BMP2 inhibits cell proliferation by downregulating EZH2 in gastric cancer

Abstract

Gastric cancer is among the most common gastrointestinal malignancies. Recent studies have suggested that bone morphogenetic protein-2 (BMP2) is related to the development and progression of various cancers. Meanwhile, evidence suggests that BMP2 might lead to epigenetic changes in gastric cancer. Thus, we investigated whether BMP2 plays a role in the development of gastric cancer via epigenetic regulation. Cell viability, colony formation, and cell cycle assays were performed to assess the effect of recombinant human BMP2 (rhBMP2) in gastric cancer cells. LDN-193189 and Noggins were used as antagonists of the canonical BMP-SMAD signaling pathway. The protein levels were determined using a western blot analysis. Lentiviral vectors with EZH2 shRNA or EZH2 overexpression were used to mediate the role of EZH2 and the relationship between BMP2 and EZH2 in gastric cancer. We found that rhBMP2 inhibits cell proliferation by arresting the cell cycle in HGC-27 and SNU-216 gastric cancer cells. Neither LDN-193189 nor Noggins, antagonists of the canonical BMP-SMAD signaling pathway, can reverse the effect of rhBMP2 on gastric cancer. Molecularly, rhBMP2 downregulates the expression of EZH2 and H3K27me3, leading to increases in P16 and P21 and decreases in CDK2, CDK4, and CDK6. Altogether, in this study, we demonstrate that BMP2 serves as a tumor suppressor in gastric cancer cells by downregulating EZH2 and H3K27me3 through the non-SMAD BMP pathway, suggesting that BMP2 might be a new therapeutic target for gastric cancer treatment. Abbreviations: BMP: bone morphogenetic protein; TGF-β: transforming growth factor-beta; EZH2: enhancer of zeste homolog 2; H3K27me3: trimethylation histone H3 lysine 27; HRECs: human retinal endothelial cells; PcG: polycomb group; PRC: polycomb repressive complexes.

Keywords: BMP2; EZH2; cell cycle; cell proliferation; gastric cancer.

Figure 1.

Effect of rhBMP2 on HGC-27/SNU-216 proliferation, colony formation, and the cell cycle. (a) The colony formation capacity of HGC-27/SNU-216 cells was reduced after culture with rhBMP2 (100 ng/ml) for 2 weeks. (b) Cell viability was measured by a CCK8 assay, which showed a difference after incubation with rhBMP2 (100 ng/ml) for 4 days compared to the control group. (c) Flow cytometric analysis of the cell cycle shows cell cycle arrest after incubation with rhBMP2 (100 ng/ml) for 48 h (*P < 0.05, **P < 0.01).

Figure 2.

Effects of the BMP receptor antagonists LDN-193189 and Noggins on HGC-27/SNU-216.(a, c) The colony formation capacity of HGC-27/SNU-216 cells was reduced after culture with rhBMP2 (100 ng/ml) or rhBMP2 with LDN-193189 (0.5 nM/ml)/Noggins (1 μg/ml) for 2 weeks. (b, d) Cell viability of HGC-27/SNU-216 cell lines was measured by a CCK8 assay after culturing with rhBMP2 (100 ng/ml) or rhBMP2 with LDN-193189 (0.5 nM/ml)/Noggins (1 μg/ml) for 4 days, which showed significant differences between the control group and rhBMP2 group, rhBMP2 group and rhBMP2 with LDN-193189/Noggins group (*P < 0.05, **P < 0.01).

Figure 3.

BMP2 regulates the expression of EZH2. (a, b) EZH2, H3K27me3, and BMI1 were measured by western blot analyses of HGC-27 and SNU-216 cells after the treatment with rhBMP-2 for 48 h, and α-Tubulin was used as an internal control. (c) Stable knockdown of EZH2 inhibited the proliferation of HGC-27 and SNU-216 cells as measured by a CCK-8 assay. (d, e) EZH2, p16, p21, CDK2, CDK4, and CDK6 were measured by a western blot analysis of HGC-27 and SNU-216 cells and both the EZH2 knockdown and NC groups. (f) Stable knockdown of EZH2 in HGC-27 and SNU-216 cells inhibited colony formation. (g) Stable knockdown of EZH2 arrested the cell cycle as measured by a flow cytometric analysis (*P < 0.05, **P < 0.01).

Figure 4.

Upregulation of EZH2 reduces the inhibition of HGC-27/SNU-216 by rhBMP2. (a, b) Cell viability was measured by a CCK8 assay after the upregulation of EZH2 in HGC-27 and SNU-216 cells compared with the control group with or without the rhBMP2 treatment. (c) Colony formation capacity of HGC-27/HGC-27-EZH2 and SNU-216/SNU-216-EZH2 cell lines after culture with rhBMP2 (100 ng/ml) or PBS for 2 weeks. (d, e, f, g) Expression of H3K27me3, P16, and P21 was measured by a western blot analysis of EZH2-overexpressing cells compared with control cells with or without the rhBMP2 treatment. α-Tubulin was used as an internal control. (h, i) Images and weights of xenograft tumors 21 days after the inoculation of HGC-27 or SNU-216 cells treated with 100 μl rhBMP2 (20 μg/ml) or PBS.