RESULTS: Here we report that the treatment of RCC cell lines with azacitidine suppressed cell proliferation in all 15 lines tested. A marked response to azacitidine therapy (>50% reduction in colony formation assay) was detected in the three cell lines with VHL promoter methylation but some RCC cell lines without VHL TSG methylation also demonstrated a similar response suggesting that multiple methylated TSGs might determine the response to demethylating therapies. To identify novel candidate methylated TSGs implicated in RCC we undertook a combined analysis of copy number and CpG methylation array data. Candidate novel epigenetically inactivated TSGs were further prioritised by expression analysis of RCC cell lines pre and post-azacitidine therapy and comparative expression analysis of tumour/normal pairs. Thus, with subsequent investigation two candidate genes were found to be methylated in more than 25% of our series and in the TCGA methylation dataset for 199 RCC samples: RGS7 (25.6% and 35.2% of tumours respectively) and NEFM in (25.6% and 30.2%). In addition three candidate genes were methylated in >10% of both datasets (TMEM74 (15.4% and 14.6%), GCM2 (41.0% and 14.6%) and AEBP1 (30.8% and 13.1%)). Methylation of GCM2 (P = 0.0324), NEFM (P = 0.0024) and RGS7 (P = 0.0067) was associated with prognosis.
CONCLUSIONS: These findings provide preclinical evidence that treatment with demethylating agents such as azacitidine might be useful for the treatment of advanced RCC and further insights into the role of epigenetic changes in the pathogenesis of RCC.