Abstract 448: The novel role of ecdysoneless (ECD) in maintain ER Ca2+ homeostasis

Abstract

Abstract Background and Significance: Calcium (Ca2+) is an essential signal transduction element involved in the regulation of several cellular activities such as gene transcription, proliferation and apoptosis. Endoplasmic Reticulum (ER) is the Ca2+ reservoir inside the cell that strictly orchestrate Ca2+ gradient across the Plasma Membrane(PM). Importantly, intracellular Ca2+ flux, is a key to orderly regulate cell proliferation, if altered can promote uncontrolled proliferation leading to cancer. Ca2+ entry through the [Ca2+]PM channels during [Ca2+]ER depletion is called the Store Operated Ca2+ Entry (SOCE). Impairment of endogenous SOCE leads to sustained [Ca2+]ER depletion and results in accumulation of unfolded proteins in the ER (known as ER stress). Understanding the basic molecular mechanism regulating PM to ER Ca2+ flow and developing novel strategies to target ER Ca2+ signaling could be beneficial from cancer therapeutic perspective. We have identified Ecdysoneless (ECD), an evolutionary conserved novel cell cycle regulator protein. ECD is overexpressed in several cancers, including breast and pancreatic cancers and its overexpression in breast cancer correlates with poor prognosis and short survival of patients. Recently our lab demonstrates that ECD is a negative regulator of ER stress; however, there is no mechanistic insights of how ECD senses and acts to maintain ER homeostasis. Experimental Design: We have utilized mouse embryonic fibroblasts (MEFs) from Tet(O)-FhECD-IRES-eGFP-rtTA and Ecdflox/flox mice transgenic mice to regulate ECD levels in cells to assess the role of ECD in Ca2+ signaling in a highly controlled system. Structured illumination microscopy(SIM) and biochemical fractionation techniques were performed to identify localization of ECD. Live cell imaging of cells stained with Ca2+ binding dyes like Fluo4-AM and Fluo5N-AM were used to access [Ca2+]Cyto and [Ca2+]ER levels respectively. Fluorescence resonance energy transfer (FRET) and immunoprecipitation methods were conducted to analyze SOCE pathway. Western blotting was used to assess the levels of various proteins involved in Ca2+ signaling. Results: We demonstrate several novel findings: (i) ECD is a smooth ER associated protein; (ii) ECD regulates assembly of ER-PM protein complex to drive Ca2+ entry through SOCE into the ER, maintaining [Ca2+]ER homeostasis; iii) ECD is overexpressed upon Ca2+ depletion and provides survival benefit to cells; v) ECD regulates Ca2+ mediated cell proliferation via CaMKII phosphorylation. Conclusion: Our results demonstrate ECD is a sensor of ER stress, induced by Ca2+deprivation, and overexpressed ECD promotes cell proliferation and survival under ER stress. These findings suggest ECD could be a potential target of Cancer therapy. Citation Format: Aniruddha Sarkar, Irfana Saleem, Sameer Mirza, Keshore Bidasee, Rajini Rao, Hamid Band, Vimla Band. The novel role of ecdysoneless (ECD) in maintain ER Ca2+ homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 448.