Copper activates HIF-1α/GPER/VEGF signalling in cancer cells

Abstract

Copper promotes tumor angiogenesis, nevertheless the mechanisms involved remain to be fully understood. We have recently demonstrated that the G-protein estrogen receptor (GPER) cooperates with hypoxia inducible factor-1α (HIF-1α) toward the regulation of the pro-angiogenic factor VEGF. Here, we show that copper sulfate (CuSO4) induces the expression of HIF-1α as well as GPER and VEGF in breast and hepatic cancer cells through the activation of the EGFR/ERK/c-fos transduction pathway. Worthy, the copper chelating agent TEPA and the ROS scavenger NAC prevented the aforementioned stimulatory effects. We also ascertained that HIF-1α and GPER are required for the transcriptional activation of VEGF induced by CuSO4. In addition, in human endothelial cells, the conditioned medium from breast cancer cells treated with CuSO4 promoted cell migration and tube formation through HIF-1α and GPER. The present results provide novel insights into the molecular mechanisms involved by copper in triggering angiogenesis and tumor progression. Our data broaden the therapeutic potential of copper chelating agents against tumor angiogenesis and progression.

Keywords: GPER; HIF-1α; Pathology Section; VEGF; angiogenesis; cancer; copper.

Figure 1. CuSO₄ induces the mRNA expression of HIF-1α and VEGF

mRNA expression of HIF-1α A. and VEGF B. in SkBr3 and HepG2 cells treated with increasing concentrations of CuSO₄ for 8 hours, as evaluated by real-time PCR. CuSO₄ (200 μM) induces the mRNA expression of HIF-1α C. and VEGF D. in a time-dependent manner. In SkBr3 and HepG2 cells treated with 200 μM CuSO₄ for 8 hours, the mRNA induction of HIF-1α E. and VEGF F. is abrogated in the presence of the copper chelating agent TEPA (50 μM) and the ROS scavenger NAC (300 μM). Values are normalized to the 18S expression and shown as fold changes of the mRNA expression induced by CuSO₄ compared to cells treated with vehicle (−). G. The transactivation of a VEGF promoter plasmid (pVEGF) observed in SkBr3 and HepG2 cells treated with 200 μM CuSO₄ for 12 hours is prevented by TEPA (50 μM) and NAC (300 μM). The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle (−) were set as 1-fold induction upon which the activities induced by CuSO₄ treatment were calculated. Each data point represents the mean ± SD of three independent experiments performed in triplicate. (○), (●) p < 0.05 for cells receiving vehicle (−) versus CuSO₄ treatment.

Figure 2. CuSO₄ rescues the inhibitory effects of TEPA on CoCl₂-induced transcription of HIF-1α and VEGF

In SkBr3 and HepG2 cells, the up-regulation of HIF-1α A. and VEGF B. mRNA expression induced upon CoCl₂ treatment (100 μM for 8 hours) is no longer evident in the presence of TEPA (50 μM) but rescued using CoCl₂ (100 μM for 8 hours) in combination with 200 μM CuSO₄, as determined by real-time PCR. Values are normalized to the 18S expression and shown as fold changes of mRNA expression induced by treatments respect to cells treated with vehicle (−). C. The transactivation of a VEGF promoter plasmid (pVEGF) observed in SkBr3 and HepG2 cells treated with 100 μM CoCl₂ for 12 hours is prevented by TEPA (50 μM) and rescued using CoCl₂ (100 μM for 12 hours) in combination with 200 μM CuSO₄. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle (−) were set as 1-fold induction upon which the activities induced by CoCl₂ treatment were calculated. Each data point represents the mean ± SD of three independent experiments performed in triplicate. (○), (●) p < 0.05 for cells receiving vehicle (−) versus treatments.

Figure 3. CuSO₄ induces the mRNA expression of GPER

mRNA expression of GPER in SkBr3 and HepG2 cells treated with increasing concentrations of CuSO₄ for 8 hours, as evaluated by real-time PCR A. CuSO₄ (200 μM) induces the mRNA expression of GPER in a time-dependent manner B. The increase in GPER mRNA observed treating SkBr3 and HepG2 cells for 8 hours with 200 μM CuSO₄ is abrogated in the presence of TEPA (50 μM) and NAC (300 μM) C. The transactivation of a GPER promoter plasmid (pGPER) observed in SkBr3 and HepG2 cells treated with 200 μM CuSO₄ for 12 hours is prevented by TEPA (50 μM) and NAC (300 μM) D. The mRNA induction of GPER observed in SkBr3 and HepG2 cells treated with 100 μM CoCl₂ for 8 hours is abrogated in the presence of TEPA (50 μM) and rescued using CoCl₂ (100 μM for 8 hours) in combination with 200 μM CuSO₄, as determined by real-time PCR E. The transactivation of a GPER promoter plasmid (pGPER) observed in SkBr3 and HepG2 cells treated with 100 μM CoCl₂ for 12 hours is prevented by TEPA (50 μM) and rescued using CoCl₂ (100 μM for 12 hours) in combination with 200 μM CuSO₄ F. Dose-response increase of c-fos mRNA expression in SkBr3 and HepG2 cells treated with CuSO₄ for 8 hours, as evaluated by real-time PCR G. CuSO₄ (200 μM) induces the mRNA expression of c-fos in a time-dependent manner H. The mRNA increase of c-fos observed treating SkBr3 and HepG2 cells for 8 hours with 200 μM CuSO₄ is abrogated in the presence of TEPA (50 μM) and NAC (300 μM) I. The transactivation of c-fos (fos-luc) and AP-1 (AP-1luc) reporter plasmids observed in SkBr3 cells treated with 200 μM CuSO₄ for 12 hours is prevented by TEPA (50 μM) and NAC (300 μM) J. In transfection assays, the luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle (−) were set as 1-fold induction upon which the activities induced by treatments were calculated. In RNA experiments, values are normalized to the 18S expression and shown as fold changes of mRNA expression induced by treatments compared to cells treated with vehicle (−). Each data point represents the mean ± SD of three independent experiments performed in triplicate. (○), (●) p < 0.05 for cells receiving vehicle (−) versus treatments.

Figure 4. CuSO₄ induces the protein expression of c-fos, HIF-1α and GPER

Up-regulation of c-fos, HIF-1α and GPER protein expression in SkBr3 and HepG2 cells treated with 200 μM CuSO₄ for 8 hours A., B. The induction of c-fos, HIF-1α and GPER protein expression observed upon treatment with 200 μM CuSO₄ for 8 hours is abolished in the presence of TEPA (50 μM) and NAC (300 μM) C., D. Results shown are representative of three independent experiments. Side panels show densitometric analysis of the blots normalized to β-actin. (▩), (●), (○), p < 0.05 for cells receiving vehicle (−) versus CuSO₄ treatment.

Figure 5. CuSO₄ induces EGFR and ERK activation

The exposure to 200 μM CuSO₄ induces EGFR (Tyr 1173) and ERK1/2 phosphorylation in SkBr3 and HepG2 cells A., B. The activation of EGFR and ERK1/2 observed in SkBr3 and HepG2 cells treated with 200 μM CuSO₄ for 30 min is abrogated in the presence of the EGFR inhibitor AG1478 (AG, 10 μM) and the MEK inhibitor PD98059 (PD, 10 μM) C., D. as well as TEPA (50 μM) and NAC (300 μM) E., F. Side panels show densitometric analysis of the blots normalized to EGFR or ERK2. Each data point represents the mean ± SD of three independent experiments. (○), (●) p < 0.05 for cells receiving vehicle (−) versus CuSO₄ treatment.

Figure 6. The EGFR/ERK transduction pathway is involved in the stimulatory responses induced by CuSO₄

The mRNA increase of c-fos A., HIF-1α B., GPER C. and VEGF D. observed in SkBr3 and HepG2 cells upon treatment with 200 μM CuSO₄ for 8 hours is prevented by AG (10 μM) and PD (10 μM), as evaluated by real-time PCR. Values are normalized to the 18S expression and shown as fold changes of mRNA expression induced by CuSO₄ compared to cells treated with vehicle (−). The transactivation of c-fos, AP-1, GPER and VEGF reporter plasmids induced in SkBr3 cells upon treatment with 200 μM CuSO₄ for 12 hours is abolished using AG (10 μM) and PD (10 μM) E., F. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle (−) were set as 1-fold induction upon which the activities induced by CuSO₄ treatment were calculated. Each data point represents the mean ± SD of three independent experiments performed in triplicate. The up-regulation of c-fos, HIF-1α and GPER protein expression observed in SkBr3 G. and HepG2 H. cells treated with 200 μM CuSO₄ for 8 hours is abolished in the presence of AG (10 μM) and PD (10 μM) G., H. Results shown are representative of three independent experiments. Side panels show densitometric analysis of the blots normalized to β-actin. (), (●), (○), p < 0.05 for cells receiving vehicle (−) versus CuSO₄ treatment.

Figure 7. CuSO₄ induces VEGF protein expression as evaluated by immunofluorescence assay

SkBr3 cells were treated for 12 hours with vehicle (panels 1-3), 200 μM CuSO₄ alone (panels 4-6) or in combination with TEPA (50 μM) (panels 7-9), NAC (300 μM) (panels 10-12), AG (10 μM) (panels 13-15) and PD (10 μM) (panels 16-18). VEGF accumulation is shown by the green signal, nuclei were stained by DAPI (blue signal). The slides were imaged on the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT). Images shown are representative of three independent experiments. Fluorescence intensities for the green channel were quantified in 10 random fields for each condition and results are expressed as fold change of relative fluorescence units (RFU) over the vehicle-treated cells (as indicated in the lower panel). Values are mean ± SD of three independent experiments. (○) p < 0.05 for cells receiving vehicle (−) versus CuSO₄ treatment.

Figure 8. c-fos is involved in the up-regulation of HIF-1α, GPER and VEGF induced by CuSO₄

Evaluation of HIF-1α and GPER protein expression in SkBr3 and HepG2 cells transfected for 24 hours with a vector or a plasmid encoding for a dominant negative form of c-fos (DN/c-fos) and then treated with 200 μM CuSO₄ for 8 hours (A., B.). Side panels show densitometric analysis of the blots normalized to β-actin. Each data point represents the mean ± SD of three independent experiments. Evaluation of VEGF protein expression by immunofluorescence assay in SkBr3 cells transfected for 24 hours with a vector (panels 1-6) or a plasmid encoding for a dominant negative form of c-fos (DN/c-fos) (panels 7-12) and then treated with vehicle or 200 μM CuSO₄ for 12 hours, as indicated. VEGF accumulation is shown by the green signal, nuclei were stained by DAPI (blue signal). The slides were imaged on the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT). Images shown are representative of three independent experiments C. Fluorescence intensities for the green channel were quantified in 10 random fields for each condition and results are expressed as fold change of relative fluorescence units (RFU) over the vehicle-treated cells D. Values are mean ± SD of three independent experiments. (○), (●) p < 0.05 for cells receiving vehicle (−) versus CuSO₄ treatment.

Figure 9. HIF-1α is involved in the up-regulation of GPER and VEGF induced by CuSO₄

Evaluation of GPER protein expression in SkBr3 and HepG2 cells transfected with shRNA or shHIF-1α for 24 hours and then treated with 200 μM CuSO₄ for 8 hours A., C. Side panels show densitometric analysis of the blots normalized to β-actin. Efficacy of HIF-1α silencing in SkBr3 and HepG2. Each data point represents the mean ± SD of three independent experiments B., D. E.-H. The transactivation of the GPER (pGPER) E. and VEGF (pVEGF) G. promoter plasmids observed in SkBr3 cells treated with 200 μM CuSO₄ for 12 hours is abrogated silencing the expression of HIF-1α. (F, H) Efficacy of HIF-1α silencing. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle were set as 1-fold induction, upon which the activities induced by treatments were calculated. Each data point represents the mean ± SD of three independent experiments performed in triplicate. I. Evaluation of VEGF protein expression by immunofluorescence assay in SkBr3 cells transfected for 24 hours with shRNA (panels 1-6) or shHIF-1α (panels 7-12) and treated with 200 μM CuSO₄ for 12 hours, as indicated. VEGF accumulation is shown by the green signal, nuclei were stained by DAPI (blue signal). The slides were imaged on the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT). Images shown are representative of three independent experiments. J. Fluorescence intensities for the green channel were quantified in 10 random fields for each condition and results are expressed as fold change of relative fluorescence units (RFU) over the vehicle-treated cells. Values are mean ± SD of three independent experiments. K. Efficacy of HIF-1α silencing. (○) p < 0.05 for cells receiving vehicle versus CuSO₄ treatment.

Figure 10. GPER is involved in VEGF protein increase induced by CuSO₄

Evaluation of VEGF protein expression by immunofluorescence assay in SkBr3 cells transfected for 24 hours with shRNA (panels 1-6) or shGPER (panels 7-12) and treated with 200 μM CuSO₄ for 12 hours, as indicated. VEGF accumulation is evidenced by the green signal, nuclei were stained by DAPI (blue signal). The slides were imaged on the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT). Images shown are representative of three independent experiments A. Fluorescence intensities for the green channel were quantified in 10 random fields for each condition and results are expressed as fold change of relative fluorescence units (RFU) over the vehicle-treated cells B. Values are mean ± SD of three independent experiments. Efficacy of GPER silencing C. The transactivation of the VEGF (pVEGF) promoter plasmid observed in SkBr3 cells treated with 200 μM CuSO₄ for 12 hours is abrogated silencing the expression of GPER D. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle were set as 1-fold induction, upon which the activities induced by treatments were calculated. Efficacy of GPER silencing E. Each data point represents the mean ± SD of three independent experiments performed in triplicate. (○) p < 0.05 for cells receiving vehicle (−) versus CuSO₄ treatment.

Figure 11. HIF-1α and GPER contribute to the endothelial tube formation triggered by CuSO₄

Tube formation in HUVECs cultured for 2 hours in medium collected from SkBr3 cells which were transfected for 24 hours with shRNA A., shHIF-1α B. or shGPER C. and then treated for 18 hours with vehicle or 200 μM CuSO₄, as indicated. C. In HUVECs cultured in conditioned medium from SkBr3 cells that were transfected with shGPER and treated with 200 μM CuSO_4, tube formation is rescued adding 10 ng/mL VEGF for 2 hours. Data are representative of three independent experiments performed in triplicate. Quantification of the number of tubes D., total tube length E. and number of branching points F. observed in HUVECs, as indicated. Data are representative of three independent experiments performed in triplicate. (○) p < 0.05 for cells receiving medium from SkBr3 cells treated with vehicle versus cells receiving medium from SkBr3 cells treated with CuSO₄. Efficacy of HIF-1α G. and GPER H. silencing in SkBr3 cells.

Figure 12. HIF-1α and GPER contribute to the endothelial cell migration induced by CuSO₄

Cell migration in HUVECs cultured for 24 hours in medium collected from SkBr3 cells which were transfected for 24 hours with control shRNA A., shHIF-1α B. or shGPER C. and then treated for 18 hours with vehicle or 200 μM CuSO₄, as indicated. C. In HUVECs cultured in medium from SkBr3 cells which were transfected with shGPER and treated with 200 μM CuSO_4, cell migration is rescued adding 10 ng/mL VEGF for 36 hours. Data are representative of three independent experiments performed in triplicate. Efficacy of HIF-1α D. and GPER E. silencing in SkBr3 cells.