Identification of novel tumor suppressor genes involved in the development of cutaneous malignant melanoma
Final Report Abstract
Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven to be a strategy for genome-wide discovery of genes containing inactivating mutations in colon and prostate cancers. Here, we present the first study of inhibition of the nonsense-mediated mRNA decay (NMD) pathway in melanoma. We used a combination of emetine and actinomycin D treatment to stabilise mRNAs containing premature termination codons (PTCs), followed by microarray analysis and sequencing to identify novel tumour suppressor genes (TSGs) in a panel of 12 melanoma cell lines. Stringent analysis of the array data was used to select 35 candidate genes for sequencing. Of these, 4 (11%) were found to carry PTCs, including ARHGEF17, DENND2D, FGFR3, and RB1. RB1 mutations have previously been described in melanoma, the other three genes represent potentially novel melanoma TSGs. ARHGEF17 showed a G1865A mutation leading to W622X in a cell line derived from a mucosal melanoma, in RB1 a C1411T base change resulting In Q471X was discovered in a cell line derived from an acral melanoma and the FGFR3 and DENND2D genes had Intronic insertions leading to PTCs in cell lines derived from superficially spreading melanomas. The fact that a subset of those genes is located on chromosomes 1 and 11 could be a step further toward identifying long suggested, but yet not found TSGs in those regions. We conclude that although the false positive rate is high, due to the lack of DNA mismatch repair gene defects, the GINI protocol is capable of identifying PTCs in melanoma cell lines and, thus, one approach to discover novel TSGs in melanoma. Although GINI gives a high number of false positives, the advent of next generation high throughput sequencers makes it possible to rapidly screen a much larger number of candidate genes for mutations. Novel melanoma TSGs discovered by this study will form the basis for better understanding the molecular pathways underlying this disease. Such knowledge may allow the development of targeted therapies to treat melanoma.
Publications
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"Identification of ARHGEF17, DENND2D, FGFR3, and RBI mutations in melanoma by inhibition of nonsense-mediated mRNA decay." Genes Chromosomes Cancer. 2008 Dec;47(12):1076-85
Bloethner S, Mould A, Stark M, Hayward NK
