The Mitochondrial Calcium Uniporter in Pancreatic Cancer Development, Metastasis, and Treatment

点击翻译按钮获取中文摘要

Summary: Pancreatic adenocarcinoma (PDAC) is a particularly lethal form of cancer which kills over 40,000 Americans every year. PDAC is most often diagnosed when disease is advanced, with apparent metastases that often lead to death. Patient outcomes are further negatively impacted by a typically 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. We hypothesize that the mitochondrial calcium uniporter (MCU), a mitochondrial membrane ion channel, may contribute to cancer development, metastasis, and tumor maintenance, and may therefore present a viable anticancer target. This protein is required for the low-level, constitutive transfer of Ca2+ from the endoplasmic reticulum to the mitochondria that cancer cells appear to be dependent upon for survival. Previous studies have been hampered by the lack of suitably selective, cell-permeable pharmacological agents to block this pathway. We thereby propose to examine the role of MCU in PDAC development, metastasis, and tumor maintenance through the use of genetic approaches and novel animal and cell culture models. We will use genetic editing techniques, such as the Cre/lox and CRISPR/Cas9 systems, to observe the role of MCU in these processes. We will knock out MCU during early development in the murine genetic model of PDAC, the KPCY mouse, to observe the role of this protein in tumor development. We will also assay proliferation, cellular metabolism, oxygen consumption rates, and mitochondrial calcium flux (via electrophysiology and mitochondrial calcium uptake assays) in cell lines and 3D cultures generated from this model, as well as patient-derived cell lines and the established human PDAC cell line, Panc-1. We will re- express MCU in the knockout cells, and we will use CRISPR/Cas9 to knock out MCU in MCU wild-type cells to ensure effects are MCU-dependent. To observe the role of MCU in metastatic colonization, we will use a tail-vein metastasis model with Panc-1 cells expressing luciferase in NOD/SCID mice, as well as transwell invasion, gel degradation assays, and Western blotting for metastasis-associated markers of epithelial to mesenchymal transition, such as cadherins and matrix metalloproteases. We will also quantify metastasis from the KPCY model using the YFP reporter gene. To observe the role of MCU in tumor maintenance and thus establish its therapeutic potential in more advanced disease, we will use an inducible CRISPR/Cas9 knockout cell culture model of murine PDAC in vitro and an orthotopic model to observe the effects of acute MCU ablation in already growing tumors and cells as a method to simulate pharmacological inhibition. These studies will elucidate the role of MCU in PDAC development, metastasis, and maintenance. Significantly, the findings resultant from this work may inform future development of screening methods or treatment modalities in PDAC, potentially saving lives.