Reduced ⁶⁴Cu uptake and tumor growth inhibition by knockdown of human copper transporter 1 in xenograft mouse model of prostate cancer

Copper is an element required for cell proliferation and angiogenesis. Human prostate cancer xenografts with increased ⁶⁴Cu radioactivity were visualized previously by PET using ⁶⁴CuCl2 as a radiotracer (⁶⁴CuCl2 PET). This study aimed to determine whether the increased tumor ⁶⁴Cu radioactivity was due to increased cellular uptake of ⁶⁴Cu mediated by human copper transporter 1 (hCtr1) or simply due to nonspecific binding of ionic ⁶⁴CuCl2 to tumor tissue. In addition, the functional role of hCtr1 in proliferation of prostate cancer cells and tumor growth was also assessed. Methods: A lentiviral vector encoding short-hairpin RNA specific for hCtr1 (Lenti-hCtr1- shRNA) was constructed for RNA interference-mediated knockdown of hCtr1 expression in prostate cancer cells. The degree of hCtr1 knockdown was determined by Western blot, and the effect of hCtr1 knockdown on copper uptake and proliferation were examined in vitro by cellular ⁶⁴Cu uptake and cell proliferation assays. The effects of hCtr1 knockdown on tumor uptake of ⁶⁴Cu were determined by PET quantification and tissue radioactivity assay. The effects of hCtr1 knockdown on tumor growth were assessed by PET/CT and tumor size measurement with a caliper. Results: RNA interference-mediated knockdown of hCtr1 was associated with the reduced cellular uptake of ⁶⁴Cu and the suppression of prostate cancer cell proliferation in vitro. At 24 h after intravenous injection of the tracer ⁶⁴CuCl2, the ⁶⁴Cu uptake by the tumors with knockdown of hCtr1 (4.02 ± 0.31 percentage injected dose per gram [%ID/g] in Lenti-hCtr1-shRNA-PC-3 and 2.30 ± 0.59 %ID/g in Lenti-hCtr1-shRNA-DU- 145) was significantly lower than the ⁶⁴Cu uptake by the control tumors without knockdown of hCtr1 (7.21 ± 1.48 %ID/g in Lenti-SCR-shRNA-PC-3 and 5.57 ± 1.20 % ID/g in Lenti-SCR-shRNA-DU-145, P , 0.001) by PET quantification. Moreover, the volumes of prostate cancer xenograft tumors with knockdown of hCtr1 (179 ± 111 mm3 for Lenti-hCtr1-shRNAPC- 3 or 39 ± 22 mm3 for Lenti-hCtr1-shRNA-DU-145) were significantly smaller than those without knockdown of hCtr1 (536 ± 191 mm3 for Lenti- SCR-shRNA-PC-3 or 208 ± 104 mm3 for Lenti-SCR-shRNA-DU- 145, P > 0.01). Conclusion: Overall, data indicated that hCtr1 is a promising theranostic target, which can be further developed for metabolic imaging of prostate cancer using ⁶⁴CuCl2 PET/CT and personalized cancer therapy targeting copper metabolism.