title: Germline and somatic DNA methylation and epigenetic regulation of KILLIN in renal cell carcinoma.
abstract: We recently identified germline methylation of KILLIN, a novel p53-regulated tumor suppressor proximal to PTEN, in >1/3 Cowden or Cowden syndrome-like (CS/CSL) individuals who are PTEN mutation negative. Individuals with germline KILLIN methylation had increased risks of renal cell carcinoma (RCC) over those with PTEN mutations. Therefore, we tested the hypothesis that KILLIN may be a RCC susceptibility gene, silenced by germline methylation. We found germline hypermethylation by combined bisulfite restriction analysis in at least one of the four CpG-rich regions in 23/41 (56%) RCC patients compared to 0/50 controls (P < 0.0001). Of the 23, 11 (48%) demonstrated methylation in the -598 to -890 bp region in respect to the KILLIN transcription start site. Furthermore, 19 of 20 advanced RCC showed somatic hypermethylation upstream of KILLIN, with the majority hypermethylated at more than one CpG island (13/19 vs. 3/23 with germline methylation, P < 0.0001). qRT-PCR revealed that methylation significantly downregulates KILLIN expression (P = 0.05), and demethylation treatment by 5-aza-2'deoxycytidine significantly increased KILLIN expression in all RCC cell lines while only increasing PTEN expression in one line. Furthermore, targeted in vitro methylation revealed a significant decrease in KILLIN promoter activity only. These data reveal differential epigenetic regulation by DNA promoter methylation of this bidirectional promoter. In summary, we have identified KILLIN as a potential novel cancer predisposition gene for nonsyndromic clear-cell RCC, and the epigenetic mechanism of KILLIN inactivation in both the germline and somatic setting suggests the potential for treatment with demethylating agents.
authors.affiliation: Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: Kristi L
authors.initials: KL
authors.lastname: Bennett
authors.forename: Rebecca
authors.initials: R
authors.lastname: Campbell
authors.forename: Shireen
authors.initials: S
authors.lastname: Ganapathi
authors.forename: Ming
authors.initials: M
authors.lastname: Zhou
authors.forename: Brian
authors.initials: B
authors.lastname: Rini
authors.forename: Ram
authors.initials: R
authors.lastname: Ganapathi
authors.forename: Hartmut P H
authors.initials: HP
authors.lastname: Neumann
authors.forename: Charis
authors.initials: C
authors.lastname: Eng
mesh_terms: D000328:Adult; D000368:Aged; D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006223:Hamartoma Syndrome, Multiple / Q000235:genetics; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000473:pathology; D008875:Middle Aged; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics; D011401:Promoter Regions, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D055815:Young Adult
chemical_list: C554482:KLLN protein, human; D025521:Tumor Suppressor Proteins; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human