Algethami, Mashael
(2024)
Evaluation of Replication Protein A (RPA) as a prognostic and therapeutic target in breast and ovarian cancer.
PhD thesis, University of Nottingham.
Abstract
Introduction: Replication protein A is a heterotrimeric complex composed of three linked subunits encoded by unique genes: RPA1 (chromosome 17p13, 70 kDa), RPA2 (chromosome 1p36.11, 32 kDa), and RPA3 (chromosome 7p13.3, 14 kDa). RPA is a critical ssDNA binding protein that coats and protects exposed ssDNA from endogenous nucleases during repair. RPA is essential for DNA replication, repair, and recombination.
The oligosaccharide/oligonucleotide binding fold (OB-fold) is an integral structural and functional component of RPA. RPA consists of a series of OB-folds (Domains A-F). In the RPA complex, RPA1 contains four ssDNA-binding domains (DBD): A, B, C, and F. These domains are responsible for most of the DNA binding activity of the complex. Both RPA2 and RPA3 contain a single DNA-binding domain and are involved in DNA binding. To understand the role of RPA in the pathogenesis of breast and ovarian cancer, we conducted a comprehensive transcriptomic, proteomic and preclinical study.
Patients and methods: RPA1, RPA2, and RPA3 protein expression were evaluated in 4221 primary invasive breast carcinomas and 776 breast ductal carcinoma in situ (DCIS) specimens using immunohistochemistry. Transcriptomic investigations were completed using the METABRIC cohort (n=1980). RPA1, RPA2, and RPA3 protein expression were also evaluated in 331 cases of epithelial ovarian cancer (EOC). Transcriptomic investigations were completed using a publicly available online gene expression dataset of 1259 patients with ovarian cancer. Cisplatin and PARP1 inhibitor (PARPi) sensitivity were tested in RPA-deficient breast and ovarian cancer cell lines.
Results: Loss of RPA1, RPA2, and RPA3 are frequent in DCIS and linked to aggressive features including high grade and ER and PR negativity. Low RPA was also associated with poor local recurrence-free survival in DCIS. In invasive breast cancer, low RPA1, low RPA2, and low RPA3 were all associated with larger tumour size, lympho-vascular invasion, higher histological grade, high stage, ER negativity, and poor breast cancer specific survival. Pre-clinically, RPA deficient breast cancer cells were more sensitive to cisplatin therapy compared to control cells. Additionally, the PARP1 inhibitor was synthetically lethal in RPA1- and RPA2-deficient cells compared to controls. Increased Olaparib sensitivity was associated with double strand breaks, cell cycle arrest, and increased apoptosis. In ovarian cancer, overexpression of RPA was associated with aggressive clinicopathological characteristics in EOC. In addition, high RPA expression was associated with a poor prognosis in platinum-sensitive ovarian tumours. Pre-clinical assessment showed that basal levels of RPA1 and RPA2 were higher in cisplatin-resistant A2780cis and PEO4 cells compared to cisplatin-sensitive A2780 and PEO1 cells. Additionally, depletion of RPA1 and RPA2 caused substantial platinum sensitization and increased PARP inhibitor sensitivity in A2780cis and PEO4 cells compared to control cells; this increased sensitivity was associated with increased accumulation of double strand breaks, S-phase cell cycle arrest, and apoptotic cells.
Conclusions: We provide the first comprehensive evidence that loss of RPA is an early event in the pathogenesis of breast cancer and promotes aggressive phenotypes. Pre-clinically, cisplatin and Olaparib were selectively toxic to RPA-deficient breast cancer cells. Moreover, RPA expression is a crucial predictor of platinum response in EOC. Thus, a combined approach of RPA inhibition and PARP inhibition may offer a more effective treatment paradigm for ovarian cancer.
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