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Nanostraws Developed at Stanford Sample a Cell’s Contents without Damage

Reporter: Irina Robu, PhD

Cells within our bodies change over time and divide, with thousands of chemical reactions happening within cell daily. Nicholas Melosh, Associate Professor of Materials Science and Engineering, developed a new, non-destructive system for sampling cells with nanoscale straws which could help uncover mysteries about how cells function.

Currently, cells are sampled via lysing which ruptures the cell membrane which means that it can’t ever be sampled again. The sample system that Dr. Melosh invented banks on, on tiny tubes 600 times smaller than a strand of hair that allow researchers to sample a single cell at a time. The nanostraws penetrate a cell’s outer membrane, without damaging it, and draw out proteins and genetic material from the cell’s salty interior.

The Nanostraw sampling technique, according to Melosh, will knowingly impact our understanding of cell development and could result to much safer and operational medical therapies because the technique allows for long term, non-destructive monitoring. The sampling technique could also inform cancer treatments and answer questions about why some cancer cells are resistant to chemotherapy while others are not. The sampling platform on which the nanostraws are grown is tiny, similar to the size of a gumball. It’s called the Nanostraw Extraction (NEX) sampling system, and it was designed to mimic biology itself.

The goal of developing this technology was to make an impact in medical biology by providing a platform that any lab could build.

SOURCE

http://news.stanford.edu/2017/02/20/minuscule-nanostraws-sample-cells-contents-without-damage

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Heat Shock Proteins (HSP) and Molecular Chaperones

Curator: Larry H. Bernstein, MD, FCAP

 

HSP

 

Report on the VIIth International Symposium on Heat Shock Proteins in Biology & Medicine

The major themes of this meeting were: new properties of heat shock proteins (HSPs) and heat shock factor (HSF) and role in the etiology of cancer, molecular chaperones in aging, extracellular HSPs in inflammation and immunity, role of heat shock and the heat shock response in immunity and cancer, protein aggregation disorders and HSP expression, and Hsp70 in blood cell differentiation.
This symposium was the seventh symposium in a series presided over by Dr Stuart Calderwood aimed at exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes. The biochemistry and ultrastructure of molecular chaperones was not emphasized, as these topics are well represented at other meetings. The major themes were: new properties of heat shock proteins (HSPs) and heat shock factor (HSF) and role in the etiology of cancer, molecular chaperones in aging, extracellular HSPs in inflammation and immunity, role of heat shock and the heat shock response in immunity and cancer, protein aggregation disorders and HSP expression, and Hsp70 in blood cell differentiation. This report gives a thematic overview and does not include all the topics presented.
NEW PROPERTIES OF HSPS AND HSF, AND ROLE IN THE ETIOLOGY OF CANCER 

One of the exciting aspects of the meeting involved advances made in understanding the biology of Hsp90. In recent years, we have understood the molecular chaperone activities of Hsp90 mostly in terms of its biochemistry, cooperative interactions with cochaperones. However, Dr Len Neckers (NCI/NIH), the conference keynote speaker, has opened up new areas in our understanding of this chaperone by characterizing the role of posttranslational modification (PTM) in terms of phosphorylation, acetylation, and sumoylation in Hsp90 biology. One particularly intriguing possibility is that altered signaling mechanisms characteristic of cancer may target such PTMs, and this could contribute to the “addiction to chaperones” observed in malignant cells. (Also discussed later by Dr Mehdi Mollapour, SUNY Upstate Medical University).

In addition, interesting differences in properties of the two Hsp90 isoforms have been detected. Dr Wei Li (University of Southern California) has shown that Hsp90a can be released into the extracellular environment and there take part in cell regulation, mediating for instance wound healing effects. In addition, proteomic studies carried out by Thomas Prince (NCI/NIH) in the Neckers lab indicate that Hsp90β may be more dedicated to “housekeeping” molecular chaperone functions while Hsp90α may play more glamorous roles in cell regulation. These distinctions might not be anticipated based on the rather minimal sequence differences between the Hsp90s but offer keen insights into the biology of this chaperone. Finally, Dr Tim Haystead (Duke University) discussed the approach of targeting ectopically expressed Hsp90 for imaging and treatment.

Another PTM with implications in the stress response is the modification of intracellular proteins by monosaccharides of O-linked β-N- acetylglucosamine (O-GlcNAc). Dr Natasha Zachara (Johns Hopkins University School of Medicine) discussed targets for this modification and roles in cytoprotection.

The poster session was also rich in Hsp90 studies, mostly from the Neckers lab—presentations by Kristin Beebe et al. (NCI/NIH) Posttranslational modification state of Hsp90 differentially affects binding of small molecule inhibitors; Toshiki Kijima et al. (NCI/NIH), Defined interactions between HSF1 and Hsp90; T. Prince et al. (NCI/NIH) Hsp90 and tyrosine kinase inhibitors: A synergistic approach towards combating cancer; Andrew W. Truman (University of Chicago)Quantitive ptoteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperonedependent regulation of ribonucleotide reductase. Inhibition of Hsp90 via Cdomain induces temporally distinct phosphorylation patterns; and Diana M. Dunn (SUNY Upstate Medical University)Phosphorylation of human Hsp90 threonine 115 modulates chaperone function and drug sensitivity.

Hsp70 is also emerging as a factor in cell regulation, exhibiting properties beyond a narrow role in chaperoning. Dr Michael Sherman (Boston University) showed a key role for Hsp72 in mammary cancer, and this property did not seem to depend on alterations in protein folding. Instead, Hsp72 appeared to function through its co-chaperone Bag3, a major regulatory molecule in cell signaling. In addition, a presentation by Stuart Calderwood (Harvard Medical School) that included work by Jianlin Gong showed that Hsp72 is required for tumor initiation and metastasis in murine spontaneous breast cancer. These effects appeared to be partially mediated through regulation of expression of the protoooncogene cMet, a key player in invasion and metastasis in cancer. We anticipate advances in understanding of the roles of individual members of the Hsp70 family, as is currently emerging for Hsp90. The prospect of targeting Hsp70 with small molecule inhibitors was elegantly discussed by Maureen Murphy (The Wistar Institute), who introduced a novel class of drugs that could selectively kill cancer cells by inhibiting Hsp70 function. In a related topic, Dr Mathias P. Mayer (University of Heidelberg) showed a detailed analysis of the activities of inhibitors targeting various domains in Hsp70.

Dr Takanori Eguchi (Harvard Medical School) then described his studies showing an unconventional role for the extracellular protease MMP3 as a nuclear protein that could trigger molecular chaperone synthesis (HspA7) in mammary cancer. Interestingly, a role in cancer for the Hsp70 co-chaperone Hsp40 was also shown by Dr Jane Trepel (NCI/NIH).

One presentation that stood apart was that of Dr Carmen Garrido (INSERM U866) who has shown very impressive studies indicating a key role for Hsp70 in hematopoiesis, acting through the factor GATA1. This role appeared to depend on nuclear localization of Hsp70, and Dr Garrido is attempting to study the role of PTM, particularly phosphorylation in this function/localization of Hsp70. This continued the theme of HSP PTM and regulation in the cell.

MOLECULAR CHAPERONES IN AGING

A symposium on molecular chaperones in aging was organized by Dr Shelley Buffenstein (University of Texas Health Science Center San Antonio). This symposium featured some fascinating studies on the naked mole rat (NMR), a rodent with a remarkable lifespan based on size (32 years compared to 3 years in the comparably sized mouse). This has permitted comparative biology studies that have uncovered important aspects of the aging process in mammals. Dr Buffenstein showed that one aspect of the proteotoxic response was enhanced in NMR—proteasome activity that was resistant to oxidative stress as well as conventional proteasome inhibitors. Such proteasome resistance appeared to be conferred by Hsp70 and Hsp40. Karl Rodriguez (also from the UTHSC San Antonio) stressed the importance of Hsp25 in the longevity of NMR. This small HSP is expressed to very high levels in this organism. Kenneth B. Storey (Carleton University) finally gave an encyclopedic presentation entitled “HeatShock Proteins and Hypometabolism in Nature”, discussing the multiple roles of chaperones in hibernation and other processes involving a step down in metabolism.

PROTEIN AGGREGATION DISORDERS AND HSP EXPRESSION

Michael Sherman (Boston University) chaired a lively and highly diverse session on protein aggregation disorders and HSPs. Gary Jones (Maynooth University) discussed his studies on the roles of Hsp104, Hsp70, and Hsp40 in prion propagation in yeast, concentrating on Hsp70. The Hsp complex was able to dissolve prions in yeast. Daniel Kaganovich (Hebrew University) then continued in a yeast theme, discussing a further strategy for resolving proteotoxic stress involving asymmetric cell division in which damaged proteins and mitochondria remain with the mother cell after mitosis. Nava Zaarur (Boston University) then discussed the role of aggresome particles in resolving aggregated proteins, in this case in eucaryotes. Alberto Macario (University of Maryland School of Medicine) discussed the role of chaperonins in proteotoxic disorders dealing with the effects of a pathogenic mutation of human CCT5 on its intrinsic properties. Dr Elaine C. Lee (University of Connecticut) discussed another type of stress. She showed significant roles for chaperones in osmotic stress responses of Caenorhabditis elegans models of polyglutamine diseases.

EXTRACELLULAR HSPS, INFLAMMATION, AND IMMUNITY

Although it is now generally accepted that HSPs can escape the confines of the cell, many questions still remain regarding their extracellular properties, particularly with regard to their immune effects. These questions include: whether HSPs are mostly immunostimulatory or immunosuppressive, whether they can induce sterile inflammation, and what structures on the immune cells recognize the HSPs. Dr Cristina Bonorino (Pontifícia Universidade Católica do Rio Grande do Sul) chaired a symposium “HSP as modulators of immunity: prokaryotic meets eukaryotic” featuring presentations by Robert Binder (University of Pittsburgh), Eckhart R. Podack (University of Miami), Renata Pasqualini (University of New Mexico Medical School), and Cristina Bonorino. In short, the talks indicated that while the prokaryotic chaperone DNA-PK can be immunosppressive and prolong the lifetime of transplanted tissues and reduce the morbidity of arthritis (Drs Bonorino and Kamal Moudgil (University of Maryland School of Medicine)), HSPs can also be immunostimulatory and act as cancer vaccines when associated with cancer antigens (Drs Binder and Podack). In the discussion, it was stressed that these effects may be related to HSP dose, with low doses of HSP antigen complex favoring immunity while higher doses may lead to immunoregulatory effects (Dr Binder). Most parties agreed that much future study is required to resolve all these issues. It was also suggested, inspired by the presentation of Dr Neckers, that HSP PTMs might also be playing roles in shading the immune effects of HSPs (Dr Bonorino). In the next session, Drs Shawn Wang (Virginia Commonwealth University School of Medicine) and John Subjeck (Roswell Park Cancer Institute) discussed the molecular foundations of their highly effective large HSP vaccines that are now in clinical trial for tumor immunotherapy. They indicated that the high avidity for antigen of the larger HSPs might be key for effectiveness. Although the nature of HSP receptors is still not fully resolved, Ayesha Murshid (Harvard Medical School) made a strong case for the scavenger receptor SREC-I as a key molecule in the effects of HSPs on immune cells. As many of the HSPs are in large families, it has not been clear whether all members of Hsp90 or Hsp70 can function outside the cell. Dr Wei Li (University of Southern California) showed that HSP90 family member Hsp90α is the major secreted factor while Dr John Williams (University of Chester) showed potent extracellular effects for human HSP70 isoform HSPA1A. Extracellular roles are not restricted to Hsp90, and Edward O’Brien (Libin Cardiovascular Institute of Alberta/University of Calgary) discussed the extracellular role of heat shock protein 27 (HSPB1) in inflammatory vascular disease. Another lively issue is whether HSPs are released as free proteins, packaged in exosomes, or whether both forms co-exist. This issue was discussed by Monika Fleshner (University of Colorado) and Antonio De Maio (University of California San Diego). Dr De Maio brought up the interesting scenario of Hsp70 binding directly to lipid membranes and perhaps forming membrane channels (Ryan White, University of Maryland).

HSPs are evidently not the only types of stress proteins that can function in the extracellular milieu, as indicated by Dr Michael A. Lynes (University of Connecticut). In a presentation entitled Therapeutic manipulation of the stress response during inflammatory disease, Dr Lynes showed a significant role for extracellular metallothionen in inflammatory bowel disease. Along those lines, Dr George Perdrizet (University of California San Diego) discussed the use of hyperbaric oxygen for enhanced wound healing in diabetic neuropathy, showing impressive clinical findings.

see more at — doi:  10.1007/s12192-014-0562-z

Lens Intermediate Filaments

 The ocular lens assembles two separate Intermediate Filament systems sequentially with differentiation. Canonical 8–11 nm IFs composed of Vimentin are assembled in lens epithelial cells and younger fiber cells, while the fiber cell-specific Beaded Filaments are switched on as fiber cell elongation initiates. Some of the key features of both filament systems are reviewed. Actin filaments and microtubules are essential to the most elemental functions of eukaryotic cells. These filamentous structures are assembled from proteins derived from small, highly conserved gene families. Though tissue specificity exists in the expression of some actin and tubulin family members, they are generally expressed in a ubiquitous manner, and are required for eukaryotic cell survival and replication. In contrast, the family of proteins that comprise the cytoplasmic Intermediate Filaments (IFs) is one of the largest in the human genome with greater than 60 members. IFs are generally not required for cell survival, and are absent from single cell eukaryotes, suggesting a more recent appearance on the evolutionary stage, and a less-essential role in the biology of the cell.
The IF family also differs sharply from actins and tubulins in that there is great variation in both size and sequence among the IF proteins, with sequence identity falling below 30% between more distant members of the human IF family. However, despite the large number of IF proteins available for the construction of IFs, any given cell typically expresses only 1–3 IF proteins, with expression tightly restricted to cell type and state of differentiation. This suggests a considerable degree of cell-specific specialization.
While IF proteins show considerable sequence and size variation, they are unified into a family on the basis of three major features:
1. Predicted domain structure (see figure 1): Algorithms that predict coiled-coil structure show a common predicted domain structure consisting of a) head and tail domains which are quite variable in both size and sequence, and b) a central rod domain where the size (~310 amino acids) and predicted secondary structure is strongly conserved. The rod domain consists of large regions of alpha helical structure (coil domains) interrupted by short non-helical regions (“linkers”) that connect the coil domains. The size, number, and placement of linkers and coils are well-conserved. Moreover, the coil domains exhibit a heptad repeat pattern where the 1, 4 positions in the heptad are dominated by hydrophobic amino acids. Because the 1,4 positions are aligned on one  side of the helix, they form a largely hydrophobic “stripe” that runs along one side of the alpha helix. This stripe mediates the dimerization of two matched coil domains. The hydrophobic stripe gently twists around the axis of the helix, giving rise to a supercoiling of two alpha helices, hence the “coiled-coil”.
The requisite hydrophobicity at the 1, 4 positions of the heptad can be conferred by any of several amino acids, thus the central rod domain of IF proteins, while showing conserved secondary structure, also exhibits a generally high degree of sequence variation. The exceptions to this are two short motifs found at either end of the central rod domain, commonly called the Helix Initiation Motif (HIM) and the Helix termination Motif (HTM). At these two sites the primary sequence among IF proteins is well conserved. Not surprisingly, the HIM and HTM motifs are intolerant of mutations, with the majority of known IF diseases arising from point mutations in these sites (http://www.interfil.org).
2. Conserved gene structure: Sequence analysis of IF proteins has allowed clustering of IF proteins into several major classes. Sequence conservation in the rod domain is high within a class (typically greater than 70%) but low between classes. Analysis of the IF genes shows that there is conservation of gene structure as well within the IF family, with the number and placement of introns and exons well conserved, especially in the central rod domain. The degree of gene structure conservation correlates well with the degree of primary sequence conservation, and reinforces the grouping of IF proteins into classes on the basis of primary sequence.
The Type I and II IF classes are called cytokeratins, and these comprise the IFs of epithelia. These begin assembly as an obligate heterodimer of one Type I and one Type II cytokeratin. The Type III IF proteins include vimentin, desmin, GFAP, and peripherin, and these are commonly found in tissues of mesenchymal origin. While Type III IF proteins can heterodimerize, they are more commonly found as homomeric filaments. The Type IV IF proteins are the neurofilament proteins Heavy (NFH), Medium (NFM) and Light (NFL), which assemble collectively into the IFs of neurons.
3. IF proteins form 8–11 nm diameter IFs. Ultimately, despite the differences in head/ tail size and sequence, and variation in the rod domain sequence, all cytoplasmic IF proteins typically assemble into 8–11 nm IFs.
The mechanism by which vimentin is removed as the cell transitions to the organelle-free state is unknown. In cells undergoing mitosis, vimentin IFs are routinely dismantled by phosphorlylation (Inagaki, Nishi et al. 1987), a modification that causes the relatively stable IF polymer break up into smaller subunits, thought to be tetramers. These are subsequently reassembled after cell division is complete. However, vimentin in lens fiber cells appears to removed, and not simply dismantled. IFs are known to be among the first targets of calcium activated proteases (calpains) in cells that are damaged, and many investigators have demonstrated the calcium-activated degradation of both vimentin and BFs in lens(Yoshida, Murachi et al. 1984; Truscott, Marcantonio et al. 1990; Marcantonio and Duncan 1991; Bettelheim, Qin et al. 1995; Andersson, Sjostrand et al. 1996; Sanderson, Marcantonio et al. 1996; Sanderson, Marcantonio et al. 2000). The dismantling of organelles implies a potential release of calcium from organelles in which it is otherwise routinely sequestered. Whether this release occurs, and whether it alters cytoplasmic calcium levels to a degree that would activate those calpains present in the fiber cell is not known.
Caspases activated in the apoptotic cascade can target conserved sites in IF proteins(Caulin, Salvesen et al. 1997). The elimination of organelles from the fiber cell represents an incomplete apoptotic event, and thus those enzymes responsible for organelle elimination may also represent a viable mechanism for explanation of vimentin’s suggested disappearance(Oshima 2002; Omary, Coulombe et al. 2004).
The loss or reduction of vimentin levels does not leave the mature lens fiber cell devoid of an IF system, however. In a manner that emulates IF switching seen in stratified epithelia, a second generation IF system is switched on in the lens as vimentin is being switched off. It is here where the story of the lens IF system takes the most unusual turn yet described in the IF field.
The initial recognition that the mature lens fiber cells departed from the IF dogma was made when Maisel and co-workers noted the presence of “Beaded Chain Filaments” or Beaded Filaments (BFs) in an electron microscopic analysis of chick lens homogenates (Maisel and Perry 1972; Maisel 1977; Bradley and Maisel 1978; Bradley, Ireland et al. 1979). These studies noted a clearly filamentous structure that was distinct from thin filaments, microtubules, and IFs, which at that time were emerging as the universal cytoskeletal structures common to essentially all vertebrate cells. Speculation emerged that these structures were thin filaments with bound alpha crystallin particles, or nucleosomes on DNA, but these explanations were ruled out experimentally (Bloemendal, Berbers et al. 1984; Ireland and Maisel 1984).
Consistent with the emerging role of IFAPs in modulating and adapting IF function is the observation that fiber cell vimentin IFs interact with the N Cadherin-gamma catenin complex (Leonard, Chan et al. 2008), lengsin(Wyatt, Gao et al. 2008), MIP(Lindsey Rose, Gourdie et al. 2006), periplakin(Yoon and FitzGerald 2008), tropomodulin(Fischer, Quinlan et al. 2003) and possibly other complexes which are present in lens(Bagchi, Katar et al. 2002; Straub, Boda et al. 2003; Bagchi, Katar et al. 2004). The number of candidate linker proteins demonstrated in lens leads to the expectation that the BF and IF are likely to accomplish multiple functions, and that these may be modulated as differentiation progresses, and as the fiber cell proteome changes, either by expression or proteolysis. Similarly, the small heat shock proteins, whose chaperone function appears essential to IF/BF assembly and maintenance, must be considered as critical parts of the biology of IFs in lens. Mutations in the small heat shock proteins have been shown to precipitate a failure in the IF systems in many tissues, and in lens specifically, and to subsequently emulate IF diseases (FitzGerald and Graham 1991; Nicholl and Quinlan 1994; Carter, Hutcheson et al. 1995; Vicart, Caron et al. 1998; Muchowski, Valdez et al. 1999; Perng, Cairns et al. 1999; Perng, Muchowski et al. 1999; Evgrafov, Mersiyanova et al. 2004; Treweek, Rekas et al. 2005; Song, Hanson et al. 2008). The growing multiplicity of IF interactions underscores the need to expect that failure in “IF function” in the lens can result from failure in a wide spectrum of proteins that affect assembly, phosphorylation, proteolytic modification, stability, removal, or linkage to other cellular structures, and that “IF failure” is likely to show considerable variability in phenotype.

Morphological characterization of the AlphaA- and AlphaB-crystallin double knockout mouse lens    Edited by Harry Maisel

Daniel L BoyleLarry TakemotoJames P Brady and Eric F Wawrousek
BMC Ophthalmology 2003; 3:3   http://dx.doi.org:/10.1186/1471-2415-3-3

Background: One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). Methods: Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. Results: Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. Conclusion: These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.

Dogfish a-Crystallin Sequences COMPARISON WITH SMALL HEAT SHOCK PROTEINS AND SCHISTOSOMA EGG ANTIGEN*

Wilfried W. de JongS, Jack A. M. Leunissen, Pieter J. M. Leenen, Anneke Zweers, and Marlies Versteeg
J BIOL CHEM  1988; 263(11):5141-5149

The amino acid sequences of the a-crystallin A and B chains of the dogfish, Squalus acanthias, have been determined. Comparison with a-crystallins from other species reveals that charged amino acid replacements have been strongly avoided in the evolution of this lens protein. The homology of a-crystallins with the small heat shock proteins is pronounced throughout the major part of the proteins, starting from the position of the first intron in the a-crystallin genes, but is also detectable in the amino-terminal sequences of human, Xenopus, and Drosophila small heat shock proteins. In addition, a remarkable short sequence similarity is present only in the amino termini of dogfish aB and Drosophila HSP22. The Schistosoma egg antigen p40 turns out to have a tandomly repeated region of homology with the common sequence domain of a-crystallins and small heat shock proteins. Comparison of hydropathy profiles indicates the conservation of conformation of the common domains in these three families of proteins. Construction of phylogenetic trees suggests that the aA and aB genes apparently originated from a single ancestral small heat shock protein gene and indicates that introns have been lost during the evolution of the heat shock protein.

Acknowledgment – Maisel, H. (ed) (1985) The Ocular Lens, Marcel Dekker, New. York

 

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New subgroups of ILC immune cells discovered through single-cell RNA sequencing

Reporter: Stephen J Williams, PhD

 

SOURCE

http://ki.se/en/news/new-subgroups-of-ilc-immune-cells-discovered-through-single-cell-rna-sequencing?elqTrackId=f79885cef36049e281109c02da213910&elq=ac700a4d4374478b9d6e10e301ae6b90&elqaid=14707&elqat=1&elqCampaignId=14

Updated on 2016-02-15. Published on 2016-02-15Denna sida på svenska

Jenny Mjösberg and Rickard Sandberg are principal investigators at Karolinska Institutet’s Department of Medicine, Huddinge and Department of Cell and Molecular Biology, respectively. Credit: Stefan Zimmerman.

A relatively newly discovered group of immune cells known as ILCs have been examined in detail in a new study published in the journal Nature Immunology. By analysing the gene expression in individual tonsil cells, scientists at Karolinska Institutet have found three previously unknown subgroups of ILCs, and revealed more about how these cells function in the human body.

Innate lymphoid cells (ILCs) are a group of immune cells that have only relatively recently been discovered in humans. Most of current knowledge about ILCs stems from animal studies of e.g. inflammation or infection in the gastrointestinal tract. There is therefore an urgent need to learn more about these cells in humans.

Previous studies have shown that ILCs are important for maintaining the barrier function of the mucosa, which serves as a first line of defence against microorganisms in the lungs, intestines and elsewhere. However, while there is growing evidence to suggest that ILCs are involved in diseases such as inflammatory bowel disease, asthma and intestinal cancer, basic research still needs to be done to ascertain exactly what part they play.

Two research groups, led by Rickard Sandberg and Jenny Mjösberg, collaborated on a study of ILCs from human tonsils. To date, three main groups of human ILCs are characterized. In this present study, the teams used a novel approach that enabled them to sort individual tonsil cells and measure their expression across thousands of  genes. This way, the researchers managed to categorise hundreds of cells, one by one, to define the types of ILCs found in the human tonsils.

Unique gene expression profiles

Rickard Sandberg, credit: Stefan Zimmerman,

“We used cluster analyses to demonstrate that ILCs congregate into ILC1, ILC2, ILC3 and NK cells, based on their unique gene expression profiles,” says Professor Sandberg at Karolinska Institutet’sDepartment of Cell and Molecular Biology, and the Stockholm branch of Ludwig Cancer Research. “Our analyses also discovered the expression of numerous genes of previously unknown function in ILCs, highlighting that these cells are likely doing more than what we previously knew.”

By analysing the gene expression profiles (or transcriptome) of individual cells, the researchers found that one of the formerly known main groups could be subdivided.

Jenny Mjösberg, credit: Stefan Zimmerman.

“We’ve identified three new subgroups of ILC3s that evince different gene expression patterns and that differ in how they react to signalling molecules and in their ability to secrete proteins,” says Dr Mjösberg at Karolinska Institutet’s Department of Medicine in Huddinge, South Stockholm. “All in all, our study has taught us a lot about this relatively uncharacterised family of cells and our data will serve as an important resource for other researchers.”

The study was financed by grants from a number of bodies, including the Swedish Research Council, the Swedish Cancer Society, the EU Framework Programme for Research and Innovation, the Swedish Society for Medical Research, the Swedish Foundation for Strategic Research and Karolinska Institutet.

Publication

The heterogeneity of human CD127+ innate lymphoid cells revealed by single-cell RNA sequencing
Åsa K. Björklund, Marianne Forkel, Simone Picelli, Viktoria Konya, Jakob Theorell, Danielle Friberg, Rickard Sandberg, Jenny Mjösberg
Nature Immunology, online 15 February 2016, doi:10.1038/ni.3368

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Medical 3D Printing and Metals in use in Medical Devices,
Presentation by Danut Dragoi, PhD

The main objective of medical 3D printing (M3DP) is to build solid / semi-solid / scaffolds / or gel structures from bio-compatible materials that can be utilized in medicine in order to correct, alleviate, support certain surgeries, or even cure some diseases based on medical / biological principles applied to human body.

Materials that replace bones are metals like Ti, Ti alloys, Tantalum, Gold, Silver, Zr and other. For replacement of teeth is traditionally used a combination of Ti-pivots and ceramic / polymers / or in some cases Hydroxylapatite (HA) coated Ti.

In order to produce a metallic object implantable in the human body, most useful technology is 3D printing of metals, commonly known as AT (addition manufacturing) technology. A definition of 3D printing is a process for making a physical object from a three-dimensional digital model, typically by laying down many successive thin layers of a material. If a printer system uses metal powders and binder instead of normal ink the printed layer by layer will develop a 3D object.

The printed object may be an orthopedic bone replacement, a tooth pivot or an artificial tooth. The picture on Slide 4 shows a Laser Sintering System (SLM) for Medical 3D Printing for metals, find specs in here.

Slide 4

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The machine shown on Slide 5 is one of the three metal printers from SLM Solutions using the technology of Selective Laser Melting, find specs in here,
Slide 5

Slide5
Feature highlight: for aerospace and medical orthopedics. Large build volume.
Material: Stainless steel, tool steel, aluminium, titanium, cobalt-chrome, inconel
Build capacity: 19.68 x 11.02 x 12.80 in. / (500 x 280 x 325 mm)
Build rate: 70 cm³/h
Resolution/Layer thickness: 20 – 200µm
Machine dimensions: 118 x 98 x 43 in.

An important aspect of metal source for M3DP is the shape of the particles, uniform size distribution and chemical purity. Using a new manufacturing approach, Zecotek, a company in Germany, link in here, developed metallic powders that can be successfully used in M3DP. Next Slide 6 shows some characteristics of this breakthrough technology.

Slide6
Slide 7

Slide7

More information on Slide 7 can be found in here.

Slide 8

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Information on Slide 8 can be found in here .
Slide 9

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Information on Slide 9 can be found in here, which is a novelty in terms of materials, the fusion for the first time between a Ti alloy and a ceramic.
Slide 10

Slide10The schematic on Slide 10 can be found in here . SLS technology is in wide use around the world due to its ability to easily make very complex geometries directly from digital CAD data. While it began as a way to build prototype parts early in the design cycle, it is increasingly being used in limited-run manufacturing to produce end-use parts. Here is how it is working. The powders are in a compartment controlled by a piston going one small step up, the roller swipes to the right a thin layer of metallic powder on the second compartment controlled by a piston that goes only one small step down, due to the fact that the printed model starts to grow up. The tip of the laser beam melts the powder or fusion the particles according with a real drawing section of the model. The process is repeated until the model is done. The key element of this technology is the laser scan device that follows exactly the drawing section of the model.

Slide 12

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Slide 12 shows a 3D printed foot that is light and well manageable for the patient. The picture can be found at this link in here. This prosthetic introduces the traces concept on light-weighting of replaceable parts for human body.
Slide 13

Slide13

Slide 13 shows a 3D printed light orthopedic pieces that are using the concept of light-weighting using traces. Their picture can be found here.

Slide 14

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Slide 14 shows tiny parts obtained with 3D printing technology, details in here.

Slide 15

Slide15

A second way to obtain solid parts is using a 3D Bioplotter, link in here .

EnvisionTec’s 3D-Bioplotter builds its products in much the same way as a traditional 3D printer. However, instead of using plastics, metals or resins, the Bioplotter uses biologic materials to form a scaffold that will be used to grow more advanced cellular cultures.

Just like a traditional 3D printer, the 3D-Bioplotter can be fed a 3D model generated in a CAD program or from a CT scan. Users can slice and hatch a 3D model to define how it will be printed. That information is then translated to code and shipped off to the Bioplotter where the real work begins.

While prototype objects in the mechanical, architectural and civil worlds can be built from a single material, in the biological world it’s rare that the desired objects have a uniform material. To meet that reality, the Bioplotter can print a model in 5 different materials making it suitable for more complex cellular assemblies.

This ability to jet different materials during a single build requires the 3D-Bioplotter to change print heads. It comes equipped with a CNC-like tool holder that can be programmed to change “print-heads” based on the material being extruded. Most bio-engineering builds favor porosity. This machine’s ability to change print heads can also help alter the flow and spacing of successive print layers to give users greater control of their models.

Slide 16

Slide16

The scaffold on slide 16 obtained with a 3D Bioploter, is useful in dentistry to augment the base of the future implantable tooth. The fixation in the picture is made of Vivos Dental’s OsteoFlux product, link see in here.
Slide 17

Slide17

Slide 17 Metals in medical dental implants, Ti becomes fused with the bone, and the tooth attached to one end of the Ti pivot, see link in here.

Slide 18

Slide18

Slide 18, Hot plasma spray bio-ceramics is the solution that doctors used for biocompatibility of an artificial jaws, link in here.

Slide 20

Slide20On slide 20 the traditional Ti casting is compared with Ti 3D printing from the powders. The advantage of 3D method is low cost and high productivity. This link in here is for traditional method, and this link here for 3D printing method.
Slide 21

Slide21Slide 21 For 3D Bioploter made by EnvisionTec we notice the usage of materials such as metal followed by post-processing sintering, Hydroxylapatite, TCP, Titanium. Using a preciptation method the machine can handle Chitosan, Collagen, 2-component system of the two possible combination: Alginate, Fibrin, PU, and Silicone. More details in here.

Slide 26

Slide26

Slide 26 shows two ultra-miniature medical pressure sensors in the eye of a needle, for details see the link in here.

Slide 27

Slide27

Slide 27 The electrodes of the bio-mems implanted on the surface of the heart are made of Gold for the electrical contact and good bio-compatibility. Classes of materials and assembly approaches that enable electronic devices with features – area coverage, mechanical properties, or geometrical forms – that would be impossible to achieve using traditional, wafer-based technologies. Examples include ’tissue-like’ bio-integrated electronics for high resolution mapping of electrophysiology in the heart and brain. The research on bio-integrated electronics can be found here.

Slide 28

Slide28

Slide 28 shows a polymeric material for determining pressure inside the eye, which is useful to monitor patients at risk from glaucoma. Again the circular electrode is made of Gold and its role is that of an antena to transmit data to a iPhone / receiver about the intraocula pressure data.
Slide 29

Slide29

The device in slide 29 is a bio-MEMS implantable for drug dosage. It has multiple micro-needles that are equivalent to a needle of a normal syringe, but painless since theyr tips do not reach the pain receptors. This picture taken from here, shows a side size of the MEMS of about 25 mm.

Slide 30

Slide30

Slide 30 lists some effects of metals in human body. Traces of heavy metals are dangerous for human body. Human body is made of light elements C,H,N,O. Heavy metals: Pb, Hg, accumulate in the body, they disrupt the metabolic processes since they are very toxic to humans. Therefore, heavy metals don’t have “+” physiological effects and Al as element is known to produce Alzheimer’s which has been implicated as a factor. According to the Alzheimer’s Society, the medical and scientific opinion is that studies have not convincingly demonstrated a causal relationship between aluminium and Alzheimer’s disease. Nevertheless, some studies, cite aluminium exposure as a risk factor for Alzheimer’s disease. Some brain plaques have been found to contain increased levels of the metal. Research in this area has been inconclusive; aluminium accumulation may be a consequence of the disease rather than a causal agent, see link in here.
Slide 31

Slide31

Slide 31 shows percent distribution of elements in human bodies, It is interesting that Ti is not making the list, see link in here.

Slide 32

Slide32

Slide 32 has Ti element circled on the Table of the elements, we notice that Zr as element was found to be a bio-compatible element too just like Ti. It is very possible from chemical point of view that all elements in Ti group have same property. The only inconvenient of elements bellow Ti is that they are heavier and their density should be adapted closer to that of human body.
Slide 33

Slide33

Slide 33 is a plot of stress (MPa) of some human implantable materials as a function of Young modulus E (GPa), their principal mechanical characteristic. There are crystalline materials such as: MgZnCa, MgZr, etc.) as well as amorphous materials bio-compatible such as: MgZnCa BMG, Ca based BMG, Sr based BMG, etc.) that have important mechanical strength that can be used in various applications. The circle in green centered on the point (75GPa, 650 MPa) is that for HydroxylApatite, which is a component of teeth and bones. Further details on this plot can be found at this link here,  .

Magnesium and its alloys are suitable materials for biomedical applications due to their low weight, high specific strength, stiffness close to bone and good biocompatibility. Specifically, because magnesium exhibits a fast biodegradability, it has attracted an increasing interest over the last years for its potential use as “biodegradable implants”. However, the main limitation is that Mg degrades too fast and that the corrosion process is accompanied by hydrogen evolution. In these conditions, magnesium implants lose their mechanical integrity before the bone heals and hydrogen gas accumulates inside the body. To overcome these limitations different methods have been pursued to decrease the corrosion rate of magnesium to acceptable levels, including the growth of coatings (conversion and deposited coatings), surface modification treatments (ion implantation, plasma surface modification, etc) or via the control of the composition and microstructure of Mg alloys themselves.

Slide 34

Slide34

Slide 34 shows two types of three point bending tests, one in which the flexural stress is plotted against displacement and second in which the stress intensity factor is plotted against the length of the crack extended beyond the notch. It is interesting that both plots can differentiate between young and aged bones. The plots can be downloaded from here,  where more experimental details and explanation can be found.

Slide 35

Slide35

Slide 35 shows the geometry for 3 point bending for fracture toughness testing. in which the stress intensity factor can be considered as a function of delta a, the depth of the notch at various values of loads. The equation of stress intensity factor can be found here.

Slide 36

Slide36

Slide 36 describes a family of stress-strain curves as function of composition for four Ti alloys. As we can see the mechanical strength of Ti alloys is well above 400 MPa, which is more than enough for replacement of bones that have a lower mechanical strength of about 175 MPa. The plot in this slide can be reviewed at this site.
Slide 37

Slide37

Slide 37 Mechanical strength of cortical bone, see link in here,  and mechanical strength of Ti alloys, seen in here.

The comparison shows a limit of elasticity of 160 MPa which is well below 400 MPa of Ti alloys or even simply Ti element which has a yield strength of 434 MPa, see link video here.
Slide 38

Slide38

Slide 38 provides information about the oxide layer on Ti binding biological tissues. Rutile and Anatase, are the two crystalline species of TiO2 formation on Ti surface. Rutile is less bio-reactive than Anatase, info in here, http://cdn.intechopen.com/pdfs-wm/33623.pdf . The metal work function changes as a consequence of the formation of the passivisation layer (the oxide), but ΔΦ is positive for rutile and negative for anatase, info in here, http://pubs.acs.org/doi/abs/10.1021/jp309827u?journalCode=jpccck .

Slide 39

Slide39

Slide 39 provides information about the crystal structures of three species of Titanium oxide: Rutile, Anatase, and Brookite. As seen from the slide, the density varies with the crystal structure. The valence of Ti in these structures is 4+, same as Carbon in many organic molecules.
Slide 40

Slide40

Slide 40 provides information about the crystal structures of Titanium monoxide. As seen from the slide, the density is the highest among all Titanium oxides. The crystal structure of Titanium monoxide is shown in this slide. The valence of Ti in these structure is 2+, that makes this oxide special in applications.
Slide 41

Slide41

Slide 41 provides information about two metals, Ti and Zr that are used in human body implantable. An explanation of why these two metals are bio-compatible is given in this slide. As we know not all metals are inert/not reactive in human body environment. As a fact bulk cubic structures of metals is less preferred such as Al, Cu, Nb, Pb, etc.. Based on a symmetry remark for living structures (carbohydrates, nucleic acids, lipids and proteins), the lower implantable metals symmetry the better. As an example Lysozyme (S.G. P43212, space group number 96) as a possible interface material with an implantable metal such as Au, Ti, Zr, admits lower space groups such as Ti ( P63/mmc. Space group number: 194). Gold is not preferred for multiple reasons too: it has a high symmetry S.G. 225 (Fm-3m) 96<225, it has has a high density 19.32 g/cc, and it is expensive.

Many metals have a degree of leachability in human body fluids except the rare/precious metals Au, Pt, Ir that are expensive as implants. The coatings of Ti with a tiny thin layer of oxide or laser coated organic ceramics, makes Ti as the best choice as human body implantable with extremely low leachability in human body fluids.
Slide 42

Slide42

Slide 42 provides crystallographic information on Ti crystal structure, unit cell size and directions.
Slide 43

Slide43

Slide 43 provides information on Zr metal as the second choice on human body implantables. The crystal structure of Zr is same as Ti, with hexagonal close packed (HCP) unit cell. The HCP cell is shown together with a body center cubic (BCC) unit and face close cubic (FCC) unit for comparison reason.
Slide 44

Slide44

Slide 44 shows the Table of major biomedical metals and alloys and their applications. More details about materials in the Table can be found here.

Slide 45

Slide45

The Table on Slide 45 shows a comparison of mechanical properties for three metal alloys. Notice the the increase of the ultimate tensile strength of Ti 64, from 434 MPa for Titanium (see slide 37) to 900 MPa for Ti 64. More data about other materials can be found here.

Slide 46

Slide46

Slide 46 lists some medical devices as they were created by the inventor Alfred Mann’s companies. Such devices are:
-rechargeable pacemaker,
-an implant for deaf people,
-an insulin pump and a
-prosthetic retina. (Mel Melcon, Los Angeles Times)
Slide 47

Slide47

Slide 47 As we imagine, the implanted devices should be coated with one of these Ti, Zr, ceramic coated Ti and Stainless Steel. Three example are given as: Ti-plates and rods, 3D printed Jaws + plasma coated HAp, Gold nano-wires.
Slide 48

Slide48

In the example on slide Slide 48, the pacemaker casing is made of titanium or a titanium alloy, electrodes are made of metal alloy insulated with polyurethan polymers, more info in here.

Slide 49

Slide49

The second device shown in slide 49 is an implant for deaf people, whose surface in contact with human body fluids is coated with Ti. More info on how this implant works can be found in here.
Slide 50Slide50The insulin pump shown in slide 50 is a schematic of the pump controlled electronically by a control algorithm device, a sensor, an electronic receiver that connects with an iPhone through an wireless channel.
Slide 51Slide51

The prosthetic retina on slide 51 is an example of a bio-MEMS based optical sensor that takes the outside image through a tiny camera, the electrical signal of the camera is sent to a receiver and then to an array of micro-electrodes tacked to the retina which send electrical impulses to the brain through the optical nerve. More details can be found in here.

Slide 52Slide52Slide 52 describes how easily available bio-compatible metal powders
can revolutionize 3D printing for medical implants. The surgical implants need to generate expected responses from neighboring cells and tissues. Cell behavior (adhesion, functional alteration, morphological changes, and proliferation) is strongly affected by the surgical implants’ surface properties. Surface topography, surface chemistry, and surface energy influence decisively the biological response to an implanted device.
The well controlled 3D printing atmosphere (neutral gases and restricted oxygen) guarantees the high purity of the 3D printed parts and preserves the materials’ properties.
The advantages of 3D printing for medical applications is thoroughly discussed in here.

Slide 53Slide53

Slide 53 shows five conclusions of the presentation, in which 1) many engineered metals are mechanically resistant in human body, but prone to certain corrosion if not coated,
2) Ti, Zr coated bio-ceramics are bio-compatible materials in human body, 3) medical devices implants and MEMS are useful as heart stent, orthopedic prosthetic, prosthetic retina, 3) M3DP has low costs, high quality, long life cycle and 4) Metal/bio-ceramic and Vivos dental’s synthetic bone for oral augmentation is a solution for today’s dental health care.
Slide 54Slide54Slide 54 shows conclusions regarding the hardware of the presentation, in which: 6) there are two types of metal 3D printing hardware for medical applications: Selective Laser Melting / Selective Laser Sintering, and 3D Bioploter (metal powder mixed with binder and further thermal treatment to remove binder and sinter the metallic matrix in a solid object that can be used as a replacement. Thank you for your attention!

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Novel Mechanisms of Resistance to Novel Agents

 

Curators: Larry H. Berstein, M.D. FACP & Stephen J. Williams, Ph.D.

For most of the history of chemotherapy drug development, predicting the possible mechanisms of drug resistance that ensued could be surmised from the drug’s pharmacologic mechanism of action. In other words, a tumor would develop resistance merely by altering the pathways/systems which the drug relied on for mechanism of action. For example, as elucidated in later chapters in this book, most cytotoxic chemotherapies like cisplatin and cyclophosphamide were developed to bind DNA and disrupt the cycling cell, thereby resulting in cell cycle arrest and eventually cell death or resulting in such a degree of genotoxicity which would result in great amount of DNA fragmentation. These DNA-damaging agents efficacy was shown to be reliant on their ability to form DNA adducts and lesions. Therefore increasing DNA repair could result in a tumor cell becoming resistant to these drugs. In addition, if drug concentration was merely decreased in these cells, by an enhanced drug efflux as seen with the ABC transporters, then there would be less drug available for these DNA adducts to be generated. A plethora of literature has been generated on this particular topic.

However in the era of chemotherapies developed against targets only expressed in tumor cells (such as Gleevec against the Bcr-Abl fusion protein in chronic myeloid leukemia), this paradigm had changed as clinical cases of resistance had rapidly developed soon after the advent of these compounds and new paradigms of resistance mechanisms were discovered.

speed of imitinib resistance

Imatinib resistance can be seen quickly after initiation of therapy

mellobcrablresistamplification

Speed of imatinib resistance a result of rapid gene amplification of BCR/ABL target, thereby decreasing imatinib efficacy

 

 

 

 

 

 

 

 

 

 

Although there are many other new mechanisms of resistance to personalized medicine agents (which are discussed later in the chapter) this post is a curation of cellular changes which are not commonly discussed in reviews of chemoresistance and separated in three main categories:

Cellular Diversity and Adaptation

Identifying Cancers and Resistance

Cancer Drug-Resistance Mechanism

p53 tumor drug resistance gene target

Variability of Gene Expression and Drug Resistance

 

Expression of microRNAs and alterations in RNA resulting in chemo-resistance

Drug-resistance Mechanism in Tumor Cells

Overexpression of miR-200c induces chemoresistance in esophageal cancers mediated through activation of the Akt signaling pathway

 

The miRNA–drug resistance connection: a new era of personalized medicine using noncoding RNA begins

 

Gene Duplication of Therapeutic Target

 

The advent of Gleevec (imatinib) had issued in a new era of chemotherapy, a personalized medicine approach by determining the and a lifesaver to chronic myeloid leukemia (CML) patients whose tumors displayed expression of the Bcr-Abl fusion gene. However it was not long before clinical resistance was seen to this therapy and, it was shown amplification of the drug target can lead to tumor outgrowth despite adequate drug exposure. le Coutre, Weisberg and Mahon23, 24, 25 all independently generated imatinib-resistant clones through serial passage of the cells in imatinib-containing media and demonstrated elevated Abl kinase activity due to a genetic amplification of the Bcr–Abl sequence. However, all of these samples were derived in vitro and may not represent a true mode of clinical resistance. Nevertheless, Gorre et al.26 obtained specimens, directly patients demonstrating imatinib resistance, and using fluorescence in situ hybridization analysis, genetic duplication of the Bcr–Abl gene was identified as one possible source of the resistance. Additional sporadic examples of amplification of the Bcr–Abl sequence have been clinically described, but the majority of patients presenting with either primary or secondary imatinib resistance fail to clinically demonstrate Abl amplification as a primary mode of treatment failure.

This is seen in the following papers:

Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL. Science. 2001 Aug 3;293(5531):876-80. Epub 2001 Jun 21.

and in another original paper by le Coutre et. al.

Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification. le Coutre P1, Tassi E, Varella-Garcia M, Barni R, Mologni L, Cabrita G, Marchesi E, Supino R, Gambacorti-Passerini C. Blood. 2000 Mar 1;95(5):1758-66

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic bcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/abl inhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 microM) 10-fold higher than the IC50 (0.1 microM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3-like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro. (Blood. 2000;95:1758-1766)

This is actually the opposite case with other personalized therapies like the EGFR inhibitor gefinitib where actually the AMPLIFICATION of the therapeutic target EGFR is correlated with better response to drug in

Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs.Ono M, Kuwano M. Clin Cancer Res. 2006 Dec 15;12(24):7242-51. Review.

Abstract

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, including EGFR, HER2/erbB2, and HER3/erbB3, is an attractive target for antitumor strategies. Aberrant EGFR signaling is correlated with progression of various malignancies, and somatic tyrosine kinase domain mutations in the EGFR gene have been discovered in patients with non-small cell lung cancer responding to EGFR-targeting small molecular agents, such as gefitinib and erlotinib. EGFR overexpression is thought to be the principal mechanism of activation in various malignant tumors. Moreover, an increased EGFR copy number is associated with improved survival in non-small cell lung cancer patients, suggesting that increased expression of mutant and/or wild-type EGFR molecules could be molecular determinants of responses to gefitinib. However, as EGFR mutations and/or gene gains are not observed in all patients who respond partially to treatment, alternative mechanisms might confer sensitivity to EGFR-targeting agents. Preclinical studies showed that sensitivity to EGFR tyrosine kinase inhibitors depends on how closely cell survival and growth signalings are coupled with EGFR, and also with HER2 and HER3, in each cancer. This review also describes a possible association between EGFR phosphorylation and drug sensitivity in cancer cells, as well as discussing the antiangiogenic effect of gefitinib in association with EGFR activation and phosphatidylinositol 3-kinase/Akt activation in vascular endothelial cells.

 

Mutant Variants of Therapeutic Target

 

resistant subclones in tissue samples and Tyrosine Kinase tumor activity

 

Mitochondrial Isocitrate Dehydrogenase and Variants

Mutational Landscape of Rare Childhood Brain Cancer: Analysis of 60 Intercranial Germ Cell Tumor Cases using NGS, SNP and Expression Array Analysis – Signaling Pathways KIT/RAS are affected by mutations in IGCTs

 

AND seen with the ALK inhibitors as well (as seen in the following papers

Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib.

Hashida S, Yamamoto H, Shien K, Miyoshi Y, Ohtsuka T, Suzawa K, Watanabe M, Maki Y, Soh J, Asano H, Tsukuda K, Miyoshi S, Toyooka S. Cancer Sci. 2015 Oct;106(10):1377-84. doi: 10.1111/cas.12749. Epub 2015 Sep 30.

In vivo imaging models of bone and brain metastases and pleural carcinomatosis with a novel human EML4-ALK lung cancer cell line.

Nanjo S, Nakagawa T, Takeuchi S, Kita K, Fukuda K, Nakada M, Uehara H, Nishihara H, Hara E, Uramoto H, Tanaka F, Yano S. Cancer Sci. 2015 Mar;106(3):244-52. doi: 10.1111/cas.12600. Epub 2015 Feb 17.

Identification of a novel HIP1-ALK fusion variant in Non-Small-Cell Lung Cancer (NSCLC) and discovery of ALK I1171 (I1171N/S) mutations in two ALK-rearranged NSCLC patients with resistance to Alectinib. Ou SH, Klempner SJ, Greenbowe JR, Azada M, Schrock AB, Ali SM, Ross JS, Stephens PJ, Miller VA.J Thorac Oncol. 2014 Dec;9(12):1821-5

Reports of chemoresistance due to variants have also been seen with the BRAF inhibitors like vemurafenib and dabrafenib:

The RAC1 P29S hotspot mutation in melanoma confers resistance to pharmacological inhibition of RAF.

Watson IR, Li L, Cabeceiras PK, Mahdavi M, Gutschner T, Genovese G, Wang G, Fang Z, Tepper JM, Stemke-Hale K, Tsai KY, Davies MA, Mills GB, Chin L.Cancer Res. 2014 Sep 1;74(17):4845-52. doi: 10.1158/0008-5472.CAN-14-1232-T. Epub 2014 Jul 23

 

 

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A Curated History of the Science Behind the Ovarian Cancer β-Blocker Trial

Curator: Stephen J. Williams, Ph.D.

 

This post is a follow-up on the two reports found in this Open Access Journal

https://pharmaceuticalintelligence.com/2015/09/16/ovarian-cancer-survival-increased-5-months-overall-with-beta-blockers-study-the-speaker/

AND

https://pharmaceuticalintelligence.com/2013/04/08/beta-blockers-help-in-better-survival-in-ovarian-cancer/

in order to explain some of the background which went into the development of these reports.

A recent paper by Anil Sood’s group at MD Anderson in Journal of Cancer: Clinical impact of selective and nonselective beta-blockers on survival in patients with ovarian cancer describes a retrospective pathologic evaluation of ovaries from patients taking various beta blockers for currently approved indications.

The history of this finding is quite interesting and, as I remember in a talk given by Dr. Sood in mid-2000’s, a microarray conducted by his lab had showed overexpression of the β2-AR (β2 adrenergic receptor in ovarian cancer cells relative to normal epithelium. At the time it appeared an interesting result however most of the cancer (and ovarian cancer) field were concentrating on the tyrosine kinase signaling pathways as potential therapeutic targets, as much promising translational research in this area was in focus at the time. As a result of this finding and noticing that sustained β-adrenergic stimulation can promote ovarian cancer cell growth (Sood, 2006), Dr. Sood’s group have been studying the effects of β-adrenergic signaling om ovarian cancer. In addition it has been shown that propanalol can block VEGF signaling and norepinephrine increased MMP2 and MMP9 expression, an effect mediated by the β2-AR.

The above re-post of a Scoop-IT describes promising results of a clinical trial for use of selective beta blockers in ovarian cancer.   As to date, there have been many clinical trials initiated in ovarian cancer and most have not met with success for example the following posts:

Good and Bad News Reported for Ovarian Cancer Therapy

a follow-up curation on the problems encountered with the PARP-inhibitor olaparib

enough is enough: Treat ‘Each Patient as an Individual’

which contains an interview with Dr. Maurie Markman (Vice President, Patient Oncology Services, and National Director for Medical Oncology, Cancer Treatment Centers of America) and Dr. Kathy D. Miller, Indiana University School of Medicine) and discusses how each patient’s ovarian cancer is genetically unique and needs to be treated as such

Therefore the mainstay therapy is still carboplatin plus a taxane (Taxotere, Abraxane). The results of this clinical trial show a 5 month improvement in survival, which for a deadly disease like ovarian cancer is a significant improvement.

First below is a SUMMARY of the paper’s methodology and findings.

Methods:

  • Four participating institutions collected retrospective patient data and pathology reports from 1425 patients diagnosed with epithelial ovarian cancer (EOC)
  • Medical records were evaluated for use of both selective and nonselective β-blockers
  • β-blockers were used for various indications however most common indication was treatment for hypertension (71% had used β1 selective blockers while rest of patients taking β blockers were given nonselective blockers for a host of other indications)
  • most patients had stage III/IV disease and in general older (median age 63 years)
  • The authors looked at overall survival (OS) however progression free survival PFS) was not calculated

Results:

  • Hypertension was associated with decreased survival (40.1 monts versus 47.4 months for normotensive patients)
  • Overall Survival for patients on any β blockers was 47.8 months versus 42.0 months for nonusers
  • Patients receiving nonselective β blockers has an OS of 94.9 months versus 38 months for EOC patients receiving β1-selective blockers
  • No effect of diabetes mellitus on survival

Authors Note on Limitations of Study:

  • Retrospective in nature
  • Lack of documentation of dosage, trade-name and duration of β-blocker use
  • Important to stratify patients on selectivity of β-blocker since Eskander et. al. found no difference of Progression Free Survival and non-selective β-blocker
  • Several β adrenergic receptor polymorphisms may exist and no downstream biomarker evaluated to determine effect on signaling; could it be a noncanonical effect?

The goal of this brief, added curation is to paint a historical picture, and highlight the scientific findings which led up to the rationale behind this clinical trial.

How the βeta Adrenergic Receptor (βAR) Became a Target for Ovarian Cancer

.

A. βAR and its signaling over-expressed in ovarian cancer

Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line.

Kimura A, Ohmichi M, Kurachi H, Ikegami H, Hayakawa J, Tasaka K, Kanda Y, Nishio Y, Jikihara H, Matsuura N, Murata Y.

Cancer Res. 1999 Oct 15;59(20):5133-42.

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor.

Rangarajan S, Enserink JM, Kuiperij HB, de Rooij J, Price LS, Schwede F, Bos JL.

J Cell Biol. 2003 Feb 17;160(4):487-93. Epub 2003 Feb 10.

B. Mechanistic Link Between Chronic Stress From Excess Adrenergic Stimulation and Angiogenesis and Metastasis

Stress-related mediators stimulate vascular endothelial growth factor secretion by two ovarian cancer cell lines.

Lutgendorf SK, Cole S, Costanzo E, Bradley S, Coffin J, Jabbari S, Rainwater K, Ritchie JM, Yang M, Sood AK.

Clin Cancer Res. 2003 Oct 1;9(12):4514-21.PMID:

Norepinephrine up-regulates the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9 in nasopharyngeal carcinoma tumor cells.

Yang EV, Sood AK, Chen M, Li Y, Eubank TD, Marsh CB, Jewell S, Flavahan NA, Morrison C, Yeh PE, Lemeshow S, Glaser R.

Cancer Res. 2006 Nov 1;66(21):10357-64.

VEGF is differentially regulated in multiple myeloma-derived cell lines by norepinephrine.

Yang EV, Donovan EL, Benson DM, Glaser R.

Brain Behav Immun. 2008 Mar;22(3):318-23. Epub 2007 Nov 5.

Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma.

Thaker PH, Han LY, Kamat AA, Arevalo JM, Takahashi R, Lu C, Jennings NB, Armaiz-Pena G, Bankson JA, Ravoori M, Merritt WM, Lin YG, Mangala LS, Kim TJ, Coleman RL, Landen CN, Li Y, Felix E, Sanguino AM, Newman RA, Lloyd M, Gershenson DM, Kundra V, Lopez-Berestein G, Lutgendorf SK, Cole SW, Sood AK.

Nat Med. 2006 Aug;12(8):939-44. Epub 2006 Jul 23.

Norepinephrine up-regulates the expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, and MMP-9 in nasopharyngeal carcinoma tumor cells.

Yang EV, Sood AK, Chen M, Li Y, Eubank TD, Marsh CB, Jewell S, Flavahan NA, Morrison C, Yeh PE, Lemeshow S, Glaser R.

Cancer Res. 2006 Nov 1;66(21):10357-64.

C. In Vivo Studies Confirm In Vitro Findings That Chronic Stress Via Adrenergic overstimulation Increases Ovarian Cancer Growth

Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma.

Thaker PH, Han LY, Kamat AA, Arevalo JM, Takahashi R, Lu C, Jennings NB, Armaiz-Pena G, Bankson JA, Ravoori M, Merritt WM, Lin YG, Mangala LS, Kim TJ, Coleman RL, Landen CN, Li Y, Felix E, Sanguino AM, Newman RA, Lloyd M, Gershenson DM, Kundra V, Lopez-Berestein G, Lutgendorf SK, Cole SW, Sood AK.

Nat Med. 2006 Aug;12(8):939-44. Epub 2006 Jul 23.

Stress hormone-mediated invasion of ovarian cancer cells.

Sood AK, Bhatty R, Kamat AA, Landen CN, Han L, Thaker PH, Li Y, Gershenson DM, Lutgendorf S, Cole SW.

Clin Cancer Res. 2006 Jan 15;12(2):369-75.

The neuroendocrine impact of chronic stress on cancer.

Thaker PH, Lutgendorf SK, Sood AK.

Cell Cycle. 2007 Feb 15;6(4):430-3. Epub 2007 Feb 9. Review.

Surgical stress promotes tumor growth in ovarian carcinoma.

Lee JW, Shahzad MM, Lin YG, Armaiz-Pena G, Mangala LS, Han HD, Kim HS, Nam EJ, Jennings NB, Halder J, Nick AM, Stone RL, Lu C, Lutgendorf SK, Cole SW, Lokshin AE, Sood AK.

Clin Cancer Res. 2009 Apr 15;15(8):2695-702. doi: 10.1158/1078-0432.CCR-08-2966. Epub 2009 Apr 7.

Sood group wanted to mimic the surgical stress after laparoscopic surgery to see if surgical stress would promote the growth of micrometasteses remaining after surgical tumor removal. Propranolol completely blocked the effects of surgical stress on tumor growth, indicating a critical role for beta-adrenergic receptor signaling in mediating the effects of surgical stress on tumor growth. In the HeyA8 and SKOV3ip1 models, surgery significantly increased microvessel density (CD31) and vascular endothelial growth factor expression, which were blocked by propranolol treatment. Tumor growth after surgery was decreased in a mouse null for βAR. Levels of cytokines G-CSF, IL-1a, IL-6, and IL-15were increased after surgery

Stress effects on FosB- and interleukin-8 (IL8)-driven ovarian cancer growth and metastasis J Biol Chem. 2010 Nov 12;285(46):35462-70. doi: 10.1074/jbc.M110.109579. Epub 2010 Sep 8.

Shahzad MM1, Arevalo JM, Armaiz-Pena GN, Lu C, Stone RL, Moreno-Smith M, Nishimura M, Lee JW, Jennings NB, Bottsford-Miller J, Vivas-Mejia P, Lutgendorf SK, Lopez-Berestein G, Bar-Eli M, Cole SW, Sood AK.

Free PMC Article

Abstract

A growing number of studies indicate that chronic stress can accelerate tumor growth due to sustained sympathetic nervous system activation. Our recent findings suggest that chronic stress is associated with increased IL8 levels. Here, we examined the molecular and biological significance of IL8 in stress-induced tumor growth. Norepinephrine (NE) treatment of ovarian cancer cells resulted in a 250-300% increase in IL8 protein and 240-320% increase in its mRNA levels. Epinephrine treatment resulted in similar increases. Moreover, NE treatment resulted in a 3.5-4-fold increase in IL8 promoter activity. These effects were blocked by propranolol. Promoter deletion analyses suggested that AP1 transcription factors might mediate catecholamine-stimulated up-regulation of IL8. siRNA inhibition studies identified FosB as the pivotal component responsible for IL8 regulation by NE. In vivo chronic stress resulted in increased tumor growth (by 221 and 235%; p < 0.01) in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells. This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoliposomes. IL8 and FosB silencing reduced microvessel density (based on CD31 staining) by 2.5- and 3.5-fold, respectively (p < 0.001). Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases. These findings may have implications for ovarian cancer management.

Dopamine blocks stress-mediated ovarian carcinoma growth.

Moreno-Smith M, Lu C, Shahzad MM, Pena GN, Allen JK, Stone RL, Mangala LS, Han HD, Kim HS, Farley D, Berestein GL, Cole SW, Lutgendorf SK, Sood AK.

Clin Cancer Res. 2011 Jun 1;17(11):3649-59. doi: 10.1158/1078-0432.CCR-10-2441. Epub 2011 Apr 29.

D. Additional mechanisms iincluding JAK/STAT modulation, prostaglandin synthesis, AKT, and Slug implicated in Stress (norepinephrine) induced increase in Ovarian Tumor Growth

Sustained adrenergic signaling leads to increased metastasis in ovarian cancer via increased PGE2 synthesis.

Nagaraja AS, Dorniak PL, Sadaoui NC, Kang Y, Lin T, Armaiz-Pena G, Wu SY, Rupaimoole R, Allen JK, Gharpure KM, Pradeep S, Zand B, Previs RA, Hansen JM, Ivan C, Rodriguez-Aguayo C, Yang P, Lopez-Berestein G, Lutgendorf SK, Cole SW, Sood AK.

Oncogene. 2015 Aug 10. doi: 10.1038/onc.2015.302. [Epub ahead of print]

The antihypertension drug doxazosin suppresses JAK/STATs phosphorylation and enhances the effects of IFN-α/γ-induced apoptosis.

Park MS, Kim BR, Kang S, Kim DY, Rho SB.

Genes Cancer. 2014 Nov;5(11-12):470-9.

hTERT mediates norepinephrine-induced Slug expression and ovarian cancer aggressiveness.

Choi MJ, Cho KH, Lee S, Bae YJ, Jeong KJ, Rha SY, Choi EJ, Park JH, Kim JM, Lee JS, Mills GB, Lee HY.

Oncogene. 2015 Jun;34(26):3402-12. doi: 10.1038/onc.2014.270. Epub 2014 Aug 25.

The antihypertension drug doxazosin inhibits tumor growth and angiogenesis by decreasing VEGFR-2/Akt/mTOR signaling and VEGF and HIF-1α expression.

Park MS, Kim BR, Dong SM, Lee SH, Kim DY, Rho SB.

Oncotarget. 2014 Jul 15;5(13):4935-44.

Meeting Abstracts on the Subject

From 2007 AACR Meeting

Neuroendocrine Modulation of Signal Transducer and Activator of Transcription-3 in Ovarian Cancer

  1. Requests for reprints:
    Anil K. Sood, Departments of Gynecologic Oncology and Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1155 Herman Pressler, CPB6.3244, Unit 1362, Houston, TX 77230-1439. Phone: 713-745-5266; Fax: 713-792-7586; E-mail: asood@mdanderson.org.

Abstract

There is growing evidence that chronic stress and other behavioral conditions are associated with cancer pathogenesis and progression, but the mechanisms involved in this association are poorly understood. We examined the effects of two mediators of stress, norepinephrine and epinephrine, on the activation of signal transducer and activator of transcription-3 (STAT3), a transcription factor that contributes to many promalignant pathways. Exposure of ovarian cancer cell lines to increasing concentrations of norepinephrine or epinephrine showed that both independently increased levels of phosphorylated STAT3 in a dose-dependent fashion. Immunolocalization and ELISA of nuclear extracts confirmed increased nuclear STAT3 in response to norepinephrine. Activation of STAT3 was inhibited by blockade of the β1- and β2-adrenergic receptors with propranolol, and by blocking protein kinase A with KT5720, but not with the α receptor blockers prazosin (α1) and/or yohimbine (α2). Catecholamine-mediated STAT3 activation was not inhibited by pretreatment with an anti–interleukin 6 (IL-6) antibody or with small interfering RNA (siRNA)–mediated decrease in IL-6 or gp130. Regarding the effects of STAT3 activation, exposure to norepinephrine resulted in an increase in invasion and matrix metalloproteinase (MMP-2 and MMP-9) production. These effects were completely blocked by STAT3-targeting siRNA. In mice, treatment with liposome-incorporated siRNA directed against STAT3 significantly reduced isoproterenol-stimulated tumor growth. These studies show IL-6–independent activation of STAT3 by norepinephrine and epinephrine, proceeding through the β1/β2-adrenergic receptors and protein kinase A, resulting in increased matrix metalloproteinase production, invasion, and in vivo tumor growth, which can be ameliorated by the down-regulation of STAT3. [Cancer Res 2007;67(21):10389–96]

From 2009 AACR Meeting

Abstract #2506: Functional \#946;2 adrenergic receptors (ADRB2) on human ovarian tumors portend worse clinical outcome

Abstract

Objective: Stress hormones such as catecholamines can augment tumor metastasis and angiogenesis; however, the prevalence and clinical significance of adrenergic receptors in human ovarian cancer is unknown and is the focus of the current study. Methods: After IRB approval, paraffin-embedded samples from 137 patients with invasive epithelial ovarian carcinoma were examined for \#946;1- and \#946;2-adrenergic receptor (ADRB1 and ADRB2, respectively) expression. Correlations with clinical outcomes were determined using parametric and non-parametric tests. Survival analyses were performed using the Kaplan-Meier method. Expression of ADRB1 and -2 was examined by quantitative RT-PCR in 15 freshly extracted human ovarian carcinoma cells. Human ovarian carcinoma cells then underwent time-variable adrenergic stimulation, and tumorigenic and angiogenic cytokine levels were examined by ELISA. Results: Sixty-six percent of the tumors had high expression of ADRB1; 80% of specimens highly expressed ADRB2. Univariate analyses demonstrated that high ADRB1 expression was associated with serous histology (p=0.03) and the presence of ascites (p=0.03), while high expression of ADRB2 was associated with advanced stage (p=0.008). Moreover, high ADRB2 expression was associated with the lower overall survival (2.2 vs. 6.5 years; p<0.001). In multivariate analysis, controlling for FIGO stage, grade, cytoreduction, age, and ADRB expression, only FIGO stage, cytoreduction status, age, and ADRB status retained statistical significance in predicting overall survival. In tumor cells freshly isolated from human ovarian cancers, 75% of samples had high expression of ADRB2 while most lacked ADRB1 compared to normal surface epithelium. Stimulation of the freshly isolated ADRB2-positive human ovarian cancer cells with norepinephrine resulted in increased levels of cAMP and increased angiogenic cytokines IL-6 and VEGF. Conclusions: ADRB2 are frequently found on human ovarian tumors and are strongly associated with poor clinical outcome. These findings support a direct mechanism by which stress hormones modulate ovarian cancer growth and metastasis as well as provide a basis for therapeutic targeting.

And from the 2015 AACR Meeting:

Abstract 3368: Sustained adrenergic signaling activates pro-inflammatory prostaglandin network in ovarian carcinoma

  1. Archana S. Nagaraja1,

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA

Abstract

Purpose: Catecholamine mediated stress effects are known to induce production of various pro-inflammatory cytokines. However, the mechanism and functional effect of adrenergic signaling in driving inflammation via pro-inflammatory metabolites is currently unknown. Here we address the functional and biological consequences of adrenergic-induced Cox2/PGE2 axis activation in ovarian cancer metastasis.

Methods: We first analyzed global metabolic changes in tumors isolated from patients with known Center for Epidemiologic Studies Depression Scale (CES-D; depressive) scores and tumoral norepinephrine (NE) levels. Beta-adrenergic receptor (ADRB) positive cells (Skov3 and HeyA8) were used to study gene and protein levels of PTGS2 (cyclooxygenase2), PTGES (prostaglandin E synthase) and metabolite PGE2 in vitro and in vivo. To study tumor-specific effects on catecholamine-derived expression of PTGS2, we used a novel DOPC delivery system of PTGS2 siRNA.

Results: Our results revealed that levels of PGs were significantly increased in patients with high depressive scores (>16). PGE2 was upregulated by 2.38 fold when compared to the low CES-D scores. A similar trend was also observed with other pro-inflammatory eicosanoids, such as 6-keto prostaglandin F1 Alpha (2.03), prostaglandin A2 (1.39) and prostaglandin E1 (1.39). Exposure to NE resulted in increased PTGS2 and PTGES (prostaglandin E2 synthase) gene expression and protein levels in Skov3 and HeyA8. PGE2 ELISA confirmed that upon treatment with NE, PGE2 levels were increased in conditioned medium from Skov3 and HeyA8 cells. Treatment with a broad ADRB agonist (isoproterenol) or ADRB2 specific agonist (terbutaline) led to increases in expression of PTGS2 and PTGES as well as PGE2 levels in supernatant. Conversely, treatment with a broad antagonist (propranolol) or an ADRB2 specific antagonist (butoxamine) in the presence of NE abrogated gene expression changes of PTGS2 and PTGES. ChIP analysis showed enrichment of Nf-kB binding to the promoter region of PTGS2 and PTGES by 2.4 and 4.0 fold respectively when Skov3ip1 cells were treated with NE. Silencing PTGS2 resulted in significantly decreased migration (40%) and invasion (25%) of Skov3 cells in the presence of NE. Importantly, in the Skov3-ip1 restraint stress orthotopic model, silencing PTGS2 abrogated stress mediated effects and decreased tumor burden by 70% compared to control siRNA with restraint stress.

Conclusion Increased adrenergic stimulation results in a pro-inflammatory milieu mediated by prostaglandins that drives tumor progression and metastasis in ovarian cancer.

Citation Format: Archana S. Nagaraja, Piotr Dorniak, Nouara Sadaoui, Guillermo Armaiz-Pena, Behrouz Zand, Sherry Y. Wu, Julie K. Allen, Rajesha Rupaimoole, Cristian Rodriguez-Aguayo, Sunila Pradeep, Lin Tan, Rebecca A. Previs, Jean M. Hansen, Peiying Yang, Garbiel Lopez-Berestein, Susan K. Lutgendorf, Steve Cole, Anil K. Sood. Sustained adrenergic signaling activates pro-inflammatory prostaglandin network in ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3368. doi:10.1158/1538-7445.AM2015-3368

Other Article in This Open Access Journal on Ovarian Cancer Include

Beta-Blockers help in better survival in ovarian cancer

Ovarian Cancer Survival Increased 5 Months Overall With Beta Blockers – Study – The Speaker

Model mimicking clinical profile of patients with ovarian cancer @ Yale School of Medicine

Preclinical study identifies ‘master’ proto-oncogene that regulates stress-induced ovarian cancer metastasis | MD Anderson Cancer Center

Beta-Blockers help in better survival in ovarian cancer

Role of Primary Cilia in Ovarian Cancer

Dasatinib in Combination With Other Drugs for Advanced, Recurrent Ovarian Cancer

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Read Full Post »


Are Cyclin D and cdk Inhibitors A Good Target for Chemotherapy?

 

Curator: Stephen J. Williams, Ph.D.

The U.S. Food and Drug Administration today granted accelerated approval to Ibrance (palbociclib) to treat advanced (metastatic) breast cancer inr postmenopausal women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer who have not yet received an endocrine-based therapy. It is to be used in combination with letrozole, another FDA-approved product used to treat certain kinds of breast cancer in postmenopausal women.

See Dr. Melvin Crasto’s blog posts on the announcement of approval of Ibrance (palbociclib) at

http://newdrugapprovals.org/2015/02/05/fda-approves-ibrance-for-postmenopausal-women-with-advanced-breast-cancer/

and about the structure and mechanism of action of palbociclib

http://newdrugapprovals.org/2014/01/05/palbociclib/

 

From the CancerNetwork at http://www.cancernetwork.com/aacr-2014/cdk-inhibitors-show-impressive-activity-advanced-breast-cancer

CDK Inhibitors Show Impressive Activity in Advanced Breast Cancer

News | April 08, 2014 | AACR 2014, Breast Cancer

By Anna Azvolinsky, PhD

Ibrance structure

 

Chemical structure of palbociclib

 

 

Palbociclib and LY2835219 are both cyclin-dependent kinase (CDK) 4/6 inhibitors. CDK4 and CDK6 are kinases that, together with cyclin D1, facilitate the transition of dividing cells from the G1 to the S (synthesis) phase of the cell cycle. Preclinical studies have shown that breast cancer cells rely on CDK4 and CDK6 for division and growth, and that selective CDK4/6 inhibitors can arrest the cells at this G1/S phase checkpoint.

The results of the phase II trial of palbociclib and phase I trial of LY2835219 both indicated that hormone receptor (HR)-positive disease appears to be the best marker to predict patient response.

LY2835219 Phase I Trial Demonstrates Early Activity

The CDK4/6 inhibitor LY2835219 has demonstrated early activity in heavily pretreated women with metastatic breast cancer. Nineteen percent of these women (9 out of 47) had a partial response and 51% (24 out of 47) had stable disease following monotherapy with the oral CDK4/6 inhibitor. Patients had received a median of seven prior therapies, and 75% had metastatic disease in the lung, liver, or brain. The median age of patients was 55 years.

All of the partial responses were in patients with HR-positive disease. The overall response rate for this patient subset was 25% (9 of 36 patients). Twenty of the patients with stable disease had HR-positive disease, with 13 patients having stable disease lasting 24 weeks or more.

Despite treatment, disease progression occurred in 23% of the patients.

These results were presented at a press briefing by Amita Patnaik, MD, associate director of clinical research at South Texas Accelerated Research Therapeutics in San Antonio, Texas, at the 2014 American Association for Cancer Research (AACR) Annual Meeting, held April 5–9, in San Diego.

The phase I trial of LY2835219 enrolled 132 patients with five different tumor types, including metastatic breast cancer. Patients received 150-mg to 200-mg doses of the oral drug every 12 hours.

The overall disease control rate was 70% for all patients and 81% among the 36 HR-positive patients.

The median progression-free survival (PFS) was 5.8 months for all patients and 9.1 months for HR-positive patients. Patnaik noted that the median PFS is still a moving target, as 18 patients, all with HR-positive disease, remain on therapy.

“The data are rather encouraging for a very heavily pretreated patient population,” said Patnaik during the press briefing.

Even though the trial was not designed to compare efficacy based on breast cancer subpopulations, the results in HR-positive tumors are particularly encouraging, according to Patnaik.

Common adverse events thought to be treatment-related were diarrhea, nausea, fatigue, vomiting, and neutropenia. These adverse events occurred in 5% or less of patients at grade 3 or 4 toxicity, except neutropenia, which occurred as a grade 3 or 4 toxicity in 11% of patients. Patnaik noted during the press briefing that the neutropenia was uncomplicated and did not result in discontinuation of therapy by any of the patients.

Palbociclib Phase II Data “Impressive”

The addition of the oral CDK4/6 inhibitor palbociclib resulted in an almost doubling of PFS in first-line treatment of postmenopausal metastatic breast cancer patients with HR-positive disease compared with a control population. The patients in this trial were not previously treated for their metastatic breast cancer, unlike the patient population in the phase I LY2835219 trial.

Patients receiving the combination of palbociclib at 125 mg once daily plus letrozole at 2.5 mg once daily had a median PFS of 20.2 months compared with a median of 10.2 months for patients treated with letrozole alone (hazard ratio = 0.488; P = .0004).

Richard S. Finn, MD, assistant professor of medicine at the University of California, Los Angeles, presented the data from the phase II PALOMA-1 trial at a press briefing at the AACR Annual Meeting.

A total of 165 patients were randomized 1:1 to either the experimental arm or control arm.

Forty-three percent of patients in the combination arm had an objective response compared with 33% of patients in the control arm.

Overall survival (OS), a secondary endpoint in this trial, was encouraging but the results are still preliminary, said Finn during the press briefing. The median OS was 37.5 months in the palbociclib arm compared with 33.3 months in the letrozole alone arm (P = .21). Finn noted that long-term follow-up is necessary to establish the median OS. “This first look of the survival data is encouraging. This is a front-line study, and it is encouraging that there is early [separation] of the curves,” he said.

No new toxicities were reported since the interim trial results. Common adverse events included leukopenia, neutropenia, and fatigue. The neutropenia could be quickly resolved and was uncomplicated and not accompanied by fever, said Finn.

Palbociclib is currently being tested in two phase III clinical trials: The PALOMA-3 trial is testing the combination of palbociclib with letrozole and fulvestrant in late-stage metastatic breast cancer patients who have failed endocrine therapy. The PENELOPE-B trial is testing palbociclib in combination with standard endocrine therapy in HR-positive breast cancer patients with residual disease after neoadjuvant chemotherapy and surgery.

References

  1. Patnaik A, Rosen LS, Tolaney SM, et al. Clinical activity of LY2835219, a novel cell cycle inhibitor selective for CDK4 and CDK6, in patients with metastatic breast cancer. American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT232.
  2. Finn RS, Crown JP, Lang I, et al. Final results of a randomized phase II study of PD 0332991, a cyclin-dependent kinase (CDK)-4/6 inhibitor, in combination with letrozole vs letrozole alone for first-line treatment of ER+/HER2-advanced breast cancer (PALOMA-1; TRIO-18). American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT101.

– See more at: http://www.cancernetwork.com/aacr-2014/cdk-inhibitors-show-impressive-activity-advanced-breast-cancer#sthash.f29smjxi.dpuf

 

The Cell Cycle and Anti-Cancer Targets

 

graph_cell_cycle

 

From Cell Cycle in Cancer: Cyclacel Pharmaceuticals™ (note dotted arrows show inhibition of steps e.g. p21, p53)

For a nice video slideshow explaining a bit more on cyclins and the cell cycle please see video below:

 

Cell Cycle. 2012 Nov 1; 11(21): 3913.

doi:  10.4161/cc.22390

PMCID: PMC3507481

Cyclin-dependent kinase 4/6 inhibition in cancer therapy

Neil Johnson and Geoffrey I. Shapiro*

See the article “Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors” in volume 11 on page 2756.

See the article “CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy” in volume 11 on page 2747.

This article has been cited by other articles in PMC.

Cyclin-dependent kinases (CDKs) drive cell cycle progression and control transcriptional processes. The dysregulation of multiple CDK family members occurs commonly in human cancer; in particular, the cyclin D-CDK4/6-retinoblastoma protein (RB)-INK4 axis is universally disrupted, facilitating cancer cell proliferation and prompting long-standing interest in targeting CDK4/6 as an anticancer strategy. Most agents that have been tested inhibit multiple cell cycle and transcriptional CDKs and have carried toxicity. However, several selective and potent inhibitors of CDK4/6 have recently entered clinical trial. PD0332991, the first to be developed, resulted from the introduction of a 2-aminopyridyl substituent at the C2-position of a pyrido(2,3-d)pyrimidin-7-one backbone, affording exquisite selectivity toward CDK4/6.1 PD0332991 arrests cells in G1 phase by blocking RB phosphorylation at CDK4/6-specfic sites and does not inhibit the growth of RB-deficient cells.2 Phase I studies conducted in patients with advanced RB-expressing cancers demonstrated mild side effects and dose-limiting toxicities of neutropenia and thrombocytopenia, with prolonged stable disease in 25% of patients.3,4 In cyclin D1-translocated mantle cell lymphoma, PD0332991 extinguished CDK4/6 activity in patients’ tumors, resulting in markedly reduced proliferation, and translating to more than 1 year of stability or response in 5 of 17 cases.5

Two recent papers from the Knudsen laboratory make several important observations that will help guide the continued clinical development of CDK4/6 inhibitors. In the study by Dean et al., surgically resected patient breast tumors were grown on a tissue culture matrix in the presence or absence of PD0332991. Crucially, these cultures retained associated stromal components known to play important roles in cancer pathogenesis and therapeutic sensitivities, as well as key histological and molecular features of the primary tumor, including expression of ER, HER2 and Ki-67. Similar to results in breast cancer cell lines,6 the authors demonstrate that only RB-positive tumors have growth inhibition in response to PD0332991, irrespective of ER or HER2 status, while tumors lacking RB were completely resistant. This result underscores RB as the predominant target of CDK4/6 in breast cancer cells and the primary marker of drug response in primary patient-derived tumors. As expected, RB-negative tumors routinely demonstrated robust expression of p16INK4A; however, p16INK4A expression was not always a surrogate marker for RB loss, supporting the importance of direct screening of tumors for RB expression to select patients appropriate for CDK4/6 inhibitor clinical trials.

In the second study, McClendon et al. investigated the efficacy of PD0332991 in combination with doxorubicin in triple-negative breast cancer cell lines. Again, RB functionality was paramount in determining response to either PD0332991 monotherapy or combination treatment. In RB-deficient cancer cells, CDK4/6 inhibition had no effect in either instance. However, in RB-expressing cancer cells, CDK4/6 inhibition and doxorubicin provided a cooperative cytostatic effect, although doxorubicin-induced cytotoxicity was substantially reduced, assessed by markers for mitotic catastrophe and apoptosis. Additionally, despite cytostatic cooperativity, CDK4/6 inhibition maintained the viability of RB-proficient cells in the presence of doxorubicin, which repopulated the culture after removal of drug. These results reflect previous data demonstrating that ectopic expression of p16INK4A can protect cells from the lethal effects of DNA damaging and anti-mitotic chemotherapies.7 Similar results have been reported in MMTV-c-neu mice bearing RB-proficient HER2-driven tumors, where PD0332991 compromised carboplatin-induced regressions,8 suggesting that DNA-damaging treatments should not be combined concomitantly with CDK4/6 inhibition in RB-proficient tumors.

To combine CDK4/6 inhibition with cytotoxics, sequential treatment may be considered, in which CDK4/6 inhibition is followed by DNA damaging chemotherapy; cells relieved of G1 arrest may synchronously enter S phase, where they may be most susceptible to agents disrupting DNA synthesis. Release of myeloma cells from a prolonged PD0332991-mediated G1 block leads to S phase synchronization; interestingly, all scheduled gene expression is not completely restored (including factors critical to myeloma survival such as IRF4), further favoring apoptotic responses to cytotoxic agents.9 Furthermore, in RB-deficient tumors, CDK4/6 inhibitors may be used to maximize the therapeutic window between transformed and non-transformed cells treated with chemotherapy. In contrast to RB-deficient cancer cells, RB-proficient non-transformed cells arrested in G1 in response to PD0332991 are afforded protection from DNA damaging agents, thereby reducing associated toxicities, including bone marrow suppression.8

In summary, the current work provides evidence for RB expression as a determinant of response to CDK4/6 inhibition in primary tumors and highlights the complexity of combining agents targeting the cell cycle machinery with DNA damaging treatments.

Go to:

Notes

Dean JL, McClendon AK, Hickey TE, Butler LM, Tilley WD, Witkiewicz AK, Knudsen ES. Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors Cell Cycle 2012 11 2756 61 doi: 10.4161/cc.21195.

McClendon AK, Dean JL, Rivadeneira DB, Yu JE, Reed CA, Gao E, Farber JL, Force T, Koch WJ, Knudsen ES. CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy Cell Cycle 2012 11 2747 55 doi: 10.4161/cc.21127.

Go to:

Footnotes

Previously published online: www.landesbioscience.com/journals/cc/article/22390

Go to:

References

  1. Toogood PL, et al. J Med Chem. 2005;48:2388–406. doi: 10.1021/jm049354h. [PubMed] [Cross Ref]
  2. Fry DW, et al. Mol Cancer Ther. 2004;3:1427–38. [PubMed]
  3. Flaherty KT, et al. Clin Cancer Res. 2012;18:568–76. doi: 10.1158/1078-0432.CCR-11-0509. [PubMed] [Cross Ref]
  4. Schwartz GK, et al. Br J Cancer. 2011;104:1862–8. doi: 10.1038/bjc.2011.177. [PMC free article] [PubMed] [Cross Ref]
  5. Leonard JP, et al. Blood. 2012;119:4597–607. doi: 10.1182/blood-2011-10-388298. [PubMed] [Cross Ref]
  6. Dean JL, et al. Oncogene. 2010;29:4018–32. doi: 10.1038/onc.2010.154. [PubMed] [Cross Ref]
  7. Stone S, et al. Cancer Res. 1996;56:3199–202. [PubMed]
  8. Roberts PJ, et al. J Natl Cancer Inst. 2012;104:476–87. doi: 10.1093/jnci/djs002. [PMC free article] [PubMed] [Cross Ref]
  9. Huang X, et al. Blood. 2012;120:1095–106. doi: 10.1182/blood-2012-03-415984. [PMC free article] [PubMed] [Cross Ref]

Cell Cycle. 2012 Jul 15; 11(14): 2756–2761.

doi:  10.4161/cc.21195

PMCID: PMC3409015

Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors

Jeffry L. Dean, 1 , 2 A. Kathleen McClendon, 1 , 2 Theresa E. Hickey, 3 Lisa M. Butler, 3 Wayne D. Tilley, 3 Agnieszka K. Witkiewicz, 4 , 2 ,* and Erik S. Knudsen 1 , 2 ,*

Author information ► Copyright and License information ►

See commentary “Cyclin-dependent kinase 4/6 inhibition in cancer therapy” in volume 11 on page 3913.

This article has been cited by other articles in PMC.

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Abstract

To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.

Keywords: ER, PD0332991, breast cancer, cell cycle, ex vivo

Go to:

Introduction

Breast cancer is a highly heterogeneous disease.14 Such heterogeneity is known to influence patient response to both standard of care and experimental therapeutics. In regards to biomarker-driven treatment of breast cancers, it was initially recognized that the presence of the estrogen receptor α (ER) in a fraction of breast cancer cells was associated with the response to tamoxifen and similar anti-estrogenic therapies.5,6 Since this discovery, subsequent marker analyses and gene expression profiling studies have further divided breast cancer into a series of distinct subtypes that harbor differing and often divergent therapeutic sensitivities.13 While clearly important in considering the use of several current standard of care therapies, these markers, or molecular sub-types, do not necessarily predict the response to new therapeutic approaches that are currently undergoing clinical development. Thus, there is the continued need for functional analyses of drug response and the definition of new markers that can be used to direct treatment strategies.

Currently, all preclinical cancer models are associated with specific limitations. It is well known that cell culture models lack the tumor microenvironment known to have a significant impact on tumor biology and therapeutic response.79 Xenograft models are dependent on the host response for the engraftment of tumor cells in non-native tissues, which do not necessarily recapitulate the nuances of complex tumor milieu.10 In addition, genetically engineered mouse models, while enabling the tumor to develop in the context of the host, can develop tumors that do not mirror aspects of human disease.10 Furthermore, it remains unclear whether any preclinical model truly represents the panoply of breast cancer subtypes that are observed in the clinic. Herein, we utilized a primary human tumor explant culture approach to interrogate drug response, as well as specific determinants of therapeutic response, in an unselected series of breast cancer cases.

Cell Cycle. 2012 Jul 15; 11(14): 2747–2755.

doi:  10.4161/cc.21127

PMCID: PMC3409014

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

  1. Kathleen McClendon, 1 , † Jeffry L. Dean, 1 , † Dayana B. Rivadeneira, 1 Justine E. Yu, 1 Christopher A. Reed, 1 Erhe Gao, 2 John L. Farber, 3 Thomas Force, 2 Walter J. Koch, 2 and Erik S. Knudsen 1 ,*

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See commentary “Cyclin-dependent kinase 4/6 inhibition in cancer therapy” in volume 11 on page 3913.

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Abstract

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.

 

 

Click on Video Link for Dr. Tolaney slidepresentation of recent data with CDK4/6 inhibitor trial results https://youtu.be/NzJ_fvSxwGk

Audio and slides for this presentation are available on YouTube: http://youtu.be/NzJ_fvSxwGk

Sara Tolaney, MD, MPH, a breast oncologist with the Susan F. Smith Center for Women’s Cancers at Dana-Farber Cancer Institute, gives an overview of phase I clinical trials and some of the new drugs being tested to treat breast cancer. This talk was originally given at the Metastatic Breast Cancer Forum at Dana-Farber on Oct. 5, 2013.

A great article on current clinical trials and explanation of cdk inhibitors by Sneha Phadke, DO; Alexandra Thomas, MD at the site OncoLive

 

http://www.onclive.com/publications/contemporary-oncology/2014/november-2014/targeting-cell-cycle-progression-cdk46-inhibition-in-breast-cancer/1

 

cdk4/6 inhibitor Ibrance Has Favorable Toxicity and Adverse Event Profile

 

As discussed in earlier posts and the Introduction to this chapter on Cytotoxic Chemotherapeutics, most anti-cancer drugs developed either to target DNA, DNA replication, or the cell cycle usually have similar toxicity profile which can limit their therapeutic use. These toxicities and adverse events usually involve cell types which normally exhibit turnover in the body, such as myeloid and lymphoid and granulocytic series of blood cells, epithelial cells lining the mucosa of the GI tract, as well as follicular cells found at hair follicles. This understandably manifests itself as common toxicities seen with these types of agents such as the various cytopenias in the blood, nausea vomiting diarrhea (although there are effects on the chemoreceptor trigger zone), and alopecia.

It was felt that the cdk4/6 inhibitors would show serious side effects similar to other cytotoxic agents and this definitely may be the case as outlined below:

(Side effects of palbociclib) From navigatingcancer.com

Palbociclib may cause side effects. Tell your doctor if any of these symptoms are severe or do not go away:

  • nausea
  • diarrhea
  • vomiting
  • decreased appetite
  • tiredness
  • numbness or tingling in your arms, hands, legs, and feet
  • sore mouth or throat
  • unusual hair thinning or hair loss

Some side effects can be serious. If you experience any of these symptoms, call your doctor immediately or get emergency medical treatment:

  • fever, chills, or signs of infection
  • shortness of breath
  • sudden, sharp chest pain that may become worse with deep breathing
  • fast, irregular, or pounding heartbeat
  • rapid breathing
  • weakness
  • unusual bleeding or bruising
  • nosebleeds

The following is from FDA Drug Trials Snapshot of Ibrance™:

 

See PDF on original submission and CDER review

original FDA Ibrance submission

original FDA Ibrance submission

CDER Review Ibrance

CDER Review Ibrance

 

4.3 Preclinical Pharmacology/Toxicology

 

For full details, please see Pharmacology/Toxicology review by Dr. Wei Chen The nonclinical studies adequately support the safety of oral administration of palbociclib for the proposed indication and the recommendation from the team is for approval. Non-clinical studies of palbociclib included safety pharmacology studies, genotoxicity

studies, reproductive toxicity studies, pharmacokinetic studies, toxicokinetic studies and repeat-dose general toxicity studies which were conducted in rats and dogs. The pivotal toxicology studies were conducted in compliance with Good Laboratory Practice regulation.

 

Pharmacology:

As described above, palbociclib is an inhibitor of CDK4 and CDK6. Palbociclib modulates downstream targets of CDK4 and CDK6 in vitro and induces G1 phase cell cycle arrest and therefore acts to inhibit DNA synthesis and cell proliferation. Combination of palbociclib with anti-estrogen agents demonstrated synergistic inhibition

of cell proliferation in ER+ breast cancer cells. Palbociclib showed anti-tumor efficacy in animal tumor model studies. Safety pharmacology studies with palbociclib demonstrated adverse effects on both the respiratory and cardiovascular function of dogs at a dose of 125mg/day (four times and 50-times the human clinical exposure

respectively) based on mean unbound Cmax.

 

General toxicology:

Palbociclib was studied in single dose toxicity studies and repeated dose studies in rats and dogs. Adverse effects in the bone marrow, lymphoid tissues, and male reproductive organs were observed at clinically relevant exposures. Partial to complete reversibility of toxicities to the hematolymphopoietic and male reproductive systems was demonstrated following a recovery period (4-12 weeks), with the exception of the male reproductive organ findings in dogs. Gastrointestinal, liver, kidney, endocrine/metabolic (altered glucose metabolism), respiratory, ocular, and adrenal effects were also seen.

 

Genetic toxicology:

Palbociclib was evaluated for potential genetic toxicity in in vitro and in vivo studies. The Ames bacterial mutagenicity assay in the presence or absence of metabolic activation demonstrated non-mutagenicity. In addition, palbociclib did not induce chromosomal aberrations in cultured human peripheral blood lymphocytes in the presence or absence of metabolic activation. Palbociclib was identified as aneugenic based on kinetochore analysis of micronuclei formation in an In vitro assay in CHO-WBL cells. In addition, palbociclib was shown to induce micronucleus formation in male rats at doses 100

mg/kg/day (10x human exposure at the therapeutic dose) in an in vivo rat micronucleus assay.

 

Reproductive toxicology: No effects on estrous cycle and no reproductive toxicities were noticed in standard assays.

 

Pharmacovigilance (note please see PDF for more information)

Deaths Associated With Trials: Although a few deaths occurred during some trials no deaths were attributed to the drug.

Non-Serious Adverse Events:

(note a reviewers comment below concerning incidence of pulmonary embolism is a combination trial with letrazole)

 

fda ibrance reviewers SAE comment

 

Other article in this Open Access Journal on Cell Cycle and Cancer Include:

 

Tumor Suppressor Pathway, Hippo pathway, is responsible for Sensing Abnormal Chromosome Numbers in Cells and Triggering Cell Cycle Arrest, thus preventing Progression into Cancer

Nonhematologic Cancer Stem Cells [11.2.3]

New methods for Study of Cellular Replication, Growth, and Regulation

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com

In Focus: Targeting of Cancer Stem Cells

 

 

 

 

 

 

 

 

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