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    Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function. Schniewind B,Groth S,Sebens Müerköster S,Sipos B,Schäfer H,Kalthoff H,Fändrich F,Ungefroren H Oncogene In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in pancreatic tumor cells. 10.1038/sj.onc.1210272
    Specificity, versatility, and control of TGF-β family signaling. Derynck Rik,Budi Erine H Science signaling Encoded in mammalian cells by 33 genes, the transforming growth factor-β (TGF-β) family of secreted, homodimeric and heterodimeric proteins controls the differentiation of most, if not all, cell lineages and many aspects of cell and tissue physiology in multicellular eukaryotes. Deregulation of TGF-β family signaling leads to developmental anomalies and disease, whereas enhanced TGF-β signaling contributes to cancer and fibrosis. Here, we review the fundamentals of the signaling mechanisms that are initiated upon TGF-β ligand binding to its cell surface receptors and the dependence of the signaling responses on input from and cooperation with other signaling pathways. We discuss how cells exquisitely control the functional presentation and activation of heteromeric receptor complexes of transmembrane, dual-specificity kinases and, thus, define their context-dependent responsiveness to ligands. We also introduce the mechanisms through which proteins called Smads act as intracellular effectors of ligand-induced gene expression responses and show that the specificity and impressive versatility of Smad signaling depend on cross-talk from other pathways. Last, we discuss how non-Smad signaling mechanisms, initiated by distinct ligand-activated receptor complexes, complement Smad signaling and thus contribute to cellular responses. 10.1126/scisignal.aav5183
    Differential roles of Smad2 and Smad3 in the regulation of TGF-β1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1. Ungefroren Hendrik,Groth Stephanie,Sebens Susanne,Lehnert Hendrik,Gieseler Frank,Fändrich Fred Molecular cancer BACKGROUND:Progression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-β)/Smad signalling pathway, eventually resulting in loss of TGF-β-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-β are mediated solely or partially through the canonical Smad signalling pathway which commences with activation of receptor-regulated Smads (R-Smads) Smad2 and Smad3 by the TGF-β type I receptor. However, little is known on the relative contribution of each R-Smad, the possible existence of functional antagonism, or the crosstalk with other signalling pathways in the control of TGF-β1-induced growth inhibition and cell migration. Using genetic and pharmacologic approaches we have inhibited in PDAC cells endogenous Smad2 and Smad3, as well as a potential regulator, the small GTPase Rac1, and have analysed the consequences for TGF-β1-mediated growth inhibition and cell migration (chemokinesis). RESULTS:SiRNA-mediated silencing of Smad3 in the TGF-β responsive PDAC cell line PANC-1 reduced TGF-β1-induced growth inhibition but increased the migratory response, while silencing of Smad2 enhanced growth inhibition but decreased chemokinesis. Interestingly, siRNA-mediated silencing of the small GTPase Rac1, or ectopic expression of a dominant-negative Rac1 mutant largely mimicked the effect of Smad2 silencing on both TGF-β1-induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-β1-induction of a Smad2-specific transcriptional reporter and Smad2 C-terminal phosphorylation in PDAC cells while Smad3-specific transcriptional activity and Smad3 C-terminal phosphorylation appeared increased. Disruption of autocrine TGF-β signalling in PANC-1 cells rendered cells less susceptible to the growth-suppressive effect of Rac1 inhibition, suggesting that the decrease in "basal" proliferation upon Rac1 inhibition was caused by potentiation of autocrine TGF-β growth inhibition. CONCLUSIONS:In malignant cells with a functional TGF-β signalling pathway Rac1 antagonizes the TGF-β1 growth inhibitory response and enhances cell migration by antagonistically regulating Smad2 and Smad3 activation. This study reveals that Rac1 is prooncogenic in that it can alter TGF-β signalling at the R-Smad level from a tumour-suppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression. 10.1186/1476-4598-10-67
    Role of Smad1 and Smad4 proteins in the induction of p21WAF1,Cip1 during bone morphogenetic protein-induced growth arrest in human breast cancer cells. Pouliot F,Labrie C The Journal of endocrinology Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of cytokines. The recent observation that BMPs can inhibit breast cancer cell proliferation in vitro suggests that BMPs or the BMP pathway may hold promise as therapeutic targets for the control of breast tumor growth in women. Better to understand the mechanism of BMP-induced growth arrest we examined the effect of BMP-2 and mediators of BMP-2 action on cell proliferation and p21(Cip1) expression in breast cancer cell lines. We show here that BMP-2 potently inhibited the proliferation of breast cancer cell lines that express both Smad1 and Smad4 (CAMA-1, MCF7, MDA-MB-231, T-47D, ZR-75-1), but not that of cells that only express Smad1 (MDA-MB-468). Growth inhibition correlated with up-regulation of p21 mRNA and protein levels. Up-regulation of p21 was resistant to cycloheximide but not to actinomycin D, suggesting that it occurred at the transcriptional level. Using p21 promoter-luciferase reporter constructs we mapped the BMP-responsive region of the p21 promoter to within 211 base pairs of the transcription start site. Induction of p21 promoter activity was rapid and coincided with up-regulation of p21 mRNA and protein levels. p21 promoter activity required both Smad1 and Smad4 and was induced by either BMP-2 or constitutively active type I BMP receptors. Moreover, the C-terminal SSVS region of Smad1 was necessary for activation of the p21 promoter by BMP-2. Taken together, these results indicate that the mechanism of BMP-induced p21 promoter activation involves BMP receptors and BMP Smads. 10.1677/joe.0.1720187
    Smad regulation in TGF-beta signal transduction. Moustakas A,Souchelnytskyi S,Heldin C H Journal of cell science Smad proteins transduce signals from transforming growth factor-beta (TGF-beta) superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. Phosphorylation of receptor-activated Smads (R-Smads) leads to formation of complexes with the common mediator Smad (Co-Smad), which are imported to the nucleus. Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. Alternatively, nuclear R-Smads associate with ubiquitin ligases and promote degradation of transcriptional repressors, thus facilitating target gene regulation by TGF-beta. Smads themselves can also become ubiquitinated and are degraded by proteasomes. Finally, the inhibitory Smads (I-Smads) block phosphorylation of R-Smads by the receptors and promote ubiquitination and degradation of receptor complexes, thus inhibiting signalling.
    Role of Smad proteins in resistance to BMP-induced growth inhibition in B-cell lymphoma. Huse Kanutte,Bakkebø Maren,Wälchli Sébastien,Oksvold Morten P,Hilden Vera I,Forfang Lise,Bredahl May L,Liestøl Knut,Alizadeh Ash A,Smeland Erlend B,Myklebust June H PloS one Bone morphogenetic protein (BMP) expression and signaling are altered in a variety of cancers, but the functional impact of these alterations is uncertain. In this study we investigated the impact of expression of multiple BMPs and their signaling pathway components in human B-cell lymphoma. BMP messages, in particular BMP7, were detected in normal and malignant B cells. Addition of exogenous BMPs inhibited DNA synthesis in most lymphoma cell lines examined, but some cell lines were resistant. Tumor specimens from three out of five lymphoma patients were also resistant to BMPs, as determined by no activation of the BMP effectors Smad1/5/8. We have previously shown that BMP-7 potently induced apoptosis in normal B cells, which was in contrast to no or little inhibitory effect of this BMP in the lymphoma cells tested. BMP-resistance mechanisms were investigated by comparing sensitive and resistant cell lines. While BMP receptors are downregulated in many cancers, we documented similar receptor levels in resistant and sensitive lymphoma cells. We found a positive correlation between activation of Smad1/5/8 and inhibition of DNA synthesis. Gene expression analysis of two independent data sets showed that the levels of inhibitory Smads varied across different B-cell lymphoma. Furthermore, stable overexpression of Smad7 in two different BMP-sensitive cell lines with low endogenous levels of SMAD7, rendered them completely resistant to BMPs. This work highlights the role of Smads in determining the sensitivity to BMPs and shows that upregulation of Smad7 in cancer cells is sufficient to escape the negative effects of BMPs. 10.1371/journal.pone.0046117
    Expression of TGFbeta type-II receptor in association with markers of proliferation and apoptosis in premalignant lung lesions. Muñoz-Antonia Teresita,Muro-Cacho Carlos,Sharma Swati,Cantor Alan,Bepler Gerold Cancer BACKGROUND:Analysis of the early molecular abnormalities that play an oncogenic role in the progression of pulmonary neoplasia may lead to the identification of useful markers for early detection and prognosis. In normal squamous epithelium, transforming growth factor beta (TGFbeta) regulates cell growth and differentiation via specific membranous receptors and intracellular signaling molecules (Smads). The authors previously observed that, in head and neck squamous cell carcinoma, the expression of the TGFbeta type II receptor (TbetaR-II) decreases as tumors become less differentiated and more biologically aggressive. METHODS:In this pilot study of 48 premalignant bronchoepithelial lesions, the authors evaluated the expression of TbetaR-II and 2 proliferation markers (Ki-67 and MCM2), and the amount of early DNA fragmentation as evidence of apoptosis. RESULTS:The authors observed that the progression of premalignant lesions toward carcinoma in situ is accompanied by a decrease in TBR II expression and apoptosis and an increase in the expression of Ki-67 and MCM2. CONCLUSIONS:These results suggested that the TGFbeta pathway may be impaired early in the neoplastic process and that a combination of selected markers can provide a useful profile to detect preneoplastic changes in individuals at high risk for developing pulmonary carcinoma. 10.1002/cncr.22937
    The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling. Hanyu A,Ishidou Y,Ebisawa T,Shimanuki T,Imamura T,Miyazono K The Journal of cell biology Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (TbetaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling. 10.1083/jcb.200106023
    Role of transforming growth factor-beta signaling in cancer. de Caestecker M P,Piek E,Roberts A B Journal of the National Cancer Institute Signaling from transforming growth factor-beta (TGF-beta) through its unique transmembrane receptor serine-threonine kinases plays a complex role in carcinogenesis, having both tumor suppressor and oncogenic activities. Tumor cells often escape from the antiproliferative effects of TGF-beta by mutational inactivation or dysregulated expression of components in its signaling pathway. Decreased receptor function and altered ratios of the TGF-beta type I and type II receptors found in many tumor cells compromise the tumor suppressor activities of TGF-beta and enable its oncogenic functions. Recent identification of a family of intracellular mediators, the Smads, has provided new paradigms for understanding mechanisms of subversion of TGF-beta signaling by tumor cells. In addition, several proteins recently have been identified that can modulate the Smad-signaling pathway and may also be targets for mutation in cancer. Other pathways such as various mitogen-activated protein kinase cascades also contribute substantially to TGF-beta signaling. Understanding the interplay between these signaling cascades as well as the complex patterns of cross-talk with other signaling pathways is an important area of investigation that will ultimately contribute to understanding of the bifunctional tumor suppressor/oncogene role of TGF-beta in carcinogenesis. 10.1093/jnci/92.17.1388
    SMAD7 is a prognostic marker in patients with colorectal cancer. Boulay Jean-Louis,Mild Gabriele,Lowy Adam,Reuter Juergen,Lagrange Magali,Terracciano Luigi,Laffer Urban,Herrmann Richard,Rochlitz Christoph International journal of cancer Chromosomal region 18q21 is frequently deleted in colorectal cancer (CRC) and is associated with poor prognosis. Potential tumor suppressor mechanisms altered by 18q21 deletion include mediation of TGFbeta signaling by SMADs. Following the definition of SMAD4 deletion as a negative predictive marker for chemotherapy benefit in patients with CRC, we aimed to evaluate the clinical relevance of the deletion of other SMAD genes clustered in this region: SMAD2 and SMAD7 in 264 CRC biopsies from a previous clinical study. In contrast to SMAD2 deletion, for which no clinical relevance was observed, hazard ratios (HR) in a multivariate analysis associated with SMAD7 deletion [overall survival (OS): HR = 0.43, p = 0.0012; disease-free survival (DFS): HR = 0.50, p = 0.0033] indicated a favorable outcome for these patients. In addition, SMAD7 duplication had a hazardous effect on survival [OS: HR = 2.10, p = 0.020; DFS: HR = 2.06, p = 0.015]. Moreover, the HRs associated with one additional copy of SMAD7 were 1.76, p = 0.00024 [OS] and 1.64, p = 0.00048 [DFS] respectively, showing a graded effect of SMAD7 on patient outcome depending on gene copy number that suggests a dose-and-effect basis. Since SMAD7 blocks TGFbeta signaling, these data are consistent with the loss of SMAD7 rendering carcinoma cells more sensitive to cell growth arrest/apoptotic effect of TGFbeta, whereas gain of SMAD7 function might result in TGFbeta resistance, thereby emphasizing the role of TGFbeta in tumor suppression. 10.1002/ijc.10908
    Overexpression of Smad proteins, especially Smad7, in oral epithelial dysplasias. Chen Yuk-Kwan,Huang Anderson Hsien-Cheng,Cheng Pei-Hsun,Yang Shang-Hsun,Lin Li-Min Clinical oral investigations OBJECTIVE:Transforming growth factor β, via membrane-bound receptors and downstream Smad2-4, 7, can modulate tumorigenesis. Smad2 and Smad3 heterodimerize with Smad4, and the complex migrates to the nucleus to regulate the expression of target genes. Smad7 is a key negative regulator of this signaling pathway. This study aimed to examine Smad2-4, 7 expression and phosphorylated Smad2-3 (p-Smad2-3) in oral epithelial dysplasia and compared it with normal oral mucosa, hyperkeratosis/epithelial hyperplasia and squamous cell carcinoma (SCC). MATERIALS AND METHODS:Immunohistochemical staining of Smad2-4, 7 and p-Smad2-3, was performed for 75 samples of human oral mucosa, including hyperkeratosis/epithelial hyperplasia (n = 20), mild epithelial dysplasia (n = 11), moderate to severe epithelial dysplasia (n = 11), and SCC (n = 43). Normal buccal mucosa samples (n = 9) were also included. RESULTS:A significant increase in Smad7 expression was observed in the ascending order of samples of normal oral mucosa, hyperkeratosis/epithelial hyperplasia/mild oral epithelial dysplasia, moderate to severe oral epithelial dysplasia, and well-differentiated oral SCC/moderately to poorly differentiated oral SCC. Additionally, significant increases in Smad7 expression were noted as compared with expression of Smad2-4 and p-Smad2-3 in lesions of hyperkeratosis/epithelial hyperplasia, mild oral epithelial dysplasia, moderate to severe oral epithelial dysplasia, well-differentiated oral SCC, and moderately to poorly differentiated oral SCC. CONCLUSIONS:Our results indicate that Smad proteins, particularly Smad7, in oral epithelial dysplasia and SCC could contribute to the attenuation of Smads anti-proliferative signaling in cancer development. CLINICAL RELEVANCE:Smad7 could be a marker for risk of malignant transformation of oral epithelial dysplasia. 10.1007/s00784-012-0756-7
    Trastuzumab inhibits pituitary tumor cell growth modulating the TGFB/SMAD2/3 pathway. Petiti Juan Pablo,Sosa Liliana Del Valle,Picech Florencia,Moyano Crespo Gabriela Deisi,Arevalo Rojas Jean Zander,Pérez Pablo Anibal,Guido Carolina Beatriz,Leimgruber Carolina,Sabatino María Eugenia,García Pedro,Bengio Verónica,Papalini Francisco Roque,Estario Paula,Berhard Celina,Villarreal Marcos,Gutiérrez Silvina,De Paul Ana Lucía,Mukdsi Jorge Humberto,Torres Alicia Inés Endocrine-related cancer In pituitary adenomas, early recurrences and resistance to conventional pharmacotherapies are common, but the mechanisms involved are still not understood. The high expression of epidermal growth factor receptor 2 (HER2)/extracellular signal-regulated kinase (ERK1/2) signal observed in human pituitary adenomas, together with the low levels of the antimitogenic transforming growth factor beta receptor 2 (TBR2), encouraged us to evaluate the effect of the specific HER2 inhibition with trastuzumab on experimental pituitary tumor cell growth and its effect on the antiproliferative response to TGFB1. Trastuzumab decreased the pituitary tumor growth as well as the expression of ERK1/2 and the cell cycle regulators CCND1 and CDK4. The HER2/ERK1/2 pathway is an attractive therapeutic target, but its intricate relations with other signaling modulators still need to be unraveled. Thus, we investigated possible cross-talk with TGFB signaling, which has not yet been studied in pituitary tumors. In tumoral GH3 cells, co-incubation with trastuzumab and TGFB1 significantly decreased cell proliferation, an effect accompanied by a reduction in ERK1/2 phosphorylation, an increase of SMAD2/3 activation. In addition, through immunoprecipitation assays, a diminution of SMAD2/3-ERK1/2 and an increase SMAD2/3-TGFBR1 interactions were observed when cells were co-incubated with trastuzumab and TGFB1. These findings indicate that blocking HER2 by trastuzumab inhibited pituitary tumor growth and modulated HER2/ERK1/2 signaling and consequently the anti-mitogenic TGFB1/TBRs/SMADs cascade. The imbalance between HER2 and TGFBRs expression observed in human adenomas and the response to trastuzumab on experimental tumor growth may make the HER2/ERK1/2 pathway an attractive target for future pituitary adenoma therapy. 10.1530/ERC-18-0067
    Structural determinants of Smad function in TGF-β signaling. Macias Maria J,Martin-Malpartida Pau,Massagué Joan Trends in biochemical sciences Smad transcription factors are central to the signal transduction pathway that mediates the numerous effects of the transforming growth factor β (TGF-β) superfamily of cytokines in metazoan embryo development as well as in adult tissue regeneration and homeostasis. Although Smad proteins are conserved, recent genome-sequencing projects have revealed their sequence variation in metazoan evolution, human polymorphisms, and cancer. Structural studies of Smads bound to partner proteins and target DNA provide a framework for understanding the significance of these evolutionary and pathologic sequence variations. We synthesize the extant mutational and structural data to suggest how genetic variation in Smads may affect the structure, regulation, and function of these proteins. We also present a web application that compares Smad sequences and displays Smad protein structures and their disease-associated variants. 10.1016/j.tibs.2015.03.012
    Loss of expression, and mutations of Smad 2 and Smad 4 in human cervical cancer. Maliekal Tessy T,Antony Marie-Lue,Nair Asha,Paulmurugan Ramasamy,Karunagaran Devarajan Oncogene Mutations in Smads, intermediates of transforming growth factor-beta signaling, are known to contribute to the loss of sensitivity to transforming growth factor-beta, a common feature of many neoplastic cells. However, not much information is available on Smad alterations in cervical cancer and so we probed, for the first time, for alterations in Smad 2 and Smad 4 genes using human cervical cancer cell lines and human cervical tissue samples. Using PCR/reverse transcription-PCR, single-stranded conformation polymorphism analysis and DNA sequencing, we observed a deletion of 'G' in the L3 loop (crucial in Smad-receptor interaction) in C-33A cells, and an insertion of 'A' in codon 122 (loss of MH2 domain) from a cervical tumor sample, both of which caused frame shift and pretermination in Smad 2. In addition, a G/A transition at 31 bp upstream-nontranslated regions of exon 8 of Smad 4 was found in Bu 25TK cells. Smad 2 expression was less in some of the cervical tumor samples than that of nonmalignant samples and six cancer samples showed C-terminal deletions that abolish Smad 2 phosphorylation sites. The loss of expression of Smad 4 found in some cervical tumor samples was due to transcription loss rather than deletion of the gene. Our results highlight an important role for Smad 2 and Smad 4 in human cervical tumors. 10.1038/sj.onc.1206806
    Preserved Smad4 expression in the transforming growth factor beta signaling pathway is a favorable prognostic factor in patients with advanced gastric cancer. Xiangming C,Natsugoe S,Takao S,Hokita S,Ishigami S,Tanabe G,Baba M,Kuroshima K,Aikou T Clinical cancer research : an official journal of the American Association for Cancer Research The signals of the transforming growth factor beta (TGF-beta) superfamily are conveyed through cell surface serine/threonine kinase receptors to the intracellular mediators known as Smads. Activation of Smads causes their translocation from the cytoplasm to the nucleus, where they function to control gene expression. The present study analyzed the expression of Smad4 and TGF-beta1 to determine their prognostic significance in advanced gastric cancer. Of 249 cases of advanced gastric cancer, 41 had invaded the muscular layer, 114 had invaded the subserosal layer, and 94 had invaded the serosa. Anti-Smad4 and TGF-beta1 antibodies were used for immunohistochemical staining. Reduced expression of Smad4 was 75.1%, whereas positive expression of TGF-beta1 was 39.6% in gastric cancer. Smad4 expression was related to the depth of tumor invasion (P < 0.05), and TGF-beta1 expression correlated with tumor gross type (P < 0.05). Postoperative survival analysis indicated that patients who had a tumor with reduced Smad4 expression had a poorer clinical outcome than those with preserved expression (P < 0.05). Furthermore, in patients with TGF-beta1-positive tumors, survival rate was significantly better in patients with preserved Smad4 expression than in those with reduced Smad4 expression (P < 0.05). According to multivariate analysis, Smad4 expression acted as an independent prognostic factor. Smad4 expression, particularly in the TGF-beta pathway, is an effective predictor of outcome for patients with advanced gastric cancer.
    Loss of the Smad3 expression increases susceptibility to tumorigenicity in human gastric cancer. Han Sang-Uk,Kim Heung-Tae,Seong Do Hwan,Kim Yong-Suk,Park Yoon-Soo,Bang Yung-Jue,Yang Han-Kwang,Kim Seong-Jin Oncogene Loss of the tumor suppressive effect of transforming growth factor-beta (TGF-beta) has been commonly found at later stages in carcinogenic progression. Although the genes encoding TGF-beta receptors and Smads have been found genetically altered in certain human cancers, no mutation in Smad3 has been observed. Therefore, suppression of Smad3 expression may mediate key oncogenic properties of TGF-beta. First, we observed that 37.5% of human gastric cancer tissues showed low to undetectable levels of Smad3 and that in nine human gastric cancer cell lines examined, two showed deficient Smad3 expression. Introduction of Smad3 into human gastric cancer cells that did not express Smad3, restored TGF-beta responsiveness: induction of p21 and p15 gene expression, and growth inhibition in response to TGF-beta. Furthermore, these Smad3-expressing cells showed markedly decreased and delayed tumorigenicity in vivo. These findings suggest that Smad3 expression may have a critical role in tumor suppression in the early stages of gastric carcinogenesis. 10.1038/sj.onc.1207259
    Phospho-specific Smad3 signaling: impact on breast oncogenesis. Tarasewicz Elizabeth,Jeruss Jacqueline S Cell cycle (Georgetown, Tex.) Members of the TGFβ superfamily are known to exert a myriad of physiologic and pathologic growth controlling influences on mammary development and oncogenesis. In epithelial cells, TGFβ signaling inhibits cell growth through cytostatic and pro-apoptotic activities but can also induce cancer cell EMT and, thus, has a dichotomous role in breast cancer biology. Mechanisms governing this switch are the subject of active investigation. Smad3 is a critical intracellular mediator of TGFβ signaling regulated through phosphorylation by the TGFβ receptor complex at the C terminus. Smad3 is also a substrate for several other kinases that phosphorylate additional sites within the Smad protein. This discovery has expanded the understanding of the significance and complexity of TGFβ signaling through Smads. This review highlights recent advances revealing the critical role of phospho-specific Smad3 in malignancy and illustrates the potential prognostic and therapeutic impact of Smad3 phospho-isoforms in breast cancer. 10.4161/cc.20546
    Regulation of TGF-beta signaling and its roles in progression of tumors. Miyazono Kohei,Suzuki Hiroyuki,Imamura Takeshi Cancer science Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor of most types of cells; therefore, perturbations of TGF-beta signaling are believed to result in progression of various tumors. On the other hand, TGF-beta has been shown to act as an oncogenic cytokine through induction of extracellular matrices, angiogenesis, and immune suppression. A wide variety of effects of TGF-beta are mediated by physical interaction of signal transducer Smad proteins with various transcription factors. Among these, Runx3 plays a pivotal role in prevention of gastric cancer. TGF-beta signaling is regulated by various mechanisms in the cytoplasm and nucleus. Inhibitory Smads (I-Smads) repress TGF-beta signaling mainly by interacting with activated TGF-beta receptors. Smad ubiquitin regulatory factors (Smurfs) play important roles in facilitating the inhibitory signals induced by I-Smads. In addition, the transcriptional co-repressors c-Ski and SnoN interact with Smads, and repress transcription induced by TGF-beta. Abnormalities of these regulators of TGF-beta signaling may thus participate in the progression of various tumors. 10.1111/j.1349-7006.2003.tb01425.x
    Expression and intracellular localization of Smad proteins in human endometrial cancer. Piestrzeniewicz-Ulanska Dagmara,Brys Magdalena,Semczuk Andrzej,Jakowicki Jerzy A,Krajewska Wanda M Oncology reports The aim of our study was to examine expression of Smad proteins i.e., Smad2, Smad3 and Smad4 both as mRNA and protein as well as their intracellular localization in normal (n=13) and neoplastic (n=42) endometrial tissue specimens using RT-PCR and immunological techniques i.e., Western blot and ELISA. Two uncommon female genital tract tumours, rhabdomyosarcoma of uterine of the cervix and uterine carcinosarcoma were also included. No statistically significant differences were found in the mRNA level of the examined Smad proteins between normal and tumour tissue specimens. Smad2 and Smad3 mRNAs were detected both in uterine carcinosarcoma and rhabdomyosarcoma of the uterine cervix. However, significantly lower Smad2 and Smad4 mRNA level was noted when the depth of myometrial invasion was considered (p<0.05). In endometrial cancer as compared to normal endometrium significantly higher levels of Smad2 and Smad3 proteins, both in cytoplasmic (p=0.002; p=0.0001) and nuclear (p=0.016; p=0.0004) fractions were observed. Both in uterine carcinosarcoma and rhabdomyosarcoma of the uterine cervix Smad2, Smad3 and Smad4 proteins were not detected. Moreover, significantly elevated Smad4 protein level in cytoplasmic fraction was stated when tumour grade and depth of myometrial invasion was undertaken (p<0.05). When intracellular distribution of Smads was considered differences between cytoplasmic and nuclear localization in normal and carcinomatous endometrium was stated. In endometrial cancer decreased number of cases with Smad3 and increased number of cases with Smad4 located in nuclear fraction was found. In conclusion, the disturbances in Smad protein expression and/or differences in their intracellular distribution suggest, that TGF-beta signaling pathway via Smads may be deregulated in endometrial carcinomas.
    Bone morphogenetic protein receptor signal transduction in human disease. Gomez-Puerto Maria Catalina,Iyengar Prasanna Vasudevan,García de Vinuesa Amaya,Ten Dijke Peter,Sanchez-Duffhues Gonzalo The Journal of pathology Bone morphogenetic proteins (BMPs) are secreted cytokines that were initially discovered on the basis of their ability to induce bone. Several decades of research have now established that these proteins function in a large variety of physiopathological processes. There are about 15 BMP family members, which signal via three transmembrane type II receptors and four transmembrane type I receptors. Mechanistically, BMP binding leads to phosphorylation of the type I receptor by the type II receptor. This activated heteromeric complex triggers intracellular signaling that is initiated by phosphorylation of receptor-regulated SMAD1, 5, and 8 (also termed R-SMADs). Activated R-SMADs form heteromeric complexes with SMAD4, which engage in specific transcriptional responses. There is convergence along the signaling pathway and, besides the canonical SMAD pathway, BMP-receptor activation can also induce non-SMAD signaling. Each step in the pathway is fine-tuned by positive and negative regulation and crosstalk with other signaling pathways. For example, ligand bioavailability for the receptor can be regulated by ligand-binding proteins that sequester the ligand from interacting with receptors. Accessory co-receptors, also known as BMP type III receptors, lack intrinsic enzymatic activity but enhance BMP signaling by presenting ligands to receptors. In this review, we discuss the role of BMP receptor signaling and how corruption of this pathway contributes to cardiovascular and musculoskeletal diseases and cancer. We describe pharmacological tools to interrogate the function of BMP receptor signaling in specific biological processes and focus on how these agents can be used as drugs to inhibit or activate the function of the receptor, thereby normalizing dysregulated BMP signaling. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. 10.1002/path.5170
    Smad-binding defective mutant of transforming growth factor beta type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines. Tian Fang,Byfield Stacey DaCosta,Parks W Tony,Stuelten Christina H,Nemani Deepa,Zhang Ying E,Roberts Anita B Cancer research The role of transforming growth factor beta (TGF-beta) in carcinogenesis is complex, with tumor suppressor and pro-oncogenic activities depending on the particular tumor cell and its stage in malignant progression. We previously have demonstrated in breast cancer cell lines that Smad2/3 signaling played a dominant role in mediating tumor suppressor effects on well-differentiated breast cancer cell lines grown as xenografts and prometastatic effects on a more invasive, metastatic cell line. Our present data based on selective interference with activation of endogenous Smad2 and Smad3 by stable expression of a mutant form of the TGF-beta type I receptor (RImL45) unable to bind Smad2/3 but with a functional kinase again show that reduction in Smad2/3 signaling by expression of RImL45 enhanced the malignancy of xenografted tumors of the well-differentiated MCF10A-derived tumor cell line MCF10CA1h, resulting in formation of larger tumors with a higher proliferative index and more malignant histologic features. In contrast, expression of RImL45 in the more aggressive MCF10CA1a cell line strongly suppressed formation of lung metastases following tail vein injection. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in these cells. Using an in vitro assay, we further show that non-Smad signaling pathways, including p38 and c-Jun NH(2)-terminal kinase, cooperate with TGF-beta/Smads in enhancing migration of metastatic MCF10CA1a cells, but that, although necessary for migration, these other pathways are not sufficient for metastasis. 10.1158/0008-5472.CAN-04-0030
    Expression of Smad proteins in human colorectal cancer. Korchynskyi O,Landström M,Stoika R,Funa K,Heldin C H,ten Dijke P,Souchelnytskyi S International journal of cancer Escape from transforming growth factor-beta (TGF-beta)-induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF-beta family members. In this study, we examined the expression of receptor-activated, common-mediator and inhibitory Smads by immunohistochemistry in human colorectal cancers. We found increased expression of receptor-activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor-activated Smads, common-mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Common-mediator Smad4 and inhibitory Smads were detected in cells of both tumor and normal tissues. We observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up-regulation of receptor-activated Smad proteins in human colorectal cancers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts. 10.1002/(sici)1097-0215(19990719)82:2<197::aid-ijc8>3.0.co;2-v
    Disruption of TGFbeta signaling pathways in human pancreatic cancer cells. Simeone D M,Pham T,Logsdon C D Annals of surgery OBJECTIVE:To investigate whether transforming growth factor beta (TGFbeta) signaling is disrupted in human pancreatic cancer cells, and to study the role of TGFbeta receptors and Smad genes. SUMMARY BACKGROUND DATA:TGFbeta is a known inhibitor of pancreatic growth. Disruption of the TGFbeta signaling pathway may play a role in pancreatic cancer development. METHODS:The effect of TGFbeta on the BxPC-3, MiaPaCa-2, and PANC-1 pancreatic cancer cell lines was evaluated by [3H]thymidine incorporation and a TGFbeta-responsive reporter assay. Expression of TGFbeta receptors and Smads 2 and 3 was assessed by cross-linking assays and reverse transcriptase-polymerase chain reaction (RT-PCR). The ability to restore TGFbeta responsiveness was evaluated by transfection of TGFbeta signaling components. RESULTS:TGFbeta produced little inhibition of DNA synthesis and did not activate a TGFbeta-responsive reporter in pancreatic cancer cell lines. 125TGFbeta cross-linking and RT-PCR confirmed the presence of TGFbeta receptors and Smad2 and Smad3 transcripts. Transfection of TGFbeta receptors or Smads 2 and 3 did not restore responsiveness. However, transfection of Smad4 into the BxPC-3 pancreatic cancer cell line restored TGFbeta responsiveness. CONCLUSIONS:Pancreatic cancer cells show loss of TGFbeta responsiveness. Smads 2 and 3 and TGFbeta receptors are not defective in the cell lines studied. Transfection of Smad4 into one of the cell lines restored TGFbeta responsiveness, suggesting an important role for Smad4 in pancreatic cancer. It is likely that other, as yet unidentified genes are important in TGFbeta resistance in pancreatic cancer cells. 10.1097/00000658-200007000-00011
    SMADs: mediators and regulators of TGF-beta signaling. Kretzschmar M,Massagué J Current opinion in genetics & development The discovery of SMAD proteins has allowed the delineation of a mechanism by which TGF-beta and related growth factors convey their signals from membrane receptors all the way into the nucleus. SMADs are directly phosphorylated and activated by the receptors and then form heteromeric SMAD-SMAD complexes that move into the nucleus where they orchestrate transcriptional responses. In rapid succession, recent reports have identified different modes of SMAD regulation by phosphorylation and have defined the SMAD domains that mediate SMAD interactions, binding to DNA or transcriptional activation. The recent discovery of antagonistic SMADs and regulatory crosstalk with Ras/MAP-kinase pathways add to our rapidly expanding understanding of this major regulatory network.
    TGF-beta signaling by Smad proteins. Miyazono K,ten Dijke P,Heldin C H Advances in immunology Members of the transforming growth factor-beta (TGF-beta) family bind to type II and type I serine/threonine kinase receptors, which initiate intracellular signals through activation of Smad proteins. Receptor-regulated Smads (R-Smads) are anchored to the cell membrane by interaction with membrane-bound proteins, including Smad anchor for receptor activation (SARA). Upon ligand stimulation, R-Smads are phosphorylated by the receptors and form oligomeric complexes with common-partner Smads (Co-Smads). The oligomeric Smad complexes then translocate into the nucleus, where they regulate the transcription of target genes by direct binding to DNA, interaction with various DNA-binding proteins, and recruitment of transcriptional coactivators or corepressors. A third class of Smads, inhibitory Smads (I-Smads), inhibits the signals from the serine/threonine kinase receptors. Since the expression of I-Smads is induced by the TGF-beta superfamily proteins, Smads constitute an autoinhibitory signaling pathway. The functions of Smads are regulated by other signaling pathways, such as the MAP kinase pathway. Moreover, Smads interact with and modulate the functions of various transcription factors which are downstream targets of other signaling pathways. Loss of function of certain Smads is involved in tumorigenesis, e.g., pancreatic and colorectal cancers. Analyses by gene targeting revealed pivotal roles of Smads in early embryogenesis, angiogenesis, and immune functions in vivo. 10.1016/s0065-2776(00)75003-6
    TGF-beta signaling, Smads, and tumor suppressors. Padgett R W,Das P,Krishna S BioEssays : news and reviews in molecular, cellular and developmental biology The transforming growth factor-beta (TGF-beta) superfamily is used throughout animal development for regulating the growth and patterning of many tissue types. During the past few years, rapid progress has been made in deciphering how TGF-beta signals are transduced from outside the cell to the nucleus. This progress is based on biochemical studies in vertebrate systems and a combination of genetic studies in Drosophila and Caenorhabditis elegans. These studies have identified a novel family of signaling proteins, the Smad family. Smads can act positively and be phosphorylated by TGF-beta-like receptors or can act negatively and prevent activation of the positively acting group. The positively acting Smads translocate to the nucleus, bind DNA, and act as transcriptional activators. Thus, genetic and biochemical studies suggest a very simple signaling pathway, in which Smads are the primary downstream participant. 10.1002/(SICI)1521-1878(199805)20:5<382::AID-BIES5>3.0.CO;2-Q
    Alterations in components of the TGF-beta superfamily signaling pathways in human cancer. Levy Laurence,Hill Caroline S Cytokine & growth factor reviews Signaling by transforming growth factor-beta (TGF-beta) superfamily ligands to the nucleus is mediated by type I and type II receptors and the intracellular signal transducers, the Smads. Alteration of some of the components of these pathways has been observed in human tumors. These alterations can be deletions or mutations, or downregulation of components that act positively in the pathway, or alternatively, amplification or overexpression of inhibitors of the pathways. The selection of these alterations during tumor progression and their correlation with clinical outcomes, such as survival, risk of recurrence after tumor resection or tendency for metastatic spread, suggest that many are involved in tumor progression. Here, we review the genetic alterations and epigenetic modifications that occur in different components of the TGF-beta superfamily signaling pathways in human tumors and we discuss their correlation with clinical outcome. The evidence suggests that not all alterations of the TGF-beta superfamily signaling pathway components in human cancer have an equivalent effect on tumor progression and we discuss what implications this has for our understanding of the role of TGF-beta signaling in human cancer. 10.1016/j.cytogfr.2005.09.009
    The tumor suppressor Smad4 is required for transforming growth factor beta-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells. Deckers Martine,van Dinther Maarten,Buijs Jeroen,Que Ivo,Löwik Clemens,van der Pluijm Gabri,ten Dijke Peter Cancer research Transforming growth factor beta (TGF-beta) can act as suppressor and promoter of cancer progression. Intracellular Smad proteins (i.e., receptor regulated Smads and common mediator Smad4) play a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-beta, but their function in TGF-beta-induced invasion and metastasis is unclear. Here, we have investigated the role of Smad4 in a cellular and mouse model for TGF-beta-induced breast cancer progression. Consistent with its tumor suppressor function, specific silencing of Smad4 in NMuMG mammary gland epithelial cells using small hairpin RNA (shRNA)-expressing RNAi vectors strongly mitigated TGF-beta-induced growth inhibition and apoptosis. Smad4 knockdown also potently inhibited TGF-beta-induced epithelial to mesenchymal transition of NMuMG cells as measured by morphologic transformation from epithelial to fibroblast-like cells, formation of stress fibers, inhibition of E-cadherin expression, and gain of expression of various mesenchymal markers. Furthermore, we show that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival. Communication of MDA-MB-231 cells with the bone microenvironment, which is needed for optimal tumor cell growth and metastasis, may be affected in Smad4 knockdown cells as TGF-beta-induced expression of interleukin 11 was attenuated on Smad4 knockdown. Taken together, our results show that Smad4 plays an important role in both tumor suppression and progression of breast cancer cells. 10.1158/0008-5472.CAN-05-3560
    14-3-3zeta Cooperates with ErbB2 to promote ductal carcinoma in situ progression to invasive breast cancer by inducing epithelial-mesenchymal transition. Lu Jing,Guo Hua,Treekitkarnmongkol Warapen,Li Ping,Zhang Jian,Shi Bin,Ling Chen,Zhou Xiaoyan,Chen Tongzhen,Chiao Paul J,Feng Xinhua,Seewaldt Victoria L,Muller William J,Sahin Aysegul,Hung Mien-Chie,Yu Dihua Cancer cell ErbB2, a metastasis-promoting oncoprotein, is overexpressed in approximately 25% of invasive/metastatic breast cancers, but in 50%-60% of noninvasive ductal carcinomas in situ (DCIS). It has been puzzling how a subset of ErbB2-overexpressing DCIS develops into invasive breast cancer (IBC). We found that co-overexpression of 14-3-3zeta in ErbB2-overexpressing DCIS conferred a higher risk of progression to IBC. ErbB2 and 14-3-3zeta overexpression, respectively, increased cell migration and decreased cell adhesion, two prerequisites of tumor cell invasion. 14-3-3zeta overexpression reduced cell adhesion by activating the TGF-beta/Smads pathway that led to ZFHX1B/SIP-1 upregulation, E-cadherin loss, and epithelial-mesenchymal transition. Importantly, patients whose breast tumors overexpressed both ErbB2 and 14-3-3zeta had higher rates of metastatic recurrence and death than those whose tumors overexpressed only one. 10.1016/j.ccr.2009.08.010
    Signalling pathways involved in endocrine resistance in breast cancer and associations with epithelial to mesenchymal transition (Review). Al Saleh Sanaa,Sharaf Leyla H,Luqmani Yunus A International journal of oncology Both de novo and acquired endocrine resistance constitute a major therapeutic problem for treatment of hormone-positive breast cancer. Multiple explanatory mechanisms have been proposed through the study of cellular models which focus principally on receptor tyrosine kinase mediated signalling pathways utilizing src, PI3K, MAPK and Smads. Many of the transducing molecules, particularly nuclear transcription factors such as Snail, Twist, Snail2, ZEB, FOXC2, TCF/LEF and Goosecoid are participants in proliferation as well as invasion and metastasis, involving a process of orchestrated cellular remodeling which is being likened to the process of epithelial to mesenchymal transition that takes place during embryonic development. We review the accumulating evidence that points towards the occurrence of this phenomenon as a consequence of the loss of endocrine control, with both processes being similarly characterized by depletion of cell adhesion proteins, E-cadherin, catenins and cytokeratins, increased association with the extracellular matrix through induction of metalloproteinases, fibronectin and collagen, and a switch to a mobile vimentin-based cytoskeletal structure with loss of apical basal polarity. 10.3892/ijo.2011.942
    Bone morphogenetic protein signaling and growth suppression in colon cancer. Beck Stayce E,Jung Barbara H,Fiorino Antonio,Gomez Jessica,Rosario Eunice Del,Cabrera Betty L,Huang Sherry C,Chow Jimmy Y C,Carethers John M American journal of physiology. Gastrointestinal and liver physiology Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily, which utilize BMP receptors and intracellular SMADs to transduce their signals to regulate cell differentiation, proliferation, and apoptosis. Because mutations in BMP receptor type IA (BMPRIA) and SMAD4 are found in the germline of patients with the colon cancer predisposition syndrome juvenile polyposis, and because the contribution of BMP in colon cancers is largely unknown, we examined colon cancer cells and tissues for evidence of BMP signaling and determined its growth effects. We determined the presence and functionality of BMPR1A by examining BMP-induced phosphorylation and nuclear translocation of SMAD1; transcriptional activity via a BMP-specific luciferase reporter; and growth characteristics by cell cycle analysis, cell growth, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolic assays. These assays were also performed after transfection with a dominant negative (DN) BMPR1A construct. In SMAD4-null SW480 cells, we examined BMP effects on cellular wound assays as well as BMP-induced transcription in the presence of transfected SMAD4. We also determined the expression of BMPR1A, BMP ligands, and phospho-SMAD1 in primary human colon cancer specimens. We found intact BMP signaling and modest growth suppression in HCT116 and two derivative cell lines and, surprisingly, growth suppression in SMAD4-null SW480 cells. BMP-induced SMAD signaling and BMPR1A-mediated growth suppression were reversed with DN BMPR1A transfection. BMP2 slowed wound closure, and transfection of SMAD4 into SW480 cells did not change BMP-specific transcriptional activity over controls due to receptor stimulation by endogenously produced ligand. We found no cell cycle alterations with BMP treatment in the HCT116 and derivative cell lines, but there was an increased G1 fraction in SW480 cells that was not due to increased p21 transcription. In human colon cancer specimens, BMP2 and BMP7 ligands, BMPRIA, and phospho-SMAD1 were expressed. In conclusion, BMP signaling is intact and growth suppressive in human colon cancer cells. In addition to SMADs, BMP may utilize SMAD4-independent pathways for growth suppression in colon cancers. 10.1152/ajpgi.00482.2005
    Crosstalk of TGF-β and estrogen receptor signaling in breast cancer. Band Arja M,Laiho Marikki Journal of mammary gland biology and neoplasia Estrogen receptor-α (ERα) and transforming growth factor (TGF)-β signaling pathways are major regulators during mammary gland development, function and tumorigenesis. Predominantly, they have opposing roles in proliferation and apoptosis. While ERα signaling supports growth and differentiation and is antiapoptotic, mammary gland epithelia cells are very sensitive to TGF-β-induced cell cycle arrest and apoptosis. Their regulatory pathways intersect, and ERα blocks TGF-β pathway by multiple means, including direct interactions of its signaling components, Smads. However, relatively little is known of the dysfunction of their interactions in cancer. A better understanding would help to develop new strategies for breast cancer treatment. 10.1007/s10911-011-9203-7
    Switching Roles of TGF-β in Cancer Development: Implications for Therapeutic Target and Biomarker Studies. Sun Nan,Taguchi Ayumu,Hanash Samir Journal of clinical medicine TGF-β induces complicated and even opposite responses in numerous biological processes, e.g., tumor suppression in pre-malignant cells and metastasis promotion in cancer cells. However, the cellular contextual determinants of these different TGF-β roles remain elusive, and the driver genes triggering the determinants' changes have not been identified. Recently, however, several findings have provided new insights on the contextual determinants of Smads in TGF-β's biological processes. These novel switches and their effectors may serve as prognostic biomarkers and therapeutic targets of TGF-β-mediated cancer progression. 10.3390/jcm5120109
    BMP-6 over-expression in prostate cancer is associated with increased Id-1 protein and a more invasive phenotype. Darby S,Cross S S,Brown N J,Hamdy F C,Robson C N The Journal of pathology Bone morphogenetic protein-6 (BMP-6) has been strongly implicated in prostate cancer development and bone metastasis. Our previous data showed that BMP-6 mRNA was absent in patients with benign prostatic hyperplasia, but evident in primary tumours with established secondary skeletal metastases. To examine the role of BMP-6 in prostate cancer progression, we have developed a BMP-6-regulatable, doxycycline-inducible gene expression system. BMP-6 induction by doxycycline addition led to increased levels of BMP-6 RNA and protein, associated with nuclear translocation of SMADs and activation of the downstream target gene Id-1. BMP-6 protein did not enhance the proliferation rate of PC3M cells but did significantly increase the rate of migration and invasion in both PC3M and DU145 cells. Increased metalloproteinase (MMP-1 and MMP-9) mRNA levels were also observed following BMP-6 induction. Luciferase reporter assays confirmed BMP-6-mediated activation of MMP-1 and MMP-9 promoters, indicating direct transcriptional activation of MMPs by BMP-6. BMP-6 stimulation also led to an increase in phosphorylation levels of MAPK proteins. We next examined the effects of BMP-6 on the downstream gene Id-1 in a cohort of prostate cancer patients. A tissue microarray (TMA) was constructed and samples stained for BMP-6 and Id-1 expression. We observed a significant increase in the intensity of staining of epithelial BMP-6 in the cancer cases compared to the benign cases (Mann-Whitney U test, p < 0.0005) and in the intensity of staining of epithelial Id-1 in the cancer cases compared to the benign cases (Mann-Whitney U test, p = 0.015). We further observed a significant positive correlation between epithelial staining for Id-1 and BMP-6 (p = 0.001) across all samples for both benign and cancer cases. These data demonstrate that BMP-6 promotes migration and invasion of prostate cancer cells, potentially through activation of Id-1 and MMP activation. 10.1002/path.2292
    Transforming growth factor β signaling pathway: A promising therapeutic target for cancer. Chen Yuhong,Di Cuixia,Zhang Xuetian,Wang Jing,Wang Fang,Yan Jun-Fang,Xu Caipeng,Zhang Jinhua,Zhang Qianjing,Li Hongyan,Yang Hongying,Zhang Hong Journal of cellular physiology Transforming growth factor β (TGF-β) is part of the transforming growth factor β superfamily which is involved in many physiological processes and closely related to the carcinogenesis. Here, we discuss the TGF-β structure, function, and its canonical Smads signaling pathway. Importantly, TGF-β has been proved that it plays both tumor suppressor as well as an activator role in tumor progression. In an early stage, TGF-β inhibits cell proliferation and is involved in cell apoptosis. In an advanced tumor, TGF-β signaling pathway induces tumor invasion and metastasis through promoting angiogenesis, epithelial-mesenchymal transition, and immune escape. Furthermore, we are centered on updated research results into the inhibitors as drugs which have been studied in preclinical or clinical trials in tumor carcinogenesis to prevent the TGF-β synthesis and block its signaling pathways such as antibodies, antisense molecules, and small-molecule tyrosine kinase inhibitors. Thus, it is highlighting the crucial role of TGF-β in tumor therapy and may provide opportunities for the new antitumor strategies in patients with cancer. 10.1002/jcp.29108
    Receptor-regulated Smads in TGF-beta signaling. Liu Fang Frontiers in bioscience : a journal and virtual library Transforming growth factor beta (TGF-beta) and related polypeptides, including activins and bone morphogenetic proteins (BMPs), constitute the largest cytokine family, possessing fascinating features. TGF-beta and related peptides are multifunctional growth factors and they regulate many aspects of cellular processes such as proliferation, differentiation, adhesion and apoptosis. These evolutionarily conserved cytokines play an essential role in the development and homeostasis of virtually every tissue in organisms ranging from fruit flies to humans. Accordingly, inactivating mutations in several components of the TGF-beta signaling pathways have been found to cause a number of human disorders. The TGF-beta family members signal through cell surface serine/threonine kinase receptors. A family of proteins, designated as Smads (mammalian homologues of Drosophila Mad and C. elegans Sma), transduces the TGF-beta signal from cell surface to the nucleus. Upon activation, the TGF-beta type I receptor phosphorylates Smad2 and Smad3, which then form complexes with Smad4 and accumulate in the nucleus to regulate transcription of a variety of genes that encode crucial determinants of cell fate, such as cell cycle components, differentiation factors and cell adhesion molecules. Although Smad2 and Smad3 are highly homologous and share some overlapping activities, they have distinct functions and are regulated differentially. This review is primarily focused on our understanding of the similar as well as distinct function and regulation of Smad2 and Smad3 in TGF-beta signaling, their physiological roles revealed by knockout studies and their tumor suppressive functions.
    Transforming growth factor-beta signaling in cancer invasion and metastasis. Leivonen Suvi-Katri,Kähäri Veli-Matti International journal of cancer Transforming growth factor-beta (TGF-beta) family members are polypeptides with dual tumor suppressive and oncogenic effects. They signal through serine/threonine kinase receptor complexes, which phosphorylate cytoplasmic mediators, the Smads. Upon phosphorylation, Smads translocate to the nucleus and associate with transcriptional coactivators or corepressors, and regulate the transcriptional activation of various TGF-beta responsive genes. In addition, TGF-beta activates cellular mitogen-activated protein kinase signaling pathways, which crosstalk with Smad signaling and regulate growth, survival and motility of cells. During tumorigenesis, malignantly transformed cells often lose the response to the tumor suppressive effects of TGF-beta, which, in turn, starts to act as an autocrine tumor promoting factor by enhancing cancer invasion and metastasis. In this review, we summarize current view on the role of TGF-beta signaling in tumorigenesis, with emphasis on cancer invasion and metastasis. On the basis of these recent observations, we discuss new therapeutic strategies targeting TGF-beta signaling at distinct levels as a basis for inhibiting tumor growth, angiogenesis, invasion and metastasis. 10.1002/ijc.23113
    A New Switch for TGFβ in Cancer. Yeh Hsi-Wen,Lee Szu-Shuo,Chang Chieh-Yu,Lang Yaw-Dong,Jou Yuh-Shan Cancer research The TGFβ cytokine plays dichotomous roles during tumor progression. In normal and premalignant cancer cells, the TGFβ signaling pathway inhibits proliferation and promotes cell-cycle arrest and apoptosis. However, the activation of this pathway in late-stage cancer cells could facilitate the epithelial-to-mesenchymal transition, stemness, and mobile features to enhance tumorigenesis and metastasis. The opposite functions of TGFβ signaling during tumor progression make it a challenging target to develop anticancer interventions. Nevertheless, the recent discovery of cellular contextual determinants, especially the binding partners of the transcription modulators Smads, is critical to switch TGFβ responses from proapoptosis to prometastasis. In this review, we summarize the recently identified contextual determinants (such as PSPC1, KLF5, 14-3-3ζ, C/EBPβ, and others) and the mechanisms of how tumor cells manage the context-dependent autonomous TGFβ responses to potentiate tumor progression. With the altered expression of some contextual determinants and their effectors during tumor progression, the aberrant molecular prometastatic switch might serve as a new class of theranostic targets for developing anticancer strategies. 10.1158/0008-5472.CAN-18-2019
    Antimetastatic role of Smad4 signaling in colorectal cancer. Zhang Bixiang,Halder Sunil K,Kashikar Nilesh D,Cho Yong-Jig,Datta Arunima,Gorden D Lee,Datta Pran K Gastroenterology BACKGROUND & AIMS:Transforming growth factor (TGF)-beta signaling occurs through Smads 2/3/4, which translocate to the nucleus to regulate transcription; TGF-beta has tumor-suppressive effects in some tumor models and pro-metastatic effects in others. In patients with colorectal cancer (CRC), mutations or reduced levels of Smad4 have been correlated with reduced survival. However, the function of Smad signaling and the effects of TGF-beta-receptor kinase inhibitors have not been analyzed during CRC metastasis. We investigated the role of TGF-beta/Smad signaling in CRC progression. METHODS:We evaluated the role of TGF-beta/Smad signaling on cell proliferation, migration, invasion, tumorigenicity, and metastasis in Smad4-null colon carcinoma cell lines (MC38 and SW620) and in those that transgenically express Smad4. We also determined the effects of a TGF-beta-receptor kinase inhibitor (LY2109761) in CRC tumor progression and metastasis in mice. RESULTS:TGF-beta induced migration/invasion, tumorigenicity, and metastasis of Smad4-null MC38 and SW620 cells; incubation with LY2109761 reversed these effects. In mice, LY2109761 blocked metastasis of CRC cells to liver, inducing cancer cell expression of E-cadherin and reducing the expression of the tumorigenic proteins matrix metalloproteinase-9, nm23, urokinase plasminogen activator, and cyclooxygenase-2. Transgenic expression of Smad4 significantly reduced the oncogenic potential of MC38 and SW620 cells; in these transgenic cells, TGF-beta had tumor suppressor, rather than tumorigenic, effects. CONCLUSIONS:TGF-beta/Smad signaling suppresses progression and metastasis of CRC cells and tumors in mice. Loss of Smad4 might underlie the functional shift of TGF-beta from a tumor suppressor to a tumor promoter; inhibitors of TGF-beta signaling might be developed as CRC therapeutics. 10.1053/j.gastro.2009.11.004
    Prognostic Impact of Canonical TGF-β Signaling in Urothelial Bladder Cancer. Stojnev Slavica,Krstić Miljan,Čukuranović Kokoris Jovana,Conić Irena,Petković Ivan,Ilić Sonja,Milosević-Stevanović Jelena,Veličković Ljubinka Janković Medicina (Kaunas, Lithuania) : Dysregulation of TGF-β signaling plays multiple roles in cancer development and progression. In the canonical TGF-β pathway, TGF-β regulates the expression of hundreds of target genes via interaction with Smads, signal transducers and transcriptional modulators. We evaluated the association of TGF-β1, Smad2, and Smad4, the key components of canonical TGFβ pathway, with clinicopathologic characteristics of urothelial bladder cancer, and assessed their prognostic value in prediction of patients' outcome. : Immunohistochemical analysis of TGF-β1, Smad2, and Smad4 expression was performed on 404 urothelial bladder cancer samples, incorporated in tissue microarrays. Expression status was correlated with clinicopathological and follow-up data. The median follow-up was 61 months. : High expression of TGF-β1, Smad2, and Smad4 was detected in 68.1%, 31.7% and 45.2% of the tumors, respectively. TGF-β1 overexpression was significantly associated with high tumor grade, and advanced pathologic stage ( < 0.001, respectively). Conversely, high Smad2 and Smad4 expression was linked to low tumor grade ( = 0,003, = 0.048, respectively), and low tumor stage ( < 0.001, = 0.003, respectively). Smad2 showed an inverse correlation with variant morphology and divergent differentiation of urothelial tumors ( = 0.014). High TGF-β1 correlated directly, while Smad2 and Smad4 correlated inversely to cancer-specific death ( = 0.043, = 0.003, and = 0.022, respectively). There was a strong relationship between Smad2 and Smad4 expression ( < 0.001). Survival analyses showed that high Smad2 and Smad4 expression was associated with longer overall survival ( = 0.003, = 0.034, respectively), while in multivariate regression analysis TGF-β1 manifested as an independent predictor of poor outcome. : Unraveling the complex roles and significance of TGF-β signaling in urothelial bladder cancer might have important implications for therapy of this disease. Assessment of TGF-β pathway status in patients with urothelial bladder cancer may provide useful prognostic information, and identify patients that could have the most benefit from therapy targeting TGF-β signaling cascade. 10.3390/medicina55060302
    Targeting the Transforming Growth Factor-beta pathway inhibits human basal-like breast cancer metastasis. Ganapathy Vidya,Ge Rongrong,Grazioli Alison,Xie Wen,Banach-Petrosky Whitney,Kang Yibin,Lonning Scott,McPherson John,Yingling Jonathan M,Biswas Swati,Mundy Gregory R,Reiss Michael Molecular cancer BACKGROUND:Transforming Growth Factor beta (TGF-beta) plays an important role in tumor invasion and metastasis. We set out to investigate the possible clinical utility of TGF-beta antagonists in a human metastatic basal-like breast cancer model. We examined the effects of two types of the TGF-beta pathway antagonists (1D11, a mouse monoclonal pan-TGF-beta neutralizing antibody and LY2109761, a chemical inhibitor of TGF-beta type I and II receptor kinases) on sublines of basal cell-like MDA-MB-231 human breast carcinoma cells that preferentially metastasize to lungs (4175TR, 4173) or bones (SCP2TR, SCP25TR, 2860TR, 3847TR). RESULTS:Both 1D11 and LY2109761 effectively blocked TGF-beta-induced phosphorylation of receptor-associated Smads in all MDA-MB-231 subclones in vitro. Moreover, both antagonists inhibited TGF-beta stimulated in vitro migration and invasiveness of MDA-MB-231 subclones, indicating that these processes are partly driven by TGF-beta. In addition, both antagonists significantly reduced the metastatic burden to either lungs or bones in vivo, seemingly independently of intrinsic differences between the individual tumor cell clones. Besides inhibiting metastasis in a tumor cell autonomous manner, the TGF-beta antagonists inhibited angiogenesis associated with lung metastases and osteoclast number and activity associated with lytic bone metastases. In aggregate, these studies support the notion that TGF-beta plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-beta signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-beta pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer. CONCLUSIONS:In aggregate, these studies support the notion that TGF-beta plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-beta signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-beta pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer. 10.1186/1476-4598-9-122
    Cyclin-dependent kinases regulate the antiproliferative function of Smads. Matsuura Isao,Denissova Natalia G,Wang Guannan,He Dongming,Long Jianyin,Liu Fang Nature Transforming growth factor-beta (TGF-beta) potently inhibits cell cycle progression at the G1 phase. Smad3 has a key function in mediating the TGF-beta growth-inhibitory response. Here we show that Smad3 is a major physiological substrate of the G1 cyclin-dependent kinases CDK4 and CDK2. Except for the retinoblastoma protein family, Smad3 is the only CDK4 substrate demonstrated so far. We have mapped CDK4 and CDK2 phosphorylation sites to Thr 8, Thr 178 and Ser 212 in Smad3. Mutation of the CDK phosphorylation sites increases Smad3 transcriptional activity, leading to higher expression of the CDK inhibitor p15. Mutation of the CDK phosphorylation sites of Smad3 also increases its ability to downregulate the expression of c-myc. Using Smad3(-/-) mouse embryonic fibroblasts and other epithelial cell lines, we further show that Smad3 inhibits cell cycle progression from G1 to S phase and that mutation of the CDK phosphorylation sites in Smad3 increases this ability. Taken together, these findings indicate that CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. Because cancer cells often contain high levels of CDK activity, diminishing Smad3 activity by CDK phosphorylation may contribute to tumorigenesis and TGF-beta resistance in cancers. 10.1038/nature02650
    Expression profile of agonistic Smads in human breast cancer cells: absence of regulation by estrogens. Pouliot F,Labrie C International journal of cancer Transforming growth factor-beta1 (TGF-beta1) is a cytokine expressed by mammary cells. While TGF-beta1 can inhibit the proliferation of human breast cancer cells, many cell lines are unresponsive to it. To shed light on the mechanisms underlying resistance to TGF-beta1, we examined expression of the mediators of TGF-beta1 signaling in the mammary carcinoma cell lines MCF-7, T47D, ZR-75-1, BT-20, MDA-MB-231 and MDA-MB-468. The levels of mRNA encoding Smad2, 3 and 4 as well as the type II (TbetaRII) and type I (TbetaRI) membrane receptors were determined by Northern analysis and/or ribonuclease protection assays. Smad2 and Smad3 mRNAs were detected in all 6 cell lines examined, whereas Smad4 mRNA was not detected in MDA-MB-468 cells, which are known to harbor a homozygous deletion of the Smad4 gene. TbetaRI was expressed in all 6 cell lines, whereas TbetaRII was not detected in ZR-75-1 and T47D cells. Of the cell lines tested, only MCF-7 cells were growth-inhibited by TGF-beta1. In contrast, only MDA-MB-231 cells showed induction of the PAI-1 promotor in response to TGF-beta1. We also examined the regulation of Smad mRNA expression by estrogens and androgens in ZR-75-1 cells. Neither estradiol nor dihydrotestosterone affected Smad2, 3 or 4 mRNA levels in ZR-75-1 cells. These results indicate that the lack of response to TGF-beta1 in the breast cancer cell lines examined can be attributed to the absence of either TbetaRII or the Smad4 gene product. Moreover, we show that the proliferative and transcriptional responses to TGF-beta1 are dissociable and that Smad expression is not regulated by sex steroids in ZR-75-1 cells. 10.1002/(sici)1097-0215(19990331)81:1<98::aid-ijc17>3.0.co;2-9
    Transforming growth factor-β1 attenuates junctional adhesion molecule-A and contributes to breast cancer cell invasion. Wang Yang,Lui Wing-Yee European journal of cancer (Oxford, England : 1990) Transforming growth factor-β1 (TGF-β1) is a potent regulator in promoting the invasion and proliferation of breast cancer cells. Junctional adhesion molecule-A (JAM-A) is a tight junction protein that displays an inverse relationship to cell invasiveness in breast cancer cells. Whether TGF-β1 signaling induces alteration of JAM-A expression leading to cell invasion has not been investigated. In this study, we report that TGF-β1 down-regulated JAM-A expression via its effect on both transcriptional and post-translational regulations of JAM-A, thus inducing cell invasion. On exploring whether TGF-β1 might be the upstream regulator of JAM-A expression, we found that knockdown of TGF-β receptors and canonical Smad signaling could upregulate JAM-A level and inhibit cell invasion in MDA-MB-231 cells. TGF-β1 treatment of MCF-7 cells caused a significant reduction of JAM-A mRNA and protein and induced cell invasion. Delineating the signal mechanisms involved in TGF-β1-mediated JAM-A repression, we found that TGF-β1 significantly inhibited JAM-A gene transcription via the activation of Smads. In addition to Smad activation, we found that involvement of p54 JNK is crucial for post-translational modification of TGF-β1-mediated JAM-A protein degradation. Blockage of JNK pathway by inhibitor could attenuate TGF-β1-induced cell invasion. We provide evidences for the first time that TGF-β1 induces breast cancer cell invasion via TGF-β1-mediated control on JAM-A expression. Identification of JAM-A as a downstream target of TGF-β1 represents a crucial mechanism in cancer progression. 10.1016/j.ejca.2012.04.016
    Keeping neuroendocrine cells in check: roles for TGFbeta, Smads, and menin? Dockray G J Gut 10.1136/gut.52.9.1237
    Turning off Smads: identification of a Smad phosphatase. Hill Caroline S Developmental cell Although the activation of Smad signaling pathways downstream of TGF-beta superfamily ligands by receptor-mediated Smad phosphorylation is well understood, nothing is known about Smad phosphatases that turn off Smad activity. Recently in Genes & Development, describe pyruvate dehydrogenase phosphatase as a Smad1 phosphatase that functions in Drosophila in the Decapentaplegic pathway and in mammalian cells in the BMP pathway. 10.1016/j.devcel.2006.03.008
    A phosphatase controls the fate of receptor-regulated Smads. Schilling Stephen H,Datto Michael B,Wang Xiao-Fan Cell In this issue of Cell, Lin et al. (2006) answer one of the long-standing questions in the TGFbeta field by identifying a phosphatase, PPM1A, that directly dephosphorylates Smad2 and Smad3 to limit their activation. 10.1016/j.cell.2006.05.015
    SMAD4 and its role in pancreatic cancer. Xia Xiang,Wu Weidong,Huang Chen,Cen Gang,Jiang Tao,Cao Jun,Huang Kejian,Qiu Zhengjun Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine Transforming growth factor-β (TGF-β) regulates cell functions and has key roles in pancreatic cancer development. SMAD4, as one of the Smads family of signal transducer from TGF-β, mediates pancreatic cell proliferation and apoptosis and is specifically inactivated in half of advanced pancreatic cancers. In recent years, many advances concerning SMAD4 had tried to unravel the complex signaling mechanisms of TGF-β and its dual role of tumor-suppressive and tumor-promoting efforts in pancreatic cancer initiation and progression through SMAD4-dependent TGF-β signaling and SMAD4-independent TGF-β signaling pathways. Meanwhile, its potential prognostic value based on immunohistochemical expression in surgical sample was variably reported by several studies and short of a systematic analysis. This review aimed to discuss the structure, functions, and regulation of this principal protein and its effects in determining the progression and prognosis of pancreatic cancer. 10.1007/s13277-014-2883-z
    Smads mediate signaling of the TGFbeta superfamily in normal keratinocytes but are lost during skin chemical carcinogenesis. He W,Cao T,Smith D A,Myers T E,Wang X J Oncogene The Smads are the signaling mediators of the TGFbeta superfamily. In the present study, we examined Smad expression in mouse epidermis and chemically-induced skin tumors. Mutations in Smad2 and -4 genes were also screened. Transcripts of Smad1 through -5 were constantly expressed in the epidermis regardless of changes in TGFbeta signaling, state of differentiation and stages of carcinogenesis. Smad7 transcripts were barely detectable in keratinocytes, but were induced by TGFbeta1 treatment and in chemically-induced skin tumors. At the protein level, Smad1 was detected throughout the epidermis, whereas Smad2 through -5 exhibited greater levels in suprabasal layers than basal keratinocytes. In cultured keratinocytes, Smad2, -3 and -4 underwent nuclear translocation upon TGFbeta1 treatment. Furthermore, nuclear translocation of Smads correlated with decreased BrdU labeling in proliferative keratinocytes. Although no mutations were detected in the Smad2 and -4 genes in tumors, proteins of Smad1 through -5 were partially or completely lost in carcinomas. These data document that Smads are expressed at high levels in the epidermis and mediate signaling of the TGFbeta superfamily. During skin carcinogenesis, loss of Smad1 through -5 and overexpression of Smad7 may contribute to the loss of growth inhibition mediated by TGFbeta superfamily members, thus resulting in tumor progression. 10.1038/sj.onc.1204117
    MicroRNA-21-5p promotes proliferation of gastric cancer cells through targeting SMAD7. Jiang Yinan,Zhang Meiling,Guo Tangxi,Yang Chaogang,Zhang Chunxiao,Hao Jinjin OncoTargets and therapy Background:MicroRNAs could target multiple genes by regulating the translation or degradation of mRNAs, and are involved in functions such as signal transduction pathways affecting the physiological functions of normal or tumor cells. Methods:In this study, the expressions of miRNA-21-5p in gastric cancer tissues and SGC-7901 cells were analyzed, and the effects of miRNA-21-5p on cell proliferation, migration, invasion, and apoptosis and the expressions of target proteins SMADs in SGC-7901 cells were evaluated. Inflammatory factors interleukin 1β and tumor necrosis factor α in siRNA-transfected SGC-7901 cells were determined by enzyme-linked immunosorbent assay. Results:MiRNA-21-5p was consistently upregulated in gastric cancer tissues and SGC-7901 cells compared to normal tissues or cells. The knockdown of miRNA-21-5p with antisense oligonucleotides suppressed cell proliferation, migration, and invasion as well as inflammatory response, and promoted cell apoptosis and SMAD7 expression. Conclusion:These results indicate SMAD7 may mediate the oncogenic properties of miRNA-21-5p in gastric cancer, and miRNA-21-5p would be a promising strategy for the treatment of gastric cancer. 10.2147/OTT.S163771
    TGF-β-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancer. Yang Haiping,Wang Longqiang,Zhao Jun,Chen Yongbing,Lei Zhe,Liu Xia,Xia Wei,Guo Lingling,Zhang Hong-Tao Lung cancer (Amsterdam, Netherlands) OBJECTIVES:Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation. MATERIALS AND METHODS:Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected. RESULTS:Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively. CONCLUSIONS:We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs. 10.1016/j.lungcan.2014.12.015
    Epigenetic regulation of cancer stem cell marker CD133 by transforming growth factor-beta. You Hanning,Ding Wei,Rountree C Bart Hepatology (Baltimore, Md.) UNLABELLED:Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. CD133, a transmembrane glycoprotein, is an important cell surface marker for both stem cells and cancer stem cells in various tissues including liver. CD133 expression has been recently linked to poor prognosis in HCC patients. CD133+ liver cancer cells are characterized by resistance to chemotherapy, self-renewal, multilineage potential, increased colony formation, and in vivo cancer initiation at limited dilution. Recent studies demonstrate that CD133 expression is regulated by DNA methylation. In this study, we explored the role of transforming growth factor beta (TGFbeta), a multifunctional cytokine that plays a critical role in chronic liver injury, in the regulation of CD133 expression. TGFbeta1 is capable of up-regulating CD133 expression specifically within the Huh7 HCC cell line in a time- and dose-dependent manner. Most important, TGFbeta1-induced CD133+ Huh7 cells demonstrate increased tumor initiation in vivo. Forced expression of inhibitory Smads, including Smad6 and Smad7, attenuated TGFbeta1-induced CD133 expression. Within CD133- Huh7 cells, TGFbeta1 stimulation inhibited the expression of DNA methyltransferases (DNMT) 1 and DNMT3beta, which are critical in the maintenance of regional DNA methylation, and global DNMT activity in CD133- Huh7 cells was inhibited by TGFbeta1. DNMT3beta inhibition by TGFbeta1 was partially rescued with overexpression of inhibitory Smads. Lastly, TGFbeta1 treatment led to significant demethylation in CD133 promoter-1 in CD133- Huh7 cells. CONCLUSION:TGFbeta1 is able to regulate CD133 expression through inhibition of DNMT1 and DNMT3beta expression and subsequent demethylation of promoter-1. TGFbeta1-induced CD133+ Huh7 cells are tumorigenic. The mechanism by which TGFbeta induces CD133 expression is partially dependent on the Smads pathway. 10.1002/hep.23544
    Regulatory mechanisms for transforming growth factor beta as an autocrine inhibitor in human hepatocellular carcinoma: implications for roles of smads in its growth. Matsuzaki K,Date M,Furukawa F,Tahashi Y,Matsushita M,Sugano Y,Yamashiki N,Nakagawa T,Seki T,Nishizawa M,Fujisawa J,Inoue K Hepatology (Baltimore, Md.) Transforming growth factor beta (TGF-beta) initiates signaling through heteromeric complexes of transmembrane type I and type II serine/threonine kinase receptors. Activated TGF-beta type I receptor phosphorylates receptor-regulated Smads (2 and 3). Antagonistic Smad 7 forms stable association with the activated TGF-beta type I receptor, blocking phosphorylation of receptor-regulated Smads. On the other hand, elevated serum concentration of TGF-beta along with resistance to its growth-inhibitory effect is commonly observed in human hepatocellular carcinoma (HCC) patients. In this study, we investigated the mechanisms of resistance to tumor-derived TGF-beta in human HCC and hepatoblastoma-derived cell lines, focusing on the roles of receptor-regulated Smads and antagonistic Smad 7. HuH-7 and HepG2 cells showed poor response to TGF-beta-mediated growth inhibition. Because neutralization of TGF-beta in the medium or blockage of signal transduction pathway by inductions of dominant negative Smad 2/3 resulted in a stimulation of cell growth, tumor-derived TGF-beta signal acts on cell growth negatively. However, Smad 7 induced by TGF-beta negatively regulated Smad 2 action and rendered most Smad 2 proteins in the cytoplasm. Taken together, these results indicate that endogenous TGF-beta-mediated induction of Smad 7 results in a higher "threshold" for the antiproliferative signals mediated by receptor-regulated Smads, and can be involved in reduced responsiveness to the cytokine in some human HCC cells. 10.1053/jhep.2000.9145
    Identification of germline alterations of the mad homology 2 domain of SMAD3 and SMAD4 from the Ontario site of the breast cancer family registry (CFR). Tram Eric,Ibrahim-Zada Irada,Briollais Laurent,Knight Julia A,Andrulis Irene L,Ozcelik Hilmi Breast cancer research : BCR INTRODUCTION:A common feature of neoplastic cells is that mutations in SMADs can contribute to the loss of sensitivity to the anti-tumor effects of transforming growth factor-β (TGF-β). However, germline mutation analysis of SMAD3 and SMAD4, the principle substrates of the TGF-β signaling pathway, has not yet been conducted in breast cancer. Thus, it is currently unknown whether germline SMAD3 and SMAD4 mutations are involved in breast cancer predisposition. METHODS:We performed mutation analysis of the highly conserved mad-homology 2 (MH2) domains for both genes in genomic DNA from 408 non-BRCA1/BRCA2 breast cancer cases and 710 population controls recruited by the Ontario site of the breast cancer family registry (CFR) using denaturing high-performance liquid chromatography (DHPLC) and direct DNA sequencing. The results were interpreted in several ways. First, we adapted nucleotide diversity analysis to quantitatively assess whether the frequency of alterations differ between the two genes. Next, in silico tools were used to predict variants' effect on domain function and mRNA splicing. Finally, 37 cases or controls harboring alterations were tested for aberrant splicing using reverse-transcription polymerase chain reaction (PCR) and real-time PCR statistical comparison of germline expressions by non-parametric Mann-Whitney test of independent samples. RESULTS:We identified 27 variants including 2 novel SMAD4 coding variants c.1350G > A (p.Gln450Gln), and c.1701A > G (p.Ile525Val). There were no inactivating mutations even though c.1350G > A was predicted to affect exonic splicing enhancers. However, several additional findings were of note: 1) nucleotide diversity estimate for SMAD3 but not SMAD4 indicated that coding variants of the MH2 domain were more infrequent than expected; 2) in breast cancer cases SMAD3 was significantly over-expressed relative to controls (P < 0.05) while the case harboring SMAD4 c.1350G > A was associated with elevated germline expression (> 5-fold); 3) separate analysis using tissue expression data showed statistically significant over-expression of SMAD3 and SMAD4 in breast carcinomas. CONCLUSIONS:This study shows that inactivating germline alterations in SMAD3 and SMAD4 are rare, suggesting a limited role in driving tumorigenesis. Nevertheless, aberrant germline expressions of SMAD3 and SMAD4 may be more common in breast cancer than previously suspected and offer novel insight into their roles in predisposition and/or progression of breast cancer. 10.1186/bcr2926
    Prognostic value of the expression of Smad6 and Smad7, as inhibitory Smads of the TGF-beta superfamily, in esophageal squamous cell carcinoma. Osawa Hidenobu,Nakajima Masanobu,Kato Hiroyuki,Fukuchi Minoru,Kuwano Hiroyuki Anticancer research BACKGROUND:The transforming growth factor beta (TGF-beta) superfamily regulates cell growth in various cells. Previous studies have shown that altered expression of TGF-beta receptors, Smad2 and Smad4, as mediators of TGF-beta superfamily signaling, contributes to tumor progression in esophageal squamous cell carcinoma (SCC). The expression of Smad6 and Smad7 as negative regulators was examined, to investigate the further effects of TGF-B superfamily signaling. MATERIALS AND METHODS:Immunohistochemical staining with anti-Smad6 and anti-Smad7 polyclonal antibodies was performed on surgical specimens obtained from 115 patients with esophageal SCC. RESULTS:The expression of Smad6 and Smad7 was inversely correlated with depth of invasion in the early stages (p = 0.0003 and p = 0.0173, respectively). Moreover, there was a significant correlation between the expression of Smad6 and Smad7, and poor survival rate (p = 0.0078 and p = 0.0207, respectively). CONCLUSION:Smad6 and Smad7 expression affects the progression of early lesions of esophageal SCC and indicates a poor prognosis.
    Mutations in the tumor suppressors Smad2 and Smad4 inactivate transforming growth factor beta signaling by targeting Smads to the ubiquitin-proteasome pathway. Xu J,Attisano L Proceedings of the National Academy of Sciences of the United States of America Biological signals for transforming growth factor beta (TGF-beta) are transduced through transmembrane serine/threonine kinase receptors that signal to a family of intracellular mediators known as Smads. Smad2 and Smad4 are important for transcriptional and antiproliferative responses to TGF-beta, and their inactivation in human cancers indicates that they are tumor suppressors. A missense mutation at a conserved arginine residue in the amino-terminal MH1 domain of both Smad2 and Smad4 has been identified in tumors from patients with colorectal and pancreatic cancers, respectively. However, the mechanism whereby this mutation interferes with Smad activity is uncertain. Here we show that these mutations do not disrupt activation of Smads, including receptor-mediated phosphorylation of Smad2, Smad2/Smad4 heteromeric complex formation, and Smad nuclear translocation. In contrast, we demonstrate that the mutant Smads are degraded rapidly in comparison with their wild-type counterparts. We show that this decrease in Smad protein stability occurs through induction of Smad ubiquitination by pathways involving the UbcH5 family of ubiquitin ligases. These studies thus reveal a mechanism for tumorigenesis whereby genetic defects in Smads induce their degradation through the ubiquitin-mediated pathway. 10.1073/pnas.97.9.4820
    Dual effects of TGF-beta on ERalpha-mediated estrogenic transcriptional activity in breast cancer. Ren Yongsheng,Wu Liyu,Frost Andra R,Grizzle William,Cao Xu,Wan Mei Molecular cancer BACKGROUND:TGF-beta resistance often develops in breast cancer cells that in turn overproduce this cytokine to create a local immunosuppressive environment that fosters tumor growth and exacerbates the invasive and metastatic behavior of the tumor cells themselves. Smads-mediated cross-talk with the estrogen receptor has been implied to play an important role in development and/or progression of breast cancer. We investigated how TGF-beta regulates ERalpha-induced gene transcription and potential mechanisms of frequent TGF-beta resistance in breast cancer. METHODS:Effect of TGF-beta on ERalpha-mediated gene transcription was investigated in breast cancer cell lines using transient transfection, real-time PCR, sequential DNA precipitation, and small interfering RNA assays. The expression of Smads on both human breast cancer cell lines and ERalpha-positive human breast cancer tissue was evaluated by immunofluorescence and immunohistochemical assays. RESULTS:A complex of Smad3/4 mediates TGF-beta inhibition of ERalpha-mediated estrogenic activity of gene transcription in breast cancer cells, and Smad4 is essential and sufficient for such repression. Either overexpression of Smad3 or inhibition of Smad4 leads to the "switch" of TGF-beta from a repressor to an activator. Down-regulation and abnormal cellular distribution of Smad4 were associated with some ERalpha-positive infiltrating human breast carcinoma. There appears a dynamic change of Smad4 expression from benign breast ductal tissue to infiltrating ductal carcinoma. CONCLUSION:These results suggest that aberrant expression of Smad4 or disruption of Smad4 activity lead to the loss of TGF-beta suppression of ERalpha transactivity in breast cancer cells. 10.1186/1476-4598-8-111
    MiR-23a promotes TGF-β1-induced EMT and tumor metastasis in breast cancer cells by directly targeting CDH1 and activating Wnt/β-catenin signaling. Ma Fei,Li Wenjie,Liu Chunxiao,Li Wei,Yu Haining,Lei Bo,Ren Yanlv,Li Zhigao,Pang Da,Qian Cheng Oncotarget TGF-β1-induced epithelial-mesenchymal transition (EMT) has been proved to be associated with metastasis of breast cancer cells. We attempted to detect a novel mechanism that microRNAs mediated the TGF-β1-induced EMT in the process of breast cancer metastasis. Here we reported that the expression of miR-23a was higher in breast cancer cells with high metastasis ability and patients with lymph node metastasis and the treatment of TGF-β1 significantly upregulated the expression of miR-23a in breast cancer cells. We found that miR-23a was upregulated by TGF-β1 post-transcriptionally and Smads directly bound the RNA Smad binding element (R-SBE) of miR-23a. Functional studies showed that inhibition of miR-23a suppressed the TGF-β1-induced EMT, migration, invasion and metastasis of breast cancer both and . In addition, we determined that miR-23a directly targeted and suppressed CDH1, one important gene in EMT phenomenon. Notably, Wnt/β-catenin signaling was activated by the suppression of CDH1 in the miR-23a mediated process of TGF-β1-induced EMT and tumor invasion. These results demonstrate that miR-23a promotes TGF-β1-induced tumor metastasis in breast cancer by targeting CDH1 and activating Wnt/β-catenin signaling. Taken together, our results indicate a novel regulatory mechanism of TGF-β1-induced EMT and suggest that miR-23a might be a potential target in breast cancer therapy. 10.18632/oncotarget.18422
    The Role of TGF-β Signaling Regulatory MicroRNAs in the Pathogenesis of Colorectal Cancer. Moradi-Marjaneh Reyhaneh,Khazaei Majid,Ferns Gordon A,Aghaee-Bakhtiari Seyed H Current pharmaceutical design Colorectal cancer (CRC) is one of the most common cancers globally and is associated with a high mortality rate. The transforming growth factor beta (TGF-β) signaling pathway plays an important role in normal intestinal tissue function, but has also been implicated in the development of CRC. MicroRNAs (miRNAs) have also recently emerged as important regulators of cancer development and progression. They act by targeting multiple signaling pathways including the TGF-β signaling pathway. There is growing evidence demonstrating that miRNAs target various components of the TGF-β signaling pathway, including TGF-β1, TGF-β2, regulatory SMADs (SMAD1, 2, 3, 5 and 9), co-mediator SMAD4, inhibitory SMADs (SMAD6 and 7) and the TGF-β receptors, and thereby alter the proliferation and migration of CRC cells. In this review, we summarize the data concerning the interaction between TGF-β signaling pathway and miRNAs with the aim to better understanding the CRC molecular mechanisms and hence better management of this disease. 10.2174/1381612825666190110150705
    Differential expression, regulation, and induction of Smads, transforming growth factor-beta signal transduction pathway in leiomyoma, and myometrial smooth muscle cells and alteration by gonadotropin-releasing hormone analog. Xu Jingxia,Luo Xiaoping,Chegini Nasser The Journal of clinical endocrinology and metabolism The objective of this study was to further elucidate the role of TGFbeta and GnRH analog (GnRHa) in leiomyoma growth and regression. We examined the expression of Smads, TGFbeta receptor intracellular signaling molecules, in leiomyoma and myometrial smooth muscle cells (LSMC and MSMC), and determined whether TGFbeta and GnRHa differentially regulate their expression and induction in these cells. Using semiquantitative RT-PCR, Western blot analysis, and immunohistochemistry, we demonstrated that leiomyoma, myometrium, LSMC, and MSMC express receptor-activated Smad3, common Smad4, and the inhibitory Smad7 mRNA and protein and showed that TGFbeta1, in a time-dependent manner, transiently induced Smad7 expression, with Smad3 and Smad4 remaining largely unchanged. TGFbeta1 increased the rate of Smad and phosphorylated Smad3 (pSmad3) induction in both cell types. Pretreatment with TGFbeta type II receptor antisense oligonucleotide resulted in a trend toward a lower TGFbeta-induced pSmad3. GnRHa, in a dose- and time-dependent manner, increased the expression of Smad7 mRNA and the rapid induction of Smad3, Smad4, and Smad7 as well as pSmad3, which declined to control values at doses above 1 micro M in MSMC, but not in LSMC. GnRHa-induced pSamd3 was partly inhibited by a GnRH antagonist (antide). We concluded that leiomyoma, myometrium, LSMC, and MSMC express Smads, which are differentially expressed, induced, and activated by TGFbeta and are altered as a result of GnRHa treatment. These results suggest that TGFbeta and GnRHa mediate their actions through cross-talk involving Smads and most likely other signaling pathways that result in leiomyoma growth and regression. 10.1210/jc.2002-021325
    Smads are the central component in transforming growth factor-beta signaling. Padgett R W,Cho S H,Evangelista C Pharmacology & therapeutics Until recently, little was known about how transforming growth factor (TGF)-beta signals are transduced to the nucleus. With the discovery of the Smad proteins initially in Drosophila and C. elegans, the unraveling of the pathway has begun. Nine different vertebrate members also have been reported, indicating that Smads are a conserved component of the TGF-beta pathway. Currently, there are three functional classes of Smads. Class I Smads are phosphorylated by TGF-beta receptors and move to the nucleus. The Class II Smads function with Class I Smads, while Class III Smads antagonize the function of Class I Smads. New evidence shows that Smads bind specific DNA sequences and induce transcription of downstream target genes, thus placing the Smads at the center of the TGF-beta signaling pathway.
    The Notch pathway inhibits TGFβ signaling in breast cancer through HEYL-mediated crosstalk. Han Liangfeng,Diehl Adam,Nguyen Nguyen K,Korangath Preethi,Teo Weiwen,Cho Soonweng,Kominsky Scott,Huso David L,Feigenbaum Lionel,Rein Alan,Argani Pedram,Landberg Goran,Gessler Manfred,Sukumar Saraswati Cancer research Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix-loop-helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL(-/-) mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer. 10.1158/0008-5472.CAN-14-0816
    Two sides of the story? Smad4 loss in pancreatic cancer versus head-and-neck cancer. Malkoski Stephen P,Wang Xiao-Jing FEBS letters TGFβ signaling Smads (Smad2, 3, and 4) were suspected tumor suppressors soon after their discovery. Nearly two decades of research confirmed this role and revealed other divergent and cancer-specific functions including paradoxical tumor promotion effects. Although Smad4 is the most potent tumor suppressor, its functions are highly context-specific as exemplified by pancreatic cancer and head-and-neck cancer: in pancreatic cancer, Smad4 loss cannot initiate tumor formation but promotes metastases while in head-and-neck cancer Smad4 loss promotes cancer progression but also initiates tumor formation, likely through effects on genomic instability. The differing consequences of impaired Smad signaling in human cancers and the molecular mechanisms that underpin these differences will have important implications for the design and application of novel targeted therapies. 10.1016/j.febslet.2012.01.054
    Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells. Tarasewicz Elizabeth,Rivas Lisbi,Hamdan Randala,Dokic Danijela,Parimi Vamsi,Bernabe Beatriz Penalver,Thomas Alexandra,Shea Lonnie D,Jeruss Jacqueline S Cell cycle (Georgetown, Tex.) Breast cancer onset and disease progression have been linked to members of the TGFβ superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFβ in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options. 10.4161/15384101.2014.950126
    Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads. Itoh F,Asao H,Sugamura K,Heldin C H,ten Dijke P,Itoh S The EMBO journal Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads. 10.1093/emboj/20.15.4132
    TGF- β: an important mediator of allergic disease and a molecule with dual activity in cancer development. Tirado-Rodriguez Belen,Ortega Enrique,Segura-Medina Patricia,Huerta-Yepez Sara Journal of immunology research The transforming growth factor- β (TGF- β ) superfamily is a family of structurally related proteins that includes TGF- β , activins/inhibins, and bone morphogenic proteins (BMPs). Members of the TGF- β superfamily regulate cellular functions such as proliferation, apoptosis, differentiation, and migration and thus play key roles in organismal development. TGF- β is involved in several human diseases, including autoimmune disorders and vascular diseases. Activation of the TGF- β receptor induces phosphorylation of serine/threonine residues and triggers phosphorylation of intracellular effectors (Smads). Once activated, Smad proteins translocate to the nucleus and induce transcription of their target genes, regulating various processes and cellular functions. Recently, there has been an attempt to correlate the effect of TGF- β with various pathological entities such as allergic diseases and cancer, yielding a new area of research known as "allergooncology," which investigates the mechanisms by which allergic diseases may influence the progression of certain cancers. This knowledge could generate new therapeutic strategies aimed at correcting the pathologies in which TGF- β is involved. Here, we review recent studies that suggest an important role for TGF- β in both allergic disease and cancer progression. 10.1155/2014/318481
    The Smads: transcriptional regulation and mouse models. Datto M,Wang X F Cytokine & growth factor reviews The field of transforming growth factor-beta (TGF-beta) signaling sees periodic discoveries that revolutionize our thinking, redirect our experiments, and peak our excitement. One of the first such discoveries was less than a decade ago: the molecular cloning of the type I and type II TGF-beta receptors. This breakthrough defined a novel family of serine/threonine kinase receptors, which led to the description of an ever-expanding superfamily. The discovery of how these receptors are grouped on the cell surface, bind TGF-beta and are activated by specific phosphorylation events further defined the uniqueness of this system in comparison to other families of growth factor receptors. Now, once again, the TGF-beta field has been revolutionized. This time, the discovery is the Smad family of proteins. Although one can hardly imagine TGF-beta without the Smads, the cloning of the Smads and their implication in TGF-beta signaling was only four years ago. Since that time, great advances have been made in our understanding of the Smads as transcription factors, which are activated by receptor mediated phosphorylation. In addition, animal models for a loss of Smad function have provided insight into the role of specific Smads in a variety of physiologic systems. The Smad field has been growing exponentially. A comprehensive review of all aspects of the Smads, therefore, would be beyond the scope of a single review. Instead, this review highlights some of the general aspects of Smad function, and then focuses on the role of specific Smad family members in transcriptional regulation, animal physiology, and disease processes. 10.1016/s1359-6101(99)00027-1
    The expression of Smads and transforming growth factor beta receptors in leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy. Chegini Nasser,Luo Xiaoping,Ding Li,Ripley Daylene Molecular and cellular endocrinology Gene microarray analysis indicated that several components of the transforming growth factor beta receptor (TGF-betaR) signaling pathway are differentially expressed in leiomyoma and myometrium. To validate the microarray results we evaluated the expression of Smads, intracellular proteins that transmit TGF-betaR signals, in leiomyoma and matched myometrium from untreated women, and women who received gonadotropin releasing hormone analogue (GnRHa) therapy. Semi-quantitative RT-PCR, Western blotting and immunohistochemistry indicate that leiomyoma and myometrium expresses receptor-activated Smad3, common Smad4 and inhibitory Smad7, with elevated expression of Smad3, Smad4 and phosphorylated Smad3 (pSmad3) as well as TGF-betaR type I and type II in leiomyoma compared to myometrium (P<0.05). GnRHa therapy resulted in lowering of TGF-betaRs as well as Smad4 and pSmad3, with concurrent increased in Smad7 expression in both leiomyoma and myometrium compared to untreated group (P<0.05). Immunohistochemically Smads and pSmad3 were localized in cytoplasmic/nuclear compartments of leiomyoma and myometrial smooth muscle cells and connective tissue fibroblasts, with alteration in their intensity (HScores) in GnRHa-treated group. In conclusion, the results indicates that leiomyoma and myometrium express all the components of TGF-betaR and Smads, and GnRHa therapy results in alteration of their expression further supporting the importance of TGF-beta system as key regulator of leiomyoma growth.
    Immunohistochemical expression of Smads 1-6 in the 15-day gestation mouse embryo: signaling by BMPs and TGF-betas. Flanders K C,Kim E S,Roberts A B Developmental dynamics : an official publication of the American Association of Anatomists The eight mammalian Smad proteins mediate cellular signaling from members of the transforming growth factor-beta (TGF-beta), bone morphogenetic protein (BMP), and activin families. Smads 1, 5, and 8 transmit signals from BMPs, while Smads 2 and 3 transmit signals from TGF-betas and activin. Smad 4 is a common mediator of both pathways, while Smads 6 and 7 inhibit signaling. Signal transduction involves translocation of Smad complexes to the nucleus and subsequent gene activation. Little is known about the expression of endogenous Smad proteins during development. We identified commercially available Smad antibodies that specifically recognize a unique Smad protein and are suitable for immunohistochemistry. Here we compare the localization of Smads 1, 2, 3, 4, 5, and 6 in tissues of the 15-day gestation mouse embryo. Immunoreactive Smad proteins are seen in many tissues with differences in the localization being dependent upon the cell type. All tissues express Smad 4 and at least one each of the BMP-specific and TGF-beta-specific Smads, while expression of Smad 6 is more restricted. Differences are observed in the nuclear versus cytoplasmic localization among the Smads in different cell types or tissues, suggesting selective activation of Smads during this stage of development. 10.1002/1097-0177(2000)9999:9999<::AID-DVDY1096>3.0.CO;2-4
    Role of SMAD proteins in colitis-associated cancer: from known to the unknown. Chandrasinghe P,Cereser B,Moorghen M,Al Bakir I,Tabassum N,Hart A,Stebbing J,Warusavitarne J Oncogene Small mothers against decapentaplegic (SMAD) proteins are a family of signal transduction molecules in transforming growth factor β (TGFβ) ligand pathways that have been found to have a key role in the pathogenesis of inflammatory bowel disease (IBD). Long standing IBD predisposes individuals to colitis-associated colorectal cancer (CAC), an entity that possess unique characteristics compared to hereditary and sporadic cancer. The ligands of the TGFβ super family along with SMADs have also been implicated in several aspects of colorectal cancer formation. SMAD proteins are shown to be involved in a number of potentially carcinogenic mechanisms such as altering gene transcription, controlling stem cell differentiation to causing epigenetic changes. Modulation of these proteins has emerged as a novel therapeutic intervention for IBD although its effect on carcinogenesis remains elusive. This account reviews available evidence linking SMAD proteins to CAC and explores the potential areas for future research in this area. 10.1038/onc.2017.300
    TGF-β Signaling in Cancer. Syed Viqar Journal of cellular biochemistry The transforming growth factor-β (TGF-β) is a family of structurally related proteins that comprises of TGF-β, activins/inhibins, and bone morphogenic proteins (BMPs). Members of the TGF-β family control numerous cellular functions including proliferation, apoptosis, differentiation, epithelial-mesenchymal transition (EMT), and migration. The first identified member, TGF-β is implicated in several human diseases, such as vascular diseases, autoimmune disorders, and carcinogenesis. Activation of the TGF-β receptor by its ligands induces the phosphorylation of serine/threonine residues and triggers phosphorylation of the intracellular effectors, SMADs. Upon activation, SMAD proteins translocate to the nucleus and induce transcription of their target genes, regulating several cellular functions. TGF-β dysregulation has been implicated in carcinogenesis. In early stages of cancer, TGF-β exhibits tumor suppressive effects by inhibiting cell cycle progression and promoting apoptosis. However, in late stages TGF-β exerts tumor promoting effects, increasing tumor invasiveness, and metastasis. Furthermore, the TGF-β signaling pathway communicates with other signaling pathways in a synergistic or antagonistic manner and regulates cellular functions. Elevated TGF-β activity has been associated with poor clinical outcome. Given the pivotal role of TGF-β in tumor progression, this pathway is an attractive target for cancer therapy. Several therapeutic tools such as TGF-β antibodies, antisense oligonucleotides, and small molecules inhibitors of TGF-β receptor-1 (TGF-βR1) have shown immense potential to inhibit TGF-β signaling. Finally, in the interest of developing future therapies, further studies are warranted to identify novel points of convergence of TGF-β with other signaling pathways and oncogenic factors in the tumor microenvironment. 10.1002/jcb.25496
    Alteration of tumor suppressor BMP5 in sporadic colorectal cancer: a genomic and transcriptomic profiling based study. Chen Erfei,Yang Fangfang,He Hongjuan,Li Qiqi,Zhang Wei,Xing Jinliang,Zhu Ziqing,Jiang Jingjing,Wang Hua,Zhao Xiaojuan,Liu Ruitao,Lei Lei,Dong Jing,Pei Yuchen,Yang Ying,Pan Junqiang,Zhang Pan,Liu Shuzhen,Du Le,Zeng Yuan,Yang Jin Molecular cancer BACKGROUND:Although the genetic spectrum of human colorectal cancer (CRC) is mainly characterized by APC, KRAS and TP53 mutations, driver genes in tumor initiation have not been conclusively demonstrated. In this study, we aimed to identify novel markers for CRC. METHODS:We performed exome analysis of sporadic colorectal cancer (sCRC) coding regions to screen loss of function (LoF) mutation genes, and carried out systems-level approaches to confirm top rank gene in this study. RESULTS:We identified loss of BMP5 is an early event in CRC. Deep sequencing identified BMP5 was mutated in 7.7% (8/104) of sCRC samples, with 37.5% truncating mutation frequency. Notably, BMP5 negative expression and its prognostic value is uniquely significant in sCRC but not in other tumor types. Furthermore, BMP5 expression was positively correlated with E-cadherin in CRC patients and its dysregulation play a vital role in epithelial-mesenchymal transition (EMT), thus triggering tumor initiation and development. RNA sequencing identified, independent of BMP/Smads pathway, BMP5 signaled though Jak-Stat pathways to inhibit the activation of oncogene EPSTI1. CONCLUSIONS:Our result support a novel concept that the importance of BMP5 in sCRC. The tumor suppressor role of BMP5 highlights its crucial role in CRC initiation and development. 10.1186/s12943-018-0925-7
    Regulating the stability of TGFbeta receptors and Smads. Lönn Peter,Morén Anita,Raja Erna,Dahl Markus,Moustakas Aristidis Cell research Transforming growth factor beta (TGFbeta) controls cellular behavior in embryonic and adult tissues. TGFbeta binding to serine/threonine kinase receptors on the plasma membrane activates Smad molecules and additional signaling proteins that together regulate gene expression. In this review, mechanisms and models that aim at explaining the coordination between several components of the signaling network downstream of TGFbeta are presented. We discuss how the activity and duration of TGFbeta receptor/Smad signaling can be regulated by post-translational modifications that affect the stability of key proteins in the pathway. We highlight links between these mechanisms and human diseases, such as tissue fibrosis and cancer. 10.1038/cr.2008.308
    A low-frequency variant in SMAD7 modulates TGF-β signaling and confers risk for colorectal cancer in Chinese population. Li Jiaoyuan,Zou Li,Zhou Ying,Li Lu,Zhu Ying,Yang Yang,Gong Yajie,Lou Jiao,Ke Juntao,Zhang Yi,Tian Jianbo,Zou Danyi,Peng Xiating,Chang Jiang,Gong Jing,Zhong Rong,Zhou Xiaobo,Miao Xiaoping Molecular carcinogenesis The TGF-β pathway plays an essential role in regulating cell proliferation and differentiation. GWASs and candidate approaches have identified a battery of genetic variants in the TGF-β pathway contributing to colorectal cancer (CRC). However, most of the significant variants are common variants and their functions remain ambiguous. To identify causal variants with low-frequency in the TGF-β pathway contributing to CRC susceptibility in Chinese population, we performed targeted sequencing of 12 key genes in TGF-β signaling in CRC patients followed by a two-stage case-control study with a total of 5109 cases and 5169 controls. Bioinformatic annotations and biochemical experiments were applied to reveal the potential functions of significant variants. Seven low-frequency genetic variants were captured through targeted sequencing. The two stage association studies showed that missense variant rs3764482 (c. 83C>T; p. S28F) in the gene SMAD7 was consistently and significantly associated with CRC risk. Compared with the wild type, the ORs for variant allele were 1.37 (95%CI: 1.10-1.70, P = 0.005), 1.55 (95%CI: 1.30-1.86, P = 1.15 × 10 ), and 1.48 (1.29-1.70, P = 2.44 × 10 ) in stage 1, stage 2, and the combined analyses, respectively. Functional annotations revealed that the minor allele T of rs3764482 was more effective than the major allele C in blocking the TGF-β signaling and inhibiting the phosphorylation of receptor-regulated SMADs (R-SMADs). In conclusion, low-frequency coding variant rs3764482 in SMAD7 is associated with CRC risk in Chinese population. The rs3764482 variant may block the TGF-β signaling via impeding the activation of downstream genes, leading to cancer cell proliferation, thus contributing to CRC pathogenesis. 10.1002/mc.22637
    Discovery of a natural small-molecule compound that suppresses tumor EMT, stemness and metastasis by inhibiting TGFβ/BMP signaling in triple-negative breast cancer. Di Lei,Liu Li-Juan,Yan Yong-Ming,Fu Rong,Li Yi,Xu Ying,Cheng Yong-Xian,Wu Zhao-Qiu Journal of experimental & clinical cancer research : CR BACKGROUND:The transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) signaling pathways are both constitutively activated in triple-negative breast cancer (TNBC). We are interested in isolating the naturally-derived small-molecule inhibitor that could simultaneously targeting TGFβ/BMP pathways and further studying its anti-proliferative/-metastatic effects as well as the underlying mechanisms in multiple tumor models. METHODS:Multiple in vitro cell-based assays are used to examine the compound's inhibitory efficacy on TNBC cell growth, stemness, epithelial-mesenchymal transition (EMT), invasion and migration by targeting TGFβ/BMP signaling pathways. Transgenic breast cancer mouse model (MMTV-PyMT), subcutaneous xenograft and bone metastasis models are used to examine ZL170's effects on TNBC growth and metastasis potentials in vivo. RESULTS:ZL170 dose-dependently inhibits cell proliferation, EMT, stemness, invasion and migration in vitro via specifically targeting canonical TGFβ/BMP-SMADs pathways in TNBC cells. The compound significantly hinders osteolytic bone metastasis and xenograft tumor growth without inflicting toxicity on vital organs of tumor-bearing nude mice. ZL170 strongly inhibits primary tumor growth and lung metastases in MMTV-PyMT transgenic mice. ZL170-treated tumors exhibit impaired TGFβ/BMP signaling pathways in both epithelial and stromal compartments, thereby creating a suppressive tumor microenvironment characterized by reduced extracellular matrix deposition and decreased infiltration of stromal cells. CONCLUSIONS:ZL170 inhibits tumor EMT, stemness and metastasis and could be further developed as a potent anti-metastatic agent used in combination with cytotoxic drugs for treatment of TNBC and other advanced metastatic cancers. 10.1186/s13046-019-1130-2
    EGF induces epithelial-mesenchymal transition through phospho-Smad2/3-Snail signaling pathway in breast cancer cells. Kim Jinkyoung,Kong Jienan,Chang Hyeyoon,Kim Hayeon,Kim Aeree Oncotarget Epithelial-mesenchymal transition (EMT) can contribute to tumor invasion, metastasis, and resistance to chemotherapy or hormone therapy. EMT may be induced by a variety of growth factors, such as epidermal growth factor (EGF). Most studies regarding EMT have focused on TGF-β-Smads signaling. The mechanism of EGF-induced EMT via activation of the Smad2/3 in breast cancer cells, MCF-7 and MDA-MB-231, remains unclear. The expression levels of Snail, vimentin, and fibronectin were increased by EGF treatment in a time-dependent manner, while the expression level of E-cadherin was decreased. EGF-induced nuclear co-localization of phospho-Smad2/3 and Snail and cancer cell migration were inhibited by pretreatment with an ERK1/2 inhibitor, PD98059 and a phospho-Smad2 inhibitor, SB203580. Knockdown of Smad2/3 expression suppressed EGF-induced expressions of Snail, vimentin, fibronectin, and cancer cell invasion, suggesting an acquisition of the mesenchymal and migratory phenotype in less aggressive MCF-7 cells. Moreover, MDA-MB-231 cells were shown that EGF-induced EMT, and cell invasion through ERK1/2-phospho-Smad2/3-Snail signaling pathway. We have discovered that EGF-stimulated activation of Smad2/3 upregulated several key EMT markers, inhibited E-cadherin expression, promoted EMT, enhanced migration and invasion in MCF-7 and MDA-MB-231 breast cancer cells. Identification of this molecular mechanism may provide new molecular targets for the development of therapies for metastatic breast cancer. 10.18632/oncotarget.13116
    TGF-β signaling pathway and breast cancer susceptibility. Scollen Serena,Luccarini Craig,Baynes Caroline,Driver Kristy,Humphreys Manjeet K,Garcia-Closas Montserrat,Figueroa Jonine,Lissowska Jolanta,Pharoah Paul D,Easton Douglas F,Hesketh Robin,Metcalfe James C,Dunning Alison M Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology BACKGROUND:TGF-β acts as a suppressor of primary tumor initiation but has been implicated as a promoter of the later malignant stages. Here associations with risk of invasive breast cancer are assessed for single-nucleotide polymorphisms (SNP) tagging 17 genes in the canonical TGF-β ALK5/SMADs 2&3 and ALK1/SMADs 1&5 signaling pathways: LTBP1, LTBP2, LTBP4, TGFB1, TGFB2, TGFB3, TGFBR1(ALK5), ALK1, TGFBR2, Endoglin, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, and SMAD7 [Approved Human Gene Nomenclature Committee gene names: ACVRL1 (for ALK1) and ENG (for Endoglin)]. METHODS:Three-hundred-fifty-four tag SNPs (minor allele frequency > 0.05) were selected for genotyping in a staged study design using 6,703 cases and 6,840 controls from the Studies of Epidemiology and Risk Factors in Cancer Heredity (SEARCH) study. Significant associations were meta-analyzed with data from the NCI Polish Breast Cancer Study (PBCS; 1,966 cases and 2,347 controls) and published data from the Breast Cancer Association Consortium (BCAC). RESULTS:Associations of three SNPs, tagging TGFB1 (rs1982073), TGFBR1 (rs10512263), and TGFBR2 (rs4522809), were detected in SEARCH; however, associations became weaker in meta-analyses including data from PBCS and BCAC. Tumor subtype analyses indicated that the TGFB1 rs1982073 association may be confined to increased risk of developing progesterone receptor negative (PR(-)) tumors [1.18 (95% CI: 1.09-1.28), 4.1 × 10(-5) (P value for heterogeneity of ORs by PR status = 2.3 × 10(-4))]. There was no evidence for breast cancer risk associations with SNPs in the endothelial-specific pathway utilizing ALK1/SMADs 1&5 that promotes angiogenesis. CONCLUSION:Common variation in the TGF-β ALK5/SMADs 2&3 signaling pathway, which initiates signaling at the cell surface to inhibit cell proliferation, might be related to risk of specific tumor subtypes. IMPACT:The subtype specific associations require very large studies to be confirmed. 10.1158/1055-9965.EPI-11-0062
    The Smads. Hill C S The international journal of biochemistry & cell biology The Smads are a family of intracellular signalling molecules that act downstream of receptors for the transforming growth factor (TGF)-beta family of ligands. Three classes of Smads have been identified. The receptor-regulated Smads are direct substrates for the type I receptors, which are serine/threonine kinases. Once phosphorylated and activated, these Smads form hetero-oligomeric complexes with a second class of Smad, the common mediator Smads. These Smad complexes translocate to the nucleus, where they are recruited to DNA primarily by site-specific DNA binding transcription factors, and participate in regulating the transcription of target genes. Inhibitory Smads are the third identified class which antagonise the activity of the receptor-regulated Smads. Aberrant TGF-beta signalling has been associated with several human diseases such as cancer and fibrosis. The identification of the Smads as primary transducers of TGF-beta signals raises the possibility that agents directed at modulating Smad activity would have therapeutic applications.
    Control of microRNA biogenesis by TGFbeta signaling pathway-A novel role of Smads in the nucleus. Hata Akiko,Davis Brandi N Cytokine & growth factor reviews microRNAs (miRNAs) are small, non-coding RNAs that modulate diverse biological functions through the repression of target genes. miRNA profiling studies have indicated that the levels of miRNAs are altered during normal development and pathogenesis of various diseases, including cancer and cardiovascular disorders. The signaling pathways which control miRNA biogenesis and the mechanisms of regulation, however, are not well understood. Following transcription, mature miRNAs are generated through a series of coordinated processing events mediated by large protein complexes. We recently found that signal transducers of the Transforming Growth Factor beta (TGFbeta) signaling pathway, the Smads, play a regulatory role in the processing of miRNA in the nucleus. In this review, we summarize the current understanding of the regulation of miRNA biogenesis mediated by the TGFbeta signaling pathway. 10.1016/j.cytogfr.2009.10.004
    Integrative genomic analyses on GLI1: positive regulation of GLI1 by Hedgehog-GLI, TGFbeta-Smads, and RTK-PI3K-AKT signals, and negative regulation of GLI1 by Notch-CSL-HES/HEY, and GPCR-Gs-PKA signals. Katoh Yuriko,Katoh Masaru International journal of oncology GLI family members are zinc-finger transcription factors, which are involved in embryogenesis and carcinogenesis through transcription regulation of GLI1, CCND1, CCND2, FOXA2, FOXC2, RUNX2, SFRP1, and JAG2. GLI1 transcription is upregulated in a variety of human tumors, such as basal cell carcinoma, lung cancer, breast cancer, gastric cancer, pancreatic cancer, and esophageal cancer. Hedgehog signaling via Smoothened cascade and receptor tyrosine kinase (RTK) signaling via PI3K-AKT cascade induce stabilization of GLI1 protein, whereas G-protein coupled receptor (GPCR) signaling via Gs-PKA cascade induces degradation of GLI1 protein. Here we report integrative genomic analyses of the GLI1 gene. The GLI1 and ARHGAP9 genes are located in a tail-to-tail manner with overlapping 3'-ends. ARHGAP9 was expressed in bone marrow, spleen, thymus, monocytes, and macrophages, whereas GLI1 was almost undetectable in normal tissues or cells with predominant ARHGAP9 expression. Because overlapping sense and anti-sense transcripts are annealed to each other to give rise to double-stranded RNAs functioning as endogenous RNAi, GLI1 expression might be negatively regulated by ARHGAP9 transcripts. GLI-binding element with one base substitution at the +1589-bp position from the transcriptional start site (TSS) of the human GLI1 gene was completely conserved in chimpanzee GLI1, mouse Gli1, and rat Gli1 genes. Ten Smad-binding elements, double E-boxes for EMT regulators, and double N-boxes for HES/HEY family members within intron 1 of the human GLI1 gene were also conserved in mammalian GLI1 orthologs. GLI1 transcription is upregulated due to Hedgehog, and TGFbeta signaling activation, whereas GLI1 transcription is downregulated due to Snail/Slug, and Notch signaling activation. Together these facts indicate that Hedgehog, TGFbeta, and RTK signals positively regulate GLI1, and that Notch, and GsPCR signals negatively regulate the GLI1. 10.3892/ijo_00000328
    The TGF-β Signaling Regulator PMEPA1 Suppresses Prostate Cancer Metastases to Bone. Fournier Pierrick G J,Juárez Patricia,Jiang Guanglong,Clines Gregory A,Niewolna Maria,Kim Hun Soo,Walton Holly W,Peng Xiang Hong,Liu Yunlong,Mohammad Khalid S,Wells Clark D,Chirgwin John M,Guise Theresa A Cancer cell Transforming growth factor-β (TGF-β) regulates the expression of genes supporting breast cancer cells in bone, but little is known about prostate cancer bone metastases and TGF-β. Our study reveals that the TGFBR1 inhibitor SD208 effectively reduces prostate cancer bone metastases. TGF-β upregulates in prostate cancer cells a set of genes associated with cancer aggressiveness and bone metastases, and the most upregulated gene was PMEPA1. In patients, PMEPA1 expression decreased in metastatic prostate cancer and low Pmepa1 correlated with decreased metastasis-free survival. Only membrane-anchored isoforms of PMEPA1 interacted with R-SMADs and ubiquitin ligases, blocking TGF-β signaling independently of the proteasome. Interrupting this negative feedback loop by PMEPA1 knockdown increased prometastatic gene expression and bone metastases in a mouse prostate cancer model. 10.1016/j.ccell.2015.04.009
    Glabridin inhibits cancer stem cell-like properties of human breast cancer cells: An epigenetic regulation of miR-148a/SMAd2 signaling. Jiang Fei,Li Yuan,Mu Juan,Hu Chunyan,Zhou Ming,Wang Xingxing,Si Lu,Ning Shilong,Li Zhong Molecular carcinogenesis In breast cancer, the cancer stem cells (CSCs) are thought to be the main cause of metastasis and recurrence. Targeting of CSCs or cancer cells with stem cell-like properties has become a new approach for the treatment of breast cancer. Glabridin (GLA), a phytochemical from the root of Glycyrrhiza glabra, exhibited effective antitumor properties in various human cancer cells. However, the roles of GLA in the regulation of CSC-like properties and the underlying molecular mechanisms remain unclear. Here, we reported that GLA attenuated the CSC-like properties through microRNA-148a (miR-148a)/transforming growth factor beta (TGFβ)-SMAD2 signal pathway in vitro and in vivo. In MDA-MB-231 and Hs-578T breast cancer cell lines, GLA enhanced the expression of miR-148a through DNA demethylation. By targeting of the SMAD2-3'-UTR, miR-148a blocked the expression/activation of SMAD2, and in turn, restored the epithelial characteristics, adhesive abilities, and CSC-like properties. Furthermore, in mouse xenograft models, we also confirmed that GLA attenuated the tumor growth, mesenchymal characteristics, and CSCs-like properties via demethylation-activated miR-148a. Our findings suggested a potential treatment strategy to reduce the CSCs-like properties, and therefore enhance the effectiveness of breast cancer therapy. 10.1002/mc.22333
    microRNA-193a stimulates pancreatic cancer cell repopulation and metastasis through modulating TGF-β2/TGF-βRIII signalings. Fang Chi,Dai Chen-Yun,Mei Zhu,Jiang Ming-Jie,Gu Dian-Na,Huang Qian,Tian Ling Journal of experimental & clinical cancer research : CR BACKGROUND:Pancreatic cancer characterizes high recurrence and poor prognosis. In clinical practice, radiotherapy is widely used for pancreatic cancer treatment. However, the outcome remains undesirable due to tumor repopulation and following recurrence and metastasis after radiation. So, it is highly needed to explore the underlying molecular mechanisms and accordingly develop therapeutic strategies. Our previous studies revealed that dying cells from chemoradiation could stimulate repopulation of surviving pancreatic cancer cells. However, we still knew little how dying cells provoke pancreatic cancer cell repopulation. We herein would explore the significance of TGF-β2 changes and investigate the modulation of microRNA-193a (miR-193a), and identify their contributions to pancreatic cancer repopulation and metastasis. METHODS:In vitro and in vivo repopulation models were established to mimic the biological processes of pancreatic cancer after radiation. Western blot, real-time PCR and dual-luciferase reporter assays were accordingly used to detect miR-193a and TGF-β2/TGF-βRIII signalings at the level of molecular, cellular and experimental animal model, respectively. Flow cytometry analysis, wound healing and transwell assay, vascular endothelial cell penetration experiment, and bioluminescence imaging were employed to assessthe biological behaviors of pancreatic cancer after different treatments. Patient-derived tumor xenograft (PDX) mice models were established to evaluate the therapeutic potential of miR-193a antagonist on pancreatic cancer repopulation and metastasis after radiation. RESULTS:miR-193a was highly expressed in the irradiated pancreatic cancer dying cells, accordingly elevated the level of miR-193a in surviving cells, and further promoted pancreatic cancer repopulation and metastasis in vitro and in vivo. miR-193a accelerated pancreatic cancer cell cycle and stimulated cell proliferation and repopulation through inhibiting TGF-β2/TGF-βRIII/SMADs/E2F6/c-Myc signaling, and even destroyed normal intercellular junctions and promoted metastasis via repressing TGF-β2/TGF-βRIII/ARHGEF15/ABL2 pathway. Knockdown of miR-193a or restoration of TGF-β2/TGF-βRIII signaling in pancreatic cancer cells was found to block pancreatic cancer repopulation and metastasis after radiation. In PDX models, the treatment in combination with miR-193a antagonist and radiation was found to dramatically inhibit pancreatic cancer cell repopulation and metastasis, and further improved the survival after radiation. CONCLUSIONS:Our findings demonstrated that miR-193a stimulated pancreatic cancer cell repopulation and metastasis through modulating TGF-β2/TGF-βRIII signalings, and miR-193a might be a potential therapeutic target for pancreatic cancer repopulation and metastasis. 10.1186/s13046-018-0697-3
    Distinct roles of individual Smads in skin carcinogenesis. Bornstein Sophia,Hoot Kristina,Han Gang-Wen,Lu Shi-Long,Wang Xiao-Jing Molecular carcinogenesis Transforming growth factor beta (TGFbeta) signaling has both tumor suppression and promotion roles. Smads are transcription factors that primarily mediate intracellular signaling for the TGFbeta superfamily. Loss of Smad2 and Smad4, but not Smad3 is common in human cancers. Given the complex nature of TGFbeta signaling, dissection of the distinct role of each Smad in mediating the multiple functions of TGFbeta signaling is warranted. To further analyze Smad deregulation during carcinogenesis, Smad2, Smad3, Smad4, and Smad7 were genetically modified in murine epidermis, and each alteration resulted in distinct skin phenotypes. Based on data from human cancer samples and from experimental models, Smad2 and Smad4 mainly function as tumor suppressors in skin carcinogenesis in vivo, whereas Smad3 and Smad7 may have dual roles in cancer. This review intends to summarize recent advances in the elucidation of the roles of Smad2, Smad3, Smad4, and Smad7 in skin carcinogenesis. 10.1002/mc.20336
    Transforming growth factor-beta, Smads, and cancer. Grady William M Clinical cancer research : an official journal of the American Association for Cancer Research 10.1158/1078-0432.CCR-05-0414
    Roles of TGFβ signaling Smads in squamous cell carcinoma. Han Gangwen,Wang Xiao-Jing Cell & bioscience Smad proteins are classified in different groups based on their functions in mediating transforming growth factor β (TGFβ) superfamily components. Smad1/5/8 mainly mediate bone morphogenetic proteins (BMP) pathway and Smad2/3 mainly mediate TGFβ pathway. Smad4 functions as common Smad to mediate both pathways. Previous studies showed many members of TGFβ superfamily play a role in carcinogenesis. The current review focuses on the role of TGFβ signaling Smads in squamous cell carcinomas (SCCs). TGFβ signaling inhibits early tumor development, but promotes tumor progression in the late stage. Although Smad2, Smad3 and Smad4 are all TGFβ signaling Smads, they play different roles in SCCs. Genetically, Smad2 and Smad4 are frequently mutated or deleted in certain human cancers whereas Smad3 mutation or deletion is infrequent. Genetically engineered mouse models with these individual Smad deletions have provided important tools to identify their diversified roles in cancer. Using these models, we have shown that Smad4 functions as a potent tumor suppressor and its loss causes spontaneous SCCs development; Smad2 functions as a tumor suppressor and its loss promotes SCC formation initiated by other genetic insults but is insufficient to initiate tumor formation. In contrast, Smad3 primarily mediates TGFβ-induced inflammation. The functions of each Smad also depends on the presence/absence of its Smad partner, thus need to be interpreted in a context-specific manner. 10.1186/2045-3701-1-41
    SMAD7: a timer of tumor progression targeting TGF-β signaling. Luo Lingyu,Li Nianshuang,Lv Nonghua,Huang Deqiang Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine In the context of cancer, transforming growth factor β (TGF-β) is a cell growth suppressor; however, it is also a critical inducer of invasion and metastasis. SMAD is the important mediator of TGF-β signaling pathway, which includes receptor-regulated SMADs (R-SMADs), common-mediator SMADs (co-SMADs), and inhibitory SMADs (I-SMADs). I-SMADs block the activation of R-SMADs and co-SMADs and thus play important roles especially in the SMAD-dependent signaling. SMAD7 belongs to the I-SMADs. As an inhibitor of TGF-β signaling, SMAD7 is overexpressed in numerous cancer types and its abundance is positively correlated to the malignancy. Emerging evidence has revealed the switch-in-role of SMAD7 in cancer, from a TGF-β inhibiting protein at the early stages that facilitates proliferation to an enhancer of invasion at the late stages. This role change may be accompanied or elicited by the tumor microenvironment and/or somatic mutation. Hence, current knowledge suggests a tumor-favorable timer nature of SMAD7 in cancer progression. In this review, we summarized the advances and recent findings of SMAD7 and TGF-β signaling in cancer, followed by specific discussion on the possible factors that account for the functional changes of SMAD7. 10.1007/s13277-014-2203-7
    DRAK2 participates in a negative feedback loop to control TGF-β/Smads signaling by binding to type I TGF-β receptor. Yang Kyung-Min,Kim Wonjoo,Bae Eunjin,Gim Jungsoo,Weist Brian M,Jung Yunshin,Hyun Ja-Shil,Hernandez Jennifer B,Leem Sun-Hee,Park Taesung,Jeong Joon,Walsh Craig M,Kim Seong-Jin Cell reports TGF-β1 is a multifunctional cytokine that mediates diverse biological processes. However, the mechanisms by which the intracellular signals of TGF-β1 are terminated are not well understood. Here, we demonstrate that DRAK2 serves as a TGF-β1-inducible antagonist of TGF-β signaling. TGF-β1 stimulation rapidly induces DRAK2 expression and enhances endogenous interaction of the type I TGF-β receptor with DRAK2, thereby blocking R-Smads recruitment. Depletion of DRAK2 expression markedly augmented the intensity and the extent of TGF-β1 responses. Furthermore, a high level of DRAK2 expression was observed in basal-like and HER2-enriched breast tumors and cell lines, and depletion of DRAK2 expression suppressed the tumorigenic ability of breast cancer cells. Thus, these studies define a function for DRAK2 as an intrinsic intracellular antagonist participating in the negative feedback loop to control TGF-β1 responses, and aberrant expression of DRAK2 increases tumorigenic potential, in part, through the inhibition of TGF-β1 tumor suppressor activity. 10.1016/j.celrep.2012.09.028
    Expression and mutational analysis of TGF-beta/Smads signaling in human cervical cancers. Ki Kyung-Do,Tong Seo-Yun,Huh Chu-Yeop,Lee Jong-Min,Lee Seon-Kyung,Chi Sung-Gil Journal of gynecologic oncology OBJECTIVE:To define the molecular basis of TGF-beta1 function in cervical carcinogenesis, we explored the expression and mutational status of TGF-beta1, TGF-beta1 receptors, and Smads, the regulators of the TGF-beta1 signaling pathway, in human cervical cancers. METHODS:Expression of TGF-beta1, TGF-beta1 receptors, and Smads transcripts were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR), and sequence alteration was analyzed using RT-PCR-single-strand conformation polymorphism (SSCP) analysis. Genomic levels of TGF-beta1, TGF-beta1 receptors and Smads was also measured by quantitative genomic PCR. RESULTS:Abnormal overexpression of TGF-beta1 and abnormal reduction of type II TGF-beta1 receptor were identified in 36% (18 of 50) and 20% (10 of 50) of cervical cancer tissues, respectively. 22% (11 of 50) in Smad2 and 14% (7 of 50) in Smad4 revealed tumor specific mRNA reduction less than a half of normal means. In addition, no evidence for sequence alterations of the gene was found by RT-PCR-SSCP analysis. CONCLUSION:Our study demonstrates that disruption of TGF-beta/Smad signaling pathway exist in human cervical cancer, suggesting that abnormal expressions of the member of TGF-beta/Smad signaling pathway might contribute to the malignant progression of human cervical tumors via suppressing the tumor suppression function of TGF-beta1 1's tumor suppression function. 10.3802/jgo.2009.20.2.117
    Role of Smads in TGFβ signaling. Heldin Carl-Henrik,Moustakas Aristidis Cell and tissue research Transforming growth factor-β (TGFβ) is the prototype for a large family of pleiotropic factors that signal via heterotetrameric complexes of type I and type II serine/threonine kinase receptors. Important intracellular mediators of TGFβ signaling are members of the Smad family. Smad2 and 3 are activated by C-terminal receptor-mediated phosphorylation, whereafter they form complexes with Smad4 and are translocated to the nucleus where they, in cooperation with other transcription factors, co-activators and co-repressors, regulate the transcription of specific genes. Smads have key roles in exerting TGFβ-induced programs leading to cell growth arrest and epithelial-mesenchymal transition. The activity and stability of Smad molecules are carefully regulated by a plethora of post-translational modifications, including phosphorylation, ubiquitination, sumoylation, acetylation and poly(ADP)-ribosylation. The Smad function has been shown to be perturbed in certain diseases such as cancer. 10.1007/s00441-011-1190-x
    TGF-beta signaling and the role of inhibitory Smads in non-small cell lung cancer. Jeon Hyo-Sung,Jen Jin Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer The signaling pathway mediated by transforming growth factor-beta (TGF-beta) participates in various biologic processes, including cell growth, differentiation, angiogenesis, apoptosis, and extracellular matrix remodeling. In the context of cancer, TGF-beta signaling can inhibit tumor growth in early-stage tumors. However, in late-stage tumors, the very same pathway promotes tumor invasiveness and metastasis. This paradoxical effect is mediated through similar to mothers against decapentaplegic or Smad protein dependent and independent mechanisms and provides an opportunity for targeted cancer therapy. This review summarizes the molecular process of TGF-beta signaling and the changes in inhibitory Smads that contribute to lung cancer progression. We also present current approaches for rational therapies that target the TGF-beta signaling pathway in cancer. 10.1097/JTO.0b013e3181ce3afd
    Posttranslational Regulation of Smads. Xu Pinglong,Lin Xia,Feng Xin-Hua Cold Spring Harbor perspectives in biology Transforming growth factor β (TGF-β) family signaling dictates highly complex programs of gene expression responses, which are extensively regulated at multiple levels and vary depending on the physiological context. The formation, activation, and destruction of two major functional complexes in the TGF-β signaling pathway (i.e., the TGF-β receptor complexes and the Smad complexes that act as central mediators of TGF-β signaling) are direct targets for posttranslational regulation. Dysfunction of these complexes often leads or contributes to pathogenesis in cancer and fibrosis and in cardiovascular, and autoimmune diseases. Here we discuss recent insights into the roles of posttranslational modifications in the functions of the receptor-activated Smads in the common Smad4 and inhibitory Smads, and in the control of the physiological responses to TGF-β. It is now evident that these modifications act as decisive factors in defining the intensity and versatility of TGF-β responsiveness. Thus, the characterization of posttranslational modifications of Smads not only sheds light on how TGF-β controls physiological and pathological processes but may also guide us to manipulate the TGF-β responses for therapeutic benefits. 10.1101/cshperspect.a022087
    Roles of Smads Family and Alternative Splicing Variants of Smad4 in Different Cancers. Ullah Irfan,Sun Weichao,Tang Liling,Feng Jianguo Journal of Cancer Transforming Growth Factor β (TGF-β) is one of the most common secretory proteins which are recognized by membrane receptors joined to transcription regulatory factor. TGF-β signals are transduced by the Smads family that regulate differentiation, proliferation, early growth, apoptosis, homeostasis, and tumor development. Functional study of TGF-β signaling pathway and Smads role is vital for certain diseases such as cancer. Alternative splicing produces a diverse range of protein isoforms with unique function and the ability to react differently with various pharmaceutical products. This review organizes to describe the general study of Smads family, the process of alternative splicing, the general aspect of alternative splicing of Smad4 in cancer and the possible use of spliceoforms for the diagnosis and therapeutic purpose. The main aim and objective of this article are to highlight some particular mechanisms involving in alternatives splicing of cancer and also to demonstrate new evidence about alternative splicing in different steps given cancer initiation and progression. 10.7150/jca.20906