Methylation associated inactivation of RASSF1A from region 3p21.3 in lung,breast and ovarian tumours

Previously we analysed overlapping homozygous deletions in lung and breast tumours/tumour lines and defined a small region of 120 kb (part of LCTSGR1) at 3p21.3 that contained putative lung and breast cancer tumour suppressor gene(s) (TSG), Eight genes including RASSF1 were isolated from the minimal region. However, extensive mutation analysis in lung tumours and tumour li nes revealed only rare inactivating mutations. Recently, de novo methylatio n at a CpG island associated with isoform A of RASSF1 (RASSF1A) was reporte d in lung tumours and tumour lines. To investigate RASSF1A as a candidate T SG for various cancers, we investigated: (a) RASSF1A methylation status in a large series of primary tumour and tumour lines; (b) chromosome 3p allele loss in lung tumours and (c) RASSF1 mutation analysis in breast tumours, R ASSF1A promoter region CpG island methylation was detected in 72% of SCLC, 34% of NSCLC, 9% of breast, 10% of ovarian and 0% of primary cervical tumou rs and in 72% SCLC, 36% NSCLC, 80% of breast and 40% of ovarian tumour line s. In view of the lower frequency of RASSF1 methylation in primary breast c ancers me proceeded to RASSF1 mutation analysis in 40 breast cancers. No mu tations were detected, but six single nucleotide polymorphisms were identif ied. Twenty of 26 SCLC tumours with 3p21.3 allelic loss had RASSF1A methyla tion, while only six out of 22 NSCLC with 3p21.3 allele loss had RASSF1A me thylation (P = 0.0012), one out of five ovarian and none out of six cervica l tumours with 3p21.3 loss had RASSF1A methylation. These results suggest t hat (a) RASSF1A inactivation by two hits (methylation and loss) is a critic al step in SCLC tumourigenesis and (b) RASSF1A inactivation is of lesser im portance in NSCLC, breast, ovarian and cervical cancers in which other gene s within LCTSGR1 are likely to be implicated.