Loss of tumor suppressor gene function can occur as a result of epigenetic silencing of large chromosomal regions, referred to as long-range epigenetic silencing (LRES) and genome-wide analyses have revealed that LRES is present in many cancer types. Here we utilise Illumina Beadchip methylation array analysis to identify LRES across 800 kilobases of chromosome 5q31 in colorectal adenomas and carcinomas (n=34) relative to normal colonic epithelial DNA (n=6). This region encompasses 53 individual protocadherin (PCDH) genes divided amongst 3 gene clusters. Hypermethylation within these gene clusters is asynchronous; whilst most PCDH hypermethylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the adenoma-carcinoma transition. PCDHGC3 was hypermethylated in 17/28 carcinomas (60.7%) according to methylation array analysis. Quantitative real-time RT-PCR showed that PCDHGC3 is the highest expressed PCDH in normal colonic epithelium, and that there was a strong reciprocal relationship between PCDHGC3 methylation and expression in carcinomas (R = -0.84). PCDH LRES patterns are reflected in colorectal tumor cell-lines; adenoma cell-lines are not methylated at PCDHGC3 and show abundant expression at the mRNA and protein level, whilst expression is suppressed in hypermethylated carcinoma cell-lines (R = -0.73). Short-interfering RNA mediated reduction of PCDHGC3 led to a decrease of apoptosis in RG/C2 adenoma cells, and over-expression of PCDHGC3 in HCT116 cells resulted in reduction of colony formation, consistent with tumor suppressor capabilities for PCDHGC3. Further functional analysis showed that PCDHGC3 can suppress Wnt and mammalian target of rapamycin (mTOR) signaling in colorectal cancer cell lines. Together, our data suggest that the PCDH LRES is an important tumor suppressor locus in colorectal cancer, and that PCDHGC3 may be a strong marker and driver for the adenoma-carcinoma transition.
|Translated title of the contribution||Long-range epigenetic silencing of chromosome 5q31 protocadherins is involved in early and late stages of colorectal tumorigenesis through modulation of oncogenic pathways|
|Number of pages||11|
|Early online date||16 Jan 2012|
|Publication status||Published - 4 Oct 2012|