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

描述（由申请人提供）：多种证据表明含铂（Pt）的药物可以进入细胞，分布到各种亚细胞区室，并通过转运体从细胞输出，转运体进化为管理铜（Cu）稳态。我们和其他人已经证明，主要的Cu内流转运体CTR1介导顺铂（DDP）、卡铂和奥沙利铂进入哺乳动物肿瘤细胞。在体外试验中，敲除CTR1的两个等位基因会损害Pt药物积累并导致耐药性；在异种移植模型中，它也使肿瘤对DDP治疗完全无反应。因此，无论是否存在其他Pt药物内流机制，CTR1都是肿瘤对这些药物敏感性的关键决定因素。和铜一样，铂类药物触发CTR1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药物在肿瘤中的积累的新策略。我们的假设是，这可以通过确定CTR1运输含Pt药物的机制，并确定在Pt药物暴露下调节CTR1运输、内吞和降解的因素来实现。具体目的是：1)确定CTR1转运含Pt药物的机制，包括Pt药物是通过CTR1形成的孔道进入肿瘤细胞，还是结合胞外结构域后通过胞吞作用进入肿瘤细胞，如果通过这两种途径进入肿瘤细胞，它们对细胞毒性的贡献是否不同；b)确定暴露于Pt药物后控制细胞内CTR1运输及其内吞作用和随后降解的机制，包括CTR1中介导其传递到质膜和从质膜恢复的基元，其磷酸化和泛素化以及Cu伴侣ATOX1控制Pt药物诱导的CTR1降解的机制；c)确定获得性铂耐药细胞中CTR1的功能出现了什么问题，包括糖基化是否存在缺陷，导致转运功能丧失，或者耐药细胞中控制CTR1运输的途径是否存在缺陷，导致向质膜的递送不足。含pt药物仍然是最重要和广泛使用的一类化疗药物。我们已经成功地利用DDP与Cu内流转运体CTR1相互作用的详细研究结果来确定改善这些药物治疗指数的策略，这些药物目前正在进入I期临床试验。该建议的创新之处在于，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，并将最先进的技术应用于对CTR1介导DDP转运的机制的仔细解剖，有望确定进一步提高含Pt药物的疗效和选择性的策略。

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.