CCN6 Modulates BMP Signaling via the Smad-Independent TAK1/p38 Pathway, Acting to Suppress Metastasis of Breast Cancer

Authors' Affiliations: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan.
Cancer Research (Impact Factor: 9.33). 07/2012; 72(18):4818-28. DOI: 10.1158/0008-5472.CAN-12-0154
Source: PubMed


CCN6 (WISP3) is an extracellular matrix protein that exerts tumor suppressive functions in breast cancer, where its decreased expression is a feature of advanced disease. However, neither its role nor mechanism of action in breast cancer metastasis has been established. Bone morphogenetic proteins (BMPs), which constitute ligands of the TGF-β superfamily, are multifunctional cytokines that induce epithelial-mesenchymal transition, cell invasion, and metastasis. In this study, we identify a CCN6-BMP4-TAK1 kinase signaling pathway that controls the ability of the p38 MAP kinase to regulate acinar morphogenesis and invasion of breast cells. ShRNA-mediated attenuation of CCN6 in human mammary epithelial cells led to BMP4 upregulation as a major response to exposure to the TGF-β superfamily. CCN6 attenuation also induced BMP4-mediated activation of the Smad-independent TAK1 and p38 kinases. Conversely, ectopic expression of CCN6 in breast cancer cells antagonized BMP4-mediated TAK1/p38 activation and invasive capacity, both by binding BMP4 protein as well as decreasing BMP4 protein levels. Effects on BMP4 and p38 were confirmed in vivo where they correlated with decreased metastasis. In clinical specimens, we found that CCN6 expression was inversely associated with BMP4 and phospho-p38 levels in 69% of invasive breast carcinomas examined, consistent with the functional results. Together our findings identify a novel modifier pathway through which CCN6 acts to limit breast cancer invasion and metastasis. Cancer Res; 72(18); 4818-28. ©2012 AACR.

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Available from: Anupama Pal, Apr 26, 2014
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    • "Moreover, CCN6 was shown to inhibit invasion and metastasis of breast cancer in vivo. The mechanism of CCN6-inhibited breast cancer progression was shown to be mediated by the BMP4/TAK1/p38 pathway, which could induce epithelial-mesenchymal transition, cell invasion, and metastasis [88]. Decline of CCN6 protein expression was demonstrated to sufficiently activate the phosphatidylinositol 3-kinase/Akt signaling pathway, thus promoting growth factor-independent survival that is triggered by resistance to detachment-induced cell death (anoikis) [95]. "
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    ABSTRACT: The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors.
    Full-text · Article · Jan 2014
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    • "Previous data from us and others showed that BMP4 is able to reduce the growth of breast cancer cells whilst inducing cell migration and invasion [6,10,11]. Here we utilized two different 3D culture systems to evaluate whether these phenotypes persist under more physiological culture conditions and further explored the mechanisms of BMP4-induced changes in cell proliferation and mobility. "
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    ABSTRACT: Bone morphogenetic protein 4 (BMP4) belongs to the transforming growth factor beta (TGF-beta) family of proteins. BMPs regulate cell proliferation, differentiation and motility, and have also been reported to be involved in cancer pathogenesis. We have previously shown that BMP4 reduces breast cancer cell proliferation through G1 cell cycle arrest and simultaneously induces migration in a subset of these cell lines. Here we examined the effects of BMP4 in a more physiological environment, in a 3D culture system. We used two different 3D culture systems; Matrigel, a basement membrane extract from mouse sarcoma cells, and a synthetic polyethylene glycol (PEG) gel. AlamarBlue reagent was used for cell proliferation measurements and immunofluorescence was used to determine cell polarity. Expression of cell cycle regulators was examined by Western blot and matrix metalloproteinase (MMP) expression by qRT-PCR. The MCF-10A normal breast epithelial cells formed round acini with correct apicobasal localization of alpha6 integrin in Matrigel whereas irregular structures were seen in PEG gel. The two 3D matrices also supported dissimilar morphology for the breast cancer cells. In PEG gel, BMP4 inhibited the growth of MCF-10A and the three breast cancer cell lines examined, thus closely resembling the 2D culture conditions, but in Matrigel, no growth inhibition was observed in MDA-MB-231 and MDA-MB-361 cells. Furthermore, BMP4 induced the expression of the cell cycle inhibitor p21 both in 2D and 3D culture, thereby partly explaining the growth arrest. Interestingly, MDA-MB-231 cells formed large branching, stellate structures in response to BMP4 treatment in Matrigel, suggestive of increased cell migration or invasion. This effect was reversed by Batimastat, a broad-spectrum MMP inhibitor, and subsequent analyses showed BMP4 to induce the expression of MMP3 and MMP14, that are thus likely to be responsible for the stellate phenotype. Taken together, our results show that Matrigel provides a more physiological environment for breast epithelial cells than PEG gel. Moreover, BMP4 partly recapitulates in 3D culture the growth suppressive abilities previously seen in 2D culture and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells.
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    • "In our previous work, we found a novel compound mutation (840delT/T1000C) of Wisp3 in Chinese PPD kindred [12] [13]; the two probands carried a substitution mutation (1000T → C, Ser334Pro) in paternal allele, and a deletion (840delT) mutation in maternal allele that caused a truncated Wisp3 protein to miss 43 residues in C-terminus [14], and we also discovered the biological behavior changes of the articular chondrocytes (ACs) separated from the patients [15]. Wisp3 also had growth-, invasion-, and angiogenesis-inhibitory functions in inflammatory breast cancer (IBC) in vitro and in vivo [16] and was a key genetic determinant of the IBC phenotype [17]. However, "
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    ABSTRACT: Wisp3 gene mutation was shown to cause spondyloepiphyseal dysplasia tarda with progressive arthropathy (SRDT-PA), but the underlying mechanism is not clear. To clarify this mechanism, we constructed the wild and mutated Wisp3 expression vectors and transfected into human chondrocytes lines C-20/A4; Wisp3 proteins subcellular localization, cell proliferation, cell apoptosis, and Wisp3-mediated gene expression were determined, and dynamic secretion of collagen in transfected chondrocytes was analyzed by (14)C-proline incorporation experiment. Mutated Wisp3 protein increased proliferation activity, decreased apoptosis of C-20/A4 cells, and aggregated abnormally in cytoplasm. Expression of collagen II was also downregulated in C-20/A4 cells transfected with mutated Wisp3. Wild type Wisp3 transfection increased intracellular collagen content and extracellular collagen secretion, but the mutated Wisp3 lost this function, and the peak phase of collagen secretion was delayed in mutated Wisp3 transfected cells. Thus abnormal protein distribution, cell proliferation, collagen synthesis, and secretion in Wisp3 mutated chondrocytes might contribute to the pathogenesis of SEDT-PA.
    Full-text · Article · Mar 2013 · International Journal of Endocrinology
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