V-ATPASE PUMPS IN PROSTATE CANCER: REGULATORY AND FUNCTIONAL STUDIES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 V-ATPase是高度保守的质子泵，对pH动态平衡非常重要。在恶性肿瘤中，V-ATPase维持细胞质和细胞内小泡管腔之间的pH梯度变化；并产生癌症进展和转移所需的酸性细胞外微环境。V-ATPase功能的缺失阻止了癌症的进展，但其中涉及的机制尚不清楚。我们的长期目标是了解癌细胞如何调节V-ATPase泵，以及V-ATPase如何帮助癌症进展到转移。 葡萄糖调节正常细胞中的V-ATPase。当葡萄糖受限或糖酵解中断时，V-ATPase分解。分解使泵失活，并被葡萄糖重排逆转。我们推测，在糖酵解主导代谢的恶性肿瘤中，葡萄糖也控制着V-ATPase的组装和活性。为了验证这一假设，我们将使用人类前列腺癌进展的细胞模型；以及人类正常前列腺癌和前列腺癌组织的原代细胞培养。前列腺的能量代谢专门用于微调糖酵解活动，这使得前列腺成为这些研究的一个有吸引力的系统。我们将通过测量暴露在葡萄糖、糖酵解抑制剂和饥饿中的细胞的可逆分解、V-ATPase活性以及细胞液和溶酶体的pH来建立葡萄糖利用和V-ATPase组装之间的相似之处(目标1)。V-ATPase缺陷细胞，作为siRNA介导的敲除和V-ATPase抑制剂处理的结果，将被用于进一步辨别V-ATPase活性和pH动态平衡之间的联系(目标2)。这些研究将为V-ATPase如何帮助癌细胞控制管腔、胞液和细胞外pH提供新的见解。

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.