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Abstract
Approximately 94% of colorectal cancers have been found with mutations in the Wnt signalling pathway. The majority of these cancers are caused by hyperactivated β-catenin dependent Wnt signalling. ROR2 is a receptor tyrosine kinase of the Wnt signalling pathway which has been shown to antagonise downstream targets of β-catenin dependent Wnt signalling. This study examines colorectal cancer cell lines, adenomas and carcinomas for evidence of ROR2 epigenetic silencing and investigates if ROR2 expression changes can alter cellular proliferation, migration and invasion.
Expression analysis of colorectal cancer cell lines found 76.7% (23/30) to have negligible ROR2 expression levels. ROR2 was also reduced in the majority of colorectal adenomas (5/6) and carcinomas (11/12) when compared to matching normal tissue. Bisulphite sequencing revealed that the ROR2 expression loss was caused by epigenetic silencing through promoter hypermethylation in colorectal cancer cell lines, adenomas and carcinomas.
Silencing of ROR2 in colorectal cancer cell lines was found to increase proliferation and migration. However, ROR2 modulation in different cell lines resulted in different expression changes amongst the Wnt target genes, suggesting that ROR2 may trigger alternate Wnt responses depending on specific genetic and phenotypic profiles.
ROR2 has been reported to be upregulated in certain cancers whilst downregulated in others. A possible cause for this disparity may be off-target binding in the antibodies as our analysis of three commercial ROR2 antibodies found that only one specifically bound to ROR2. The use of antibodies with non-specific binding can adversely affect immunohistochemistry analysis, making prior antibody validation essential. Our immunohistochemistry analysis using a validated antibody confirmed that ROR2 was downregulated in the majority of colorectal cancer cases and also found no correlation between ROR2 presence and patient survival as had been reported in a previous publication.
This study revealed epigenetic silencing of ROR2 to be an early event in colorectal neoplasia. Functional assays also found that silencing of ROR2 is associated with increased proliferation and migration in cell lines. If future research can confirm ROR2 epigenetic silencing and expression loss to be critical in driving early colorectal cancer development in a biological model more approximate to that found in colorectal adenomas, it can potentiate ROR2 as a possible therapeutic target for preventing disease progression.