Project Summary: It remains unknown why some estrogen receptor alpha (ER)-positive breast cancers are sensitive to estrogen therapy while others are resistant, and strategies for effectively utilizing estrogen therapy are not well-established. The long-term goal of this line of investigation is to maximize the clinical potential of endocrine therapies for the management of ER+ breast cancer. The overall objective of this project is to define the mechanism that controls cell fate in ER+ breast cancer in response to the estrogen 17b-estradiol. The central hypothesis is that basal estrogen-independent ER transcriptional activity caused by ER amplification, overexpression, or mutation sensitizes breast cancer cells to the cytotoxic effects of 17b-estradiol/ER-induced DNA damage. The rationale for this project is that definition of (i) the mechanism underlying therapeutic response to 17b-estradiol and (ii) tumor features that dictate response to 17b-estradiol will provide a precision medicine basis for its use and offer strategies to enhance response. The central hypothesis will be tested by pursuing three specific aims: (1) Determine how 17b-estradiol/ER-induced DNA damage and response control cell fate; (2) Determine how inhibition of the DNA damage response affects sensitivity to 17b-estradiol; (3) Define the role of ER (ESR1) mutations in dictating breast cancer response to 17b-estradiol. In the first aim, the kinetics and spatiotemporal relationship of 17b-estradiol/ER-induced transcriptional activity, DNA damage, and response will be measured in genetically engineered and estrogen-independent ER+ breast cancer cells. These studies will provide a mechanistic basis for the cytotoxic effects of 17b-estradiol. The second aim will use cell lines and patient-derived xenografts for measurement of the effects of 17b-estradiol in the context of pharmacological inhibition of poly(ADP-ribose) polymerases 1/2 (PARP) as well as homologous recombination deficiency. These studies will offer treatment strategies to enhance response to 17b-estradiol. In the third aim, engineered cells and ESR1-mutant patient-derived xenografts will be used for measurement of 17b-estradiol- induced changes in cell fate, tumor growth, and ER transcriptional activity. These studies will provide understanding of how ESR1 mutations shape cancer cell response to 17b-estradiol and provide a mechanistic basis to inform its clinical use. The proposed research is innovative because it implicates ER-induced DNA damage in the mechanism of cytotoxicity induced by 17b-estradiol therapy, enabling the development of strategies that target the DNA damage response for advanced ER+ breast cancer. Based on our clinical trial findings, this project will test the innovative concept that ER mutations sensitize ER+ breast cancer cells to 17b-estradiol. The proposed research is significant because it will reveal the root cause of 17b-estradiol- induced cytotoxicity in ER+ breast cancer, as well as expla...