rRNA transcription as potential therapeutic target in Triple Negative Breast Cancers

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Ribosomal RNA gene (rDNA) transcription that gives rise to the synthesis of the three most substantial ribosomal RNAs (rRNA) is governed by the enzyme RNA polymerase I (Pol I), which represents a crucial regulatory process for ribosomal biogenesis (RiBi). For more than a hundred years, it has been recognised that both the number and size of nucleoli, where RiBi occurs, are elevated in tumour cells. The appreciation that amplified rRNA expression is a major actor in malignancy advancement through enhanced protein production and consequent ability for cellular replication, and also via the regulation of cellular checkpoints and chromatin structure, has generated much interest in novel treatment options for neoplasia that target Pol-I transcription per se. In the current thesis, rRNA synthesis inhibition as a possible treatment option in triple negative breast cancer (TNBC) was investigated. This mechanism represents an opportunity to transform therapy for the most deadly types of this disease. This was accomplished by conducting detailed studies with the use of four novel agents that exhibit extremely selective inhibitory action on Pol I. The fact that transcription of Pol I is essential to the metastatic dissemination of tumour cells offered justification for targeting transcription of Pol I in TNBC. In contrast to ionising radiation or traditional chemotherapy, Pol I inhibitors do not damage the genome and therefore only have a modest effect on non-cancerous cells. Work was performed in order to explore the sensitivity of four cell lines, embodying different types of TNBC, to these novel Pol-I inhibitors and to distinguish possible biomarkers for patient stratification. Given the potential for these agents to enable additional studies to be performed in order to evaluate the relationship between malignancy advancement and transcription of Pol I, potential associations between the growth inhibitory efficacy of four discrete Pol-I inhibitors were appraised in an experimental model consisting of TNBC cell lines. The consequences of these drugs on cell growth, rRNA production, the cell cycle, cellular viability, rDNA chromatin and the relationships between the effectiveness of the agent and end expression of specific genes were assessed. Concurrently, the first data suggesting that PMR-116 acts to specifically inhibit Pol-I transcription in in vivo and in vitro conditions, were attained. It was also shown that Pol-I transcription inhibition can be effectively inhibited by PMR-116 from pre-assembled PICS; displacement of SL1 was recognised as the agent’s possible mode of action. In addition, a novel mechanism was identified in relation to the anti-tumour effects of CX-5461. This compound causes targeted DNA damage at rDNA loci, an area that is often amplified and which undergoes enhanced transcription within tumour cells, leading to a forceful activation of ATR-dependent DDR. Simultaneously, CX-5461 also stimulates NSP. The combination of these two pathways gives rise to selective malignancy cell necrosis. In combination, the results from this thesis have determined that Pol-I inhibition is a viable target for the future development of chemotherapeutic agents