Development of a Novel Therapeutic Strategy for Estrogen Receptor Negative Breast Cancer

Abstract

Estrogen receptor-α negative (ERα-) tumours are a highly aggressive and heterogeneous class of breast cancer (BC) with higher prevalence in younger women. ERα- BC is associated with a worse prognosis and limited therapeutic options. Therefore, discovery of new therapeutic targets is a clinical imperative. This thesis investigated three therapeutic targets for ERα- BC: the androgen receptor (AR), transcription factor AP-2β (TFAP-2β) and cyclin-dependent kinase 9 (CDK9). AR is expressed in 20-50% of ERα- BC, but preclinical evidence about its role is equivocal, indicating both proliferative and anti-proliferative effects. To inform this controversy, genomic parameters associated with differential AR-mediated growth effects were examined in two ERα-/AR+ breast cancer cell lines with differing proliferative responses to the natural AR ligand 5α-dihydrotestosterone (DHT). Comparing the AR-regulated transcriptome of MDA-MB-453 (growth stimulated by DHT) with MFM-223 (growth inhibited by DHT) breast cancer cells revealed that DHT regulates a discrete set of genes associated with distinct biological functions in the two models. DHT increased expression of genes associated with metabolism, development, and cell growth in MDA-MB-453 cells. In MFM-223 cells, DHT induced expression of genes with tumour suppressor activity. Analysis of AR cistrome showed that lack of AR enrichment at tumour suppressor genes exclusively upregulated by DHT in MFM-223 cells did not explain the inability of AR to induce transcription of these genes in MDA-MB-453 cells. We therefore hypothesised that differential DHT-regulated transcriptomes are driven by distinct interactions with AR co-regulatory proteins. Unbiased proteomic analysis of the AR interactomes identified TFAP-2β as a candidate factor of interest. High TFAP-2β expression specifically clustered molecular apocrine tumours (ERα-/AR+/HER2+) and TFAP-2β was required to sustain proliferative capacity, cell viability and expression of C-MYC and HER2 oncogenes in a representative cell line. Cistrome analysis revealed substantial co-localization of TFAP-2β and AR (at approximately 40% of total loci) following treatment with DHT. However, this interaction was not a critical determinant of oncogenic AR signalling. We conclude that TFAP-2β has AR-independent oncogenic effects and represents a novel new target for the molecular apocrine sub-type of ERα- BC. CDK9 is a transcriptional cyclin that increases RNA Polymerase II (RNAPII) activity to sustain expression of normally short-lived oncogenic and anti-apoptotic proteins. Targeting CDK9 has been hampered by poor selectivity of existing inhibitors (CDK9i). Here, we report a novel CDK9i, D-11, which exhibited high potency against CDK9 (Ki=8nM) and displayed remarkable selectivity over other CDKs and 369 human kinases. D-11 suppressed proliferation and triggered apoptosis in ERα- BC cells and these effects were ascribed to the reduction of p-RNAPII, C-MYC and MCL1 levels, indicative of targeted CDK9 inhibition. In vivo, D-11 inhibited ERα- BC tumour growth without affecting body weight or histology of normal tissues, indicating its potential for clinical translation as a treatment for ERα- BC. Overall, this thesis expands current knowledge about targeting AR in ERα- BCs, provides preclinical evidence to support TFAP-2β as a novel therapeutic target and D-11 as a highly selective, non-toxic CDK9 inhibitor ready for clinical development.