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Abstract

Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor α (ERα) account for ~70% of cases, and can be treated with targeted endocrine therapy. Endocrine therapy abrogates estrogen (E2) mediated tumor growth either by blocking the ER itself (tamoxifen, fulvestrant) or by inhibiting the enzyme responsible for E2 production (aromatase inhibitors). However, around 40% of the patients eventually relapse due to resistance development. While several advancements have been made and second-line treatments are available for relapsing patients, resistance remains an urgent clinical problem that needs to be addressed. To investigate the mechanisms underlying development of resistance to endocrine therapies, I utilized various strategies to tackle two different aspects. To identify novel drivers of resistance, I developed new resistant cell lines and investigated the early phases of the resistance process with a combination of high throughput techniques. The analysis revealed ATF3 as a putative regulator of the response to therapy and of the rewiring of cells' central processes. The role of ATF3 was validated in vitro modulating its expression through knockout, knockdown and overexpression. ATF3 was identified to be essential in controlling proliferation, cell cycle and apoptosis rate of the cells under treatment through the regulation of MAPK/AKT signaling pathways. Its role was confirmed in vivo in a xenograft mouse model and the high expression levels were verified in patient datasets, adding clinical relevance to the findings. The second aspect I investigated was the relevance of clonality in endocrine therapy resistance. To do this, I used a cellular barcoding approach to track single cells during resistance development against tamoxifen and E2 deprivation in vitro. The analysis of the barcodes complexity in resistant clones revealed cell line-specific and treatment-specific mechanisms of resistance development. The distinct barcodes composition also reflected different signaling pathways activities that indicate specific paths to resistance for the independent replicates. Overall this study elucidates key features of endocrine resistance both through the identification of ATF3 as a novel mediator of endocrine resistance and through the dissection of the mechanisms underlying the selection/adaptation of independent replicates to the endocrine treatments.