Use of integrative epigenetic and cytogenetic analyses to identify novel tumor-suppressor genes in malignant melanoma

Abstract

The objective of this study was to identify novel tumor-suppressor genes in melanoma, using an integrative genomic approach. Data from: (i) earlier reports of DNA loss and gain in malignant melanoma accompanied by comparative genomic hybridization high-definition array data of the entire human genome; (ii) microarray expression data from melanoma-derived cell lines identifying genes with significantly increased expression due to methylation using a pharmacologic demethylating strategy; and (iii) publicly available RNA expression microarray data of primary tumors and benign nevi were integrated using statistical tools to define a population of candidate tumor-suppressor genes. Twenty-seven genes were identified in areas of deletion that demonstrated diminished expression in primary melanomas relative to benign nevi and were significantly increased in expression by 5-Aza treatment. Seven genes of these 27 genes demonstrated methylation and deletion in a validation cohort of 14 separate primary tumors. These were: CHRDL1, SFRP1, TMEM47, LPL, RARRES1, PLCXD1, and KOX15. All of these genes demonstrated growth-suppressive properties with transfection into melanoma-derived cell lines. Seven putative tumor-suppressor genes in malignant melanoma were identified using a novel integrative technique.

Figure 1

Bisulfite sequencing of methylated candidate genes identified by integrative analysis. TSS= Transcriptional start site. ○ Unmethylated CpG ● Methylated CpG.

Figure 2

Gene copy number analysis. Asterisk (*) indicates statistically significant decrease in copy number (relative to 2 for autosomal genes and 1 for X-linked genes) suggestive of deletion (p<.05). Error bars represent standard deviation.

Figure 3

Transient transfection of melanoma derived cell lines with genes of interest. Cells harvested 72 hours after transfection. Cellular signal was quantitated with calcein fluorescence. Y axes represent the relative ratio of the detector signal absorption. Experiments performed in octuplicate. * indicates statistically significant difference from empty vector transfection (p<.05). Error bars represent standard deviation. A Transfection of LPL, RARRES1, and CHRDL1 results in growth inhibition of the HTB-72 cell line. B Transfection of LPL, KOX15, PLCXD1, TMEM47, RARRES1, and CHRDL1 results in growth inhibition of the HTB-70 cell line. C Transfection of LPL, PLCXD1, TMEM47, RARRES1, SFRP1 and CHRDL1 results in growth inhibition of the HTB-66 cell line. D Transfection of LPL, and SFRP1 results in growth inhibition of the A375 cell line.