cancer genetics

Cancer genetics

Luisa Lanfrancone, Giuliana Pelicci and Pier Giuseppe Pelicci

University of Perugia, Perugia, Italy

The accumulation of multiple genetic changes underlies the process of tumorigenesis, and both dominantly acting oncogenes and inactivated tumor suppressor genes co-exist in the same tumor. Individual mutations are thought to independently contribute to the kaleidoscopic transformed phenotype. Several examples have now been found of mutations in genes that, through different mechanisms, act on central control points either to ensure genome stability or to regulate the common pathways that signal cell proliferation, survival and differentiation. Mutations at these loci may have multiple, and

apparently unrelated, phenotypic consequences.

Current Opinion in Genetics and Development 1994, 4:109-l 19

Introduction

Cancer cells contain many genetic alterations that accumulate gradually during the process of tumor progression. Although the nature and order of appear- ance is not fully understood for any type of neoplasia, their cumulative number is considered crucial for the expression of the transformed phenotype 1141. The transformed phenotype abounds in structural and functional alterations that are thought to be acquired independently during the development of the tumor. Finally, there is a breakdown in the regulation of cell proliferation, cells multiply without restraint, cell-cell interactions become modified, and the neoplastic cells invade the surrounding tissue and metastasize.

No coherent picture of cancer genetics can be formed unless we know the triggering cause and the mechanism(s) responsible for the accumulation of multiple genetic lesions in the neoplastic cell, the physiological function of the various ‘cancer genes’, and the contribution each and every genetic aberration makes to the transformed phenotype of each single tumor. It is not clear whether the accumulation of multiple genetic events in the developing tumor cells is merely the consequence of random genetic changes coupled with intense selection, or whether enhanced genomic instability in the neoplastic cells favours, or is required for, the onset of these changes 121. Numer- ous hereditary syndromes predispose to cancer, and in vitro studies on non-neoplastic somatic cells from such patients have shown that they are, indeed, genetically

unstable. Genomic instability, therefore, may be an in- heritable genetic trait associated with a predisposition to cancer, and similar genetic defects may be operative even in spontaneous tumors 151.

During the past 20 years, many experimental data have been gathered that implicate loci physiologically involved in regulation of cell proliferation as being the genomic defects of cancer cells. On the basis of their biochemical activity, where known, and the type of genetic alteration that affects the neoplastic cell, ‘cancer genes’ have been divided into two groups: proto- oncogenes and tumor suppressor genes. The proto- oncogenes code for proteins that are components of the cellular signalling pathways of growth stimula- tory signals (such ‘as hormones, hormone receptors, cytoplasmic signalling proteins, and nuclear factors). Proto-oncogene alterations, such as point mutations, chromosome translocations and gene amplification, lead to a gain-of-function that accelerates cell division. Because they are able to transform cells despite the expression of residual normal alleles, the mutant alleles of proto-oncogenes are termed dominant. Tu- mor suppressor genes are less well understood, but although the proteins they encode also seem to be physiological components of cellular signalling pathways (such as receptors, cytoplasmic signalling proteins, and nuclear factors), they inhibit, rather than favour, cell proliferation by arresting the progression through the cell cycle, blocking differentiation, or in- ducing senescence or death. As would be expected from their growth-suppressing function, genetic alter-

Abbreviations AT-ataxia-telangectasia; FAP-familial adenomatous polyposis; FCC-familial colon cancer;

(F)MTC-(familial) medullary thyroid carcinoma; CADD-growth arrest and DNA damage inducible; HNPCC-hereditary non-polyposis colorectal cancer; MDM-murine double minute;

MEN-multiple endocrine neoplasia; NF-neurofibromatosis; PML-promyelccytic leukemia; RAR-retinoic acid receptor; RR-retinoblastoma gene; RXR-retinoid X receptors; VHL-von Hippel-Lindau; XP-xeroderma pigmentosum.

8 Current Biology Ltd ISSN 0959-437X 109

110 Oncogenes and cell proliferation

ations in tumor suppressor genes (point mutations or deletions) lead to loss-of-function and are considered to be recessive at the cellular level 12-41.

This review will deal with some of the most important findings made during the past twelve months.

The control of genomic stability and the transmission of ‘cancer susceptibility

Increased genomic instability in cancer cells It has been clear for some time, from the high number of chromosome aberrations encountered in virtually all types of neoplasias, that abundant genetic defects accumulate in neoplastic cells. Only recently, however, has ‘it been shown that in some tumors, as high a frequency as one mutation in every l&30 kb of the genome can be found early in tumor development KP-8.1. Using microsatellite analysis or a similar ap- proach, three research groups have identified diffuse genetic alterations in a high percentage (12-28%) of randomly selected colon cancers 16.-S*]. The fact that microsatellite alterations have been encountered in both the superficial and profound parts of colon carcinomas, as well as in preneoplastic lesions (e.g. colon adenomas), indicates that they are probably acquired early during tumor development [@I. These alterations are unlikely to be specific to colon cancers, since other groups have already reported microsatellite alterations in other types of tumors (e.g. ovary carcinoma and multiple myeloma), though with a lower incidence 19-111. The build-up of mutations in the tumor cell genome could derive from one or more genes that, when defective, cause genomic instability at numerous loci.

Increased genomic instability is a genetic trait There is much unequivocal experimental evidence to demonstrate that genomic instability is not the consequence of a transformed phenotype, but a recessive, independently acquired genetic trait. First, the frequency of the phenomenon of gene amplification, studied as a type of genomic instability, is very low (<log) in normal diploid fibroblasts, whereas it is high (10-5-103) ‘n 1 preneoplastic and neoplastic cell lines 112,131. Second, the ability to amplify is suppressed in somatic cell hybrids of highly tumorigenic cells and normal cells 114’1. Third, the three recessive phenotypes - that is immortal, tumorigenic, and amplification potential - that can be identified by analyzing malignant and normal somatic cell hybrids, segregate independently 114*1. It would seem, therefore, that normal cells contain one or more genes that modulate the stability of the genome, and that alterations in these genes increase genomic instability.

Genes controlling genomic stability: ~53 Two recent papers have established a correlation between the expression levels of the tumor suppressor

gene ~5.3 1151 and the ability of a cell to undergo gene amplification [16’,17’1. Three types of non-tumorigenic cells have been studied for their potential to amplify the endogenous genes: fibroblasts with wild type ~53, p53+/+; fibroblasts heterozygous for mutant ~53, PT.?+/-, from mice heterozygous for a null ~5.3 allele or from patients with the Li-Fraumeni syndrome car- rying an heterozygous mutated ~53 allele; and fibroblasts homozygous for mutant ~53, p53/-, from mice deficient in ~53 or Li-Fraumeni fibroblasts which have lost the remaining wild type allele during in vitro cul- ture. Whereas no gene amplification can be detected in p53+/+ and p53+/- cells, it is increased in p53-/- cells (with a frequency of 10-5-10-Q The acquired ability to amplify appears to be a direct consequence of the loss of ~53, because gene amplification was no longer detectable when the wild-type ~53 was reintroduced in p53-/- cells 116’,17*1.

The mechanism by which ~53 controls genome stability is very probably linked to its function as a cell cycle control protein 1181. An important function of the cell cycle is to control the response to genetic damage and external signals. At least two stages of the cell cycle are regulated in response to DNA damage: the Gl-S and G2-M transitions. These transitions are control points (checkpoints) where the integrity of the genetic mate- rial is checked and, though not essential for cell viability, they increase the fidelity of the division process. Following damage to DNA, for example after yirradi- ation, both prokaryotic and eukaryotic cells pause be- fore proceeding from the Gl to the S phase (Gl arrest) or from the G2 to the M phase (G2 arrest>. It is thought that this slowing of the cell cycle allows the damaged DNA to be repaired, so avoiding replication of a faulty DNA template (Gl arrest) and segregation of damaged chromosomes (G2 arrest), which limits the propagation of hereditable genetic errors. There is evidence that ~53 is essential for the Gl-S checkpoint: progression from the Gl to the S phase is frequently blocked in cells that constitutively express transfected wild type ~5.3 1191, ~53 protein levels are increased in cells that undergo a y-irradiation-induced Gl arrest, and cells deficient in ~53 lose the Gl arrest, but are arrested in G2 120,211. Correlations between ~53 protein levels, amplification capacity of endogenous genes, and alterations in G1-S transition, further support the idea that ~53 regulates genome stability through its Gl-S checkpoint function. In fact, p5.3-/- cells acquire gene amplification capacity, but are unable to slow Gl-S progression in response to genotoxic agents 117’1. Therefore, ~53 appears to be an important component of a DNA damage responsive control function between the Gl and S phases.

The putative function of ~53 in the control of genome integrity agrees well with its presumed role in both spontaneous tumorigenesis and predisposition to cancer. Alterations in the ~53 gene are probably the most frequently encountered defects in human tumors (up to 50% of all tumors) 1151. The type of genetic alteration found in ~53 varies enormously in different tumors, including loss of both alleles, loss of one al-

Cancer genetics Lanfrancone. Pelicci and Pelicci 111

lele and mutation in the other, and mutations in only one allele. In all cases, the alterations are thought to result in the loss of normal p53 function, and when normal and mutated ~53 genes co-exist, the mutant protein appears to be able to bind and inactivate the remaining wild type product (dominant negative mutation) 122,231. In line with this view, mice that express mutant ~53 genes, and mice with a p53+/- or p53-/- genotype, develop tumors with greater frequency than their p53+/+ littermates 124,251. Similarly, subjects that carry a mutation in the germline ~53 (Li-Fraumeni syndrome) have a higher risk of developing various rnesenchymal and epithelial neoplasias at multiple sites early in life, and are apparently able to transmit the predisposition to cancer as a dominant trait 1261. The heterozygous mutant ~53 allele may predispose individ- uals affected by the Li-Fraumeni syndrome to develop cancer by conferring them with a greater probability of acquiring genomic rearrangements, including loss of the wild-type ~5.3 allele, which may further acceler- ate the accumulation of genetic lesions. Interestingly, cultured Li-Fraumeni fibroblasts tend to inactivate the normal ~5.3 allele and accumulate structural chromosome aberrations at an elevated rate 116’,17’1. Tumors that arise in subjects with the Li-Fraumeni syndrome are homozygous for the ~5.3 mutation, and in some, the tumors seem to be associated with exposure to carcinogens, including ionizing radiation 1261.

Genes involved in the ‘~53 signalling pathway’ Tumcrig:. lit cells with structurally and functionally wild type p53 have the potential to amplify at a high frequency 117.1, and this suggests that other proteins involved in the p53 signalling pathway, or an alternative pathway, may modulate the frequency of gene amplification. The p53 protein binds to DNA in a sequence- specific manner 1271, behaves as a transcription factor (with an activator function on some genes that contain p53-binding sites, and with an inhibiting function on other, TATA-controlled genes 1282911, and interacts physically with other cell factors 122,27,281. Numerous other gene products, including those that bind p53 and the p53 target genes, may, therefore, be implicated in the p53 signalling pathway.

The growth arrest and DNA damage inducible protein, GADD45, like ~53, is induced by ionizing radiations. This response to ionizing radiation is greatly reduced in cells from patients with ataxia-telangectasia (AT), a human autosomal recessive disorder associated with a markedly increased incidence of cancer. As with p53-/- cells, there is no ionizing radiation induced Gl arrest or increase in p53 protein levels in AT cells. GADD45 could be a p53 target gene, as demonstrated by the fact that GADD45 expression is induced by ionizing radiation only if p53 is intact, and that the wild type, but not the mutant, p53 binds specifically to the GADD45 gene 1301. Probably the gene(s) that are defec-

tive in AT are required for activating the p5YGADD45 signalling pathway.

The WAFl gene has been isolated recently in a subtrac- tive hybridization screen to identify endogenous genes regulated by ~53. WAFl expression is activated by wild type, but not mutated, p53 and is probably mediated by direct interaction of p53 with a p53-binding site within the transcription regulatory region of the WAFl gene 1311. The WAFl gene product is a 21 kDa protein that corresponds to an inhibitor of the Gl cyclin-dependent kinases which was simultaneously and independently identified by a number of research groups working on the characterization of multi-protein complexes con- taining cyclin dependent kinases (Cc&s), cyclins and ~21 132-341. Cdks work by inactivating negative regu- lators of the cell cycle (for example the retinoblastoma gene product, pRb) whose functional state varies during the different cell cycle phases, partly as a result of the type of cyclin they associate with, which is thought to be crucial in the regulation of both the Gl and G2 checkpoints. The WAFl gene product (also known as Cipl, Cdil and ~21) associates with Cdk2, a specific Cdk that complexes with Gl cyclins (Dl, D3 and E) and is functionally implicated in G1-S progression. The WAFl gene product functions as a potent inhibitor of Cdk2 activity on several substrates, including pRb. It has been demonstrated in a number of ways that the WAFl gene product is a p53 target, active along the same pathway as ~53: it is not expressed in p53-/- cells, its expression is increased by UV irradiation and this response is lost in p5+/- cells, and it induces growth arrest 131,341. The induction of ~21 by p53 and the consequent inhibition of cyclin-dependent kinase activity may be crucial for p53-induced Gl arrest in normal cells, and alterations in this pathway in cancer cells with inaqtivated p53 alleles could contribute to growth deregulation. Establishing whether genetic alterations in ~21 are, by themselves, able to contribute to the development of tumors should be a high priority aim.

The murine double minute 2 (MDM2) gene product binds ~53, inhibits its function as a transcription factor 1351, and is frequently amplified in human sarcomas 1361. Interestingly, sarcomas with MDM2 amplifications do not carry ~53 mutations 1361. The oncogenic activity of MDM2 may lie in its capacity to bind p53 Ufl and, when constitutively expressed, to functionally inactivate p53 1381 through mechanisms similar to those adopted by virally encoded oncoproteins, such as the simian virus 40 T antigen, adenovirus ElB and human papilloma virus E6.

Replication protein A (RPA) is a multisubunit complex able to bind single-stranded DNA. It is involved in DNA replication and, perhaps, DNA excision repair. The p53 protein interacts with RPA, and interferes negatively with its capacity to bind single-stranded DNA in vitro 139’-41*1. The capacity of p53 to regulate RPA and, potentially, to interfere with replication, may be one of

112 Oncogenes, and cell proliferation

the mechanisms underlying the control by ~53 of the entry to the S phase.

The familial colon carcinoma gene Other p53-independent pathways could be implicated in the genesis of increased genetic instability in tumor cells. The gene responsible for the hereditary non-polyposis colorectal cancer (HNPCC), termed the familial colon cancer (FCC) gene, is a candidate [8*,42*1. HNPCC is one of the two principal condi- tions that predispose a subject to colorectal and other types of cancer - the other being familial adenomatous polyposis (FAP), characterized by thousands of benign tumors lining the entire large intestine 151. HNPCC accounts for 4-13O41 of all colon cancers, but cannot be distinguished from sporadic cases at physi- cal examination. When microsatellite alteration analysis is carried out on colorectal cancer DNAs from HNPCC subjects, the incidence was found to be 13% in the sporadic cases and 7080% in the familial cases, which seems to indicate that genomic instability is a component of the familial cancer phenotype 18’1. The p53 protein is probably not responsible for this phenotype, since the presence of p53 mutations is inversely cor- related with the genomic instability phenotype. FCC, a gene on chromosome 2~15-16, is a candidate, as it has been implicated in the pathogenesis of predisposition to colon cancer in HNPCC by genetic linkage analysis 18’,42*1. The FCCgene is probably not a typical suppressor gene, as no loss of FCC-linked anonymous markers has been documented in colon cancer DNA. Further, on the basis of the high frequency and type of mutation (mostly small deletions) that arise in HPNCC tumor DNAs, FCC would seem to be a gene encoding a factor involved in the replication/repair mechanisms that, when defective, cause increased genomic instability. A candidate FCCgene (hMSH?l has recently been identified as a human homolog of the bacterial MufS and yeast MSH mismatch repair genes. Somatic and germline mutations of the gene have been identified in colon cancer cells with diffuse microsatellite alterations 143,441. Although the biochemical activity of the hMSH2 gene product has not yet been demonstrated, colon cancer cells with diffuse microsatellite alterations have been shown to possesss a defect in mismatch repair 1451.

Genes that regulate DNA repair/transcription processes It is not surprising that genes which take part in the process of DNA synthesis and repair should increase genetic instability when they mutate, and also be ca- pable of transmitting a predisposition to cancer 151. There is a group of rare human recessive disorders thought to derive from defects in DNA repair that are characterized by an intrinsic susceptibility to malig- nancy and cellular hypersensitivity to the action of DNA-damaging agents: AT, xeroderma pigmentosum (XP), Fanconi anemia, Bloom syndrome and Cock- ayne syndrome. DNA repair is a process programmed

to remove DNA lesions that accumulate following exposure to toxic agents like radiations or chemicals. If the DNA lesions were not eliminated, they would interfere with both replication and transcription, and so compromise cell viability and/or cell division fidelity. All of these hereditary disorders, with the exception of Bloom syndrome, are genetically very heterogenous, which means that many different genes can lead to similar clinical and cellular phenotypes. For example, genetic complementation analysis has revealed eight complementation groups (A-H) in XI’, one of the genetically best studied disorders. Some genes involved in the pathogenesis of XP and other DNA repair disorders have been identified and, of these, some are homologous to yeast genes that take part in DNA repair. The ERCC2 gene, which is able to correct the group D XI’ (XI’-D) cell phenotype, can complement yeast RAD3 mutations 1461, the XPGC gene, which corrects the XP-G defect, is homologous with yeast RAD2 1471, and the XI’-C complementary XPCCis gene homologous with RAD4 1481. Certain of the genes involved in these disorders are homologous to DNA helicases, for example ERCC2, ERCCG (which corrects the repair defect of Cockayne syndrome group B cells), and ERCC3 (which specifically corrects the XI’-B cell phenotype) 146,491.

The physiological function of these genes, and the effects exerted by their mutations, are probably more complex than just an involvement in the DNA repair process. The phenotype of all these hereditary disorders is very complex; xP, for example, may manifest pigmentation abnormalities and progressive neurologi- cal degeneration. It is worth noting that the ERCC-3 gene product has been demonstrated to be part of the basal transcriptional factor TFIIH 150’1, thereby establishing an important functional connection between transcription processes and DNA repair, and suggesting that subtle alterations in general transcription may be implicated in certain hereditary disorders that, among other phenotypical defects, predispose to cancer.

Other cancer susceptibility genes

A number of cancer susceptibility syndromes are known to be caused by heterozygotic defects in a variety of tumor suppressor genes other than ~53: FAP, neurofibromatosis type 1 (NFI), retinoblastoma (R/3) and Wilms tumor (WT-I) genes (for review, see 13,411. However, little is known about the function of these tumor suppressor genes, or the mechanisms of tumor promotion. An important step forward in understanding their physiological function has come from the demonstration that the FAP protein associates with the a and j3 catenins, thereby establishing a vital link between FAP, cell adhesion and (possibly) tumor initiation 151,521. The a and p catenins have been implicated as acting in the signalling pathway of cadherins, transmembrane proteins involved in mediating adhe-

Cancer genetics Lanfrancone, Pelicci and Pelicci 113

sion and communication between cells of the epithelial layers and anchorage to the cytoskeletal actin.

Cancer predisposition due to alterations in tumor suppressor genes follows an apparently dominant pattern of inheritance. The rate-limiting event for tumor development is thought to be the inactivation of the normal allele. This interpretation of the genetics of tumor suppressor genes is based on the retinoblastoma model, but is (probably) limited. In some cancer predisposing syndromes, such as FAR there is no evidence of loss of the hereditary heterozygosis in the cancer cells [1,31, and the monoallelic inactivation of some tumor suppressor genes, such as ~53, appears not to be recessive at the cellular level [W,54*1 suggesting that the heterozygotic defect of some tumor suppressor genes may be sufficient to produce the phenotypic effect. Ad- ditional putative tumor suppressor genes, thought to be responsible for other cancer predisposition syndromes, have been identified very recently. They provide new clues for the understanding of the genetics of cancer predisposition and mechanisms of tumorigenesis.

Neurofibromatosis type 2 (NF2) The NF2 syndrome predisposes a sufferer to the development of schwannomas or meningiomas, most frequently bilateral schwannomas on the vestibular branch of the eighth cranial nerve 151. The NF2 gene (also called SCH or Merlin) was identified from chromosome 22q12, and encodes a product with similarities to proteins (such as moesin, ezrin, radixin, erythrocyte protein 4.1, talin) that have been proposed to act as links between the cell membrane and the cytoskeleton 155,561. The NF2 gene appears to follow the pattern of a ‘recessive tumor suppressor’ gene. Deletions as small as 1 bp, which result in truncation of the NF2 protein, have been found in the germline of NF2 family members. NF2 mutations segregate with the disease, and have also been found in both NF2 and sporadic meningiomas. As the fP2 mutations are associated with the loss of the wild type allele in meningiomas, NF2 may also be implicated in the pathogenesis of sporadic tumors, especially those with a high frequency of chromosome 22 monosomy, such as pheocromocytomas, gliomas, colon and breast cancers. Other tumor-predisposing alleles, among them ~53, RB or, more recently h!Fl (which has been shown to be altered in such unrelated tumors as astrocytomas, myelodisplastic syndrome, malignant melanomas and neuroblastomas [57,581), have already been ascribed a role in the pathogenesis of spontaneous tumors.

Multiple endocrine neoplasia type 2A (MENZA) The MEN2A syndrome is associated with endocrine tumors: bilateral medullary thyroid carcinoma (MTC), phoeocromocytoma and parathyroid hyperplasia. MTC may also occur alone, usually at an earlier age than in MEN2A, in a familial form (FMTC) 151. The MEN2A gene has been mapped to a chromosome lOq11.2 region that contains the ret locus, and ret mutations have

been found at one allele in the somatic cells of both MEN2A asymptomatic and affected family members, as well as in MENZA and FMTC tumor DNA 159,601. Notably, the other ret allele remains structurally un- altered and is expressed. Mutations in ret have not been found in either sporadic MTC or phoeocromo- cytomas, nor in the germline of patients with the MEN2B, a related hereditary cancer syndrome. These findings suggest that the inherited mutant allele acts as a dominant oncogene during MEN2A tumorigenesis, and that a somatic mutation in the second allele is not required for tumorigenesis. This is supported by the biochemical function of the Ret protein and the type of abnormalities present in MEN2A and FMTC patients. Ret is, in fact, a receptor tyrosine kinase (with unknown ligand), and was first described as a transforming oncogene activated by rearrangement during transformation of NIH3T3 cells. Ret is also activated by rearrangements that juxtapose its transmembrane and tyrosine kinase domains with other domains encoded by 5’ sequence, as seen in 25% of papillary thyroid carcinomas. The Ret abnormalities in MEN2A and FMTC patients are due to the mutation of one of the cysteines in the Ret extracellular domain. In 19 of 20 cases studied 1591, the mutation affected the same cysteine. Since the MENZA patients develop early hyperplasia of both thyroid C cells (from which MTC arises) and of adrenal medulla chromaffin cells (from which phoeocromocytoma derives), the mutated germline ret allele may suf- fice to provide a proliferative stimulus that, in its turn, increases the mutations that favour MEN2A/FMTC tumor progression. Alternatively, Ret could act as a tumor suppressor gene in normal C cells; the mutation would then reduce the dosage of the wild type allele or create a dominant negative allele. Further research on the biochemical and biological activities of the MEN2A/FMTC ret mutants should help clarify the picture.

The von Hippel-Lindau syndrome The von Hippel-Lindau (VI-IL) syndrome predisposes to retinal hemangioblastomas, renal cell carcinomas and pheocromocytomas. Rearrangements in the VI-IL gene, which was identified from chromosome 3p25- 26, mostly occur by intragenic deletions in both the constitutional DNA of unrelated VHL patients and in sporadic renal cell carcinomas. The partial sequence of the predicted VI-IL gene product shows no homology to other proteins 1611.

Chromosome translocations

Chromosome translocations differ from the other types of genomic instability discussed so far, both in the mechanism through which they are determined, and in their contribution to the process of neoplastic transformation 162,631. Firstly, they are specific for certain types of neoplasia, such as leukemias, lymphomas, sarcomas, and mesenchymal neoplasms (see below).

114 Oncogenq and cell proliferation

The high specificity of the translocations allows them to be used as diagnostic markers; for example, the t(15;17) of acute promyelocytic leukemia, the t(9;22) of chronic myelogenous leukemia, and the t(8;14) of follicular lymphomas. Secondly, chromosome translocations are molecular events that occur at preferential sites in the genome and have modest molecular heterogeneity. Such site specificity suggests that the mechanisms responsible for the genesis are equally specific. This is the case in many of the translocations associated with B cell and T cell lymphoid neoplasias, where one of the two translocation breakpoints is situated in one of the loci encoding either the immunoglobulins or the T-cell receptor. It appears that, in this type of recombination, errors in recombinase activity or class switch enzymes, which normally play a part in the regulation of expression mechanisms (DNA rearrangements) of the immunoglobulin and T-cell receptor loci in lymphoid cells, contribute to the lymphoid tumor-associated chromosome recombination mechanism. Third, chromosome translocations are mostly solitary alterations unassociated with other chromosome aberrations. The t(15;17), for example, is the sole chromosome anomaly in 90% of cases, which contrasts with the situation encountered in most carcinomas where the main problem is to distinguish the pathogeneti- tally essential, primary karyotypic alterations from sec- ondary alterations that may only represent cytogenetic noise 1641. On the basis of the mechanism through which translocations activate the genes implicated in chromosome breaksites, they can be roughly divided into two groups: those that induce alterations in the expression of the involved gene, and those that induce abnormalities resulting in mutant proteins.

Recombinations with the immunoglobulin and T-ceil receptor genes As a consequence of chromosome recombination, one of many cell genes can become juxtaposed with one of the immunoglobulin or T-cell receptor loci, and this results in altered transcriptional activity of the translo- cated gene. Some of the genes involved in this group of translocations are probably proto-oncogenes whose inappropriate expression contributes directly to triggering cell growth. This is the case with certain translocations, mainly those associated with lymphomas: t(11;14), t(8;14), and t(14;18) [651. The translocation t(11;14) results from the juxtaposition of the- gene that encodes the heavy immunoglobulin chain (IgH) on chromosome 14 and sequences from chromosome 11. The putative oncogene of this translocation, termed DRADI, is homologous with the cyclin DI gene, a member of the cyclin gene family which codes for proteins implicated in cell cycle progression [651. The t(8;14) oncogene is c-myc on chromosome 8, which is also involved in positively regulating cell proliferation, and whose expression is deregulated as a consequence of its juxtaposition with the IgH locus on chromosome 14 i651. In the t(14;18), the IgH locus is in juxtaposition with the bci-2 locus, which encodes a cell death

suppressor activity protein [65]. Support for their role in lymphomagenesis and cell proliferation control, comes from the fact that malignant lymphomas develop in both myc and bc12 transgenic mice, probably secondar- ily to developmental alterations in the lymphoid cells in the pretumor animals: there is an increased number of resting B cells, due to prolonged cell survival, in bc12 transgenic mice, and an expanded population of cycling pre-B cells in myc transgenic mice 166,671. Other genes that combine with immunoglobulin and T-cell receptor genes have been identified recently: 1~1-1 and tul-1, that encode helix-loop-helix proteins, are involved in the t(7;19) and t(1;14) of acute T-cell leukemia 1621; the HOX-13 homeobox gene, implicated in the t(10;14) of T-acute lymphoblastic leukemia [621; and the BCL-6 zinc-finger gene that takes part in translocations affecting chromosome 3q27 in large cell lymphomas 1681.

Fusion proteins In another group of chromosome translocations, which occur mainly in leukemias, recombination fuses two genes, one of which is always a transcription factor. In the t(1;19> of acute lymphoblastic leukemias, the E2A gene, located on the chromosome 19 break- point, fuses with the PBX gene on chromosome 1, and the resulting chimeric protein retains the transcriptional truns-activation domain of the E2A protein and the putative DNA-binding homeobox domain of the PBX protein 162,631. The t(15;17) fuses the PML gene on chromosome 15 with the retinoic acid a receptor (RARa) gene on chromosome 17, which encodes a protein with a zinc finger DNA-binding domain 162,631. There are many other examples: a homolog of the Drosophila melanogaster developmental gene tritbo- YU.X from llq23 fuses with a locus from 4q21 during the t(4;ll) of acute mixed-lineage leukemia 169-711; the t(8;21) of the M2-acute myeloid leukemia and the t(3;21) of myelodisplasia fuse a protein, AMLl, that is homologous with the D. melanogaster runt segmenta- tion gene product, with a putative zinc-finger transcription factor on chromosome 8 or with the EAP small nuclear protein from chromosome 3 172,731; in the MCacute myeloid leukemia, the transcription factor, CBFB/PEB2B, fuses with a myosin heavy chain during the pericentric inversion of chromosome I6 1741; in the t(I6;21) of acute myeloid leukemias, the putative transcription factor ERG gene, an e&related gene, fuses to an (as yet> unidentified sequence from chromosome I6 1751; and in the t(11;22) of Ewing sarcoma, a fusion gene is formed between the e&like FL11 and EWS genes [761.

There is accumulating evidence that these proteins are involved in multiple nuclear signalling pathways that converge to regulate fundamental processes, among them differentiation and survival. If induction of terminal differentiation and apoptotic cell death is one of the mechanisms that physiologically limits the potential of a stem cell to proliferate, then genes involved in leukemia-associated translocations should be con-

Cancer genetics Lanfrancone, Pelicci and Pelicci 115

sidered as being tumor suppressor genes and their tumorigenic conversion mechanism would be expected to be loss-of-function mutations. The uninvolved alleles appear to be intact and expressed in all cases of this type of translocations, and the fusion proteins would be expected to be dominant negative mutations.

The v-ErbA model The oncogenes v-erbA and v-erbB are present in the avian erythroblastosis virus, and co-operate in caus- ing erythroleukemia in chickens. The v-erbB product induces the expansion of undifferentiated erythroid precursors that are, however, scarcely tumorigenic because still sensitive to differentiative stimuli. The v-erbA product potentiates the transforming effect of v-erbB by efficiently blocking erythroblast differentiation [41.

The v-erbA oncogene is a mutated form of the c-erbA proto-oncogene that encodes one of the thyroid hormone receptors. This protein is a member of the nuclear hormone receptor family that includes steroid, retinoid and vitamin D receptors. Members of this family function as ligand-induced transcription factors by directly binding to specific response elements in the promoter region of target genes. Their binding to response elements in vitro, and their activation of target gene expression in vivo, are markedly enhanced by heterodimerization with auxiliary proteins, one class of which are the retinoid X receptors 0000, which are themselves transcription factors of a signalling pathway activated by a recently identified retinoid, 9cis-retinoic acid 177-791. The c-erbA product functions both as an activator and as a repressor of transcription. In the absence of ligand, it binds target genes and represses or inhibits transcriptional activation, whereas in the presence of ligand, it stimulates transcription. The v-ErbA protein acts as a constitutive repressor, in that it does not bind the lig- and and does not truns-activate 1801. However, v-ErbA can also interfere with the action of several members of the steroid/retinoid family, and its capacity to repress the action of RARs correlates with its oncogenic activity [Sl]. The mechanism through which v-ErbA exerts a dominant negative activity on the thyroid hormone receptor, RARa and other nuclear receptor signalling pathways, relies mainly on its capacity to heterodimer- ize with RXR. The v-ErbA/RXR heterodimer is a strong inhibitor of the RXR response to 9 cis-retinoic acid and greatly increases the capacity of v-ErbA to bind specific responsive elements, and so efficiently compete for binding to a subset of c-ErbA and RAR target genes 182-841. Furthermore, dimerization of v-ErbA with RXR could influence signalling pathways in which RXR is a cofactor (RAR, thyroid hormone receptor, vitamin D receptor), by mechanisms other than specific binding to responsive elements, for example via an RXR-se- questering mechanism. It is worth noting that, physiologically, RXR interferes with the activated protein-l CAP-11 signalling pathway by boosting the activities of RARs and c-ErbA anti-APl, and v-ErbA plays a dominant negative role in this mechanism too [851.

Because it is able to associate with other nuclear factors and to bind to the DNA itself, v-ErbA has the potential to interfere negatively with multiple nuclear signalling pathways. Amplification of the phenotypic effects of the activating mutation(s) in v-erbA is therefore the direct consequence of the fact that nuclear hormone receptors do not function autonomously in gene regulation, but rather as part of a complex, combinatorial control network whose effect is to integrate diverse signalling pathways. The result of this is that the action of one class of receptors is profoundly influenced by other members of the same family, or even other factors.

The acute promyelocytic leukemia PML/RARa protein The acute promyelocytic leukemia PML/RARa protein interferes with differentiation and survival of myeloid hematopoietic precursors. The multiple dominant negative mechanism of v-ErbA could provide a model for the mechanism of action of some transcription factor mutants generated by acute leukemia-associated translocations, and has important implications for the transformation process in these neoplasias. Like v- ErbA, these transcription factors contain functional domains for protein interaction and sequence-specific DNA binding and can, in consequence, take part in multiple interactions with other nuclear factors and target genes. Their mutants could, therefore, interfere (maybe negatively) with multiple nuclear signalling pathways, and the effects of a single mutation could be amplified strongly at the phenotypic level.

The acute promyelocytic leukemia PML&ARa could be one such mutant. The t(15;17) fuses a putative transcription factor, PML, with a member of the RAR family, RARa. Important domains of the wild type proteins are retained ifi the fusion protein: the PML putative DNA-binding domain and dimerization interface, the RARa DNA-binding domain, and RXR dimerization and retinoic acid binding domains. PMYRARa is, therefore, a potentially multifunctional protein and, in fact, it has the capacity to bind retinoic acid target genes in both a ligand-dependent and ligand-independent manner, as well as to dimerize with wild type PML, PMYRARa and RXR. Whether it is able to bind to (still poorly understood) putative PML DNA target sequences is, for now, unknown [861.

Because of its capacity to bind RXR, PMYRARa could interfere with multiple signalling pathways, probably by reducing the RXR pool available to other hormone receptors. In vitro, excess PML/RARa prevents both vitamin D receptor binding to responsive elements and activation of a reporter gene f871. The many biochemical activities of I~MWRARa correlate well with its multiple biological activities. When expressed in hematopoietic myeloid precursor cell lines, it induces a block in vitamin D induced differentiation, enhances sensitivity to retinoic acid, and protects cells from the apoptotic death provoked by a growth factor deprivation, without greatly affecting proliferation rhythm 188.1. Al-

116 Oncogenes and cell proliferation

though the biological activities of PMKRARa correlate well with important characteristics of the acute promyelocytic leukemia phenotype, little is known of the underlying biochemical mechanism.

.

The acute lymphoblastic leukemia EZA-PBX protein The acuie lymphoblastic leukemia E2A-PBX protein interferes with proliferation and survival of lymphoid hematopoietic precursors. This fusion protein may act as a chimeric transcription factor, as the trans-activation domain of E2A is fused to the carboxyl terminus of PBXl, which contains its DNA-binding homeodomain. E2A-PBX could, therefore, interact with binding sites for PBXl in specific gene promoters 162,631. Despite the lack of information on the biochemistxy of the E2A&PBX fusion protein, recent in vim data suggest that its biological effects are also very complex. An- imals that express the E2A-PBXl transgene develop clonal lymphoid malignancies. Neverthless, the pretumor E2A-PBX animals have severe lymphopaenia, owing to high levels of apoptotic cell death, suggesting that E2A-PBX also induces severe alterations in lymphoid development 18Pl. Despite the lymphopaenia, the number of mitotically active lymphoid cells is increased, suggesting that E2A-PBXl is a protein involved in the regulation of both cell proliferation and cell survival.

For neither the effect of PBX/2A on cell proliferation and survival, nor for the effect of PMYRARa on survival and differentiation, is it known whether one phenotype is the consequence of the other, or whether the two fusion proteins act concomitantly on independent signalling pathways.

In the case of ~53, which also exerts effects on cell proliferation and survival, regulation of the two pathways appears to be the consequence of the function of p53 in processing the effects of certain external stimuli on the cell cycle. The ~53 protein is required both for the Gl block by radiation and anticancer drugs, as already mentioned, and for activating apoptosis induced by the same agents 153’,54*,901. The genotoxic stimuli in cells that express p53 apparently result in pausing during the cell cycle, or in induction of apoptotic cell death. For example, cell death is favoured when cells expressing ~53 are forced to progress through the cell cycle by activated c-Myc 1911 or ElA genes 192,931.

Biologically, the three signalling pathways of cell survival, differentiation and proliferation are probably in- tegrated. Elegant single-cell experiments have demonstrated that, when provided with a bc12 survival stimulus, haematopoietic cells undergo terminal differentiation independently of any external differentiation stimulus and in the absence of cell proliferation [94*1.

Acknowledgements

We thank Riccardo DaIht Faverd and Pier Paoio DiFiore for stim- ulating discussions and criticism of a first dmft of this manuscript.

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CHEN H-W, PRIVAL~KY LM: The c&-A Oncogcnc Represses the Actions of Both Rctinoid X and Rctinoid A Receptors but Dots So by Distinct Mechanisms. Mol Cell Blol 1993, 13:5970-5980.

BAR~~NO B, BUGGE TH, BAR’IUNEK P, VIVANCO-RUIZ MD, SONNTAG-BUCK V, BEUC H, ZENKE M, STUNNENUERG HG: Uniigandcd T3R. But Not Its Oncogcnic Variant, verbA, Suppresses RAR-Dependent Transactivation by litrating Out RXR. EMBO / 1993, 12:13431354.

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Gene M-PBXl Inducts Pro~icration. Apoptosis, and Maiig- nant Lymphomas in Transgcnic Mice. Cell 1993, 74:833-843.

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90. LOWE SW, RULEY HE, JACKS T, HOUSMAN DE: p53-Dcpendcnt Apoptosis Modulates the Cytototdcity of Anticancer Agents. Cell 1993, 74~957-967.

91. EVAN GI, WYLUE AH, GILBERT CS, DT~LEWOOD TD, LAND H, BROOKS M, WATERS CM, PENN LZ, HANCOCK DC: Induc- tion of Apoptosis in Fibroblasts by c-myc Protein. Cell 1992, 69119-128.

92. LOWE SW, RULEY HE: Stabilization of the p53 ‘Dtmor Sup pressor is Induced by Adcnovirus 5 ElA and Accompanies Apoptosis. Genes L&u 1993, 7:535-545.

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mcnt of a Multipotent Hcmopoictic Ccii Line in the Absence of Added Growth Factors. CeU 1993, 74:82H32.

This paper demonstrates that extended survival of hematopoietic precursor cells is accompanied by muitiiineage differentiation, and that differentiation occurs without ceii ProtiferJtion.

This paper shows that the expression of the leukemia-specific PMYRARa protein in hematopoietic precursor cell lines induces a block of differentiation by vitamin D. increases sensitivity to retinoic acid and reduces apoptotic cell death.

L Lanfrancone, G Pelicci and PG Pelicci, laboratorio di Biologia Molecolare, Policlinico, lnstito Medicina Intema e Scienze Onco- logiche, Monteluce. Vii Brunamonti, 06100 Perugia, Italy.

cancer genetics

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