Restin suppressed epithelial-mesenchymal transition and tumor metastasis in breast cancer cells through upregulating mir-200a/b expression via association with p73

Abstract

Background: Restin belongs to MAGE superfamily and is known as MAGE H1. Restin was firstly cloned from HL-60 cells treated with all-trans retinoic acid (ATRA). Previous studies showed a pro-apoptotic role of Restin in several cell lines. However, little information is available on its expression patterns and functions in vivo. Our study was performed to detect if Restin plays a role in breast cancer cells in vitro and in vivo.

Methods and results: Real-time PCR and western blot were conducted to detect Restin expression in multiple breast cancer cell lines and Restin level was negatively related with cell motility. Restin overexpression and knockdown stable cell lines were established by transducing lentivirus into MCF-7 and MDA-MB-231 cells. Cell morphology, wound closure assay, transwell migration and invasion assays were performed to detect if Restin inhibited EMT. Our data showed that Restin overexpressed cells exhibited classical epithelial cell morphology, and Restin overexpression resulted in activation of epithelial markers and suppression of mesenchymal markers, and inhibition of cell migration and invasion. Tumor xenograft model was used to characterize the biological functions of Restin in vivo. We found that Restin overexpression led to reduced lung metastasis. Real-time PCR, western blot, luciferase assay and ChIP assay were performed to identify the potential targets of Restin and the underlying molecular mechanisms. Among several master regulators of EMT, only ZEB1/2 levels were dramatically inhibited by Restin. Unexpectedly, Restin indirectly regulated ZEB1/2 expression at post-transcriptional level. We further identified mir-200a/b, well-characterized mediators controlling ZEB1/2 expression, were transcriptionally activated by Restin and the regulation was dependent on the p53 binding site in mir-200b/a/429 promoter. Further mechanical studies demonstrated Restin interacted with p73, one of p53 family members, which contributed to Restin-mediated activation of mir-200a/b and suppression of ZEB1/2.

Conclusions: Taken together, our results suggest that Restin inhibits EMT and tumor metastasis by controlling the expression of the tumor metastasis suppressor mir-200a/b via association with p73. Our findings not only establish a mechanistic link between Restin, EMT and tumor metastasis, but also provide strong evidence supporting the notion that MAGE Group II proteins may exert a tumor suppressive effect in vivo.

Figure 1

Restin expression level was negatively related with tumor cell mobility. (A) Restin overexpression failed to inhibit cell proliferation determined by [³H] thymidine incorporation assay. MCF-7 and MDA-MB-231 (2 x 10⁴) cells were seeded onto 24-well plates. [³H] thymidine (1 mCi/well) was added 4 hours before the termination of experiment. Data were represented as the means ± S.M. of three independent experiments. mRNA (B) and protein (C) levels of Restin were determined by RT-PCR and western blot. MCF-10A, HMEC, MCF-7, MDA-MB-231 and MDA-MB-451 cells (1 x 10⁵) were seeded onto 6-well plates. Values were expressed as means ± S.M. of at least three independent experiments. (D) Restin expression in MCF-7 cells upon TGF-β treatment was determined by western blot. MCF-7 cells (5 x 10⁴) were seeded onto 6-well plates and TGF-β (5 ng/ml) were added for 6 days. Tubulin was used as a loading control. Results presented here are representative of three different experiments.

Figure 2

Restin prevented EMT in breast cancer cells. (A) Morphologic changes in MDA-MB-231 cells upon Restin overexpression. Phase-contrast cell images were taken (×40 magnification). mRNA (B) and protein levels (C) of epithelial markers (E-cadherin and ZO-1) and mesenchymal markers (Fibronectin, N-cadherin and Vimentin) in MDA-MB-231 cells (Control and Restin overexpression) were determined by real-time PCR and western blot. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 vs Control group. Tubulin was used as a loading control. Results presented here were representative of three different experiments. (D) Morphologic changes in MCF-7 cells upon Restin knockdown plus TGF-β treatment. MCF-7 cells (si-Control and si-Restin) were seeded onto 6-well plates and TGF-β (5 ng/ml) were added for 6 days. Phase-contrast cell images were taken (×40 magnification). (E) Protein levels of epithelial markers (E-cadherin and ZO-1) and mesenchymal markers (Fibronectin, N-cadherin and Vimentin) in MCF-7 (si-Control and si-Restin) cells were determined by western blot. Tubulin was used as a loading control. Results presented here were representative of three different experiments. Restin: Restin overexpression; si-Restin: Restin knockdown.

Figure 3

Restin overexpression inhibited migration and invasiveness of breast cancer cells. (A) Scratch wound healing assay of MDA-MB-231 cells. 5 × 10⁵ MDA-MB-231 cells (Control and Restin overexpression) were seeded onto 6-well plates. The 6-well plates were scratched (time 0) and 20 random fields of view per well were photographed after 12 h of scratch. Representative pictures from 3 independent experiments were shown. The motility of MDA-MB-231 and MDA-MB-451 cells was evaluated by transwell migration (B) and Matrigel invasion assays (C) as described in the Materials and Methods. Migration was quantitated by counting 10 fields at a magnification of × 400. Data were presented as the average number of migrated cells from three independent experiments. * p < 0.05 vs Control group. (D) Adhesion capacity of MDA-MB-231 and MDA-MB-451 cells was measured by cell attachment assay. MDA-MB-231 and MDA-MB-451 cells (Control and Restin overexpression) were seeded onto 96-well plates pretreated with fibronectin for 30 min at a density of 2 × 10³ cells/well in triplicate. Cells were allowed to adhere for 2 h and adherent cells were stained with crystal violet and quantified using the absorbance as a measurement at 550 nm. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 vs Control.

Figure 4

Restin overexpression inhibited lung metastasis in vivo. (A) The volume of primary tumors was measured after the transplantation of Control and Restin overexpressed MDA-MB-231 cells. 1 × 10⁵ MDA-MB-231 cells were subcutaneously injected into the mammary fat pad of immunocompromised NOD/SCID mice (n = 5). Tumor growth was monitored every 4 days. Data were shown as mean size ± S.M. of tumors in five mice per cell line. Animal experiments were repeated three times. (B) Xenograft tumor size from NOD/SCID mice grafted with Control and Restin-overexpressed cells. (C) The metastatic nodules in the lungs were counted following the orthotopic transplantation of Control and Restin overexpressed MDA-MB-231 cells. Values were expressed as means ± S.M. in five mice. * p < 0.05 vs Control. (D) Representative HE staining of lung metastases. Arrows indicate micrometastases.

Figure 5

Restin downregulated ZEB1/2 expression at the post-transcriptional level. RNA (A) and protein levels (B) of FOXC2, Twist, ZEB1, ZEB2, Slug and Snail in Control and Restin overexpressed MDA-MB-231 cells were determined by real-time PCR and western blot. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 vs Control group. (C) Expression level of above proteins in si-Control and si-Restin MCF-7 cells treated with TGF-β was determined by western blot. Tubulin was used as a loading control. Results presented here were representative of three different experiments. (D) MCF-7 stable cells (si-Control and si-Restin) were seed onto 6-well plates and transfected with control and ZEB1 siRNAs (si-ZEB1). 48 h posttransfection, proteins were collected and western blot was performed to detect protein levels of E-cadherin, Fibronectin and Vimentin. Tubulin was used as a loading control. Result presented here was a representative of three different experiments. (E) ZEB1 promoter activity was determined by luciferase reporter assay. HEK293 cells were seeded in 24-well plates and transfected with Restin overexpression lentivirus and ZEB1 promoter plasmids. The firefly luciferase activity was normalized to that of the renilla luciferase. (F) ZEB1 3’UTR activity was determined in HEK293 cells by luciferase reporter assay upon Restin overexpression. HEK293 cells were seeded in 24-well plates and transfected with ZEB1 3’UTR plasmids and different dose of Restin overexpression lentivirus. The firefly luciferase activity was normalized to that of the renilla luciferase. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 relative to cells without addition of lentivirus (−), ^# p < 0.05 relative to Control group.

Figure 6

mir-200b/a was responsible for Restin-mediated downregulation of ZEB1. RT-PCR was performed to detect mir-200a and mir-200b levels in Restin overexpressed MDA-MB-231 cells (A) and Restin knockdown MCF-7 cells (B). Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 relative to Control or si-Con group. (C) mir-200b/a/429 promoter activity was determined in HEK293 cells by luciferase reporter assay. HEK293 cells were seeded ontn 24-well plates and transfected with mir-200b/a/429 promoter plasmids and different dose of Restin overexpression lentivirus. The firefly luciferase activity was normalized to that of the renilla luciferase. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 relative to cells without addition of lentivirus (−), ^# p < 0.05 relative to their Control group. (D) ZEB1 3’UTR (WT and mutant) activity was determined by luciferase reporter assay upon Restin overexpression. WT and mutant ZEB1 3’UTR with deletion of mir-200 binding sites were transfected into HEK293 cells. 24 h later, different dose of Restin overexpression lentivirus were added. The firefly luciferase activity was normalized to that of the renilla luciferase. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 relative to cells without addition of Restin lentivirus.

Figure 7

Restin activated mir-200 transcription in a p73-dependent manner. (A) A schematic diagram of the human mir-200b/a/429-luciferase constructs (WT and its truncations) was shown. (B) A series of mir-200b/a/429 promoter activities were determined by luciferase reporter assay. Restin overexpression lentivirus were added into 293T cells and mir-200b/a/429 promoter truncation plasmids were transiently transfected. * p < 0.05 relative to Control lentivirus. (C) mir-200b/a/429 promoter (WT and p53 mutant) activities were measured by luciferase reporter assay. * p < 0.05 relative to Control lentivirus. (D) Western blot was performed to detect p53 and p73 levels in MCF-7, MDA-MB-231 and MDA-MB-157 cells. (E) Co-immunoprecipitation assay was performed to detect the exogenous interaction between Restin and p73. HERK-293 cells were transiently transfected with Flag-tagged Restin and His-tagged p73 plasmids. (Upper panel) Cell extracts were immunoprecipitated with mouse IgG or anti-His antibody and then blotted with anti-Flag antibodies. (Lower panel) Cell extracts were immunoprecipitated with mouse IgG or anti-Flag antibody and then blotted with anti-His antibody. (F) MDA-MB-231 stable cells (Control and Restin overexpression) were seed onto 24-well plates and co-transfected with Control or p73 siRNAs (si-Con and si-p73) and mir-200b/a/429 promoter construct. * p < 0.05 relative to si-Con group, # p < 0.05 relative to Control cells. (G) MDA-MB-231 stable cells (Control and Restin overexpression) were seed onto 6-well plates and transfected with control and p73 siRNAs (si-Con and si-p73). RT-PCR was performed to detect mir-200a and mir-200b expression levels. * p < 0.05 relative to si-Con group, # p < 0.05 relative to Control cells. (H) MDA-MB-231 stable cells (Control and Restin overexpression) were seed onto 6-well plates and transfected with control and p73 siRNAs (si-p73). Western blot was performed to detect ZEB1 and ZEB2 levels.