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Title: The role of the RNA binding protein LARP-1 in the post-transcriptional regulation of cisplatin resistant ovarian cancer
Author: Stavraka, Chara
ISNI:       0000 0004 9349 845X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy accounting for over 4000 deaths annually in the UK. Due to the lack of a validated screening tool, patients usually present with advanced disease and develop resistance to platinum-based chemotherapy. Resensitising resistant tumour cells to cisplatin remains an unmet clinical need. The RNA-binding protein LARP1 is highly expressed in ovarian cancer and its depletion by RNA interference (RNAi) was found to restore platinum sensitivity in cisplatin-resistant EOC cell lines showing a synergistic anti-tumour effect with cisplatin. Using immunoprecipitation followed by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) in cisplatin sensitive (OVCAR3) and resistant (OVCAR8) ovarian cancer cell lines before and after cisplatin treatment, I identified PABP1, and YB-1 as strong, RNA-dependent LARP1 interactors in both cell lines. Upon cisplatin treatment, the interaction of LARP1 with YB-1 was preserved only in the resistant cell line and was further investigated as both proteins are known for promoting cisplatin resistance. In untreated OVCAR8 and OVCAR3 cells, LARP1 and YB-1 are predominantly cytoplasmic but accumulate in the nucleus upon cisplatin-induced genotoxic stress. LARP1 and YB-1 are both in complex with, and regulate the mRNA transcripts of genes linked to cisplatin resistance such as the efflux ATPase pump ATP7B, the DNA damage binding protein 2 (DDB2) and the pro-survival factor BCL2. Both LARP1 and YB-1 act on their targets by regulating their mRNA abundance with LARP1 showing a greater effect. Using the Surface Sensing of Translation (SUnSET) method, LARP1 was found to play a fundamental role in maintaining protein synthesis during genotoxic stress. I conclude that LARP1, via its RNA-mediated interaction with YB-1, is a key post-transcriptional regulator of genes involved in pre-and post-target mechanisms of cisplatin resistance. Furthermore, it plays a vital role in preserving “de novo” protein synthesis and consequently cell survival during cisplatin induced genotoxic stress. Its diverse and fundamental functions make it a promising therapeutic target.
Supervisor: Buluwela, Laki ; Mura, Manuela ; Ghaem-Maghami, Sadaf Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral