Functional characterisation of EFA6R, a possible biomarker for Epithelial Ovarian Cancer

Abstract

Background: EFA6R functions as a guanine nucleotide exchange factor for Arf6 -a Ras-like GTPase protein that potently regulates tumour progression. EFA6R is consistently expressed in healthy ovarian epithelium, but is drastically downregulated in the vast majority of Epithelial Ovarian Cancers (EOC). Therefore, EFA6R could be a novel biomarker for EOC.Aim: 1. Study EFA6R expression in Epithelial Ovarian Cancer (EOC) cell lines and tissues. 2. Identify the mechanisms of EFA6R downregulation and its role in mediating cellular phenotype. 3. Delineate the expression, localisation and cellular functions of EFA6R isoforms. Results: In EOC Tissue cDNA array, 73% of 192 samples exhibited little or significantly less EFA6R expression than healthy samples. In tissue microarrays containing 80 individual cases of different grades, EFA6R expression was lost with tumour progression. Similar downregulation was observed in tissue lysates (6/7 samples) and 10/10 OC cell lines. In SKOV-3 cells treated with demethylation agent 5-aza-2'deoxycytidine (5-Aza-Cdr) EFA6R expression was restored at mRNA and protein level; ~10-fold increase in EFA6R expression corresponded with significant attenuation of cell migration (~60% decrease in migration observed in Ibidi wound healing assay and ~ 5-fold decrease in migration and invasion in transwell assay). Exogenous expression of EFA6R plasmid DNA constructs showed that EFA6R localisation to the plasma membrane requires the PH and to a lesser extent, the CC domain. There, it functions as an Arf6-specific GEF, modulating actin stress fibres. Endogenous expression of EFA6R was observed in HEK293 and ReN cells. In the former, it potentially regulates bintegrin expression and in the latter, it plays a part in neuronal differentiation.Conclusion: EFA6R loss of function can be attributed to epigenetic mechanisms. Its downregulation, increases migration and metastasis through Arf6-independent pathways. One of the EFA6R isoforms has limited expression in some cell lines where it may play an important physiological role Contents