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

描述(申请人提供)：多条证据表明，含铂的药物可以进入细胞，被分配到不同的亚细胞室，并通过进化为管理铜(铜)稳态的转运体从细胞输出。我们和其他人已经证明，主要的铜内流转运体CTR1介导顺铂(DDP)、卡铂和奥沙利铂进入哺乳动物肿瘤细胞。CTR1的两个等位基因的敲除会损害铂药物的积累，并在体外测试时导致耐药性；在异种移植模型中，它还使体内的肿瘤对DDP治疗完全没有反应。因此，无论存在什么其他铂药物内流机制，CTR1都是肿瘤对这些药物敏感性的关键决定因素。与铜一样，铂类药物会引发CTR1的快速内吞和降解，因此这些药物会限制自己的摄取。该项目的总体目标是确定选择性增强铂药物在肿瘤中积累的新策略。我们的假设是，这可以通过确定CTR1运输含铂药物的机制和确定调节CTR1运输、内吞和降解的因素来响应铂药物的暴露而实现。其具体目的是：1)确定CTR1转运含铂药物的机制，包括铂药物是通过CTR1形成的孔道进入肿瘤细胞，还是通过与胞外结构域结合后的内吞进入肿瘤细胞，如果通过这两条途径进入肿瘤细胞，它们对细胞毒性的贡献是否不同；b)确定控制CTR1在细胞内的转运及其内吞作用的机制，以及接触铂药物后的降解，包括CTR1中介导其转运到质膜和从质膜恢复的基序、其磷酸化和泛素化以及铜伴侣蛋白ATOX1控制铂药物诱导的CTR1降解的机制；C)确定具有获得性铂耐药的细胞中CTR1功能的错误之处，包括是否存在使转运功能失效的糖基化缺陷，或者控制耐药细胞中CTR1运输的途径缺陷，从而导致不能向质膜输送。含铂类药物仍然是最重要和应用最广泛的一类化疗药物之一。我们已经成功地利用DDP与铜流入转运体CTR1相互作用的详细研究结果，确定了改善这些药物的治疗指数的策略，目前正在进入I期临床试验。这一建议具有创新性，因为它挑战了该领域的教条，引入了关于铂药物如何进入细胞的新概念，并将最先进的技术引入仔细剖析CTR1介导顺铂转运的机制，有望确定进一步提高含铂药物的有效性和选择性的策略。

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.