Madhusudan, Srinivasan(Shoqafi, Ahmed)2025-07-102025-01-02Shoqafi, Ahmed (2025) Evaluation of DNA repair biomarkers in epithelial ovarian and breast cancers. PhD thesis, University of Nottingham.https://eprints.nottingham.ac.uk/id/eprint/80298https://hdl.handle.net/20.500.14154/75794DNA damage signalling and repair pathways play a crucial role in the regulation of genomic stability. Impaired DNA repair promotes genomic instability, a key route to the development of cancers. Cells have developed signalling pathways that are activated in response to DNA damage to detect and repair the damage that has occurred. Ataxia-Telangiectasia Mutated (ATM) and Ataxia-Telangiectasia and Rad3-related (ATR) kinases are activated in the presence of DNA damage and initiate a series of phosphorylation events that lead to the activation of downstream effectors. Some of these effectors include p53 and BRCA1, which regulate cell cycle, DNA repair, and apoptosis. The major DNA repair pathways are nucleotide excision repair (NER), which removes bulky DNA adducts; base excision repair (BER), which is involved in repairing small, non-helix-distorting base lesions; mismatch repair (MMR), which corrects replication errors and double-strand break repair which include homologous recombination (HR) and non-homologous end joining (NHEJ). All these pathways are regulated in a manner that is specific to the type of damage that needs to be repaired so that repair is as accurate as possible. Since conventional therapies for advanced epithelial ovarian cancer (EOC) and breast cancers have been found to be less effective, there has been a shift to search for new therapeutic approaches that aim at targeting the DNA repair pathways. The host laboratory, using whole-exome sequencing in platinum-sensitive (PEO1, A2780) and platinum-resistant (PEO4, A2780cis) ovarian cancer cell lines, identified TP73 and POLE as potential predictors of platinum resistance. In the current study, I have investigated the role of TP73 and POLE in EOC and breast cancers in detail. TP73 is a member of the TP53 family of transcription factors that are involved in DNA repair, cell growth, migration, and death. In 331 EOC samples, I observed that high protein expression of TP73 was associated with higher tumour grade, late-stage disease and shorter progression free survival (PFS). In the large publicly available clinical cohort (n=522) and the cancer genome atlas (TCGA) ovarian cohort (n=182), TP73 transcript was upregulated in tumours compared to normal tissues and associated with shorter PFS. Preclinically, I have shown that overexpression of TP73 in A2780 platinum-sensitive ovarian cancer cells enhanced cell proliferation, invasion and increased DNA repair capacity. In clinical breast cancers, analysis of TP73 expression in 1,369 invasive breast cancers and 317 DCIS cases revealed that high cytoplasmic TP73 expression is significantly associated with aggressive disease features, including high tumour grade, ER negativity, triple-negative phenotype, and poor breast cancer-specific survival, particularly in the TP53 mutant subgroup. These findings highlight the prognostic and predictive significance of TP73 in EOC and breast cancers. POLE has roles during DNA replication and repair pathways. I have investigated POLE expression in EOC and breast cancers. Immunohistochemical analysis of 331 EOC samples revealed that 75% exhibited low nuclear POLE expression, while 25% showed high expression. High POLE levels were significantly associated with higher tumour grade, poor progression free survival (PFS) and overall survival (OS). The transcriptomic levels of POLE were analysed in patients with EOC, revealing that high POLE mRNA expression was significantly associated with poor progression free survival (PFS) and overall survival (OS) (All p=<0,05). Functional studies in platinum-resistant OVCAR 4 cells demonstrated that POLE knockdown increased cisplatin sensitivity, which was associated with double-strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. The data supports the role of POLE in predicting response to platinum chemotherapy in EOC. The study of 1,480 invasive breast cancer cases revealed that exhibited low nuclear POLE expression, which was associated with aggressive tumour features, poorer breast cancer specific survival (BCSS), and reduced response to endocrine therapy in ER+ and luminal subtypes. These findings suggest that POLE may be a predictive factor in ER+ breast cancers. Taken together, the data provides evidence for the role of TP73 and POLE as potential biomarkers in EOC and breast cancers.251enDNA repairDNA damageTP73POLEBreastOvarianResistantSensitiveThesis