Mutations in genes such as FLCN and VHL are implicated in the development of renal cell carcinoma (RCC). However, DNA methylation and transcriptional silencing at gene promoters can also be involved, as is the case with RASSF1A, which is rarely mutated but often inactivated by promoter methylation in sporadic RCC (see Morris & Maher, 2010 for a review). A study by Ricketts et al. (2012) has now taken this concept further by revealing novel genes that also appear to be methylated in sporadic kidney cancer.
The authors used the Illumina Infinium HumanMethylation27 BeadChip array to directly assay the DNA methylation status of >14,000 genes in 38 samples of sporadic RCC and 9 normal kidney samples. Aberrant hyper- and hypo-methylation was observed in several (but not all) tumours when compared to control samples. Closer analysis identified a number of genes that were both highly methylated and had a functional relevance to cancer. In addition, this technique was verified using bisulphite sequencing and combined bisulfite restriction analysis (CoBRA).
Cluster analysis was used to group these genes into 5 categories depending on their level of methylation, and no association was observed between these groups and VHL mutation status. 6 genes (SLC34A2, OVOL1, DLEC1, TMPRSS2, SST and BMP4) were then investigated further and were shown to be methylated in a variety of RCC cell lines. These genes were also re-expressed in these cells after the addition of 5-Aza-2’-deoxycytidine, a DNA demethylating agent. Furthermore, the 6 genes exhibited both tumour-specific methylation and reduced expression when compared to normal kidney controls.
To investigate the functional impact of these genes, siRNA was used to knockdown OVOL1, SST and DLEC1 in HEK293 cells. Upon knockdown, anchorage-independent growth was significantly increased, especially with the OVOL1 knockdown. Moreover, real-time RT-PCR demonstrated that OVOL1 knockdown led to an increase in the expression of the proto-oncogene MYC (when compared to siRNA controls). OVOL1 is a zinc-finger transcription factor that is a downstream target of TGF-β signalling, and it has not been previously associated with human cancer.
In summary, this study has implicated a number of genes in the development of sporadic RCC, which could be used to develop novel biomarkers and (epigenetic) therapies. Khoo et al. (2003) noted that FLCN could also be inactivated by DNA methylation in sporadic RCC. Consequently, could DNA methylation play a role in BHD syndrome, for example, where no known FLCN mutations have been identified?
- Khoo SK, Kahnoski K, Sugimura J, Petillo D, Chen J, Shockley K, Ludlow J, Knapp R, Giraud S, Richard S, Nordenskjöld M, Teh BT. Inactivation of BHD in sporadic renal tumors. Cancer Res. 2003 Aug 1;63(15):4583-7. PMID: 12907635
- Morris MR, Maher ER. Epigenetics of renal cell carcinoma: the path towards new diagnostics and therapeutics. Genome Med. 2010 Sep 3;2(9):59. PMID: 20815920
- Ricketts CJ, Morris MR, Gentle D, Brown M, Wake N, Woodward ER, Clarke N, Latif F, Maher ER. Genome-wide CpG island methylation analysis implicates novel genes in the pathogenesis of renal cell carcinoma. Epigenetics. 2012 Mar;7(3):278-90. PMID: 22430804
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