Epigenetic Inactivation of the RASSF 1 A 3 p 21 . 3 Tumor Suppressor Gene in Both Clear Cell and Papillary Renal Cell Carcinoma 1

Renal cell carcinoma (RCC), the most common adult kidney neoplasm, is histopathologically heterogeneous, with most sporadic RCCs ( 80%) classified as clear cell (CC) tumors. Chromosome 3p allele loss is the most frequent genetic alteration in RCC but is associated specifically with sporadic and hereditary forms of clear cell RCC (CCRCC) and is not a feature of non-CC-RCC, such as papillary (chromophilic) RCC. The VHL tumor suppressor gene (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 inactivation is not sufficient for CC-RCC tumorigenesis, and inactivation of 3p12–p21 TSG(s) appears to be necessary in CC-RCC irrespective of VHL gene inactivation status. Recently, we demonstrated that the candidate 3p21 TSG, RASSF1A, is hypermethylated in most small cell lung cancers. We have now investigated the role of RASSF1A inactivation in primary RCC tumors. RASSF1A promoter methylation was detected in 23% (32 of 138) of primary CC-RCC tumors. In CC-RCC cell lines, RASSF1A methylation was associated with silencing of RASSF1A expression and restoration of expression after treatment with 5 -azacytidine. The frequency of RASSF1A methylation was similar in CC-RCC with and without VHL gene inactivation (24% versus 21%), and there was no association between epigenetic silencing of the RASSF1A and VHL TSGs, because 0 of 6 tumors with VHL hypermethylation had RASSF1A methylation, and VHL was not methylated in 26 CC-RCCs with RASSF1A methylation. Although 3p allele loss has been reported rarely in papillary RCC, we identified RASSF1A methylation 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) there is no relationship between epigenetic silencing of RASSF1A and VHL inactivation status in CC-RCC. Fiftyfour CC-RCCs analyzed for RASSF1A methylation were informative for 3p21 allele loss, and 20% (7 of 35) with 3p21 allele loss demonstrated RASSF1A methylation. All informative CC-RCCs with 3p21 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 previous studies reporting infrequent 3p21 allele loss in this tumor type illustrates how the systematic identification of all major human cancer genes will require detailed analysis of the cancer genome and epigenome. INTRODUCTION RCC accounts for 2% of adult malignancies and is the most common adult kidney neoplasm. Histopathologically, most sporadic RCCs ( 80%) are classified as CC-RCCs, and papillary (chromophilic) tumors are the most common form of non-CC-RCC (1). Deletions of the short arm of chromosome 3 are the most widely observed genetic aberration in RCC. Although 3p allele loss is reported in 45–90% of sporadic RCCs, it is associated specifically with hereditary and sporadic CC-RCCs and is infrequent in non-CC-papillary RCC (2–8). Germ-line mutations in the VHL TSG are associated with a high risk of CC-RCCs, and somatic inactivation of the VHL gene (by loss, mutation, or epigenetic silencing) occurs in up to 70% of CCRCCs (9–14). However, VHL gene mutations are rare in non-CCRCCs. In contrast, germ-line mutations in the MET proto-oncogene cause hereditary papillary (chromophilic) RCCs, and somatic MET gene mutations are rare in CC-RCCs (15). Deletions of 3p are frequent in many adult cancers including lung, breast, ovary, testicular, and head and neck carcinomas (16). VHL TSG inactivation does not occur in these tumor types; therefore, several TSGs map to 3p. In addition to the FHIT TSG at 3p14, the finding of homozygous deletions in lung and breast cancers has mapped additional TSGs to 3p21 and 3p12. In detailed 3p alleleloss studies of CC-RCCs, we found evidence that loss of 3p12–p21 TSGs is required for tumor development in CC-RCCs with and without VHL gene inactivation, and that VHL inactivation alone was not sufficient for tumorigenesis (14, 17, 18). Furthermore, chromosome transfer experiments demonstrated that 3p fragments not containing the VHL gene (e.g., 3p12–p14 and 3p21–p22) can suppress RCC tumorigenicity (19, 20). Within 3p12–p21, several distinct regions are proposed to contain TSGs. The FHIT gene has been reported to suppress RCC tumorigenicity in nude mouse assays, and reduced expression and hemizygous deletions involving FHIT may occur in the majority of CC-RCCs (21–24). However, the role of FHIT in RCC tumorigenesis has been questioned (25), and 3p allele loss analysis in CC-RCCs suggests a primary role for 3p21 TSG(s) (14, 18, 26). A critical 120-kb interval for a lung and breast cancer TSG was identified by overlapping homozygous deletions (27, 28). We identified eight genes within this interval, and although mutation analysis of these candidate TSGs in lung tumors and tumor lines revealed only rare inactivating mutations, we and others have demonstrated promoter methylation and absent expression of RASSF1A in many lung cancer cell lines and tumors (29–31). In view of the evidence for a 3p21 TSG in CC-RCCs, we have investigated RASSF1A as a candidate gene in RCC. Received 5/21/01; accepted 7/31/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Medical Research Council, the Cancer Research Campaign, and the Association for International Cancer Research. A. M. is supported by the University of Antioquia, Medellin, Colombia, and a fellowship from COLCIENCIAS. S. H. is supported by Fundacao para a Ciencia e a Technologia. 2 To whom requests for reprints should be addressed, at Section of Medical and Molecular Genetics, Department of Paediatrics and Child Health, University of Birmingham, The Medical School, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 44-121-627-2741; Fax: 44-121-414-2538; E-mail: E.R. Maher@bham.ac. uk. 3 The abbreviations used are: RCC, renal cell carcinoma; CC-RCC, clear cell RCC; VHL, von Hippel-Lindau; TSG, tumor suppressor gene; RT-PCR, reverse transcriptionPCR; LOH, loss of heterozygosity.