DESCRIPTION (provided by applicant): Our long term goal is to identify antiestrogen-resistant mechanisms operative in human breast cancer in order to improve breast cancer therapy. Our studies are novel and show that the catabolic process of autophagy (also referred to as macroautophagy), which is induced by antiestrogen treatment of estrogen receptor positive (ER+) breast cancer cells, facilitates breast cancer cell survival and contributes to the development of antiestrogen resistance. We further demonstrate that the tumor suppressor protein Rb plays a pivotal role in the survival of breast cancer cells undergoing antiestrogen-induced macroautophagy, while high-level ceramide and its downstream effector BNIP3 affects autophagocytic cell death and apoptosis of antiestrogen- treated breast cancer cells. We hypothesize that (1) antiestrogen-induced macroautophagy is a cell survival mechanism dependent on active Rb and low-level ceramide levels;and (2) surviving cells undergoing antiestrogen-induced macroautophagy can be effectively killed by blocking macroautophagsome function or by treating with agents that raise intracellular ceramide levels to a threshold that induces expression of the pro- apoptotic protein BNIP3. Our aims are (1) to determine how Rb regulates antiestrogen-induced macroautophagy and cell survival versus ACD II and apoptosis in ER+ breast cancer cells;(2) to determine if the level of ceramide determines the fate of antiestrogen-treated cells undergoing macroautophagy (ACD II/apoptosis or survival) by regulating the levels of pRb and BNIP3;and (3) to determine if inhibitors of macroautophagosome function increase the cytotoxic effects of antiestrogen therapies in vivo by increasing cellular levels of ceramide and inducing BNIP3. We will utilize a combination of in vitro-based somatic cell genetic approaches, siRNA targeting, and in vivo tumor growth studies to study the survival mode of macroautophagy. Analysis of cell death will include a determination of caspase activation, cleaved PARP, cleaved lamin A, ceramide levels, and BNIP3 levels and subcellular localization. This study will further our understanding of how macroautophagy and macroautophagosme function is regulated in antiestrogen- sensitive and -resistant ER+ breast cancer cells. Such knowledge is critical to devising treatment strategies that suppress autophagy-mediated survival and restore antiestrogen sensitivity in breast cancer patients. Project Narrative: The expression of antiestrogen resistance is the most common impediment to the successful treatment of estrogen receptor-positive breast cancer. This study proposes that breast cancer cell survival and antiestrogen resistance is dependent on activated autophagosomes, organelles within the cytoplasm of breast cancer cells, and that blocking their function will restore sensitivity to antiestrogen treatment and promote breast cancer cell death.
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