Mechanisms of tumorigenesis and therapy resistance in breast cancer

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. Breast cancer treatments are subtype-based but eventually cancer cells become resistant, with tumour relapse remaining a significant challenge for patient management. This thesis addresses tumour suppressor genes that drive breast tumorigenesis and potential mechanisms of therapy resistance. The first section of the thesis describes a framework for the identification and validation of novel tumour suppressor genes that were identified in an in vivo genome-wide CRISPR/Cas9-screen in Trp53+/– heterozygous mice. The genes that encode for the scaffold protein Axin1 and the regulatory subunit from the protein kinase A complex, Prkar1a were identified as tumour suppressor genes that collaborate with loss of Trp53 in mammary tumorigenesis. Axin1- and Prkar1a-deleted organoids showed a morphological change with an increased proliferative capacity. RNAseq analysis revealed potential deregulation of cellular energetics in Axin1 mutant organoids. In addition, direct in vivo genome editing via intraductal injection of lentiviral vectors engineered to express dual short-guide RNAs validated Axin1 and Prkar1a as potent tumour suppressor genes that cooperate with loss of Trp53 to accelerate tumorigenesis each combination gave rise to hormone receptor negative tumours. In the second part of this thesis, mass cytometry was applied to breast cancer patient-derived xenografts (PDXs) to understand changes in cell survival and signal transduction pathways at the single cell level. Mass cytometry can detect up to 40 different protein markers via antibodies conjugated to heavy metals. Two primary questions were investigated: (a) what is the proportion of cells within a tumour that respond to treatment and (b) what molecular changes contribute to therapy resistance and tumour relapse? Short- and long-term dual therapy consisting of ABT-199 plus fulvestrant for ER+ PDX models or S63845 plus