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 ...

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...