Embryonic genes in cancer

Embryonic genes in cancer Roser Calvo a and Harry A. Drabkin b " Hospital Universitari Germans Trias i Pujol, Medical Oncology Service, Badalona (Barcelona), Spain Division of Medical Oncology, University of Colorado Health Sciences Center, Denver, CO 80262, USA

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renal cancers [4]. Observations such as these led to the concept of specific 'gatekeeper' genes, whose inactivation (tumour suppressor genes) or activation (proto-oncogenes) in particular cell types is necessary for tumour development. These are, in essence, rate-limiting mutations effecting key regulatory pathways which occur in conjunction with other genetic and epi-genetic mutational events. Like APC mutations in colon cancer, Ptc is a good example of the 'gatekeeper' of basal cell carcinoma (BCC). Interestingly, many of the genes in the hh and interacting Wnt/wg pathways are either oncogenes or tumour suppressor genes [5] (Fig. 1). This is the case for the oncogenes GUI and Wnt (homologues of cubitus interruptus (ci) and wingless (wg), respectively) [6,7], or the tumour suppressor genes PTCH (human ptc) and APC (see below). Indeed, GUI was first identified by and named for its involvement in glioma formation [8], and Wntl is known to be involved in murine mammary tumourigenesis [9]. These genes, along with the Smad genes, which mediate TGFp1 signalling, are involved in the formation of a range of tumour types.

The connection between developmental genes and cancer has been a topic of great interest as the processes of proliferation, differentiation and tumourigenesis have long been thought to be inter-related. Pioneered by genome-wide mutational screens in Drosophila, and more recently coupled to powerful molecular technologies, many genes responsible for developmental alterations have now been incontrovertibly linked to human cancer. Among these are genes of the segment polarity class, the majority of which have been shown to encode components of the hedgehog (hh) and wingless (wg) pathways. Genes such as patched (ptc), a key regulator in Drosophila embryonic development, provide important examples of how normal development and tumourigenesis intertwine.

Oncogenes and tumour suppressor genes in embryonic development and cancer The notion that specific tumours within cancer syndromes share mutations of critical genes with their sporadic counterparts was established over a decade ago. Indeed, the discovery of mutations in the retinoblastoma gene (RE) in both hereditary and sporadic tumours [1] was followed by the observation of mutations in the adenomatous polyposis coli (APC) gene, not only in sporadic polyps [2] but in those patients affected by the familial adenomatous polyposis (FAP) syndrome, in which patients develop multiple adenomas and cancer. Likewise, RET, previously the only proto-oncogene found to be responsible for an inherited cancer syndrome, the multiple endocrine neoplasia (MEN), was also found to be mutated in sporadic medullary thyroid tumours [3]. More recently, activating mutations in the receptor tyrosine kinase, MET, have been shown to be responsible for some forms of hereditary and spontaneous papillary

Hedgehog signalling pathway: the patched gene in development and cancer The hh signalling pathway is a fundamental signal transduction system in embryonic development, being responsible for the patterning of a range of embryonic and adult tissues in both the fly and vertebrates. Hedgehog, which has three homologues in mammals (Sonic, Desert and Indian), is a unique cholesterol-tethered membrane ligand which binds to its receptor, patched. Dysregulation of this pathway results in the formation of several tumour types [6] and dysmorphology syndromes. From genetic screens, ptc and smoothened (smo) were identified as genes acting upstream of fused (fu), suppressor of fused (su (fit)), costal-2 (cos-2) and ci (homol207

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Fig. 1. Segment polarity gene pathways include oncogenes (bold) and tumour suppressor genes (underlined). The mammalian ortologues that act in HH signaling are shown. WNTs signal through mammalian DFZ2 homologues (HFZs) and cause the release of P-catenin/arm from the destruction complex. Free P-catenin/arm translocates into the nucleus and stimulates cell division, perhaps working as other mitogens through the activation of cyclin E/CDK2. TGF-P and BMPs signal through the TGF-P receptors (TGFBR) and through SMAD proteins (DPC4 and others). TGF-P inhibits cell division through the repression of phosphorylation of pRB (see text for details). Figure is modified from [5]. •<,

ogous to the vertebrate Gli genes). In Drosophila, known downstream genetic targets of the hh pathway include wg (homologous to the vertebrate Wnt genes), decapentaplegic (dpp, a member of the TGF{} superfamily most homologous to the vertebrate bone morphogenetic proteins, BMPs), as well as ptc itself. The term 'genetic targets' includes genes which are both directly (cell-autonomous) as well as indirectly regulated. Recently, several other genes have been shown to be responsive to hh signalling such as hip (for hedgehog interacting protein) [10] and WSB1/SWIP-1 [11]. Tumour formation occurs when mutation of regulatory genes leads specifically to the activation of downstream hh responsive genes. Ptc was first identified in a search for genes essential for embryonic development in flies [12]. Ptc is a component of the hh receptor complex acting as a repressive component of the pathway. Hedgehog protein (hh) must overcome pfc-induced repression to activate hh target genes. The characterisation of another Drosophila segment polarity gene, smo, added an important clue to the understanding of the hh pathway and the way in which ptc functions. It is now clear that in the absence of hh, ptc interacts with the seven-membrane spanning protein, smo (smoothened), rendering it inactive. However, when hh binds to ptc, inhibition of smo signalling is released initiating a signalling cascade that includes the intracellular components of the hh pathway, fu,

su (fu), cos-2 and the ci gene product. The end result is the transcription of downstream target genes, ptc, wg, and dpp (Fig. 2). Thus, in the absence of ptc function, there is constitutive signalling from smo, which results in the expression of downstream target genes. The recent identification of mutations in the human PTCH gene in BCCs indicated that HH signalling was important in human cutaneous carcinogenesis. Indeed, PTCH was identified as the locus responsible for the Nevoid Basal Cell Carcinoma Syndrome (NBCCS) or Gorlin's syndrome [13-15], an autosomally inherited disorder in which patients have multiple BCCs, various malformations, and other tumours including medulloblastoma, ovarian fibroma, meningioma, fibrosarcoma, rhabdomyosarcoma and cardiac fibroma. One PTCH allele is mutated in the germline of these patients [1,16-18], and the basal cell carcinomas in NBCCS individuals arise with inactivation of the remaining PTCH allele, consistent with PTCH acting as a tumour suppressor gene. The finding of PTCH mutations in a proportion of sporadic BCCs, in many cases with both alleles inactivated by either mutation or loss of heterozygosity (LOH) [19], supports this putative function. The estimated frequency of PTCH mutations in sporadic BCCs ranges from 12-38%, although this may be a low estimate based on the mutation detection method. To date, the molecular analysis of these

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mutations shows that most give rise to a truncated form of the protein. In addition to mutations in the PTCH gene, LOH in the region of chromosome 9q encompassing PTCH is observed in upwards of 50% ofBCCs[20]. It is speculated that BCCs arise through the inappropriate activation of the Shh pathway in the nonfollicular epithelium caused in many cases by loss of PTCH function in these human skin cells. Given that constitutive activation of smo is likely to upregulate transcription of hh target genes in the same way as PTCH inactivation, it is not surprising that activating mutations in the smo gene have been detected in 1020% of sporadic BCCs [21]. Since hedgehog itself is primarily responsible for activation of this pathway, it is feasible that it also may be mutated in associated tumours. Indeed, a single recurrent mutation in Shh was initially reported in a range of tumour types including BCC [22] but subsequent studies failed to detect this mutation, suggesting that it is extremely rare [23,24]. Other tumours that carry PTCH mutations include sporadic medulloblastomas and other primitive neuro-

ectodermal tumours [24] as well as the benign skin lesions tricoepitheliomas, esophageal squamous cell carcinomas and transitional cell carcinomas of the bladder [25-27]. Interestingly, in the mouse loss of only one copy (haploinsufficiency) appears to be sufficient for medulloblastoma development [28].

Wnt genes in growth control, development and carcinogenesis Among the most striking links between oncogenesis and development are those provided by wnt genes. In particular, the discovery that activating mutations in P-catenin are associated with a variety of human cancers has fuelled an extraordinary explosion of interest into the relationship between Wnt signalling and oncogenesis. In recent years, major advances have been made in understanding the wingless /wnt signalling pathway in Drosophila and in vertebrates [29-32]. Wnt genes are sources of differentiation-inducing signals during normal development events, but they also have the potential

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Fig. 2. The hedgehog signalling pathway, (a) In the absence of hedgehog (hh), the patched protein (ptc) inhibits signalling by smoothened (smo) and the intracellular components of the hh pathway (Fused [Fu], Suppressor of Fused [Su(Fu)], Costal-2 [Cos2], and Cubitus interruptus [Ci]), form a multimeric complex that associates with microtubules. The full-length activating form of the transcription factor encoded by Ci (Ci 155 ) is cleaved to yield a repressing form (Ci 75 ). The activity of protein kinase A (PKA) appears to promote Ci cleavage, possibly by phosphorylating Ci directly, (b) When hh binds to Ptc, the inhibition of smo is released. Smo activity promotes the dissociation of the complex of segment polarity proteins (Fu, Su(Fu), Cos2, Ci), normally associated with the microtubules. Cleavage of Ci is blocked and the full-length form of the protein (Ci 155 ) associates with the co-activator CBP to activate transcription of target genes. In vertebrates the role of Ci is achieved by complex interactions involving three Gli genes.

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cinoma and melanoma cell lines contain elevated levels of P-catenin; in many of these cell lines the increased stability of P-catenin may be attributed to point mutations in its amino terminus region that change the serine and threonine residues, thereby blocking its phosphorylation and subsequent degradation [33,34]. The W7VTs (human homologues of Drosophila wg), with a critical role in the formation of many differentiated cell types [31], are genes expressed in cell-specific patterns in early development. WNTs signal through mammalian homologues of the frizzled (fz) gene family (HFZs) of receptors [35]. A prevailing model of the wnt signalling cascade is summarised in Fig. 4 [36]. In the absence of a wnt signal, P-catenin/arm level is low due to the degradation promoted by glycogen synthase kinase 3P (GSK3) and APC. In the presence of a wnt signal, a fz receptor is activated and it, in turn, activates dishevelled (dsh). dsh then inactivates the GSK3 kinase, resulting in high levels of free P-catenin/arm in the cytoplasm upon its release from the destruction complex. P-Catenin then enters the nucleus where it interacts with the transcription factors TCF/LEF-1 (T-Cell Factor in mammals and Xenopus and Lymphoid Enhancer Factor-1 in Drosophila) to modulate transcription of wnt-responsive genes. The end result is the stimulation of cell division. Four proteins have been identified that directly promote the degradation of P-catenin: GSK3, Axin, APC, and P-TrCP/Slimb. These proteins comprise

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Ubiquitination sites Fig. 3. Structure of P-catenin. The central domain consists of 12 imperfect repeats, called arm repeats, that interact in an overlapping and mutually exclusive manner with APC, TCF, E-cadherin and Fascin. The amino-terminus contains multiple phosphorylation sites which are correlated with downregulation of P-catenin. Mutation of these potential phosphorylation sites or deletion of the amino-terminus produces a more stable and hence constitutively active B-catenin protein.The carboxyl terminus contains a transcriptional activation domain.

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to promote carcinogenesis through local effects on cell proliferation, particularly in the mammary gland. Indeed, mis-regulation of wnt signalling not only can cause developmental defects but is also implicated in the genesis of several human cancers [29]. At the core of the Wnt pathway is P-catenin, a multifunctional protein with independent roles in cadherin-mediated cell adhesion and Wnt signal transduction. P-catenin is central to the wnt signalling pathway and its activity is controlled by a large number of binding partners that affect its stability and localisation, ultimately modulating several developmental processes such as gene expression and cell adhesion. Activating mutations in P-catenin and in components regulating its stability contribute to the formation of certain tumours. P-Catenin displays a high degree of homology with the Drosophila segment polarity gene armadillo {arm). Its primary structure comprises an amino terminus, important for regulating the stability of P-catenin; a carboxy terminus, which functions as a transcriptional activation domain, and a central domain that consists of 12 repeats (referred to as arm repeats), through which P-catenin will interact with its binding partners APC, TCF, E-cad and Fascin (Fig. 3). The amino terminus domain of P-catenin contains a series of serine and threonine residues, which may be phosphorylated. Phosphorylation of these residues is thought to be the signal for the degradation of P-catenin by the ubiquitinproteasome pathway. Interestingly, several colon car-

Embryonic genes in cancer

In the presence of a Wnt/Wg signal

Fig. 4. Wnt/P-catenin pathway, (a) In the absence if a Wnt signal, Dishevelled (Dsh) is inactive (Dshj) and Dmsophila Zeste-white 3 or its mammalian homologue glycogen synthase kinase 3 (Zw3/GSK3) is active. GSK3 phosphorylates P-catenin. This phosphorylation is facilitated by Axin and APC. Phosphorylation of P-catenin targets it for ubiquitination by P-TrCP resulting in the proteasomal degradation of P-catenin. Meanwhile, some members of the TCF/LEF family of transcription factors are bound to their DNA-binding site in the nucleus acting as repressors through interaction with the co-repressors, Groucho, CBP and CtBP. (b) In contrast, in the presence of a Wnt signal, Dishevelled becomes activated (Dslu). Dsh, and GBP inhibit phosphorylation of P-catenin by GSK3. P-Catenin fails to be phosphoiylated and thus is no longer targeted into the ubiquitin-proteasome pathway. Instead, p-catenin accumulates in the cytoplasm and enters the nucleus. Nuclear P-catenin binds TCF/LEF and activates transcription of target genes.

the 'destruction' complex and act to maintain low steady-state levels of P-catenin in the cell. GSK3, the central player in this destruction complex, encodes a Ser/Thr kinase which phosphorylates the aminoterminal serine and threonine residues of P-catenin and thereby targets it for destruction, thus acting as a negative regulator of the Wnt pathway. APC was first identified as a tumour suppressor as mutations predispose individuals to develop colon carcinomas. It was later shown that APC binds directly to P-catenin and GSK3 [37]. Several colorectal carcinoma cell lines contain mutant APC and elevated levels of P-catenin; overexpression of wild-type APC in these cell lines significantly reduces the level of free P-catenin, suggesting that APC is a negative regulator of the wnt signalling pathway. Elevated free P-catenin in the cytoplasm, as is the case when the cell receives a wnt signal, leads to the nuclear accumulation of P-catenin where it associates with transcription factors of the TCF/LEF class and acts as a transcriptional activator. Interestingly, in the absence of P-catenin, TCF still can bind to its target DNA. In such a case, however, TCF not only fails to activate transcription of target genes but can function as a transcriptional repres-

sor by its binding of transcriptional repressors such as groucho and the related TLEs (transducin-Iike enhancer of split). In addition to its role in regulating gene expression, P-catenin can also affect cell adhesion. By binding to both E-cadherin (E-cad) and a-catenin simultaneously, P-catenin links the adherens junction to the cytoskeleton of the cell. However, the degree by which wnt signalling effects cell adhesion through p-catenin is not yet clear. Certain Wnt ligands, such as Wnt5a, affect cell migration and inter-cellular adhesion in a calcium dependent manner through a seemingly non-P-catenin mechanism. In an indirect fashion, wnt signalling appears to modulate cell adhesion since the mouse E-cad promoter contains a TCF-binding site and it has been shown [38] that activation of the wnt signalling pathway leads to transcriptional activation of E-cad. Whether this effect is mediated directly through the TCF/P-catenin complex remains to be seen. APC may also have an important role in cell adhesion [39], and like ptc, it is a cytoplasmic molecule that controls a complex signalling cascade. It seems reasonable to speculate that the first effects of the lack of proper response of

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In the absence of a Wol/Wg signal

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TGF-P pathway disruption in cancer Besides WNTs, the hh signalling pathway influences cellular proliferation via a different family of effector proteins, the transforming growth factor-^ (TGF-P) family and related cytokines —such as activins and bone morphogenetic proteins (BMPs) [43]. These molecules, by controlling the expression of cell cycle regulators, cell adhesion molecules, and differentiation factors, regulate cell fate and are therefore important for the development and maintenance of most tissues [44]. Since the TGF-p cytokines were first identified as inhibitors of epithelial cell proliferation [45], manifested by Gl-phase arrest, terminal differentiation, or induction of apoptosis, it has been anticipated that understanding the mechanism of TGF-p action would also shed light on the events leading to neoplastic transformation. Members of the TGF-p family of ligands (mammalian homologues of Drosophila dpp) signal through heteromeric complexes of two transmembrane serine/threonine kinases, the type I and type II receptors [46], which will interact with the SMAD proteins, mediators of the TGF-P signalling. Upon their phosphorylation in their carboxy-terminal domain by receptors, SMADs become activated; they

then associate to a second group of collaborating SMAD proteins (co-SMAD) and move into the nucleus, where they interact with transcription factors, such as FAST-1, and stimulate target gene expression. Thus, SMAD proteins directly transmit TGF-P signals from the cell surface receptors to the nucleus (Fig. 5). The only known member of the co-SMAD group in vertebrates is smad 4, originally identified as the product of the deleted in pancreatic cancer locus 4 (DPC4) tumour suppressor gene [47], which is mutated or deleted in a high proportion of pancreatic cancers and in a smaller proportion of other cancers. Given the growth inhibitory effects of the TGF-P signalling pathway, it is reasonable to speculate that its disruption may predispose to or cause cancer. This idea has been confirmed in recent studies. Inactivation of the type II TGF-P receptor has been detected in some tumour types and may be associated with the RER+ (replication error) phenotype [48]. These data suggest that type II TGF-P receptor may function as a tumour suppressor gene. A significant number of inactivating mutations have also been found in SMAD genes derived from human cancers, smad 4/DPC4 is either deleted or mutated in a large proportion of pancreatic cancers [47]. smad 2 is also mutated in a number of colon and head and neck carcinomas [49-51]. The majority of the identified missense mutations map within the carboxy-terminal domain of the protein, which not only mediates receptor-mediated phosphorylation and activation of SMADs but contributes essential proteinprotein interactions to the SMAD pathway and therefore trans-activation functions. Thus, tumourigenic or developmentally inactivating missense mutations in the carboxy-terminal domain abolish specific SMAD interactions that are vital for the pathway. Tumourderived missense mutations have also been identified in the amino-terminal domain of the protein. This region is involved in the process of self-inhibition of SMADs, which occur through direct interaction between the amino-terminal and carboxy-terminal domains. Tumour-derived mutations in the aminotenninal domain of smad 2 or smad 4/DPC4 increase their affinity for their respective carboxy-terminal domains, thereby preventing smad 2-smad 4 association and TGF-P signalling [52]. In addition, many cancers, including most pancreatic tumours, show allelic loss at a site on chromosome 18q that contains the genes DPC4 and DCC {deleted in colorectal cancer), a tumour suppressor for colorectal cancer. Finally, TGF-P signals can regulate the retinoblastoma protein (pRB) in the nucleus through the regulation of pl5/pl6. Diverse inactivating mutations in the RB gene occur in many types of cancer, and func-

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these proteins to signals from neighbouring cells may be abnormal adhesion of the cells, resulting in abnormally proliferating clusters of cells from which other genetic hits can occur, leading to tumour progression. Wnt-1 was originally identified as a preferred integration site for the mouse mammary tumour virus in breast carcinomas [40]. Although much evidence points to the possibility of Wnt-1 acting as an oncogene, mutations in Wnt-1 have not been linked to cancer in humans. However, since mutations in several components of the Wnt/P-catenin pathway are implicated in tumour formation, it is likely that Wnt-1, or other wnt proteins capable of activating the Wnt/p-catenin pathway, may act as oncogenes in humans. In addition, a number of studies provide evidence that WntSa is able to antagonise the Wnt pathway [41]. The ability of WntSa to suppress transformation by Wnt-1 in vitro suggests that WntSa may act as a tumour suppressor. However, the role of WntSa in carcinogenesis remains unclear, since current in vivo evidence appears to conflict with the concept of WntSa as a tumour suppressor, given the decrease in cell growth and proliferation observed in mice after removal of the WntSa function, suggesting a positive role for this gene in the regulation of cell growth and differentiation [42].

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Fig. 5. TGF-P signalling pathway. The TGF-p family of ligands binds to and activates distinct combinations of type I and type n serine-threonine kinase receptors leading to a transient association with specific receptor-activatable SMADs. These SMADs (Smad 1, 5, 8, 2, and 3, in vertebrates) become phosphorylated (p) by the activated type I receptors, then associate with a co-SMAD (e.g. Smad 4) and move into the nucleus. In the nucleus the SMAD complexes associate with DNA-binding transcriptions factors (e.g. FAST-1, or in this hypothetical setting, A, B, and C) leading to active transcriptional complexes and stimulation of target gene expression. The combination of activated target genes and the particular cellular environment will determine the kind of biological response. (Figure is adapted from [46].)

tional pRB can suppress the tumourigenicity of these cells. Whereas the phosphorylated pRB protein forms complexes with the E2F transcription factor and activates genes required for cells to pass the restriction point in the Gl phase of the cell cycle [53], underphosphorylated pRB prevents progression through the cell cycle. TGF-P may inhibit growth by preventing pRB phosphorylation. The pRB is normally phosphorylated by at least two cyclin/cyclin dependent kinases (CDK) complexes, which in turn can be inhibited by other proteins such as pl5 and pi6, both identified as tumour suppressor genes. Such repressors, pl5 and pi6, through their interaction with the cyclin D-CDK4/6 complex, prevent pRB phosphorylation and cell cycle progression, helping to explain why the loss of p l 5 / p l 6 is a common event in tumours. TGF-p is known to affect the expression and/or activity of pl5/pl6, potential effectors of TGF-P-mediated cell cycle arrest [54]. In addition, the CDK inhibitor p21 can block the activity of both CDK2 and CDK4/6. DNA damage due to chemical agents or radiation leads to the accumulation of p53 protein. p53 induces the expression of p21 and prevents the cell from entering S phase with a damaged

genome. Cells that cannot repair their DNA can be induced to undergo apoptosis.

Homeoboxes, homeodomains and cancer Another group of genes first isolated as determinants of development, the homeobox-encoding genes, have also been shown to have either oncogenic or tumour suppressor potential. Homeobox genes control anterior-posterior cell fates in multiple tissues of organisms as different as flies, mice, worms and vertebrates [55]. Individual genes within the homeobox gene family share a 183-nucleotide DNA segment termed the homeobox [56]. The homeobox encodes a 61-amino acid protein segment called the homeodomain, a region able to bind specific DNA sequences. Homeodomain proteins have been shown to be transcription factors, with either positive or negative effects on the expression of target genes, which will lead to the formation of characteristic structures along the body axis. The search for homeobox downstream targets is currently an important avenue in the investigation of homeobox gene func-

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Fig. 6. Hox genes nomenclature system. Hox genes in vertebrates are clustered in four complexes (A-D). Based on sequence similarity Hox genes can be sorted into 13 'paralog' groups, each group having, in most cases, a representative in each complex. The order of paralogs along the chromosome is maintained in the four complexes. Genes within the complexes are numbered according to the paralog group they belong, from 1 (at the 3' end) to 13 (at the 5' end). In several cases a representative of a paralog group is absent from a complex, in which case the corresponding gene number is omitted (blank spaces in the figure). H = nomenclature for human. M = nomenclature for mouse. The genes required farthest anterior in the developing animal are at the 3' end of the complex. Genes with more posterior domains of action are located farther 5' in the complex. Thus, in the figure, paralog groups are demarcated by 'anterior' and 'posterior'. Drosophila counterpart homeotic genes are also shown.

tion. Few homeobox target genes are known in flies and almost none in vertebrates, so the mechanism by which homeobox genes control morphogenesis remains incompletely understood. Most Hox targets identified to date have been found in the visceral mesoderm of Drosophila, where they are required for the development of the midgut constrictions. A hierarchy of target genes regulated by the Hox genes Ultrabithorax (Ubx) and abdominal A (abdA) to dictate morphogenetic decisions in the midgut has been identified. In particular, these target molecules include WG and DPP proteins (components involved in transducing wg and dpp signals), and the transcription factor Teashirt (Tsh) [57]. To date, a plethora of mammalian homeobox genes have been reported, among which 38 are located in four clusters (A-D) and are referred to as 'Hox' genes. Each cluster contains 9 to 11 genes which are numbered beginning with those located at the 3' end of the complex (Fig. 6). In cancer, the deregulation of homeobox genes has been most convincingly demonstrated in leukemia. For example, the pre-B cell t(l;19) translocation fuses E2A with the homeobox gene, Pbxl, and the T-cell t(10;14) results in overexpression of Hoxll

[58-60]. In acute myelogenous leukaemia, the human Trithorax or MLL gene, which normally helps to maintain Hox gene expression, is a frequent target of chromosomal rearrangements. Recently, Joh et al. [61], demonstrated that a chimeric MLL-LTG9 protein led to the inhibition of HoxA7, HoxB7 and HoxC9 expression in mouse 32Dcl3 myeloid cells. In an experimental setting, a high proportion of mice transplanted with bone marrow cells overexpressing either HoxB8, A9, A10 or B3 eventually developed AML [62-64]. In solid tumours, rearrangements of Hox genes have not been reported. However, expression surveys have noted differences between normal and tumour samples in kidney, colon and lung carcinomas [65-68]. In melanomas, Card et al. [69] demonstrated that HOXB7 was constitutively expressed in 25/25 melanoma cell lines and that antisense HOXB7 inhibited cellular proliferation and the expression of the basic fibroblast growth factor (bFGF). In the gut, the caudal homologue, Cdx-2, functions as a tumour suppressor gene. Not only is its activity downregulated by mutant Ras [70] but mice lacking Cdx-2 develop multiple gastrointestinal polyps [71]. Moreover, in one case of a human colon carcinoma both

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Embryonic genes in cancer

CDX2 alleles were mutated [72]. While expression and alterations of Cdx-2 were previously confined to the gut, the human CDX2 gene has recently been involved in a t(12;13) chromosomal translocation along with TEL [73,74] . Further analysis of HOX genes in different cancer types combined with other interactive components such as the WAT genes will lead not only to a better understanding of carcinogenesis but will provide important prognostic and diagnostic molecular markers. Also, since these pathways are interactive during normal development, an integrated approach to their analysis should be particularly informative.

The cyclooxygenase (COX) enzymes catalyse a key step in the conversion of arachidonate to PGH2 (prostaglandin H2), the immediate substrate for a series of cell specific prostaglandin and thromboxane synthesis. Prostaglandins play key roles in several biologic processes such as regulation of immune function, kidney development, reproductive biology, and gastrointestinal integrity [75]. COX-1 and COX-2, the two COX isoforms, differ mainly in their expression patterns; while COX-1 is expressed in most tissues, COX-2 is absent but is induced by numerous physiological stimuli. Cyclooxygenase activity can be inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), the prototype of which is aspirin. Although it has been suggested that COX-1 is the 'housekeeping' isoform of cyclooxygenase, and that COX-2 acts in a pro-inflammatory fashion, being rapidly inducible in response to numerous stimuli, the pro-inflammatory role of COX-2 has recently been questioned since it has been demonstrated that COX-2 generated prostaglandins may actually enhance resolution of inflammation [76]. Phenotypic analysis of Coxl and Coxl null mice yields information about the role of cyclooxygenases in development, and it is clear that the role of these genes in a developing organism is much more complicated than was once thought. Indeed, given that COX-1 generated prostaglandins appear to be cytoprotective to the gastric mucosa, it was hypothesised that Coxl null mice might exhibit gastric pathology. Surprisingly, disruption of Coxl not only did not result in gastrointestinal abnormalities but null mice were generally healthy [77]. In contrast, Cox2 null mice show reproductive anomalies and defects in kidney development [78-80]. Some of the same processes occur in both uterine

decidualisation (sometimes referred to as pseudomalignant state) and cancer, allowing COX-2 parallels to be established between cancer and development. Indeed, during blastocyst implantation and decidualisation, a vascular network must be established to support the nutritional needs of the developing embryo. In a similar way, expansion of a tumour mass requires a vascular network to support its metabolic demands. There is evidence that COX-2 generated prostaglandins participate in angiogenesis, common to both development and cancer. It has also been demonstrated that maximal tumour expansion occurs when the tumour escapes immunologic surveillance. Like tumour cells, the blastocyst, also considered foreign material, must escape immunologic surveillance to survive. In this regard, COX-2 generated prostaglandins have been demonstrated to be immunosuppressive. There is ample genetic and pharmacologic evidence to implicate COX-2 in neoplasia. It has been found that NSAIDs, inhibitors of cyclooxygenase, are chemopreventive for colon cancer. If NSAIDs do reduce the risk of developing colon cancer, by what mechanism do they achieve this effect? Certainly, the COX enzymes are known targets of NSAIDs. If COX were shown to contribute to tumour growth, then one might expect that levels of downstream metabolites (i.e. prostaglandins) would be increased in tumours, and that there would be abnormal COX expression. Consistent with this hypothesis, COX-2 is overexpressed in 50% of benign polyps and 8 0 85% of adenocarcinomas [81]. Mice heterozygous for an APC mutant allele develop hundreds of intestinal polyps. Interestingly, offspring from cox2 null by ApcA116 matings exhibit an 86% reduction in polyp number when compared to offspring from control animals [82], thus providing genetic evidence that COX-2 contributes to tumour formation or growth. The precise contribution of COX-2 to neoplastic growth has not been elucidated. However, there is some evidence that COX-2 may blunt the apoptotic response in tumour cells and play a direct role in tumour cell growth. Additionally, there is evidence that COX-2 may indirectly modulate tumour expansion since it has been demonstrated that COX-2 induces angiogenesis in vitro and can also downregulate natural killer T-cell function.

Summary Over the past several years, great strides have been made in our understanding of the mechanisms and functions of many signalling pathways involved in

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both development and oncogenesis. It is now clear that some proteins/genes essential for cell differentiation can also provide signals for growth control, suggesting that these genes may be mutated in tumours; this knowledge can help further the search for new targets for chemotherapy. However, many questions about how dysregulation of signalling pathways leads to neoplasia are still unanswered. With the dramatic increase in the number of researchers examining those questions, answers will not be far away. References

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