A Mechanistic Investigation of the Relationship Between Extramural Vascular Invasion (EMVI) and CpG Island Methylator Phenotype (CIMP) in Rectal Cancer

Abstract

Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer death worldwide. Each year, one million people will develop CRC, and 40-50% will die within five years. Furthermore, rectal and distal sigmoid cancers are known to present at a later stage and have a poorer prognosis than other colonic cancers. Rectal cancers that demonstrate pathological extramural vascular invasion (EMVI-positive) are known to have a poorer prognosis than those that do not (EMIV-negative), and EMVI has is acknowledged as an important risk factor for systemic recurrence, local recurrence and death. Additionally, EMVI status influences the need for pre- and post-operative chemoradiation (CRT); which may influence survival outcomes. Aberrant DNA methylation is emerging as a carcinogenic mechanism and potential biomarker in colorectal cancer. This study investigates the association between hypermethylation and EMVI in vivo and in vitro. Firstly, the in vivo associations between hypermethylation, EMVI, and clinical and histopathological outcomes are examined. Secondly, an investigation of the effects of demethylation on invasive colorectal cell lines in vitro aims to illuminate the genetic and cellular mechanisms that underlie methylation-dependent pathological cellular behaviour. Finally, highlighted biologic mechanisms are investigated in vivo to discover if there is an association with EMVI and survival outcomes. By these means the axis of association between hypermethylation, EMVI, and clinical outcomes is investigated. The investigation is conducted within the framework of consensus molecular subtyping in colorectal cancer, and in concordance with current methodologies of assessing DNA methylation status. The primary findings demonstrate that EMVI is associated with hypermethylation in vivo, but that there is no direct correlation between hypermethylation and disease-free (DFS) or overall survival (OS). In vitro, demethylation of hypermethylated colorectal cancer cells, by means of established demethylating agent 5-azacytidine and putative demethylator RRx-001, reduces their propensity to migrate and invade. Demethylation in vitro is also associated with changes in the expression of the metalloproteinases involved in the metabolism of the basement membrane and the epithelial-to-mesenchymal transition and tumour metastasis, notably MMP2 and TIMP2. Changes in expression were confirmed at transcriptomal and proteomic levels in response to demethylation. In vivo, MMP2 expression was shown to be statistically significantly associated with EMVI, DFS, and OS, and was also independently predictive of EMVI, raising the possibility that it could act as a diagnostic and predictive biomarker in rectal cancers. These findings indicate a mechanistic association between hypermethylation and EMVI, mediated by methylation-dependent expression of metalloproteinases. Metalloproteinase expression, specifically MMP2, may act as a potential biomarker for EMVI and correlates to survival outcomes in rectal cancer.