The copper (Cu) exporter ATP7B is over-expressed in many tumors with acquired resistance to Cu, cisplatin (DDP) and carboplatin. The ability of ATP7B to mediate resistance to the platinum (Pt)-containing drugs has now been demonstrated in multiple experimental systems in this and other laboratories, and the importance of ATP7B expression to the survival of patients treated with the Pt drugs has been suggested by the results of several clinical studies. ATP7B is one of only 3 genes that unequivocally produce cisplatin (DDP) resistance when over-expressed, the others being another Cu exporter, ATP7A, and metallothionein II. The overall goal of this project is to determine the mechanism by which ATP7B mediates resistance to the platinum-containing drug in ovarian carcinoma cells and to identify strategies for overcoming this resistance. Our prior work has demonstrated that, at clinically relevant concentrations, the Pt drugs enter cells, are distributed to various subcellular compartments and exported from cells by transporters and chaperones that have evolved to control Cu homeostasis. These proteins protect Cu from oxidation and reaction with thiols and sequester it into subcellular compartments for loading onto Cu-dependent enzymes. We posit that they do the same thing for the platinum drugs, on the one hand protecting them from neutralization by reaction with thiols but on the other hand sequestering them into the secretory export pathway and thus limiting their toxicity. This is a novel concept that provides an explanation for several long-standing questions about how the Pt drugs escape detoxification by cytoplasmic thiols before they reach DNA and why the extent of Pt DNA adduct formation is not directly linked to the extent of whole cell Pt accumulation. Our hypothesis is that, as it does for Cu, ATP7B sequesters DDP into vesicles of the secretory pathway that are then exported from the cell.
The specific aims are focused on 2 different approaches to validating this hypothesis that are based on new information about the ATP7B molecule. They are to: 1) identify the components of the ATP7B molecule that are essential to its ability to mediate DDP resistance in human ovarian carcinoma cells;and, 2) determine the extent to which ATP7B-mediated resistance to DDP relies on the interaction of ATP7B with proteins that regulate its ability to load Cu into the secretory pathway or control movement of vesicles to and from the cell surface in this pathway. This will include analysis of the participation of: the intracellular chloride channel ClC-4 that binds to ATP7B and is known to regulate vesicle pH and the loading of Cu onto ceruloplasmin by ATP7B, and dynactin that links vesicles to the motor protein dynein and microtubules and whose p62 subunit is known to interact with ATP7B in a Cu-dependent manner. A careful dissection of the mechanism by which ATP7B mediates the efflux of Pt drugs is expected to identify strategies for increasing the efficacy and selectivity of the Pt containing drugs which remain one of the most important and widely used class of anticancer agents.
The copper efflux transporter ATP7B is one of the few genes that when over-expressed unequivocally produces resistance in many types of tumor cells and while a good deal is known about the biochemistry of this transporter, how it mediates resistance to the Pt drugs remains unknown. We have developed a sharply focused hypothesis regarding this mechanism, and will use the extraordinarily powerful tool of molecular dissection of this single molecule to identify the key components required to produce the resistant phenotype. Our successful identification of strategies to increase the therapeutic index of cisplatin, based on a molecular level investigation of the copper influx transporter CTR1, indicates that through analysis of the mechanisms by which ATP7B mediates Pt drug resistance will yield strategies for increasing the efficacy and selectivity of this very important class of anticancer agents.
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