title: Strategies on the use of Nucleolar Function Inhibitors in cancer therapy
Creators: name: Moreno Laporta, Lucia
Publisher: name: University of Liverpool Repository
Year: 2023

description: Kidney cancer is the 7th most common cancer in the UK with over 13,300 new cases and about 4,709 deaths per annum (1). Renal cell carcinoma (RCC) accounts for 85% of kidney cancers and stage at diagnosis for this type of kidney cancer is strongly associated with patient outcome with barely 12% 5-year survival rate in patients diagnosed with advanced RCC (2). Patients with metastatic RCC are often treated with systemic treatment, including chemotherapy, targeted therapy and immunotherapy, however, narrowed therapeutic indices limit their use as the high toxicity severely affects the patients’ quality of life and ability to follow through treatment regimens (3–5). The present thesis proposes a strategy to sensitise RCC cells to the effects of nucleolar function inhibitors (NFIs), particularly considering that changes in the morphology of the nucleoli are associated with high aggressiveness and poor prognosis of RCC patients (6), and the role that dysregulated nucleolar activity plays on increased proliferation of cancer cells (7). Given the fact that nucleolar function is modulated by cellular pathways for which targeted therapies have already been developed and are currently used to treat RCC, such as the mechanistic target of rapamycin (mTOR) pathway (8,9), the aims of this research are to determine whether mTOR inhibitors effectively modulate nucleolar function of RCC in vitro, to identify potential candidates for target-sensitised chemotherapy, and finally, to assess the effect of the drug combinations proposed. Microplate-based ribosomal RNA (rRNA) synthesis analysis using click chemistry revealed that ATP-competitive inhibitor Torin 1, but not the allosteric mTOR inhibitors Rapamycin, Temsirolimus and Everolimus, inhibits nucleolar function in ACHN and UoK111 cells. This was consistent with inhibition of cell viability and induction of cell cycle arrest achieved only by Torin 1 in the same cell lines, shown with MTT assay and flow cytometry, respectively. While treatment with both allosteric and ATP-competitive inhibitors of mTOR effectively reduced mTOR activity, as exemplified by the inhibition of the phosphorylation of mTOR complex 1 (mTORC1) substrate S6K and the changes in the phosphorylation patterns of 4EBP1 observed using Western Blot, only Torin 1 inhibited the phosphorylation of the RNA Polymerase I (RNA Pol I) transcription factor TIF-IA, which is a downstream target of mTOR, suggesting that decrease cell viability of RCC cells in the presence of Torin 1 might be mediated by the ability of this compound to modulate the nucleolar function. Subsequent study of the effects of the combination of Actinomycin D (ActD) and ATP-competitive mTOR inhibitors on cell viability and rRNA synthesis performed with MTT assay and microplate-based rRNA synthesis analysis using click chemistry and isobolographic analysis of the drug interactions, suggest that ATP-competitive mTOR inhibitors can sensitise RCC cells to the cytotoxic effects of ActD. Ultimately, the findings described in this thesis present the opportunity for a novel strategy for the treatment of RCC, targeting the nucleolus, which plays an important role in RCC, while taking advantage of the existing therapeutic agents. Specifically, we propose that combination of ATP-competitive mTOR inhibitors with the NFI ActD sensitises RCC cells to the cytotoxic effects of ActD, a strategy we termed target-sensitised chemotherapy.