Smad6 determines BMP-regulated invasive behaviour of breast cancer cells in a zebrafish xenograft model

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.

TGF-β Family Signaling in Tumor Suppression and Cancer Progression

“…Alterations that cause persistent high levels of Smad7 blunt TGF-b signaling and have been described in endometrial carcinomas and thyroid follicular tumors (Cerutti et al 2003;Dowdy et al 2005). Similarly, increased Smad6 expression also attenuates TGF-b family signaling, preventing its tumor-suppression function in pancreatic or breast cancer (Kleeff et al 1999;de Boeck et al 2016). As reported above for Smad4 depletion in genetic models, Smad7 overexpression in immune cells has been associated with chronic inflammation in the colonic mucosa.…”

Embryonic zebrafish xenograft assay of human cancer metastasis

“…The extra-uterine development of hundreds of eggs also permits a greater number of studies in genetically identical organisms. Since the first reported xenotransplantation of human cells into zebrafish ( Lee et al , 2005), many laboratories have shown that zebrafish embryos are useful for the study of other facets of tumour biology including cancer-induced angiogenesis ( Haldi et al , 2006); cancer cell invasion and metastasis ( de Boeck et al , 2016; Marques et al , 2009); cancer stem cell growth ( Bansal et al , 2014; Chen et al , 2017); interaction of cancer cells with the host ( Feng & Martin, 2015); and drug screening ( Corkery et al , 2011; Gibert et al , 2013). Importantly, the development of human tumours and their response to chemotherapeutic treatment in zebrafish embryos is comparable to that observed in mouse xenograft assays ( Fior et al , 2017).…”

Light‐Triggered Cancer Cell Specific Targeting and Liposomal Drug Delivery in a Zebrafish Xenograft Model

“…This includes the development of embryonic zebrafish xenograft models to study the pathogenesis of human cancers,41–43 including human breast cancers 44,45. Here, MDA‐MB‐231 breast cancer cells, stably expressing GFP, were microinjected into the circulation of 2‐day old zebrafish larvae via the duct of Cuvier and quickly accumulated (<1 h post injection [hpi]) within the caudal hematopoietic tissue (CHT) 46. One hour after injection of cancer cells, either fluorescently labeled E PEG ‐ or F PEG ‐liposomes (4 mol% PEG 5000 , 1 mol% DOPE‐LR probe) were injected (1 m m , 3 nL) into circulation via the posterior cardinal vein (PCV).…”