PhD ThesisHER2 protein overexpression in breast cancer patients is a predictor of poor prognosis and resistance to therapies. Despite significant advances in the development of targeted therapies and improvements in the 5-year survival rate of metastatic HER2 positive breast cancer patients, new approaches are needed to better understand the disease at an early stage in order to identify means to inhibit its progression. An inducible breast cancer transformation system allows examination of early molecular changes at high temporal resolution. Here, we show that HER2 overexpression to similar levels as those observed in a subtype of HER2 breast cancer patients is sufficient to induce transformation of MCF10A cells. We found that HER2 activation generated gross morphological changes in 3D cell culture, increased anchorage-independent growth of cells and altered the transcriptional programme of various genes associated with oncogenic transformation. Global phosphoproteomic analysis during early transformation uncovered numerous signalling changes associated with cancer upon HER2 overexpression. Candidate pathways included chromatin regulators, in addition to known cascades such as MAPK, focal adhesion, mTOR, and HER signalling pathways. To understand the effect of kinase signalling on chromatin accessibility landscape, we performed ATAC-seq on acini isolated from 3D cell culture. This enables elucidation of HER2 induced signalling effects on chromatin architecture and its contribution to transformation at temporal resolution. Uniquely, we identify that HER2 overexpression promotes reprogramming-associated heterogeneity, with a subset of cells acquiring a stem-like phenotype, expressing breast stem and cancer stem cell markers, making them likely targets for malignant transformation. Our preliminary data show that this population of cells, which counterintuitively enriches for relatively low HER2 protein abundance, possesses transformational drive, resulting in increased anchorage-independent growth in vitro compared to cells not enriching for stem markers. Our data provide a discovery platform for signalling to chromatin pathways in HER2-driven cancers, offering an opportunity for biomarker discovery and identification of novel drug targets
