ECDYSONELESS, A NOVEL REGULATOR OF CALCIUM INFLUX

Background and Significance Cell proliferation produces two cells from one and is required for cell growth, and is most critical in embryonic development as well as in normal adult organ function, whereas uncontrolled cell proliferation is a hallmark of cancer. Consequently, understanding the regulatory controls of cell cycle progression is of utmost importance. Ecdysoneless (ECD), an evolutionary conserved protein, was first identified in Drosophila melanogaster regulating larval development. Our lab established the importance of mammalian ECD by demonstrating that Ecd germline knockout mice is embryonically lethal, and deletion in cells causes G 1 /G 0 arrest, which can be rescued by exogenous ECD, affirming the requirement of ECD for normal mammalian cell cycle progression. Importantly, ECD is overexpressed in ErbB2‐positive breast cancers and its overexpression correlates to poor prognosis and short survival. We also demonstrated the co‐oncogenic role of ECD with Ras to induce oncogenic transformation of mammary cells. Recently, our lab has also elucidated that ECD binds to a co‐chaperonic complex called R2TP, and this interaction is required for cell cycle progression. Hypothesis Based on our preliminary and published studies, we anticipate that ECD is a regulator of Ca 2+ homeostasis by modulating [Ca 2+ ] ER ATPase and providing cell survival and proliferation advantage to cells. We also hypothesize that upregulation of ECD in tumor cells provides survival benefit during endoplasmic reticulum (ER) stress due to low calcium microenvironment. Experimental Design We have utilized our mouse embryonic fibroblasts (MEFs) from Tet(O)‐FhECD‐IRES‐eGFP‐rtTA transgenic mice. These cells can be induced by doxycycline to overexpress ECD protein. In addition, we have generated MEFs from Ecd flox/flox mice and treatment of these MEFs with adenovirus CRE can be used to completely knockout ECD in cellular system. In addition, we have defined selective siRNA/shRNA against Ecd and generated mammary epithelial cells expressing these siRNA/shRNA to knockdown Ecd. Using these cellular reagents, we first performed Structured illumination microscopy(SIM) to localize ECD, then we performed biochemical fractionation. Use of above mentioned cells allowed assessment of effect of ECD on calcium signaling by live cell imaging. Western blotting was used to assess the levels of various proteins involved in calcium signaling. Results Using ECD‐overexpressing, Ecd knockout and Ecd knockdown cells and various techniques mentioned above, we demonstrate several novel findings: i) ECD as well as R2TP complex components are localized to smooth endoplasmic reticulum (SER); ii) ECD regulates levels of intracellular Ca 2+ ; iii) ECD regulates CaMKII phosphorylation; iv) ECD protein provides survival benefit to cells upon Ca 2+ starvation; v) ECD regulates Ca 2+ mediated cell proliferation Conclusion Our results demonstrate ECD protein is localized to SER and regulates levels of intracellular Ca 2+ and CaMKII phosphorylation. Significantly, ECD provides survival benefits to cells upon Ca 2+ starvation mediated ER stress and regulates Ca 2+ mediated cell proliferation. A successful outcome of these studies will provide new mechanistic insights into oncogenic roles of ECD in the context of calcium signaling and could provide a rationale to target calcium signaling pathways in ECD‐high ErbB2‐positive breast cancers. Support or Funding Information U.S ARMY CDMRP W810X WH‐14‐1‐0567 UNMC Graduate Assistantships/Fellowships 2016‐2018