can be used to differentiate between PR and CR in rare cases (e.g., after treatment to differentiate between residual benign lesions and residual malignant lesions in tumor types such as germ cell tumors).

Baseline documentation of “Target” and “Non-Target” lesions

  • All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs should be identified as target lesions and recorded and measured at baseline.
  • Target lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repeated measurements (either by imaging techniques or clinically).
  • A sum of the longest diameter (LD) for all target lesions will be calculated and reported as the baseline sum LD. The baseline sum LD will be used as reference by which to characterize the objective tumor response.
  • All other lesions (or sites of disease) should be identified as non-target lesions and should also be recorded at baseline. Measurements of these lesions are not required, but the presence or absence of each should be noted throughout follow-up.

Response Criteria

Evaluation of target lesions

  • Complete Response (CR): Disappearance of all target lesions
  • Partial Response (PR): At least a 30% decrease in the sum of the LD of target lesions, taking as reference the baseline sum LD
  • Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started
  • Progressive Disease (PD): At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions

Evaluation of non-target lesions

  • Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level
  • Incomplete Response/ Stable Disease (SD): Persistence of one or more non-target lesion(s) or/and maintenance of tumor marker level above the normal limits
  • Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions

Evaluation of best overall response

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for PD the smallest measurements recorded since the treatment started). In general, the patient’s best response assignment will depend on the achievement of both measurement and confirmation criteria

  • Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having “symptomatic deterioration”. Every effort should be made to document the objective progression even after discontinuation of treatment.
  • In some circumstances it may be difficult to distinguish residual disease from normal tissue. When the evaluation of complete response depends on this determination, it is recommended that the residual lesion be investigated (fine needle aspirate/biopsy) to confirm the complete response status.

Duration of stable disease

  • SD is measured from the start of the treatment until the criteria for disease progression are met, taking as reference the smallest measurements recorded since the treatment started.
  • The clinical relevance of the duration of SD varies for different tumor types and grades. Therefore, it is highly recommended that the protocol specify the minimal time interval required between two measurements for determination of SD. This time interval should take into account the expected clinical benefit that such a status may bring to the population under study.

Response review

  • For trials where the response rate is the primary endpoint it is strongly recommended that all responses be reviewed by an expert(s) independent of the study at the study’s completion. Simultaneous review of the patients’ files and radiological images is the best approach.

Tumor microenvironment

The tumor microenvironment is the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, other cells, signaling molecules, and the extracellular matrix(ECM).[1] The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesisand inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells, such as in immuno-editing. The tumor microenvironment has been shown to contribute to tumour heterogeneity. In one of its earliest forms, this concept of interplay between the tumor and its microenvironment can be seen in Stephen Paget‘s “seed and soil” theory where he postulated that metastases of a particular type of cancer (“the seed”) often metastasizes to certain sites (“the soil”) based on the similarity of the environments of the original and secondary tumor sites.[2] Later, experiments by Halachmi and Witz in mice showed that for the same cancer cell line, inoculation in mice (where the tumor microenvironment could affect the cancer) created greater tumorigenicity than the same strain inoculated in in vitro culture.[3][4]

80-90% of cancer are carcinomas, or cancers that form in the epithelial tissue.[5] This tissue is not vascularized, which prevents tumors from growing greater than 2mm in diameter without recruiting new blood vessels to feed itself.[6] The process of angiogenesis is dysregulated to feed the cancer cells, and as a result the vasculature formed differs from that of normal tissue.

The enhanced permeability and retention effect (EPR effect) is the observation that the vasculature of tumors is often leaky and accumulates molecules in the blood stream to a greater extent than normal tissue. This effect linked to inflammation is not only seen in tumors, but in hypoxic area of cardiac muscles following a myocardial infarction (MI or heart attack).[7][8] This leaky vasculature is thought to have several causes, including a dearth of pericytes and a malformed basement membrane.[8

The tumor microenvironment is often hypoxic. As the tumor mass increases, the interior of the tumor grows farther away from existing blood supply. While angiogenesis can reduce this affect, the partial pressure of oxygen is below 5 mm Hg (venous blood has a partial pressure of oxygen at 40 mm Hg) in more than 50% of locally advanced solid tumors.[9][10] The hypoxic environment leads to genetic instability, which is associated with cancer progression, via downregulating nucleotide excision repair (NER) and mismatch repair (MMR) pathways.[11] Hypoxia also causes the upregulation of hypoxia-inducible factor 1 alpha (HIF1-α), which induces angiogenesis, and is associated with poorer prognosis and the activation of genes associated with metastasis.[10]

While a lack of oxygen can cause glycolytic behavior in cells, tumor cells have also been shown to undergo aerobic glycolysis as well, in which they preferentially produce lactate from glucose even when there is abundant oxygen. This phenomenon is called the Warburg effect, in honor of its discoverer, Otto Warburg.[12] No matter the cause, this leaves the extracellular microenvironment acidic (pH 6.5-6.9), while the cancer cells themselves are able to remain neutral (ph 7.2-7.4). It has been shown that this induces greater cell migration in vivo and in vitro, possibly by promoting degradation of the ECM.[13][14]

The stroma of a carcinoma is the connective tissue below the basal lamina. This includes fibroblasts, ECM, immune cells, and other cells and molecules. The stroma surrounding a tumor often reacts to the intrusion via inflammation, similar to how it might with a wound, leading cancer to be called “wounds that do not heal.”[15] Inflammation can encourage angiogenesis, speed the cell cycle, and prevent cell death, all of which augments tumor growth.

Carcinoma associated fibroblasts (CAFs) are a heterogenous group of fibroblasts whose function is pirated by cancer cells and then contribute toward carcinogenesis[17] These cells usually are derived from the normal fibroblasts in the surrounding stroma but can also come from pericytes, smooth muscle cells, fibrocytesmesenchymal stem cells (MSCs, often derived from bone marrow), or via epithelial-mesenchymal transition (EMT) or endothelial-mesenchymal transition (EndMT).[18][19][20] Unlike their normal counterparts, CAFs do not retard cancer growth in vitro.[21] Beyond simply lacking the ability of tumor inhibition, CAFs also perform several functions which support tumor growth, such as secreting vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), and other pro-angiogenic signals to induce angiogenesis.[9] CAFs can also secrete transforming growth factor beta (TGF-β), which is associated with EMT, a process by which cancer cells can metastasize,[22] and is associated with inhibiting cytotoxic T cells and natural killer T cells.[23] As fibroblasts, CAFs are able to rework the ECM to include more paracrine survival signals such as IGF-1 and IGF-2, thus promoting survival of the surrounding cancer cells.[17] CAFs are also associated with the Reverse Warburg Effect where the CAFs perform aerobic glycolysis and feed lactate to the cancer cells.[17]

Several markers are used to identify CAFs including expression of α smooth muscle actin (αSMA), vimentinplatelet-derived growth factor receptor α (PDGFR-α), platelet-derived growth factor receptor β (PDGFR-β), fibroblast specific protein 1 (FSP-1), and fibroblast activation protein (FAP).[19] None of the factors can be used to differentiate CAFs from all other cells by itself.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of cells of myelogenous origin with the potential to repress T cell responses. They regulate wound repair and inflammation and are rapidly expanded in cancer, correlating with that signs of inflammation are seen in most if not all tumor sites.[24][25] Tumors can produce exosomes that stimulate inflammation via MDSCs.[26][27] This group of cells include some tumor associated macrophages (TAMs).[24] TAMs are a central component in the strong link between chronic inflammation and cancer. TAMs are recruited to the tumor as a response to cancer associated inflammation.[28] Unlike normal macrophages, TAMs lack cytotoxic activity.[29] TAMs have been induced in vitro by exposing macrophage progenitors to different immune regulatory cytokines, such as interleukin 4(IL-4) and interleukin 13 (IL-13).[17] TAMs gather in necrotic regions of tumors where they have been associated with hiding the cancer cells from normal immune cells by secreting interleukin 10 (IL-10),[30] aiding angiogenesis by secreting vascular endothelial growth factor(VEGF) and nitric oxide synthase(NOS),[9] supporting tumor growth by secreting epidermal growth factor (EGF)[30] and remodeling the ECM.[9] TAMs show sluggish NF-κB activation, which allows for the smoldering inflammation seen in cancer.[31] An increased amount of TAMs is associated with worse prognosis.[32][33] TAMs represent a potential target for novel cancer therapies.

TAMs have recently been associating with using exosomes (vesicles used by mammalian cells to secrete intracellular contents) to deliver invasion-potentiating microRNA (miRNA) into cancerous cells, specifically breast cancer cells.[26][34]

Fibroblasts are in charge of laying down most of the collagens, elastin, glycosaminoglycans, proteoglycans (e.g. perlecan), and glycoproteins in the ECM. As many fibroblasts are transformed into CAFs during carcinogenesis, this reduces the amount of ECM produced and the ECM that is produced can be malformed, like collagen being loosely woven and non-planar, even curved.[37] In addition, CAFs produce matrixmatrix metalloproteinases (MMP), which cleave the proteins within the ECM.[9] CAFs are also able to disrupt the ECM via force, generating a track that a carcinoma cell can follow directly behind.[38] In either case, destruction of the ECM allows cancer cells to escape from their in situ location and intravasate into the blood stream where they can metastasize systematically. It can also provide passage for endothelial cells to complete angiogenesis to the tumor site.

Destruction of the ECM also modulates the signaling cascades controlled by the interaction of cell-surface receptors and the ECM, and it also reveals binding sites previously hidden, like the integrin alpha-v beta-3(αVβ3) on the surface of melanoma cells can be ligated to rescue the cells from apoptosis after degradation of collagen.[39][40] In addition, the degradation products may have downstream effects as well that can increase tumorigenicity of cancer cells and can serve as potential biomarkers.[39] The destruction of the ECM also releases the cytokines and growth factors stored therein (for example, VEGF, basic fibroblast growth factor (bFGF), insulin-like growth factors (IGF1 and IGF2), TGF-β, EGF, heparin-binding EGF-like growth factor (HB-EGF), and tumor necrosis factor (TNF), which can increase the growth of the tumor.[37][41]Cleavage of ECM components can also release cytokines that inhibit tumorigenesis, such as degradation of certain types of collagen can form endostatin, restin, canstatin, & tumstatin, which have antiangiogenic functions.[37]

Stiffening of the ECM is associated with tumor progression.[42] This stiffening may be partially attributed to CAFs secreting lysyl oxidase (LOX), an enzyme that cross-links the collagen IV found in the ECM.[19][43]

Numerous high throughput screens for cancer therapeutics are performed in vitro on cancer cell lines without the accompanying microenvironment, but current studies are also investigating the effects of supportive stroma cells on the biology of cancer cells and their resistance to therapy.[44] These studies revealed that there are interesting therapeutic targets in the microenvironment like integrins or chemokines.[44] These were missed by initial screens for anti-cancer drugs and might also help explain why so few initially identified drugs are highly potent in vivo.

Much effort has been devoted into developing nanocarrier vehicles (~20-200 nm in diameter) for transportation of drugs and other therapeutic molecules, so that these therapies can be targeted to selectively extravasate through tumor vasculature via the EPR effect. Using a nanocarrier is now considered the gold standard of targeted cancer therapy because it targets almost all tumors besides those few that are hypovascularized, like prostate and pancreatic tumors.[8][45] These efforts include protein capsids[46] and liposomes.[47] However, as some important, normal tissues, like the liver and kidneys, also have fenestrated endothelium, great care must be taken with using the correct size (10-100 nm, with greater retention in tumors seen in using larger nanocarriers) and charge (anionic or neutral).[8] Lymphatic vessels do not usually develop with the tumor, leading to increased interstitial fluid pressure, which made abrogate the journey of these nanocarriers to the tumor.[8][48]

Bevacizumab is clinically approved to treat a variety of cancer by targeting VEGF-A, which is produced by both CAFs and TAMs, thus slowing angiogenesis. Many other small molecule inhibitors exist that block the receptors for the growth factors released, thus making the cancer cell deaf to much of the paracrine signaling produced by CAFs and TAMs. These inhibitors include SunitinibPazopanibSorafenib, and Axitinib, all of which inhibit platelet derived growth factor receptors (PDGF-Rs) and VEGF receptors (VEGFRs). Cannabidiol, a cannabis derivate without psychoactive side effects, has also been shown to inhibit the expression of VEGF in Kaposi’s sarcoma cells.[49]

Natalizumab is a monoclonal antibody that was designed to target one of the molecules responsible for cell adhesion (integrin VLA-4) and has promising in vitro activity in B cell lymphomas and leukemias.[44]

Also, Trabectedin is known to have immunomodulatory effects that inhibit TAMs.[30]

Current formulations of liposomes encapsulating anti-cancer drugs for selective uptake to tumors via the EPR effect include: Doxil and Myocet, both of which encapsulate doxorubicin (a DNA intercalator and common chemotherapeutic); DaunoXome, which encapsulates daunorubicin (another DNA intercalator similar to doxorubicin); and Onco-TCS, which encapsulates vincristine (a molecule which constitutively induces formation of microtubules, dysregulating cell division). Another novel utilization of the EPR effect comes from Protein-bound paclitaxel (marketed under the trade name Abraxane) where paclitaxel (a molecule which dysregulates cell division via stabilization of microtubules) is bound to albumin to add bulk and aid delivery.

  1. Michael J. Duffy The biochemistry of metastasis Advances in Clinical Chemistry, Volume 32 1996, Pages 135–160
  2. Fabienne Danhier, Olivier Feron, Véronique Préat To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery Journal of Controlled Release, Volume 148, Issue 2, 1 December 2010, Pages 135–146
  3. Cynthia E. Weber, Paul C. Kuo The tumor microenvironment Surgical Oncology, Volume 21, Issue 3, September 2012, Pages 172–177
  4. Mikhail V. Blagosklonny Antiangiogenic therapy and tumor progression Cancer Cell, Volume 5, Issue 1, January 2004, Pages 13–17
  5. Ranjit S. Bindra, Peter M. Glazer Genetic instability and the tumor microenvironment: towards the concept of microenvironment-induced mutagenesis Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 569, Issues 1–2, 6 January 2005, Pages 75–85
  6. Robert A. Gatenby & Robert J. Gillies Why do cancers have high aerobic glycolysis? Nature Reviews Cancer Volume 4, November 2004, Pages 891-899
  7. Veronica Estrella, Tingan Chen, Mark Lloyd, Jonathan Wojtkowiak, Heather H. Cornnell, Arig Ibrahim-Hashim, Kate Bailey, Yoganand Balagurunathan, Jennifer M. Rothberg, Bonnie F. Sloane, Joseph Johnson, Robert A. Gatenby, and Robert J. Gillies Acidity Generated by the Tumor Microenvironment Drives Local Invasion Cancer Research, Volume 73, Issue 5, 1 March 2013, Pages 1524-1535
  8. Joydeb Kumar Kundu, Young-Joon Surh Inflammation: Gearing the journey to cancer Mutation Research/Reviews in Mutation Research, Volume 659, Issues 1–2, July–August 2008, Pages 15–30
  9. Kati Räsänen, Antti Vaheri Activation of fibroblasts in cancer stroma Experimental Cell Research, Volume 316, Issue 17, 15 October 2010, Pages 2713–2722
  10. Timothy Marsh, Kristian Pietras, Sandra S. McAllister Fibroblasts as architects of cancer pathogenesis Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease Online 30 October 2012
  11. Michael Quante, Shui Ping Tu, Hiroyuki Tomita, Tamas Gonda, Sophie S.W. Wang, Shigeo Takashi, Gwang Ho Baik, Wataru Shibata, Bethany DiPrete, Kelly S. Betz, Richard Friedman, Andrea Varro, Benjamin Tycko, & Timothy C. Wang Bone Marrow-Derived Myofibroblasts Contribute to the Mesenchymal Stem Cell Niche and Promote Tumor Growth Cancer Cell, Volume 19, Issue 2, 15 February 2011, Pages 257–272

Pathology Outlines _ Nat Pernick, MD
Copyright: (c) 2002-2015,, Inc.

Immunohistochemistry basics

Reviewer: Nat Pernick, M.D. (see Reviewers page)
Revised: 21 March 2014, last major update August 2013
Copyright: (c) 2002-2013,, Inc.


  • Immunohistochemistry (IHC) is a tool for surgical pathology and research
  • Diagnosis should be based on H&E morphology, with confirmation by immunohistochemistry or molecular testing; it is dangerous to use immunohistochemistry alone to make the diagnosis
  • A stain / result is not just positive or negative; focus on the types of cells that are immunoreactive and determine if they are tumor cells, inflammatory cells, normal cells or stromal cells; comparing the results to an H&E stained section or a negative control of the same block may be helpful (Am J Surg Pathol 2007;31:1627J Clin Pathol 2011;64:466)
  • After you identify the type of cell staining, it is helpful to note the percentage of these cells staining, the intensity of staining (weak, 1+, 2+, 3+, 4+) and the pattern of staining (membranous, cytoplasmic, nuclear, dot-like)
  • The pattern of immunoreactivity should follow the anatomic distribution of the antigen before it is called positive / immunoreactive
  • Reference: CAP Laboratory Improvement Programs: Principles of Analytic Validation of Immunohistochemical Assays

Common errors

  • Not using a positive or negative control; they are helpful in interpreting the staining pattern, particularly if it is heavy or weak
  • Other sources of error are ectopic antigen expression (may be due to abundant endogenous biotin, Hum Pathol 2011;42:369), cross reactions (Mod Pathol 2012;25:231), less specificity than thought (Int J Clin Exp Pathol 2012;5:137), use of the wrong secondary antibody (EJN Blog) or rarely the wrong primary antibody

Immunohistochemistry – common panels


  • Epithelial markers: low molecular weight keratin (CAM 5.2), AE1-AE3 cytokeratin cocktail, CK7, CK20, CEA and EMA
  • Alternative epithelial markers on sarcomatoid carcinoma include p63, MOC-31 and TTF1 (Mod Pathol 2005;18:1471)
  • Melanocytic markers: S100 (also a mesenchymal marker), HMB45, MelanA / Mart1
  • Lymphoid markers: CD3 (T cells), CD20 (B cells), CD15 (Hodgkin), CD30 (Hodgkin & other) and various others
  • Histiocytic markers: CD68, lysozyme, CD1a (Langerhans cells)
  • Neuroendocrine markers: neuron specific enolase, chromogranin, synaptophysin, CD56 and CD57
  • Mesenchymal markers: vimentin (non-specific), factor XIIIa (fibrous histiocytoma), factor VIII (vessels), CD31 (vessels), CD34 (vessels), D2-40 (lymphatics), HHF35 (actin), smooth muscle actin and desmin
  • Cell proliferation / apoptosis markers: Ki-67, bcl2

Tissue of origin / unknown primary

PubMed Search: tissue of origin[title]


IHC stains examples

IHC stains examples

CD Markers


CD: cluster designation or cluster of differentiation; a protocol to identify and investigate cell surface molecules

Nomenclature proposed in 1982 at First International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA)

A classification system for monoclonal antibodies generated by laboratories worldwide against cell surface molecules, initially on leukocytes, but now also from other cell types

Data is collated and analyzed by cluster analysis based on pattern of binding to leukocytes or other cell types

Must be at least two monoclonal antibodies for each antigen

“w” indicates that the CD is not well characterized or is represented by only one monoclonal antibody

Current CD markers range from CD1 to CD363

Interpretation should be based on cellular distribution of staining (i.e. membranous, cytoplasmic, nuclear), proportion of positively stained cells, staining intensity and cutoff levels



CD molecules have various functions, including receptors or ligands; also cell adhesion, antigen presentation

Although commonly used by pathologists to characterize cells, they most likely also have an important (although sometimes unknown) function in cell physiology

Enzyme cytochemistry


  • Detects enzymatic activity in cytoplasm
  • Advantage over immunocytochemistry is determination of enzyme’s intracellular localization and intensity of catalytic activity (for research purposes)
  • Flow cytometry and immunocytochemistry are often preferred to determine presence of enzyme molecule (but not catalytic activity or localization)
  • Enzyme product unites with coupler, which produces localized color at site of enzyme activity
  • Fresh smears are preferred, especially for myeloperoxidase; if not possible, store unstained slides away from light


  • Simultaneous capture: reagent in incubation medium combines with reaction product (example: diazo method for alkaline phosphatase)
  • Post-incubation coupling: insoluble reaction product is coupled with a colored or opaque substance (example: Rutenburg and Seligman method for acid phosphatase)
  • Self-colored substrate reaction: water-soluble dye is made insoluble when enzyme removes a hydrophilic group, leading to colored precipitate at site of enzyme activity
  • Intramolecular rearrangement: produces a colored insoluble precipitate at sites of enzyme activity of an otherwise colorless substrate (University of Iowa)

Molecular Pathology

Authors: Zubair W. Baloch, M.D., Ph.D., Joshua Bradish, M.D., Betty Chung, D.O., M.P.H., M.A., Rodney E. Shackelford, D.O., Ph.D.; Editors: Liang Cheng, M.D., Gregory A. Hosler, M.D., Ph.D. (see Reviewers page)

DNA purificationintroductionanalyzing puritybasic protocolanion exchange chromatographycesium chloride density gradient centrifugationcommercial DNA extraction machinesethanol precipitationorganic extractionPCR inhibitorsRNA purificationsilica adsorptiontissue preparation

DNA sequencing: 
historyMaxam-Gilbert sequencingSanger sequencingcapillary electrophoresisother innovationsreal timepyroseqencingnanotechnologyRoche 454 FLX pyrosequencerIllumina Genome AnalyzerHeliScope Sequencer

generalprobesprotocolprobe patternsimages

Microarray: introductionhistorybasicsconsiderationserrors
variations: antibodybead basedcellularCGHsolid phasetissue (TMA)

definitionhistorybasicsTaq polymerasereaction stagesthermocyclersapplications
variations: generalmethylation specificmultiplexnestedreal-timereverse transcriptase  

Nat Pernick and PO group

Nat Pernick and PO group

Contact us (248/646-0325) with any questions

Pathology Outline

Below is a comprehensive categorization of the diseases documented in this site. Click on a section name, to view the Cases. For many of the diseases we have also provided descriptions in PDF format. To read the disease description, click on the PDF icon next to the disease name.

A > Z
by Location
by Treatment
by Image Type
Tumor Types
Bone Tumors
Soft Tissue Tumors

There are some important issues related to the pathology of cancer that need to be addressed. The two references above are both valuable for a source useful for reference to the characterization and methods of identification of most tumors. The second is concerned with soft tissue and bone sarcomas.
PathologyOutlines is also an excellent source for soft tissue and bone sarcomas.

It is important to realize that despite enormous progress in the molecular biology of carcinomas and sarcomas, there is a characteristic natural history and clinical presentation, and the ability to distinguish within types is related to differential expression that is related to metabolic characteristics and tissue differentiation.  The pathologist uses a system of morphological grading based on the nuclear to cytoplasmic ratio, the loss of architecture (such a glandular anaplasia) that is important, but not sufficient.  The staging is based on regional lymph node extension, and to distant metastasis.  In addition, the use of cell differentiation markers and immunohistochemical staining is essential.

However, the field is now being rewritten in a large way that will not have a significant effect for perhaps a decade by the clinical pathology and molecular diagnostics measurement of miRNAs and lncRNAs, that are measureable in tissue and in serum, and the expanded use of mass spectrometry, and MS combined with optical methods. This is important for the differentiation of types of malignant expression within cell types, and will be important for matching malignancy to pharmaceutical targets.  Despite the use of the term cancer targeting, the reality has been that single chemotherapy has not been sufficient in the treatment of advanced disease.  This I attribute to the complexity of the interactions between affected dysregulated pathways. The same problem has been encountered in the multiple hit progression of infection to systemic inflammatory response to sepsis to multiple organ failure. The assumption that there is a magic bullet has been an illusion.  This is where the mathematical modeling has become important because we are dealing with more than one major variable:

  1. Local control
  2. Cell-like cell interactions
  3. Cell-unlike cell interactions
  4. Level of disruption involving cell migration
  5. The level of mitochondrial respiration
  6. The degree of loss of apoptosis

There is also a consideration of age, sex, and endocrine factors.  This is particularly illustrated in the case of childhood malignancies, such as neuroblastoma.  In the case of bone tumors, it is not widely recognized that there is a relationship between muscle and bone in the remodeling process, and a relationship between the type of neoplasm and the anatomical location, and also a relationship to the loss of remodeling control after age 65 years.

This is illustrated by the classification of bone tumors as – periosteal and endosteal, and as epiphyseal, metaphyseal, and diaphyseal (for long bones).
The parosteal sarcoma may be fibrosarcoma or osteosarcoma, and may be derived from a fibrous dysplasia, a myositis ossificans, a fibroxanthoma, a lipoma, or malignant transformation in an osteochondroma.  The prognosis for such a cancer after a local wide excision is far better than a cancer within the bone.  This was the case 40 years ago, long before modern molecular diagnostics.  In the case of epiphyseal lesions, one expects the cancer to be dictated by cartilaginous origin, but it also can arise from a cystic lesion at the joint margin. The growth and development of bone and the greatest activity of growth in length of a long bone is at the growth plate, in the metaphysis.  This also happens to be the site most affected by scurvy, rickets (articular cartilage and metaphysis), and by hyperparathyroidism. The metaphysis is where the cartilage is converted to calcified bone matrix, which is remodeled by the removal of bone by osteoblasts and the laying down of bone by osteoblasts.  Stable bone at equilibrium is maintained by osteocytes.  The circulation in bone is in Volkmann’s canals.  What types of tumors do we find at the metaphysis? Malignant Giant Cell Tumor and Osteosarcoma.  Chondrosarcomas may arise there also from an enchondroma, a benign tumor within the bone at the metaphysis, or an osteochondroma.  Perhaps the most wild type of bone tumor is the malignant combined giant cell and osteogenic sarcoma that arises in Paget’s disease.  This is a disease that occurs in older age which is characterized by a loss of control of bone remodeling resulting in the rapid remodeling of bone with the generation of a primitive bone that can be called – pumice bone. It is easily fractured.  Bone remodels to a peak in the late 30’s, and then slows down, but occurs throughout life. The bone becomes more compact.  In remodeling bone is removed by osteoclasts and bone is added by osteoblasts.  A single osteoclast removes 100 microns of bone that is replaced by 100 osteoblasts adding 1 micron each.  This dynamic was measured by Dr. Lent C. Johnson.

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Hypoxia Inducible Factor 1 (HIF-1)

Writer and Curator: Larry H Bernstein, MD, FCAP

7.9  Hypoxia Inducible Factor 1 (HIF-1)

7.9.1 Hypoxia and mitochondrial oxidative metabolism

7.9.2 Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

7.9.3 Hypoxia-Inducible Factors in Physiology and Medicine

7.9.4 Hypoxia-inducible factor 1. Regulator of mitochondrial metabolism and mediator of ischemic preconditioning

7.9.5 Regulation of cancer cell metabolism by hypoxia-inducible factor 1

7.9.6 Coming up for air. HIF-1 and mitochondrial oxygen consumption

7.9.7 HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption

7.9.8 HIF-1. upstream and downstream of cancer metabolism

7.9.9 In Vivo HIF-Mediated Reductive Carboxylation

7.9.10 Evaluation of HIF-1 inhibitors as anticancer agents



7.9.1 Hypoxia and mitochondrial oxidative metabolism

Solaini G1Baracca ALenaz GSgarbi G.
Biochim Biophys Acta. 2010 Jun-Jul; 1797(6-7):1171-7

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondria’s central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O2] sensors, convey signals to HIF-1directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F1F0 ATPase in the hypoxia-induced mitochondrial autophagy.

Over the last two decades a defective mitochondrial function associated with hypoxia has been invoked in many diverse complex disorders, such as type 2 diabetes [1] and [2], Alzheimer’s disease [3] and [4], cardiac ischemia/reperfusion injury [5] and [6], tissue inflammation [7], and cancer [8][9][10],[11] and [12].

The [O2] in air-saturated aqueous buffer at 37 °C is approx. 200 μM [13]; however, mitochondria in vivo are exposed to a considerably lower [O2] that varies with tissue and physiological state. Under physiological conditions, most human resting cells experience some 5% oxygen tension, however the [O2] gradient occurring between the extracellular environment and mitochondria, where oxygen is consumed by cytochrome c oxidase, results in a significantly lower [O2] exposition of mitochondria. Below this oxygen level, most mammalian tissues are exposed to hypoxic conditions  [14]. These may arise in normal development, or as a consequence of pathophysiological conditions where there is a reduced oxygen supply due to a respiratory insufficiency or to a defective vasculature. Such conditions include inflammatory diseases, diabetes, ischemic disorders (cerebral or cardiovascular), and solid tumors. Mitochondria consume the greatest amount (some 85–90%) of oxygen in cells to allow oxidative phosphorylation (OXPHOS), which is the primary metabolic pathway for ATP production. Therefore hypoxia will hamper this metabolic pathway, and if the oxygen level is very low, insufficient ATP availability might result in cell death [15].

When cells are exposed to an atmosphere with reduced oxygen concentration, cells readily “respond” by inducing adaptive reactions for their survival through the AMP-activated protein kinase (AMPK) pathway (see for a recent review [16]) which inter alia increases glycolysis driven by enhanced catalytic efficiency of some enzymes, including phosphofructokinase-1 and pyruvate kinase (of note, this oxidative flux is thermodynamically allowed due to both reduced phosphorylation potential [ATP]/([ADP][Pi]) and the physiological redox state of the cell). However, this is particularly efficient only in the short term, therefore cells respond to prolonged hypoxia also by stimulation of hypoxia-inducible factors (HIFs: HIF-1 being the mostly studied), which are heterodimeric transcription factors composed of α and β subunits, first described by Semenza and Wang [17]. These HIFs in the presence of hypoxic oxygen levels are activated through a complex mechanism in which the oxygen tension is critical (see below). Afterwards HIFs bind to hypoxia-responsive elements, activating the transcription of more than two hundred genes that allow cells to adapt to the hypoxic environment [18] and [19].

Several excellent reviews appeared in the last few years describing the array of changes induced by oxygen deficiency in both isolated cells and animal tissues. In in vivo models, a coordinated regulation of tissue perfusion through vasoactive molecules such as nitric oxide and the action of carotid bodies rapidly respond to changes in oxygen demand [20][21][22][23] and [24]. Within isolated cells, hypoxia induces significant metabolic changes due to both variation of metabolites level and activation/inhibition of enzymes and transporters; the most important intracellular effects induced by different pathways are expertly described elsewhere (for recent reviews, see [25][26] and [27]). It is reasonable to suppose that the type of cells and both the severity and duration of hypoxia may determine which pathways are activated/depressed and their timing of onset [3][6][10][12][23] and [28]. These pathways will eventually lead to preferential translation of key proteins required for adaptation and survival to hypoxic stress. Although in the past two decades, the discovery of HIF-1 by Gregg Semenza et al. provided a molecular platform to investigate the mechanism underlying responses to oxygen deprivation, the molecular and cellular biology of hypoxia has still to be completely elucidated. This review summarizes recent experimental data concerned with mitochondrial structure and function adaptation to hypoxia and evaluates it in light of the main structural and functional parameters defining the mitochondrial bioenergetics. Since mitochondria contain an inhibitor protein, IF1, whose action on the F1F0 ATPase has been considered for decades of critical importance in hypoxia/ischemia, particular notice will be dedicated to analyze molecular aspects of IF1 regulation of the enzyme and its possible role in the metabolic changes induced by low oxygen levels in cells.

Mechanism(s) of HIF-1 activation

HIF-1 consists of an oxygen-sensitive HIF-1α subunit that heterodimerizes with the HIF-1β subunit to bind DNA. In high O2 tension, HIF-1α is oxidized (hydroxylated) by prolyl hydroxylases (PHDs) using α-ketoglutarate derived from the tricarboxylic acid (TCA) cycle. The hydroxylated HIF-1α subunit interacts with the von Hippel–Lindau protein, a critical member of an E3 ubiquitin ligase complex that polyubiquitylates HIF. This is then catabolized by proteasomes, such that HIF-1α is continuously synthesized and degraded under normoxic conditions [18]. Under hypoxia, HIF-1α hydroxylation does not occur, thereby stabilizing HIF-1 (Fig. 1). The active HIF-1 complex in turn binds to a core hypoxia response element in a wide array of genes involved in a diversity of biological processes, and directly transactivates glycolytic enzyme genes [29]. Notably, O2 concentration, multiple mitochondrial products, including the TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, hence the cellular responses to O2 depletion [30] and [31]. Incidentally, impaired TCA cycle flux, particularly if it is caused by succinate dehydrogenase dysfunction, results in decreased or loss of energy production from both the electron-transport chain and the Krebs cycle, and also in overproduction of free radicals [32]. This leads to severe early-onset neurodegeneration or, as it occurs in individuals carrying mutations in the non-catalytic subunits of the same enzyme, to tumors such as phaeochromocytoma and paraganglioma. However, impairment of the TCA cycle may be relevant also for the metabolic changes occurring in mitochondria exposed to hypoxia, since accumulation of succinate has been reported to inhibit PHDs [33]. It has to be noticed that some authors believe reactive oxygen species (ROS) to be essential to activate HIF-1 [34], but others challenge this idea [35], therefore the role of mitochondrial ROS in the regulation of HIF-1 under hypoxia is still controversial [36]. Moreover, the contribution of functional mitochondria to HIF-1 regulation has also been questioned by others [37][38] and [39].

Major mitochondrial changes in hypoxia

Major mitochondrial changes in hypoxia

Fig. 1. Major mitochondrial changes in hypoxia. Hypoxia could decrease electron-transport rate determining Δψm reduction, increased ROS generation, and enhanced NO synthase. One (or more) of these factors likely contributes to HIF stabilization, that in turn induces metabolic adaptation of both hypoxic cells and mitophagy. The decreased Δψm could also induce an active binding of IF1, which might change mitochondrial morphology and/or dynamics, and inhibit mitophagy. Solid lines indicate well established hypoxic changes in cells, whilst dotted lines indicate changes not yet stated. Inset, relationships between extracellular O2concentration and oxygen tension.

Oxygen is a major determinant of cell metabolism and gene expression, and as cellular O2 levels decrease, either during isolated hypoxia or ischemia-associated hypoxia, metabolism and gene expression profiles in the cells are significantly altered. Low oxygen reduces OXPHOS and Krebs cycle rates, and participates in the generation of nitric oxide (NO), which also contributes to decrease respiration rate [23] and [40]. However, oxygen is also central in the generation of reactive oxygen species, which can participate in cell signaling processes or can induce irreversible cellular damage and death [41].

As specified above, cells adapt to oxygen reduction by inducing active HIF, whose major effect on cells energy homeostasis is the inactivation of anabolism, activation of anaerobic glycolysis, and inhibition of the mitochondrial aerobic metabolism: the TCA cycle, and OXPHOS. Since OXPHOS supplies the majority of ATP required for cellular processes, low oxygen tension will severely reduce cell energy availability. This occurs through several mechanisms: first, reduced oxygen tension decreases the respiration rate, due first to nonsaturating substrate for cytochrome c oxidase (COX), secondarily, to allosteric modulation of COX[42]. As a consequence, the phosphorylation potential decreases, with enhancement of the glycolysis rate primarily due to allosteric increase of phosphofructokinase activity; glycolysis however is poorly efficient and produces lactate in proportion of 0.5 mol/mol ATP, which eventually drops cellular pH if cells are not well perfused, as it occurs under defective vasculature or ischemic conditions  [6]. Besides this “spontaneous” (thermodynamically-driven) shift from aerobic to anaerobic metabolism which is mediated by the kinetic changes of most enzymes, the HIF-1 factor activates transcription of genes encoding glucose transporters and glycolytic enzymes to further increase flux of reducing equivalents from glucose to lactate[43] and [44]. Second, HIF-1 coordinates two different actions on the mitochondrial phase of glucose oxidation: it activates transcription of the PDK1 gene encoding a kinase that phosphorylates and inactivates pyruvate dehydrogenase, thereby shunting away pyruvate from the mitochondria by preventing its oxidative decarboxylation to acetyl-CoA [45] and [46]. Moreover, HIF-1 induces a switch in the composition of cytochrome c oxidase from COX4-1 to COX4-2 isoform, which enhances the specific activity of the enzyme. As a result, both respiration rate and ATP level of hypoxic cells carrying the COX4-2 isoform of cytochrome c oxidase were found significantly increased with respect to the same cells carrying the COX4-1 isoform [47]. Incidentally, HIF-1 can also increase the expression of carbonic anhydrase 9, which catalyses the reversible hydration of CO2 to HCO3 and H+, therefore contributing to pH regulation.

Effects of hypoxia on mitochondrial structure and dynamics

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. The fusion/fission machineries are modulated in response to changes in the metabolic conditions of the cell, therefore one should expect that hypoxia affect mitochondrial dynamics. Oxygen availability to cells decreases glucose oxidation, whereas oxygen shortage consumes glucose faster in an attempt to produce ATP via the less efficient anaerobic glycolysis to lactate (Pasteur effect). Under these conditions, mitochondria are not fueled with substrates (acetyl-CoA and O2), inducing major changes of structure, function, and dynamics (for a recent review see [48]). Concerning structure and dynamics, one of the first correlates that emerge is that impairment of mitochondrial fusion leads to mitochondrial depolarization, loss of mtDNA that may be accompanied by altered respiration rate, and impaired distribution of the mitochondria within cells [49][50] and [51]. Indeed, exposure of cortical neurons to moderate hypoxic conditions for several hours, significantly altered mitochondrial morphology, decreased mitochondrial size and reduced mitochondrial mean velocity. Since these effects were either prevented by exposing the neurons to inhibitors of nitric oxide synthase or mimicked by NO donors in normoxia, the involvement of an NO-mediated pathway was suggested [52]. Mitochondrial motility was also found inhibited and controlled locally by the [ADP]/[ATP] ratio [53]. Interestingly, the author used an original approach in which mitochondria were visualized using tetramethylrhodamineethylester and their movements were followed by applying single-particle tracking.

Of notice in this chapter is that enzymes controlling mitochondrial morphology regulators provide a platform through which cellular signals are transduced within the cell in order to affect mitochondrial function [54]. Accordingly, one might expect that besides other mitochondrial factors [30] and [55] playing roles in HIF stabilization, also mitochondrial morphology might reasonably be associated with HIF stabilization. In order to better define the mechanisms involved in the morphology changes of mitochondria and in their dynamics when cells experience hypoxic conditions, these pioneering studies should be corroborated by and extended to observations on other types of cells focusing also on single proteins involved in both mitochondrial fusion/fission and motion.

Effects of hypoxia on the respiratory chain complexes

O2 is the terminal acceptor of electrons from cytochrome c oxidase (Complex IV), which has a very high affinity for it, being the oxygen concentration for half-maximal respiratory rate at pH 7.4 approximately 0.7 µM [56]. Measurements of mitochondrial oxidative phosphorylation indicated that it is not dependent on oxygen concentration up to at least 20 µM at pH 7.0 and the oxygen dependence becomes markedly greater as the pH is more alkaline [56]. Similarly, Moncada et al. [57] found that the rate of O2 consumption remained constant until [O2] fell below 15 µM. Accordingly, most reports in the literature consider hypoxic conditions occurring in cells at 5–0.5% O2, a range corresponding to 46–4.6 µM O2 in the cells culture medium (see Fig. 1 inset). Since between the extracellular environment and mitochondria an oxygen pressure gradient is established [58], the O2 concentration experienced by Complex IV falls in the range affecting its kinetics, as reported above.

Under these conditions, a number of changes on the OXPHOS machinery components, mostly mediated by HIF-1 have been found. Thus, Semenza et al. [59] and others thereafter [46] reported that activation of HIF-1α induces pyruvate dehydrogenase kinase, which inhibits pyruvate dehydrogenase, suggesting that respiration is decreased by substrate limitation. Besides, other HIF-1 dependent mechanisms capable to affect respiration rate have been reported. First, the subunit composition of COX is altered in hypoxic cells by increased degradation of the COX4-1 subunit, which optimizes COX activity under aerobic conditions, and increased expression of the COX4-2 subunit, which optimizes COX activity under hypoxic conditions [29]. On the other hand, direct assay of respiration rate in cells exposed to hypoxia resulted in a significant reduction of respiration [60]. According with the evidence of Zhang et al., the respiration rate decrease has to be ascribed to mitochondrial autophagy, due to HIF-1-mediated expression of BNIP3. This interpretation is in line with preliminary results obtained in our laboratory where the assay of the citrate synthase activity of cells exposed to different oxygen tensions was performed. Fig. 2 shows the citrate synthase activity, which is taken as an index of the mitochondrial mass [11], with respect to oxygen tension: [O2] and mitochondrial mass are directly linked.

Citrate synthase activity

Citrate synthase activity

Fig. 2. Citrate synthase activity. Human primary fibroblasts, obtained from skin biopsies of 5 healthy donors, were seeded at a density of 8,000 cells/cm2 in high glucose Dulbecco’s Modified Eagle Medium, DMEM (25 mM glucose, 110 mg/l pyruvate, and 4 mM glutamine) supplemented with 15% Foetal Bovine Serum (FBS). 18 h later, cell culture dishes were washed once with Hank’s Balanced Salt Solution (HBSS) and the medium was replaced with DMEM containing 5 mM glucose, 110 mg/l pyruvate, and 4 mM glutamine supplemented with 15% FBS. Cell culture dishes were then placed into an INVIVO2 humidified hypoxia workstation (Ruskinn Technologies, Bridgend, UK) for 72 h changing the medium at 48 h, and oxygen partial pressure (tension) conditions were: 20%, 4%, 2%, 1% and 0.5%. Cells were subsequently collected within the workstation with trypsin-EDTA (0.25%), washed with PBS and resuspended in a buffer containing 10 mM Tris/HCl, 0.1 M KCl, 5 mM KH2PO4, 1 mM EGTA, 3 mM EDTA, and 2 mM MgCl2 pH 7.4 (all the solutions were preconditioned to the appropriate oxygen tension condition). The citrate synthase activity was assayed essentially by incubating 40 µg of cells with 0.02% Triton X-100, and monitoring the reaction by measuring spectrophotometrically the rate of free coenzyme A released, as described in [90]. Enzymatic activity was expressed as nmol/min/mg of protein. Three independent experiments were carried out and assays were performed in either duplicate or triplicate.

However, the observations of Semenza et al. must be seen in relation with data reported by Moncada et al.[57] and confirmed by others [61] in which it is clearly shown that when cells (various cell lines) experience hypoxic conditions, nitric oxide synthases (NOSs) are activated, therefore NO is released. As already mentioned above, NO is a strong competitor of O2 for cytochrome c oxidase, whose apparent Km results increased, hence reduction of mitochondrial cytochromes and all the other redox centres of the respiratory chain occurs. In addition, very recent data indicate a potential de-activation of Complex I when oxygen is lacking, as it occurs in prolonged hypoxia [62]. According to Hagen et al. [63] the NO-dependent inhibition of cytochrome c oxidase should allow “saved” O2 to redistribute within the cell to be used by other enzymes, including PHDs which inactivate HIF. Therefore, unless NO inhibition of cytochrome c oxidase occurs only when [O2] is very low, inhibition of mitochondrial oxygen consumption creates the paradox of a situation in which the cell may fail to register hypoxia. It has been tempted to solve this paradox, but to date only hypotheses have been proposed [23] and [26]. Interestingly, recent observations on yeast cells exposed to hypoxia revealed abnormal protein carbonylation and protein tyrosine nitration that were ascribed to increased mitochondrially generated superoxide radicals and NO, two species typically produced at low oxygen levels, that combine to form ONOO [64]. Based on these studies a possible explanation has been proposed for the above paradox.

Finally, it has to be noticed that the mitochondrial respiratory deficiency observed in cardiomyocytes of dogs in which experimental heart failure had been induced lies in the supermolecular assembly rather than in the individual components of the electron-transport chain [65]. This observation is particularly intriguing since loss of respirasomes is thought to facilitate ROS generation in mitochondria [66], therefore supercomplexes disassembly might explain the paradox of reduced [O2] and the enhanced ROS found in hypoxic cells. Specifically, hypoxia could reduce mitochondrial fusion by impairing mitochondrial membrane potential, which in turn could induce supercomplexes disassembly, increasing ROS production[11].

Complex III and ROS production

It has been estimated that, under normoxic physiological conditions, 1–2% of electron flow through the mitochondrial respiratory chain gives rise to ROS [67] and [68]. It is now recognized that the major sites of ROS production are within Complexes I and III, being prevalent the contribution of Complex I [69] (Fig. 3). It might be expected that hypoxia would decrease ROS production, due to the low level of O2 and to the diminished mitochondrial respiration [6] and [46], but ROS level is paradoxically increased. Indeed, about a decade ago, Chandel et al. [70] provided good evidence that mitochondrial reactive oxygen species trigger hypoxia-induced transcription, and a few years later the same group [71] showed that ROS generated at Complex III of the mitochondrial respiratory chain stabilize HIF-1α during hypoxia (Fig. 1 and Fig. 3). Although others have proposed mechanisms indicating a key role of mitochondria in HIF-1α regulation during hypoxia (for reviews see [64] and [72]), the contribution of mitochondria to HIF-1 regulation has been questioned by others [35][36] and [37]. Results of Gong and Agani [35] for instance show that inhibition of electron-transport Complexes I, III, and IV, as well as inhibition of mitochondrial F0F1 ATPase, prevents HIF-1α expression and that mitochondrial reactive oxygen species are not involved in HIF-1α regulation during hypoxia. Concurrently, Tuttle et al. [73], by means of a non invasive, spectroscopic approach, could find no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize HIF-1α under moderate hypoxia. The same authors found the levels of HIF-1α comparable in both normal and ρ0 cells (i.e. cells lacking mitochondrial DNA). On the contrary, experiments carried out on genetic models consisting of either cells lacking cytochrome c or ρ0 cells both could evidence the essential role of mitochondrial respiration to stabilize HIF-1α [74]. Thus, cytochrome c null cells, being incapable to respire, exposed to moderate hypoxia (1.5% O2) prevented oxidation of ubiquinol and generation of the ubisemiquinone radical, thus eliminating superoxide formation at Complex III [71]. Concurrently, ρ0 cells lacking electron transport, exposed 4 h to moderate hypoxia failed to stabilize HIF-1α, suggesting the essential role of the respiratory chain for the cellular sensing of low O2 levels. In addition, recent evidence obtained on genetic manipulated cells (i.e. cytochrome b deficient cybrids) showed increased ROS levels and stabilized HIF-1α protein during hypoxia [75]. Moreover, RNA interference of the Complex III subunit Rieske iron sulfur protein in the cytochrome b deficient cells, abolished ROS generation at the Qo site of Complex III, preventing HIF-1α stabilization. These observations, substantiated by experiments with MitoQ, an efficient mitochondria-targeted antioxidant, strongly support the involvement of mitochondrial ROS in regulating HIF-1α. Nonetheless, collectively, the available data do not allow to definitely state the precise role of mitochondrial ROS in regulating HIF-1α, but the pathway stabilizing HIF-1α appears undoubtedly mitochondria-dependent [30].

Overview of mitochondrial electron and proton flux in hypoxia

Overview of mitochondrial electron and proton flux in hypoxia

Overview of mitochondrial electron and proton flux in hypoxia

Fig. 3. Overview of mitochondrial electron and proton flux in hypoxia. Electrons released from reduced cofactors (NADH and FADH2) under normoxia flow through the redox centres of the respiratory chain (r.c.) to molecular oxygen (blue dotted line), to which a proton flux from the mitochondrial matrix to the intermembrane space is coupled (blue arrows). Protons then flow back to the matrix through the F0 sector of the ATP synthase complex, driving ATP synthesis. ATP is carried to the cell cytosol by the adenine nucleotide translocator (blue arrows). Under moderate to severe hypoxia, electrons escape the r.c. redox centres and reduce molecular oxygen to the superoxide anion radical before reaching the cytochrome c (red arrow). Under these conditions, to maintain an appropriate Δψm, ATP produced by cytosolic glycolysis enters the mitochondria where it is hydrolyzed by the F1F0ATPase with extrusion of protons from the mitochondrial matrix (red arrows).

Hypoxia and ATP synthase

The F1F0 ATPase (ATP synthase) is the enzyme responsible of catalysing ADP phosphorylation as the last step of OXPHOS. It is a rotary motor using the proton motive force across the mitochondrial inner membrane to drive the synthesis of ATP [76]. It is a reversible enzyme with ATP synthesis or hydrolysis taking place in the F1 sector at the matrix side of the membrane, chemical catalysis being coupled to H+transport through the transmembrane F0 sector.

Under normoxia the enzyme synthesizes ATP, but when mitochondria experience hypoxic conditions the mitochondrial membrane potential (Δψm) decreases below its endogenous steady-state level (some 140 mV, negative inside the matrix [77]) and the F1F0 ATPase may work in the reversal mode: it hydrolyses ATP (produced by anaerobic glycolysis) and uses the energy released to pump protons from the mitochondrial matrix to the intermembrane space, concurring with the adenine nucleotide translocator (i.e. in hypoxia it exchanges cytosolic ATP4− for matrix ADP3−) to maintain the physiological Δψm ( Fig. 3). Since under conditions of limited oxygen availability the decline in cytoplasmic high energy phosphates is mainly due to hydrolysis by the ATP synthase working in reverse [6] and [78], the enzyme must be strictly regulated in order to avoid ATP dissipation. This is achieved by a natural protein, the H+ψm-dependent IF1, that binds to the catalytic F1 sector at low pH and low Δψm (such as it occurs in hypoxia/ischemia) [79]. IF1 binding to the ATP synthase results in a rapid and reversible inhibition of the enzyme [80], which could reach about 50% of maximal activity (for recent reviews see [6] and [81]).

Besides this widely studied effect, IF1 appears to be associated with ROS production and mitochondrial autophagy (mitophagy). This is a mechanism involving the catabolic degradation of macromolecules and organelles via the lysosomal pathway that contributes to housekeeping and regenerate metabolites. Autophagic degradation is involved in the regulation of the ageing process and in several human diseases, such as myocardial ischemia/reperfusion [82], Alzheimer’s Disease, Huntington diseases, and inflammatory diseases (for recent reviews see [83] and [84], and, as mentioned above, it promotes cell survival by reducing ROS and mtDNA damage under hypoxic conditions.

Campanella et al. [81] reported that, in HeLa cells under normoxic conditions, basal autophagic activity varies in relation to the expression levels of IF1. Accordingly, cells overexpressing IF1 result in ROS production similar to controls, conversely cells in which IF1 expression is suppressed show an enhanced ROS production. In parallel, the latter cells show activation of the mitophagy pathway (Fig. 1), therefore suggesting that variations in IF1 expression level may play a significant role in defining two particularly important parameters in the context of the current review: rates of ROS generation and mitophagy. Thus, the hypoxia-induced enhanced expression level of IF1[81] should be associated with a decrease of both ROS production and autophagy, which is in apparent conflict with the hypoxia-induced ROS increase and with the HIF-1-dependent mitochondrial autophagy shown by Zhang et al. [60] as an adaptive metabolic response to hypoxia. However, in the experiments of Zhang et al. the cells were exposed to hypoxia for 48 h, whereas the F1F0-ATPase inhibitor exerts a prompt action on the enzyme and to our knowledge, it has never been reported whether its action persists during prolonged hypoxic expositions. Pertinent with this problem is the very recent observation that IEX-1 (immediate early response gene X-1), a stress-inducible gene that suppresses production of ROS and protects cells from apoptosis [85], targets the mitochondrial F1F0-ATPase inhibitor for degradation, reducing ROS by decreasing Δψm. It has to be noticed that the experiments described were carried out under normal oxygen availability, but it does not seem reasonable to rule out IEX-1 from playing a role under stress conditions as those induced by hypoxia in cells, therefore this issue might deserve an investigation also at low oxygen levels.

In conclusion, data are still emerging regarding the regulation of mitochondrial function by the F1F0 ATPase within hypoxic responses in different cellular and physiological contexts. Given the broad pathophysiological role of hypoxic cellular modulation, an understanding of the subtle tuning among different effectors of the ATP synthase is desirable to eventually target future therapeutics most effectively. Our laboratory is actually involved in carrying out investigations to clarify this context.

Conclusions and perspectives

The mitochondria are important cellular platforms that both propagate and initiate intracellular signals that lead to overall cellular and metabolic responses. During the last decades, a significant amount of relevant data has been obtained on the identification of mechanisms of cellular adaptation to hypoxia. In hypoxic cells there is an enhanced transcription and synthesis of several glycolytic pathway enzymes/transporters and reduction of synthesis of proteins involved in mitochondrial catabolism. Although well defined kinetic parameters of reactions in hypoxia are lacking, it is usually assumed that these transcriptional changes lead to metabolic flux modification. The required biochemical experimentation has been scarcely addressed until now and only in few of the molecular and cellular biology studies the transporter and enzyme kinetic parameters and flux rate have been determined, leaving some uncertainties.

Central to mitochondrial function and ROS generation is an electrochemical proton gradient across the mitochondrial inner membrane that is established by the proton pumping activity of the respiratory chain, and that is strictly linked to the F1F0-ATPase function. Evaluation of the mitochondrial membrane potential in hypoxia has only been studied using semiquantitative methods based on measurements of the fluorescence intensity of probes taken up by cells experiencing normal or hypoxic conditions. However, this approach is intrinsically incorrect due to the different capability that molecular oxygen has to quench fluorescence [86] and [87] and to the uncertain concentration the probe attains within mitochondria, whose mass may be reduced by a half in hypoxia [60]. In addition, the uncertainty about measurement of mitochondrial superoxide radical and H2O2 formation in vivo [88] hampers studies on the role of mitochondrial ROS in hypoxic oxidative damage, redox signaling, and HIF-1 stabilization.

The duration and severity of hypoxic stress differentially activate the responses discussed throughout and lead to substantial phenotypic variations amongst tissues and cell models, which are not consistently and definitely known. Certainly, understanding whether a hierarchy among hypoxia response mechanisms exists and which are the precise timing and conditions of each mechanism to activate, will improve our knowledge of the biochemical mechanisms underlying hypoxia in cells, which eventually may contribute to define therapeutic targets in hypoxia-associated diseases. To this aim it might be worth investigating the hypoxia-induced structural organization of both the respiratory chain enzymes in supramolecular complexes and the assembly of the ATP synthase to form oligomers affecting ROS production [65] and inner mitochondrial membrane structure [89], respectively.

7.9.2 Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

DR WisePS WardJES ShayJR CrossJJ Gruber, UM Sachdeva, et al.
Proc Nat Acad Sci Oct 27, 2011; 108(49):19611–19616

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutamine-derived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions.

Citrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O2), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (45). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD+, which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.

This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (67). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabolism based on his observation that hypoxic yeast cells preferred to convert glucose into lactic acid rather than burning it in an oxidative fashion. The molecular basis for this shift in mammalian cells has been linked to the activity of the transcription factor HIF1 (810). Stabilization of the labile HIF1α subunit occurs in hypoxia. It can also occur in normoxia through several mechanisms including loss of the von Hippel-Lindau tumor suppressor (VHL), a common occurrence in renal carcinoma (11). Although hypoxia and/or HIF1α stabilization is a common feature of multiple cancers, to date the source of citrate in the setting of hypoxia or HIF activation has not been determined.

Here, we study the sources of hypoxic citrate synthesis in a glioblastoma cell line that proliferates in profound hypoxia (0.5% O2). Glucose uptake and conversion to lactic acid increased in hypoxia. However, glucose conversion into citrate dramatically declined. Glutamine consumption remained constant in hypoxia, and hypoxic cells were addicted to the use of glutamine in hypoxia as a source of α-ketoglutarate. Glutamine provided the major carbon source for citrate synthesis during hypoxia. However, the TCA cycle-dependent conversion of glutamine into citric acid was significantly suppressed. In contrast, there was a relative increase in glutamine-dependent citrate production in hypoxia that resulted from carboxylation of α-ketoglutarate. This reductive synthesis required the presence of mitochondrial isocitrate dehydrogenase 2 (IDH2). In confirmation of the reverse flux through IDH2, the increased reductive metabolism of glutamine-derived α-ketoglutarate in hypoxia was associated with increased synthesis of 2HG. Finally, constitutive HIF1α-expressing cells also demonstrated significant reductive-carboxylation-dependent synthesis of citrate in normoxia and a relative defect in the oxidative conversion of glutamine into citrate. Collectively, the data demonstrate that mitochondrial glutamine metabolism can be rerouted through IDH2-dependent citrate synthesis in support of hypoxic cell growth.

Some Cancer Cells Can Proliferate at 0.5% O2 Despite a Sharp Decline in Glucose-Dependent Citrate Synthesis.

At 21% O2, cancer cells have been shown to synthesize citrate by condensing glucose-derived acetyl-CoA with glutamine-derived oxaloacetate through the activity of the canonical TCA cycle enzyme citrate synthase (4). In contrast, less is known regarding the synthesis of citrate by cells that can continue proliferating in hypoxia. The glioblastoma cell line SF188 is able to proliferate at 0.5% O2 (Fig. 1A), a level of hypoxia that is sufficient to stabilize HIF1α (Fig. 1B) and predicted to limit respiration (1213). Consistent with previous observations in hypoxic cells, we found that SF188 cells demonstrated increased lactate production when incubated in hypoxia (Fig. 1C), and the ratio of lactate produced to glucose consumed increased demonstrating an increase in the rate of anaerobic glycolysis. When glucose-derived carbon in the form of pyruvate is converted to lactate, it is diverted away from subsequent metabolism that can contribute to citrate production. However, we observed that SF188 cells incubated in hypoxia maintain their intracellular citrate to ∼75% of the level maintained under normoxia (Fig. 1D). This prompted an investigation of how proliferating cells maintain citrate production under hypoxia.

SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis.

SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis.

Fig. 1. SF188 glioblastoma cells proliferate at 0.5% O2 despite a profound reduction in glucose-dependent citrate synthesis. (A) SF188 cells were plated in complete medium equilibrated with 21% O2 (Normoxia) or 0.5% O2 (Hypoxia), total viable cells were counted 24 h and 48 h later (Day 1 and Day 2), and population doublings were calculated. Data are the mean ± SEM of four independent experiments. (B) Western blot demonstrates stabilized HIF1α protein in cells cultured in hypoxia compared with normoxia. (C) Cells were grown in normoxia or hypoxia for 24 h, after which culture medium was collected. Medium glucose and lactate levels were measured and compared with the levels in fresh medium. (D) Cells were cultured for 24 h as in C. Intracellular metabolism was then quenched with 80% MeOH prechilled to −80 °C that was spiked with a 13C-labeled citrate as an internal standard. Metabolites were then extracted, and intracellular citrate levels were analyzed with GC-MS and normalized to cell number. Data for C and D are the mean ± SEM of three independent experiments. (E) Model depicting the pathway for cit+2 production from [U-13C]glucose. Glucose uniformly 13C-labeled will generate pyruvate+3. Pyruvate+3 can be oxidatively decarboxylated by PDH to produce acetyl-CoA+2, which can condense with unlabeled oxaloacetate to produce cit+2. (F) Cells were cultured for 24 h as in C and D, followed by an additional 4 h of culture in glucose-deficient medium supplemented with 10 mM [U-13C]glucose. Intracellular metabolites were then extracted, and 13C-enrichment in cellular citrate was analyzed by GC-MS and normalized to the total citrate pool size. Data are the mean ± SD of three independent cultures from a representative of two independent experiments. *P < 0.05, ***P < 0.001.

Increased glucose uptake and glycolytic metabolism are critical elements of the metabolic response to hypoxia. To evaluate the contributions made by glucose to the citrate pool under normoxia or hypoxia, SF188 cells incubated in normoxia or hypoxia were cultured in medium containing 10 mM [U-13C]glucose. Following a 4-h labeling period, cellular metabolites were extracted and analyzed for isotopic enrichment by gas chromatography-mass spectrometry (GC-MS). In normoxia, the major 13C-enriched citrate species found was citrate enriched with two 13C atoms (cit+2), which can arise from the NAD+-dependent decarboxylation of pyruvate+3 to acetyl-CoA+2 by PDH, followed by the condensation of acetyl-CoA+2 with unenriched oxaloacetate (Fig. 1 E and F). Compared with the accumulation of cit+2, we observed minimal accumulation of cit+3 and cit+5 under normoxia. Cit+3 arises from pyruvate carboxylase (PC)-dependent conversion of pyruvate+3 to oxaloacetate+3, followed by the condensation of oxaloacetate+3 with unenriched acetyl-CoA. Cit+5 arises when PC-generated oxaloacetate+3 condenses with PDH-generated acetyl-CoA+2. The lack of cit+3 and cit+5 accumulation is consistent with PC activity not playing a major role in citrate production in normoxic SF188 cells, as reported (4).

In hypoxic cells, the major citrate species observed was unenriched. Cit+2, cit+3, and cit+5 all constituted minor fractions of the total citrate pool, consistent with glucose carbon not being incorporated into citrate through either PDH or PC-mediated metabolism under hypoxic conditions (Fig. 1F). These data demonstrate that in contrast to normoxic cells, where a large percentage of citrate production depends on glucose-derived carbon, hypoxic cells significantly reduce their rate of citrate production from glucose.

Glutamine Carbon Metabolism Is Required for Viability in Hypoxia.

In addition to glucose, we have previously reported that glutamine can contribute to citrate production during cell growth under normoxic conditions (4). Surprisingly, under hypoxic conditions, we observed that SF188 cells retained their high rate of glutamine consumption (Fig. 2A). Moreover, hypoxic cells cultured in glutamine-deficient medium displayed a significant loss of viability (Fig. 2B). In normoxia, the requirement for glutamine to maintain viability of SF188 cells can be satisfied by α-ketoglutarate, the downstream metabolite of glutamine that is devoid of nitrogenous groups (14). α-ketoglutarate cannot fulfill glutamine’s roles as a nitrogen source for nonessential amino acid synthesis or as an amide donor for nucleotide or hexosamine synthesis, but can be metabolized through the oxidative TCA cycle to regenerate oxaloacetate, and subsequently condense with glucose-derived acetyl-CoA to produce citrate. To test whether the restoration of carbon from glutamine metabolism in the form of α-ketoglutarate could rescue the viability defect of glutamine-starved SF188 cells even under hypoxia, SF188 cells incubated in hypoxia were cultured in glutamine-deficient medium supplemented with a cell-penetrant form of α-ketoglutarate (dimethyl α-ketoglutarate). The addition of dimethyl α-ketoglutarate rescued the defect in cell viability observed upon glutamine withdrawal (Fig. 2B). These data demonstrate that, even under hypoxic conditions, when the ability of glutamine to replenish oxaloacetate through oxidative TCA cycle metabolism is diminished, SF188 cells retain their requirement for glutamine as the carbon backbone for α-ketoglutarate. This result raised the possibility that glutamine could be the carbon source for citrate production through an alternative, nonoxidative, pathway in hypoxia.

Glutamine carbon is required for hypoxic cell viability

Glutamine carbon is required for hypoxic cell viability

Glutamine carbon is required for hypoxic cell viability

Fig. 2. Glutamine carbon is required for hypoxic cell viability and contributes to increased citrate production through reductive carboxylation relative to oxidative metabolism in hypoxia. (A) SF188 cells were cultured for 24 h in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2(Hypoxia). Culture medium was then removed from cells and analyzed for glutamine levels which were compared with the glutamine levels in fresh medium. Data are the mean ± SEM of three independent experiments. (B) The requirement for glutamine to maintain hypoxic cell viability can be satisfied by α-ketoglutarate. Cells were cultured in complete medium equilibrated with 0.5% O2 for 24 h, followed by an additional 48 h at 0.5% O2 in either complete medium (+Gln), glutamine-deficient medium (−Gln), or glutamine-deficient medium supplemented with 7 mM dimethyl α-ketoglutarate (−Gln +αKG). All medium was preconditioned in 0.5% O2. Cell viability was determined by trypan blue dye exclusion. Data are the mean and range from two independent experiments. (C) Model depicting the pathways for cit+4 and cit+5 production from [U-13C]glutamine (glutamine+5). Glutamine+5 is catabolized to α-ketoglutarate+5, which can then contribute to citrate production by two divergent pathways. Oxidative metabolism produces oxaloacetate+4, which can condense with unlabeled acetyl-CoA to produce cit+4. Alternatively, reductive carboxylation produces isocitrate+5, which can isomerize to cit+5. (D) Glutamine contributes to citrate production through increased reductive carboxylation relative to oxidative metabolism in hypoxic proliferating cancer cells. Cells were cultured for 24 h as in A, followed by 4 h of culture in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. 13C enrichment in cellular citrate was quantitated with GC-MS. Data are the mean ± SD of three independent cultures from a representative of three independent experiments. **P < 0.01.

Cells Proliferating in Hypoxia Maintain Levels of Additional Metabolites Through Reductive Carboxylation.

Previous work has documented that, in normoxic conditions, SF188 cells use glutamine as the primary anaplerotic substrate, maintaining the pool sizes of TCA cycle intermediates through oxidative metabolism (4). Surprisingly, we found that, when incubated in hypoxia, SF188 cells largely maintained their levels of aspartate (in equilibrium with oxaloacetate), malate, and fumarate (Fig. 3A). To distinguish how glutamine carbon contributes to these metabolites in normoxia and hypoxia, SF188 cells incubated in normoxia or hypoxia were cultured in medium containing 4 mM [U-13C]glutamine. After a 4-h labeling period, metabolites were extracted and the intracellular pools of aspartate, malate, and fumarate were analyzed by GC-MS.

In normoxia, the majority of the enriched intracellular asparatate, malate, and fumarate were the +4 species, which arise through oxidative metabolism of glutamine-derived α-ketoglutarate (Fig. 3 B and C). The +3 species, which can be derived from the citrate generated by the reductive carboxylation of glutamine-derived α-ketoglutarate, constituted a significantly lower percentage of the total aspartate, malate, and fumarate pools. By contrast, in hypoxia, the +3 species constituted a larger percentage of the total aspartate, malate, and fumarate pools than they did in normoxia. These data demonstrate that, in addition to citrate, hypoxic cells preferentially synthesize oxaloacetate, malate, and fumarate through the pathway of reductive carboxylation rather than the oxidative TCA cycle.

IDH2 Is Critical in Hypoxia for Reductive Metabolism of Glutamine and for Cell Proliferation.

We hypothesized that the relative increase in reductive carboxylation we observed in hypoxia could arise from the suppression of α-ketoglutarate oxidation through the TCA cycle. Consistent with this, we found that α-ketoglutarate levels increased in SF188 cells following 24 h in hypoxia (Fig. 4A). Surprisingly, we also found that levels of the closely related metabolite 2-hydroxyglutarate (2HG) increased in hypoxia, concomitant with the increase in α-ketoglutarate under these conditions. 2HG can arise from the noncarboxylating reduction of α-ketoglutarate (Fig. 4B). Recent work has found that specific cancer-associated mutations in the active sites of either IDH1 or IDH2 lead to a 10- to 100-fold enhancement in this activity facilitating 2HG production (1517), but SF188 cells lack IDH1/2 mutations. However, 2HG levels are also substantially elevated in the inborn error of metabolism 2HG aciduria, and the majority of patients with this disease lack IDH1/2 mutations. As 2HG has been demonstrated to arise in these patients from mitochondrial α-ketoglutarate (18), we hypothesized that both the increased reductive carboxylation of glutamine-derived α-ketoglutarate to citrate and the increased 2HG accumulation we observed in hypoxia could arise from increased reductive metabolism by wild-type IDH2 in the mitochondria.

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2

Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2

Fig. 4. Reductive carboxylation of glutamine-derived α-ketoglutarate to citrate in hypoxic cancer cells is dependent on mitochondrial IDH2. (A) α-ketoglutarate and 2HG increase in hypoxia. SF188 cells were cultured in complete medium equilibrated with either 21% O2 (Normoxia) or 0.5% O2 (Hypoxia) for 24 h. Intracellular metabolites were then extracted, cell extracts spiked with a 13C-labeled citrate as an internal standard, and intracellular α-ketoglutarate and 2HG levels were analyzed with GC-MS. Data shown are the mean ± SEM of three independent experiments. (B) Model for reductive metabolism from glutamine-derived α-ketoglutarate. Glutamine+5 is catabolized to α-ketoglutarate+5. Carboxylation of α-ketoglutarate+5 followed by reduction of the carboxylated intermediate (reductive carboxylation) will produce isocitrate+5, which can then isomerize to cit+5. In contrast, reductive activity on α-ketoglutarate+5 that is uncoupled from carboxylation will produce 2HG+5. (C) IDH2 is required for reductive metabolism of glutamine-derived α-ketoglutarate in hypoxia. SF188 cells transfected with a siRNA against IDH2 (siIDH2) or nontargeting negative control (siCTRL) were cultured for 2 d in complete medium equilibrated with 0.5% O2. (Upper) Cells were then cultured at 0.5% O2 for an additional 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. 13C enrichment in intracellular citrate and 2HG was determined and normalized to the relevant metabolite total pool size. (Lower) Cells transfected and cultured in parallel at 0.5% O2 were counted by hemacytometer (excluding nonviable cells with trypan blue staining) or harvested for protein to assess IDH2 expression by Western blot. Data shown for GC-MS and cell counts are the mean ± SD of three independent cultures from a representative experiment. **P < 0.01, ***P < 0.001.

In an experiment to test this hypothesis, SF188 cells were transfected with either siRNA directed against mitochondrial IDH2 (siIDH2) or nontargeting control, incubated in hypoxia for 2 d, and then cultured for another 4 h in hypoxia in media containing 4 mM [U-13C]glutamine. After the labeling period, metabolites were extracted and analyzed by GC-MS (Fig. 4C). Hypoxic SF188 cells transfected with siIDH2 displayed a decreased contribution of cit+5 to the total citrate pool, supporting an important role for IDH2 in the reductive carboxylation of glutamine-derived α-ketoglutarate in hypoxic conditions. The contribution of cit+4 to the total citrate pool did not decrease with siIDH2 treatment, consistent with IDH2 knockdown specifically affecting the pathway of reductive carboxylation and not other fundamental TCA cycle-regulating processes. In confirmation of reverse flux occurring through IDH2, the contribution of 2HG+5 to the total 2HG pool decreased in siIDH2-treated cells. Supporting the importance of citrate production by IDH2-mediated reductive carboxylation for hypoxic cell proliferation, siIDH2-transfected SF188 cells displayed a defect in cellular accumulation in hypoxia. Decreased expression of IDH2 protein following siIDH2 transfection was confirmed by Western blot. Collectively, these data point to the importance of mitochondrial IDH2 for the increase in reductive carboxylation flux of glutamine-derived α-ketoglutarate to maintain citrate levels in hypoxia, and to the importance of this reductive pathway for hypoxic cell proliferation.

Reprogramming of Metabolism by HIF1 in the Absence of Hypoxia Is Sufficient to Induce Increased Citrate Synthesis by Reductive Carboxylation Relative to Oxidative Metabolism.

The relative increase in the reductive metabolism of glutamine-derived α-ketoglutarate at 0.5% O2 may be explained by the decreased ability to carry out oxidative NAD+-dependent reactions as respiration is inhibited (1213). However, a shift to preferential reductive glutamine metabolism could also result from the active reprogramming of cellular metabolism by HIF1 (810), which inhibits the generation of mitochondrial acetyl-CoA necessary for the synthesis of citrate by oxidative glucose and glutamine metabolism (Fig. 5A). To better understand the role of HIF1 in reductive glutamine metabolism, we used VHL-deficient RCC4 cells, which display constitutive expression of HIF1α under normoxia (Fig. 5B). RCC4 cells expressing either a nontargeting control shRNA (shCTRL) or an shRNA directed at HIF1α (shHIF1α) were incubated in normoxia and cultured in medium with 4 mM [U-13C]glutamine. Following a 4-h labeling period, metabolites were extracted and the cellular citrate pool was analyzed by GC-MS. In shCTRL cells, which have constitutive HIF1α expression despite incubation in normoxia, the majority of the total citrate pool was constituted by the cit+5 species, with low levels of all other species including cit+4 (Fig. 5C). By contrast, in HIF1α-deficient cells the contribution of cit+5 to the total citrate pool was greatly decreased, whereas the contribution of cit+4 to the total citrate pool increased and was the most abundant citrate species. These data demonstrate that the relative enhancement of the reductive carboxylation pathway for citrate synthesis can be recapitulated by constitutive HIF1 activation in normoxia.

Reprogramming of metabolism by HIF1 in the absence of hypoxia

Reprogramming of metabolism by HIF1 in the absence of hypoxia

Reprogramming of metabolism by HIF1 in the absence of hypoxia is sufficient to induce reductive carboxylation of glutamine-derived α-ketoglutarate.

Fig. 5. Reprogramming of metabolism by HIF1 in the absence of hypoxia is sufficient to induce reductive carboxylation of glutamine-derived α-ketoglutarate. (A) Model depicting how HIF1 signaling’s inhibition of pyruvate dehydrogenase (PDH) activity and promotion of lactate dehydrogenase-A (LDH-A) activity can block the generation of mitochondrial acetyl-CoA from glucose-derived pyruvate, thereby favoring citrate synthesis from reductive carboxylation of glutamine-derived α-ketoglutarate. (B) Western blot demonstrating HIF1α protein in RCC4 VHL−/− cells in normoxia with a nontargeting shRNA (shCTRL), and the decrease in HIF1α protein in RCC4 VHL−/− cells stably expressing HIF1α shRNA (shHIF1α). (C) HIF1-induced reprogramming of glutamine metabolism. Cells from B at 21% O2 were cultured for 4 h in glutamine-deficient medium supplemented with 4 mM [U-13C]glutamine. Intracellular metabolites were then extracted, and 13C enrichment in cellular citrate was determined by GC-MS. Data shown are the mean ± SD of three independent cultures from a representative of three independent experiments. ***P < 0.001.

Compared with glucose metabolism, much less is known regarding how glutamine metabolism is altered under hypoxia. It has also remained unclear how hypoxic cells can maintain the citrate production necessary for macromolecular biosynthesis. In this report, we demonstrate that in contrast to cells at 21% O2, where citrate is predominantly synthesized through oxidative metabolism of both glucose and glutamine, reductive carboxylation of glutamine carbon becomes the major pathway of citrate synthesis in cells that can effectively proliferate at 0.5% O2. Moreover, we show that in these hypoxic cells, reductive carboxylation of glutamine-derived α-ketoglutarate is dependent on mitochondrial IDH2. Although others have previously suggested the existence of reductive carboxylation in cancer cells (1920), these studies failed to demonstrate the intracellular localization or specific IDH isoform responsible for the reductive carboxylation flux. Recently, we identified IDH2 as an isoform that contributes to reductive carboxylation in cancer cells incubated at 21% O2 (16), but remaining unclear were the physiological importance and regulation of this pathway relative to oxidative metabolism, as well as the conditions where this reductive pathway might be advantageous for proliferating cells.

Here we report that IDH2-mediated reductive carboxylation of glutamine-derived α-ketoglutarate to citrate is an important feature of cells proliferating in hypoxia. Moreover, the reliance on reductive glutamine metabolism can be recapitulated in normoxia by constitutive HIF1 activation in cells with loss of VHL. The mitochondrial NADPH/NADP+ ratio required to fuel the reductive reaction through IDH2 can arise from the increased NADH/NAD+ ratio existing in the mitochondria under hypoxic conditions (2122), with the transfer of electrons from NADH to NADP+ to generate NADPH occurring through the activity of the mitochondrial transhydrogenase (23). Our data do not exclude a complementary role for cytosolic IDH1 in impacting reductive glutamine metabolism, potentially through its oxidative function in an IDH2/IDH1 shuttle that transfers high energy electrons in the form of NADPH from mitochondria to cytosol (1624).

In further support of the increased mitochondrial reductive glutamine metabolism that we observe in hypoxia, we report here that incubation in hypoxia can lead to elevated 2HG levels in cells lacking IDH1/2 mutations. 2HG production from glutamine-derived α-ketoglutarate significantly decreased with knockdown of IDH2, supporting the conclusion that 2HG is produced in hypoxia by enhanced reverse flux of α-ketoglutarate through IDH2 in a truncated, noncarboxylating reductive reaction. However, other mechanisms may also contribute to 2HG elevation in hypoxia. These include diminished oxidative activity and/or enhanced reductive activity of the 2HG dehydrogenase, a mitochondrial enzyme that normally functions to oxidize 2HG back to α-ketoglutarate (25). The level of 2HG elevation we observe in hypoxic cells is associated with a concomitant increase in α-ketoglutarate, and is modest relative to that observed in cancers with IDH1/2 gain-of-function mutations. Nonetheless, 2HG elevation resulting from hypoxia in cells with wild-type IDH1/2 may hold promise as a cellular or serum biomarker for tissues undergoing chronic hypoxia and/or excessive glutamine metabolism.

The IDH2-dependent reductive carboxylation pathway that we propose in this report allows for continued citrate production from glutamine carbon when hypoxia and/or HIF1 activation prevents glucose carbon from contributing to citrate synthesis. Moreover, as opposed to continued oxidative TCA cycle functioning in hypoxia which can increase reactive oxygen species (ROS), reductive carboxylation of α-ketoglutarate in the mitochondria may serve as an electron sink that decreases the generation of ROS. HIF1 activity is not limited to the setting of hypoxia, as a common feature of several cancers is the normoxic stabilization of HIF1α through loss of the VHL tumor suppressor or other mechanisms. We demonstrate here that altered glutamine metabolism through a mitochondrial reductive pathway is a central aspect of hypoxic proliferating cell metabolism and HIF1-induced metabolic reprogramming. These findings are relevant for the understanding of numerous constitutive HIF1-expressing malignancies, as well as for populations, such as stem progenitor cells, which frequently proliferate in hypoxic conditions.

7.9.3 Hypoxia-Inducible Factors in Physiology and Medicine

Gregg L. Semenza
Cell. 2012 Feb 3; 148(3): 399–408.

Oxygen homeostasis represents an organizing principle for understanding metazoan evolution, development, physiology, and pathobiology. The hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis in all metazoan species. Rapid progress is being made in elucidating homeostatic roles of HIFs in many physiological systems, determining pathological consequences of HIF dysregulation in chronic diseases, and investigating potential targeting of HIFs for therapeutic purposes. Oxygen homeostasis represents an organizing principle for understanding metazoan evolution, development, physiology, and pathobiology. The hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis in all metazoan species. Rapid progress is being made in elucidating homeostatic roles of HIFs in many physiological systems, determining pathological consequences of HIF dysregulation in chronic diseases, and investigating potential targeting of HIFs for therapeutic purposes.


Oxygen is central to biology because of its utilization in the process of respiration. O2 serves as the final electron acceptor in oxidative phosphorylation, which carries with it the risk of generating reactive oxygen species (ROS) that react with cellular macromolecules and alter their biochemical or physical properties, resulting in cell dysfunction or death. As a consequence, metazoan organisms have evolved elaborate cellular metabolic and systemic physiological systems that are designed to maintain oxygen homeostasis. This review will focus on the role of hypoxia-inducible factors (HIFs) as master regulators of oxygen homeostasis and, in particular, on recent advances in understanding their roles in physiology and medicine. Due to space limitations and the remarkably pleiotropic effects of HIFs, the description of such roles will be illustrative rather than comprehensive.

O2 and Evolution, Part 1

Accumulation of O2 in Earth’s atmosphere starting ~2.5 billion years ago led to evolution of the extraordinarily efficient system of oxidative phosphorylation that transfers chemical energy stored in carbon bonds of organic molecules to the high-energy phosphate bond in ATP, which is used to power physicochemical reactions in living cells. Energy produced by mitochondrial respiration is sufficient to power the development and maintenance of multicellular organisms, which could not be sustained by energy produced by glycolysis alone (Lane and Martin, 2010). The modest dimensions of primitive metazoan species were such that O2 could diffuse from the atmosphere to all of the organism’s thousand cells, as is the case for the worm Caenorhabditis elegans. To escape the constraints placed on organismal growth by diffusion, systems designed to conduct air to cells deep within the body evolved and were sufficient for O2delivery to organisms with hundreds of thousands of cells, such as the fly Drosophila melanogaster. The final leap in body scale occurred in vertebrates and was associated with the evolution of complex respiratory, circulatory, and nervous systems designed to efficiently capture and distribute O2 to hundreds of millions of millions of cells in the case of the adult Homo sapiens.

Hypoxia-Inducible Factors

Hypoxia-inducible factor 1 (HIF-1) is expressed by all extant metazoan species analyzed (Loenarz et al., 2011). HIF-1 consists of HIF-1α and HIF-1β subunits, which each contain basic helix-loop-helix-PAS (bHLH-PAS) domains (Wang et al., 1995) that mediate heterodimerization and DNA binding (Jiang et al., 1996a). HIF-1β heterodimerizes with other bHLH-PAS proteins and is present in excess, such that HIF-1α protein levels determine HIF-1 transcriptional activity (Semenza et al., 1996).

Under well-oxygenated conditions, HIF-1α is bound by the von Hippel-Lindau (VHL) protein, which recruits an ubiquitin ligase that targets HIF-1α for proteasomal degradation (Kaelin and Ratcliffe, 2008). VHL binding is dependent upon hydroxylation of a specific proline residue in HIF-1α by the prolyl hydroxylase PHD2, which uses O2 as a substrate such that its activity is inhibited under hypoxic conditions (Epstein et al., 2001). In the reaction, one oxygen atom is inserted into the prolyl residue and the other atom is inserted into the co-substrate α-ketoglutarate, splitting it into CO2 and succinate (Kaelin and Ratcliffe, 2008). Factor inhibiting HIF-1 (FIH-1) represses HIF-1α transactivation function (Mahon et al., 2001) by hydroxylating an asparaginyl residue, using O2 and α-ketoglutarate as substrates, thereby blocking the association of HIF-1α with the p300 coactivator protein (Lando et al., 2002). Dimethyloxalylglycine (DMOG), a competitive antagonist of α-ketoglutarate, inhibits the hydroxylases and induces HIF-1-dependent transcription (Epstein et al., 2001). HIF-1 activity is also induced by iron chelators (such as desferrioxamine) and cobalt chloride, which inhibit hydroxylases by displacing Fe(II) from the catalytic center (Epstein et al., 2001).

Studies in cultured cells (Jiang et al., 1996b) and isolated, perfused, and ventilated lung preparations (Yu et al., 1998) revealed an exponential increase in HIF-1α levels at O2 concentrations less than 6% (~40 mm Hg), which is not explained by known biochemical properties of the hydroxylases. In most adult tissues, O2concentrations are in the range of 3-5% and any decrease occurs along the steep portion of the dose-response curve, allowing a graded response to hypoxia. Analyses of cultured human cells have revealed that expression of hundreds of genes was increased in response to hypoxia in a HIF-1-dependent manner (as determined by RNA interference) with direct binding of HIF-1 to the gene (as determined by chromatin immunoprecipitation [ChIP] assays); in addition, the expression of hundreds of genes was decreased in response to hypoxia in a HIF-1-dependent manner but binding of HIF-1 to these genes was not detected (Mole et al., 2009), indicating that HIF-dependent repression occurs via indirect mechanisms, which include HIF-1-dependent expression of transcriptional repressors (Yun et al., 2002) and microRNAs (Kulshreshtha et al., 2007). ChIP-seq studies have revealed that only 40% of HIF-1 binding sites are located within 2.5 kb of the transcription start site (Schödel et al., 2011).

In vertebrates, HIF-2α is a HIF-1α paralog that is also regulated by prolyl and asparaginyl hydroxylation and dimerizes with HIF-1β, but is expressed in a cell-restricted manner and plays important roles in erythropoiesis, vascularization, and pulmonary development, as described below. In D. melanogaster, the gene encoding the HIF-1α ortholog is designated similar and its paralog is designated trachealess because inactivating mutations result in defective development of the tracheal tubes (Wilk et al., 1996). In contrast, C. elegans has only a single HIF-1α homolog (Epstein et al., 2001). Thus, in both invertebrates and vertebrates, evolution of specialized systems for O2 delivery was associated with the appearance of a HIF-1α paralog.

O2 and Metabolism

The regulation of metabolism is a principal and primordial function of HIF-1. Under hypoxic conditions, HIF-1 mediates a transition from oxidative to glycolytic metabolism through its regulation of: PDK1, encoding pyruvate dehydrogenase (PDH) kinase 1, which phosphorylates and inactivates PDH, thereby inhibiting the conversion of pyruvate to acetyl coenzyme A for entry into the tricarboxylic acid cycle (Kim et al., 2006Papandreou et al., 2006); LDHA, encoding lactate dehydrogenase A, which converts pyruvate to lactate (Semenza et al. 1996); and BNIP3 (Zhang et al. 2008) and BNIP3L (Bellot et al., 2009), which mediate selective mitochondrial autophagy (Figure 1). HIF-1 also mediates a subunit switch in cytochrome coxidase that improves the efficiency of electron transfer under hypoxic conditions (Fukuda et al., 2007). An analogous subunit switch is also observed in Saccharomyces cerevisiae, although it is mediated by a completely different mechanism (yeast lack HIF-1), suggesting that it may represent a fundamental response of eukaryotic cells to hypoxia.

Regulation of Glucose Metabolism nihms-350382-f0001

Regulation of Glucose Metabolism nihms-350382-f0001

Regulation of Glucose Metabolism
Figure 1
Regulation of Glucose Metabolism

It is conventional wisdom that cells switch to glycolysis when O2 becomes limiting for mitochondrial ATP production. Yet, HIF-1α-null mouse embryo fibroblasts, which do not down-regulate respiration under hypoxic conditions, have higher ATP levels at 1% O2 than wild-type cells at 20% O2, demonstrating that under these conditions O2 is not limiting for ATP production (Zhang et al., 2008). However, the HIF-1α-null cells die under prolonged hypoxic conditions due to ROS toxicity (Kim et al. 2006Zhang et al., 2008). These studies have led to a paradigm shift with regard to our understanding of the regulation of cellular metabolism (Semenza, 2011): the purpose of this switch is to prevent excess mitochondrial generation of ROS that would otherwise occur due to the reduced efficiency of electron transfer under hypoxic conditions (Chandel et al., 1998). This may be particularly important in stem cells, in which avoidance of DNA damage is critical (Suda et al., 2011).

Role of HIFs in Development

Much of mammalian embryogenesis occurs at O2 concentrations of 1-5% and O2 functions as a morphogen (through HIFs) in many developmental systems (Dunwoodie, 2009). Mice that are homozygous for a null allele at the locus encoding HIF-1α die by embryonic day 10.5 with cardiac malformations, vascular defects, and impaired erythropoiesis, indicating that all three components of the circulatory system are dependent upon HIF-1 for normal development (Iyer et al., 1998Yoon et al., 2011). Depending on the genetic background, mice lacking HIF-2α: die by embryonic day 12.5 with vascular defects (Peng et al., 2000) or bradycardia due to deficient catecholamine production (Tian et al., 1998); die as neonates due to impaired lung maturation (Compernolle et al., 2002); or die several months after birth due to ROS-mediated multi-organ failure (Scortegagna et al., 2003). Thus, while vertebrate evolution was associated with concomitant appearance of the circulatory system and HIF-2α, both HIF-1 and HIF-2 have important roles in circulatory system development. Conditional knockout of HIF-1α in specific cell types has demonstrated important roles in chondrogenesis (Schipani et al., 2001), adipogenesis (Yun et al., 2002), B-lymphocyte development (Kojima et al., 2002), osteogenesis (Wang et al., 2007), hematopoiesis (Takubo et al., 2010), T-lymphocyte differentiation (Dang et al., 2011), and innate immunity (Zinkernagel et al., 2007). While knockout mouse experiments point to the adverse effects of HIF-1 loss-of-function on development, it is also possible that increased HIF-1 activity, induced by hypoxia in embryonic tissues as a result of abnormalities in placental blood flow, may also dysregulate development and result in congenital malformations. For example, HIF-1α has been shown to interact with, and stimulate the transcriptional activity of, Notch, which plays a key role in many developmental pathways (Gustafsson et al., 2005).

Translational Prospects

Drug discovery programs have been initiated at many pharmaceutical and biotech companies to develop prolyl hydroxylase inhibitors (PHIs) that, as described above for DMOG, induce HIF activity for treatment of disorders in which HIF mediates protective physiological responses. Local and/or short term induction of HIF activity by PHIs, gene therapy, or other means are likely to be useful novel therapies for many of the diseases described above. In the case of ischemic cardiovascular disease, local therapy is needed to provide homing signals for the recruitment of BMDACs. Chronic systemic use of PHIs must be approached with great caution: individuals with genetic mutations that constitutively activate the HIF pathway (described below) have increased incidence of cardiovascular disease and mortality (Yoon et al., 2011). On the other hand, the profound inhibition of HIF activity and vascular responses to ischemia that are associated with aging suggest that systemic replacement therapy might be contemplated as a preventive measure for subjects in whom impaired HIF responses to hypoxia can be documented. In C. elegans, VHL loss-of-function increases lifespan in a HIF-1-dependent manner (Mehta et al., 2009), providing further evidence for a mutually antagonistic relationship between HIF-1 and aging.


Cancers contain hypoxic regions as a result of high rates of cell proliferation coupled with the formation of vasculature that is structurally and functionally abnormal. Increased HIF-1α and/or HIF-2α levels in diagnostic tumor biopsies are associated with increased risk of mortality in cancers of the bladder, brain, breast, colon, cervix, endometrium, head/neck, lung, ovary, pancreas, prostate, rectum, and stomach; these results are complemented by experimental studies, which demonstrate that genetic manipulations that increase HIF-1α expression result in increased tumor growth, whereas loss of HIF activity results in decreased tumor growth (Semenza, 2010). HIFs are also activated by genetic alterations, most notably, VHL loss of function in clear cell renal carcinoma (Majmunder et al., 2010). HIFs activate transcription of genes that play key roles in critical aspects of cancer biology, including stem cell maintenance (Wang et al., 2011), cell immortalization, epithelial-mesenchymal transition (Mak et al., 2010), genetic instability (Huang et al., 2007), vascularization (Liao and Johnson, 2007), glucose metabolism (Luo et al., 2011), pH regulation (Swietach et al., 2007), immune evasion (Lukashev et al., 2007), invasion and metastasis (Chan and Giaccia, 2007), and radiation resistance (Moeller et al., 2007). Given the extensive validation of HIF-1 as a potential therapeutic target, drugs that inhibit HIF-1 have been identified and shown to have anti-cancer effects in xenograft models (Table 1Semenza, 2010).

Table 1  Drugs that Inhibit HIF-1

Process Inhibited Drug Class Prototype
HIF-1 α synthesis Cardiac glycosidemTOR inhibitorMicrotubule targeting agent

Topoisomerase I inhibitor



HIF-1 α protein stability HDAC inhibitorHSP90 inhibitorCalcineurin inhibitor

Guanylate cyclase activator



Heterodimerization Antimicrobial agent Acriflavine
DNA binding AnthracyclineQuinoxaline antibiotic DoxorubicinEchinomycin
Transactivation Proteasome inhibitorAntifungal agent BortezomibAmphotericin B
Signal transduction BCR-ABL inhibitorCyclooxygenase inhibitorEGFR inhibitor

HER2 inhibitor

ImatinibIbuprofenErlotinib, Gefitinib


Over 100 women die every day of breast cancer in the U.S. The mean PO2 is 10 mm Hg in breast cancer as compared to > 60 mm Hg in normal breast tissue and cancers with PO2 < 10 mm Hg are associated with increased risk of metastasis and patient mortality (Vaupel et al., 2004). Increased HIF-1α protein levels, as identified by immunohistochemical analysis of tumor biopsies, are associated with increased risk of metastasis and/or patient mortality in unselected breast cancer patients and in lymph node-positive, lymph node-negative, HER2+, or estrogen receptor+ subpopulations (Semenza, 2011). Metastasis is responsible for > 90% of breast cancer mortality. The requirement for HIF-1 in breast cancer metastasis has been demonstrated for both autochthonous tumors in transgenic mice (Liao et al., 2007) and orthotopic transplants in immunodeficient mice (Zhang et al., 2011Wong et al., 2011). Primary tumors direct the recruitment of bone marrow-derived cells to the lungs and other sites of metastasis (Kaplan et al., 2005). In breast cancer, hypoxia induces the expression of lysyl oxidase (LOX), a secreted protein that remodels collagen at sites of metastatic niche formation (Erler et al., 2009). In addition to LOX, breast cancers also express LOX-like proteins 2 and 4. LOX, LOXL2, and LOXL4 are all HIF-1-regulated genes and HIF-1 inhibition blocks metastatic niche formation regardless of which LOX/LOXL protein is expressed, whereas available LOX inhibitors are not effective against all LOXL proteins (Wong et al., 2011), again illustrating the role of HIF-1 as a master regulator that controls the expression of multiple genes involved in a single (patho)physiological process.

Translational Prospects

Small molecule inhibitors of HIF activity that have anti-cancer effects in mouse models have been identified (Table 1). Inhibition of HIF impairs both vascular and metabolic adaptations to hypoxia, which may decrease O2 delivery and increase O2 utilization. These drugs are likely to be useful (as components of multidrug regimens) in the treatment of a subset of cancer patients in whom high HIF activity is driving progression. As with all novel cancer therapeutics, successful translation will require the development of methods for identifying the appropriate patient cohort. Effects of combination drug therapy also need to be considered. VEGF receptor tyrosine kinase inhibitors, which induce tumor hypoxia by blocking vascularization, have been reported to increase metastasis in mouse models (Ebos et al., 2009), which may be mediated by HIF-1; if so, combined use of HIF-1 inhibitors with these drugs may prevent unintended counter-therapeutic effects.

HIF inhibitors may also be useful in the treatment of other diseases in which dysregulated HIF activity is pathogenic. Proof of principle has been established in mouse models of ocular neovascularization, a major cause of blindness in the developed world, in which systemic or intraocular injection of the HIF-1 inhibitor digoxin is therapeutic (Yoshida et al., 2010). Systemic administration of HIF inhibitors for cancer therapy would be contraindicated in patients who also have ischemic cardiovascular disease, in which HIF activity is protective. The analysis of SNPs at the HIF1A locus described above suggests that the population may include HIF hypo-responders, who are at increased risk of severe ischemic cardiovascular disease. It is also possible that HIF hyper-responders, such as individuals with hereditary erythrocytosis, are at increased risk of particularly aggressive cancer.

O2 and Evolution, Part 2

When lowlanders sojourn to high altitude, hypobaric hypoxia induces erythropoiesis, which is a relatively ineffective response because the problem is not insufficient red cells, but rather insufficient ambient O2. Chronic erythrocytosis increases the risk of heart attack, stroke, and fetal loss during pregnancy. Many high-altitude Tibetans maintain the same hemoglobin concentration as lowlanders and yet, despite severe hypoxemia, they also maintain aerobic metabolism. The basis for this remarkable evolutionary adaptation appears to have involved the selection of genetic variants at multiple loci encoding components of the oxygen sensing system, particularly HIF-2α (Beall et al., 2010Simonson et al., 2010Yi et al., 2010). Given that hereditary erythrocytosis is associated with modest HIF-2α gain-of-function, the Tibetan genotype associated with absence of an erythrocytotic response to hypoxia may encode reduced HIF-2α activity along with other alterations that increase metabolic efficiency. Delineating the molecular mechanisms underlying these metabolic adaptations may lead to novel therapies for ischemic disorders, illustrating the importance of oxygen homeostasis as a nexus where evolution, biology, and medicine converge.

7.9.4 Hypoxia-inducible factor 1. Regulator of mitochondrial metabolism and mediator of ischemic preconditioning

Semenza GL1.
Biochim Biophys Acta. 2011 Jul; 1813(7):1263-8.

Hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability by regulating gene expression. A critical cell-autonomous adaptive response to chronic hypoxia controlled by HIF-1 is reduced mitochondrial mass and/or metabolism. Exposure of HIF-1-deficient fibroblasts to chronic hypoxia results in cell death due to excessive levels of reactive oxygen species (ROS). HIF-1 reduces ROS production under hypoxic conditions by multiple mechanisms including: a subunit switch in cytochrome c oxidase from the COX4-1 to COX4-2 regulatory subunit that increases the efficiency of complex IV; induction of pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; induction of BNIP3, which triggers mitochondrial selective autophagy; and induction of microRNA-210, which blocks assembly of Fe/S clusters that are required for oxidative phosphorylation. HIF-1 is also required for ischemic preconditioning and this effect may be due in part to its induction of CD73, the enzyme that produces adenosine. HIF-1-dependent regulation of mitochondrial metabolism may also contribute to the protective effects of ischemic preconditioning.

The story of life on Earth is a tale of oxygen production and utilization. Approximately 3 billion years ago, primitive single-celled organisms evolved the capacity for photosynthesis, a biochemical process in which photons of solar energy are captured by chlorophyll and used to power the reaction of CO2 and H2O to form glucose and O2. The subsequent rise in the atmospheric O2 concentration over the next billion years set the stage for the ascendance of organisms with the capacity for respiration, a process that consumes glucose and O2 and generates CO2, H2O, and energy in the form of ATP. Some of these single-celled organisms eventually took up residence within the cytoplasm of other cells and devoted all of their effort to energy production as mitochondria. Compared to the conversion of glucose to lactate by glycolysis, the complete oxidation of glucose by respiration provided such a large increase in energy production that it made possible the evolution of multicellular organisms. Among metazoan organisms, the progressive increase in body size during evolution was accompanied by progressively more complex anatomic structures that function to ensure the adequate delivery of O2 to all cells, ultimately resulting in the sophisticated circulatory and respiratory systems of vertebrates.

All metazoan cells can sense and respond to reduced O2 availability (hypoxia). Adaptive responses to hypoxia can be cell autonomous, such as the alterations in mitochondrial metabolism that are described below, or non-cell-autonomous, such as changes in tissue vascularization (reviewed in ref. 1). Primary responses to hypoxia need to be distinguished from secondary responses to sequelae of hypoxia, such as the adaptive responses to ATP depletion that are mediated by AMP kinase (reviewed in ref 2). In contrast, recent data suggest that O2 and redox homeostasis are inextricably linked and that changes in oxygenation are inevitably associated with changes in the levels of reactive oxygen species (ROS), as will be discussed below.

HIF-1 Regulates Oxygen Homeostasis in All Metazoan Species

A key regulator of the developmental and physiological networks required for the maintenance of O2homeostasis is hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric transcription factor that is composed of an O2-regulated HIF-1α subunit and a constitutively expressed HIF-1β subunit [3,4]. HIF-1 regulates the expression of hundreds of genes through several major mechanisms. First, HIF-1 binds directly to hypoxia response elements, which are cis-acting DNA sequences located within target genes [5]. The binding of HIF-1 results in the recruitment of co-activator proteins that activate gene transcription (Fig. 1A). Only rarely does HIF-1 binding result in transcriptional repression [6]. Instead, HIF-1 represses gene expression by indirect mechanisms, which are described below. Second, among the genes activated by HIF-1 are many that encode transcription factors [7], which when synthesized can bind to and regulate (either positively or negatively) secondary batteries of target genes (Fig. 1B). Third, another group of HIF-1 target genes encode members of the Jumonji domain family of histone demethylases [8,9], which regulate gene expression by modifying chromatin structure (Fig. 1C). Fourth, HIF-1 can activate the transcription of genes encoding microRNAs [10], which bind to specific mRNA molecules and either block their translation or mediate their degradation (Fig. 1D). Fifth, the isolated HIF-1α subunit can bind to other transcription factors [11,12] and inhibit (Fig. 1E) or potentiate (Fig. 1F) their activity.

Mechanisms by which HIF-1 regulates gene expression. nihms232046f1

Mechanisms by which HIF-1 regulates gene expression. nihms232046f1

Mechanisms by which HIF-1 regulates gene expression.

Fig. 1 Mechanisms by which HIF-1 regulates gene expression. (A) Top: HIF-1 binds directly to target genes at a cis-acting hypoxia response element (HRE) and recruits coactivator proteins such as p300 to increase gene transcription.

HIF-1α and HIF-1β are present in all metazoan species, including the simple roundworm Caenorhabitis elegans [13], which consists of ~103 cells and has no specialized systems for O2 delivery. The fruit flyDrosophila melanogaster evolved tracheal tubes, which conduct air into the interior of the body from which it diffuses to surrounding cells. In vertebrates, the development of the circulatory and respiratory systems was accompanied by the appearance of HIF-2α, which is also O2-regulated and heterodimerizes with HIF-1β [14] but is only expressed in a restricted number of cell types [15], whereas HIF-1α and HIF-1β are expressed in all human and mouse tissues [16]. In Drosophila, the ubiquitiously expressed HIF-1α ortholog is designatedSimilar [17] and the paralogous gene that is expressed specifically in tracheal tubes is designated Trachealess[18].

HIF-1 Activity is Regulated by Oxygen

In the presence of O2, HIF-1α and HIF-2α are subjected to hydroxylation by prolyl-4-hydroxylase domain proteins (PHDs) that use O2 and α-ketoglutarate as substrates and generate CO2 and succinate as by-products [19]. Prolyl hydroxylation is required for binding of the von Hipple-Lindau protein, which recruits a ubiquitin-protein ligase that targets HIF-1α and HIF-2α for proteasomal degradation (Fig. 2). Under hypoxic conditions, the rate of hydroxylation declines and the non-hydroxylated proteins accumulate. HIF-1α transactivation domain function is also O2-regulated [20,21]. Factor inhibiting HIF-1 (FIH-1) represses transactivation domain function [22] by hydroxylating asparagine residue 803 in HIF-1α, thereby blocking the binding of the co-activators p300 and CBP [23].

Negative regulation of HIF-1 activity by oxygen nihms232046f2

Negative regulation of HIF-1 activity by oxygen nihms232046f2

Negative regulation of HIF-1 activity by oxygen

Fig. 2 Negative regulation of HIF-1 activity by oxygen. Top: In the presence of O2: prolyl hydroxylation of HIF-1a leads to binding of the von Hippel-Lindau protein (VHL), which recruits a ubiquitin protein-ligase that targets HIF-1a for proteasomal degradation;

When cells are acutely exposed to hypoxic conditions, the generation of ROS at complex III of the mitochondrial electron transport chain (ETC) increases and is required for the induction of HIF-1α protein levels [24]. More than a decade after these observations were first made, the precise mechanism by which hypoxia increases ROS generation and by which ROS induces HIF-1α accumulation remain unknown. However, the prolyl and asparaginyl hydroxylases contain Fe2+ in their active site and oxidation to Fe3+would block their catalytic activity. Since O2 is a substrate for the hydroxylation reaction, anoxia also results in a loss of enzyme activity. However, the concentration at which O2 becomes limiting for prolyl or asparaginyl hydroxylase activity in vivo is not known.

HIF-1 Regulates the Balance Between Oxidative and Glycolytic Metabolism

All metazoan organisms depend on mitochondrial respiration as the primary mechanism for generating sufficient amounts of ATP to maintain cellular and systemic homeostasis. Respiration, in turn, is dependent on an adequate supply of O2 to serve as the final electron acceptor in the ETC. In this process, electrons are transferred from complex I (or complex II) to complex III, then to complex IV, and finally to O2, which is reduced to water. This orderly transfer of electrons generates a proton gradient across the inner mitochondrial membrane that is used to drive the synthesis of ATP. At each step of this process, some electrons combine with O2 prematurely, resulting in the production of superoxide anion, which is reduced to hydrogen peroxide through the activity of mitochondrial superoxide dismutase. The efficiency of electron transport appears to be optimized to the physiological range of O2 concentrations, such that ATP is produced without the production of excess superoxide, hydrogen peroxide, and other ROS at levels that would result in the increased oxidation of cellular macromolecules and subsequent cellular dysfunction or death. In contrast, when O2levels are acutely increased or decreased, an imbalance between O2 and electron flow occurs, which results in increased ROS production.

MEFs require HIF-1 activity to make two critical metabolic adaptations to chronic hypoxia. First, HIF-1 activates the gene encoding pyruvate dehydrogenase (PDH) kinase 1 (PDK1), which phosphorylates and inactivates the catalytic subunit of PDH, the enzyme that converts pyruvate to acetyl coenzyme A (AcCoA) for entry into the mitochondrial tricarboxylic acid (TCA) cycle [25]. Second, HIF-1 activates the gene encoding BNIP3, a member of the Bcl-2 family of mitochondrial proteins, which triggers selective mitochondrial autophagy [26]. Interference with the induction of either of these proteins in hypoxic cells results in increased ROS production and increased cell death. Overexpression of either PDK1 or BNIP3 rescues HIF-1α-null MEFs. By shunting pyruvate away from the mitochondria, PDK1 decreases flux through the ETC and thereby counteracts the reduced efficiency of electron transport under hypoxic conditions, which would otherwise increase ROS production. PDK1 functions cooperatively with the product of another HIF-1 target gene, LDHA [27], which converts pyruvate to lactate, thereby further reducing available substrate for the PDH reaction.

PDK1 effectively reduces flux through the TCA cycle and thereby reduces flux through the ETC in cells that primarily utilize glucose as a substrate for oxidative phosphorylation. However, PDK1 is predicted to have little effect on ROS generation in cells that utilize fatty acid oxidation as their source of AcCoA. Hence another strategy to reduce ROS generation under hypoxic conditions is selective mitochondrial autophagy [26]. MEFs reduce their mitochondrial mass and O2 consumption by >50% after only two days at 1% O2. BNIP3 competes with Beclin-1 for binding to Bcl-2, thereby freeing Beclin-1 to activate autophagy. Using short hairpin RNAs to knockdown expression of BNIP3, Beclin-1, or Atg5 (another component of the autophagy machinery) phenocopied HIF-1α-null cells by preventing hypoxia-induced reductions in mitochondrial mass and O2 consumption as a result of failure to induce autophagy [26]. HIF-1-regulated expression of BNIP3L also contributes to hypoxia-induced autophagy [28]. Remarkably, mice heterozygous for the HIF-1α KO allele have a significantly increased ratio of mitochondrial:nuclear DNA in their lungs (even though this is the organ that is exposed to the highest O2 concentrations), indicating that HIF-1 regulates mitochondrial mass under physiological conditions in vivo [26]. In contrast to the selective mitochondrial autophagy that is induced in response to hypoxia as described above, autophagy (of unspecified cellular components) induced by anoxia does not require HIF-1, BNIP3, or BNIP3L, but is instead regulated by AMP kinase [29].

The multiplicity of HIF-1-mediated mechanisms identified so far by which cells regulate mitochondrial metabolism in response to changes in cellular O2 concentration (Fig. 3) suggests that this is a critical adaptive response to hypoxia. The fundamental nature of this physiological response is underscored by the fact that yeast also switch COX4 subunits in an O2-dependent manner but do so by an entirely different molecular mechanism [33], since yeast do not have a HIF-1α homologue. Thus, it appears that by convergent evolution both unicellular and multicellular eukaryotes possess mechanisms by which they modulate mitochondrial metabolism to maintain redox homeostasis despite changes in O2 availability. Indeed, it is the balance between energy, oxygen, and redox homeostasis that represents the key to life with oxygen.

Regulation of mitochondrial metabolism by HIF-1  nihms232046f3

Regulation of mitochondrial metabolism by HIF-1 nihms232046f3

Regulation of mitochondrial metabolism by HIF-1α

Fig. 3 Regulation of mitochondrial metabolism by HIF-1α. Acute hypoxia leads to increased mitochondrial generation of reactive oxygen species (ROS). Decreased O2 and increased ROS levels lead to decreased HIF-1α hydroxylation (see Fig. 2) and increased HIF-1-dependent 


7.9.5 Regulation of cancer cell metabolism by hypoxia-inducible factor 1

Semenza GL1.
Semin Cancer Biol. 2009 Feb; 19(1):12-6.

The Warburg Effect: The Re-discovery of the Importance of Aerobic Glycolysis in Tumor Cells

The induction of hypoxia-inducible factor 1 (HIF-1) activity, either as a result of intratumoral hypoxia or loss-of-function mutations in the VHL gene, leads to a dramatic reprogramming of cancer cell metabolism involving increased glucose transport into the cell, increased conversion of glucose to pyruvate, and a concomitant decrease in mitochondrial metabolism and mitochondrial mass. Blocking these adaptive metabolic responses to hypoxia leads to cell death due to toxic levels of reactive oxygen species. Targeting HIF-1 or metabolic enzymes encoded by HIF-1 target genes may represent a novel therapeutic approach to cancer.

7.9.6 Coming up for air. HIF-1 and mitochondrial oxygen consumption

Simon MC1.
Cell Metab. 2006 Mar;3(3):150-1.

Hypoxic cells induce glycolytic enzymes; this HIF-1-mediated metabolic adaptation increases glucose flux to pyruvate and produces glycolytic ATP. Two papers in this issue of Cell Metabolism (Kim et al., 2006; Papandreou et al., 2006) demonstrate that HIF-1 also influences mitochondrial function, suppressing both the TCA cycle and respiration by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 regulation in hypoxic cells promotes cell survival.

Comment on

Oxygen deprivation (hypoxia) occurs in tissues when O2 supply via the cardiovascular system fails to meet the demand of O2-consuming cells. Hypoxia occurs naturally in physiological settings (e.g., embryonic development and exercising muscle), as well as in pathophysiological conditions (e.g., myocardial infarction, inflammation, and solid tumor formation). For over a century, it has been appreciated that O2-deprived cells exhibit increased conversion of glucose to lactate (the “Pasteur effect”). Activation of the Pasteur effect during hypoxia in mammalian cells is facilitated by HIF-1, which mediates the upregulation of glycolytic enzymes that support an increase in glycolytic ATP production as mitochondria become starved for O2, the substrate for oxidative phosphorylation (Seagroves et al., 2001). Thus, mitochondrial respiration passively decreases due to O2 depletion in hypoxic tissues. However, reports by Kim et al. (2006) and Papandreou et al. (2006) in this issue of Cell Metabolism demonstrate that this critical metabolic adaptation is more complex and includes an active suppression of mitochondrial pyruvate catabolism and O2consumption by HIF-1.

Mitochondrial oxidative phosphorylation is regulated by multiple mechanisms, including substrate availability. Major substrates include O2 (the terminal electron acceptor) and pyruvate (the primary carbon source). Pyruvate, as the end product of glycolysis, is converted to acetyl-CoA by the pyruvate dehydrogenase enzymatic complex and enters the tricarboxylic acid (TCA) cycle. Pyruvate conversion into acetyl-CoA is irreversible; this therefore represents an important regulatory point in cellular energy metabolism. Pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase activity by phosphorylating its E1 subunit (Sugden and Holness, 2003). In the manuscripts by Kim et al. (2006) and Papandreou et al. (2006), the authors find that PDK1 is a HIF-1 target gene that actively regulates mitochondrial respiration by limiting pyruvate entry into the TCA cycle. By excluding pyruvate from mitochondrial metabolism, hypoxic cells accumulate pyruvate, which is then converted into lactate via lactate dehydrogenase (LDH), another HIF-1-regulated enzyme. Lactate in turn is released into the extracellular space, regenerating NAD+ for continued glycolysis by O2-starved cells (see Figure 1). This HIF-1-dependent block to mitochondrial O2 consumption promotes cell survival, especially when O2 deprivation is severe and prolonged.



Figure 1. Multiple hypoxia-induced cellular metabolic changes are regulated by HIF-1

By stimulating the expression of glucose transporters and glycolytic enzymes, HIF-1 promotes glycolysis to generate increased levels of pyruvate. In addition, HIF-1 promotes pyruvate reduction to lactate by activating lactate dehydrogenase (LDH). Pyruvate reduction to lactate regenerates NAD+, which permits continued glycolysis and ATP production by hypoxic cells. Furthermore, HIF-1 induces pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase and blocks conversion of pyruvate to acetyl CoA, resulting in decreased flux through the tricarboxylic acid (TCA) cycle. Decreased TCA cycle activity results in attenuation of oxidative phosphorylation and excessive mitochondrial reactive oxygen species (ROS) production. Because hypoxic cells already exhibit increased ROS, which have been shown to promote HIF-1 accumulation, the induction of PDK1 prevents the persistence of potentially harmful ROS levels.

Papandreou et al. demonstrate that hypoxic regulation of PDK has important implications for antitumor therapies. Recent interest has focused on cytotoxins that target hypoxic cells in tumor microenvironments, such as the drug tirapazamine (TPZ). Because intracellular O2 concentrations are decreased by mitochondrial O2 consumption, HIF-1 could protect tumor cells from TPZ-mediated cell death by maintaining intracellular O2 levels. Indeed, Papandreou et al. show that HIF-1-deficient cells grown at 2% O2 exhibit increased sensitivity to TPZ relative to wild-type cells, presumably due to higher rates of mitochondrial O2 consumption. HIF-1 inhibition in hypoxic tumor cells should have multiple therapeutic benefits, but the use of HIF-1 inhibitors in conjunction with other treatments has to be carefully evaluated for the most effective combination and sequence of drug delivery. One result of HIF-1 inhibition would be a relative decrease in intracellular O2 levels, making hypoxic cytotoxins such as TPZ more potent antitumor agents. Because PDK expression has been detected in multiple human tumor samples and appears to be induced by hypoxia (Koukourakis et al., 2005), small molecule inhibitors of HIF-1 combined with TPZ represent an attractive therapeutic approach for future clinical studies.

Hypoxic regulation of PDK1 has other important implications for cell survival during O2 depletion. Because the TCA cycle is coupled to electron transport, Kim et al. suggest that induction of the pyruvate dehydrogenase complex by PDK1 attenuates not only mitochondrial respiration but also the production of mitochondrial reactive oxygen species (ROS) in hypoxic cells. ROS are a byproduct of electron transfer to O2, and cells cultured at 1 to 5% O2 generate increased mitochondrial ROS relative to those cultured at 21% O2 (Chandel et al., 1998 and Guzy et al., 2005). In fact, hypoxia-induced mitochondrial ROS have also been shown to be necessary for the stabilization of HIF-1 in hypoxic cells (Brunelle et al., 2005Guzy et al., 2005 and Mansfield et al., 2005). However, the persistence of ROS could ultimately be lethal to tissues during chronic O2 deprivation, and PDK1 induction by HIF-1 should promote cell viability during long-term hypoxia. Kim et al. present evidence that HIF-1-deficient cells exhibit increased apoptosis after 72 hr of culture at 0.5% O2 compared to wild-type cells and that cell survival is rescued by enforced expression of exogenous PDK1. Furthermore, PDK1 reduces ROS production by the HIF-1 null cells. These findings support a novel prosurvival dimension of cellular hypoxic adaptation where PDK1 inhibits the TCA cycle, mitochondrial respiration, and chronic ROS production.

The HIF-1-mediated block to mitochondrial O2 consumption via PDK1 regulation also has implications for O2-sensing pathways by hypoxic cells. One school of thought suggests that perturbing mitochondrial O2consumption increases intracellular O2 concentrations and suppresses HIF-1 induction by promoting the activity of HIF prolyl hydroxylases, the O2-dependent enzymes that regulate HIF-1 stability (Hagen et al., 2003 and Doege et al., 2005). This model suggests that mitochondria function as “O2 sinks.” Although Papandreou et al. demonstrate that increased mitochondrial respiration due to PDK1 depletion results in decreased intracellular O2 levels (based on pimonidazole staining), these changes failed to reduce HIF-1 levels in hypoxic cells. Another model for hypoxic activation of HIF-1 describes a critical role for mitochondrial ROS in prolyl hydroxylase inhibition and HIF-1 stabilization in O2-starved cells (Brunelle et al., 2005Guzy et al., 2005 and Mansfield et al., 2005) (see Figure 1). The mitochondrial “O2 sink” hypothesis can account for some observations in the literature but fails to explain the inhibition of HIF-1 stabilization by ROS scavengers (Chandel et al., 1998Brunelle et al., 2005Guzy et al., 2005 and Sanjuán-Pla et al., 2005). While the relationship between HIF-1 stability, mitochondrial metabolism, ROS, and intracellular O2 redistribution will continue to be debated for some time, these most recent findings shed new light on findings by Louis Pasteur over a century ago.

Selected reading

Brunelle et al., 2005

J.K. Brunelle, E.L. Bell, N.M. Quesada, K. Vercauteren, V. Tiranti, M. Zeviani, R.C. Scarpulla, N.S. Chandel

Cell Metab., 1 (2005), pp. 409–414

Article  PDF (324 K) View Record in Scopus Citing articles (357)

Chandel et al., 1998

N.S. Chandel, E. Maltepe, E. Goldwasser, C.E. Mathieu, M.C. Simon, P.T. Schumacker

Proc. Natl. Acad. Sci. USA, 95 (1998), pp. 11715–11720

View Record in Scopus Full Text via CrossRef Citing articles (973)

Doege et al., 2005Doege, S. Heine, I. Jensen, W. Jelkmann, E. Metzen

Blood, 106 (2005), pp. 2311–2317

View Record in Scopus Full Text via CrossRef Citing articles (84)

Guzy et al., 2005

R.D. Guzy, B. Hoyos, E. Robin, H. Chen, L. Liu, K.D. Mansfield, M.C. Simon, U. Hammerling, P.T. Schumacker

Cell Metab., 1 (2005), pp. 401–408

Article  PDF (510 K) View Record in Scopus Citing articles (593)

Hagen et al., 2003

Hagen, C.T. Taylor, F. Lam, S. Moncada

Science, 302 (2003), pp. 1975–1978

View Record in Scopus Full Text via CrossRef Citing articles (450)

7.9.7 HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption

Papandreou I1Cairns RAFontana LLim ALDenko NC.
Cell Metab. 2006 Mar; 3(3):187-97.

The HIF-1 transcription factor drives hypoxic gene expression changes that are thought to be adaptive for cells exposed to a reduced-oxygen environment. For example, HIF-1 induces the expression of glycolytic genes. It is presumed that increased glycolysis is necessary to produce energy when low oxygen will not support oxidative phosphorylation at the mitochondria. However, we find that while HIF-1 stimulates glycolysis, it also actively represses mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 phosphorylates and inhibits pyruvate dehydrogenase from using pyruvate to fuel the mitochondrial TCA cycle. This causes a drop in mitochondrial oxygen consumption and results in a relative increase in intracellular oxygen tension. We show by genetic means that HIF-1-dependent block to oxygen utilization results in increased oxygen availability, decreased cell death when total oxygen is limiting, and reduced cell death in response to the hypoxic cytotoxin tirapazamine.

Comment in

Tissue hypoxia results when supply of oxygen from the bloodstream does not meet demand from the cells in the tissue. Such a supply-demand mismatch can occur in physiologic conditions such as the exercising muscle, in the pathologic condition such as the ischemic heart, or in the tumor microenvironment (Hockel and Vaupel, 2001 and Semenza, 2004). In either the physiologic circumstance or pathologic conditions, there is a molecular response from the cell in which a program of gene expression changes is initiated by the hypoxia-inducible factor-1 (HIF-1) transcription factor. This program of gene expression changes is thought to help the cells adapt to the stressful environment. For example, HIF-1-dependent expression of erythropoietin and angiogenic compounds results in increased blood vessel formation for delivery of a richer supply of oxygenated blood to the hypoxic tissue. Additionally, HIF-1 induction of glycolytic enzymes allows for production of energy when the mitochondria are starved of oxygen as a substrate for oxidative phosphorylation. We now find that this metabolic adaptation is more complex, with HIF-1 not only regulating the supply of oxygen from the bloodstream, but also actively regulating the oxygen demand of the tissue by reducing the activity of the major cellular consumer of oxygen, the mitochondria.

Perhaps the best-studied example of chronic hypoxia is the hypoxia associated with the tumor microenvironment (Brown and Giaccia, 1998). The tumor suffers from poor oxygen supply through a chaotic jumble of blood vessels that are unable to adequately perfuse the tumor cells. The oxygen tension within the tumor is also a function of the demand within the tissue, with oxygen consumption influencing the extent of tumor hypoxia (Gulledge and Dewhirst, 1996 and Papandreou et al., 2005b). The net result is that a large fraction of the tumor cells are hypoxic. Oxygen tensions within the tumor range from near normal at the capillary wall, to near zero in the perinecrotic regions. This perfusion-limited hypoxia is a potent microenvironmental stress during tumor evolution (Graeber et al., 1996 and Hockel and Vaupel, 2001) and an important variable capable of predicting for poor patient outcome. (Brizel et al., 1996Cairns and Hill, 2004Hockel et al., 1996 and Nordsmark and Overgaard, 2004).

The HIF-1 transcription factor was first identified based on its ability to activate the erythropoetin gene in response to hypoxia (Wang and Semenza, 1993). Since then, it is has been shown to be activated by hypoxia in many cells and tissues, where it can induce hypoxia-responsive target genes such as VEGF and Glut1 (Airley et al., 2001 and Kimura et al., 2004). The connection between HIF-regulation and human cancer was directly linked when it was discovered that the VHL tumor suppressor gene was part of the molecular complex responsible for the oxic degradation of HIF-1α (Maxwell et al., 1999). In normoxia, a family of prolyl hydroxylase enzymes uses molecular oxygen as a substrate and modifies HIF-1α and HIF2α by hydroxylation of prolines 564 and 402 (Bruick and McKnight, 2001 and Epstein et al., 2001). VHL then recognizes the modified HIF-α proteins, acts as an E3-type of ubiquitin ligase, and along with elongins B and C is responsible for the polyubiquitination of HIF-αs and their proteosomal degradation (Bruick and McKnight, 2001Chan et al., 2002Ivan et al., 2001 and Jaakkola et al., 2001). Mutations in VHL lead to constitutive HIF-1 gene expression, and predispose humans to cancer. The ability to recognize modified HIF-αs is at least partly responsible for VHL activity as a tumor suppressor, as introduction of nondegradable HIF-2α is capable of overcoming the growth–inhibitory activity of wild-type (wt) VHL in renal cancer cells (Kondo et al., 2003).

Mitochondrial function can be regulated by PDK1 expression. Mitochondrial oxidative phosphorylation (OXPHOS) is regulated by several mechanisms, including substrate availability (Brown, 1992). The major substrates for OXPHOS are oxygen, which is the terminal electron acceptor, and pyruvate, which is the primary carbon source. Pyruvate is the end product of glycolysis and is converted to acetyl-CoA through the activity of the pyruvate dehydrogenase complex of enzymes. The acetyl-CoA then directly enters the TCA cycle at citrate synthase where it is combined with oxaloacetate to generate citrate. In metazoans, the conversion of pyruvate to acetyl-CoA is irreversible and therefore represents a critical regulatory point in cellular energy metabolism. Pyruvate dehydrogenase is regulated by three known mechanisms: it is inhibited by acetyl-CoA and NADH, it is stimulated by reduced energy in the cell, and it is inhibited by regulatory phosphorylation of its E1 subunit by pyruvate dehydrogenase kinase (PDK) (Holness and Sugden, 2003 and Sugden and Holness, 2003). There are four members of the PDK family in vertebrates, each with specific tissue distributions (Roche et al., 2001). PDK expression has been observed in human tumor biopsies (Koukourakis et al., 2005), and we have reported that PDK3 is hypoxia-inducible in some cell types (Denko et al., 2003). In this manuscript, we find that PDK1 is also a hypoxia-responsive protein that actively regulates the function of the mitochondria under hypoxic conditions by reducing pyruvate entry into the TCA cycle. By excluding pyruvate from mitochondrial consumption, PDK1 induction may increase the conversion of pyruvate to lactate, which is in turn shunted to the extracellular space, regenerating NAD for continued glycolysis.

Identification of HIF-dependent mitochondrial proteins through genomic and bioinformatics approaches

In order to help elucidate the role of HIF-1α in regulating metabolism, we undertook a genomic search for genes that were regulated by HIF-1 in tumor cells exposed to hypoxia in vitro. We used genetically matched human RCC4 cells that had lost VHL during tumorigenesis and displayed constitutive HIF-1 activity, and a cell line engineered to re-express VHL to establish hypoxia-dependent HIF activation. These cells were treated with 18 hr of stringent hypoxia (<0.01% oxygen), and microarray analysis performed. Using a strict 2.5-fold elevation as our cutoff, we identified 173 genes that were regulated by hypoxia and/or VHL status (Table S1 in the Supplemental Data available with this article online). We used the pattern of expression in these experiments to identify putative HIF-regulated genes—ones that were constitutively elevated in the parent RCC4s independent of hypoxia, downregulated in the RCC4VHL cells under normoxia, and elevated in response to hypoxia. Of the 173 hypoxia and VHL-regulated genes, 74 fit the putative HIF-1 target pattern. The open reading frames of these genes were run through a pair of bioinformatics engines in order to predict subcellular localization, and 10 proteins scored as mitochondrial on at least one engine. The genes, fold induction, and mitochondrial scores are listed in Table 1.

HIF-1 downregulates mitochondrial oxygen consumption

Having identified several putative HIF-1 responsive gene products that had the potential to regulate mitochondrial function, we then directly measured mitochondrial oxygen consumption in cells exposed to long-term hypoxia. While other groups have studied mitochondrial function under acute hypoxia (Chandel et al., 1997), this is one of the first descriptions of mitochondrial function after long-term hypoxia where there have been extensive hypoxia-induced gene expression changes. Figure 1A is an example of the primary oxygen trace from a Clark electrode showing a drop in oxygen concentration in cell suspensions of primary fibroblasts taken from normoxic and hypoxic cultures. The slope of the curve is a direct measure of the total cellular oxygen consumption rate. Exposure of either primary human or immortalized mouse fibroblasts to 24 hr of hypoxia resulted in a reduction of this rate by approximately 50% (Figures 1A and 1B). In these experiments, the oxygen consumption can be stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide 3-chloro phenylhydrazone) and was completely inhibited by 2 mM potassium cyanide. We determined that the change in total cellular oxygen consumption was due to changes in mitochondrial activity by the use of the cell-permeable poison of mitochondrial complex 3, Antimycin A. Figure 1C shows that the difference in the normoxic and hypoxic oxygen consumption in murine fibroblasts is entirely due to the Antimycin-sensitive mitochondrial consumption. The kinetics with which mitochondrial function slows in hypoxic tumor cells also suggests that it is due to gene expression changes because it takes over 6 hr to achieve maximal reduction, and the reversal of this repression requires at least another 6 hr of reoxygenation (Figure 1D). These effects are not likely due to proliferation or toxicity of the treatments as these conditions are not growth inhibitory or toxic to the cells (Papandreou et al., 2005a).

Since we had predicted from the gene expression data that the mitochondrial oxygen consumption changes were due to HIF-1-mediated expression changes, we tested several genetically matched systems to determine what role HIF-1 played in the process (Figure 2). We first tested the cell lines that had been used for microarray analysis and found that the parental RCC4 cells had reduced mitochondrial oxygen consumption when compared to the VHL-reintroduced cells. Oxygen consumption in the parental cells was insensitive to hypoxia, while it was reduced by hypoxia in the wild-type VHL-transfected cell lines. Interestingly, stable introduction of a tumor-derived mutant VHL (Y98H) that cannot degrade HIF was also unable to restore oxygen consumption. These results indicate that increased expression of HIF-1 is sufficient to reduce oxygen consumption (Figure 2A). We also investigated whether HIF-1 induction was required for the observed reduction in oxygen consumption in hypoxia using two genetically matched systems. We measured normoxic and hypoxic oxygen consumption in murine fibroblasts derived from wild-type or HIF-1α null embryos (Figure 2B) and from human RKO tumor cells and RKO cells constitutively expressing ShRNAs directed against the HIF-1α gene (Figures 2C and 4C). Neither of the HIF-deficient cell systems was able to reduce oxygen consumption in response to hypoxia. These data from the HIF-overexpressing RCC cells and the HIF-deficient cells indicate that HIF-1 is both necessary and sufficient for reducing mitochondrial oxygen consumption in hypoxia.

HIF-dependent mitochondrial changes are functional, not structural

Because addition of CCCP could increase oxygen consumption even in the hypoxia-treated cells, we hypothesized that the hypoxic inhibition was a regulated activity, not a structural change in the mitochondria in response to hypoxic stress. We confirmed this interpretation by examining several additional mitochondrial characteristics in hypoxic cells such as mitochondrial morphology, quantity, and membrane potential. We examined morphology by visual inspection of both the transiently transfected mitochondrially localized DsRed protein and the endogenous mitochondrial protein cytochrome C. Both markers were indistinguishable in the parental RCC4 and the RCC4VHL cells (Figure 3A). Likewise, we measured the mitochondrial membrane potential with the functional dye rhodamine 123 and found that it was identical in the matched RCC4 cells and the matched HIF wt and knockout (KO) cells when cultured in normoxia or hypoxia (Figure 3B). Finally, we determined that the quantity of mitochondria per cell was not altered in response to HIF or hypoxia by showing that the amount of the mitochondrial marker protein HSP60 was identical in the RCC4 and HIF cell lines (Figure 3C)

PDK1 is a HIF-1 inducible target protein

After examination of the list of putative HIF-regulated mitochondrial target genes, we hypothesized that PDK1 could mediate the functional changes that we observed in hypoxia. We therefore investigated PDK1 protein expression in response to HIF and hypoxia in the genetically matched cell systems. Figure 4A shows that in the RCC4 cells PDK1 and the HIF-target gene BNip3 (Greijer et al., 2005 and Papandreou et al., 2005a) were both induced by hypoxia in a VHL-dependent manner, with the expression of PDK1 inversely matching the oxygen consumption measured in Figure 1 above. Likewise, the HIF wt MEFs show oxygen-dependent induction of PDK1 and BNip3, while the HIF KO MEFs did not show any expression of either of these proteins under any oxygen conditions (Figure 4B). Finally, the parental RKO cells were able to induce PDK1 and the HIF target gene BNip3L in response to hypoxia, while the HIF-depleted ShRNA RKO cells could not induce either protein (Figure 4C). Therefore, in all three cell types, the HIF-1-dependent regulation of oxygen consumption seen in Figure 2, corresponds to the HIF-1-dependent induction of PDK1 seen in Figure 4.

In order to determine if PDK1 was a direct HIF-1 target gene, we analyzed the genomic sequence flanking the 5′ end of the gene for possible HIF-1 binding sites based on the consensus core HRE element (A/G)CGTG (Caro, 2001). Several such sites exist within the first 400 bases upstream, so we generated reporter constructs by fusing the genomic sequence from −400 to +30 of the start site of transcription to the firefly luciferase gene. In transfection experiments, the chimeric construct showed significant induction by either cotransfection with a constitutively active HIF proline mutant (P402A/P564G) (Chan et al., 2002) or exposure of the transfected cells to 0.5% oxygen (Figure 4D). Most noteworthy, when the reporter gene was transfected into the HIF-1α null cells, it did not show induction when the cells were cultured in hypoxia, but it did show induction when cotransfected with expression HIF-1α plasmid. We then generated deletions down to the first 36 bases upstream of transcription and found that even this short sequence was responsive to HIF-1 (Figure 4D). Analysis of this small fragment showed only one consensus HRE site located in an inverted orientation in the 5′ untranslated region. We synthesized and cloned a mutant promoter fragment in which the core element ACGTG was replaced with AAAAG, and this construct lost over 90% of its hypoxic induction. These experiments suggest that it is this HRE within the proximal 5′ UTR that HIF-1 uses to transactivate the endogenous PDK1 gene in response to hypoxia.

PDK1 is responsible for the HIF-dependent mitochondrial oxygen consumption changes

In order to directly test if PDK1 was the HIF-1 target gene responsible for the hypoxic reduction in mitochondrial oxygen consumption, we generated RKO cell lines with either knockdown or overexpression of PDK1 and measured the oxygen consumption in these derivatives. The PDK1 ShRNA stable knockdown line was generated as a pool of clones cotransfected with pSUPER ShPDK1 and pTK-hygro resistance gene. After selection for growth in hygromycin, the cells were tested by Western blot for the level of PDK1 protein expression. We found that normoxic PDK1 is reduced by 75%, however, there was measurable expression of PDK1 in these cells in response to hypoxia (Figure 5A). When we measured the corresponding oxygen consumption in these cells, we found a change commensurate with the level of PDK1. The knockdown cells show elevated baseline oxygen consumption, and partial reduction in this activity in response to hypoxia. Therefore, reduction of PDK1 expression by genetic means increased mitochondrial oxygen consumption in both normoxic and hypoxic conditions. Interestingly, these cells still induced HIF-1α (Figure 5A) and HIF-1 target genes such as BNip3L in response to hypoxia (data not shown), suggesting that altered PDK1 levels do not alter HIF-1α function.



PDK1 expression directly regulates cellular oxygen consumption rate

Figure 5. PDK1 expression directly regulates cellular oxygen consumption rate

  1. A)Western blot of RKO cell and ShRNAPDK1RKO cell lysates after exposure to 24 hr of normoxia or 0.5% O2. Blots were probed for HIF 1α, PDK1, and tubulin as a loading control.
  2. B)Oxygen consumption rate in RKO and ShRNAPDK1RKO cells after exposure to 24 hr of normoxia or 0.5% O2.
  3. C)Western blot of RKOiresGUS cell and RKOiresPDK1 cell lysates after exposure to 24 hr of normoxia or 0.5% O2. Blots were probed for HIF 1α, PDK1, and tubulin as a loading control.
  4. D)Oxygen consumption rate in RKOiresGUS and RKOiresPDK1 cells after exposure to 24 hr of normoxia or 0.5% O2.
  5. E)Model describing the interconnected effects of HIF-1 target gene activation on hypoxic cell metabolism. Reduced oxygen conditions causes HIF-1 to coordinately induce the enzymes shown in boxes. HIF-1 activation results in increased glucose transporter expression to increase intracellular glucose flux, induction of glycolytic enzymes increases the conversion of glucose to pyruvate generating energy and NADH, induction of PDK1 decreases mitochondrial utilization of pyruvate and oxygen, and induction of LDH increases the removal of excess pyruvate as lactate and also regenerates NAD+ for increased glycolysis.

For all graphs, the error bars represent the standard error of the mean.

We also determined if overexpression of PDK1 could lead to reduced mitochondrial oxygen consumption. A separate culture of RKO cells was transfected with a PDK1-IRES-puro expression plasmid and selected for resistance to puromycin. The pool of puromycin resistant cells was tested for PDK1 expression by Western blot. These cells showed a modest increase in PDK1 expression under control conditions when compared to the cells transfected with GUS-IRES-puro, with an additional increase in PDK1 protein in response to hypoxia (Figure 5C). The corresponding oxygen consumption measurements showed that the mitochondria is very sensitive to changes in the levels of PDK1, as even this slight increase was able to significantly reduce oxygen consumption in the normoxic PDK1-puro cultures. Further increase in PDK1 levels with hypoxia further reduced oxygen consumption in both cultures (Figure 5D). The model describing the relationship between hypoxia, HIF-1, PDK1, and intermediate metabolism is described inFigure 5E.

Altering oxygen consumption alters intracellular oxygen tension and sensitivity to hypoxia-dependent cell killing

The intracellular concentration of oxygen is a net result of the rate at which oxygen diffuses into the cell and the rate at which it is consumed. We hypothesized that the rate at which oxygen was consumed within the cell would significantly affect its steady-state intracellular concentrations. We tested this hypothesis in vitro using the hypoxic marker drug pimonidazole (Bennewith and Durand, 2004). We plated high density cultures of HIF wild-type and HIF knockout cells and placed these cultures in normoxic, 2% oxygen, and anoxic incubators for overnight treatment. The overnight treatment gives the cells time to adapt to the hypoxic conditions and establish altered oxygen consumption profiles. Pimonidozole was then added for the last 4 hr of the growth of the culture. Pimonidazole binding was detected after fixation of the cells using an FITC labeled anti-pimonidazole antibody and it was quantitated by flow cytometry. The quantity of the bound drug is a direct indication of the oxygen concentration within the cell (Bennewith and Durand, 2004). The histograms in Figure 6A show that the HIF-1 knockout and wild-type cells show similar staining in the cells grown in 0% oxygen. However, the cells treated with 2% oxygen show the consequence of the genetic removal of HIF-1. The HIF-proficient cells showed relatively less pimonidazole binding at 2% when compared to the 0% culture, while the HIF-deficient cells showed identical binding between the cells at 2% and those at 0%. We interpret these results to mean that the HIF-deficient cells have greater oxygen consumption, and this has lowered the intracellular oxygenation from the ambient 2% to close to zero intracellularly. The HIF-proficient cells reduced their oxygen consumption rate so that the rate of diffusion into the cell is greater than the rate of consumption.

Figure 6. HIF-dependent decrease in oxygen consumption raises intracellular oxygen concentration, protects when oxygen is limiting, and decreases sensitivity to tirapazamine in vitro

  1. A)Pimonidazole was used to determine the intracellular oxygen concentration of cells in culture. HIF wt and HIF KO MEFs were grown at high density and exposed to 2% O2or anoxia for 24 hr in glass dishes. For the last 4 hr of treatment, cells were exposed to 60 μg/ml pimonidazole. Pimonidazole binding was quantitated by flow cytometry after binding of an FITC conjugated anti-pimo mAb. Results are representative of two independent experiments.
  2. B)HIF1α reduces oxygen consumption and protects cells when total oxygen is limited. HIF wt and HIF KO cells were plated at high density and sealed in aluminum jigs at <0.02% oxygen. At the indicated times, cells were harvested, and dead cells were quantitated by trypan blue exclusion. Note both cell lines are equally sensitive to anoxia-induced apoptosis, so the death of the HIF null cells indicates that the increased oxygen consumption removed any residual oxygen in the jig and resulted in anoxia-induced death.
  3. C)PDK1 is responsible for HIF-1’s adaptive response when oxygen is limiting. A similar jig experiment was performed to measure survival in the parental RKO, the RKO ShRNAHIF1α, and the RKOShPDK1 cells. Cell death by trypan blue uptake was measured 48 hr after the jigs were sealed.
  4. D)HIF status alters sensitivity to TPZ in vitro. HIF wt and HIF KO MEFs were grown at high density in glass dishes and exposed to 21%, 2%, and <0.01% O2conditions for 18 hr in the presence of varying concentrations of Tirapazamine. After exposure, cells were harvested and replated under normoxia to determine clonogenic viability. Survival is calculated relative to the plating efficiency of cells exposed to 0 μM TPZ for each oxygen concentration.
  5. E)Cell density alters sensitivity to TPZ. HIF wt and HIF KO MEFs were grown at varying cell densities in glass dishes and exposed to 2% O2in the presence of 10 μM TPZ for 18 hr. After the exposure, survival was determined as described in (C).

For all graphs, the error bars represent the standard error of the mean.

HIF-induced PDK1 can reduce the total amount of oxygen consumed per cell. The reduction in the amount of oxygen consumed could be significant if there is a finite amount of oxygen available, as would be the case in the hours following a blood vessel occlusion. The tissue that is fed by the vessel would benefit from being economical with the oxygen that is present. We experimentally modeled such an event using aluminum jigs that could be sealed with defined amounts of cells and oxygen present (Siim et al., 1996). We placed 10 × 106 wild-type or HIF null cells in the sealed jig at 0.02% oxygen, waited for the cells to consume the remaining oxygen, and measured cell viability. We have previously shown that these two cell types are resistant to mild hypoxia and equally sensitive to anoxia-induced apoptosis (Papandreou et al., 2005a). Therefore, any death in this experiment would be the result of the cells consuming the small amount of remaining oxygen and dying in response to anoxia. We found that in sealed jigs, the wild-type cells are more able to adapt to the limited oxygen supply by reducing consumption. The HIF null cells continued to consume oxygen, reached anoxic levels, and started to lose viability within 36 hr (Figure 6B). This is a secondary adaptive effect of HIF1. We confirmed that PDK1 was responsible for this difference by performing a similar experiment using the parental RKO cells, the RKOShRNAHIF1α and the RKOShRNAPDK1 cells. We found similar results in which both the cells with HIF1α knockdown and PDK1 knockdown were sensitive to the long-term effects of being sealed in a jig with a defined amount of oxygen (Figure 6c). Note that the RKOShPDK1 cells are even more sensitive than the RKOShHIF1α cells, presumably because they have higher basal oxygen consumption rates (Figure 5B).

Because HIF-1 can help cells adapt to hypoxia and maintain some intracellular oxygen level, it may also protect tumor cells from killing by the hypoxic cytotoxin tirapazamine (TPZ). TPZ toxicity is very oxygen dependent, especially at oxygen levels between 1%–4% (Koch, 1993). We therefore tested the relative sensitivity of the HIF wt and HIF KO cells to TPZ killing in high density cultures (Figure 6D). We exposed the cells to the indicated concentrations of drug and oxygen concentrations overnight. The cells were then harvested and replated to determine reproductive viability by colony formation. Both cell types were equally resistant to TPZ at 21% oxygen, while both cell types are equally sensitive to TPZ in anoxic conditions where intracellular oxygen levels are equivalent (Figure 6A). The identical sensitivity of both cell types in anoxia indicates that both cell types are equally competent in repairing the TPZ-induced DNA damage that is presumed to be responsible for its toxicity. However, in 2% oxygen cultures, the HIF null cells displayed a significantly greater sensitivity to the drug than the wild-type cells. This suggests that the increased oxygen consumption rate in the HIF-deficient cells is sufficient to lower the intracellular oxygen concentration relative to that in the HIF-proficient cells. The lower oxygen level is significant enough to dramatically sensitize these cells to killing by TPZ.

If the increased sensitivity to TPZ in the HIF ko cells is determined by intracellular oxygen consumption differences, then this effect should also be cell-density dependent. We showed that this is indeed the case in Figure 6E where oxygen and TPZ concentrations were held constant, and increased cell density lead to increased TPZ toxicity. The effect was much more pronounced in the HIF KO cells, although the HIF wt cells showed some increased toxicity in the highest density cultures, consistent with the fact they were still consuming some oxygen, even with HIF present (Figure 1). The in vitro TPZ survival data is therefore consistent with our hypothesis that control of oxygen consumption can regulate intracellular oxygen concentration, and suggests that increased oxygen consumption could sensitize cells to hypoxia-dependent therapy.


The findings presented here show that HIF-1 is actively responsible for regulating energy production in hypoxic cells by an additional, previously unrecognized mechanism. It has been shown that HIF-1 induces the enzymes responsible for glycolysis when it was presumed that low oxygen did not support efficient oxidative phosphorylation (Iyer et al., 1998 and Seagroves et al., 2001). The use of glucose to generate ATP is capable of satisfying the energy requirements of a cell if glucose is in excess (Papandreou et al., 2005a). We now find that at the same time that glycolysis is increasing, mitochondrial respiration is decreasing. However, the decreased respiration is not because there is not enough oxygen present to act as a substrate for oxidative phosphorylation, but because the flow of pyruvate into the TCA cycle has been reduced by the activity of pyruvate dehydrogenase kinase. Other reports have suggested that oxygen utilization is shifted in cells exposed to hypoxia, but these reports have focused on other regulators such as nitric oxide synthase (Hagen et al., 2003). NO can reduce oxygen consumption through direct inhibition of cytochrome oxidase, but this effect seems to be more significant at physiologic oxygen concentrations, not at severe levels seen in the tumor (Palacios-Callender et al., 2004).

7.9.8 HIF-1. upstream and downstream of cancer metabolism

Semenza GL1.
Curr Opin Genet Dev. 2010 Feb; 20(1):51-6

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression. Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression.

Metastatic cancer is characterized by reprogramming of cellular metabolism leading to increased uptake of glucose for use as both an anabolic and catabolic substrate. Increased glucose uptake is such a reliable feature that it is utilized clinically to detect metastases by positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) with a sensitivity of ~90% [1]. As with all aspects of cancer biology, the details of metabolic reprogramming differ widely among individual tumors. However, the role of specific signaling pathways and transcription factors in this process is now understood in considerable detail. This review will focus on the involvement of hypoxia-inducible factor 1 (HIF-1) in both mediating metabolic reprogramming and responding to metabolic alterations. The placement of HIF-1 both upstream and downstream of cancer metabolism results in a feed-forward mechanism that may play a major role in the development of the invasive, metastatic, and lethal cancer phenotype.

O2 concentrations are significantly reduced in many human cancers compared to the surrounding normal tissue. The median PO2 in breast cancers is ~10 mm Hg, as compared to ~65 mm Hg in normal breast tissue [2]. Reduced O2 availability induces HIF-1, which regulates the transcription of hundreds of genes [3*,4*] that encode proteins involved in every aspect of cancer biology, including: cell immortalization and stem cell maintenance; genetic instability; glucose and energy metabolism; vascularization; autocrine growth factor signaling; invasion and metastasis; immune evasion; and resistance to chemotherapy and radiation therapy [5].

HIF-1 is a transcription factor that consists of an O2-regulated HIF-1α and a constitutively expressed HIF-1β subunit [6]. In well-oxygenated cells, HIF-1α is hydroxylated on proline residue 402 (Pro-402) and/or Pro-564 by prolyl hydroxylase domain protein 2 (PHD2), which uses O2 and α-ketoglutarate as substrates in a reaction that generates CO2 and succinate as byproducts [7]. Prolyl-hydroxylated HIF-1α is bound by the von Hippel-Lindau tumor suppressor protein (VHL), which recruits an E3-ubiquitin ligase that targets HIF-1α for proteasomal degradation (Figure 1A). Asparagine 803 in the transactivation domain is hydroxylated in well-oxygenated cells by factor inhibiting HIF-1 (FIH-1), which blocks the binding of the coactivators p300 and CBP [7]. Under hypoxic conditions, the prolyl and asparaginyl hydroxylation reactions are inhibited by substrate (O2) deprivation and/or the mitochondrial generation of reactive oxygen species (ROS), which may oxidize Fe(II) present in the catalytic center of the hydroxylases [8].

HIF-1 and metabolism  nihms156580f1

HIF-1 and metabolism nihms156580f1

HIF-1 and metabolism

Figure 1 HIF-1 and metabolism. (A) Regulation of HIF-1α protein synthesis and stability and HIF-1-dependent metabolic reprogramming. The rate of translation of HIF-1α mRNA into protein in cancer cells is dependent upon the activity of the mammalian 

The finding that acute changes in PO2 increase mitochondrial ROS production suggests that cellular respiration is optimized at physiological PO2 to limit ROS generation and that any deviation in PO2 — up or down — results in increased ROS generation. If hypoxia persists, induction of HIF-1 leads to adaptive mechanisms to reduce ROS and re-establish homeostasis, as described below. Prolyl and asparaginyl hydroxylation provide a molecular mechanism by which changes in cellular oxygenation can be transduced to the nucleus as changes in HIF-1 activity. This review will focus on recent advances in our understanding of the role of HIF-1 in controlling glucose and energy metabolism, but it should be appreciated that any increase in HIF-1 activity that leads to changes in cell metabolism will also affect many other critical aspects of cancer biology [5] that will not be addressed here.

HIF-1 target genes involved in glucose and energy metabolism

HIF-1 activates the transcription of SLC2A1 and SLC2A3, which encode the glucose transporters GLUT1 and GLUT3, respectively, as well as HK1 and HK2, which encode hexokinase, the first enzyme of the Embden-Meyerhoff (glycolytic) pathway [9]. Once taken up by GLUT and phosphorylated by HK, FDG cannot be metabolized further; thus, FDG-PET signal is determined by FDG delivery to tissue (i.e. perfusion) and GLUT/HK expression/activity. Unlike FDG, glucose is further metabolized to pyruvate by the action of the glycolytic enzymes, which are all encoded by HIF-1 target genes (Figure 1A). Glycolytic intermediates are also utilized for nucleotide and lipid synthesis [10]. Lactate dehydrogenase A (LDHA), which converts pyruvate to lactate, and monocarboxylate transporter 4 (MCT4), which transports lactate out of the cell (Figure 1B), are also regulated by HIF-1 [9,11]. Remarkably, lactate produced by hypoxic cancer cells can be taken up by non-hypoxic cells and used as a respiratory substrate [12**].

Pyruvate represents a critical metabolic control point, as it can be converted to acetyl coenzyme A (AcCoA) by pyruvate dehydrogenase (PDH) for entry into the tricarboxylic acid (TCA) cycle or it can be converted to lactate by LDHA (Figure 1B). Pyruvate dehydrogenase kinase (PDK), which phosphorylates and inactivates the catalytic domain of PDH, is encoded by four genes and PDK1 is activated by HIF-1 [13,14]. (Further studies are required to determine whether PDK2PDK3, or PDK4 is regulated by HIF-1.) As a result of PDK1 activation, pyruvate is actively shunted away from the mitochondria, which reduces flux through the TCA cycle, thereby reducing delivery of NADH and FADH2 to the electron transport chain. This is a critical adaptive response to hypoxia, because in HIF-1α–null mouse embryo fibroblasts (MEFs), PDK1 expression is not induced by hypoxia and the cells die due to excess ROS production, which can be ameliorated by forced expression of PDK1 [13]. MYC, which is activated in ~40% of human cancers, cooperates with HIF-1 to activate transcription of PDK1, thereby amplifying the hypoxic response [15]. Pharmacological inhibition of HIF-1 or PDK1 activity increases O2 consumption by cancer cells and increases the efficacy of a hypoxia-specific cytotoxin [16].

Hypoxia also induces mitochondrial autophagy in many human cancer cell lines through HIF-1-dependent expression of BNIP3 and a related BH3 domain protein, BNIP3L [19**]. Autocrine signaling through the platelet-derived growth factor receptor in cancer cells increases HIF-1 activity and thereby increases autophagy and cell survival under hypoxic conditions [21]. Autophagy may also occur in a HIF-1-independent manner in response to other physiological stimuli that are associated with hypoxic conditions, such as a decrease in the cellular ATP:AMP ratio, which activates AMP kinase signaling [22].

In clear cell renal carcinoma, VHL loss of function (LoF) results in constitutive HIF-1 activation, which is associated with impaired mitochondrial biogenesis that results from HIF-1-dependent expression of MXI1, which blocks MYC-dependent expression of PGC-1β, a coactivator that is required for mitochondrial biogenesis [23]. Inhibition of wild type MYC activity in renal cell carcinoma contrasts with the synergistic effect of HIF-1 and oncogenic MYC in activating PDK1 transcription [24].

Genetic and metabolic activators of HIF-1

Hypoxia plays a critical role in cancer progression [2,5] but not all cancer cells are hypoxic and a growing number of O2-independent mechanisms have been identified by which HIF-1 is induced [5]. Several mechanisms that are particularly relevant to cancer metabolism are described below.

Activation of mTOR

Alterations in mitochondrial metabolism

NAD+ levels

It is of interest that the NAD+-dependent deacetylase sirtuin 1 (SIRT1) was found to bind to, deacetylate, and increase transcriptional activation by HIF-2α but not HIF-1α [42**]. Another NAD+-dependent enzyme is poly(ADP-ribose) polymerase 1 (PARP1), which was recently shown to bind to HIF-1α and promote transactivation through a mechanism that required the enzymatic activity of PARP1 [43]. Thus, transactivation mediated by both HIF-1α and HIF-2α can be modulated according to NAD+ levels.

Nitric oxide

Increased expression of nitric oxide (NO) synthase isoforms and increased levels of NO have been shown to increase HIF-1α protein stability in human oral squamous cell carcinoma [44]. In prostate cancer, nuclear co-localization of endothelial NO synthase, estrogen receptor β, HIF-1α, and HIF-2α was associated with aggressive disease and the proteins were found to form chromatin complexes on the promoter of TERT gene encoding telomerase [45**]. The NOS2 gene encoding inducible NO synthase is HIF-1 regulated [5], suggesting another possible feed-forward mechanism.

7.9.9 In Vivo HIF-Mediated Reductive Carboxylation

Gameiro PA1Yang JMetelo AMPérez-Carro R, et al.
Cell Metab. 2013 Mar 5; 17(3):372-85.

Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [1–13C] glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.

Cancer cells undergo fundamental changes in their metabolism to support rapid growth, adapt to limited nutrient resources, and compete for these supplies with surrounding normal cells. One of the metabolic hallmarks of cancer is the activation of glycolysis and lactate production even in the presence of adequate oxygen. This is termed the Warburg effect, and efforts in cancer biology have revealed some of the molecular mechanisms responsible for this phenotype (Cairns et al., 2011). More recently, 13C isotopic studies have elucidated the complementary switch of glutamine metabolism that supports efficient carbon utilization for anabolism and growth (DeBerardinis and Cheng, 2010). Acetyl-CoA is a central biosynthetic precursor for lipid synthesis, being generated from glucose-derived citrate in well-oxygenated cells (Hatzivassiliou et al., 2005). Warburg-like cells, and those exposed to hypoxia, divert glucose to lactate, raising the question of how the tricarboxylic acid (TCA) cycle is supplied with acetyl-CoA to support lipogenesis. We and others demonstrated, using 13C isotopic tracers, that cells under hypoxic conditions or defective mitochondria primarily utilize glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) (Filipp et al., 2012Metallo et al., 2012;Mullen et al., 2012Wise et al., 2011).

The transcription factors hypoxia inducible factors 1α and 2α (HIF-1α, HIF-2α) have been established as master regulators of the hypoxic program and tumor phenotype (Gordan and Simon, 2007Semenza, 2010). In addition to tumor-associated hypoxia, HIF can be directly activated by cancer-associated mutations. The von Hippel-Lindau (VHL) tumor suppressor is inactivated in the majority of sporadic clear-cell renal carcinomas (RCC), with VHL-deficient RCC cells exhibiting constitutive HIF-1α and/or HIF-2α activity irrespective of oxygen availability (Kim and Kaelin, 2003). Previously, we showed that VHL-deficient cells also relied on RC for lipid synthesis even under normoxia. Moreover, metabolic profiling of two isogenic clones that differ in pVHL expression (WT8 and PRC3) suggested that reintroduction of wild-type VHL can restore glucose utilization for lipogenesis (Metallo et al., 2012). The VHL tumor suppressor protein (pVHL) has been reported to have several functions other than the well-studied targeting of HIF. Specifically, it has been reported that pVHL regulates the large subunit of RNA polymerase (Pol) II (Mikhaylova et al., 2008), p53 (Roe et al., 2006), and the Wnt signaling regulator Jade-1. VHL has also been implicated in regulation of NF-κB signaling, tubulin polymerization, cilia biogenesis, and proper assembly of extracellular fibronectin (Chitalia et al., 2008Kim and Kaelin, 2003Ohh et al., 1998Thoma et al., 2007Yang et al., 2007). Hypoxia inactivates the α-ketoglutarate-dependent HIF prolyl hydroxylases, leading to stabilization of HIF. In addition to this well-established function, oxygen tension regulates a larger family of α-ketoglutarate-dependent cellular oxygenases, leading to posttranslational modification of several substrates, among which are chromatin modifiers (Melvin and Rocha, 2012). It is therefore conceivable that the effect of hypoxia on RC that was reported previously may be mediated by signaling mechanisms independent of the disruption of the pVHL-HIF interaction. Here we (1) demonstrate that HIF is necessary and sufficient for RC, (2) provide insights into the molecular mechanisms that link HIF to RC, (3) detected RC activity in vivo in human VHL-deficient RCC cells growing as tumors in nude mice, (4) provide evidence that the reductive phenotype ofVHL-deficient cells renders them sensitive to glutamine restriction in vitro, and (5) show that inhibition of glutaminase suppresses growth of VHL-deficient cells in nude mice. These observations lay the ground for metabolism-based therapeutic strategies for targeting HIF-driven tumors (such as RCC) and possibly the hypoxic compartment of solid tumors in general.

Functional Interaction between pVHL and HIF Is Necessary to Inhibit RC

Figure 1  HIF Inactivation Is Necessary for Downregulation of Reductive Carboxylation by pVHL

We observed a concurrent regulation in glucose metabolism in the different VHL mutants. Reintroduction of wild-type or type 2C pVHL mutant, which can meditate HIF-α destruction, stimulated glucose oxidation via pyruvate dehydrogenase (PDH), as determined by the degree of 13C-labeled TCA cycle metabolites (M2 enrichment) (Figures 1D and 1E). In contrast, reintroduction of an HIF nonbinding Type 2B pVHL mutant failed to stimulate glucose oxidation, resembling the phenotype observed in VHL-deficient cells (Figures 1D and 1E). Additional evidence for the overall glucose utilization was obtained from the enrichment of M3 isotopomers using [U13-C6]glucose (Figure S1A), which shows a lower contribution of glucose-derived carbons to the TCA cycle in VHL-deficient RCC cells (via pyruvate carboxylase and/or continued TCA cycling).

To test the effect of HIF activation on the overall glutamine incorporation in the TCA cycle, we labeled an isogenic pair of VHL-deficient and VHL-reconstituted UMRC2 cells with [U-13C5]glutamine, which generates M4 fumarate, M4 malate, M4 aspartate, and M4 citrate isotopomers through glutamine oxidation. As seen in Figure S1BVHL-deficient/VHL-positive UMRC2 cells exhibit similar enrichment of M4 fumarate, M4 malate, and M4 asparate (but not citrate) showing that VHL-deficient cells upregulate reductive carboxylation without compromising oxidative metabolism from glutamine. …  Labeled carbon derived from [5-13C1]glutamine can be incorporated into fatty acids exclusively through RC, and the labeled carbon cannot be transferred to palmitate through the oxidative TCA cycle (Figure 1B, red carbons). Tracer incorporation from [5-13C1]glutamine occurs in the one carbon (C1) of acetyl-CoA, which results in labeling of palmitate at M1, M2, M3, M4, M5, M6, M7, and M8 mass isotopomers. In contrast, lipogenic acetyl-CoA molecules originating from [U-13C6]glucose are fully labeled, and the labeled palmitate is represented by M2, M4, M6, M8, M10, M12, M14, and M16 mass isotopomers.

Figure 2 HIF Inactivation Is Necessary for Downregulation of Reductive Lipogenesis by pVHL

To determine the specific contribution from glucose oxidation or glutamine reduction to lipogenic acetyl-CoA, we performed isotopomer spectral analysis (ISA) of palmitate labeling patterns. ISA indicates that wild-type pVHL or pVHL L188V mutant-reconstituted UMRC2 cells relied mainly on glucose oxidation to produce lipogenic acetyl-CoA, while UMRC2 cells reconstituted with a pVHL mutant defective in HIF inactivation (Y112N or Y98N) primarily employed RC. Upon disruption of the pVHL-HIF interaction, glutamine becomes the preferred substrate for lipogenesis, supplying 70%–80% of the lipogenic acetyl-CoA (Figure 2C). This is not a cell-line-specific phenomenon, but it applies to VHL-deficient human RCC cells in general; the same changes are observed in 786-O cells reconstituted with wild-type pVHL or mutant pVHL or infected with vector only as control (Figure S2).

HIF Is Sufficient to Induce RC (reductive carboxylation) from Glutamine in RCC Cells

As shown in Figure 3C, reintroduction of wild-type VHLinto 786-O cells suppressed RC, whereas the expression of the constitutively active HIF-2α mutant was sufficient to stimulate this reaction, restoring the M1 enrichment of TCA cycle metabolites observed in VHL-deficient 786-O cells. Expression of HIF-2α P-A also led to a concomitant decrease in glucose oxidation, corroborating the metabolic alterations observed in glutamine metabolism (Figures 3D and 3E).

Figure 3 Expression of HIF-2α Is Sufficient to Induce Reductive Carboxylation and Lipogenesis from Glutamine in RCC Cells

Expression of HIF-2α P-A in 786-O cells phenocopied the loss-of-VHL with regards to glutamine reduction for lipogenesis (Figure 3G), suggesting that HIF-2α can induce the glutamine-to-lipid pathway in RCC cells per se. Although reintroduction of wild-type VHL restored glucose oxidation in UMRC2 and UMRC3 cells (Figures S3B–S3I), HIF-2α P-A expression did not measurably affect the contribution of each substrate to the TCA cycle or lipid synthesis in these RCC cells (data not shown). UMRC2 and UMRC3 cells endogenously express both HIF-1α and HIF-2α, whereas 786-O cells exclusively express HIF-2α. There is compelling evidence suggesting, at least in RCC cells, that HIF-α isoforms have overlapping—but also distinct—functions and their roles in regulating bioenergetic processes remain an area of active investigation. Overall, HIF-1α has an antiproliferative effect, and its expression in vitro leads to rapid death of RCC cells while HIF-2α promotes tumor growth (Keith et al., 2011Raval et al., 2005).

Metabolic Flux Analysis Shows Net Reversion of the IDH Flux upon HIF Activation

To determine absolute fluxes in RCC cells, we employed 13C metabolic flux analysis (MFA) as previously described (Metallo et al., 2012). Herein, we performed MFA using a combined model of [U-13C6]glucose and [1-13C1]glutamine tracer data sets from the 786-O derived isogenic clones PRC3 (VHL−/ −)/WT8 (VHL+) cells, which show a robust metabolic regulation by reintroduction of pVHL. To this end, we first determined specific glucose/glutamine consumption and lactate/glutamate secretion rates. As expected, PRC3 exhibited increased glucose consumption and lactate production when compared to WT8 counterparts (Figure 4A). While PRC3 exhibited both higher glutamine consumption and glutamate production rates than WT8 (Figure 4A), the net carbon influx was higher in PRC3 cells (Figure 4B). Importantly, the fitted data show that the flux of citrate to α-ketoglutarate was negative in PRC3 cells (Figure 4C). This indicates that the net (forward plus reverse) flux of isocitrate dehydrogenase and aconitase (IDH + ACO) is toward citrate production. The exchange flux was also higher in PRC3 than WT8 cells, whereas the PDH flux was lower in PRC3 cells. In agreement with the tracer data, these MFA results strongly suggest that the reverse IDH + ACO fluxes surpass the forward flux in VHL-deficient cells. The estimated ATP citrate lyase (ACLY) flux was also lower in PRC3 than in WT8 cells. Furthermore, the malate dehydrogenase (MDH) flux was negative, reflecting a net conversion of oxaloacetate into malate in VHL-deficient cells (Figure 4C). This indicates an increased flux through the reductive pathway downstream of IDH, ACO, and ACLY. Additionally, some TCA cycle flux estimates downstream of α-ketoglutarate were not significantly different between PRC and WT8 (Table S1). This shows that VHL-deficient cells maintain glutamine oxidation while upregulating reductive carboxylation (Figure S1B). This finding is in agreement with the higher glutamine uptake observed in VHL-deficient cells. Table S1 shows the metabolic network and complete MFA results. …

Addition of citrate in the medium, in contrast to acetate, led to an increase in the citrate-to-α-ketoglutarate ratio (Figure 5L) and absolute citrate levels (Figure S4H) not only in VHL-deficient but alsoVHL-reconstituted cells. The ability of exogenous citrate, but not acetate, to also affect RC in VHL-reconstituted cells may be explained by compartmentalization differences or by allosteric inhibition of citrate synthase (Lehninger, 2005); that is, the ability of acetate to raise the intracellular levels of citrate may be limited in (VHL-reconstituted) cells that exhibit high endogenous levels of citrate. Whatever the mechanism, the results imply that increasing the pools of intracellular citrate has a direct biochemical effect in cells with regards to their reliance on RC. Finally, we assayed the transcript and protein levels of enzymes involved in the reductive utilization of glutamine and did not observe significant differences between VHL-deficient andVHL-reconstituted UMRC2 cells (Figures S4I and S4J), suggesting that HIF does not promote RC by direct transactivation of these enzymes. The IDH1/IDH2 equilibrium is defined as follows:


Figure 5 Regulation of HIF-Mediated Reductive Carboxylation by Citrate Levels

We sought to investigate whether HIF could affect the driving force of the IDH reaction by also enhancing NADPH production. We did not observe a significant alteration of the NADP+/NADPH ratio between VHL-deficient and VHL-positive cells in the cell lysate (Figure S4I). Yet, we determined the ratio of the free dinucleotides using the measured ratios of suitable oxidized (α-ketoglutarate) and reduced (isocitrate/citrate) metabolites that are linked to the NADP-dependent IDH enzymes. The determined ratios (Figure S4J) are in close agreement with the values initially reported by the Krebs lab (Veech et al., 1969) and showed that HIF-expressing UMRC2 cells exhibit a higher NADP+/NADPH ratio. Collectively, these data strongly suggest that HIF-regulated citrate levels modulate the reductive flux to maintain adequate lipogenesis.

Reductive Carboxylation from Glutamine Is Detectable In Vivo

Figure 6 Evidence for Reductive Carboxylation Activity In Vivo

Loss of VHL Renders RCC Cells Sensitive to Glutamine Deprivation

We hypothesized that VHL deficiency results in cell addiction to glutamine for proliferation. We treated the isogenic clones PRC3 (VHL-deficient cells) and WT8 (VHL-reconstituted cells) with the glutaminase inhibitor 968 (Wang et al., 2010a). VHL-deficient PRC3 cells were more sensitive to treatment with 968, compared to the VHL-reconstituted WT8 cells (Figure 7A). To confirm that this is not only a cell-line-specific phenomenon, we also cultured UMRC2 cells in the presence of 968 or diluent control and showed selective sensitivity of VHL-deficient cells (Figure 7B).

Figure 7 VHL-Deficient Cells and Tumors Are Sensitive to Glutamine Deprivation

(A–E) Cell proliferation is normalized to the corresponding cell type grown in 1 mM glutamine-containing medium. Effect of treatment with glutaminase (GLS) inhibitor 968 in PRC3/WT8 (A) and UMRC2 cells (B). Rescue of GLS inhibition with dimethyl alpha-ketoglutarate (DM-Akg; 4 mM) or acetate (4 mM) in PRC3/WT8 clonal cells (C) and polyclonal 786-O cells (D). Effect of GLS inhibitor BPTES in UMRC2 cells (E). Student’s t test compares VHL-reconstituted cells to control cells in (A), (B), and (E) and DM-Akg or acetate-rescued cells to correspondent control cells treated with 968 only in (C) and (D) (asterisk in parenthesis indicates comparison between VHL-reconstituted to control cells). Error bars represent SEM.

(F) GLS inhibitor BPTES suppresses growth of human UMRC3 RCC cells as xenografts in nu/nu mice. When the tumors reached 100mm3, injections with BPTES or vehicle control were carried out daily for 14 days (n = 12). BPTES treatment decreases tumor size and mass (see insert). Student’s t test compares control to BPTES-treated mice (F). Error bars represent SEM.

(G) Diagram showing the regulation of reductive carboxylation by HIF.

In summary, our findings show that HIF is necessary and sufficient to promote RC from glutamine. By inhibiting glucose oxidation in the TCA cycle and reducing citrate levels, HIF shifts the IDH reaction toward RC to support citrate production and lipogenesis (Figure 7G). The reductive flux is active in vivo, fuels tumor growth, and can potentially be targeted pharmacologically. Understanding the significance of reductive glutamine metabolism in tumors may lead to metabolism-based therapeutic strategies.

Along with others, we reported that hypoxia and loss of VHL engage cells in reductive carboxylation (RC) from glutamine to support citrate and lipid synthesis (Filipp et al., 2012Metallo et al., 2012Wise et al., 2011). Wise et al. (2011) suggested that inactivation of HIF in VHL-deficient cells leads to reduction of RC. These observations raise the hypothesis that HIF, which is induced by hypoxia and is constitutively active inVHL-deficient cells, mediates RC. In our current work, we provide mechanistic insights that link HIF to RC. First, we demonstrate that polyclonal reconstitution of VHL in several human VHL-deficient RCC cell lines inhibits RC and restores glucose oxidation. Second, the VHL mutational analysis demonstrates that the ability of pVHL to mitigate reductive lipogenesis is mediated by HIF and is not the outcome of previously reported, HIF-independent pVHL function(s). Third, to prove our hypothesis we showed that constitutive expression of a VHL-independent HIF mutant is sufficient to phenocopy the reductive phenotype observed in VHL-deficient cells. In addition, we showed that RC is not a mere in vitro phenomenon, but it can be detected in vivo in human tumors growing as mouse xenografts. Lastly, treatment of VHL-deficient human xenografts with glutaminase inhibitors led to suppression of their growth as tumors.

7.9.10 Evaluation of HIF-1 inhibitors as anticancer agents

Semenza GL1.
Drug Discov Today. 2007 Oct; 12(19-20):853-9

Hypoxia-inducible factor 1 (HIF-1) regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, genetic instability, vascularization, metabolic reprogramming, autocrine growth factor signaling, invasion/metastasis, and treatment failure. In animal models, HIF-1 overexpression is associated with increased tumor growth, vascularization, and metastasis, whereas HIF-1 loss-of-function has the opposite effect, thus validating HIF-1 as a target. In further support of this conclusion, immunohistochemical detection of HIF-1α overexpression in biopsy sections is a prognostic factor in many cancers. A growing number of novel anticancer agents have been shown to inhibit HIF-1 through a variety of molecular mechanisms. Determining which combination of drugs to administer to any given patient remains a major obstacle to improving cancer treatment outcomes.

Aurelian Udristioiu


Aurelian Udristioiu

Lab Director at Emergency County Hospital Targu Jiu

Mechanisms that control T cell metabolic reprogramming are now coming to light, and many of the same oncogenes importance in cancer metabolism are also crucial to drive T cell metabolic transformations, most notably Myc, hypoxia inducible factor (HIF)1a, estrogen-related receptor (ERR) a, and the mTOR pathway.
The proto-oncogenic transcription factor, Myc, is known to promote transcription of genes for the cell cycle, as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role in inducing the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor (HIF)1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions

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