Characterizing novel molecular mechanisms involved in breast cancer tumourigenesis/progression and tamoxifen resistance

Abstract

Breast cancer is the most common cancer among women. Breast cancer tumorigenesis could be due to loss/mutation of BRCA1, yet the mechanisms underlying BRCA1-associated tumorigenesis are not well understood. Tamxoifen stands in the first-line for treatment of oestrogen receptor (ER) positive breast cancers but drug resistance significantly compromises its clinical efficacy in preventing cancer progression and improving survival. The mechanism of tamoxifen resistance is not fully understood and there is no clinical in-use biomarker that can reliably predict tamoxifen response. My PhD study aims at characterizing novel molecular pathways that are involved in breast cancer tumorigenesis/progression and tamoxifen resistance, focusing on two gaps in breast cancer research: 1) the role of BRCA1-mediated epigenetic regulation of gene expression; 2) identifying biomarker that can be used for prediction of tamoxifen resistance. In chapter 3, regulation of tumour suppressor FOXO3a by BRCA1 through EZH2-mediated epigenetic mechanism is studied. Knockdown or overexpression of BRCA1 suppresses or induces FOXO3a expression at both mRNA and protein levels, respectively. In addition, FOXO3a gene expression is negatively regulated by EZH2 in basal cell lines where BRCA1 is suppressed or mutated but not in BRCA1-wild-type MCF-7 cells, as chemical inhibition or siRNA-mediated depletion of EZH2 enhanced FOXO3a expression in basal cell lines. Moreover, ChIP assays showed that DNMT1, DNMT3a and DNMT3b and H3K27me3 are recruited to FOXO3a promoter in basal cell lines but not in MCF-7, and depletion of BRCA1 in MCF-7 induces the deposition of DNMTs and H3K27me3 to FOXO3a promoter. It is also found BRCA1 suppressed cancer cell proliferation at least partially through FOXO3a. Consistent with the in vitro data, FOXO3a promoter methylation scores are significantly higher in BRCA1-muated tumours comparing with non-BRCA1 mutated tumours. These data collectively suggest that BRCA1 can supress promoter methylation and enhance transcription of FOXO3a indirectly through binding to EZH2 and restraining its methyltransferase activity. The findings could contribute to better understanding of BRCA1-associated tumorigenesis and the regulation of FOXO3a in breast cancer. In chapter 4, the role of BQ323636.1, a splice variant of NCOR2 in tamoxifen resistance is studied and its efficacy in predicting tamoxifen resistance in breast cancer patients is also evaluated. It was found that overexpression of BQ323636.1 conferred resistance to tamoxifen in both breast cancer cell lines and orthotopic mouse model. Mechanistic studies showed that BQ323636.1 overexpression compromised the suppressive role of NCOR2 in regulating oestrogen-response element activity shown by luciferase assay and rescues the transcriptional suppression of tamoxifen on ER-target genes. Co- immunoprecipitation also showed that BQ323636.1 could bind to NCOR2 and inhibit the formation of co-repressor complex, which could explain how BQ323636.1 interferes with functions of NCOR2. Unlike NOCR2 that is localized in the nucleus, BQ323636.1 is found to be expressed in both nucleus and cytoplasm while nuclear BQ323636.1 level is significantly higher in tamoxifen resistant cells. More importantly, in human breast cancer tissue microarray, nuclear BQ323636.1 overexpression is significantly associated with tamoxifen resistance as well as patients’ survival. The findings lead to the proposal of using BQ323636.1 as a biomarker for prediction of tamoxifen resistance.