This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. V-ATPases are highly conserved proton pumps important for pH homeostasis. In malignant tumors, V-ATPases sustain an altered pH gradient between the cytoplasm and the lumen of intracellular vesicles;and generate the acid extracellular microenvironment necessary for cancer progression and metastasis. Lack of V-ATPase function blocks cancer progression but the mechanism involved is not known. Our long term goal is to understand how cancer cells regulate V-ATPase pumps and how V-ATPases assist in cancer progression to metastasis. Glucose regulates V-ATPases in normal cells. When glucose is limiting or glycolysis is interrupted the V-ATPases disassemble. Disassembly inactivates the pump and is reversed by glucose readdition. We postulate that glucose also controls V-ATPase assembly and activity in malignant tumors where glycolysis dominates metabolism. To test this hypothesis we will use a cell model of human prostate cancer progression;and human primary cell cultures of normal prostate and prostate cancer tissue. The energy metabolism of the prostate is specialized for fine tuning glycolytic activity which makes the prostate an attractive system for these studies. We will establish parallels between glucose utilization and V-ATPase assembly by measuring reversible disassembly, V-ATPase activity, and cytosolic and lysosomal pH in cells exposed to glucose, glycolytic inhibitors, and starvation (Aim 1). V-ATPase deficient cells, as a result of siRNA-mediated knockdown and treatment with V-ATPase inhibitors, will be used to further discern connections between V-ATPase activity and pH homeostasis (Aim 2). These studies will lend new insight into how V-ATPases aid cancer cells in controlling luminal, cytosolic, and extracellular pH.
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