Control of the cellular pharmacology of the platinum-containing drugs by CTR1

描述（由申请方提供）：多条证据表明，含铂（Pt）药物可进入细胞，分布至各种亚细胞区室，并通过转运蛋白从细胞中输出，转运蛋白进化为管理铜（Cu）稳态。我们和其他人已经表明，主要的铜内流转运蛋白CTR 1介导顺铂（DDP），卡铂和奥沙利铂进入哺乳动物肿瘤细胞的进口。CTR 1的两个等位基因的敲除会损害Pt药物蓄积，并在体外测试时导致耐药性;它还使肿瘤在异种移植模型中对体内DDP治疗完全无反应。因此，无论可能存在什么其他Pt药物流入机制，CTR 1都是肿瘤对这些药物敏感性的关键决定因素。与Cu一样，Pt药物触发CTR 1的快速内吞和降解，因此这些药物限制了它们自身的摄取。该项目的总体目标是确定选择性增强Pt药物在肿瘤中积聚的新策略。这是我们的假设，这可以通过确定CTR 1运输含铂药物的机制，并确定调节CTR 1运输，内吞作用和降解的因素，以响应铂药物暴露来实现。具体目标是：1）确定CTR 1转运含Pt药物的机制，包括Pt药物是通过CTR 1形成的孔进入肿瘤细胞还是结合细胞外结构域后通过内吞进入肿瘤细胞，如果是通过两种途径进入，是否对细胞毒性做出不同的贡献; B）确定控制CTR 1在细胞内运输及其内吞作用和随后在暴露于Pt药物（包括CTR 1中的基序）后降解的机制介导其递送至质膜和从质膜回收、其磷酸化和泛素化以及Cu伴侣ATOX 1控制Pt药物诱导的CTR 1降解的机制; c）、确定获得性铂耐药细胞中CTR 1功能的问题，包括是否存在使转运功能失效的糖基化缺陷或控制CTR 1运输的途径缺陷在耐药细胞中，导致向质膜的递送不足。含铂类药物是目前最重要、应用最广泛的化疗药物之一。我们已经成功地使用DDP与Cu内流转运蛋白CTR 1相互作用的详细研究结果，以确定改善这些药物治疗指数的策略，目前正在进入I期临床试验。该建议是创新的，因为它挑战了该领域的教条，引入了关于Pt药物如何进入细胞的新概念，并将最先进的技术带到了CTR 1介导DDP转运的机制的仔细剖析中，这可以预期将确定进一步增加含Pt药物的功效和选择性的策略。

DESCRIPTION (provided by applicant): Multiple lines of evidence indicate that the platinum (Pt)-containing drugs can enter cells, be distributed to various subcellular compartments and exported from cells via transporters that evolved to manage copper (Cu) homeostasis. We and others have shown that the major Cu influx transporter CTR1 mediates the import of cisplatin (DDP), carboplatin and oxaliplatin into mammalian tumor cells. Knockout of both alleles of CTR1 impairs Pt drug accumulation and results in resistance when tested in vitro; it also renders tumors completely unresponsive to DDP treatment in vivo in a xenograft model. Thus, irrespective of what other Pt drug influx mechanisms might exist, CTR1 is a key determinant of the sensitivity of tumors to these drugs. Like Cu, the Pt drugs trigger the rapid endocytosis and degradation of CTR1 and thus these drugs limit their own uptake. It is the overall goal of this project to identify novel strategies for selectively enhancing Pt drug accumulation in tumors. It is our hypothesis that this can be achieved by determining the mechanism by which CTR1 transports the Pt-containing drugs and identifying the factors that modulate CTR1 trafficking, endocytosis and degradation in response to Pt drug exposure. The specific aims are to: 1) determine the mechanism by which CTR1 transports the Pt-containing drugs including whether the Pt drugs enter tumor cells by transiting the pore formed by CTR1 or by endocytosis after binding to the extracellular domain, and if they enter by both routes, whether they make different contributions to cytotoxicity; b) determine the mechanism that controls the trafficking of CTR1 within the cell and its endocytosis and subsequent degradation following exposure to the Pt drugs including the motifs in CTR1 that mediate its delivery to and recovery from the plasma membrane, its phosphorylation and ubiquitination and the mechanism by which the Cu chaperone ATOX1 controls Pt drug- induced degradation of CTR1; c) determine what is wrong with CTR1 function in cells with acquired Pt drug resistance including whether there is a defect in glycosylation that disables the transport function or defects in the pathways that control trafficking of CTR1 in resistant cells that result in inadequate delivery to the plasma membrane. The Pt-containing drugs remain one of the most important and widely used class of chemotherapeutic agents. We have already succeeded in using the results of detailed studies of the interaction of DDP with the Cu influx transporter CTR1 to identify a strategy for improving the therapeutic index of these agents that is currently entering a Phase I clinical trial. This proposal is innovative in that it challenges dogma in the field, introduces new concepts regarding how the Pt drugs enter cells, and brings state-of-the-art technology to a careful dissection of the mechanism by which CTR1 mediates the transport of DDP that can be expected to identify strategies for further increasing the efficacy and selectivity of the Pt containing drugs.