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.