Abstract 2282: Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer

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Abstract Molecular classification of breast cancers into receptor-based subtypes, including estrogen positive or triple negative breast cancers, establish targets that guide individualized treatment for patients (Journal of clinical oncology:27, 1153-1154, 2009). However, drug resistance observed for breast cancer raises the challegens for the breast cancer treatment (CA: a cancer journal for clinicians 59, 303-313, 2009). Targeting the death receptor-5 (DR-5) to induce breast cancer apoptosis is a promising strategy for breast cancer treatment (Immunity 3, 673-682, 1995, Breast Cancer Res Treat 113, 217-230, 2009). TRA-8 is a DR-5 specific agonistic antibody (Nature medicine 7, 954-960, 2001), and TRA-8 can induce apoptosis in breast cancer cells (Clinical cancer research: 9, 3731-3741, 2003). TRA-8 signal DR-5-mediated apoptotic signal via formation of the death inducing signaling complex (DISC) at the DR-5 death domain (Immunity 12, 599-609, 2000). Calmodulin (CaM) is a mediator of calcium signaling and has been shown to play a role in regulating DR-5 mediated apoptosis (Biochem Cell Biol 87, 919-926, 2009). Understanding the mechanism of DR-5-CaM interaction and the role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation, may unveil CaM-DR-5 binding as a potential target to modulate DR-5 mediated apoptosis. In this study we characterize an interaction between CaM and DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. Complex-immunoprecipitation of DR-5 from MCF-7 and MDA-MB-231 lysates demonstrated endogenous CaM and DR-5 form a complex. Native PAGE of recombinant 6xHis-SUMO-CaM incubated with MCF-7 and MDA-MB-231 lysates showed the formation of a CaM and DR-5 complex. CaM pull down of DR-5 from MCF-7 and MDA-MB231 lysates in the presence of 1 mM Ca2+ or 2 mM EGTA showed CaM/DR-5 interaction is calcium dependent. TRA-8 activation of DR-5 resulted in CaM recruitment into the DR-5 mediated DISC. Additionally, Either EGTA or CaM antagonist trifluopherazine (TFP) attenuated DR-5 mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. We present evidence for the Ca2+ dependent CaM and DR-5 interaction and a role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation in breast cancer cells. Results from this study provide the basis for the further characterization of CaM-DR5 interactions and its important role in cellular activities, which could lead to novel strategies for effectively regulating DR-5-mediated apoptosis in breast cancer. Citation Format: Romone M. Fancy, Hong Wang, Tong Zhou, Yuhua Song. Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2282. doi:10.1158/1538-7445.AM2014-2282