Abstract
BackgroundColorectal cancer (CRC) is closely linked to Wnt signalling, with 94 % of cases exhibiting a Wnt related mutation. ROR2 is a receptor tyrosine kinase that is thought to repress β-catenin dependent Wnt signalling. Our study aims to determine if ROR2 is epigenetically silenced in CRC and determine if in vitro silencing of ROR2 potentiates Wnt signalling, and alters the proliferative, migratory or invasive potential of cells.MethodsROR2 expression was examined in CRC cell lines and patient adenomas using qRT-PCR, while COBRA and bisulphite sequencing was used to analyse ROR2 promoter methylation. 258 patient primary tumour samples from publicly available databases were also examined for ROR2 expression and methylation. In addition, the functional effects of ROR2 modulation were investigated in HCT116 cells following ROR2 siRNA knockdown and in RKO and SW620 cells following ectopic ROR2 expression.ResultsReduced ROR2 expression was found to correlate with ROR2 promoter hypermethylation in colorectal cancer cell lines, carcinomas and adenomas. ROR2 expression was downregulated in 76.7 % (23/30) of CRC cell lines with increasing ROR2 promoter hypermethylation correlating with progressively lower expression. Analysis of 239 primary tumour samples from a publicly available cohort also found a significant correlation between reduced ROR2 expression and increased promoter methylation. Methylation analysis of 88 adenomas and 47 normal mucosa samples found greater percentage of adenoma samples to be methylated. Additional analysis also revealed that adenoma samples with reduced ROR2 expression also possessed ROR2 promoter hypermethylation. ROR2 knockdown in the CRC cell line HCT116 significantly decreased expression of the β-catenin independent Wnt targets genes JNK and NFATC1, increased cellular proliferation and migration but decreased invasion. When ROR2 was ectopically expressed in RKO and SW620 cells, there was no significant change to either cellular proliferation or migration.ConclusionROR2 is frequently epigenetically inactivated by promoter hypermethylation in the early stages of colorectal neoplasia and this may contribute to colorectal cancer progression by increasing cellular proliferation and migration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2576-7) contains supplementary material, which is available to authorized users.
Highlights
Colorectal cancer (CRC) is closely linked to Wnt signalling, with 94 % of cases exhibiting a Wnt related mutation
Bisulphite sequencing revealed that C170 and HCT116 cell lines, which had the highest levels of receptor 2 (ROR2) expression, had little to no methylation across the examined promoter molecules
Our study has found that ROR2 promoter hypermethylation and subsequent expression loss is an early event in CRC progression that first occurs in non-invasive adenomas
Summary
Colorectal cancer (CRC) is closely linked to Wnt signalling, with 94 % of cases exhibiting a Wnt related mutation. Consistent with its reported antagonism of β-catenin dependent Wnt signals, a 2010 study found ROR2 to be silenced in colorectal cancer, resulting in increased cellular proliferation [27]. Other reports in colorectal cancer, melanoma and osteosarcoma have found elevated ROR2 expression in tumours compared to normal tissue [28,29,30,31,32]. These conflicting reports have raised questions as to the role ROR2 plays in cancer and presents the possibility that the downstream effects of ROR2 are dependent on other Wnt genes and the cellular context of the cancer itself [33,34,35]
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