TGF--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--induced invasion of M4 cells, while leaving basal invasion intact. In line with this, TGF-was found to strongly induce MMP2 and MMP9 expression in a Smad3-and Smad4-dependent manner. This collagenembedded spheroid system therefore offers a valuable screening model for TGF-/Smad-and MMP2-and MMP9-dependent breast cancer invasion.
BackgroundThe transforming growth factor (TGF)-β superfamily comprises cytokines such as TGF-β and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-β are associated with poor outcome, whereas inhibition of TGF-β-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells.MethodsIn this study, we investigated the effect of BMP-7 on TGF-β-induced invasion in a 3 dimensional invasion assay.ResultsBMP-7 inhibited TGF-β-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-β-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-β-induced expression of integrin αvβ3 in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin β3 negatively affected TGF-β-induced invasion. On the other hand, overexpression of integrin β3 counteracted the inhibitory effect of BMP7 on TGF-β-induced invasion.ConclusionThus, BMP-7 may exert anti-invasive actions by inhibiting TGF-β-induced expression of integrin β3.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-011-0058-0) contains supplementary material, which is available to authorized users.
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 stage 1,2 . Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4 . The mechanisms by which TGF-β induces invasion are not well understood.TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes 5 . In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways 6 .To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells 7 , the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice 8 , and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung 9 . This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid 10 . Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures 11 . This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy.
IntroductionDepending on their stage of development or differentiation, B cells express specific chemokine receptors enabling them to respond to chemokines that control homing to the bone marrow (BM) and secondary lymphoid organs. [1][2][3] Here they are provided with signals crucial for progression through the consecutive stages of B-cell development and differentiation. The chemokine stromal cellderived factor-1 (SDF-1, also known as CXCL12) plays an important role in lymphocyte trafficking. SDF-1, originally identified as a growth-stimulatory factor for pre-B cells, 4 is constitutively expressed by BM stromal cells. [5][6][7] Its cognate receptor, the 7-transmembrane G-protein-coupled receptor CXCR4, is broadly expressed by cells of the immune system and mediates SDF-1-induced migration of hematopoietic progenitors and lymphocytes. [1][2][3]8,9 In the B-cell lineage, CXCR4 is prominently expressed by pre-B cells and controls localization in cellular niches in the BM obligatory for B-cell development. 3,8 Furthermore, CXCR4 expression by mature B cells is required for proper germinal center organization, which is essential for antigen-specific B-cell differentiation. 10 In addition, SDF-1 controls plasma cell migration, 6 and plasma cells require CXCR4 expression to localize in the BM compartment. 11,12 Multiple myeloma (MM), an incurable hematologic malignancy with a median survival of 3 to 4 years, is characterized by the expansion of malignant plasma cells in the BM. These tumor cells also express CXCR4, 13 and SDF-1 controls their ␣41-mediated adhesion to VCAM-1, fibronectin, and endothelial cells, as well as their transendothelial migration and homing/retention in the BM tumor microenvironment. [13][14][15][16][17][18][19] MM cells are critically dependent on the BM microenvironment, where cytokines produced by BM stromal cells provide them with proliferation and survival signals required for their expansion. 16 Thus, by controlling homing and retention of MM cells in the BM, SDF-1 plays an important role in the pathogenesis of MM.The important role of chemokine-induced migration in B-cell development and differentiation, as well as in the pathogenesis of MM, prompted us to explore the underlying signaling pathways. Recently, we have established an important role for Bruton tyrosine kinase (Btk) and phospholipase C-␥2 (PLC␥2) in chemokineinduced integrin-mediated migration and homing of B cells. 20 Importantly, however, Btk is not expressed in plasma cells and MM cells. [21][22][23] Several members of the Ras superfamily of small GTPases have also been implicated in chemokine-controlled B-cell migration. [24][25][26][27] This includes members of the Rho family 24,25 and members of the Ras family, such as Ras and Rap1. 26,27 Ral, another member of the Ras family of small GTPases, has been shown to mediate a wide variety of cellular responses, including regulation of cytoskeletal rearrangements and cell motility. 28 Methods MaterialsMonoclonal antibodies used were mouse anti-RalA (IgG2a), anti-Rap1 (IgG1)...
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