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REVIEWS MODELS OF CANCER Cell lineage and cell death: Caenorhabditis elegans and cancer research Malia B. Potts* and Scott Cameron*‡ Abstract | Cancer is a complex disease in which cells have circumvented normal restraints on tissue growth and have acquired complex abnormalities in their genomes, posing a considerable challenge to identifying the pathways and mechanisms that drive fundamental aspects of the malignant phenotype. Genetic analyses of the normal development of the nematode Caenorhabditis elegans have revealed evolutionarily conserved mechanisms through which individual cells establish their fates, and how they make and execute the decision to survive or undergo programmed cell death. The pathways identified through these studies have mammalian counterparts that are co-opted by malignant cells. Effective cancer drugs now target some of these pathways, and more are likely to be discovered. *Departments of Pediatrics and Molecular Biology, Division of Pediatric Haematology-Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390‑9148, USA. ‡ Present address: Novartis Institutes for Biomedical Research, Oncology Translational Medicine, 220 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. Correspondence to S.C. e‑mail: johnscott.cameron@ novartis.com doi:10.1038/nrc2984 Published online 2 December 2010; corrected online 25 February 2011 In multicellular organisms, a remarkable array of spe cific cell types must be generated to build an animal, and the inappropriate survival or proliferation of an individual cell can ultimately be deleterious to the organ ism as a whole1. Thus, mechanisms have evolved that precisely control cell fates and kill unwanted cells2. The breakdown of these mechanisms can contribute to the development of cancer 3. Although cell fate decisions have long been appreci ated as a part of normal development, the involvement of cell death in this process initially seemed intellec tually unsatisfactory — death is on some level the break down of normality, so how could it be reproducible in a way that can be defined experimentally? And what might be the purpose of generating many cells the only apparent function of which is to die? In this Review, we describe how studies of cell lineage and programmed cell death in the nematode Caenorhabditis elegans have revealed basic mechanisms of cell death regulation that are important in human cancer, and how this research is contributing to the development of new approaches to cancer therapy. C. elegans and cell lineage It is remarkable that studying a 1 mm-long, transparent worm that is not susceptible to cancer has led to tremen dous advances in our understanding of human diseases. A single defining aspect of C. elegans biology provided a foundation for these advances: its simple and essen tially invariant cell lineage4,5. This aspect of its biology, together with classical and modern tools for the genetic analysis of living animals, is a unifying theme of research with this organism. The effects of discoveries made with this worm extend broadly in biology because many fun damental processes that control animal development are evolutionarily conserved. In C. elegans, the term cell lineage refers to the pat tern of divisions from the zygote to the adult, and also to the fates adopted by individual cells at each point in the lineage. Knowledge of this pattern allows the study of cell fate determination with unparalleled precision. The adult hermaphrodite has 959 somatic cells; there fore, mutants with an abnormal number of cells can be readily identified and the causes of the abnormality (lack of cell death or change in cell fate specification, for example) defined by simply following the cell divisions as the mutants develop on a microscope slide. Helpfully, mutants with cell lineage defects often also have obvi ous defects in function or morphology that are easier to recognize than an abnormal number of cell nuclei. Screens for additional mutants with similar or opposite morphological or functional defects have then been used to discover additional genes that drive or regulate the processes involved. Below, we discuss the results from such screens to discover the mechanism of programmed cell death and highlight accumulating evidence show ing that the genes that control the death decision in single somatic cells of C. elegans are evolutionarily con served and co-opted by malignant cells to ensure their survival. 50 | JANUARY 2011 | VOLUME 11 www.nature.com/reviews/cancer © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS At a glance •The invariant nature of the development of cell lineages in the nematode worm Caenorhabditis elegans has made it a useful model for understanding the mechanisms of cell lineage control and cell death. •Many of the genes that regulate cell death and lineage development have homologues in mammals. •Mapping cell lineage development in the nematode has been particularly informative for understanding the regulation of haematopoiesis in mammals, with many of the genes that regulate cell lineage being deregulated in leukaemia and lymphoma. •Further understanding of the regulation of cell fate in C. elegans and mammals should identify new targets for anticancer drug development. Programmed cell death Programmed cell death, or apoptosis, is crucially impor tant in the genesis of cancer and in the success or failure of cancer therapy. Groundbreaking work using C. elegans helped to expose apoptosis as a genetically controlled process, and studies in C. elegans have continued to illuminate the identities and functions of the molecules involved, paving the way for the development and testing of new cancer therapeutics that are designed to induce apoptosis in cancer cells. The focus on programmed cell death in cancer began with the important work of Kerr, Wyllie and Currie6. Their remarkable 1972 paper described the hallmark morphological features of apoptotic cells and the importance of apoptosis in normal and malignant tissues. The authors recognized that tissue and tumour growth reflected an imbalance between the generation of new cells by mitosis and the loss of cells by apop tosis, and that cancer therapies such as radiation and chemotherapy drugs induced apoptosis. They specu lated that apoptosis might be controlled in some man ner by the genome, but the challenge of identifying the mechanisms that execute a process affecting a tiny proportion of cells in a tissue at any given time must have seemed daunting. At about this time, John Sulston and Bob Horvitz in Sydney Brenner’s laboratory had begun describing the C. elegans cell lineage. They observed that certain cells were born, only to die shortly after birth, with an invari ant morphology 4,5. Why and how do these deaths occur, and do mammalian lineages have similar deaths? The role of death in cell lineage. Why bother with a cell division that generates two cells, one of which undergoes programmed cell death, rather than just generating the intended single cell? One possibil ity is that the process of division is necessary for the proper segregation of cell fate determinants. Another possibility is that this is an efficient mechanism that has not been selected against during evolution (FIG. 1). Programmed cell death is used to eliminate cells that adopt different fates in males and hermaphrodites, or that are needed in one part of the animal but not in another 5,7. Using programmed cell death in these ways allows a single cell lineage programme to operate in both sexes or in different parts of the animal with only minor modifications. It is not yet possible to examine the cell lineages of mammalian cells with the precision that is available for C. elegans, but there are similarities in the mechanisms that underlie cell fate decisions, and it seems likely that mammalian lineages might use programmed cell death in ways similar to those described in C. elegans. Indeed, there are compelling examples of conserved mechanisms of cell lineage control between C. elegans and haemato poietic lineage factors8–12, and apoptosis is a prominent and unexplained aspect of the initial attempts to follow mammalian haematopoietic lineages in vitro13. From early observations of cell death to mechanisms. The reproducible morphology and pattern of deaths in C. elegans provided a platform for sophisticated genetic screens that identified the molecules that are responsi ble for cell death. The identification and analysis of the genes discovered in these screens revealed a genetically encoded mechanism of cellular suicide (FIG. 2). Important discoveries included the demonstration that CED‑3, a protease (more specifically, a caspase), and CED‑4, which was later discovered to be conserved in mammals in the form of a scaffolding protein APAF1, functioned cell autonomously and were essential for cell death2,14,15. CED‑9 antagonized the functions of CED‑3 and CED‑4 to prevent cell death16,17. The deaths of somatic cells are induced by egg-laying defective 1 (EGL‑1)18,19, which was originally identified in screens for egg-laying-defective mutant worms. As discussed below, the transcriptional regulation of egl‑1 expression has turned out to be a crucial step for the cell death decision, and BH3‑only proteins serve similar functions in humans. The iden tification of CED‑9 as a homologue of BCL‑2 (REF. 17) has been cited as the ‘Aha!’ moment of discovery in the work on programmed cell death20, as it suggested that the mechanism was evolutionarily conserved in animals. The conservation of this crucial function in cell death was supported by experiments in which human BCL‑2 expression rescued the cell death phenotype that is evi dent in ced‑9 mutants16,21. Moreover, because mutations affecting BCL‑2 cause lymphoma by preventing the death of lymphocytes22, it became obvious that defects in pro grammed cell death contributed to human disease. This was further underpinned by discovering that mice that are deficient for the pro-apoptotic BH3‑only proteincoding gene Bad develop lymphoma, and that mice that are deficient in Bid develop a myeloproliferative disor der that can progress to leukaemia23,24. Thus, the genetic analysis of programmed cell death in C. elegans revealed the mechanism underlying a fundamental process of development that is abnormal in cancer cells. Cell death in humans: elaborating on the simple ele‑ gance of the worm. Each of the four genes that consti tutes the canonical, core pathway of programmed cell death in C. elegans has one or more homologous genes in mammals (FIG. 2). In worms and mammals, cell death can be initiated by transcriptional activation of BH3‑only genes18, which activate the adaptor CED‑4 or APAF1, and this is antagonized by the pro-survival Bcl‑2 fam ily members19. CED‑4 or APAF1 then participate in NATURE REVIEWS | CANCER VOLUME 11 | JANUARY 2011 | 51 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS the activation of the caspases, which cleave multiple targets to kill the cell25. Despite the obvious homology between the human and nematode apoptosis path ways, some divergence is also apparent. In contrast to humans, cytochrome c is not required for the activation of nematode CED‑3 by CED‑4 in vitro, and CED‑4 lacks the WD domain repeats that mediate interaction with cytochrome c in the apoptosome of mammals26–28. These and other data suggest that the mitochondrial release of cytochrome c is not part of cell killing in C. elegans. In a second important difference from humans, caspase inhibition by inhibitor of apoptosis (IAP) proteins is not apparently used as a mechanism of regulating pro grammed cell death in nematodes. In this regard C. ele‑ gans also differs from Drosophila melanogaster, which relies heavily on antagonism of IAP function rather than the generation of BH3‑only proteins to initiate cell death29,30. Therefore, mammalian apoptotic pathways are Figure 1 | The role of programmed cell death in cell lineage. a | Cell death is often used to eliminate cells that are the non-essential spatial homologues of cells that are required in other parts of the body. For example, the individual cell lineages that generate the mature ventral nerve cord along the anterior–posterior body axis of Caenorhabditis elegans are very similar to one another, and programmed cell death is used to kill off cells that are not needed in certain body regions4. Specifically, six neurons generated by the P3–P8 lineages in the midregion of the ventral nerve cord regulate the laying of eggs through the vulva in the middle of the animal; the corresponding cells that are generated by the anterior and posterior lineages undergo programmed cell death4,129 (arrows). Similarly, two cells in the posterior that undergo programmed cell death are analogous to more anterior neurons that survive and extend their axons posteriorly (arrows). Presumably, there is no purpose served in having posterior neurons extend posterior axons. b | Often a sex-specific cell type is initially produced in both sexes but is induced to die specifically in the sex in which it is not needed. For example, the male-specific cephalic (CEM) neurons survive in males and undergo programmed cell death in hermaphrodites; the hermaphrodite-specific neurons (HSNs) survive in hermaphrodites and die in males, and this is controlled by tra‑1 (REFS 5,7). AMso, amphid socket cell; P, twelve postembryonic blast cells that give rise to ventral cord motorneurons; PHB, neuron type, phasmid; W, postembryonic neuroblast. more complex than those of C. elegans, and it is impor tant to note that the simple, sequential model in which programmed cell death is initiated by the transcription of egl‑1 in most cells of C. elegans is incomplete, as recent data suggest important roles for additional regulation downstream of egl‑1 transcription31–33. An important area of current research is to understand how regulatory pathways that might impinge on the cell death pathway at multiple points interact to result in a consistent output of survival or programmed cell death. Transcription factors specify cell death Just as the core apoptotic pathway is conserved from worms to humans, it is also clear that the regulatory pathways that control the cell death decision of indi vidual cells of C. elegans are conserved in mammals. Moreover, these regulatory pathways are often dereg ulated in cancer cells to ensure their survival (FIG. 3; TABLE 1). This raises the intriguing possibility that tran scriptional repression of apoptosis by lineage specific transcription factors is a crucial function that is ena bled by the mutation of specific tumour suppressors and proto-oncogenes. ces genes and acute lymphoblastic leukaemia. In genetic screens in C. elegans aimed at identifying genes that are involved in the regulation of programmed cell death in the serotonergic nervous system, some mutants were identified that affected the deaths of only a small number of cells. For example, ces‑1 gain-of-function mutations and ces‑2 loss-of-function mutations result in the survival of specific groups of cells in the pharynx 34. Other deaths occur normally in these mutants, and for this reason the genes involved were termed cell death specification (ces). These data suggest that the ces genes participate in the decision process that must pre cede the execution of death. Cloning of the ces genes revealed that they encode transcription factors: Ces‑2 is the homologue of the PAR family bZIP protein hepatic leukaemia factor (HLF)35, and ces‑1 is a homologue of SLUG, a zinc finger protein and Snail family member 36,37. Given the central role of transcription in many cell fate decisions, this finding strengthens the idea that pro grammed cell death is a cell fate — little different from other cell fates. In NSM sister cells, egl‑1 transcription is controlled by competition between CES‑1 (a repressor of transcription) and a heterodimeric helix–loop–helix 2 (HLH‑2)–HLH‑3 basic HLH (bHLH) transcription fac tor complex (an activator) for specific binding sites on the egl‑1 promoter 36,37. CES‑2 is a repressor that inhibits ces‑1 activity in the NSM sisters; the gain-of-function mutations in ces‑1 that cause NSM cell survival are pro moter mutations that probably disrupt CES‑2 binding to the ces‑1 promoter 36. Some children with acute lymphoblastic leukaemia (ALL) have a chromosomal translocation that couples the DNA binding domain of HLF to the transcrip tional activation domain of E2A, and brings the fusion gene under the control of the E2A promoter, which is expressed in lymphocytes38–40. How this fusion protein promoted leukaemia was unclear, but the discovery that 52 | JANUARY 2011 | VOLUME 11 www.nature.com/reviews/cancer © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS HLF was homologous to CES‑2 suggested a detailed model, which was subsequently supported by an ele gant series of papers38,41–43: the fusion converts HLF from a repressor to an activator able to prevent cell death. Consistent with a function that was predicted by the analysis of ces‑2, the E2A–HLF fusion maintains the transcription of the ces‑1 homologue SLUG, which probably represses the transcription of the BH3‑only gene PUMA (also known as BBC3), ensuring the sur vival of the malignant lymphoblasts38,41–43. Therefore, genetic pathways that regulate programmed cell death or the survival of specific cells of C. elegans can be evolutionarily conserved and altered by mutation to promote the survival of human cancer cells. Inhibition of this pathway in the malignant cells can induce their death38, suggesting that a better understanding of these pathways might be therapeutically useful. As noted above, many divisions in the C. elegans lin eage are asymmetric and generate one cell that survives and one cell that undergoes programmed cell death. Recent work has shown that the ces genes can cou ple asymmetric division and programmed cell death during the pharyngeal neurosecretory-motor (NSM) neuroblast divisions44. When observing cell lineages in which a daughter cell dies, it is normally obvious dur ing the division which cell will die — the dying cell is smaller than the sister cell that survives. The asymme try in size of the newly generated NSM neurons and the NSM sisters is not a consequence of the death process, because the asymmetry is preserved in egl‑1 mutants in which death is prevented. However, in ces‑1 and ces‑2 mutants this asymmetry is disrupted44. Moreover, the axis of NSM neuroblast division is often abnormal in ces‑2 and ces‑1 mutants. The positioning of the mitotic spindle and the orientation of the division axis is closely linked to the fates established by the daughters of pro genitor cells in mammals and model organisms45,46, and defects in this process are also observed in malignant cells. Elucidating the mechanism through which the Ces proteins influence the asymmetric division of the NSM neuroblasts and a better understanding of how this is coupled to programmed cell death might sug gest mechanisms through which malignant cells can self-renew. tra‑1 and GLI: cell fate and hedgehog signalling. tra‑1 encodes a transcription factor that is the terminal, glo bal regulator of sex determination in C. elegans, and it promotes hermaphrodite fates7,47. Analysis of semidominant egl‑1 mutations revealed promoter muta tions that disrupt TRA‑1 repression of egl‑1 expression, leading to the inappropriate activation of egl‑1 and the death of two neurons that are essential for egg laying in hermaphrodites, the hermaphrodite-specific neurons (HSNs)18. tra‑1 loss-of-function mutants are transformed to a male phenotype, with approximately one-third of cells in the animals adopting different fates4,48. The dis covery that TRA‑1 directly regulates cell death as one of its many functions poses a considerable challenge for the recognition and analysis of genes that directly regulate programmed cell death decisions in C. elegans Figure 2 | The programmed cell death pathway of C. elegans is evolutionarily conserved. The products of four genes are required for essentially all of the programmed cell deaths of somatic cells of Caenorhabditis elegans, and the pathway is evolutionarily conserved. Human cells typically have multiple family members of each protein class. There is added complexity in the apoptotic pathways of human cells that is not shown in the figure: these include the release of cytochrome c from mitochondria; the inhibitor of apoptosis proteins functioning as an additional inhibitor of caspase activity in addition to Bcl‑2 family members; and the extrinsic and death receptor-mediated pathways serving as the primary mechanism of activating apoptosis in some circumstances. There is strong evidence that defects in apoptosis contribute to human cancer, including the loss of BH3 domain pro-apoptotic molecules, activation of Bcl‑2 family anti-apoptotic molecules and defects in the upstream regulatory mechanisms that control the apoptosis cascade. For reasons that are unclear, the evidence is substantially weaker that mutations affecting APAF1 or the caspase genes contribute broadly to human cancers, suggesting that blocking apoptosis upstream of mitochondria offers a greater advantage during the evolution of a tumour. MOMP, mitochondrial outer membrane permeabilization. and in human cancers, because an abnormality in cell death may be mistakenly presumed to be a secondary consequence of an altered cell fate. In contrast to its pro-survival function in the HSN neurons, TRA‑1 eliminates male-specific cephalic (CEM) neurons in the hermaphrodite through indirect activation of egl‑1 and ced‑3 (REFS 33,49,50) (FIG. 1). In the absence of TRA‑1, the Bar homeodomain transcription factor CEH‑30 is activated by UNC‑86 (a POU domain transcription factor), LSR‑1 (a leucyl-tRNA synthetase) NATURE REVIEWS | CANCER VOLUME 11 | JANUARY 2011 | 53 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS and UNC‑132 (which has partial homology to PIM1 but lacks a kinase domain)33. CEH‑30 represses egl‑1 and ced‑3 through interaction with the general transcrip tional repressor UNC‑37 (a Groucho homologue) and promotes CEM survival in males49. In hermaphrodites, TRA‑1 represses ceh‑30, causing derepression of egl‑1 and ced‑3 and death of the CEMs33,49,50. The mechanisms through which CEH‑30 represses egl‑1 and ced‑3 are not yet clear. Notably, homologues of several of the anti-apoptotic genes identified from studies of CEM neurons in C. elegans have oncogenic properties in mammals. For example, the POU domain transcription fac tors contribute to oncogenesis in multiple cancers, including neuroepitheliomas, Ewing’s sarcomas, testicular germ cell tumours, melanomas and breast cancers51–54. Overexpression of the leucyl-tRNA syn thetase LARS1 has been shown to promote lung can cer cell growth and migration55, and overexpression of Groucho homologues has been observed in multiple tumour types56. PIM1 is a well-established oncogene, but whether unc‑132 shares functional similarities to PIM1 that are relevant to tumorigenesis remains to be determined33,57. The human homologues of TRA‑1 are the GLI onco genic zinc finger transcription factors that respond to Hedgehog (Hh) signalling 58. Consistent with its role in normal embryonic development, recent data suggest Figure 3 | Cell death is specified by transcription factors in C. elegans, and their human homologues are oncogenes. The transcription factors and pathways that determine cell death or survival are shown, as well as the locations of the cells that they affect. Only factors that directly control the transcription of the cell death pathway genes are included. The human homologues of the Caenorhabditis elegans genes are shown, with mammalian oncogenes in grey. HSN, hermaphrodite-specific neurons; NSM, pharyngeal neurosecretory-motor neurons; P, twelve postembryonic blast cells that give rise to ventral cord motorneurons; VC, ventral cord. that Hh functions non-cell autonomously in many cancers59, and can promote the survival of cancer cells partly by affecting the transcriptional regulation of a Bcl‑2 family member in tumour cells — as TRA‑1 does in the HSN neurons in C. elegans. Stromal Hh signalling in the Eμ-Myc mouse model of B cell lymphoma acti vates GLI in the lymphoma cells and maintains expres sion of BCL‑2, preventing apoptosis; inhibition of Hh signalling induces apoptosis60. This mechanism might be conserved in human non-Hodgkin’s lymphoma and multiple myeloma60. In primary keratinocytes, GLI proteins directly regulate the transcription of BCL2 (REFS 61,62). Together, these data suggest that the direct regulation of apoptosis is a conserved function of GLI family members that promotes the survival of cancer cells. It is not yet clear how many cancers depend on Hh activation of BCL2 expression for their survival, or whether the Hh pathway inhibitors that are currently in clinical trials might kill cancer cells by interrupting this survival pathway. Oncoproteins and programmed cell death Of the 13 genes that directly specify the cell death or survival of individual cells in C. elegans, at least nine are the homologues of human oncogenes (FIG. 3) . These include Hox proteins; Hox cofactors, including a Trithorax group regulator that is required for Hox expression; a Pax family member protein, which directly regulates ced‑9; and a Caudal homologue, which directly regulates transcription of ced‑3. In each case there is initial evidence that the oncogenic forms of these transcription factors directly regulate cell survival in mammals. Moreover, for a Six family homeodomain protein that directly regulates egl‑1, there is good evi dence that human homologues are involved in cancer and regulate apoptosis, but the mechanisms involved that promote malignancy are unclear. Six family homeodomain. Genetic screens using animals in which cell type-specific reporters drive green fluores cent protein expression have been a powerful tool for identifying genes that regulate the survival of specific cell types. A recent screen using transgenic C. elegans worms that express a reporter gene for a single neuron, M4, identified a point mutation in a regulatory, noncoding region of egl‑1 that resulted in the survival of the M4 neuron63. The mutation disrupted a predicted binding site for a Six family homeodomain transcrip tion factor, CEH‑34, suggesting that the site was required for the transcriptional activation of egl‑1 and the death of the M4 neuron. Indeed, loss-of-function mutations in ceh‑34 were also identified in the screen. CEH‑34 directly regulates egl‑1 transcription in the M4 neurons as part of an evolutionarily conserved complex with the eyes absent homologue EYA‑1. Several Six family members have been studied in human cancers and, as would be expected for transcription factors, their roles in promoting malignancy seem to be context dependent. These roles include the regulation of cell survival and cell death through mechanisms that have not yet been elucidated64. 54 | JANUARY 2011 | VOLUME 11 www.nature.com/reviews/cancer © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS Table 1 | Human oncogenes homologous to the cell death specification genes of C. elegans C. elegans gene Cell death role Human homologue Mutation type in cancer Cancer type Refs ces‑2 Inhibit HLF Gain of function ALL tra-1 Inhibit Gli Gain of function Many 38,39,42,117,118 lin‑39 Inhibit Hox Gain of function ALL, AML and others 99,112,121–124 ceh-20 Promote or inhibit Pbx Gain of function ALL 99,112,121–124 unc‑62 Inhibit Meis Overexpressed Leukaemia 99,112,121–124 lin‑59 Inhibit MLL Gain of function ALL and AML 99,112,121–124 egl‑38 Inhibit Pax Gain of function Rhabdomyosarcoma pal‑1 Promote Cdx Loss of function Gastrointestinal cancers 126–128 pal‑1 Promote Cdx Gain of function Leukaemia through Hox upregulation 126–128 119,120 125 ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; Cdx, caudal-type homeobox transcription factor; C. elegans, Caenorhabditis elegans; Gli; glioma associated oncogene homologue; HLF, hepatic leukaemia factor; Hox, homeobox; MLL, mixed lineage leukaemia; Pax, paired box gene; Pbx, pre-B cell leukaemia transcription factor. Hox genes. Like TRA‑1 and GLI, Hox protein complexes have diverse roles in development that include the direct regulation of programmed cell death. A mutation affect ing the Hox gene lin‑39 was the first one isolated in the genetic screens in C. elegans that led to the elucidation of the programmed cell death pathway 2,65. Consistent with its classical role in animal development, the Hox complex in C. elegans includes six genes that determine the pattern of cell fates along the body axis65–71. The lin‑39 gene is required for the survival of six neurons in the mid-body of the animal65, and the more posterior Hox gene mab‑5 is required for the deaths of two cells in the posterior 67. In lin‑39 mutants, the six neurons in the mid-body are homeotically transformed and adopt the fate of their more anterior and posterior sister cells, which undergo programmed cell death65. But what is the mechanism responsible for this transformation? Hox proteins often function together with TALE fam ily homeodomain cofactors, as this increases the DNA binding affinity and selectivity of Hox proteins. These TALE family members include CEH‑20 and UNC‑62 in C. elegans and their respective mammalian homologues, Pbx and Meis72–78. In the mid-body, LIN‑39, CEH‑20 and UNC‑62 are all required for the repression of egl‑1 to ensure the survival of the six neurons; a LIN39–CEH‑20 complex probably represses egl‑1 through multiple regulatory sites79. The death of the two cells in the posterior of the nema tode occurs through a similar mechanism, which involves MAB‑5 and CEH‑20 — although with a curious twist. In the case of one of the two cells, a MAB‑5–CEH‑20 complex binds to a single site in the egl‑1 regulatory region to activate transcription and induce programmed cell death74. Remarkably, the death of the second cell, which is also dependent on MAB‑5 and CEH‑20 for survival, is regulated indirectly through an unknown mechanism. This indicates that the survival or death of even very closely related cell types can be controlled through distinct mechanisms, and is a striking demon stration of the kinds of discoveries made possible by the known and essentially invariant lineage of C. elegans. It is not clear why two distinct mechanisms are required to regulate the death of these two similar cells. Hox protein complexes can therefore activate or repress the transcription of a cell death gene to ensure the survival or programmed cell death of individual cells. For lin‑39 and mab‑5 this role is consistent with their specification of fates along the body axis by determining regional patterns of programmed cell death. Abnormal activation of Hox genes is common in human leukaemias, and their role in normal and malig nant haematopoiesis has been intensively studied. The C. elegans Trithorax group chromatin-modifying protein LIN‑59 is required for Hox gene expression, similar to the MLL protein in human leukaemias79 (BOX 1). Might the core cell death pathway genes be direct targets of Hox proteins in leukaemia cells? The C. elegans data, and similar data from Drosophila80,81, indicate that the regulation of programmed cell death by Hox genes is context dependent — Hox genes are broadly expressed but only small numbers of cells are dependent on Hox function for their survival. There is some evidence that this context-dependent survival role might be conserved in human leukaemias. Inhibition of HOXA9 function in human leukaemia cells induces apoptosis specifically in leukaemias that harbour activating mutations in MLL82. The mechanism underlying this dependence has not yet been elucidated, but the model organism data suggest that direct repression of apoptotic pathway genes such as BH3 domain-encoding genes is a possibility. egl‑38 and Pax‑2 regulate ced‑9 (BCL2). Two pairedbox homeodomain transcription factors directly regulate apoptosis in C. elegans, egl‑38 and pax‑2 (REF. 32). Unlike the ces, tra‑1 and Hox genes, egl‑38 and pax‑2 ensure the survival of many cells in the developing animal, includ ing somatic cells and developing germ cells. Moreover, egl‑38 and pax‑2 prevent programmed cell death by stimulating the transcription of the BCL2 homologue ced‑9, rather than by repressing egl‑1 (REF. 32). Pax genes are subject to translocation and can become fused to Forkhead family genes by the t(2;13) and t(1;13) chromosomal translocations in childhood rhabdomyosarcoma. These fusion proteins inhibit pro grammed cell death in rhabdomyosarcoma cells through an unknown mechanism83. Translocations involving Pax NATURE REVIEWS | CANCER VOLUME 11 | JANUARY 2011 | 55 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS genes and the immunoglobulin enhancer also occur in some non-Hodgkin’s lymphomas84–88. Pax genes are broadly expressed in a large collection of tumour cell lines, including the NCI60 cell lines, and inhibi tion of individual Pax genes in many of these cell lines induces programmed cell death89. How Pax expression prevents programmed cell death in these tumour lines is unknown, although direct stimulation of BCL2L1 expression by PAX3 has been reported90; this mecha nism would be consistent with the pathway defined in C. elegans. Germline cell death and DNA damage Germ cell development in C. elegans proceeds through regulated but stochastic proliferation and differen tiation, rather than through the essentially invariant cell lineage that is characteristic of somatic cells. In a typical gonad, around 50% of the germ cells undergo programmed cell death in a process that is depend ent on ced‑4 and ced‑3, but not egl‑1 (REF. 91) , and that relies on the pro-apoptotic activity of the RB pathway genes lin‑35, dpl‑1, efl‑1 and efl‑2 (REF. 92). DNA damage increases germ cell death in a dosedependent manner and induces cell cycle arrest in the proliferating germ line, whereas somatic cells are pro foundly resistant 93. Damage-induced germ cell death, in contrast to normal homeostatic germ cell death, is controlled by egl‑1, a second BH3 domain-encoding gene ced‑13 and ced‑9, and it also requires ced‑4 and ced‑3. Furthermore, the TP53 homologue cep‑1 is required for damage-induced apoptosis, but not cell cycle arrest, in the germ line, as are lin‑35, dpl‑1 and efl‑2 (REF. 92). Recent work has also revealed a new and surprising mechanism through which HIF1A, which encodes the α-subunit of the hypoxia-inducible factor, can inhibit apoptosis. The HIF1A homologue activates the expression of a tyrosinase in two head neurons, which act on the C. elegans germ line through cep‑1 to inhibit apoptosis94. Thus, the C. elegans germ line is a simple but powerful genetic context in which conserved aspects of the DNA-damage response and Box 1 | Hox genes and cancer Hox function can be activated by chromosomal translocations, including those that directly affect individual Hox genes, Pbx family cofactors, or the Trithorax homologue MLL. MLL encodes a clinically important component of a chromatin-modifying complex that directly binds and activates Hox gene expression96–100. Ten percent of adults and 70% of infants with acute myeloid leukaemia (AML) or acute lymphocytic leukaemia (ALL) have translocations affecting MLL101–103, and 2–5% of children or adults with ALL have translocations affecting PBX1 (REFS 102,104); direct activation of Hox genes by translocation also occurs but is less common. Leukaemias with increased Hox gene activity are considerably more difficult to cure101,105. Individual Hox genes have defined roles in normal haematopoiesis, as revealed by gene disruptions in mice, although these experiments probably underestimate their importance because of compensatory changes in Hox gene expression in the knockout animals106–110. Individual Hox genes also have defined roles in malignant haematopoiesis, and HOXA9 in particular mediates some of the essential properties of the leukaemic stem cell in MLL-dependent AML111–115. The Pbx and Meis cofactors are probably essential for leukaemogenesis, as is the motif in the Hox proteins that mediates interaction with Pbx proteins112,116, suggesting that complexes between these proteins regulate at least some genes that are crucial for the development of leukaemia. the TP53 and RB tumour suppressors can be studied, topics that are greatly important in cancer biology but that cannot be as easily addressed in the somatic tissues of C. elegans 95. Looking forwards to improve cancer therapy Elucidating the programmed cell death pathways of C. elegans has immensely improved our understand ing of how current cancer therapy works and has paved the way for developing new therapeutic strategies. Unexpectedly, many of the lineage-specific regulatory pathways controlling cell viability in C. elegans are evo lutionarily conserved in mammalian cell lineages and can be mutated in cells, resulting in aberrant survival. There seems to be a remarkable correlation between the transcriptional regulators of cell death in C. elegans and haematopoietic malignancies, including leukaemia. This may reflect the fairly extensive knowledge of the factors that control haematopoietic cell lineages, which are the best understood of the mammalian lineage programmes. Alternatively, transcriptional programmes directed by oncogenic transcription factors that are the products of chromosomal translocations often drive haematopoietic malignancies, and suppression of apoptosis by the fusion proteins may have a more important role in the develop ment of blood cancers. The comparatively much more complex genomes of solid tumours has made it difficult to identify chromosomal translocations that contribute to malignancy, but new genomics methods are now identifying an increasing number of recurrent fusion proteins. As our knowledge of mammalian lineage pro grammes develops, it will be interesting to see whether they too have transcription factors that directly regulate apoptosis as one aspect of the control of cell fate, which we predict will be the case. We are now in an exciting new era in which we are beginning to apply our knowledge of the biological pathways that control cell fate and programmed cell death to develop new cancer drugs. Looking forwards, we can anticipate the approval of therapies that directly target the apoptotic and cell fate pathways in human cancers. To effectively apply these new drugs we need to develop a comprehensive understanding of the mecha nisms that cancer cells depend on to avoid apoptosis and to promote self renewal and proliferation; develop strategies for identifying tumours and patients with specific classes of defects in the pathways involved; and generate molecules that target pathway components to kill cancer cells. 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Acknowledgements The authors would like to acknowledge Matt Porteus, Jim Amatruda, John Abrams and Bob Horvitz for their contributions to the manuscript. S.C. would also like to particularly thank Matt and Jim for their friendship. Competing interests statement The authors declare no competing financial interests. FURTHER INFORMATION WormAtlas: http://www.wormatlas.org/ Wormbase: http://www.wormbase.org/ ALL LINKS ARE ACTIVE IN THE ONLINE PDF www.nature.com/reviews/cancer © 2011 Macmillan Publishers Limited. All rights reserved