Delineating early transformational events in HER2 positive breast cancer using an inducible MCF10A cell line.

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

The role of intratumour heterogeneity and chromosomal instability in cancer

Increasing evidence supports the existence of intratumour heterogeneity in many solid and haematological tumour types, with potential clinical implications for both cancer diagnosis and treatment. Multi-region whole-exome sequencing of surgically resected non-small cell lung cancer (NSCLC) tumours demonstrated intratumour spatial and temporal heterogeneity in the mutational burden, copy number aberrations, and mutational signatures identified in these tumours. Furthermore, heterogeneity of mutations, including driver mutations, was also demonstrated in pre-invasive lung adenocarcinoma in situ lesions, suggesting that clonal evolution may be a feature of the early stages of cancer development. Whilst deciphering the clonal landscape of tumours may rely on multi-region and repeated tissue sampling, this remains challenging outside the context of clinical studies, and is not routine clinical practice. A non-invasive alternative may be the use of circulating biomarkers, such as circulating cell-free tumour DNA (cfDNA). Truncal and branch mutations were identified in cfDNA from patients with early stage NSCLC using different approaches. The detection of low frequency branch mutations, which are predicted to be subclonal in origin and may be potentially involved in the emergence of therapeutic resistance and tumour progression, were difficult to identify in cfDNA. Further studies are required to develop effective strategies for clonal and subclonal mutation detection in cfDNA, and to determine the utility of such biomarkers in representing the tumour genomic landscape, and in tracking tumour evolution in time. Chromosomal instability (CIN), describes an increased rate of numerical and structural chromosome aberrations, and is a known driver of intercellular genetic tumour heterogeneity. CIN has been shown to be associated with drug resistance and poor clinical outcome in several cancer types. However, in oestrogen receptor (ER)-negative breast cancer it has previously been shown that extreme CIN is associated with improved clinical outcome, consistent with a negative impact of CIN on cellular fitness and growth. This paradoxical relationship was further validated in a large breast cancer cohort study, in which extreme CIN was associated with improved outcome in patients with ER-negative cancer (p trend = 0.03). A similar relationship was seen in ERnegative/ human epidermal growth factor receptor (HER2)-negative cancers (p trend = 0.007). Identifying such patients may help distinguish good from poor prognostic groups, and therefore support treatment and risk stratification. We are yet to uncover the true extent of intratumour heterogeneity and CIN in different cancer types, their relevance to clinical outcome, and how we may be able to overcome or exploit these features for the therapeutic gain and benefit of patients with cancer. Longitudinal studies employing serial tissue and circulating biomarker sampling have the potential to address these questions, and to truly define the breadth of genetic diversity in different tumour types and its relevance to patient outcome