Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. A mutational analysis of the Protein Tyrosine Kinase (PTK) gene family in cutaneous metastatic melanoma identified 30 somatic mutations in the kinase domain of 19 PTKs. The whole of the coding region of these 19 PTKs was further evaluated for somatic mutations in a total of 79 melanoma samples. This analysis revealed novel ERBB4 mutations in 19% of melanoma patients and that an additional two kinases (FLT1 and PTK2B) are mutated in 10% of melanomas. Seven missense mutations in the most commonly altered PTK (ERBB4) were examined and found to increase kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA-mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies might lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas.Malignant melanoma is the most fatal skin cancer 1,2 . To develop personalized treatments for advanced disease, it is important to identify genetic alterations leading to melanoma. Protein tyrosine kinases (PTKs) are frequently mutated in cancer (http://www.sanger.ac.uk/genetics/CGP/Census/), and since they are amenable to pharmacologic inhibition 3,4 , further analysis of the PTK gene family may identify new therapeutic strategies. In this study, we used high-throughput gene sequencing to analyze the entire PTK gene family in melanoma, and have identified many novel somatic alterations.We initially sequenced the coding exons comprising the kinase domains of all 86 members of this gene superfamily in 29 melanomas (Supplementary Table 1). A total of 593 exons were extracted from genomic databases and amplified by polymerase chain reaction (PCR) * To whom correspondence should be addressed: National Human Genome Research Institute, 50 South Drive, MSC 8000, Building 50, Room 5140, Bethesda MD 20892-8000, Phone: 301-451-2628, Fax: 301-480-9864, samuelsy@mail.nih.gov. Author contributions T.DP. and Y.S. designed the study; J.R.W. and S.A.R. collected and analyzed the melanoma samples, N.S.A., J.C.C., K.E.Y., J.C.L., NISC., P.C and Y.S. analyzed the genetic data; T.D.P., X.W. and K.E.Y., performed and analyzed the functional data. All authors contributed to the final version of the paper. NIH Public Access Author ManuscriptNat Genet. Author manuscript; available in PMC 2010 July 6. Table 2) and directly sequenced with dye-terminator chemistry. We determined whether a mutation was somatic (i.e., tumor-specific) by examining the sequence of the gene in genomic DNA from normal tissue of the relevant patient. From the ~12 Mb of sequence information obtained, we identified 19 genes containing a total of 30 somatic mutations within their kinase domains. All coding exons of these 19 genes were then analyzed for mutations in a total of 79 melanoma samples using specific primers (Supplementary Table 3).We identified 99 non-synonymous, somatic mutations in ...

An additional tumor suppressor gene on chromosome 9p telomeric to the CDKN2A/B locus has long been postulated to exist. Using Affymetrix 250K single nucleotide polymorphism arrays to screen for copy number changes in glioblastoma multiforme (GBM), we detected a high frequency of deletions of the PTPRD gene, which encodes a receptor protein tyrosine phosphatase at chromosome 9p23-24.1. Missense and nonsense mutations of PTPRD were identified in a subset of the samples lacking deletions, including an inherited mutation with somatic loss of the wild-type allele. We then sequenced the gene in melanoma and identified 10 somatic mutations in 7 of 57 tumors (12%). Reconstitution of PTPRD expression in GBM and melanoma cells harboring deletions or mutations led to growth suppression and apoptosis that was alleviated by both the somatic and constitutional mutations. These data implicate PTPRD in the pathogenesis of tumors of neuroectodermal origin and, when taken together with other recent reports of PTPRD mutations in adenocarcinoma of the colon and lung, suggest that PTPRD may be one of a select group of tumor suppressor genes that are inactivated in a wide range of common human tumor types. [Cancer Res 2008; 68(24):10300-6]

A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.MMPs are proteolytic enzymes that degrade components of extracellular matrix and basement membranes 1 . MMPs have been associated with cancer metastasis 2,3 , and small molecule inhibitors of MMPs were tested as potential anticancer agents. However, clinical trials using these inhibitors showed no effect and, occasionally, accelerated tumor growth 4,5 . In contrast to the idea that MMP activity promotes melanoma progression, mouse models suggested that MMPs can have an antitumor role 6-8 . In particular, an increase in skin tumor incidence was seen in MMP-8-deficient mice 6 . These findings suggest that an in-depth analysis of the specific role of individual MMPs in particular cancer types is warranted. We systematically addressed © 2009 Nature America, Inc. All rights reserved.Correspondence should be addressed to Y.S. (samuelsy@mail.nih.gov).. AUTHOR CONTRIBUTIONS L.H.P. and Y.S. designed the study; J.R.W., P.F., A.C.F. and S.A.R. collected and analyzed the melanoma samples, A.S.B., J.C.C., N.S.A., P.B., P.P.-G., S.D., C.W., C.E.B., J. Table 2 online) and sequenced with dye terminator chemistry. To determine whether a given mutation was somatic, we sequenced the gene in genomic DNA from matched normal tissue. From the ∼5.5 Mb of sequence information obtained, we identified eight MMP genes containing somatic mutations (Table 1). Genes found to have one or more nonsynonymous mutations were then screened for mutations in an additional 47 melanomas. Through this approach, we identified 28 somatic mutations in eight genes, affecting 23% of the melanoma tumors analyzed (Table 1 and Supplementary Fig. 1 online).In seven tumors, both alleles of the MMP gene were affected, a characteristic associated with tumor suppressor genes. In addition, 6 of the 28 mutations were nonsense or splice-site alterations, which were predicted to result in aberrant or truncated proteins. Most tumors with MMP gene mutations also contained mutations in NRAS or BRAF. The clinical information associated with melanoma tumors containing MMP mutations is described in Supplementary Table 3 online.The observed somatic mutations could be either 'driver' mutations that have a functional role underlying neoplasia or nonfunctional 'passenger' changes. In the eight genes found to be mutated, 28 nonsynonymous (N) and 5 synonymous (S) somatic mutations were identified, yielding a N:S ratio of 28:5, significantly higher than the N:S ratio of 2:1 predicted for nonselected passenger mutations 9 (P < 0.026), suggesting that these are driver mutations. The ratio of C>T mutations compared to other nucleotide s...

SummaryMicrophthalmia-associated transcription factor (MITF) is involved in melanocyte cell development, pigmentation and neoplasia. To determine whether MITF is somatically mutated in melanoma, we compared the sequence of MITF from primary and metastatic lesions to patient-matched normal DNA. In the 50 metastatic melanoma tumor lines analysed, we discovered four samples that had genomic amplifications of MITF and four that had MITF mutations in the regions encoding the transactivation, DNA binding or basic, helix-loophelix domains. Sequence analysis for SOX10, a transcription factor, which both acts upstream of MITF and synergizes with MITF, identified an additional three samples with frameshift or nonsense mutations. Microphthalmia-associated transcription factor and SOX10 were found to be mutated in a mutually exclusive fashion, possibly suggesting disruption in a common genetic pathway. Taken together we found that over 20% of the metastatic melanoma cases had alterations in the MITF pathway. We show that the MITF pathway is also altered in primary melanomas: 2/26 demonstrated mutations in MITF and 6/55 demonstrated mutations in SOX10. Our findings suggest that altered MITF function during melanomagenesis can be achieved by MITF amplification, MITF single base substitutions or by mutation of its regulator SOX10.

The transcription factor SOX10 is essential for survival and proper differentiation of neural crest cell lineages, where it plays an important role in the generation and maintenance of melanocytes. SOX10 is also highly expressed in melanoma tumors, but a role in disease progression has not been established. Here we report that melanoma tumor cell lines require wild-type SOX10 expression for proliferation, and SOX10 haploinsufficiency reduces melanoma initiation in the metabotropic glutamate receptor 1 (Grm1Tg) transgenic mouse model. Stable SOX10 knockdown in human melanoma cells arrested cell growth, altered cellular morphology, and induced senescence. Melanoma cells with stable loss of SOX10 were arrested in the G1 phase of the cell cycle, with reduced expression in the melanocyte determining factor MITF, elevated expression of p21WAF1 and p27KIP2, hypophosphorylated RB and reduced levels of its binding partner E2F1. Since cell cycle dysregulation is a core event in neoplastic transformation, the role for SOX10 in maintaining cell cycle control in melanocytes suggests a rational new direction for targeted treatment or prevention of melanoma.

Summary Hypoxia and HIF1α signaling direct tissue-specific gene responses regulating tumor progression, invasion and metastasis. By integrating HIF1α knockdown and hypoxia-induced gene expression changes, this study identifies a melanocyte-specific, HIF1α-dependent/hypoxia-responsive gene expression signature. Integration of these gene expression changes with HIF1α ChIP-Seq analysis identifies 81 HIF1α direct target genes in melanocytes. The expression levels for ten of the HIF1α direct targets – GAPDH, PKM, PPAT, DARS, DTWD1, SEH1L, ZNF292, RLF, AGTRAP, and GPC6 – are significantly correlated with reduced time of Disease Free Status (DFS) in melanoma by logistic regression (P-value =0.0013) and ROC curve analysis (AUC= 0.826, P-value<0.0001). This HIF1α-regulated profile defines a melanocyte-specific response under hypoxia, and demonstrates the role of HIF1α as an invasive cell state gatekeeper in regulating cellular metabolism, chromatin and transcriptional regulation, vascularization and invasion.

BackgroundThe ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them.ResultsIn this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3_MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3_MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3_MCS6. Consistent with its in vitro activity, we also show that ERBB3_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3_MCS6 expression in vivo in zebrafish.ConclusionsTaken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3_MCS6.

Summary The complex genetic changes underlying metastatic melanoma need to be deciphered to develop new and effective therapeutics. Previously, genome-wide microarray analyses of human melanoma identified two reciprocal gene expression programs, including transcripts regulated by either transforming growth factor, beta 1 (TGFβ1) pathways or microphthalmia-associated transcription factor (MITF)/SRY-box containing gene 10 (SOX10) pathways. We extended this knowledge by discovering that melanoma cell lines with these two expression programs exhibit distinctive microRNA (miRNA) expression patterns. We also demonstrated that hypoxia-inducible factor 1 alpha (HIF1A) is increased in TGFβ1 pathway-expressing melanoma cells and that HIF1A upregulates miR-210, miR-218, miR-224, and miR-452. Reduced expression of these four miRNAs in TGFβ1 pathway-expressing melanoma cells arrests the cell cycle, while their overexpression in mouse melanoma cells increases the expression of the hypoxic response gene Bnip3. Taken together, these data suggest that HIF1A may regulate some of the gene expression and biological behavior of TGFβ1 pathway-expressing melanoma cells, in part via alterations in these four miRNAs.