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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 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。V-ATPase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的发生。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。 糖代谢调节正常细胞中的V-ATPase。当葡萄糖受限或糖酵解中断时，组成V-ATPase的两个结构域(V1和Vo)分解。分解使泵失活，并被葡萄糖重排逆转。我们推测，在以厌氧糖酵解为主代谢的恶性肿瘤中，葡萄糖也控制着V-ATPase的组装和活性(Warburg效应)。为了验证这一新的假设，我们将使用人类前列腺癌进展的细胞模型；以及人类正常前列腺癌和前列腺癌组织的原代细胞培养。V-ATPase在前列腺中含量丰富，而前列腺的能量代谢高度专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，将被用来进一步识别V-ATPase活性、pH动态平衡和抗氧化性之间的联系(目标2)。这些研究将为V-ATPase如何帮助前列腺癌细胞控制管腔、胞液和细胞外pH提供新的见解。它们可能导致开发更有效的治疗策略来控制V-ATPase产生的异常pH梯度，而这种异常pH梯度对于肿瘤的发生、癌症的进展、转移和耐药性是必不可少的。

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.