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

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 <i>PADI2</i> mRNA expression with overall survival using both platforms (RNA-seq cohort p=0.008 and microarray cohort p=0.0112). Low expression of <i>PADI2</i> 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 <i>ARHGEF10L</i> and 0.75-fold decrease in <i>FZD5</i> expression upon PADI2 overexpression. In this thesis, The Cancer Genome Atlas expression studies of <i>PADI2</i> showed that higher <i>PADI2</i> confers decreased survival of EOC patients. Conversely, <i>PADI2</i> overexpression <i>in vitro</i>, induced apoptosis/autophagy and decreased proliferation/cellular aggregation possibly via deregulating <i>FZD5</i> and <i>ARHGEF10L</i>. 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.

Name dc:type.qualificationname

Doctoral Thesis

Level dc:type.qualificationlevel

Student thesis

Grantor dc:publisher.institution

University of Northampton

Year dc:date.issued

2019

Author dc:creator

• Albalbeisi, Nermin

Advisors dc:contributor.advisor

• Machado, Lee Richard
• Anthony, Karen

• PADI2
• ovarian cancer
• calcium
• cisplatin
• apoptosis
• proliferation
• autophagy
• aggregation
• spheroid
• necrosis
• CRISPR/Cas9

Language dc:language

eng

Identifier

oai:pure.atira.dk:studenttheses/ffa34581-e47c-4776-97f3-98d3c43783ba

OAI identifier oai:identifier

oai:pure.atira.dk:studenttheses/ffa34581-e47c-4776-97f3-98d3c43783ba