Preston L. Adams

Copper Transporter 2 Regulates Endocytosis and Controls Tumor Growth and Sensitivity to Cisplatin In Vivo

Copper transporter 2 (CTR2) is one of the four copper transporters in mammalian cells that influence the cellular pharmacology of cisplatin and carboplatin. CTR2 was knocked down using a short hairpin RNA interference. Robust expression of CTR2 was observed in parental tumors grown in vivo, whereas no staining was found in the tumors formed from cells in which CTR2 had been knocked down. Knockdown of CTR2 reduced growth rate by 5.8-fold, increased the frequency of apoptotic cells, and decreased the vascular density, but it did not change copper content. Knockdown of CTR2 increased the tumor accumulation of cis-diamminedichloroplatinum(II) [cisplatin (cDDP)] by 9.1-fold and greatly increased its therapeutic efficacy. Because altered endocytosis has been implicated in cDDP resistance, uptake of dextran was used to quantify the rate of macropinocytosis. Knockdown of CTR2 increased dextran uptake 2.5-fold without reducing exocytosis. Inhibition of macropinocytosis with either amiloride or wortmannin blocked the increase in macropinocytosis mediated by CTR2 knockdown. Stimulation of macropinocytosis by platelet-derived growth factor coordinately increased dextran and cDDP uptake. Knockdown of CTR2 was associated with activation of the Rac1 and cdc42 GTPases that control macropinocytosis but not activation of the phosphoinositide-3 kinase pathway. We conclude that CTR2 is required for optimal tumor growth and that it is an unusually strong regulator of cisplatin accumulation and cytotoxicity. CTR2 regulates the transport of cDDP in part through control of the rate of macropinocytosis via activation of Rac1 and cdc42. Selective knockdown of CTR2 in tumors offers a strategy for enhancing the efficacy of cDDP.

The role of the methionines and histidines in the transmembrane domain of mammalian copper transporter 1 in the cellular accumulation of cisplatin.

Mammalian copper transporter 1 (CTR1) is a high-affinity copper influx transporter that also mediates the uptake of platinum-containing chemotherapeutic agents including cisplatin (cDDP). Methionines 150, 154, and histidine 139 have been proposed to form a series of stacked rings in the pore formed by the CTR1 homotrimer, each of which is required for maximal copper transport. To examine the mechanism by which hCTR1 also transports cDDP, variant forms of hCTR1 in which methionines 150 and 154 were converted to isoleucines or in which histidine 139 was converted to alanine were re-expressed in cells in which both alleles of CTR1 had been knocked out. Each of these conversions disabled copper transport and increased cellular resistance to the cytotoxic effect of copper. In contrast, conversion of the methionines increased the uptake and cytotoxicity of cDDP well above that attained with wild-type hCTR1. Conversion of His139 to alanine did not impair cDDP uptake and actually enhanced cytotoxicity. Thus, although Met150 and Met154 facilitate the movement of copper through the pore, they serve to obstruct the passage of cDDP. None of the modifications altered the ability of cDDP to trigger the degradation of hCTR1, indicating that cDDP must interact with hCTR1 at other sites as well. Although both copper and cDDP may rely on a series of transchelation reactions to pass through the hCTR1 trimeric complex, the details of the molecular interactions must be different, which provides a potential basis for selective pharmacological modulation of copper versus cDDP cytotoxicity.

Regulation of Copper Transporter 2 Expression by Copper and Cisplatin in Human Ovarian Carcinoma Cells

Down-regulation of copper transporter 1 (CTR1) reduces uptake and sensitivity, whereas down-regulation of CTR2 enhances both. Cisplatin (DDP) triggers the rapid degradation of CTR1 and thus limits its own accumulation. We sought to determine the effect of DDP and copper on the expression of CTR2. Changes in CTR1 and CTR2 mRNA and protein levels in human ovarian carcinoma 2008 cells and ATOX1(+/+) and ATOX1(−/−) mouse embryo fibroblasts in response to exposure to DDP and copper were measured by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and deconvolution microscopy. DDP triggered rapid degradation of CTR1 in 2008 human ovarian cancer cells. However, it increased the expression of CTR2 mRNA and protein levels. Expression of CTR2 was heavily modulated by changes in intracellular copper concentration; copper depletion produced rapid disappearance of CTR2, whereas excess copper increased the level of CTR2 protein. This increase was associated with an increase in CTR2 mRNA and prolongation of the CTR2 half-life. Consistent with prior observations that short hairpin RNA interference-mediated knockdown of CTR2 enhanced DDP uptake and tumor cell kill, reduction of CTR2 by copper starvation also enhanced DDP uptake and cytotoxicity. Comparison of the ability of copper and DDP to modulate the expression of CTR1 in ATOX1(+/+) and ATOX1(−/−) indicated that ATOX1 participates in the regulation of CTR2 expression. Unlike CTR1, the expression of CTR2 is increased rather than decreased by DDP. Therefore, these two copper transporters have opposite effects on DDP sensitivity. CTR2 expression is regulated by copper availability via the copper-dependent regulator ATOX1.

The Role of the N-terminus of Mammalian Copper Transporter 1 in the Cellular Accumulation of Cisplatin

The mammalian copper transporter 1 (CTR1) is responsible for the uptake of copper (Cu) from the extracellular space, and has been shown to play a major role in the initial accumulation of platinum-based drugs. In this study we re-expressed wild type and structural variants of hCTR1 in mouse embryo fibroblasts in which both alleles of mCTR1 had been knocked out (CTR1(-/-)) to examine the role of the N-terminal extracellular domain of hCTR1 in the accumulation of cisplatin (cDDP). Deletion of either the first 45 amino acids or just the (40)MXXM(45) motif in the N-terminal domain did not alter subcellular distribution or the amount of protein in the plasma membrane but it eliminated the ability of hCTR1 to mediate the uptake of Cu. In contrast it only partially reduced cDDP transport capacity. Neither of these structural changes prevented cDDP from triggering the rapid degradation of hCTR1. However, they did alter the potency of the cDDP that achieved cell entry, possibly reflecting the fact that hCTR1 may mediate the transport of cDDP both through the pore it forms in the plasma membrane and via endocytosis. We conclude that cDDP interacts with hCTR1 both at (40)MXXM(45) and at sites outside the N-terminal domain that produce the conformational changes that trigger degradation.

The CXXC motifs in the metal binding domains are required for ATP7B to mediate resistance to cisplatin.

The copper (Cu) exporter ATP7B mediates resistance to cisplatin (cDDP) but details of the mechanism are unknown. We explored the role of the CXXC motifs in the metal binding domains (MBDs) of ATP7B by investigating binding of cDDP to the sixth metal binding domain (MBD6) or a variant in which the CXXC motif was converted to SXXS. Platinum measurement showed that cDDP bound to wild type MBD6 but not to the SXXS variant. Wild type ATP7B rendered ovarian 2008 cells resistant to cDDP. In 2008 and in HEK293T cells, wild type ATP7B trafficked from TGN to peripheral locations in response to Cu or cDDP. A variant in which the CXXC motifs in all 6 MBDs were converted to SXXS localized correctly to the TGN but failed to traffic when exposed to either Cu or cDDP. Deletion of either the first 5 MBDs or all 6 MBDs resulted in failure to localize to the TGN. Neither the SXXS variant nor the deletion variant was able to mediate resistance to cDDP. We conclude that cDDP binds to the CXXC motifs of ATP7B and that this interaction is essential to the trafficking of ATP7B and to its ability to mediate resistance to cDDP.

Sec61β Controls Sensitivity to Platinum-Containing Chemotherapeutic Agents through Modulation of the Copper-Transporting ATPase ATP7A

The Sec61 protein translocon is a multimeric complex that transports proteins across lipid bilayers. We discovered that the Sec61β subunit modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum drugs. To investigate the mechanism, expression of Sec61β was constitutively knocked down in 2008 ovarian cancer cells. Sec61β knockdown (KD) resulted in 8-, 16.8-, and 9-fold resistance to cisplatin (cDDP), carboplatin, and oxaliplatin, respectively. Sec61β KD reduced the cellular accumulation of cDDP to 67% of that in parental cells. Baseline copper levels, copper uptake, and copper cytotoxicity were also reduced. Because copper transporters and chaperones regulate platinum drug accumulation and efflux, their expression in 2008 Sec61β-KD cells was analyzed; ATP7A was found to be 2- to 3-fold overexpressed, whereas there was no change in ATP7B, ATOX1, CTR1, or CTR2 levels. Cells lacking ATP7A did not exhibit increased cDDP resistance upon knockdown of Sec61β. Sec61β-KD cells also exhibited altered ATP7A cellular distribution. We conclude that Sec61β modulates the cytotoxicity of many chemotherapeutic agents, with the largest effect being on the platinum drugs. This modulation occurs through effects of Sec61β on the expression and distribution of ATP7A, which was shown previously to control platinum drug sequestration and cytotoxicity.

The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B

The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 μM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.