The balance of cell surface and soluble type III TGF-? receptor regulates BMP signaling in normal and cancerous mammary epithelial cells.
ABSTRACT: Bone morphogenetic proteins (BMPs) are members of the TGF-? superfamily that are over-expressed in breast cancer, with context dependent effects on breast cancer pathogenesis. The type III TGF-? receptor (T?RIII) mediates BMP signaling. While T?RIII expression is lost during breast cancer progression, the role of T?RIII in regulating BMP signaling in normal mammary epithelium and breast cancer cells has not been examined. Restoring T?RIII expression in a 4T1 murine syngeneic model of breast cancer suppressed Smad1/5/8 phosphorylation and inhibited the expression of the BMP transcriptional targets, Id1 and Smad6, in vivo. Similarly, restoring T?RIII expression in human breast cancer cell lines or treatment with sT?RIII inhibited BMP-induced Smad1/5/8 phosphorylation and BMP-stimulated migration and invasion. In normal mammary epithelial cells, shRNA-mediated silencing of T?RIII, T?RIII over-expression, or treatment with sT?RIII inhibited BMP-mediated phosphorylation of Smad1/5/8 and BMP induced migration. Inhibition of T?RIII shedding through treatment with TAPI-2 or expression of a non-shedding T?RIII mutant rescued T?RIII mediated inhibition of BMP induced Smad1/5/8 phosphorylation and BMP induced migration and/or invasion in both in normal mammary epithelial cells and breast cancer cells. Conversely, expression of a T?RIII mutant, which exhibited increased shedding, significantly reduced BMP-mediated Smad1/5/8 phosphorylation, migration, and invasion. These data demonstrate that T?RIII regulates BMP-mediated signaling and biological effects, primarily through the ligand sequestration effects of sT?RIII in normal and cancerous mammary epithelial cells and suggest that the ratio of membrane bound versus sT?RIII plays an important role in mediating these effects.
Project description:The type III transforming growth factor ? (TGF-?) receptor (T?RIII), also known as betaglycan, is the most abundantly expressed TGF-? receptor. T?RIII suppresses breast cancer progression by inhibiting migration, invasion, metastasis, and angiogenesis. T?RIII binds TGF-? ligands, with membrane-bound T?RIII presenting ligand to enhance TGF-? signaling. However, T?RIII can also undergo ectodomain shedding, releasing soluble T?RIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound T?RIII on TGF-? signaling and breast cancer biology, we defined T?RIII mutants with impaired (?Shed-T?RIII) or enhanced ectodomain shedding (SS-T?RIII). Inhibiting ectodomain shedding of T?RIII increased TGF-? responsiveness and abrogated T?RIII's ability to inhibit breast cancer cell migration and invasion. Conversely, expressing SS-T?RIII, which increased soluble T?RIII production, decreased TGF-? signaling and increased T?RIII-mediated inhibition of breast cancer cell migration and invasion. Of importance, SS-T?RIII-mediated increases in soluble T?RIII production also reduced breast cancer metastasis in vivo. Taken together, these studies suggest that the ratio of soluble T?RIII to membrane-bound T?RIII is an important determinant for regulation of T?RIII- and TGF-?-mediated signaling and biology.
Project description:During the course of breast cancer progression, normally dormant tumour-promoting effects of transforming growth factor beta (TGFbeta), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro-migratory TGFbeta 'switch' in mammary epithelial cells in vitro, we found that TGFbeta stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFbeta receptor, ALK5, as evidenced by studies using short hairpin RNA-resistant ALK5 mutants in ALK5-depleted cells and in vitro kinase assays. Functionally, Smad1/5 co-depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFbeta-stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFbeta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGFbeta switch in mammary epithelial cells.
Project description:The type III TGF-? receptor (T?RIII) is a TGF-? co-receptor that presents ligand to the type II TGF-? receptor to initiate signaling. T?RIII also undergoes ectodomain shedding to release a soluble form (sT?RIII) that can bind ligand, sequestering it away from cell surface receptors. We have previously identified a T?RIII extracellular mutant that has enhanced ectodomain shedding ("super shedding (SS)"-T?RIII-SS). Here, we utilize T?RIII-SS to study the balance of cell surface and soluble T?RIII in the context of lung cancer. We demonstrate that expressing T?RIII-SS in lung cancer cell models induces epithelial-to-mesenchymal transition (EMT) and that these T?RIII-SS (EMT) cells are less migratory, invasive and adhesive and more resistant to gemcitabine. Moreover, T?RIII-SS (EMT) cells exhibit decreased tumorigenicity but increased growth rate in vitro and in vivo. These studies suggest that the balance of cell surface and soluble T?RIII may regulate a dichotomous role for T?RIII during cancer progression.
Project description:The type III TGF-? receptor (T?RIII or betagylcan) is a TGF-? superfamily coreceptor with emerging roles in regulating TGF-? superfamily signaling and cancer progression. Alterations in TGF-? superfamily signaling are common in colon cancer; however, the role of T?RIII has not been examined. Although T?RIII expression is frequently lost at the message and protein level in human cancers and suppresses cancer progression in these contexts, here we demonstrate that, in colon cancer, T?RIII messenger RNA expression is not significantly altered and T?RIII expression is more frequently increased at the protein level, suggesting a distinct role for T?RIII in colon cancer. Increasing T?RIII expression in colon cancer model systems enhanced ligand-mediated phosphorylation of p38 and the Smad proteins, while switching TGF-? and BMP-2 from inhibitors to stimulators of colon cancer cell proliferation, inhibiting ligand-induced p21 and p27 expression. In addition, increasing T?RIII expression increased ligand-stimulated anchorage-independent growth, a resistance to ligand- and chemotherapy-induced apoptosis, cell migration and modestly increased tumorigenicity in vivo. In a reciprocal manner, silencing endogenous T?RIII expression decreased colon cancer cell migration. These data support a model whereby T?RIII mediates TGF-? superfamily ligand-induced colon cancer progression and support a context-dependent role for T?RIII in regulating cancer progression.
Project description:Bone morphogenetic protein 2 (BMP2) and BMP4 are key regulators of the fate and differentiation of human mammary epithelial stem cells (SCs), as well as of their niches, and are involved in breast cancer development. We established that MCF10A immature mammary epithelial cells reliably reproduce the BMP response that we previously identified in human primary epithelial SCs. In this model, we observed that BMP2 promotes luminal progenitor commitment and expansion, whereas BMP4 prevents lineage differentiation. Environmental pollutants are known to promote cancer development, possibly by providing cells with stem-like features and by modifying their niches. Bisphenols, in particular, were shown to increase the risk of developing breast cancer. Here, we demonstrate that chronic exposure to low doses of bisphenol A (BPA) or benzo(a)pyrene (B(a)P) alone has little effect on SCs properties of MCF10A cells. Conversely, we show that this exposure affects the response of immature epithelial cells to BMP2 and BMP4. Furthermore, the modifications triggered in MCF10A cells on exposure to pollutants appeared to be predominantly mediated by altering the expression and localization of type-1 receptors and by pre-activating BMP signaling, through the phosphorylation of small mothers against decapentaplegic 1/5/8 (SMAD1/5/8). By analyzing stem and progenitor properties, we reveal that BPA prevents the maintenance of SC features prompted by BMP4, whereas promoting cell differentiation towards a myoepithelial phenotype. Inversely, B(a)P prevents BMP2-mediated luminal progenitor commitment and expansion, leading to the retention of stem-like properties. Overall, our data indicate that BPA and B(a)P distinctly alter the fate and differentiation potential of mammary epithelial SCs by modulating BMP signaling.
Project description:The TGF-? pathway plays a major role in tumor progression through regulation of epithelial and stromal cell signaling. Dysfunction of the pathway can lead to carcinoma progression and metastasis. To gain insight into the stromal role of the TGF-? pathway in breast cancer, we performed laser capture microdissection (LCM) from breast cancer patients and reduction mammoplasty patients. Microdissected tumor stroma and normal breast stroma were examined for gene expression. Expression of the TGF-? type III receptor (TGFBR3) was greatly decreased in the tumor stroma compared to control healthy breast tissue. These results demonstrated a 44-fold decrease in TGFBR3 mRNA in tumor stroma in comparison to control tissue. We investigated publicly available databases, and have identified that TGFBR3 mRNA levels are decreased in tumor stroma. We next investigated fibroblast cell lines derived from cancerous and normal breast tissue and found that in addition to mRNA levels, T?RIII protein levels were significantly reduced. Having previously identified that cancer-associated fibroblasts secrete greater levels of tumor promoting cytokines, we investigated the consequences of soluble-T?RIII (sT?RIII) on fibroblasts. Fibroblast conditioned medium was analyzed for 102 human secreted cytokines and distinct changes in response to sT?RIII were observed. Next, we used the fibroblast-conditioned medium to stimulate human monocyte cell line THP-1. These results indicate a distinct transcriptional response depending on sT?RIII treatment and whether it was derived from normal or cancerous breast tissue. We conclude that the effect of T?RIII has distinct roles not only in cancer-associated fibroblasts but that sT?RIII has distinct paracrine functions in the tumor microenvironment.
Project description:We have previously observed that tropomyosin receptor kinase B (TrkB) induces breast cancer metastasis by activating both the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) and phosphatidylinositol-3-Kinase (PI3K)/AKT signaling pathways and inhibiting runt-related transcription factor 3 (RUNX3) and kelch-like ECH-associated protein 1 (KEAP1). These studies indicated that TrkB expression is crucial to the pathogenesis of breast cancer. However, how TrkB regulates bone morphogenetic protein (BMP) signaling and tumor suppression is largely unknown. Herein, we report that TrkB is a key regulator of BMP-mediated tumor suppression. TrkB enhances the metastatic potential of cancer cells by promoting cell anchorage-independent growth, migration, and suppressing BMP-2-mediated growth inhibition. TrkB inhibits the BMP-mediated activation of SMAD family member 1 (SMAD1) by promoting the formation of the TrkB/BMP type II receptor complex and suppresses RUNX3 by depleting BMP receptor I (BMPRI) expression. In addition, the knockdown of TrkB restored the tumor-inhibitory effect of BMP-2 via the activation of SMAD1. Moreover, the TrkB kinase activity was required for its effect on BMP signaling. Our study identified a unique role of TrkB in the regulation of BMP-mediated growth inhibition and BMP-2-induced RUNX3 expression.
Project description:Bone morphogenetic proteins (BMPs) have been implicated in the control of proliferation, tissue formation, and differentiation. BMPs regulate the biology of stem and progenitor cells and can promote cellular differentiation, depending on the cell type and context. Although the BMP pathway is known to be involved in early embryonic development of the mammary gland via mesenchymal cells, its role in later epithelial cellular differentiation has not been examined. The majority of the mammary gland development occurs post-natal, and its final functional differentiation is characterized by the emergence of alveolar cells that produce milk proteins. Here, we tested the hypothesis that bone morphogenetic protein receptor 1A (BMPR1A) function was required for mammary epithelial cell differentiation. We found that the BMPR1A-SMAD1/5/8 pathway was predominantly active in undifferentiated mammary epithelial cells, compared with differentiated cells. Reduction of BMPR1A mRNA and protein, using short hairpin RNA, resulted in a reduction of SMAD1/5/8 phosphorylation in undifferentiated cells, indicating an impact on this pathway. When the expression of the BMPR1A gene knocked down in undifferentiated cells, this also prevented beta-casein production during differentiation of the mammary epithelial cells by lactogenic hormone stimulation. Addition of Noggin, a BMP antagonist, also prevented beta-casein expression. Together, this demonstrated that BMP-BMPR1A-SMAD1/5/8 signal transduction is required for beta-casein production, a marker of alveolar cell differentiation. This evidence functionally identifies BMPR1A as a potential new regulator of mammary epithelial alveolar cell differentiation.
Project description:More efficient therapies that target multiple molecular mechanisms are needed for the treatment of incurable bone metastases. Halofuginone is a plant alkaloid-derivative with antiangiogenic and antiproliferative effects. Here we demonstrate that halofuginone is an effective therapy for the treatment of bone metastases, through multiple actions that include inhibition of TGF? and BMP-signaling. Halofuginone blocked TGF-?-signaling in MDA-MB-231 and PC3 cells showed by inhibition of TGF-?-induced Smad-reporter, phosphorylation of Smad-proteins, and expression of TGF-?-regulated metastatic genes. Halofuginone increased inhibitory Smad7-mRNA and reduced TGF-?-receptor II protein. Proline supplementation but not Smad7-knockdown reversed halofuginone-inhibition of TGF-?-signaling. Halofuginone also decreased BMP-signaling. Treatment of MDA-MB-231 and PC3 cells with halofuginone reduced the BMP-Smad-reporter (BRE)4, Smad1/5/8-phosphorylation and mRNA of the BMP-regulated gene Id-1. Halofuginone decreased immunostaining of phospho-Smad2/3 and phospho-Smad1/5/8 in cancer cells in vivo. Furthermore, halofuginone decreased tumor-take and growth of orthotopic-tumors. Mice with breast or prostate bone metastases treated with halofuginone had significantly less osteolysis than control mice. Combined treatment with halofuginone and zoledronic-acid significantly reduced osteolytic area more than either treatment alone. Thus, halofuginone reduces breast and prostate cancer bone metastases in mice and combined with treatment currently approved by the FDA is an effective treatment for this devastating complication of breast and prostate-cancer.
Project description:The type III TGF-? receptor (T?RIII) is a ubiquitous co-receptor for TGF-? superfamily ligands with roles in suppressing cancer progression, in part through suppressing cell motility. Here we demonstrate that T?RIII promotes epithelial cell adhesion to fibronectin in a ?-arrestin2 dependent and TGF-?/BMP independent manner by complexing with active integrin ?5?1, and mediating ?-arrestin2-dependent ?5?1 internalization and trafficking to nascent focal adhesions. T?RIII-mediated integrin ?5?1 trafficking regulates cell adhesion and fibronectin fibrillogenesis in epithelial cells, as well as ?5 localization in breast cancer patients. We further demonstrate that increased T?RIII expression correlates with increased ?5 localization at sites of cell-cell adhesion in breast cancer patients, while higher T?RIII expression is a strong predictor of overall survival in breast cancer patients. These data support a novel, clinically relevant role for T?RIII in regulating integrin ?5 localization, reveal a novel crosstalk mechanism between the integrin and TGF-? superfamily signaling pathways and identify ?-arrestin2 as a regulator of ?5?1 trafficking.