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Cellular Senescence and Cancer

December 1, 2002

Chaired By

 Ronald DePinho, MD of Harvard Medical School
 Charles Sherr, MD, PhD of St Jude Children’s Research Hospital

Meeting Description

The topic of the 2002 Forbeck Forum pertains to the growth abnormalities that are characteristic of all types of tumors. Normal cells have limited life spans while tumor cells acquire genetic changes that result in immortalization and infinite growth capacity. Studies in the past decade have begun to unravel the molecular mechanisms responsible for this uncontrolled growth. These studies have provided many insights into how cancers start since the genes that cause this unrestricted growth are so commonly mutated in human cancers. In addition, since normal cells in the body are not immortalized, blocking these immortalizing steps could be selectively toxic to tumor cells. Thus, identification of proteins in cells responsible for keeping tumor cells growing indefinitely also provides potential novel targets for the design of new therapeutic agents in the treatment of cancer. Such agents might be broadly useful in a wide range of cancers. Discussions at this forum focused on what is known about the molecular mechanisms of cellular immortalization that leads to the development of tumor cells and will extend to concepts of how this information can be used to prevent or better treat cancers.

Meeting Summary

This exciting and highly productive Forum focused on cellular senescence – a biological response governed by known cancer-relevant pathways and thought to be integral to the suppression of cancer and the response to anti-cancer agents. Investigators from diverse areas discussed the cellular senescence mechanism from the molecular, cellular and organismal perspectives.

Numerous outstanding questions were discussed including: Does senescence represent an effective mammalian tumor suppressor mechanism on one hand yet drives the age related pathologies on the other? Are there species-specific differences in mice and humans or does this relate to experimental design? What role does the telomere plays in suppressing or fueling chromosomal instability, and how does this influence the initiation and progress of cancer in the organism? What are the natures of the signals emanating from the telomere and how is this signal mediated by damage signaling pathways in normal and neoplastic cells? How is telomerase regulated? How does cellular senescence pathways influence the biological impact of oncogenic lesions such as Myc, and can we forge a link to the core cell cycle machinery? A discussion of these issues generated more questions than answers and the level of discussion was so robust that most speakers found it challenging to get past the first few slides of their talk.

Dr. Judith Campisi of Lawrence Berkeley National Laboratory focused on the issue of the cellular senescence is an example of evolutionary antagonistic pleiotropy and presented evidence that cellular senescence in cultured cells is driven by a process linked to accumulated oxidative stress. She also reviewed the evidence that strongly suggests that senescent cells accumulate in normal tissues, and that these cells may provide a permissive microenvironment for epithelial carcinogenesis. Such senescent microenvironment cells secrete proteases among other factors that have been linked to tumor progression.

Dr. Titia de Lange of the Rockefeller University discussed how abnormal telomere structure activates a senescent response in mouse and human cells. She emphasized that, although there is general agreement on the involvement of p53 in the senescence signaling pathway, the data are less clear on the role of p16. Furthermore, the telomere damage signaling pathway appears to be different in human and mouse cells as reflected in the response of these cultured cells to alterations in telomere structure brought about by the expression of aberrant telomere binding proteins.

Dr. Ron DePinho of the Dana- Farber Cancer Institute and Harvard Medical School presented his work on engineered mice harboring defective tumor suppressor pathways and critically shortened telomeres and how such alterations impact on processing of aging and cancer. He presented data showing that advancing age, telomere attrition, and accompanying genomic instability cooperate to compromise the overall health and well-being of mammals on the level of tissue stem cells. The telomerase knockout mouse has provided a model to dissect the complex role of telomeres in cancer pathogenesis. Cancer, particularly epithelial carcinomas, is among the most common aspects of aging in humans and telomere erosion has been cited as a risk factor in the genesis of certain human tumor types. In line with this view, late generation mTERC null mice exhibit an age- and generation dependent increase in cytogenetic abnormalities and a significant increase in the incidence of spontaneous cancers. To examine the genetic interactions between telomere dysfunction and key checkpoint pathways in relation to tumor formation, the cellular response to telomere dysfunction was examined against the backdrop of various tumor suppressor mutations. In p53-/- (but not Ink4a/Arf or Atm-/-) mice, carcinomas emerged as the largest class of clinically apparent tumors, greatly exceeding sarcomas and lymphomas. These epithelial cancers emerged with complex cytogenetic profiles similar to that seen in human carcinomas, pointing to telomere dysfunction as a mechanism driving chromosomal instability. Acquisition of fully malignant phenotype including metastases may require telomerase-mediated telomere maintenance as a late event in the evolution of these cancers.

Dr. Steve Elledge of Baylor College of Medicine provided an overview of the cellular response to double strand breaks in DNA. He discussed the relationship of this response to the process of telomere erosion that occurs when cells have undergone extensive proliferation, such as during the early stages of tumorigenesis. He then discussed how cells can overcome the defects in telomere erosion by inducing the catalytic subunit of human telomerase and described a number of genes he recently identified in genetic screens that repress telomerase expression in normal cells. Many of these were linked to tumor suppressor pathways, furthering the link between telomerase and cancer.

Dr. Gerard Evan of University of California presented his elegant mouse models of cancer designed to understand the immense complexity of the tumor phenotype and the underlying genotype. In particular, he delineated the issues surrounding the concept of tumor establishment and maintenance. He focused on the possibility that cancers require only a very restricted complement of interlocking mutations but which can only be acquired by a circuitous and protracted evolutionary process. A case in point is the Myc oncoprotein. Deregulation of expression of the c-Myc protein represents an archetypical proliferation-deregulating oncogenic lesion found in most human cancers. However, the potent pro-apoptotic activity of c-Myc means that its deregulation can only be accommodated in cells in which cell death is being potently suppressed. They have explored the mechanisms by which c- Myc induces apoptosis and identified a novel p53-independent pathway by which c-Myc directly influences mitochondrial integrity. To explore the role of c-Myc-induced apoptosis in limiting c-Myc oncogenesis in vivo, they constructed mice that harbor a switchable c- Myc protein targeted to specific tissues. These animals allow the direct examination in vivo of both the immediate and the delayed consequences of acute activation of the c-myc oncogene in different somatic settings. Activation of c-Myc targeted to pancreatic ß cells using the insulin promoter rapidly (16 hours) induces ~100% sustained ß cell proliferation in all islets, in the absence of any other growth-promoting lesion. However, such ß cell proliferation is accompanied by overwhelming apoptosis that rapidly leads to islet involution and concomitant acute diabetes. The clear implication is that ß cell neoplasia cannot arise without early suppression of apoptosis. To confirm the predicted oncogenic synergy between c-myc and suppression of apoptosis in ß cells, we co-expressed the anti-apoptotic Bcl-xL protein in ß cells together with switchable c-Myc. In this case, activation of c- Myc triggers rapid, progressive and inexorable ß cell neoplasia that is immediately accompanied by profound angiogenesis and local invasion. Similar oncogenic results are obtained upon activation of c- Myc in ß cells lacking the tumor suppressors p19ARF or p53, although each type of anti-apoptotic lesion has its own distinct suite of attendant characteristics. Thus, inhibition of c-Myc induced apoptosis, either through the mitochondrial or ARF/p53 pathway, is sufficient to enable Myc to induce a state resembling full malignancy. Subsequent deactivation of the switchable Myc oncoprotein triggers rapid and complete regression of all ß cell adenomas, indicating that Myc is required both to induce and to maintain the neoplastic state. He concluded with an outline of the implications of these data both for our understanding of the evolution of tumors and for identification of useful therapeutic targets.

Dr. William Hahn of the Dana Farber Cancer Institute presented a provocative series of results showing that the rate limiting, telomerase catalytic subunit, hTERT, is expressed in primary, pre-senescent human fibroblasts, previously believed to lack hTERT and telomerase expression, during transit through the cell cycle. Disruption of this expression of telomerase inhibits cell proliferation, induces early entry into replicative senescence and alters the maintenance of the 3’ single-stranded telomeric overhang. These observations support the view that telomerase and telomere structure is dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.

Dr. Gregory Hannon of Cold Spring Harbor Laboratory provided exciting new insights on RNAi in mammalian cells that build on the use of biochemical systems from Drosophila and genetic studies in plants and invertebrates. Together, these efforts have begun to reveal a mechanistic basis for RNA interference and related phenomena. The canonical model involves a two-step mechanism that includes an RNAseIII family nuclease, Dicer, which initiates RNAi by processing dsRNA silencing triggers into small RNAs of ~22 nt in length. These enter an effector complex RISC, which seeks out and degrades homologous substrates. Genetic studies of Dicer-null animals (i.e., C. elegans) have suggested roles for the RNAi machinery in the regulation of endogenous genes. Specifically, Dicer and components of the RISC complex have been implicated in processing of and in gene regulation by endogenously encoded small hairpin RNAs, known collectively as microRNAs (miRNAs). The Hannon group exploited these observations to test the possibility that miRNAs might be remodeled to regulate genes of interest. They demonstrated that expression of shRNAs from RNA polymerase III or RNA polymerase II promoters results in silencing of homologous genes and have recently extended these findings to living animals. They continue to pursue parallel paths toward a deeper understanding of the underlying mechanism of RNAi and toward expanding the applications of RNAi as a tool for investigating gene function in mammals.

Dr. Jacqueline Lees of MIT Center for Cancer Research reviewed the complexity of the RB-E2F signaling network in normal and cancer cells. Through the analysis of various E2f mutant mouse strains and the resultant E2F-deficient mouse embryonic fibroblasts (MEFs) has demonstrated that E2F3 as a key inducer of cellular proliferation. E2F3 acts in a dose-dependent manner to induce the cell cycle dependent expression of almost all known E2F-responsive genes. The reduced expression of one or more of these E2F3-regulated genes delays passage through the G1/S transition and significantly reduces the rate of DNA synthesis. As a result, E2F3-deficient MEFs have a major defect in their ability to reenter the cell cycle in response to mitogen stimulation and proliferate at a markedly reduced rate relative to wild-type controls. They have identified additional changes in the E2F3-deficient cells that are typically associated with cellular stresses such as senescence and oncogenic challenge. These include increased expression of p16INK4A, p21CIP1 and p19ARF. The increased expression of p19ARF is particularly surprising given the prevailing view that p19ARF is an E2Fresponsive gene. In addition, p53 appears to be activated in the E2F3-deficient cells, revealing significant cross-talk between the E2F and the p53 pathways. We have generated E2f3:p19ARF, E2f3:p16INK4A and E2f3:p53 compound mutant mice to investigate how this interplay is mediated and how it contributes to both cell cycle control and tumorigenesis. She presented considerable progress in establishing the properties of the double mutant MEFs (DKOs). The homozygous mutation of either p19ARF or p16INK4A does not affect either the up-regulation of p21CIP1 or the proliferation defect of the E2F3-deficient cells. In contrast, the inactivation of p53 specifically suppresses the inappropriate expression of p21CIP1, but not p16INK4A or p19ARF, indicating that the changes in p21 expression are predominantly p53- dependent. Surprisingly, the inactivation of p53 and resulting reduction in p21 levels did not suppress the proliferation defect of the E2F3-deficient cells. Moreover, E2f3:p19ARF, E2f3:p16INK4A and E2f3:p53 DKOs that have been transformed with rasV12 show similar impaired proliferation when tested in either focus formation or soft agar colony assays. Importantly, however, E2F3-loss did not affect the well-documented, immortalized phenotypes of the p19ARF, p16INK4A and p53 mutant MEFs. She therefore concluded that p19ARF, p16INK4A or p53 act upstream of E2F3 in the control of cellular proliferation but downstream of E2F3 in the control of cellular senescence. Her program continues to investigate how E2F3-loss results in the activation of p53 and the induced expression of p16INK4A and p19ARF.

Dr. Scott Lowe of the Cold Spring Harbor Laboratory presented the idea that the process of cellular senescence can be conceptualized much like apoptosis, in that both processes involve defined programs that can eliminate damaged cells following stress. With this analogy in mind, Dr. Lowe discussed several issues, including: (1) factors that influence the decision to apoptose or senesce; 2) how different stress signals are integrated into a common arrest program; and (3) the molecular mechanisms that drive senescent cells into an apparently irreversible cell cycle arrest. He also discussed how disruption of specific nodes in tumor suppressor networks can influence cellular responses to cancer chemotherapy. In particular, Lowe and colleagues have shown that a senescence program controlled by p53 and p16INK4a contributes to treatment outcome in vivo, and that these proteins act in a cooperative manner to engage the cell-cycle arrest program. In parallel, they have also identified an important role for the Rb tumor suppressor in the process by which senescent cells are maintained in a state that is non-responsive to mitogens.

Dr. Jerry Shay of the UT Southwestern Medical Center used the example of neuroblastoma (IVS) to discuss the relationship between telomeres, senescence and telomerase. Two important concepts emerged, first telomerase is not needed for the initiation of cancer but a mechanism to maintain telomeres is required for the long term progression of all human cancers. Other topics covered by Dr. Shay included: 1. Is progressive telomere shortening in human cells a mechanism to prevent cancer or a cancer-initiating mechanism leading to increased genomic instability? 2. What is the evidence that telomere shortening is important in chronic human diseases leading to increased susceptibility to cancer? 3. What are the most promising approaches for inhibiting telomerase for use in cancer therapeutics? 4. What is the evidence for alternatives to telomerase for telomere-maintenance and how can we investigate these mechanisms? In particular, he reviewed evidence that cell grown in typical culture conditions are exposed to a continuous state of oxidative stress that may not be representative of cells in vivo. This oxidative stress leads to increased ROS that may be sensed by p16 leading to a growth arrest state that mimics replicative senescence. Many “stressors”, including oligonucleotide-based cancer therapies, ectopic over-expression of genes, irradiation, as well as inadequate culture conditions can induced this premature senescence (stasis, culture shock) state. He emphasized that these effects have largely been misinterpreted as fundamental biological mechanisms involved in regulating cellular senescence that are telomere length independent. He articulated the view that the analysis of the mechanisms of signal transduction, regulation of gene expression, proliferation, senescence and death may be compromised by the failure to consider the environment in which the cells are propagated.

Dr. Charles Sherr of St Jude Children’s Research Hospital discussed the family of INK4 protein in cell cycle regulation and cancer suppression. He reported that, among the family of four INK4 proteins, only one (p16INK4a) has been frequently linked to cellular senescence and tumor suppression. In mice, the four proteins are expressed in different patterns, with p18INK4c and p19INK4d being detected during development in utero, and with p16INK4a and p15INK4b being induced in cultured cells in response to stress, and in mice in haphazard patterns as they age. The so-called “Rb pathway” (p16 — cyclin D/Cdks — Rb family) is not linear, because p27 and p21 are normally sequestered into cyclin D/Cdk complexes and these Cdk inhibitors are “mobilized” by INK4 proteins to block cyclin E/Cdk2 and cyclin A/Cdk2 activity. Finally, both INK4a and ARF are actively suppressed during embryonic development.

Although ARF is an E2F-responsive gene, it is not periodically expressed during the cell cycle, implying that its promoter is insulated from responding to transient signals. Although some have suggested that Myc and E2F1 regulate apoptosis by inducing ARF (and even that Myc may depend upon E2F1 for this activity), our unpublished results reveal that Myc-induced apoptosis does not require E2F1 activity.

Dr. Maarten van Lohuizen of the Netherlands Cancer Institute presented his studies on the regulation of INK4a/ARF tumor suppressor locus in normal and neoplastic cells. The INK4a/ARF tumor suppressors are now well established as an important cancer prevention mechanism by halting cell cycle progression upon different kinds of stress signals, such as activation of oncogenes. The INK4a/ARF locus is under stringent control (strongly repressed in normal cells) and there is evidence for different ‘threshold levels’ for Arf in mediating ‘stasis/senescence’ arrest and suppression of oncogenic transformation. Therefore, important questions to be answered further are how is the INK4a/ARF locus regulated and do we know all the downstream effectors of INK4a/ARF signaling? He and his colleagues approached these issues by generating INK4a/Arf reporter constructs and by developing genetic screens in primary cells to bypass stasis/senescence. Another relatively unexplored issue of interest is a possible role for the INK4a/ARF fail-safe during development. Such connections are suggested by the transcriptional regulation of INK4a/ARF by developmental regulators such as Polycomb repressors and TBX2/3. These connections are of special interest to cancer in light of the emerging role for Polycomb repressors in controlling the balance between differentiation and proliferation (self renewal) of precursor cells/stem cells.

Dr. Karen H. Vousden reviewed the p53 response indicating that cell cycle arrest and apoptosis reflect separable functions of p53 that can be controlled independently. Several mechanisms that contribute to the choice of response induced by p53 have been identified, including differential expression of cell cycle arrest and apoptotic target genes, and selective inhibition of expression of cell cycle arrest targets. p53 mediated induction of proteins with anti-apoptotic activity may also contribute to the overall choice of response. The apoptotic activity of p53 is key for tumor suppression, and is likely to be an effective activity to target for restoration by new therapies. It is therefore of interest to understand the mechanisms that determine the outcome to p53 activation. A therapeutic approach in tumors that retain wild type p53 is to inhibit HDM2 - the ubiquitin ligase for p53 - and so stabilize p53. It remains unclear, however, how tumor cells will respond to such treatment. Small molecules with this activity would, to some extent, mimic the function of HMD2-binding proteins like ARF and L11. Interestingly, their preliminary work has suggested that p53 responses induced by ARF and L11 are not the same, and understanding the basis for these differences will contribute to the development of new therapeutics.

In closing, the Forum resulted in an extremely robust exchange among the participants. Several attendees remarked on how effective the Forum was in communicating new unpublished information and ‘out-of-the-box’ thinking that will no doubt stimulate new lines of basic research and new opportunities for cancer intervention. The regulation of telomeres and telomerase play a critical role in determining genomic stability and replicative lifespan. The work discussed at the forum will encourage the participants to re-investigate some of the paradigms that describe how telomere maintenance regulates replicative senescence. Further studies promise to incorporate observations not easily explained by prior models of telomere function and to help define how this knowledge can be exploited to better understand and treat cancer.

Forum Participants

Alison Bertuch, MD, PhD
Baylor College of Medicine
 Forbeck Scholar

Judith Campisi, PhD
Lawrence Berkeley National Laboratory

Titia de Lange, PhD
Rockefeller University

Ronald DePinho, MD
Harvard Medical School

Steven Elledge, PhD
Baylor College of Medicine

Gerrard Evan, PhD
University of California San Francisco

William Hahn, MD
Harvard Medical School

Gregory Hannon
Cold Spring Harbor Laboratory

Jan Karlseder, PhD
The Salk Institute
 Forbeck Scholar

Jacqueline A. Lees
MIT Center for Cancer Research

Scott M. Lowe, PhD
Cold Spring Harbor Laboratory

Masashi Narita, MD, PhD
Cold Spring Harbor Laboratory
 Forbeck Scholar

Jerry W. Shay, PhD
UT Southwestern Medical Center

Charles Sherr, MD, PhD
St Jude Children's Research Hospital

Maarten van Lohuizen
The Netherlands Cancer Institute

Karen Vousden, PhD
National Cancer Institute