Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma

Renal cell carcinoma (RCC), the most common adult kidney neoplasm, is histo pathologically heterogeneous, with most sporadic RCCs (similar to 80%) clas sified as clear cell (CC) tumors. Chromosome 3p allele loss is the most fre quent genetic alteration in RCC but is associated specifically with sporadi c and hereditary forms of clear cell RCC (CC-RCC) and is not a feature of n on-CC-RCC, such as papillary (chromophilic) RCC. The VHL tumor suppressor g ene (TSG) maps to chromosome 3p25, and somatic inactivation of the VHL gene occurs in up to 70% of CC-RCC tumors and cell lines. However, VHL inactiva tion is not sufficient for CC-RCC tumorigenesis, and inactivation of 3p12-p 21 TSG(s) appears to be necessary in CC-RCC irrespective of VHL gene inacti vation status. Recently, we demonstrated that the candidate 3p21 TSG, RASSF 1A, is hypermethylated in most small cell lung cancers. We have now investi gated the role of RASSF1A inactivation in primary RCC tumors. RASSF1A promo ter methylation was detected in 23% (32 of 138) of primary CC-RCC tumors. I n CC-RCC cell lines, RASSF1A methylation was associated with silencing of R ASSF1A expression and restoration of expression after treatment with 5'-aza cytidine. The frequency of RASSF1A methylation was similar in CC-RCC with a nd without VHL gene inactivation (24% versus 21%), and there was no associa tion between epigenetic silencing of the RASSF1A and VHL TSGs, because 0 of 6 tumors with VHL hypermethylation had RASSF1A methylation, and VHL was no t methylated in 26 CC-RCCs with RASSF1A methylation. Although 3p allele los s has been reported rarely in papillary RCC, we identified RASSF1A methylat ion in 44% (12 of 27) of papillary RCCs analyzed. Thus: (a) inactivation of RASSF1A is a frequent event in both CC-RCC and papillary RCC tumors; (b) t here is no relationship between epigenetic silencing of RASSF1A and VHL ina ctivation status in CC-RCC. Fifty-four CC-RCCs analyzed for RASSF1A methyla tion were informative for 3p21 allele loss, and 20% (7 of 35) with 3p21 all ele loss demonstrated RASSF1A methylation. All informative CC-RCCs with 3p2 1 allele loss and no RASSF1A methylation also demonstrated allele losses at other regions of 3p so that tumorigenesis in these cases may result from: (a) haploinsufficiency of RASSF1A; (b) inactivation of other 3p21 TSGs; or (c) inactivation of 3p TSGs from outside of 3p21. RASSF1A is the first TSG to be inactivated frequently in both papillary and CC-RCCs. The finding of frequent epigenetic inactivation of RASSF1A in papillary RCCs despite previ ous studies reporting infrequent 3p21 allele loss in this tumor type illust rates how the systematic identification of all major human cancer genes wil l require detailed analysis of the cancer genome and epigenome.