title: The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system.
abstract: Transforming growth factor-β (TGF-β) has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stages. In contrast to the mechanisms by which TGF-β induces growth arrest, the pathways that mediate tumor invasion are not well understood. Here, we describe a TGF-β-dependent invasion assay system consisting of spheroids of MCF10A1 normal breast epithelial cells (M1) and RAS-transformed (pre-)malignant derivatives (M2 and M4) embedded in collagen gels. Both basal and TGF-β-induced invasion of these cell lines was found to correlate with their tumorigenic potential; M4 showing the most aggressive behavior and M1 showing the least. Basal invasion was strongly inhibited by the TGF-β receptor kinase inhibitor SB-431542, indicating the involvement of autocrine TGF-β or TGF-β-like activity. TGF-β-induced invasion in premalignant M2 and highly malignant M4 cells was also inhibited upon specific knockdown of Smad3 or Smad4. Interestingly, both a broad spectrum matrix metalloproteinase (MMP) inhibitor and a selective MMP2 and MMP9 inhibitor mitigated TGF-β-induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-β was found to strongly induce MMP2 and MMP9 expression in a Smad3- and Smad4-dependent manner. This collagen-embedded spheroid system therefore offers a valuable screening model for TGF-β/Smad- and MMP2- and MMP9-dependent breast cancer invasion.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Eliza
authors.initials: E
authors.lastname: Wiercinska
authors.forename: Hildegonda P H
authors.initials: HP
authors.lastname: Naber
authors.forename: Evangelia
authors.initials: E
authors.lastname: Pardali
authors.forename: Gabri
authors.initials: G
authors.lastname: van der Pluijm
authors.forename: Hans
authors.initials: H
authors.lastname: van Dam
authors.forename: Peter
authors.initials: P
authors.lastname: ten Dijke
mesh_terms: D001549:Benzamides / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004149:Dioxoles / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness / Q000235:genetics* / Q000517:prevention & control; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000187:drug effects / Q000473:pathology; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000235:genetics
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004149:Dioxoles; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9