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First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Ateequllah Hayat is first author on ‘ Low HER2 expression in normal breast epithelium enables dedifferentiation and malignant transformation via chromatin opening’, published in DMM. Ateequllah conducted the research described in this article while a PhD student in Gabriella Ficz's lab at Queen Mary University of London, London, UK, and is now a lecturer in drug development at St George's, University of London, London, UK, investigating transcriptomic/epigenomic changes in cancer resistance.

Overexpression of the HER2 protein in breast cancer patients is a predictor of poor prognosis and resistance to therapies. We used an inducible breast cancer transformation system that allows investigation of early molecular changes. HER2 overexpression to similar levels as those observed in a subtype of HER2-positive breast cancer patients induced transformation of MCF10A cells and resulted in gross morphological changes, increased anchorage-independent growth of cells, and altered the transcriptional programme of genes associated with oncogenic transformation. Global phosphoproteomic analysis during HER2 induction predominantly detected an increase in protein phosphorylation. Intriguingly, this correlated with chromatin opening, as measured by ATAC-seq on acini isolated from 3D cell culture. HER2 overexpression resulted in opening of many distal regulatory regions and promoted reprogramming-associated heterogeneity. We found that a subset of cells acquired a dedifferentiated breast stem-like phenotype, making them likely candidates for malignant transformation. Our data show that this population of cells, which counterintuitively enriches for relatively low HER2 protein abundance and increased chromatin accessibility, possesses transformational drive, resulting in increased anchorage-independent growth in vitro compared to cells not displaying a stem-like phenotype.

HER2 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.

Drug resistance is a major obstacle to the long-term effectiveness of cancer therapies. Approximately 70% of breast cancer patients relapse after 5 years of treatment, and the lack of biomarkers associated with drug resistance translates to poor prognosis in clinic. Previous research has utilised omics approaches to uncover biomarkers driving drug resistance, with a strong emphasis on genetic mutations. Here, we identified a nine-marker signature associated with resistance to lapatinib in a HER2-positive breast cancer model using a target discovery approach by employing an integrative multi-omics strategy, combining ATAC-seq, RNA-seq, and proteomics. We found that 7 markers in the drug resistance-signature had not been previously found to be implicated in HER2 positive breast cancer. We counterintuitively found that drug resistant cells have restrictive chromatin accessibility with reduced gene expression associated with limited total proteome changes. However, upon closer look, we identified that the drug resistance-signature had increased chromatin accessibility closer to the transcriptional start sites of those genes and are highly differentially expressed across the three datasets. Our data show that despite the overall transcriptional and proteomic landscape showing limited changes, there are several markers that are highly expressed, which correlate with increased anchorage-independent and invasive phenotype in vitro in lapatinib resistant cells compared to cancer cells. Our results demonstrate that disease aggressiveness can be related to reduced chromatin and gene expression dynamics. We anticipate that the resistant signature identified here using integrative target discovery approach can be applied to complex, representative models and validated before they can be targeted by suitable therapeutic agents.Competing Interest StatementThe authors have declared no competing interest.

Transcription factors (TFs) are proteins that affect gene expression by binding to regulatory regions of DNA in a sequence specific manner. The binding of TFs to DNA is controlled by many factors, including the DNA sequence, concentration of TF, chromatin accessibility and co-factors. Here, we systematically investigated the binding mechanism of hundreds of TFs by analysing ChIP-seq data with our explainable statistical model, ChIPanalyser. This tool uses as inputs the DNA sequence binding motif; the capacity to distinguish between strong and weak binding sites; the concentration of TF; and chromatin accessibility. We asked whether TFs preferred to bind to DNA in open or dense chromatin conformation and found that approximately one third of TFs are predicted to bind the genome in a DNA accessibility independent fashion. Our model predicted this to be the case when the TF binds to its strongest binding regions in the genome, and only a small number of TFs have the capacity to bind dense chromatin at their weakest binding regions, such as CTCF USF2 and CEBPB. Our study demonstrated that the binding of hundreds of human and mouse TFs is predicted by ChIPanalyser with high accuracy and showed that many TFs can bind dense chromatin.