MicroRNA-200b targets protein kinase Cα and suppresses triple-negative breast cancer metastasis

Abstract

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and lacks effective targeted therapies. The microRNA-200 (miR-200) family is found to inhibit or promote breast cancer metastasis; however, the underlying mechanism is not well understood. This study was performed to investigate the effect and mechanism of miR-200b on TNBC metastasis and identify targets for developing more efficient treatment for TNBC. We found that miR-200 family expression levels are significantly lower in highly migratory TNBC cells and metastatic TNBC tumors than other types of breast cancer cells and tumors. Ectopically expressing a single member (miR-200b) of the miR-200 family drastically reduces TNBC cell migration and inhibits tumor metastasis in an orthotopic mouse mammary xenograft tumor model. We identified protein kinase Cα (PKCα) as a new direct target of miR-200b and found that PKCα protein levels are inversely correlated with miR-200b levels in 12 kinds of breast cancer cells. Inhibiting PKCα activity or knocking down PKCα levels significantly reduces TNBC cell migration. In contrast, forced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration and tumor metastasis. Further mechanistic studies revealed that PKCα downregulation by miR-200b results in a significant decrease of Rac1 activation in TNBC cells. These results show that loss of miR-200b expression plays a crucial role in TNBC aggressiveness and that miR-200b suppresses TNBC cell migration and tumor metastasis by targeting PKCα. Our findings suggest that miR-200b and PKCα may serve as promising therapeutic targets for metastatic TNBC.

Fig. 1 .

The miR-200 family levels in breast cancer cells are inversely correlated with their migratory capabilities. (A) Q-PCR analysis of miR-200 expression levels in breast cancer cells. The levels of miR-200 family are expressed relative to that of MCF-7 cells and are presented as mean ± SD (n = 3). *P < 0.05, compared with other types of breast cancer cells. (B) Comparison of TNBC cell migration by wound healing assay. Scale bar = 100 µm. Similar results were obtained in two repeated experiments.

Fig. 2.

Stably expressing miR-200b in basal mesenchymal-like TNBC cells drastically reduces cell migration and inhibits mammary tumor metastasis. (A) Wound healing cell migration assay for GFP control and miR-200b stably expressing cells. (B) Representative images of GFP immunohistochemistry staining of lung sections from mice with mammary fat pad injection of MDA-MB-231-GFP or MDA-MB-231-GFP-200b cells as described in Materials and methods. Brownish color inside the foci indicates GFP-positive staining. (C) Quantifications of GFP-positive immunohistochemistry staining foci in lung sections from mice with mammary fat pad injection of MDA-MB-231-GFP or MDA-MB-231-GFP-200b cells. (D) Representative overlaid images of GFP fluorescence (green color) and nuclear DAPI (blue color) staining in lung sections from mice with mammary fat pad injection of MDA-MB-231-GFP or MDA-MB-231-GFP-200b cells. Lung sections were first stained with DAPI, then viewed and photographed under a fluorescence microscope. Scale bar = 100 µm.

Fig. 3.

PKCα is a direct target of miR-200b. (A) Western blot analysis of PKCα protein levels in 12 kinds of breast cancer cells. (B) Western blot analysis of PKCα protein levels in GFP control and miR-200b stably expressing cells. (C, D) Quantifications of PKCα 3′UTR wild-type and mutant-type vector luciferase reporter activity in GFP control and miR-200b stably expressing cells. The luciferase reporter activity (mean ± SD, n = 3) is expressed relative to control cells. *P < 0.05, compared with control cell group. Similar results were obtained in two repeated experiments.

Fig. 4.

Inhibiting PKCα activity or knocking down PKCα expression reduces basal mesenchymal-like TNBC cell migration. (A) Effect of GO6976 (1 µM) treatment on basal mesenchymal-like TNBC cell migration determined by wound healing assay. (B) Western blot analysis of PKCα, PKCβI and ZEB1 protein level in control and PKCα shRNA knockdown cells. (C) Wound healing cell migration assay for control and PKCα shRNA knockdown cells. Scale bar = 100 µm. Similar results were obtained in two repeated experiments.

Fig. 5.

Forced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration and tumor metastasis. (A) Western blot analysis of PKCα, ZEB1 and E-cadherin levels in MDA-MB-231-GFP, MDA-MB-231-GFP-200b-pLenti6.3 and MDA-MB-231-GFP-200b-pLenti6.3-PKCα cells. (B) Wound healing cell migration assay for MDA-MB-231-GFP-200b-pLenti6.3 and MDA-MB-231-GFP-200b-pLenti6.3-PKCα cells. Scale bar = 100 µm. (C) Representative images of H&E staining of lung sections from mice with mammary fat pad injection of MDA-MB-231-GFP-200b-pLenti6.3 or MDA-MB-231-GFP-200b-pLenti6.3-PKCα cells. Scale bar = 100 µm. (D) Representative overlaid images of immunofluorescence staining of GFP (red color) with nuclear DAPI staining (blue color) in lung sections from mice with mammary fat pad injection of MDA-MB-231-GFP-200b-pLenti6.3 or MDA-MB-231-GFP-200b-pLenti6.3-PKCα cells. Scale bar = 50 µm. (E) Representative overlaid images of GFP direct fluorescence (green color) with nuclear DAPI staining (blue color) in lung sections from mice with mammary fat pad injection of MDA-MB-231-GFP-200b-pLenti6.3 or MDA-MB-231-GFP-200b-pLenti6.3-PKCα cells. Scale bar = 50 µm.

Fig. 6.

Downregulation of PKCα by miR-200b reduces activation of the Rho GTPase Rac1. Rac1-GTP pulldown assay and quantifications for GFP control and miR-200b stably expressing cells (A and B), for GFP controls cells treated with dimethyl sulfoxide or GO6976 (1 µM) (C and D), for shRNA vector control and PKCα shRNA cells (E), and for miR-200b stably expressing alone and PKCα-miR-200b double stably expressing cells (F). Subconfluent cells were serum starved 24h, and then incubated in fresh medium supplemented with 5% fetal bovine serum for 1h and collected for Rac1-GTP pulldown assay. Rac1-GTP and total Rac1 levels were quantified using ImageJ software and the quantifications are presented as the ratio of Rac1-GTP levels divided by the corresponding total Rac1 levels (mean ± SD, n = 3) relative to that of control cells. *P < 0.05, compared with control cell or dimethyl-sulfoxide-treated group.