Copper transporter 2 regulates endocytosis and controls tumor growth and sensitivity to cisplatin in vivo

Abstract

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.

Fig. 1.

Expression of CTR2 and growth rate of CTR1(−/−) and CTR2(kd) tumors. A, Western blot analysis of CTR2 levels in CTR1(+/+), CTR1(−/−), CTR(+/+) CTR2(kd), and CTR(−/−) CTR2(kd) cells. B, immunohistochemical staining of CTR1(−/−) and CTR(−/−) CTR2(kd) tumors for expression of CTR2 (brown). C, tumor volume as a function of time; ■, CTR1(−/−) tumors; □, CTR1(−/−) CTR2(kd) tumors. Vertical bars, S.E.M.

Fig. 2.

Immunohistochemical characterization of proliferation, apoptosis, and vessel density in CTR1(−/−) and CTR(−/−) CTR2(kd) tumors. A, Ki67 staining for proliferation; numerical quantification of Ki67-positive cells per five high-power fields. B, TUNEL staining for apoptotic nuclei; numerical quantification of TUNEL-positive nuclei per five high-power fields. C, immunohistochemical staining for CD31; numerical quantification of vessel density. Vertical bars, ± S.E.M. *, p < 0.02.

Fig. 3.

Effect of knocking down CTR2 on responsiveness to cDDP in vivo. Tumor volume as a function of time with (□) or without (♦) intraperitoneal injection of 10 mg/kg cDDP. A, CTR1(−/−) tumors; B, CTR1(−/−) CTR2(kd) tumors. Vertical bars, ± S.E.M.

Fig. 4.

Effect of CTR2 on whole cell accumulation and efflux of Texas red- labeled dextran. A, dextran accumulation CTR1(+/+), CTR1(+/+) CTR2(kd), CTR1(−/−) and CTR1(−/−) CTR2(kd) cells. B, dextran accumulation in cells after a 1 h exposure to 100 μM BCS. C, dextran content as a function of efflux time in ♢, CTR1(+/+); □, CTR1(+/+) CTR2(kd); ▴, CTR1(−/−); and ■, CTR1(−/−) CTR2(kd) cells. Vertical bars, ± S.E.M. *p < 0.04.

Fig. 5.

Effect of amiloride, wortmannin, and PDGF on the CTR2 regulation of cDDP accumulation. A, accumulation of platinum after a 30-min exposure to cDDP without (■) or with (□) inhibition of macropinocytosis by amiloride. B, accumulation of platinum after a 1-h exposure to cDDP without (■) or with (□) inhibition of macropinocytosis by wortmannin. C, 30-min dextran accumulation with and without PDGF pretreatment. D, effect of PDGF pretreatment on uptake of cDDP after an exposure to 30 μM cDDP for 1 h. Vertical bars, S.E.M. *, p < 0.001.

Fig. 6.

CTR2 activates the GTPases that control macropinocytosis. A, relative levels of total and phosphorylated Rac1 and cdc42 in CTR1(+/+), CTR1(+/+) CTR2(kd), CTR1(−/−), and CTR1(−/−) CTR2(kd) cells. B, relative levels of phosphorylated Akt.