Apoptosis and Cancer: Basic Mechanisms and Therapeutic Opportunities in the Postgenomic Era1

[CANCER RESEARCH 63, 263–268, January 1, 2003] Meeting Report

Frank Stenner-Liewen2 and John C. Reed University of the Saarland, Hamburg/Saar, D 66424, Germany [F. S-L.] and The Burnham Institute, La Jolla, California 92037 [J. C. R.]

Death in Paradise. Apoptosis researchers gathered on the Island of genes implicated in apoptosis regulation is reflective of the complex Hawaii for an AACR Special Conference devoted to apoptosis and biology of the multicellular mammalian organisms and suggests that cell cancer. The meeting was organized by John C. Reed of the Burnham type and tissue-specific mechanisms have evolved for achieving exquis- Institute and Scott Lowe of the CSHL3 and was designed to explore itely precise control over cell life and death decisions. This abundant the latest developments in understanding of apoptosis mechanisms of diversity of apoptosis-relevant genes also suggests opportunities for care- relevance to tumor biology. fully fine tuning therapeutic strategies designed to either enhance or Dysregulation of apoptosis occurs commonly in cancers and has been inhibit apoptosis for amelioration of disease. Dr. Reed illustrated a few implicated in many events relevant to the pathogenesis and progression of examples of how improved understanding of apoptosis mechanisms has tumors, including: (a) cell accumulation caused by failure of normal cell begun to create opportunities for drug discoveries and new diagnostics for turnover mechanisms (programmed cell death); (b) creating a permissive cancer, including: (a) small molecule antagonists of antiapoptotic Bcl-2 environment for genetic instability and oncogene activation, events that family proteins that mimic endogenous inhibitors of these proteins might otherwise kill cells; (c) promoting resistance to immune cell attack; (BH3 peptides); (b) inhibitors of IAP family proteins based on mimics of (d) contributing to resistance to the cytotoxic effects of chemotherapy and SMAC and other endogenous antagonists of these antiapoptotic proteins; and radiation; (e) allowing for tumor cell survival in the face of hypoxia, a (c) new antiapoptotic genes whose expression correlates with risk of relapse topic of relevance to antiangiogenesis-based therapies for cancer; and (f) after local therapy (surgery or radiotherapy) for early stage solid tumors. Dr. Hengartner’s presentation focused on his recent work studying fostering tumor metastasis by allowing cells to survive in a detached the response of germ cells in C. elegans to X-irradiation. Although the (suspended) state. Although much information has been gained about the somatic cells of the nematode are strikingly resistant to X-irradiation, molecules underlying the phenomenon of apoptosis in the past decade, the germ cells undergo apoptosis in response to genotoxic injury, new discoveries continue to teach us that we have much yet to learn, providing a genetically retraceable model system for understanding particularly where mechanisms of tumor resistance to apoptosis are con- mechanisms that link DNA damage to apoptotic responses. In re- cerned and with respect to new strategies for restoring apoptosis sensitivity. sponse to X-irradiation, germ cells of C. elegans up-regulate expres- Comparative Genomics. The opening evening of presentations fo- sion of EGL1, a BH3 only proapoptotic protein and antagonist of the cused on apoptosis from a genomics perspective. Lectures were given by Bcl-2 homologue CED9. A screen for worms in which germ cells Drs. John Reed (Burnham Institute), Michael Hengartner (University of failed to undergo apoptosis after X-irradiation revealed a requirement Zurich), and John Abrahms (University of Texas, Southwestern) describ- for the HUS1 gene. The HUS1 protein accumulates in nuclei after ing mechanisms of apoptosis in Homo sapiens, Caenorhabditis elegans, DNA damage, localizing to chromosomes and participating in a and Drosophila melanogaster, respectively. Dr. Reed organized the hu- multiprotein complex that includes RAD1, RAD9, and possibly man genes relevant to apoptosis according to their domain families, RAD17. HUS1-deficient animals failed to up-regulate EGL1 mRNA introducing the caspases, CARD, death domain, death effector domain, in their germ cells after X-irradiation. Ablation of EGL1 reduces but Bcl-2, and IAP family proteins. Over 100 genes belonging to these does not abolish X-irradiation-induced apoptosis in germ cells of C. protein domain families have been recognized in the human genome, elegans, however, suggesting that EGL1 represents only one of the suggesting that a minimum of 0.25% of all recognized human genes is genes relevant to apoptosis induction under these circumstances. devoted to regulation of programmed cell death. The diversity of human Interestingly, the worm homologue of p53 is relevant to this pathway and is required for X-irradiation-induced apoptosis but not cell cycle Received 5/8/02; accepted 10/31/02. arrest in germ cells of C. elegans adults. In addition, ablation of p53 The costs of publication of this article were defrayed in part by the payment of page in C. elegans does not prevent induction of EGL1 mRNA in germ charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cells in response to X-irradiation. Thus, the targets of p53 which link 1 This conference, organized by the American Association for Cancer Research, was DNA damage to apoptosis induction remain to be elucidated. held February 13–17, 2002, in Waikoloa, Hawaii. John Abrahms (University of Texas, Southwestern) reviewed the cen- 2 To whom requests for reprints should be addressed, at the University of the Saarland, Innere Medizin I, Geb. 40, 66424 Hamburg/Saar, Germany. Phone: 49-6841-1623013; tral role of IAP family proteins in the regulation of cell death in the fruit E-mail: [email protected]. fly D. melanogaster. IAPs represent a family of antiapoptotic proteins 3 The abbreviations used are: CSHL, Cold Spring Harbor Laboratory; VDAC, voltage- dependent anion channel; IAP, inhibitor of apoptosis; SKL, Sickle; RPR, Reaper; TRAIL, that directly bind and inhibit certain caspase family cell death proteases. tumor necrosis factor-related apoptosis-inducing ligand; TNF, tumor necrosis factor; The IAPs, in turn, are antagonized by several proapoptotic IAP-binding DIAP, Drosophila inhibitor of apoptosis protein; INK4a, inhibitor of CDK4; DAXX, proteins, including RPR, Grim, and Hid in the fly. The genes encoding death-associated protein; PML, promyelocytic leukemia protein; PODs, PML oncogenic domains; BAX, Bcl associated X protein; EGL1, egg-laying defective 1 protein; OMI, these IAP antagonists are all located near each other on the same chro- mitochondrial serine protease HtrA2/Omi; BIR, baculovirus IAP repeat; FLIP, FLICE mosome. The human genome contains functionally homologous genes, inhibitory proteins; ATM, atxia telangiectasia mutated; SMAC, second mitrochondria- but humans and flies use different strategies for achieving regulation of derived activator of caspase; CARD, caspase activation and recruitment domain; UCSD, University of California, San Diego; NF-␬B, nuclear factor-␬B; NACHT, nucleotide- IAPs by their respective antagonists. In Drosophila, expression of IAP binding domain; IKK, I␬B kinase; LRR, leucine-rich repeat; AR, androgen receptor; ER, antagonists such as RPR, Grim, and Hid is inducible, placing the bulk of estrogen receptor; LPS, lipopolysaccharide; ARF, ADP ribosylation factor; RB, retino- blastoma; LBD, ligand-binding domain; POD, PML oncogenic domain; XIAP, X-linked regulation at a transcriptional level. In contrast, regulation of IAP antag- inhibitor of apoptosis protein; HLF, hepatic leukemia factor. onists in humans occurs at a post-transcriptional level, where these 263

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2003 American Association for Cancer Research. APOPTOSIS AND CANCER proteins are initially sequestered in mitochondria, undergoing release into of apoptosis in vitro and in animal models. The tat-BH4 peptides, e.g., the cytosolic in response to specific types of apoptotic stimuli. However, reduce apoptosis in the small intestine after X-irradiation of mice, sug- Dr. Abrahms also presented evidence suggesting a new facet to cell death gesting possible therapeutic opportunities. induction by IAP antagonists. At least some of the IAP antagonists have Dr. Thompson (University of Pennsylvania) addressed the connec- an additional function which allows them to target mitochondria, pro- tion between mitochondria, metabolism, and the mechanism of the moting cell death through an undefined mechanism. It could be that Bcl-2 family genes. He reviewed evidence that on growth factor Drosophila IAP antagonists such as RPR interact with Bcl-2 family withdrawal from factor-dependent lymphoid cells, rates of glucose proteins on the surface of mitochondria, promoting changes in mitochon- transport into cells decline, along with reductions in the rate of drial membrane permeability which unmask caspase activators, thereby glycolysis, ATP levels, and oxygen consumption. Cells overexpress- triggering mitochondria-dependent pathways for cell death. Additional ing Bcl-XL display marked resistance to growth factor deprivation as work on this interesting facet of IAP antagonist function will likely well as glucose deprivation. These cells suffer less severe reductions provide new insights into the similarities and differences in apoptosis in intracellular ATP concentrations and can survive for days to weeks regulation in flies versus humans. in the absence of glucose. Similarly, a double knockout of genes Among the transcription factors which induce expression of IAP encoding proapoptotic Bax and Bak results in murine cells which can antagonist RPR is p53. During screens for p53-inducible genes, Dr. survive for weeks without nutrients in culture. Dr. Thompson argued Abrahms discovered a new IAP antagonist called SKL. This new protein that defects in the cell death pathway controlled by Bcl-2 family has been recently identified through alternative strategies by several proteins can allow cancer cells to survive under suboptimal condi- groups and is encoded by a gene adjacent to the chromosomal interval tions. He proposed that an alternative mechanism for promoting tumor that encodes RPR, Hid, and Grim. The SKL protein contains a conserved cell growth, thus, would be to simply increase nutrient uptake and N-terminal motif which is responsible for binding IAPs. The SKL gene proposed that dysregulation of the PTEN/AKT pathway represents contains a p53-binding site motif similar to that found in the RPR gene. one such opportunity for tumor cells. Dysregulation of AKT activity SKL is among 12 additional genes that are induced by X-irradiation in a results in increased production of the GLUT-1 glucose transporter and p53-dependent manner. Analysis of these other p53-inducible genes is marked increases in the rate of glycolosis. An intriguing idea is that likely to provide new insights into mechanisms of genotoxic stress responses. this alteration in metabolic pathways found in some cancer cells might Bcl-2 Family Proteins. A session devoted to inhibition of death somehow be exploited for therapeutic purposes. included three talks on Bcl-2 family proteins by Drs. Susan Corey Dr. Douglas Green (La Jolla Institute of Allergy & Immunology) (Walter & Elisa Hall Institute), Yoshihide Tsujimoto (Osaka Univer- used the Drosophila Snyder cells (S2) in conjunction with siRNA to sity), and Craig Thompson (University of Pennsylvania), as well as a study the requirements for certain genes during apoptosis induced by presentation by Dr. Douglas Green (La Jolla Institute for Allergy & UV radiation, etoposide, and other cell death stimuli. Using siRNA to Immunology) on mechanisms of mitochondria-dependent cell death. ablate expression of cytochrome c in these cells, Dr. Green provided Dr. Corey’s presentation focused on the BH3-only proapoptotic pro- evidence that, at least in Drosophila cells, cytochrome c is not tein Bim, an antagonist of Bcl-2. She presented evidence from Bim required for apoptosis. This finding suggests important differences in knockout mice suggesting that Bim is a tumor suppressor in vivo. the apoptosis mechanisms of flies and humans. Dr. Green speculated Mating Myc/IgH mice with Bim knockouts results in faster onset of that determinants exposed on the surface of mitochondria in Drosoph- lymphomas. Bim knockout mice also accumulate more T and B ila cells may substitute for cytochrome c, allowing for activation of lymphocytes, as well as myeloid cells, and have increased levels of the Apaf homologue Dark and triggering caspase activation. circulating immunoglobulins attributable to excessive numbers of Apoptosis and Genotoxic Injury. Several presentations addressed plasma cells. Cells from these animals display increased resistance to mechanisms of cellular responses to chemotherapy and radiation, a topic certain apoptotic stimuli, including microtubule-binding anticancer of great interest to cancer researchers. Dr. Junying Yuan (Harvard), e.g., drugs, which is of interest because Bim associates with microtubules addressed the role of the LKB1 kinase as a mediator of p53-dependent and is released on microtubule disruption to interact with Bcl-2-family cell death in the small intestine. Homozygous mutations inactivate the proteins on the surface of mitochondria. Of keen interest were the LKB1 gene in patients with Peutz-Jaeger syndrome. LKB1 associates results of experiments in which Bcl-2 knockout mice were bred with with microtubules and may participate in a pathway that somehow senses Bim knockouts. The lack of Bim complements the lack of Bcl-2, microtubule disruption. Overexpression of active LKB1 kinase induces producing nearly normal animals. In contrast to the phenotype of apoptosis in a p53-dependent manner. Moreover, p53 knockout cells are Bcl-2 knockout mice, which are runted, display shortened ears, de- insensitive to LKB1-induced apoptosis, and gene transfection of p53 into velop polysystic kidney disease and premature graying of their hair, as these cells restores sensitivity. Dr. Yuan speculated that p53 induces well as lymphoid system collapse, the double knockout of Bcl-2 and expression of a gene which somehow modifies LKB1-dependent apop- Bim results in mice which are essentially normal. These data suggest tosis pathways, e.g., p53 might induce expression of a LKB1 substrate. that Bim and Bcl-2 counteract each other in a delicate balance that is Additional presentations by Drs. Andre Gudkov (University of Illinois), important, at least, for T and B lymphocyte, myeloid cells, nephro- Klaus Debatin (University of Ulm), Waifik El Deiry (University of genic progenitors, and melanocytes in vivo. Pennsylvania), Yuri Labeznik (CSHL), and Tak Mak (Toronto) further Dr. Tsujimoto (Osaka University) focused most of his remarks on addressed mechanisms of p53-dependent pathways of relevance to DNA- interactions of Bcl-2 and Bcl-XL with VDAC, which resides in the outer damaging anticancer drugs. Dr. El Deiry demonstrated previously that membrane of mitochondria. In an in vitro reconstitution system using p53 induces expression of the TNF family death receptor, DR5. Using unilammellar liposomes, Dr. Tsujimoto et al. have obtained evidence that DR5 knockout mice, he showed evidence of tissue-specific requirement

Bcl-XL closes the VDAC channel. In contrast, recombinant Bax protein for DR5 for apoptosis induced by X-irradiation, e.g., DR5 appears to be causes VDAC pore opening, generating channels of sufficient size to required for optimal apoptotic responses in the thymus but not the spleen allow leakage of cytochrome c. The structure of this Bax/VDAC pore is after X-irradiation. Dr. El Deiry also presented evidence of yet another ␣ presently undefined. The first -helix of the Bcl-XL protein, called the Bcl-2 family member whose expression is induced by p53. He showed BH4 domain, is responsible for interactions with VDAC, according to Dr. that expression of Bid is up-regulated by 3–5-fold after induction of p53 Tsujimoto. Using synthetic peptides that fuse the HIV tat membrane in a model cell line. Consensus p53 binding sites are found in the penetration sequence with BH4, his group has demonstrated suppression Bid-encoding genes of humans and mice, and p53 can bind in vitro to 264

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2003 American Association for Cancer Research. APOPTOSIS AND CANCER these DNA sites. Continuing with the theme of tissue specificity and p53 interesting data regarding Fas resistance mechanisms in tumors. He target genes, Dr. El Deiry showed that although X-irradiation induces showed that although Fas induces caspase-8 activation in some cancer increases in Bid in the red pulp of spleen, the p53 target gene PUMA is cell lines that overexpress Bcl-2 or Bcl-XL, these cells nevertheless induced in the white pulp. Both Bid and PUMA are BH3-only proapop- remain viable. Dr. Peter showed that Bcl-XL in collaboration with the totic members of the Bcl-2 family. The functional importance of Bid for death effector domain protein bifunctional apoptosis regulator protein p53-induced apoptosis was suggested by experiments using murine em- can sequester active caspase-8 on the surface of mitochondria, evi- bryonic fibroblasts cells that are deficient in Bid, revealing resistance to dently preventing it from reaching substrates and thereby squelching doxorubcin-induced apoptosis, but it remains to be determined whether apoptosis. These findings reveal a postreceptor mechanism for thwart- Bid is critical for apoptosis in vivo. Finally, Dr. El Deiry presented ing apoptosis induction by TNF family death receptors. provocative evidence suggesting that p53 may also directly induce tran- Continuing the theme of resistance to TNF family death receptors, scription of the caspase-6 gene, at least in some types of tumor cells. Ralph Schwall (Genentech) showed that a colon cancer cell line lacking Thus, the list of p53-dependent targets potentially involved in p53- expression of Bax and Bak displays resistance to TRAIL-induced apop- mediated apoptosis grew significantly at the meeting. tosis. These and other data suggest that some types of cells require Dr. Tak Mak (Toronto) presented data on Chk2 knockout mice. These mitochondrial participation in the cell death pathways induced by TNF mice are viable and do not spontaneously produce tumors but do exhibit family death receptors and raised concerns about whether TRAIL will be increased rates of tumorigenesis when treated with chemical carcinogens, effective against chemoresistant cancers that have developed defects in such as 7,12-dimethylbenz(a)anthracene. Dr. Mak presented a model in the mitochondrial pathway. Nevertheless, TRAIL has antitumor activity which DNA damage activates the ATM kinase, in turn activating Chk2. in animal models and displays synergy with cytotoxic anticancer drugs in He suggested that both ATM and Chk2 are important for activating p53 vivo. With regards to underlying mechanisms of TRAIL synergy with for apoptosis induction, with ATM phosphorylating serine 15 and Chk2 cytotoxic drugs, Dr. Schwall showed that pretreatment of a Bax-deficient phosphorylating serine 20 on the p53 protein. Chk2 knockout mice colon cancer cell line with camptothecin induced up-regulation of Bak exhibit resistance to apoptosis induction in certain tissues after X-irradi- and DR5, thereby sensitizing these cells to TRAIL. Thus, clinical trials ation, lending further support to the hypothesis that Chk2 plays an with this biological agent are eagerly awaited. important role in p53-induced apoptosis in vivo. Steven Frisch (Burnham Institute) focused on mechanisms of anoikis, Dr. Yoichi Taya (National Cancer Center Research Institute, To- the process by which epithelial cells undergo apoptosis on detachment kyo, Japan) continued the theme of regulation of p53 by phosphoryl- from extracellular matrix. He showed that cell detachment plays an ation, presenting his evidence for a role of phosphorylation at serine important role in controlling the subcellular location of Fas-associated 46. Dr. Taya suggested that phosphorylation at serine 46 dictates the death domain, an adapter protein essential for signaling by TNF family difference between p53-induced cell cycle arrest versus apoptosis. He death receptors. Dr. Frisch presented evidence that Fas-associated death presented evidence that only when p53 is phosphorylated on serine 46 domain is located in the nucleus of attached cells, undergoing transloca- are certain genes induced, among which is the gene encoding p53 tion into the cytosol on detachment. Efforts are under way to elucidate the apoptosis inducing protein 1. This protein targets mitochondria, in- mechanisms responsible, but protein phosphorylation may play a role. ducing depolarization of these organelles and contributing to apopto- These interesting findings are relevant to the mechanisms of tumor sis. His group is attempting to purify the serine 46 kinase and has metastasis and warrant further exploration. identified a protein complex that appears to contain casein kinase-2␣ Endogenous Inhibitors of Caspases. IAP family proteins are con- and -2␤. Additional work is ongoing to determine whether this kinase served throughout metazoan evolution and contain at least one copy of complex is critical for some aspects of p53-induced apoptosis. the BIR domain. Some members of this protein family have been Additionally on the theme of genotoxic stress responses and mech- shown to directly bind caspases, thereby suppressing apoptosis. Over- anisms of chemotherapy-induced apoptosis, Yuri Labeznik (CSHL) expression of certain IAP family genes has been documented recently used siRNA techniques to generate evidence that caspase-2 may play in human cancers, peaking interest in this family of antiapoptotic an important role in apoptosis induction in at least one tumor cell line genes. Several speakers addressed the molecular mechanisms by after treatment with etoposide. An engineered caspase-2 cDNA, which IAPs function as caspase inhibitors, as well as explorations of which is resistant to the siRNA molecule, was capable of restoring endogenous IAP antagonists, including Drs. Guy Salvesen (Burnham sensitivity to chemotherapeutic drugs in this particular tumor model. Institute), Emad Alnemri (Thomas Jefferson University), Julian Details of the mechanism by which caspase-2 may participate in Downward (Imperial Cancer Research Institute), John Abrahms (Uni- chemotherapy-induced apoptosis remain unclear. versity of Texas, Southwestern), Herman Steller (Rockefeller Univer- Death Receptor Signaling. TNF family death receptors represent sity), and Klaus Michael Debatin (Ulm University). important initiators of apoptotic pathways, which are capable in many Dr. Salvesen reviewed the three-dimensional crystal structure of the XIAP types of cells of triggering apoptosis through a mitochondria-indepen- (BIR2)-caspase-3 complex. The structure reveals the inhibitory mechanism dent cell death pathway. Several presentations were devoted to this by which XIAP suppresses caspase-3 and may have important implications family of cytokine receptors, including presentations by Drs. Marcus for generating strategies that might allow disassociation of IAPs from their Peter (University of Chicago), Ralph Schwall (Genentech), Shigekazu caspase targets, thereby promoting apoptosis of tumor cells. Nagata (Osaka University), and Steven Frisch (Burnham Institute). Dr. Steller (Rockefeller University) presented evidence that a Ras- Dr. Peter presented evidence that the protein c-FLIP can be both an dependent pathway in the fly suppresses the proapoptotic effects of inhibitor and enhancer of Fas-induced apoptosis, suggesting that a IAP antagonist Hid in Drosophila. He presented evidence that the delicate balance between the levels of caspase-8 and FLIP determines Ras-pathway triggers activity of kinases that phosphorylate and inac- the ultimate sensitivity of cells to apoptosis induction by death recep- tivate the Hid protein. It remains to be determined whether analogous tors. He showed, using an inducible dimerization system, that dimer- proteins exist in mammalian cells. Genetic screens in the fly also ization of FLIP can induce caspase-8 activation. Dr. Peter also showed revealed mutant versions of the DIAP1, which are resistant to sup- that antisense-mediated reductions in FLIP decrease rather than in- pression by various IAP antagonists, including RPR, Hid, and Grim. crease in sensitivity of HeLa cells to Fas-involved apoptosis. These Most of these mutations map to either the BIR1 or BIR2 domains. findings suggest that previous models of the role of FLIP in tumor cell Interestingly, some mutant versions of DIAP1 display resistance biology may have been overly simplified. Dr. Peter also presented against RPR and Grim but not Hid and vice versa. These and other 265

data prompted Dr. Steller to speculate that the interactions among mutations in Nod2 are associated with Crohn’s disease, an inflamma- IAPs and their antagonists may be more complex than originally tory bowel disease. Mutations often affect the LRR region, which is appreciated. He introduced data about the important role of ubiquiti- speculated to represent a ligand-binding domain (LBD) for bacteria- nation in controlling the levels of IAPs. In the fly, DIAP1 and DIAP2 derived molecules, such as LPS. Dr. Tschopp (University of Lau- contain RING domains, which are speculated to interact with ubiq- sanne) talked about a similar protein, NALP1 (NAC), which also uitin-conjugating enzymes, by analogy to their human counterparts. contains a CARD, NACHT, and LRRs but in addition carries a newly Dr. Steller presented evidence that RPR, Grim, or Hid interactions recognized domain called the PAAD (PYK, PYRIN; DAPIN) domain. with IAPs may influence their functions as E3 ligases. Thus, these Dr. Tschopp suggested that the CARD domain of this protein interacts IAP antagonists may trigger ubiquitination and destruction of IAPs. with the CARD of procaspase-5, whereas the PAAD domain interacts Alternatively, the E3 ubiquitin ligase activity of IAPs may be impor- with the adapter protein ASC, connecting it to procaspase-1 via a tant for their functions as antiapoptotic proteins, allowing them to not CARD–CARD interaction. He presented evidence of a large multi- only bind but also induce degradation of active caspases. Regardless protein complex that is induced in macrophages in response to LPS, of the specific biochemical mechanism, genetic screens in the fly in which he termed the “inflammosome.” Thus, structurally similar the Steller Lab have revealed genetic modifiers of the DIAP pathway, molecules may be involved in activation either of NF-␬B or caspases. which include the E2 protein, ubiquitin-conjugating enzyme-D1. Interestingly, the caspases activated by NALP-1 (NAC) are involved Dr. Alnemri (Thomas Jefferson University) reviewed his laborato- in inflammation through their ability to cleave and activate proinflam- ry’s efforts to identify IAP antagonists in mammalian cells. Using matory cytokines, such as prointerleukin-1␤ and prointerleukin-18, proteonomics approaches, they have identified three proteins that can rather than participating in apoptosis. Dr. Tschopp also reviewed the interact with the XIAP. Two of these have recently been identified, recent evidence that hereditary mutations in the PAAD family protein including SMAC (Diablo) and OMI (HTRA2). Dr. Alnemri reviewed known as Cryopyrin are causitory in the autoinflammatory disease the insights which have come from solving three-dimensional struc- syndromes of familial cold urticaria syndrome and Muckle-Wells tures of IAP BIR domains complexed to SMAC or OMI, presenting syndrome. Mutations associated with these inflammatory disorders for the first time the crystal structure of OMI in a complex with the affect the NACHT domain, and Dr. Tschopp suggested that such BIR3 domain of XIAP. The IAP antagonists share a conserved tet- mutations allow for spontaneous oligomerization of the Cryopyrin

rapeptide motif in their NH2 terminus, which binds to a crevice on the protein, triggering caspase-1 activation. BIR domain, apparently competing with caspases for interaction at Dr. Tak Mak continued the theme of exploring the interface be- this site. Mutations within this IAP binding abrogate the effects of tween inflammation and cancer in his work on Bcl-10, an adapter OMI on IAP-mediated suppression of caspases. Interestingly, both protein that links certain CARD family proteins to the IKK complex, SMAC and OMI are sequestered in mitochondria. Their internal promoting NF-␬B induction through unclear mechanisms. The Bcl-10 IAP-binding motif is revealed on cleavage by mitochondrial proteases protein interacts with MALT, also known as para-caspase. Chromo- during import of these proteins into these organelles. These structural somal translocations involving either the Bcl-10 gene on chromosome studies reveal a clear path forward with respect to drug discovery and 1 or the MALT gene on chromosome 14 have been identified in hint that small molecule antagonists of IAPs which mimic the tet- gastric lymphomas associated with chronic Helicobacter pylori infec- rapeptide structure found in SMAC and OMI may be forthcoming. tion. Dr. Mak suggested a model in which stimulation of B cells by In this regard, Dr. Klaus Michael Debatin (Ulm University) pre- Helicobacter pylori antigens sets the stage for chromosomal rear- sented proof-of-concept data in which a heptapeptide representing the rangements, which then trigger activation of Bcl-10 or para-caspase, ␬ IAP-binding NH2 terminus of SMAC was used to promote apoptosis leading to constitutive activation of NF- B and thereby emulating of cancer cells in vitro, demonstrating synergy with cytotoxic anti- some of the survival signals which these cells receive through their cancer drugs. In an in vivo orthotopic model of glioma, Dr. Debatin antigen receptors. Dr. Mak presented evidence that Bcl-10 knockout showed that a tat-SMAC peptide collaborated with TRAIL in induc- mice have a defect in T- and B-cell antigen receptor signaling into the ing potent tumor regression. NF-␬B pathway. T cells from these animals, e.g., fail to proliferate in Dr. Julian Downward Imperial Cancer Research Fund, London, Eng- response to anti-CD3 and anti-CD28 antibodies and also exhibit land (ICFR) added weight to the evidence of an important role for certain defects in production of interleukin-2 and expression of interleukin-2 IAP antagonists in apoptosis induction. Using siRNA to down-regulate receptors. Alternative pathways for NF-␬B induction remain intact in OMI expression, he showed that OMI but not SMAC plays an important Bcl-10 knockout cells, including LPS, TNF, and interleukin-1. Thus, role in UV irradiation-induced apoptosis in the U205 tumor cell line. the Bcl-10/para-caspase pathway may be important in immune re- Interestingly, OMI is a serine protease, and it has been speculated that sponses, as well as lymphogenesis, making it an attractive pharma- release of this protease from mitochondria into the cytosol may contribute ceutical target for autoimmune diseases and lymphoid malignancies. to caspase-independent cell death. Additional work on this alternative Dr. Michael Karin (UCSD) reviewed the evidence that elevated function for OMI is required before firm conclusions can be drawn. levels of NF-␬B are found in several types of tumors. He introduced Transcriptional Control of Apoptosis. NF-␬B family transcrip- both the classical NF-␬B pathway and an alternative pathway in tion factors have emerged as important contributors to the antiapo- which the protein p100 is inducibly cleaved to produce a p52 protein ptotic state of some cancers. This family of transcription factors has that can partner with Rel-B to activate NF-␬B target genes. Although been shown to directly induce the expression of several antiapoptotic TNF␣, interleukin-1, and LPS induce the classic pathway, this alter-

genes, including Bcl-XL, BFL1, and cIAP2. Several presentations native pathway involving inducible p100 cleavage appears to be were devoted to pathways of relevance to NF-␬B induction, including activated by lymphotoxin-␤, B-LyS (BAFF), and possibly T- and presentations by Drs. Gabriel Nun˜ez (University Michigan) and Mi- B-cell antigen receptors. There was speculation at the meeting that chael Karin (UCSD). Dr. Nun˜ez’s remarks focused on the proteins perhaps MALT (para-caspase) could play a role in this inducible Nod1 and Nod2. These proteins contain a CARD domain followed by cleavage of p100, although no direct evidence is available to date. It a nucleotide-binding domain (NACHT) and several leucine rich re- is intriguing, however, that Drosophila appears to use a caspase called peats (LRRs). The CARD domains of these proteins interact with DRONC to link TOLL-like receptors involved in innate immunity to Cardiak (RIP2), an adapter protein that communicates directly with a NF-␬B activation pathway. Dr. Karin also presented evidence that the IKK complex. Dr. Nun˜ez reviewed recent evidence that hereditary induction of NF-␬B may be insufficient for protection from apoptosis. 266

In one model system in vitro, e.g., parallel activation of p38 MAP from ecdysone-induced cell death. Using DNA microarrays, Dr. Bae- kinase was found to be essential for NF-␬B-mediated induction of hrecke has obtained evidence that expression of several proapoptotic antiapoptotic genes. Thus, combinatorial pathway activation may play genes is induced by ecdysone in the salivary gland of developing flies, an important role in dictating whether NF-␬B provides effective including RPR, Hid, DBorg-2, DARK, DRONC, drICE, as well as survival signals and may also tip the balance between the effects of APG-9, a homologue of a yeast gene required for production of NF-␬B on expression of inflammatory versus antiapoptotic genes. autophagic vacuoles. Although the relevance to tumor biology of this The subject of transcriptional regulation of apoptosis continued with autophagic pathway for cell death remains unclear, it has been re- presentations by Drs. Barbara Osborne (University of Massachusetts) on ported previously that Bcl-2 interacts in mammalian cells with Beclin, Notch, Stavros Manolagas (University of Arkansas) on steroid hormone a protein implicated in regulation of autophagy. Future studies of this receptors, and Eric Baehrecke (University of Maryland) on steroid re- interesting cell destruction mechanism appear likely to provide new ceptors. Dr. Osborne reviewed mechanisms of Notch signaling, showing insights into caspase-independent pathways for cell death. how these transmembrane receptors become cleaved by proteases and Continuing the theme of transcription factors regulating apoptosis, Dr. releasing a cytosolic domain that translocates into the nucleus and regu- Thomas Look (Dana-Farber Cancer Institute) presented data on SLUG, a lates transcription in collaboration with other proteins. She presented homologue of the C. elegans CES-1, which has been implicated in evidence of deregulated expression of certain Notch family members in transcriptional regulation of a proapoptotic BH3-only member of the human cancers. In T lymphocytes, Notch-1 plays a role in suppressing Bcl-2 family called EGL-1. In C. elegans, CES-1 (a zinc-finger transcrip- apoptosis induced by NUR77 (TR3), an orphan member of the retinoid/ tion factor) suppresses expression of EGL-1, reducing apoptosis in a steroid family of transcription factors. Antisense and other types of particular type of cell in this organism. Expression of CES-1 is sup- experiments have revealed a critical need for NUR77 in activation- pressed by CES-2 (a b-Zip family transcription factor) in the worm. Dr. induced apoptosis of T cells. In some systems, NUR77 induces apoptosis Look demonstrated that an analogous system appears to exist in mam- through transcriptional mechanisms, but recent reports indicate NUR77 malian cells, in which HLF (a b-Zip family protein) suppresses expres- can also have extranuclear functions as a promoter of mitochondria- sion of SLUG, resulting in an antiapoptotic state. Chromosomal translo- dependent apoptosis, which can be distinguished from its role as a cations involving HLF in combination with E2F occur in pre-B-cell acute transcription factor. Investigations are under way in the Osborne lab to lymphocytic leukemia, dysregulating this pathway and suppressing ap- delineate the mechanisms by which NUR77 and Notch family proteins optosis. Furthermore, SLUG(Ϫ/Ϫ) mice are more sensitive to X-irradi- regulate apoptosis pathways. ation than wild-type littermates. A search by Dr. Look for the relevant Dr. Stavros Manolagas (University of Arkansas) focused his remarks targets of the SLUG protein revealed a role for BAX. Dr. Look demon- on nonclassical mechanisms of signaling by estrogen receptors (ERs) and strated that SLUG-deficient cells (derived from knockout mice) contain androgen receptors (ARs) in the cytoplasm. ER and AR can form com- higher and more sustained levels of BAX mRNA after X-irradiation, plexes with Src and Shc, activating a protein kinase pathway involving compared with wild-type cells. Expression of several other proapoptotic MAP kinase kinase, extracellular signal-regulated kinases, and inducing Bcl family members, including Harakiri, Bcl-xL/Bcl-2 associated death pro- ELK-1 activation. He showed that some small molecule ligands of ER␣ moter, Bcl-2 interacting mediator of cell death, Bik-like killer protein, and can induce the kinase pathway without activating transcription. The LBD BH3 interacting domain death agonist, was not changed. Further functional of the steroid receptors seems to be sufficient for this kinase-inducing analysis of the role of BAX in apoptosis regulation by SLUG is under way. activity. Targeting the LBD to membranes potently activates the MAP Apoptosis Regulation by Oncogenes and Tumor Suppressor kinase kinase/extracellular signal-regulated kinase pathway, whereas tar- Genes. The relation of oncogenes and tumor suppressor genes to geting the LBD to the nucleus does not. Dr. Manolagas demonstrated, the core cell death machinery was a topic of considerable emphasis at the interestingly, that a variety of small molecule ligands of estrogen and ARs meeting, including presentations by Drs. Jean Wang (UCSD) on the RB can promote the kinase pathway in a gender-independent manner with protein, Gerard Evan (University of California, San Francisco) on c-Myc, certain androgens stimulating ER␣ and conversely estrogens stimulating Joseph Nevins (Duke University) on E2F, Pier Paolo Pandolfi (Memorial AR. He calls these small molecule ligands “activators of nongenotrophic Sloan Kettering Cancer Center) on PML, and Scott Lowe (CSHL) on p53 estrogen-like signaling” (ANGELS). Data from a variety of in vivo and p16 INK4a. Gerard Evan (University of California, San Francisco) studies were presented by Dr. Manolagas addressing tissue-specific ef- discussed the role of the c-Myc oncogene in apoptosis regulation and fects of synthetic hormone ligands on survival of osteoblasts and oste- tumorigenesis. The c-Myc protein is known for its activity as both a oclasts in bone, in the absence of concomitant effects on uterus, seminal stimulator of cell proliferation and an inducer of apoptosis. Effective vesicles, and other sex hormone-dependent tissue. His data on novel tumorigenesis by c-Myc typically requires collaboration with antiapo- activators of nongenotrophic estrogen-like signaling suggested possibil- ptotic proteins that nullify its cell death promoting activity, while leaving ities for selectively preserving bone density, while avoiding trophic its proliferative functions intact. Dr. Evan presented data from Myc/ER effects on cancer prone tissues. transgenic mice, which express a conditional form of the Myc protein Dr. Eric Baehrecke (University of Maryland) presented data on whose activity is dependent on binding of small molecule drug Tamox- steroid regulation of programmed cell death during Drosophila mor- ifen to the ER portion of the fusion protein. Expression of Myc/ER in vivo phogenesis. This elegant system has provided a model for genetic in pancreatic islets promotes cell proliferation and apoptosis. Dr. Evan analysis of a steroid-dependent pathway triggering programmed cell demonstrated that loss of p53 or ARF nullifies the apoptotic function of death. The steroid hormone ecdysone interacts with a specific member Myc, promoting tumorigenesis. Of significance, expression of Myc in a of the retinoid/steroid of transcription factors, controlling a genetic p53- or ARF-null background is sufficient to produce tumors which are program that results in cell death in the salivary gland during pupa- invasive, angiogenic, and genetically unstable. These data imply that tion. Dr. Baehrecke reviewed evidence for a role in the E74 and E93 even as little as two genetic lesions can be sufficient to provide tumors genes in salivary gland dysfunction induced by ecdysone. Interest- with the full panoply of attributes needed for aggressive behavior. Data ingly, morphological analysis of the dying cells revealed a role for were presented that overexpression of Bcl-XL in pancreatic islets together autophagy, where lysosomes fuse with other organelles and digest with Myc/ER also generates adenomas, where Bcl-XL suppresses very cellular constituents. Dr. Baehrecke provided evidence of a caspase- potently the proapoptotic effects of c-Myc. In this animal model, turning independent pathway, through studies in which baculovirus p35 was off Myc by withdrawal of Tamoxifen is sufficient to cause complete expressed in salivary gland cells but found to be insufficient for rescue regression of adenomas. These findings demonstrate the reversibility of 267

neoplastic process and show that continuous activity of Myc is required PML also interacts with p63 and p73 (members of the p53 family) and to drive neoplastic cell expansion in vivo. similarly enhances their transactivation functions. However, PML Dr. Joseph Nevins (Duke University) continued with the theme of may also regulate apoptosis through additional mechanisms which do mechanisms of Myc-induced apoptosis, focusing on the role of E2F not require p53 family proteins, as evidenced by its ability to affect family transcription factors. Previous studies have demonstrated re- translocation of the proapoptotic DAXX protein to PODs. DAXX is a duced apoptosis in response to Myc expression in cells which lack putative transcriptional transrepressor which somehow enhances ap- E2F-1, after serum withdrawal. Dr. Nevins demonstrated, in contrast, optosis induction by Fas/TNF family death receptors and which that E2F-3 knockout cells undergo apoptosis normally in response to interacts physically with PML. Genetic ablation of the PML gene Myc. Similarly, although overexpression of either E2F-1 or E2F-3 in results in increased resistance of cells to Fas-induced apoptosis, al- cultured cells can drive S phase entry, only E2F-1 is capable of though the specific mechanisms remain to be clarified. Regardless, promoting apoptosis. These findings provide evidence that only se- these studies of PML reveal an interesting link between nuclear lected members of the E2F family, of which there are seven in the structures and the regulation of apoptosis pathways. human genome, induce apoptosis. E2F-1 induces increases in p53 Dr. Scott Lowe (CSHL) concluded the conference with a presen- protein. Dr. Nevins reviewed data which have demonstrated a path- tation on the role of defective apoptosis mechanisms and chemore- way in which E2F-1 induces expression of p19ARF, in turn suppress- sistance in cancer. Using a variety of elegant in vivo models, Dr. Lowe ing expression of MDM2, which then permits accumulation of p53. provided evidence that suppression of apoptosis by overexpression of He presented evidence that DNA-damaging agents induce elevations Bcl-2 has a profound influence on in vivo resistance of lymphoma in E2F-1 protein levels without concomitant increases in E2F-1 cells to antineoplastic drugs. Dr. Lowe used the E␮-Myc transgenic mRNA. This effect is caused by a prolongation of the half-life of the mouse model of B-cell lymphoma to explore the effects of alterations protein and is selective for E2F-1, in as much as levels of E2F-2 and in various tumor suppressor genes and proto-oncogenes on responses E2F-3 do not change in response to DNA damage. Elevations in to chemotherapy in vivo. As expected, overexpressing Bcl-2 in E␮- E2F-1 after genotoxic injury require ATM, based on experiments with Myc lymphomas by retroviral transduction suppressed apoptosis in- cell lines lacking this important kinase involved in a pathway notify- duction after treatment with DNA-damaging drug cyclophosphamide, ing cells of damaged DNA. Dr. Nevins showed that E2F-1 is a correlating with failure of involved lymph nodes to shrink. However, substrate for both ATM and the closely related kinase ATM and despite the paucity of cell death, Dr. Lowe provided evidence that Rad3-related protein kinase, whereas E2F-2, E2F-3, and other E2F these Myc/Bcl-2 tumors do not generally progress, apparently because family members are not. Phosphorylation of E2F-1 at serine 31 of DNA damage-induced replicative senescence. Studies with genes reduced ubiquitination and turnover of E2F protein, resulting in its of the INK4a locus suggested a role for both the p16 cyclin-kinase accumulation and induction of p19ARF. The specific target genes inhibitor and p19ARF regulator of the Mdm2/p53 pathway in post- responsible for E2F-1-induced apoptosis may include APAF-1, which chemotherapy-induced senescence. Thus, even if apoptosis is sup- was demonstrated recently to contain E2F-1-binding sites in its pro- pressed, anticancer drugs may induce replicative senescence, thereby moter and to be directly induced by this transcription factor. preventing further tumor growth. Homozygous disruption in INK4a Dr. Jean Wang (UCSD) addressed the role of the RB tumor sup- genes (p16/p19) or p53 was shown to ablate the senescence response, pressor gene in tumorigenisis and apoptosis regulation. She presented resulting in failure of chemotherapy in this transgenic mouse model. evidence that RB plays a role in suppressing both cell proliferation Therapeutic Implications. Overall, the meeting showed that tre- and apoptosis. Cells from RB knockout mice display increased apo- mendous progress has been made in recent years toward understanding ptosis in response to certain cell death stimuli. Dr. Wang demonstrated that the RB protein is cleaved by caspases, generating an the molecular basis of apoptosis regulation in normalcy and its dysregu- apparent dominant-negative form of the RB protein that promotes lation in cancer. Importantly, from an understanding of these basic apoptosis. Using gene knock-in technology, Dr. Wang demonstrated mechanisms, a variety of new strategies for combating cancer is begin- that mice carrying a noncleavable RB mutant are protected from ning to emerge, e.g., the discovery of endogenous antagonists of anti- apoptosis induction by LPS, TNF, and axotomy-induced neuronal apoptotic Bcl-2 and IAP family proteins and elucidation of peptidyl- apoptosis but are not resistant to X-irradiation or doxorubicin. These motifs in these antagonists that are sufficient for overcoming apoptosis findings suggest a specific role for RB in regulating certain pathways suppression reveals a path forward for production of nonpeptidyl small for apoptosis, with mechanisms remaining unclear at this time. molecule antagonists that could be used for treatment of cancer. Small ␬ Pier Paolo Pandolfi (Memorial Sloan Kettering Cancer Center) molecule inhibitors of the NF- B-activating kinases (IKKs) could also be addressed the role of the tumor suppressor PML in apoptosis regula- useful for restoring apoptosis sensitivity in cancers, as well as perhaps tion. The PML protein resides within subdomains of the nucleus drugs that antagonize para-caspase, if evidence continues to mount im- called PODs. PML somehow promotes apoptosis in a general way, plicating this novel protease in NF-␬B induction in some types of tumors. although the mechanisms remain unclear. Dr. Pandolfi presented Biologicals for the treatment of cancer, such as TRAIL, also appear to be evidence that defects in the expression or structure of the PML protein quite promising, suggesting the possibility of tapping into mitochondria- are very common in human cancers, including many solid tumors. independent apoptosis pathways and thereby triggering alternative cell PML also becomes deregulated through chromosomal translocations death pathways in chemo-resistance cancers. In addition to these new with the retinoic acid receptor-␣ gene in acute promyelomonocytic strategies for inducing apoptosis of cancer cells, it should not be forgotten leukemias. The fusion of PML with retinoic acid receptor-␣ disrupts that already, several agents are in human clinical trials which touch on POD formation, resulting in an apoptosis-resistant state. Dr. Pendolfi some of the same mechanisms mentioned above, including: (a) Bcl-2 demonstrated that SUMOlyation of PML is required its proper target- antisense oligonucleotides currently in Phase III trials for chemo- ing to PODs and for recruitment for other proteins into these struc- refractory malignancies; (b) proteasome-inhibiting drugs that (among tures, including the proapoptotic protein DAXX. He showed that PML other things) prevent IkB degradation and thereby thwart NF-␬B activa- recruits p53 into PODs after genotoxic injury and proposed the tion; and (c) p53 and E1a gene therapies, which trigger expression of formation of a ternary complex containing p53, PML, and CREB apoptosis-inducing genes and sensitize tumor cells to radiation. Pros- binding protein, which results in acetylation of p53, rendering p53 pects, therefore, seem bright for applying information about apoptosis competent to transactivate target genes. Dr. Pandolfi indicated that mechanisms for improving treatment of cancer in the near future. 268

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2003 American Association for Cancer Research. Apoptosis and Cancer: Basic Mechanisms and Therapeutic Opportunities in the Postgenomic Era

Frank Stenner-Liewen and John C. Reed

Cancer Res 2003;63:263-268.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/63/1/263

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/63/1/263. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.