Characterising the Role of the Calcium-dependent Citrullinating Enzyme Peptidyl Arginine Deiminase 2 in Ovarian Cancer

Abstract

Epithelial ovarian cancer (EOC) represents the fifth most common cause of cancer mortality among women worldwide and accounts for the highest fatalities amongst gynaecological malignancies. The dysregulation of calcium-dependent peptidyl arginine deiminase 2 (PADI2) plays a key role in the tumorigenesis of several cancers; however, the role of PADI2 in the pathogenesis of EOC is yet to be investigated. Using RNA-seq and microarray data from primary serous ovarian cancers (The Cancer Genome Atlas data set) combined with survival data, the expression of PADI2 was assessed using each platform (n=262 and n=564). Kaplan-Meier analysis and Log-rank tests showed an association of PADI2 mRNA expression with overall survival using both platforms (RNA-seq cohort p=0.008 and microarray cohort p=0.0112). Low expression of PADI2 was associated with improved survival. Expression studies were used to examine the overexpression and knockout (CRISPR/Cas9 editing) of PADI2 expression, respectively, in the human-derived high-grade serous OVCAR-4 and mouse-derived EOC ID8-Luc2 cell lines. In OVCAR-4 cells, PADI2 overexpression reduced proliferation (22%) and cellular aggregation (94%), while increasing apoptosis (34%) and autophagy (38%), in a Ca2+- and citrullination-dependent manner, at 72 hours. PADI2 overexpression also induced cisplatin cytotoxicity by 10% at 72 hours. In ID8-Luc2 cells, PADI2 overexpression significantly decreased cisplatin cytotoxicity by 24%, independently of exogenous Ca2+ supplementation or induced citrullination, at 72 hours. In addition, qRT-PCR validation of TCGA gene co-expression data indicated that PADI2 overexpression correlated with expression of a number of genes. This was confirmed in functional studies where there was a 1.69-fold increase in ARHGEF10L and 0.75-fold decrease in FZD5 expression upon PADI2 overexpression. In this thesis, The Cancer Genome Atlas expression studies of PADI2 showed that higher PADI2 confers decreased survival of EOC patients. Conversely, PADI2 overexpression in vitro, induced apoptosis/autophagy and decreased proliferation/cellular aggregation possibly via deregulating FZD5 and ARHGEF10L. Collectively, this work suggests that PADI2 may serve as a potential therapeutic target in human EOC. Codon based selection analyses were used to test for evidence of positive selection in PADI2. The phylogenetic maximum likelihood analysis of PADI2 orthologues retrieved from 31 species indicated that there was no evidence of positive selection in PADI2 codons, but numerous residues were under evolutionary constraint.

Date of Award	Oct 2019
Original language	English
Awarding Institution	University of Northampton
Supervisor	Ian Livingstone (Supervisor), Lee Machado (Supervisor) & Karen Anthony (Supervisor)