Copper Transporter 2 (CTR2) as a regulator of cisplatin accumulation and sensitivity

Platinum(Pt)-containing cancer drugs are highly polar molecules that do not diffuse across lipid membranes; thus, their uptake into tumor cells must involve a transport process. Cells selected for resistance to these drugs uniformly exhibit impaired drug accumulation. The copper (Cu) transport pathway has been demonstrated to be responsible for the majority of Pt-drug accumulation and cellular trafficking. The overall goal of this dissertation was to determine whether Cu transporter 2 (CTR2) plays a role in the cellular accumulation of cisplatin (DDP), and if so, whether it influences the sensitivity of cells to DDP. This was accomplished through the study of a CTR2 knockdown model system. It was discovered that the loss of CTR2 protein expression leads to increased DDP accumulation and sensitivity both in vitro and in vivo. Additionally, it was determined that CTR2 is required for optimal tumor growth, as CTR2kd tumors demonstrated increased the frequency of apoptotic cells and reduced vascular density. Once CTR2 was established as a regulator of DDP accumulation and sensitivity, the investigations went on to focus on how CTR2 expression and degradation is controlled by DDP, and Cu. Cu and DDP exposure were shown to increase CTR2 levels. This increase was associated with an increase in CTR2 mRNA and prolongation of CTR2 half-life. Cu starvation triggered rapid degradation of CTR2, which was dependent on proteosomal activity and the status of the copper chaperone Atox1. Consistent with the observations previously made, reduction of CTR2 by Cu starvation also enhanced DDP uptake and cytotoxicity. During the course of these studies, the unique observation was made that CTR2 is partially localized in the nucleus of cells. Finally, the mechanism by which decreased CTR2 levels lead to increased accumulation of DDP was explored. CTR2 knockdown did not change the rate of efflux of or the amount of vesicular DDP. Decreased CTR2 levels, due to knockdown or degradation, triggered the up-regulation of cellular macropinocytosis and activation of the GTPases necessary for endocytosis. Inhibition of endocytosis blocked the increased accumulation of DDP in CTR2kd cells, suggesting that CTR2 limits Pt-drug accumulation through the regulation of endocytosi