Profiling epigenetic inactivation of tumor suppressor genes in tumors and plasma from cutaneous melanoma patients.

Aberrant methylation of CpG islands in promoter regions of tumor suppressor genes (TSG) has been demonstrated in epithelial origin tumors. However, the methylation profiling of tumor-related gene promoter regions in cutaneous melanoma tumors has not been reported. Seven known or candidate TSGs that are frequently hypermethylated in carcinomas were assessed by methylation-specific polymerase chain reaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanoma tumors. Four TSGs were frequently hypermethylated in 86 metastatic tumor specimens: retinoic acid receptor-beta2 (RAR-beta2) (70%), RAS association domain family protein 1A (RASSF1A) (57%), and O6-methylguanine DNA methylatransferase (MGMT) (34%), and death-associated protein kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK was significantly lower in primary melanomas (n=20) compared to metastatic melanomas. However, hypermethylation of RAR-beta2 was 70% in both primary and metastatic melanomas. Cell lines had hypermethylation profiles similar to those of metastatic melanomas. The analysis of these four markers of metastatic tumors demonstrated that 97% had > or =1 gene(s) and 59% had > or =2 genes hypermethylated. The methylation of genes was verified by bisulfite sequencing. The mRNA transcripts could be re-expressed in melanoma cell lines having hypermethylated genes following treatment with 5'-aza 2'-deoxycytidine (5Aza-dC). Analysis of melanoma patients' plasma (preoperative blood; n=31) demonstrated circulating hypermethylated MGMT, RAR-beta2, and RASSF1A DNA for at least one of the markers in 29% of the patients. Our findings indicate that the incidence of TSG hypermethylation increases during tumor progression. Methylation of TSG may play a significant role in cutaneous melanoma progression.

Epigenetic inactivation of RAS association domain family protein 1 (RASSF1A) in malignant cutaneous melanoma.

Recent findings have shown the inactivation of a Ras effector homologue gene referred to as the Ras association domain family 1 (RASSF1) gene, which is a potential human tumor suppressor gene located on chromosome 3p21.3. Hypermethylation of the CpG island promoter region of a major alternative transcript of this gene, RASSF1A, has been suggested to play a key role in pathogenesis of various carcinomas. There is limited analysis of inactivation of RASSF1A in tumors other than carcinomas. Hypermethylation of two regions of the RASSF1A CpG island was investigated in metastatic cutaneous melanomas using methylation-specific PCR; region 1 is located upstream, and region 2 is located within the first exon (1alpha) of the open reading frame of the RASSF1A transcript. Eleven melanoma cell lines and 44 melanoma tumors were examined. Methylation of RASSF1A CpG island promoter region 1 was detected in 7 (64%) cell lines and 18 (41%) tumors, and methylation of region 2 was detected in 9 (82%) cell lines and 22 (50%) tumors. Overall, RASSF1A gene hypermethylation was detected in 55% of the melanoma tumors. No methylation was detected in normal skin tissues or healthy donor lymphocytes. All cell lines that showed methylation at promoter region 1 were also methylated at promoter region 2. Hypermethylation of both CpG island regions correlated with no expression of the RASSF1A gene. RASSF1A transcripts could be reexpressed in cell lines after treatment with 5'-aza-2'-deoxycytidine. Our findings indicate that the RASSF1A gene is turned off in a significant number of melanomas and that CpG promoter region hypermethylation may play a role in the transcriptional inactivation of the RASSF1A gene in malignant melanoma.