Understanding the functional involvement of ERɑ and hypoxia in the pathophysiology of breast cancers with different molecular subtypes

Breast cancer is the leading cause of cancer-related mortality for women. Dysregulated RNA polymerase III (Pol III) transcription of tRNA is significantly implicated in cancer progression. Oestrogen receptor alpha (ERɑ) potentiates ~75% of breast tumours, and resistance to endocrine therapies that target ERɑ activity is a major clinical problem urgently requiring significant advances in research to improve survival outcomes for women with endocrine-resistant disease. The ERɑ is a prolific transcription factor that upregulates many pro-tumorigenic genes, including tRNAs in response to hormone activation. This thesis sought to investigate the mechanism driving ERɑ-dependent tRNA expression. Analysis of public ChIP-seq data showed ERɑ was physically associated with ~50% of tRNA loci in breast cancer cells. ERɑ recruitment to tRNA genes was mediated by protein-protein interactions of ERɑ with Pol III-specific TFIIIC, determined by qPLEX-RIME and coimmunoprecipitation. FOXA1, a modulator of ERɑ activity, was enriched at tRNA promoters, suggesting FOXA1 may facilitate ERɑ recruitment to Pol III-transcribed genes and hormone-dependent activation of transcription. Further exploration of this ERɑ-FOXA1-Pol III axis could lead to novel and necessary developments in the treatment of advanced ERɑ-driven breast cancer.

Altered Na+ homeostasis is a critical determinant of breast cancer progression. Intratumoral hypoxia is linked to disruption of many cellular processes, including ion transport, which has significant implications in therapy resistance and advanced disease. This thesis aimed to delineate hypoxia-dependent changes in Na+ transport. Optimisation of reference genes (RGs) for studying alterations in gene expression in hypoxic breast cancer cell lines by RT-qPCR identified RPLP1 and RPL27 as suitable RGs for such investigations. RNA-seq and RT-qPCR found hypoxia enhanced Na+ transporter gene expression in ERɑ+ breast cancer cell lines, particularly by upregulating Na+/K+- ATPase (NKA) and epithelial Na+ channel (ENaC), highlighting a new mechanism by which hypoxia may contribute to breast cancer progression and therapy resistance. Conversely, voltage gated Na+ channels (VGSCs) were not affected by low O2 tension, but ERɑ was shown to mediate expression of some VGSC isoforms. Understanding changes in Na+ handling in advanced breast cancer is imperative and may result in the development or repurposing of targeted therapies aimed at modulating Na+ transport to improve breast cancer outcomes.