Heregulin-β1 Activates NF-E2-related Factor 2 and Induces Manganese Superoxide Dismutase Expression in Human Breast Cancer Cells via Protein Kinase B and Extracellular Signal-regulated Protein Kinase Signaling Pathways

Abstract

Heregulin-β1, a ligand of ErbB-2 and ErbB-3/4 receptors, has been reported to potentiate oncogenicity and metastatic potential of breast cancer cells. In the present work, treatment of human mammary cancer (MCF-7) cells with heregulin-β1 resulted in enhanced cell migration and expression of manganese superoxide dismutase (MnSOD) and its mRNA transcript. Silencing of MnSOD abrogated clonogenicity and migrative ability of MCF-7 cells. Heregulin-β1 treatment also increased nuclear translocation, antioxidant response element binding and transcriptional activity of NF-E2-related factor 2 (Nrf2). A dominant-negative mutant of Nrf2 abrogated heregulin-β1-induced MnSOD expression. Treatment with heregulin-β1 caused activation of protein kinase B (Akt) and extracellular signal-regulated protein kinase (ERK). The pharmacological inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase 1/2, which are upstream of Akt and ERK, respectively, attenuated heregulin-β1-induced MnSOD expression and nuclear localization of Nrf2. In conclusion, heregulin-1 induces upregulation of MnSOD and activation of Nrf2 via the Akt and ERK signaling in MCF-7 cells, which may confer metastatic potential and invasiveness of these cells.

Keywords: Breast cancer; Heregulin-β1; Manganese superoxide dismutase; NF-E2-related factor 2.

Figure 1. Heregulin-β1 stimulates the migration of MCF-7 cells and induces expression of manganese superoxide dismutase (MnSOD).

(A) MCF-7 cells were treated with heregulin-β1 for 24 hours and subjected to the wound healing assay. (B) mRNA expression correlation data between neuregulin-1 (NRG1) and MnSOD in the metastatic breast cancer (TCGA provisional) were obtained by using the cBioportal web interface. (C) MCF-7 cells were treated with heregulin-β1 (50 ng/mL) for indicated time periods. The expression of MnSOD mRNA and protein was measured by RT-PCR and Western blot analyses, respectively as described in Materials and Methods. (D). MCF-7 cells were treated with 0, 1, 10, or 50 ng/mL of heregulin-β1 for 8 hours (for mRNA) and 24 hours (for protein). The expression of MnSOD mRNA and protein was measured by RT-PCR and Western blot analyses, respectively. (E) Immunocytochemical analysis was performed using an anti-MnSOD antibody after the treatment of MCF-7 cells with 50 ng/mL heregulin-β1 for 24 hours. Images of the cellular fluorescence were acquired using a confocal laser-scanning microscope as described under Materials and Methods; (a) Vehicle-control cells. (b) Cells treated with 50 ng/mL heregulin-β1 for 24 hours. Scale bar, 100 μm. (F) MCF-7 cells treated with heregulin-β1 (0, 1, 10, or 50 ng/mL) for 24 hours, and the catalytic activity of MnSOD was measured as described in Materials and Methods. *Significantly different from the control without heregulin-β1 treatment (P < 0.05).

Figure 2. Heregulin-β1 up-regulates manganese superoxide dismutase (MnSOD) expression through activation of extracellular signal-regulated protein kinase (ERK) and protein kinase B (Akt), and activates NF-E2-related factor 2 (Nrf2).

(A, B) MCF-7 cells were preincubated with U0126 (0, 0.1, 1, or 5 μM) or LY294002 (0, 1, 5, or 20 μM) and exposed to heregulin-β1 (50 ng/mL) for 8 hours. The protein from cell lysates was resolved by SDS-PAGE and analyzed by Western blot analysis to measure the phosphoryation of ERK (A) or Akt (B). (C, D) The effects of pharmacologic inhibitors of ERK and Akt on expression of MnSOD were assessed by Western blot analysis after exposure to heregulin-β1 for 24 hours. Treatment conditions and other detials are same as above. (E) MCF-7 cells were transfected transiently with dominant-negative (DN)-ERK or control plasmid (CEP). After transfected with each vectors for 12 hours using DOTAP reagent, MCF-7 cells were subjected to heregulin-β1 treatment for 24 h. The expression of MnSOD was monitored by Western blot analysis. (G) MCF-7 cells were transfected transiently with hemaglutinin-tagged full-length Akt (HA-Akt) or kinase-dead Akt (KD-Akt). After transfection with each vector for 12 h using DOTAP reagent, MCF-7 cells were treated with heregulin-β1 for additional 24 hours. Proteins from cell lysates were resolved by SDS-PAGE and analyzed by Western blot analysis. (H) Nuclear extracts from MCF-7 cells were prepared after treatment with heregulin-β1 (50 ng/mL) for indicated time periods. Immunoblots of nuclear lysates from treated MCF-7 cells were probed with the Nrf2 specific antibody. (I) Immunocytochemical analysis was performed using anti-Nrf2 antibody after the treatment of MCF-7 cells with 50 ng/mL of heregulin-β1 for 4 hours. Cells were stained with propidium iodide and analyzed by confocal microscopy. Scale bar, 50 μm. (J) Heregulin-β1 induces DNA binding activity of Nrf2 in MCF-7 cells. MCF-7 cells were treated with heregulin-β1 (50 ng/mL) for indicated time periods. The DNA-binding activity of Nrf2 in MCF-7 cells stimulated with heregulin-β1 was measured by electrophoretic mobility shift assay (EMSA). The nuclear extract isolated from heregulin-β1-treated cells was used for EMSA as described under Materials and Methods. (K) The heregulin-β1-mediated increase in the transcriptional activation of antioxidant response elements (ARE) was measured by the luciferase reporter gene assay. After overnight transfection, cells were exposed to 50 ng/mL heregulin-β1 for 8 hours, and treated with reporter lysis buffer for the measurement of the luciferase activity.

Figure 3. Heregulin-β1-induced nuclear translocation of NF-E2-related factor 2 (Nrf2) is mediated by extracellular signal-regulated protein kinase (ERK) and protein kinase B (Akt).

(A, B) MCF-7 cells were preincubated with U0126 (5 μM) or LY294002 (20 μM) and exposed to heregulin-β1 (50 ng/mL) for 8 hours for immunofluorescence (A) and Western blot (B) analyses of Nrf2. Scale bar, 100 μm. (C) Effects of dominant negative mutant Nrf2 on manganese superoxide dismutase (MnSOD) expression.

Figure 4. Silencing of manganese superoxide dismutase (MnSOD) abolishes the migrative capability (A) and clonogenicity of (B) MCF-7 cells.

Cells were silenced for the MnSOD gene with specific siRNA and cell migration and colony formation were measured as described in Materials and Methods. *Significantly different between the groups compared (P < 0.05); ***Significantly different between the groups compared (P < 0.001).

Figure 5. Proposed scheme for the role of heregulin-β1 in breast cancer progression.

MnSOD, manganese superoxide dismutase; ERK, extracellular signal-regulated protein kinase; Akt, protein kinase B; Nrf2, NF-E2-related factor 2; ROS, reactive oxygen species.