Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients.

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Approximately 30% of women diagnosed with ERα breast cancer relapse with metastatic disease following adjuvant treatment with endocrine therapies 1,2 . The connection between acquisition of drug resistance and invasive potential is poorly understood. In this study, we demonstrate that the type II keratin topological associating domain (TAD) 3 undergoes epigenetic reprogramming in cells that develop resistance to aromatase inhibitors (AI), leading to keratin 80 (KRT80) upregulation. In agreement, an increased number of KRT80-positive cells are observed at relapse in vivo while KRT80 expression associates with poor outcome using several clinical endpoints. KRT80 expression is driven by de novo enhancer activation by sterol regulatory element-binding protein 1 4 (SREBP1). KRT80 upregulation directly promotes cytoskeletal rearrangements at the leading edge, increased focal adhesion maturation and cellular stiffening, which collectively promote cancer cell invasion. Shear-wave elasticity imaging of prospective patients shows that KRT80 levels correlate with stiffer tumors in vivo. Collectively, our data uncover an unpredicted and potentially targetable direct link between epigenetic and cytoskeletal reprogramming promoting cell invasion in response to chronic AI treatment. MainAI treatment is standard of care for breast cancer (BC), yet BC cells frequently display drug-resistance and stronger metastatic potential at relapse, suggesting that chronic exposure to endocrine treatment might contribute in shaping the invasive potential, as suggested by previous in vitro studies 5 . The mechanism/s, order of events and molecular players mediating these phenomena are not well understood but it is likely that they involve cytoskeletal re-arrangements as they are essential for cancer invasion and metastasis 6 . One possibility is that endocrine therapies (ET) might indirectly promote invasive potential by selecting for interrelated phenotypes during tumor evolution 7-9 . Alternatively, AI treatment may directly contribute to the activation of invasive transcriptional programs. Chronic exposure to ET leads to coordinated activation and decommissioning of regulatory regions such as enhancer and promoters as shown by global changes in the localization of epigenetic marks

microenvironment. Neutralising VEGF by avastin effectively abrogates metastasis induced by the ISX-BRD4 complex. In NSCLC carcinoma samples, significantly increased ISX expression was noted, correlating with distinct clinical metastatic features and poor prognosis. Conclusion These results suggest that the ISX-BRD4 axis mediates EMT signalling and exerts significant regulatory effects on tumour initiation and metastasis. Introduction Breast cancer (BC) is the most common cancer type and the second most frequent cause of cancer related death in women. 70% of all BC cases contain variable amounts of oestrogen receptor-alpha (ERa) positive cells. ERa is central to BC pathogenesis and serves as the target of endocrine therapies (ET). ERa-positive BC is typically subdivided in two 'intrinsic' molecular subtypes (luminal A and luminal B) characterised by distinct prognosis, highlighting functional inter-patient heterogeneity. Recent analyses demonstrate that patient-to-patient heterogeneity is more pervasive (reflected by histological, genetic architecture and transcriptional differences) ultimately influencing long-term response to endocrine treatment. Additionally, the presence of genetic intra-tumour heterogeneity has also now been extensively documented in several cancer types, demonstrating the role of clonal evolution in cancer. Parallel to genetic evolution, phenotypic/functional changes driven by epigenetic mechanisms can also contribute to breast cancer progression and ET resistance in cell lines. Nevertheless, little is known about the epigenome of BC patients, its influence on intra-tumour phenotypic heterogeneity and its role in breast cancer progression. Material and methods Here we show the results of a systematic investigation of the epigenetic landscape of ERa positive primary and metastatic breast cancer from 47 individuals. Our results represent the first large scale topographic mapping of the active regulatory landscape of longitudinal ERa-positive BC. Using H3K27ac we mapped active promoters and enhancers across treatment naïve primary and endocrine treated metastatic patients. We used bioinformatic approaches to deconvolute the complex regulatory landscape and identified inter-and intra-patient epigenetic heterogeneity. Results and discussions We mined promoters and enhancers from clinically relevant breast cancer samples for potential regulatory drivers identifying YY1 as a novel key player in ERapositive BC. Finally, we demonstrate that epigenetic mapping can efficiently estimate phenotypic heterogeneity changes throughout BC progression. Conclusion Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients. PO-355