SACM - United Kingdom
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Item Restricted Evaluation of Replication Protein A (RPA) as a Prognostic and Therapeutic Target in Breast and Ovarian Cancer(University of Nottingham, 2024-02-14) Algethami, Mashael Abdulaziz; Madhusudan, SrinivasanIntroduction: 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.23 0Item Restricted The Role of Prolactin Receptor in Ovarian Cancer(2023) Mohammed, Rean; El-Bahrawy, Mona; McNeish, IainOvarian cancer is the most lethal gynaecological malignancy affecting women. Surgery and chemotherapy are the main treatments, but the development of resistance to chemotherapy remains the main obstacle to treatment, necessitating the development of other therapeutic modalities. Prolactin (PRL) is a polypeptide hormone which has various function in vertebrates, including lactation, reproduction, mammary cell growth and differentiation and immune functions. Prolactin receptor (PRLR) and ligands are expressed in many different types of cells, and a link between prolactin and cancer has been suspected for many years. Research studies have demonstrated a role for PRL and PRLR, most importantly in breast and ovarian cancers, but also in a variety of other cancers. This study examines the effect of PRLR knockdown and antagonism on cell viability, apoptosis and responses to chemotherapy in a number of high grade serous carcinoma (HGSC) cell lines, namely SKOV3, COV362, ES-2, PEA-1, PEO-1, PEO-4 and one breast carcinoma cell line MCF-7. Using immunocytochemistry, Western blotting and RTqPCR, the expression of PRLR and its ligands was studied in the cell lines. The results confirmed that all the cell lines that were studied expressed PRLR, and at least one of the ligands at different levels. Using the PRLR antagonist G129R alone and combined with cisplatin had no effect on cell viability or apoptosis in SKOV-3, COV362, ES-2 and MCF-7. The stable knockdown of PRLR in cell lines was established in the ovarian carcinoma cell line PEO-1 and the breast carcinoma cell line MCF-7. The mRNA from cells with stable knockdown of PRLR of PEO1, and MCF-7 cells was sent for RNA sequencing in order to study the changes in gene expression on knocking down PRLR, with the aim of understanding the role of PRLR in tumour cell biology further. Several interesting pathways were affected, including early/late oestrogen response and MYC target V1 pathways. Selected genes from these pathways were explored further. My results showed that all cell lines that were used in this project expressed PRLR and its ligands, but the ligands varied from one cell line to another. G129R antagonist had no effect on cell viability or apoptotic activity in any of the cell lines when used alone and no additive response when it was used along with cisplatin in all cell lines. The RNA-seq that resulted from knocking down PRLR in MCF-7 showed three interesting pathways. Selected genes from early/late oestrogen response, including ESR1, DHRS2, TFAP2C, SLC7A5, XBP1, ADCY9, SULT2B1 and CCND1, were validated by RTqPCR in PEO-1, OVCAR-3 and MCF-7. The expression of ESR1, DHRS2, SLC7A5, ADCY9 and CCND1 was aligned with the results of RNA-seq in siRNA PRLR MCF-7, but TFAP2C, XBP-1 and SULT2B showed no significant changes. However, there was no significant change in the expression in knockdown PEO-1 and OVCAR-3, apart from ADCY9, which was significantly up-regulated in knockdown PEO-1, while SULT2B1 were significantly down-regulated. Different systems and control mechanisms may therefore be applied to study different organs as well as different mechanisms between cell types within the same organ.36 0