Genetic Alterations in Signaling Pathways in Melanoma Frank G. Haluska,1HensinTsao,2 Helen Wu,1Frank S. Haluska,1Alexander Lazar,3 and Vikas Goel1 Abstract Alterations in the RAS signaling cascade are almost uniformly present in melanoma. RAS itself is only infrequently mutated in melanoma although downstream of RAS lie BRAF on the mitogen- activated protein kinase pathway and PTEN on the protein kinase B/Akt pathway.These genes are often altered in melanomas; indeed, the most frequent target of mutation in melanomas is BRAF, which is mutated in f60% to 70% of superficial spreading melanomas.These mutations occur in abackgroundthatisnotnormal,withtheCDKN2A locus also typically being mutated.We review herein the data that suggest that the distribution of the signaling mutations is important. In gener- al, melanomas carry a mutated NRAS,amutatedBRAF, or concurrent BRAF and PTEN muta- tions.These data support the hypothesis that the biochemical functions of RAS are portioned by mutations in the pathways lying downstream. Moreover, these mutations have no apparent rela- tionship to the patterns of alteration of CDKN2A and its downstream effectors.Thus, the data also suggest that successful exploitation of mutations in melanoma will be dependent on understand- ing not only mutations and their frequency but their genetic context as well. The development of rational treatments for melanoma will More important at present in our thinking about targeted depend on our taking advantage of the molecular basis of its therapy for melanoma is the RAS pathway. The RAS gene itself clinical features. The necessary understanding of the molecular is infrequently mutated in melanoma. However, the RAS genetics underlying melanoma is gradually emerging. It has protein does several important functions, and downstream of become clear that alterations in two genes and their down- it lie BRAF on the mitogen-activated protein kinase (MAPK) stream effectors are almost uniformly present in melanoma. pathway and phosphatase and tensin homologue (PTEN)on The first of these is the CDKN2A locus, and the second is the the protein kinase B/Akt pathway. These genes are frequently pathway that includes RAS. Understanding the role alterations altered in melanomas and it seems that the RAS-PTEN/BRAF in these pathways play in melanoma and understanding their axis is almost always abnormal. Mutated targets on this interactions may provide key insights that will lead to pathway are appealing therapeutic targets. therapeutic interventions that are rationally designed and In this review, we show that the distribution of these implemented. mutations is important. We have shown that melanomas carry The CDKN2A locus encodes two tumor suppressor genes, either a mutated RAS or mutated BRAF and PTEN (1). The CDKN2A/p16 and p14ARF, which play central roles in the lesson is that the latter two abnormalities cooperate in control of cell cycle progression and checkpoint control. The oncogenesis and that this may have important implications p16-CDK4-pRB pathway in melanoma is almost always for planning therapeutic strategies. abnormal. CDKN2A also encodes the p53-controlling p14ARF, and disruption of CDKN2A therefore usually results in both loss of pRB and p53 control. However, altered tumor Signaling Mutations in Melanoma suppressor loci, with resultant loss of function of the encoded proteins, are problematic therapeutic targets. Efforts to develop RAS mutations in melanoma. RAS genes are among the most small-molecule CDK inhibitors, essentially mimics of p16, are frequently mutated genes in human cancers, but of course ongoing but progress thus far has been slow. different malignancies display different frequencies and spectra of mutations in NRAS, HRAS, and KRAS. Human melanomas carry mutations almost exclusively in NRAS, with 90% of mutations localizing to codon 61 (1). HRAS and KRAS are less Authors’ Affiliations:1Division of Hematology/Oncology and 2Department of frequently mutated. Albino and Fountain (2) report that 24% Dermatology, Massashusetts General Hospital, Boston, Massachusetts and of cultured metastatic and 12% of noncultured primary and 3University of Texas M.D. Anderson Cancer Center, Houston,Texas metastatic melanomas carry NRAS alterations, although others Received 11/16/05; accepted 12/21/05. report higher frequencies in primary tumors (3) and have Grant support: NIH grants RO1CA 095798-01A1and P50 CA 93683-01 (F.G. Haluska). suggested that mutations correlate with metastases or disease Presented at the First International Conference on Innovations and Challenges in progression. Melanoma, July 15-16, 2005, Cambridge, Massachusetts. RAS controls several biochemical processes. The best Requests for reprints: Frank G. Haluska, New England Medical Center, 750 understood pathway that involves RAS is the receptor tyrosine Washington St., Boston, MA 02111. Phone: 617-636-2626; Fax: 617-636-4367; E-mail: [email protected]. kinase-MAPK pathway (4). This pathway includes BRAF and F 2006 American Association for Cancer Research. contributes to control of cellular proliferation by RAS, in doi:10.1158/1078-0432.CCR-05-2518 particular the control of malignant cell proliferation by www.aacrjournals.org 2301s Clin Cancer Res 2006;12(7 Suppl) April 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Ta b l e 1. BRAF, NRAS,andPTEN/MMAC1 mutations in melanoma cell lines Cell line NRAS * PTEN/MMAC1 BRAF exon 15 Mutation Effect Mutation Effect Mutation Effect MGH-MC-2 G35A G12D wt WT wt WT Mel-SW C180A Q61K wt WT wt WT MGH-PO-1 C180A Q61K wt WT wt WT SK-Mel-l19 A181G Q61R wt WT wt WT SK-Mel-30 C180A Q61K wt WT wt WT Mel J A181T Q61L wt WT wt WT K19 C180A Q61K wt WT wt WT HS 9 4 0 A18 1G Q 61R w t WT w t WT SK-Mel-63 C180A Q61K wt WT wt WT HS944 C180A Q61K Del exon 2 Frameshift/premature stop wt WT SK-Mel-39 wt WT 546insA Frameshift/premature stop T1796A V599E MGH-BO-1 wt WT Del exon 2 Frameshift/premature stop T1796A V599E Sk Mel37 wt WT Del exon 2 Frameshift/premature stop T1796A V599E UACC 903 wt WT T226G Tyr76 Stop T1796A V599E RU wt WT IVS3del+1!+4 Possible splice variant T1796A V599E MM455 wt WT Del exon 6 Frameshift/premature stop T1796A V599E SK-Mel-28 wt WT A499G T167A T1796Ac V599E SK-Mel-131 wt WT IVS5+2T!A Possible splice variant T1796A V599E MEL-11 wt WT wt No proteinb T1796A V599E WM1158 wt WT wt No proteinb T1796A V599E WM239A wt WT wt No proteinb TG1796-97AT V599D WM1799 wt WT Failed PCR No proteinb T1796A V599E SK-Mel-23 wt WT Del Gene No predicted product wt WT ML wt WT Del Gene No predicted product wt WT MGH-MC-1 wt WT wt WT T1796A V599E K1-Mel wt WT wt WT T1796A V599E MEL-31 wt WT wt WT T1796A V599E WM164 wt WT wt WT T1796A V599E MH-12 wt WT wt WT TG1796-97AT V599D MH-2 wt WT wt WT T1796A V599E K-16 wt WT wt WT T1796A V599E A375 wt WT wt WT T1796Ac V599E HS939T wt WT wt WT T1796A V599E MH-17 wt WT wt WT T1796A V599E Mel-M wt WT wt WT T1796A V599E K4 wt WT wt WT T1796A V599E Mel-S wt WT wt WT T1796A V599E K2 wt WT wt WT T1796A V599E WM115 wt WT wt WT T1796A V599E MM608 wt WT wt WT T1796Ac V599E *NRAS, PTEN/MMAC1 status on some cell lines published (30). cBoth alleles show 599E. bNo protein byWestern blot. activated RAS. RAS also controls apoptosis (5). This response is mice engineered to carry an activated RAS develop melanoma? controlled through the phosphatidylinositol 3-kinase-PTEN- In the Cdkn2aÀ/À background, the answer is yes (6, 7). This Akt pathway as RAS directly binds the catalytic subunit of mouse model of melanoma most closely replicates the human phosphatidylinositol 3-kinase. The downstream consequence genetic findings. There are several other models that are less of this is phosphorylation of Akt and protection against well characterized but the RAS/Cdkn2a model is the most apoptosis. faithful replica of human data and shows the importance of Because RAS pathway abnormalities in melanoma occur these pathways in melanoma genesis. almost uniformly with CDKN2A alterations, understanding this The PTEN tumor suppressor in melanoma. PTEN is another interaction is crucial. If melanomas carry RAS mutations, do important element in signal transduction altered in human Clin Cancer Res 2006;12(7 Suppl) April 1, 2006 2302s www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Genetic Alterations in Melanoma melanomas. PTEN was identified as a tumor suppressor uncultured metastases for mutations in codons 12, 13, and 61 candidate from the region of chromosome 10q23-24 frequently of NRAS, KRAS, and HRAS. They found a total of 11 NRAS altered in gliomas and melanomas. Cytogenetic evidence shows mutations [11 of 53 (21%); 1 at codon 12 and 10 at codon 61] that 10q loss is an early and frequent event in melanomas in our cell lines. From our uncultured samples, we found two (8–11). Although initial work showed no mutations in a small NRAS mutations (2 of 17; 12%) in codon 61. The authors number of melanomas, we showed PTEN loss in 30% of found no mutations in the other RAS genes. melanoma cell lines (12) as did others (13). We have recently PTEN mutations in human melanomas. We also screened for reviewed the role of PTEN in melanoma in detail (14). mutations in exon 1 through 9 of PTEN in 45 melanoma cell PTEN encodes a protein with extensive homology to dual lines, 17 paired cutaneous melanoma and peripheral blood specificity protein phosphatases and, like RAS, it is implicated samples, and germ-line DNA from 28 melanoma patients with in the pathways that control apoptosis through protein kinase a family history of melanoma.