Abstract
The transforming growth factor-β (TGF-β) family is known to play critical roles in cancer progression. While the dual role of TGF-β is well described, the function of bone morphogenetic proteins (BMPs) is unclear. In this study, we established the involvement of Smad6, a BMP-specific inhibitory Smad, in breast cancer cell invasion. We show that stable overexpression of Smad6 in breast cancer MCF10A M2 cells inhibits BMP signalling, thereby mitigating BMP6-induced suppression of mesenchymal marker expression. Using a zebrafish xenograft model, we demonstrate that overexpression of Smad6 potentiates invasion of MCF10A M2 cells and enhances the aggressiveness of breast cancer MDA-MB-231 cells in vivo, whereas a reversed phenotype is observed after Smad6 knockdown. Interestingly, BMP6 pre-treatment of MDA-MB-231 cells induced cluster formation at the invasive site in the zebrafish. BMP6 also stimulated cluster formation of MDA-MB-231 cells co-cultured on Human Microvascular Endothelial Cells (HMEC)-1 in vitro. Electron microscopy illustrated an induction of cell-cell contact by BMP6. The clinical relevance of our findings is highlighted by a correlation of high Smad6 expression with poor distant metastasis free survival in ER-negative cancer patients. Collectively, our data strongly indicates the involvement of Smad6 and BMP signalling in breast cancer cell invasion in vivo.
Highlights
(a,b) Western blot analysis of total protein from control (CO) and Flag-Smad[6] stable expression (S6) MCF10A M2 cell lines treated with 50 ng/ml BMP6 or 0.5 ng/ml transforming growth factor (TGF)-β for 1 hr (a, *aspecific bands) and 50 ng/ml BMP6 or 5 ng/ml TGF-β for 24 hrs (b). (c) Gelatin zymogram showing MMP9 activity in conditioned medium of mock and TGF-β treated MCF10A M2 cells. (d,e) Representative images of 6 dpi zebrafish larvae showing the invasion of control (CO) (d) and Smad[6] overexpression (S6) (e) MCF10A M2 cells. (f) Quantification of invasive cluster numbers in CO and Smad6 overexpression (S6) MCF10A M2 injected zebrafish larvae
Stable Flag-Smad[6] overexpression showed inhibition on BMP6-induced phosphorylation of Smad[1], but only minor attenuation on TGF-β -induced phosphorylation of Smad[2], illustrating specific blocking of bone morphogenetic proteins (BMPs) signalling in these cells (Fig. 1a)
TGF-β -induced expression of Fibronectin and MMP9 was enhanced by Flag-Smad[6] overexpression (Fig. 1b,c). These results indicated that overexpression of Smad[6] could potentially alter the balance of TGF-β and BMP signalling in breast cancer cells, which may change their invasive properties by elevating the expression of TGF-β responding genes
Summary
(a,b) Western blot analysis of total protein from control (CO) and Flag-Smad[6] stable expression (S6) MCF10A M2 cell lines treated with 50 ng/ml BMP6 or 0.5 ng/ml TGF-β for 1 hr (a, *aspecific bands) and 50 ng/ml BMP6 or 5 ng/ml TGF-β for 24 hrs (b). (c) Gelatin zymogram showing MMP9 activity in conditioned medium of mock and TGF-β treated MCF10A M2 cells. (d,e) Representative images of 6 dpi zebrafish larvae showing the invasion of control (CO) (d) and Smad[6] overexpression (S6) (e) MCF10A M2 cells. (f) Quantification of invasive cluster numbers in CO and S6 MCF10A M2 injected zebrafish larvae. (d,e) Representative images of 6 dpi zebrafish larvae showing the invasion of control (CO) (d) and Smad[6] overexpression (S6) (e) MCF10A M2 cells. Increased expression of negative regulators of BMP signalling was found in highly metastatic cells[24,25]. To study the early stages of tumour cell dissemination and metastasis we make use of a zebrafish breast cancer xenograft model. By performing Duct of Cuvier implantation of breast cancer cells in zebrafish embryos, we can monitor the effect of manipulating BMP signalling on invasion and micro-metastasis. We inhibited BMP signalling in the human breast cancer cell lines through stable overexpression of inhibitory Smad[6] and found that Smad[6], and the inhibition of BMP signalling, significantly enhances breast cancer cell invasion in vivo
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