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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 V-ATP酶是高度保守的质子泵，对pH稳态很重要。在恶性肿瘤中，V-ATP酶维持细胞质和细胞内囊泡腔之间改变的pH梯度;并产生癌症进展和转移所必需的酸性细胞外微环境。缺乏V-ATP酶功能会阻止癌症进展，但其机制尚不清楚。V-ATP酶缺陷细胞中异常的pH稳态可能会阻碍某些癌症特异性事件。 我们的长期目标是了解癌细胞如何调节V-ATP酶泵以及V-ATP酶如何帮助癌症进展到转移。 葡萄糖代谢调节正常细胞中的V-ATP酶。当葡萄糖受限或糖酵解中断时，包含V-ATP酶的两个结构域（V1和Vo）分解。胰岛素泵失活，并通过葡萄糖再灌注逆转。 我们推测，葡萄糖也控制V-ATP酶组装和活性的恶性肿瘤中，无氧糖酵解占主导地位的代谢（瓦尔堡效应）。为了检验这一新的假设，我们将使用人类前列腺癌进展的细胞模型;以及正常前列腺和前列腺癌组织的人类原代细胞培养物。V-ATP酶在前列腺中是丰富的，并且前列腺的能量代谢高度专门用于微调糖酵解活性，这使得前列腺成为这些研究的有吸引力的系统。我们将通过测量暴露于葡萄糖、糖酵解抑制剂和饥饿的细胞中的可逆分解、V-ATP酶活性以及胞质和溶酶体pH来建立葡萄糖利用和V-ATP酶组装之间的平行关系（目的1）。由于siRNA介导的敲低和用V-ATP酶抑制剂处理，V-ATP酶缺陷细胞将用于进一步辨别V-ATP酶活性、pH稳态和抗氧化应激性之间的联系（目的2）。这些研究将为V-ATP酶如何帮助前列腺癌细胞控制管腔，胞质和细胞外pH值提供新的见解。它们可能导致更有效的治疗策略的发展，以控制V-ATP酶产生的异常pH梯度，这是肿瘤发生，癌症进展，转移和耐药性所必需的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. Aberrant pH homeostasis in V-ATPase-deficient cells likely hinders some cancer-specific events. 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 metabolism regulates V-ATPases in normal cells. When glucose is limiting or glycolysis is interrupted the two domains comprising V-ATPases (V1 and Vo) 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 anaerobic glycolysis dominates metabolism (Warburg effect). To test this novel hypothesis we will use a cell model of human prostate cancer progression; and human primary cell cultures of normal prostate and prostate cancer tissue. V-ATPases are abundant in the prostate and the energy metabolism of the prostate is highly 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, pH homeostasis, and resistance to oxidative stress (Aim 2). These studies will lend new insight into how V-ATPases aid prostate cancer cells in controlling luminal, cytosolic, and extracellular pH. They may lead to the development of more effective therapeutic strategies to control the V-ATPase-generated abnormal pH gradients, which are essential for tumorigenesis, cancer progression, metastasis, and drug resistance.