Pancreatic adenocarcinoma (PDAC) is a particularly lethal form of cancer that kills over 40,000 Americans every year. PDAC is most often diagnosed when disease is advanced, with metastases that lead to death. Patient outcomes are further negatively-impacted by a typical poor response to currently-available treatments. It is thus critical to develop a stronger understanding of the processes which lead to PDAC development and metastasis, as well as to determine novel, more-efficacious targets for therapies. Low-level, constitutive endoplasmic reticulum (ER)-to-mitochondria transfer of calcium is required for optimal bioenergetics and cancer-cell survival. We hypothesize that this pathway contributes to pancreatic cancer development, metastasis, and tumor maintenance, and may therefore present a viable anticancer target. The ER-localized IP3R calcium-release ion channel and the mitochondrial calcium-uniporter ion channel, MCU, mediate calcium transfer between the two organelles at membrane-contact sites. However, it has been impossible to target this pathway in vivo because of the lack of selective, cell-permeable pharmacological agents against these ion channels. We therefore propose to examine the role of ER-to-mitochondria calcium transfer in PDAC development, metastasis, and tumor maintenance through the use of novel animal and cell-culture models. We will genetically delete MCU during early development in a murine genetic-model of PDAC, the KPCY mouse, to observe the role of this protein in tumor development. In addition, we will use tumor cells as well as genetically- modified cells using Cre/lox and CRISPR/Cas9 systems, as well as patient-derived cell lines and the established human PDAC cell line, Panc-1. We will assay proliferation, cellular bioenergetics, oxygen consumption rates, and mitochondrial calcium homeostasis, using biochemical, cell biological and biophysical approaches, including electrophysiology, live-cell imaging and fluorimetry, to define the role of ER-to- mitochondria calcium transfer in these processes. To determine the role of MCU in metastasis, we will quantify metastasis in the KPCY model using the sensitive YFP-reporter gene, and we will use an in vivo tail-vein metastasis model with genetically-modified Panc-1 cells expressing luciferase in NOD/SCID mice, as well as in vitro transwell-invasion and gel-degradation assays, and biochemical and morphological assessment of metastasis-associated markers of epithelial-to-mesenchymal transition. To observe the role of ER-to- mitochondria calcium transfer in tumor maintenance and thus its therapeutic potential for more advanced disease, we will use an inducible CRISPR/Cas9 cell-culture model of murine PDAC in vitro and an in vivo inducible orthotopic model to observe the effects of acute MCU ablation in already-growing tumors and cells as a method to simulate profound pharmacological inhibition. These studies will elucidate the role of ER-to- mitochondria calcium transfer in ...