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CpG island arrays represent a high-throughput epigenomic discovery platform to identify global disease-specific promoter hypermethylation candidates along bladder cancer progression. DNA obtained from 10 pairs of invasive bladder tumours were profiled vs their respective normal urothelium using differential methylation hybridisation on custom-made CpG arrays (n=12 288 clones). Promoter hypermethylation of 84 clones was simultaneously shown in at least 70% of the tumours. SOX9 was selected for further validation by bisulphite genomic sequencing and methylation-specific polymerase chain reaction in bladder cancer cells (n=11) and primary bladder tumours (n=101). Hypermethylation was observed in bladder cancer cells and associated with lack of gene expression, being restored in vitro by a demethylating agent. In primary bladder tumours, SOX9 hypermethylation was present in 56.4% of the cases. Moreover, SOX9 hypermethylation was significantly associated with tumour grade and overall survival. Thus, this high-throughput epigenomic strategy has served to identify novel hypermethylated candidates in bladder cancer. In vitro analyses supported the role of methylation in silencing SOX9 gene. The association of SOX9 hypermethylation with tumour progression and clinical outcome suggests its relevant clinical implications at stratifying patients affected with bladder cancer.

Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays