PLS3 promoter methylation and plastin expression in colorectal cancer

Abstract

Colorectal cancer (CRC) is one of the most prevalent malignancies in the Western world. It is a heterogeneous disease that begins from a benign polyp and progresses to invasive carcinoma via at least three distinct pathways. Prognosis of newly diagnosed cases mainly relies on the Dukes’ classification system, yet the 5-year survival rate of patients with CRC varies from 90% to 10% with tumour progression. The pursuit of biomarkers that can accurately predict patient prognosis has intensified in recent years. In our laboratory, we have previously shown that positive lamin A/C expression is a potential risk biomarker in CRC. Subsequent downstream investigations revealed that CRC cells overexpressing lamin A were significantly more motile compared to their GFP-only counterparts. This increase in motility was, in part, due to upregulation of the actin-bundling protein T-plastin (encoded by the PLS3 gene). The main aims of this thesis were to investigate PLS3 promoter methylation and plastin expression in CRC, using a combination of both epigenetic and immunohistochemical approaches. PLS3 promoter methylation was studied in CRC cell lines and a large cohort of CRC patients from the Netherlands Cohort Study on Diet and Cancer (NLCS) tissue archive. T-plastin expression was supressed because of promoter methylation and its expression was restored upon treatment with the DNA methylation inhibitor 5-aza-2’deoxycitidine (p = 0.01; p = 0.05). Furthermore, T-plastin was methylated in ~50% of CRC patients but methylation status did not provide any prognostic value. However, there was an association between both methylation status and sex (p = 0.01) and methylation status and tumour location (p = 0.05). The expression of plastin proteins in CRC was studied by raising polyclonal antibodies against two plastin isoforms and determining their specificity using immunoblotting and peptide inhibition assays. The most promising antibody, Dann1a, was used to analyse the expression of plastin by immunohistochemistry, using patient sections obtained from the NLCS archive. Strikingly, plastin was absent from normal colonic mucosa but was aberrantly expressed in 80% of CRC patients and was frequently observed at the invasive front of the tumour. Finally, we identified and validated a candidate gene network using Ingenuity Pathway Analysis, based on the over expression of GFP-lamin A in CRC cells. This data suggests that lamin A is a master regulator of a pathway linked to cancer and cell motility. These studies will stimulate further studies into the role of specific plastins in epithelial cancers and provide a framework for the development of novel drugable targets for treatment of CRC.