The anti-tumor effects of Mfn2 in breast cancer are dependent on promoter DNA methylation, the P21Ras motif and PKA phosphorylation site

Abstract

Mitofusin 2 (Mfn2) is expressed in numerous human tissues and serves a pivotal role in cell proliferation. However, Mfn2 is considered as an anti-tumor gene, and is silenced in human malignant tumors, including those of breast cancer. However, the mechanisms contributing to Mfn2 silencing and the mechanism of its anti-tumor function in breast cancer remain unclear. In the present study, hypoexpression of Mfn2, and hypermethylation of its promoter, was confirmed in human breast cancer cells and in breast cancer tissues by reverse transcription-quantitative polymerase chain reaction (PCR) and methylation specific PCR, respectively. Chemical demethylation treatment with 5-aza-2'-deoxycytidine upregulated the mRNA expression level of Mfn2 in MCF-7 cells in a dose-dependent manner. In addition, overexpression of Mfn2 repressed the proliferation, migration and invasion of MCF-7 cells, mediated by inhibition of the Ras-extracellular signal-regulated kinase (ERK)1/2 signaling pathway. However, overexpression of Mfn2 with deletion of the p21^Ras motif (Mfn2^ΔRas) and protein kinase A (PKA) phosphorylation site (Mfn2^ΔPKA) partially reduced the anti-tumor function of Mfn2, and inhibited the Ras-ERK1/2 signaling pathway. Taken together, the present study confirmed the anti-tumor effects of Mfn2 in human breast cancer and clarified that the mechanism of its anti-tumor functions includes promoter DNA methylation, the P21^Ras binding site and PKA phosphorylation.

Keywords: DNA methylation; Ras-extracellular signal-regulated kinase 1/2 signaling pathway; human breast cancer; mitofusin 2.

Figure 1.

mRNA expression and promoter methylation status of Mfn2 in human breast cancer. (A) mRNA expression of Mfn2 in human breast cancer relative to the matched adjacent non-tumor tissues from 7 patients (P1-P7) were analyzed by RT-qPCR with GAPDH as an internal control. *P<0.05 vs. matched adjacent non-tumor tissue. (B) Methylation statuses of Mfn2 promoter in breast cancer and adjacent non-tumor tissues were analyzed by methylation-specific polymerase chain reaction. (C) Methylation statuses of the Mfn2 promoter in MCF-7 cells treated with 5-aza-CdR at 5, 10 and 15 µM for 5 days. (D) The mRNA expression of Mfn2 in MCF-7 cells treated with 5, 10 and 15 µM 5-aza-CdR, determined by RT-qPCR with GAPDH as an internal control. The results are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. untreated cells. Mnf2, mitofusin 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; 5-aza-CdR, 5-aza-2′-deoxycytidine; m, methylated PCR product; u, unmethylated PCR product.

Figure 2.

Proliferation, migration and invasion abilities of MCF-7 cells transfected with N1, Mfn2, Mfn2^ΔRas and Mfn2^ΔPKA plasmids. (A) The mRNA level of Mfn2 in each group was detected by reverse transcription-quantitative polymerase chain reaction 24 h post-transfection, using GAPDH as internal control. The data are presented as expression relative to that of N1. (B) Mfn2 protein expression was detected by western blotting 48 h post-transfection. Quantification of the protein band was analyzed by Image Pro Plus software. (C) Cells of each group were seeded into 96 plates at 7.0×10³/well. Cell proliferation was detected using a Cell counting kit-8 assay for 3 days. The data are representative of 3 experiments. (D) Cells were seeded in a 25 cm² tissue culture flask and harvested at 80% confluence for cell cycle distribution analysis by flow cytometry. (E) Migration activity was detected using a scratch wound assay. ×100, magnification. The bar graph demonstrates the fold change of wound distance relative to N1. (F) Invasion ability was analyzed using a transwell assay with Matrigel. The cells attached to the lower surface of the insert were stained with crystal violet, imaged and counted in 5 random fields of view (×200, magnification). The data represent the mean ± standard deviation of the number of cells relative to the N1 group in 3 experiments. *P<0.05 and **P<0.01 vs. N1 group; ^#P<0.05 vs. Mfn2 group. N1, EGFP-N1 plasmid; Mfn2, mitofusin 2; Mfn2^ΔRas, Mfn2 ORF lacking the p21^Ras coding sequence; Mfn2^ΔPKA, Mfn2 ORF lacking the protein kinase A phosphorylation site coding sequence.

Figure 3.

Expression of p-ERK and Cyclin D1 in MCF-7 cells transfected with N1, Mfn2, Mfn2^ΔRas and Mfn2^ΔPKA plasmids. (A) Protein expression levels of ERK and p-REK in determined by western blotting, using GAPDH as a control. The results are expressed as the mean ± standard deviation. *P<0.05 vs. N1 group; ^#P<0.05 vs. Mfn2 group. (B) mRNA expression of Cyclin D1 determined by reverse transcription-quantitative polymerase chain reaction, relative to the N1 group, using GAPDH as an internal control. (C) Relative protein expression levels of Cyclin D1 determined by western blotting using β-actin as a control. Showed. Results are expressed as the mean ± standard deviation of 3 experiments, relative to the N1 group. *P<0.05 vs. N1 group; ^#P<0.05 vs. Mfn2 group. p-, phosphorylated; ERK1/2, extracellular signal-regulated kinase 1/2; N1, EGFP-N1 plasmid; Mfn2, mitofusin 2; Mfn2^ΔRas, Mfn2 ORF lacking the p21^Ras coding sequence; Mfn2^ΔPKA, Mfn2 ORF lacking the protein kinase A phosphorylation site coding sequence.