Oncogenes in melanoma - Nature

Oncogene (2003) 22, 3087–3091

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Oncogenes in melanoma David Polsky*,1 and Carlos Cordon-Cardo2 1

Oncology Section of the Skin and Cancer Unit, Department of Dermatology, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA; 2Division of Molecular Pathology, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

Transformation of normal melanocytes into melanoma cells is accomplished by the activation of growth stimulatory pathways, typically leading to cellular proliferation, and the inactivation of apoptotic and tumor suppressor pathways. Small molecule inhibitors of proteins in the growth stimulatory pathways are under active investigation, and their application to melanoma patients would represent a new treatement strategy to inhibit cell proliferation or induce cell death. We provide a general overview of the mechanisms of oncogene activation and the functions of oncogenes. Lastly, we review oncogenic events in melanoma. Oncogene (2003) 22, 3087–3091. doi:10.1038/sj.onc.1206449 Keywords: melanoma; oncogenes; review; proliferation; transformation

categories: proto-oncogenes and tumor suppressor genes (also known as growth suppressor genes). Activation of proto-oncogenes by point mutation, amplification, translocation, or even insertion of noneukaryotic sequences, yields oncogenes that have as their main characteristic a ‘gain’ of function. They have also been described as ‘dominant’ and ‘positive’ phenomena. Inactivation of tumor suppressor genes occurs mainly through an allelic deletion followed by a point mutation of the contralateral allele. These events have also been described as ‘recessive’ and ‘negative’. Alterations in proto-oncogenes and tumor suppressor genes seem equally prevalent among human cancers. The focus of this section will be on proto-oncogenes and oncogenic events associated with cutaneous melanoma. Mechanisms of oncogene activation

Introduction The coordinated balance of four critical programs, cell division, differentiation, senescence, and apoptosis, maintains cellular homeostasis. Cancer constitutes an abnormal cellular growth, uncoordinated with that of the normal tissue, which persists in the same excessive manner after cessation of the initial transformation event (Willis, 1952). A combination of genetic (somatic or hereditary) and epigenetic alterations constitutes the basis for the instability characteristic of neoplastic transformation. Environmental insults, producing somatic mutations, are the most frequent abnormalities identified in human cancer. In melanocytic neoplasms, ultraviolet (UV) radiation from sunlight has been implicated as the primary environmental carcinogen, although UV signature mutations have not yet been identified commonly. In addition, germline mutations in the tumor suppressor gene INK4A, or oncogene CDK4 have been identified in familial melanoma. It is therefore conceivable to view melanoma as fundamentally a genetic disease entailing germline and somatic mutations. In addition, epigenetic alterations, such as promoter methylation or protein degradation, are also considered suppressive or dominant events involved in tumorigenesis and tumor progression. Target genes implicated in cellular transformation and tumor progression have been divided into two *Correspondence: D Polsky; E-mail: [email protected]

Different kinds of genomic changes can produce oncogenic effects (Tabin et al., 1982; Varmus, 1985). Some oncogenes have point mutations that alter the function of the encoded protein. In other cases, a genetic translocation places an active promoter region or transcriptional regulatory element next to a protooncogene (Hodgson and Maher, 1997). Consequently, the normal control mechanisms that constrain that gene are undermined, the oncogene is continually expressed, and cellular proliferation results. For example, the t(8;14)(q24;32) translocation for Burkitt lymphoma places the MYC gene under the control of the very active promoter for the heavy-chain immunoglobulin cluster. Thus, the Myc protein is overproduced, and the cell proliferation genes are expressed. In other cases, the breakpoints of a translocation occur within introns of two genes, and, after joining, a single reading frame is established with genetic information from both genes resulting in a fusion, chimeric protein with oncogenic potential. If such a fusion protein is under the control of an active promoter, then the oncogenic protein is synthesized (Rubie et al., 1997). For example, the translocation represented by the Ph chromosome creates a chimeric gene that specifies segments from the BCR and ABL genes and is under the control of the BCR promoter. The ABL portion of the chimeric gene encodes tyrosine protein kinase. Thus, as part of the fusion protein, tyrosine protein kinase activity is continuously present, the signal transduction pathway is turned on, and cell proliferation persists (Pasternak, 1999).

Oncogenes in melanoma D Polsky and C Cordon-Cardo

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Overproduction of oncogenic proteins often occurs when there is repeated replication (DNA amplification) of a specific chromosome region. The mechanism that brings about localized chromosome DNA amplification is not fully understood (Hodgson and Maher, 1997). When the amplification process generates a set of repeated DNA segments that are confined to a region of a chromosome, a distinctive repetitive chromosome banding pattern, called a homogeneous staining region (HSR), is produced. Alternatively, multiple DNA elements formed by repeated replication of a chromosome region are released into the nucleoplasm. After DNA staining, these extrachromosomal DNA components have a joined double-dot appearance and are called double minutes (DMs). A DM is a pair of circular, 1 Mb DNA fragments. Since they do not have centromeres, DMs are randomly distributed to daughter cells. In human tumors, about 95% of cases of amplification are DMs and the rest are HSRs. In either instance, the end result is often an enormous increase in the number of copies of an oncogene and, as a result, the overproduction of an oncogenic protein. Amplification of oncogenes occurs in a variety of cancers (Pasternak, 1999), including melanoma (see below). In summary, proto-oncogenes may be mutated to promote cell transformation by a variety of mechanisms: (a) point mutations may alter the function of the gene product and produce a transforming protein (e.g. RAS and RAF in melanoma); (b) translocations may activate the proto-oncogene (e.g. MYC), or result in fusion genes encoding novel products (e.g. BCR–ABL in chronic myeloid leukemia); (c) gene amplification may lead to increased expression of the oncoprotein (e.g. HDM2 in osteosarcoma); and (d) overexpression of the protooncogene product(s) in general, because of increased transcriptional activity (e.g. growth factor/growth factor receptor autocrine loops, see below). Functions of Oncogenes Oncogenes were first demonstrated in RNA tumor viruses, and further research revealed that they are derived from normal cellular genes called proto-oncogenes (Varmus, 1985). These proto-oncogenes have a role in normal cellular homeostasis, but when altered as described above become activated tumor-specific oncogenes. More than 70 oncogenes have been identified as participating in cellular proliferation, differentiation, senescence, and apoptosis. The functions of proto-oncogenes can be classified into broad groups such as: (1) growth factors and their receptors; (2) signal transducers; and (3) nuclear protooncogenes, including transcription factors. Growth factors and their receptors Overexpression of certain growth factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) have been reported to be involved in melanoma as autocrine and paracrine loops together with their Oncogene

corresponding receptors (FGF-R, PDGF-R, and EGFR, respectively) (Satyamoorthy and Herlyn, 2002). Other growth factors described as overexpressed in some neoplasms, including melanoma, are the insulinlike growth factors 1 and 2 (IGF-1, -2) and the transforming growth factors a and b (TGF-a, -b) along with their receptors (IGF-R and TGF-R, respectively) (Berking et al., 2001; Satyamoorthy et al., 2001). Examples of other growth factor/growth factor receptor proto-oncogenes include neurotrophins/Trk receptors (Innominato et al., 2001), Kit ligand/Kit receptor in gastrointestinal stromal tumors (GIST) (Tuveson et al., 2001), and vascular endothelial growth factor (VEGF)/VEGF receptors. In addition, activating point mutations in growth factor receptors have also been observed. The transmembrane receptor tyrosine kinases RET and MET may be mutated in inherited cancers (multiple endocrine neoplasia type 2 (MEN2) and familial papillary renal cancer. RET and MET mutations result in abnormal tyrosine kinase activity of the receptor protein. Signal transducers Proto-oncogene signal transducers may fall into different groups: (a) membrane-associated/guanine nucleotide-binding proteins (RAS, GSP, and GIP); (b) membrane-associated/cytoplasmic protein tyrosine kinases (ABL, SRC, and FGR), and (c) cytoplasmic protein serine-threonine kinases (RAF1, MOS, and COT). Oncogenes of the RAS family (Ha (Harvey)RAS-1, K (Kirsten)-RAS-2, and N (neuroblastoma)RAS) each encode a 21-kDa protein (p21), which shares homology with G-proteins. Oncogenic Ras proteins have sustained mutations that render them constitutively active by maintaining the proteins in the GTPbound activated state. Activating RAS mutations are frequent in human cancers (e.g. colon, pancreas, bladder, and lung), although the particular RAS gene involved varies between cancer types. Although it seems clear that RAS has an important role in the control of cellular proliferation, the precise details of the mechanism of action are not completely known. Further complexity is provided by the discovery of GTPaseactivating proteins (GAPs) that may modulate RAS activation, and the suggestion that the neurofibromatosis type 1 gene (NF1) product may be a GAP protein. Nuclear proto-oncogenes Nuclear proto-oncogenes include members of the MYC family (c-MYC, N-MYC, and L-MYC), which have been widely studied, and amplification of myc genes has been found in a variety of tumor types including lung (c-MYC, N-MYC, L-MYC), colon (c-MYC), breast (c-MYC), and neuroblastoma (N-MYC). The MYC protein complexes with a second protein, MAX, to form a functional heterodimer that regulates expression of target genes. Other nuclear oncoproteins include FOS and JUN, which also bind together to form a functional heterodimer.


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Oncogenic events in melanoma The number of studies dealing with oncogene mutations utilizing clinical samples of cutaneous melanomas is quite limited. In addition to the already listed reports dealing with overexpression of certain growth factor receptors/ growth factor molecules (see above), the most commonly activated oncogenes in melanoma are those belonging to the RAS/MAPK signaling pathway, which mediate cellular responses to signals from growth factor receptors. RAS Studies conducted by Albino and collaborators revealed that 24% of cultured malignant melanomas had activated RAS genes, with N-RAS being activated 10 times as frequently as Harvey (Ha)-RAS. However, only 5– 6% of noncultured primary and metastatic melanomas were found to contain mutated N-RAS. RAS mutations were not found in any precursor lesions analysed, including dysplastic nevi (2). However, more recent analyses utilizing novel technologies have reported that N-RAS mutations are frequent events even in some nevi and early-stage melanomas. More specifically, Papp et al. (1999) reported that 56% of congenital nevi harbored activating N-RAS point mutations. Bastian et al. (2000) in a study of Spitz nevi found H-RAS copy number increases on chromosome 11p, which were highly associated with oncogenic point mutations of HRAS alleles. Demunter et al. (2001) reported N-RAS mutations in 33% of primary and 26% of metastatic clinical melanoma samples. In an earlier study, van Elsas et al. (1996) observed that 15% of cutaneous melanomas carry mutated N-RAS alleles. It can be concluded that the role of RAS mutations are more frequent events than previously postulated, occurring even in benign melanocytic neoplasms, such as nevi. In that respect, it is of interest to point out that different mutated RAS alleles are also found commonly in early stages of other neoplasms, including K-RAS mutations in benign colonic polyps (Kinzler and Vogelstein, 1996) and H-RAS mutations in papillary superficial tumors of the urinary bladder (Ooi et al., 1994). Moreover, in a murine model, when melanocyte-specific expression of a mutated Ha-Ras was combined with the Ink4a null genotype, the mice developed spontaneous, cutaneous melanomas with high frequency (Chin et al., 1997). The human and murine data suggest that RAS activation contributes not only to growth advantage and transformation, but also to tumor maintenance. B-RAF More recently, mutations affecting the serine/threonine kinase B-RAF have been reported in several tumor types, with high rates in melanoma (Davies et al., 2002). In a cohort of primarily cell lines, these authors found two activating B-RAF point mutations leading to increased kinase activity in 66% of melanoma samples. RAF kinase activity impacts on the MAPK pathway conferring mitogenic responses and growth advantage to

these cells. The primary B-RAF mutation found in melanoma was the V599E substitution, which occurred in the absence of RAS mutations, suggesting that it was sufficient to deregulate this pathway. In a more recent study, Pollock et al. (2003) analysed microdissected tissues of melanoma and melanocytic nevi for mutations in B-RAF exon 15 (Pollock et al., 2003, #806). In 41/60 (68%) metastases, 4/5 (80%) primary melanomas, and 63/77 (82%) nevi, they observed mutations resulting in the V599E substitution. Finding such a high frequency of B-RAF mutations in nevi, which are benign melanocytic proliferations, has led to the speculation that B-RAF activation alone is not sufficient for melanocyte transformation, but that it may be a first step in the process. Cyclin D1 Among the downstream effectors of the RAS/MAPK signaling cascade, Cyclin D1 is an important regulator of the G1/S cell cycle transition, contributing to the phosphorylation of the retinoblastoma protein (pRB) by binding to cyclin-dependent kinase 4 (CDK4). Cyclin D1 is overexpressed in several neoplasms including parathyroid, breast, and sarcomas. In melanoma, amplification of Cyclin D1 has been identified mostly in acral melanomas (those occurring on the hands and feet) (44%) and less frequently in other subtypes (Sauter et al., 2002). Acral melanomas appear distinct from other subtypes in that they are frequently characterized by amplifications in one or two loci, in contrast to other types of melanoma in which amplifications are generally lacking. Strong evidence supporting an oncogenic role for Cyclin D1 came from experiments demonstrating that antisense treatment of mice bearing melanoma xenografts overexpressing Cyclin D1 led to apoptosis and tumor shrinkage (Sauter et al., 2002). CDK4 As mentioned above, the Cyclin D1/CDK4 complex mediates phosphorylation of pRB in the mid-G1 phase of the cell cycle. Binding of this complex to the p16INK4A tumor suppressor inhibits this phosphorylation step. Shortly after the discovery of p16INK4A as a melanoma susceptibility gene, a point mutation in CDK4 producing an amino-acid substitution of arginine to cysteine at residue 24 (R24C) was detected in SKMEL-29 cells (Wolfel et al., 1995), as well as in three melanoma families (Zuo et al., 1996; Soufir et al., 1998). This mutation interferes with the binding of CDK4 to p16, but not to cyclin D. This is one of the few instances, together with the RET gene, in which an activated oncogene is carried as a germline mutation. In addition, murine models using ‘knock-in’ technology produced Cdk4R24C/R24C animals that were susceptible to melanoma development (Sotillo et al., 2001; Rane et al., 2002). c-MYC Extra c-MYC oncogene copies have been reported in four out of eight primary and 11 out of 33 melanoma Oncogene


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metastases. However, none of the 12 nevi revealed these changes (Kraehn et al., 2001). As critical as the activities of MYC alleles might be, their role in melanoma is uncertain at this point, as other studies have drawn conflicting conclusions (Boni et al., 1998; Chana et al., 2001). HDM2 The protein product encoded by HDM2, the human homolog of the murine Mdm2, binds to the transcriptional activation domain of p53, blocking its ability to regulate target genes and targeting p53 for proteosomemediated enzymatic degradation by ubiquitination. HDM2 may also have p53-independent functions, but its oncogenic potential is likely derived from disrupting the critical tumor suppressor activity of p53. HDM2 is amplified in many tumor types, especially sarcomas (Momand et al., 1998). In melanoma, overexpression of HDM2 has been observed in the absence of amplification (Polsky et al., 2001). As transcription of HDM2 is controlled by both p53 and factors upregulated by growth factor receptor signaling (Ries et al., 2000), it was initially unclear whether HDM2 was acting as an oncogene in melanoma, or a marker of p53 activation. More recently, overexpression of HDM2 was shown to correlate with improved clinical outcomes in melanoma patients, independent of tumor thickness, the most important prognostic marker in localized disease (Polsky et al., 2002 #782). Therefore, it is unlikely that HDM2 is driving an oncogenic process in melanoma. Finally, a recent study has determined that frequent chromosomal gains in melanoma map to 1q10 and 6q21, representing orphan regions for other potential activated oncogenes (Mertens et al., 1997). The recent implementation of high-throughput technologies aimed at global molecular profiling is beginning to identify candidate oncogenes and tumor suppressor genes not previously implicated in melanoma (Bittner et al., 2000; Clark et al., 2000). A recent observation from these studies is that alterations of the Wnt signaling pathway have been identified and associated with human melanoma progression. More specifically, the Wnt5a gene was found to be upregulated in melanoma and Wnt5a expression was directly correlated with PKC activation as well as with increasing tumor grade. These observations support a role for Wnt5a activation in melanoma, affecting cell motility and invasion (Weeraratna et al., 2002).

pathway has been demonstrated in several assays (Meier et al., 2000), including the growth inhibition of melanoma cell lines by antisense oligonucleotides against the bFGF mRNA or by synthetic peptides that bind to the bFGF receptor and act as antagonists (Becker et al., 1989; Wang and Becker, 1997). Another less well-described growth factor is MGSA/GRO. The three MGSA/GRO genes alpha, beta, and gamma are members of the chemokine family of growth factors. First isolated from the supernatants of a human melanoma cell line (Richmond et al., 1983), and later detected in melanoma primary cell cultures, these factors stimulate melanocyte growth, angiogenesis, and neutrophil chemotaxis. In addition, overexpression of MGSA/GRO in normal melanocytes leads to anchorage-independent growth in soft agar and tumors in nude mice (Owen et al., 1997). Also, blocking the binding of MGSA/GRO to melanoma cells led to growth inhibition that could be overcome by an excess of MGSA/GRO (Hayashi et al., 1997). Melanomas produce many other growth factors that appear to be important for various aspects of their malignant phenotype (Satyamoorthy and Herlyn, 2002). Inhibitors of apoptosis Traditionally, the protein products of oncogenes have included cytokines and growth factors, growth factor receptors, and proteins involved in signaling pathways that promote cell division. More recently, genes that inhibit apoptosis have also been identified as oncogenes. The prototype of these genes is BCL-2. Originally identified at the chromosomal breakpoint in follicular lymphoma, this novel protein was found to inhibit cell death rather than promote cell growth. BCL-2 belongs to a family of intracellular proteins whose role is to regulate caspase activation that leads to DNA fragmentation and cell death (reviewed in Cory and Adams, 2002). In melanoma, BCL-2 has been reported to be overexpressed in primary and metastatic lesions, and that this phenotype is associated with tumor progression (Leiter et al., 2000). Currently, an experimental trial utilizing BCL-2 antisense technology is in progress. Another gene identified as overexpressed in patients with advanced melanoma is the so-called ‘survivin’. Inhibition of this gene in vitro has been linked to deregulation of apoptosis and increased sensitivity to cytotoxic drugs in melanoma cells (Grossman et al., 2001).

Growth factors Conclusions One of the characteristics of melanocyte transformation is that these cells acquire the ability to produce their own growth factors to form autocrine stimulatory loops (reviewed in Halaban, 1996). The best-described mediator of this effect in melanoma is bFGF. Melanocytes are dependent for their survival on signaling via the bFGF receptor. Melanomas appear to activate a normally repressed bFGF gene, leading to low level, constitutive, and autocrine stimulation via the bFGF receptor (Halaban, 1991). The importance of this Oncogene

There is a growing body of evidence demonstrating that oncogenes are important contributors to the deregulation of cell proliferation and apoptotic pathways in melanoma. High-throughput, expression-profiling assays that detect the overexpression of specific genes are well suited for the discovery of new oncogenes in melanoma. This is encouraging, as these genes represent attractive targets for small molecule inhibitors that may prove to be effective in the management of this difficult disease.


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