ABSTRACT
Background: Genome-wide studies identified pan-cancer genes and shared biological networks affected by epigenetic dysregulation among diverse tumor entities. Here, we systematically screened for hypermethylation of DNA damage repair (DDR) genes in a comprehensive candidate-approach and exemplarily identify and validate candidate DDR genes as targets of epigenetic inactivation unique to bladder cancer (BLCA), which may serve as non-invasive biomarkers. Methods: Genome-wide DNA methylation datasets (2755 CpG probes of n = 7819 tumor and n = 659 normal samples) of the TCGA network covering 32 tumor entities were analyzed in silico for 177 DDR genes. Genes of interest were defined as differentially methylated between normal and cancerous tissues proximal to transcription start sites. The lead candidate gene was validated by methylation-specific PCR (MSP) and/or bisulfite-pyrosequencing in different human cell lines (n = 36), in primary BLCA tissues (n = 43), and in voided urine samples (n = 74) of BLCA patients. Urines from healthy donors and patients with urological benign and malignant diseases were included as controls (n = 78). mRNA expression was determined using qRT-PCR in vitro before (n = 5) and after decitabine treatment (n = 2). Protein expression was assessed by immunohistochemistry (n = 42). R 3.2.0. was used for statistical data acquisition and SPSS 21.0 for statistical analysis. Results: Overall, 39 DDR genes were hypermethylated in human cancers. Most exclusively and frequently methylated (37%) in primary BLCA was RBBP8, encoding endonuclease CtIP. RBBP8 hypermethylation predicted longer overall survival (OS) and was found in 2/4 bladder cancer cell lines but not in any of 33 cancer cell lines from entities with another origin like prostate. RBBP8 methylation was inversely correlated with RBBP8 mRNA and nuclear protein expression while RBBP8 was re-expressed after in vitro demethylation. RBBP8 methylation was associated with histological grade in primary BLCA and urine samples. RBBP8 methylation was detectable in urine samples of bladder cancer patients achieving a sensitivity of 52%, at 91% specificity. Conclusions: RBBP8 was identified as almost exclusively hypermethylated in BLCA. RBBP8/CtIP has a proven role in homologous recombination-mediated DNA double-strand break repair known to sensitize cancer cells for PARP1 inhibitors. Since RBBP8 methylation was detectable in urines, it may be a complementary marker of high specificity in urine for BLCA detection.
