4087

SMAD2

TGF-beta and its signaling mediators, Smad2, -3, and -4, are involved with tumor suppression and promotion functions. Smad4-/- mouse epidermis develops spontaneous skin squamous cell carcinomas (SCCs), and Smad3-/- mice are resistant to carcinogen-induced skin cancer; however, the role of Smad2 in skin carcinogenesis has not been explored. In the present study, we found that Smad2 and Smad4, but not Smad3, were frequently lost in human SCCs. Mice with keratinocyte-specific Smad2 deletion exhibited accelerated formation and malignant progression of chemically induced skin tumors compared with WT mice. Consistent with the loss of Smad2 in poorly differentiated human SCCs, Smad2-/- tumors were poorly differentiated and underwent epithelial-mesenchymal transition (EMT) prior to spontaneous Smad4 loss. Reduced E-cadherin and activation of its transcriptional repressor Snail were also found in Smad2-/- mouse epidermis and occurred more frequently in Smad2-negative human SCCs than in Smad2-positive SCCs. Knocking down Snail abrogated Smad2 loss-associated EMT, suggesting that Snail upregulation is a major mediator of Smad2 loss-associated EMT. Furthermore, Smad2 loss led to a significant increase in Smad4 binding to the Snail promoter, and knocking down either Smad3 or Smad4 in keratinocytes abrogated Smad2 loss-associated Snail overexpression. Our data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss-associated EMT during skin carcinogenesis.

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-beta1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-beta1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-beta1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-beta1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-beta1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-beta1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.CI - Copyright (c) 2010 Wiley-Liss, Inc.

The versatile cytokine transforming growth factor beta (TGF-beta) regulates cellular growth, differentiation, and migration during embryonic development and adult tissue homeostasis. Activation of TGF-beta receptors leads to phosphorylation of Smad2 and Smad3, which oligomerize with Smad4 and accumulate in the nucleus where they recognize gene regulatory regions and orchestrate transcription. Termination of Smad-activated transcription involves Smad dephosphorylation, nuclear export, or ubiquitin-mediated degradation. In an unbiased proteomic screen, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a Smad-interacting partner. PARP-1 dissociates Smad complexes from DNA by ADP-ribosylating Smad3 and Smad4, which attenuates Smad-specific gene responses and TGF-beta-induced epithelial-mesenchymal transition. Thus, our results identify ADP-ribosylation of Smad proteins by PARP-1 as a key step in controlling the strength and duration of Smad-mediated transcription.CI - Copyright (c) 2010 Elsevier Inc. All rights reserved.

Smad2 and Smad3, the intracellular mediators of transforming growth factor beta (TGF-beta) signaling, are directly phosphorylated by the activated type I receptor kinase, and then shuttle from the cytoplasm into the nucleus to regulate target gene expression. Here, we report that the 70-kDa heat-shock protein (HSP70) interacts with Smad2 and decreases TGF-beta signal transduction. Ectopic expression of HSP70 prevents receptor-dependent phosphorylation and nuclear translocation of Smad2, and blocks TGF-beta-induced epithelial-mesenchymal transition (EMT) in HaCat cells. Our findings reveal an essential role of HSP70 in TGF-beta-induced epithelial-mesenchymal transition (EMT) by impeding Smad2 phosphorylation.CI - Copyright (c) 2011. Published by Elsevier Ltd.

MicroRNAs (miRNAs) are important regulators of gastric cancer development and progression. miR-148a is one of the most frequently and highly downregulated miRNAs in gastric cancer and is associated with advanced clinical stage and poor prognosis. In this study, we investigated the role of miR-148a in gastric cancer metastasis. Levels of miR-148a were determined by qRT-PCR in 60 gastric cancer samples. Cell migration and invasion assays were performed in a stably expressing miRNA-148a gastric cancer cell line established using a lentivirus expression system. epithelial-mesenchymal transition (EMT) was evaluated using qRT-PCR and Western Blots to detect epithelial marker E-cadherin and mesenchymal marker, vimentin. Luciferase reporter assays were used to identify downstream targets and biological function of miR-148a. Gastric cancer tissue had significantly lower expression of miR-148a compared to non-tumor tissue. Low miR-148a levels were associated with lymph node metastasis, N stage, and blood vessel invasion. miR-148a overexpression inhibited metastasis of gastric cancer cells. miR-148a overexpression also downregulated vimentin expression and upregulated E-cadherin expression, suggesting that miR-148a inhibited EMT. Finally, the SMAD2 gene was identified as the direct and functional target of miR-148a. MiR-148a suppresses gastric cancer metastasis and EMT, likely via SMAD2. Restoration of miR-148a expression could have important implications in gastric cancer therapy.

epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is a major pathologic change in the development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. ERK1/2 pathway has been reported to play a key role in the carcinogenesis, cancer metastasis, and multiple fibrotic diseases. We hypothesized that ERK1/2 signaling could cross-interact with transforming growth factor beta2 (TGFbeta2)/Smad and Notch signaling pathways in the regulation of EMT in RPE cells. Here, we demonstrated that ERK1/2 signaling was activated in TGFbeta2-induced EMT in human RPE cells, while blockade of the canonical TGFbeta2/Smad2/3 signaling with SB431542 could not inhibit TGFbeta2-induced the activation of ERK1/2. Meanwhile, blockade of ERK1/2 signaling with a specific MEK/ERK1/2 inhibitor U0126 strongly prevented TGFbeta2-induced the downregulation of P-cadherin, and the upregulation of alpha-SMA, collagen type IV, N-cadherin and fibronectin in RPE cells. In addition, we also identified that blockade of ERK1/2 signaling could inhibit not only the canonical TGFbeta/Smad signaling, but also the Jagged/Notch pathway. Finally, we found that blockade of Notch pathway with a specific inhibitor DAPT could inhibit TGFbeta2-induced the activation of ERK1/2 pathway conversely. Therefore, our study provides evidence that ERK1/2 signaling can cross-interact with the canonical TGFbeta/Smad and the Jagged/Notch signaling pathways in RPE cells EMT. ERK1/2 inhibitor may have therapeutic value in the prevention and treatment of PVR and other fibrotic diseases.

TGF-beta/Smad signaling induces epithelial-mesenchymal transition (EMT) and tumor metastasis. As essential mediators in this pathway, Smad2 and Smad3 have been extensively studied and found to promote EMT and the subsequent mobility as well as invasiveness of lung cancer cells. In the present study, we determined that miR-136 directly targeted Smad2 and Smad3, leading to reduced migration and invasiveness of lung adenocarcinoma (ADC) cell lines, accompanied by increased epithelial markers as well as decreased mesenchymal markers. Moreover, ectopic expression of either Smad2 or Smad3 partially restored the malignant phenotype of ADC cells overexpressing miR-136. Taken together, our data demonstrate that miR-136 may play a tumor-suppressive role by repressing EMT and prometastatic traits via targeting Smad2 and Smad3. The potent antimetastasis property of miR-136 and its multitarget mechanism provide new insights in developing novel therapeutic approaches.

Metastasis is the major cause for the high mortality rate of pancreatic cancer. Human embryonic stem cell (hESC) associated genes frequently correlate with malignant disease progression. Recent studies have demonstrated that the embryonic protein Nodal, which plays a critical role during embryonic development, is re-expressed in several types of tumors and promotes cancers progression. However, little is known about the role of Nodal in pancreatic cancer. Here, we show that Nodal expression is upregulated in human pancreatic cancer tissues. Moreover, Nodal expression levels correlate well with the grade of pancreatic cancer differentiation. In addition, we present clear evidence that Nodal induces signal transduction through the Smad2/3-dependent pathway in vitro. Furthermore, we show that Nodal promotes pancreatic cancer cell migration and invasion, induces epithelial-mesenchymal transition (EMT) and enhances the expression of matrix metalloproteinase-2 (MMP2) and CXC chemokine receptor 4 (CXCR4). Using an in vivo liver metastasis model of pancreatic cancer, we observed that blocking Nodal signaling activity with the small-molecule inhibitor SB431542 decreases the number and size of liver metastases. Taken together, our results suggest that Nodal overexpression induces a metastatic phenotype in pancreatic cancer cells, and that targeting Nodal signaling may be a promising therapeutic strategy for pancreatic cancer.

epithelial-mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-beta (TGF-beta) signaling, and TGF-beta drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-beta also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-beta type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-beta-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-beta receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-beta/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-beta receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-beta receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-beta signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-beta-induced Smad activation through differential partitioning of receptor complexes at the cell surface.

BACKGROUND/AIMS: Transforming growth factor beta (TGF-beta) plays a major role in tumorigenesis. MicroRNA-181b (miRNA-181b) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. This study aimed to confirm the relationship of miRNA-181b and the TGF-beta-Smad2/3/4 pathway with the induction of the epithelial-to-mesenchymal transition (EMT) in gastric cancer. METHODS: This study investigated the ability of TGF-beta to induce migration by wound healing and transwell invasion assays in human gastric cancer cell lines. miRNA expression was altered using miRNA-181b mimic and inhibitor in the same system. expression of miRNA-181b, the hypothetical target gene Timp3 and EMT-related markers were analyzed by real-time real-time quantitative RT-PCR. Immunoblotting was used to investigate the levels of phospho-Smad2 and Smad4. Dual-luciferase reporter assays were performed to confirm the direct binding of miRNA-181b to Timp3. RESULTS: miRNA-181b was significantly upregulated in response to TGF-beta treatment in gastric cancer cell lines. Overexpression of miR-181b mimic induced an in vitro EMT-like change to a phenotype similar to that following TGF-beta treatment alone and was reversed by miRNA-181b inhibitor. Inhibition of TGF-beta-Smad2/3 signaling with SD-208 significantly attenuated the upregulation of miRNA-181b. Knockdown of Smad4 in gastric cancer cells strongly attenuated the upregulation of miRNA-181b. Moreover, miR-181b was found to directly target the 3' untranslated region (3'UTR) of Timp3 mRNA affecting TGF-beta-induced EMT. CONCLUSIONS: Our results elucidate a novel mechanism through which the TGF-beta pathway regulates the EMT of gastric cancer cells by increasing the levels of miRNA-181b to target Timp3 via the Smad2/3/4-dependent pathway. These findings provide insights into the cellular and environmental factors regulating EMT, which may guide future studies on therapeutic strategies targeting these cells.CI - (c) 2016 The Author(s) Published by S. Karger AG, Basel.

Vasculogenic mimicry (VM) is a nonangiogenesis-dependent pathway that promotes tumor growth and disease progression. Nodal signaling has several vital roles in both embryo development and cancer progression. However, the effects of Nodal signaling on VM formation in breast cancer and its underlying mechanisms are ill-defined. We analyzed the relationship between Nodal signaling and VM formation in one hundred human breast cancer cases and the results showed that the expression of Nodal was significantly correlated with VM formation, tumor metastasis, differentiation grade, TNM stage and poor prognosis. Furthermore, up-regulation of Nodal expression promoted VM formation of breast cancer cells in vitro and in vivo. Knockdown of Nodal expression restrained VM formation. In addition, Nodal induced EMT and up-regulated the expression of Slug, Snail and c-Myc. We found that blocking the Smad2/3 pathway by administering SB431542 inhibited VM formation in breast cancer cell lines and xenografts. Taken together, Nodal signaling through the Smad2/3 pathway up-regulated Slug, Snail and c-Myc to induce EMT, thereby promoting VM formation. Our study suggests that the Nodal signaling pathway may serve as a therapeutic target to inhibit VM formation and improve prognosis in breast cancer patients.

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-beta-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.

Although progress in clinical and basic research has significantly increased our understanding of breast cancer, little is known about the molecular mechanism underlying breast cancer metastasis. Identification of effective therapeutic targets to prevent breast cancer metastasis is urgently needed. The function of miR-503-3p has been investigated in other cancers, but its role in breast cancer remains undefined. Here, we found that miR-503-3p was overexpressed in breast cancer tissue and plasma compared with adjacent normal breast tissue and with plasma from healthy individuals. Moreover, we identified miR-503-3p to be an oncogene of breast cancer cell proliferation, migration and invasion. Upregulation of miR-503-3p in breast cancer cells inhibited expression of epithelial-mesenchymal transition (EMT)-related protein SMAD2 and the epithelial marker protein E-cadherin by directly binding to their mRNA 3' untranslated region, whereas increased expression of mesenchymal marker proteins, including vimentin and N-cadherin. Taken together, our findings support a critical role for miR-503-3p in induction of breast cancer EMT and suggest that plasma miR-503-3p may be a useful diagnostic biomarker for breast cancer.CI - Copyright (c) 2016 Institute of Genetics and developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Cytosolic phospholipase A2alpha (cPLA2alpha), a key phospholipase that regulates lipid metabolism, plays an important role in tumor progression. In the present study of hepatocellular carcinoma (HCC), cPLA2alpha was overexpressed in highly metastatic HCC cell lines. Immunohistochemical staining showed increased levels of cPLA2alpha at the invasive edges of HCC, and a clinicopathological analysis of samples from 111 patients revealed that its expression level was linked with micro-vascular invasion and cirrhosis. Knockdown of cPLA2alpha inhibited migration, probably due to its role in actin polymerization. Overexpression of cPLA2alpha promoted cell migration and invasion. Based on the mechanistic analysis, our data suggested that cPLA2alpha mediate epidermal growth factor (EGF) induced epithelial-mesenchymal transition (EMT) through PI3K/AKT/ERK pathway. cPLA2alpha activity was required for the transforming growth factor-(TGF)-beta-induced EMT. However, cPLA2alpha inhibited Smad2/3 activation and promoted the activation of the PI3K/AKT/ERK pathway. A xenograft tumor transplant model confirmed the role of cPLA2alpha in HCC invasion and metastasis. Based on the mechanistic analysis, cPLA2alpha mediated both EGF- and TGF-beta-induced EMT, which are essential for HCC metastasis. cPLA2alpha is a potentially target for novel therapies of HCC.CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.

As a Ca(2+) binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-beta1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-beta1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-beta1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-beta1. On treatment with TGF-beta1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-beta1. These results indicated that CRT regulates EMT induced by TGF-beta1 through Smad signaling pathway. Finally, TGF-beta1-induced-EMT enhanced store-operated Ca(2+) influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-beta1 on thapsigargin (TG) -induced Ca(2+) release, but had failed to reduce store-operated Ca(2+) influx. The alteration of intracellular Ca(2+) concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-beta1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-beta1-induced-EMT through modulating Smad signaling. Furthermore, TGF-beta1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.CI - Copyright (c) 2017 Elsevier Ltd. All rights reserved.

The downregulation of tight junction protein CLDN6 promotes breast cancer cell migration and invasion; however, the exact mechanism underlying CLDN6 downregulation remains unclear. CLDN6 silence is associated with DNA methyltransferase 1 (DNMT1) mediated DNA methylation, and DNMT1 is regulated by the transforming growth factor beta (TGFbeta)/SMAD pathway. Therefore, we hypothesized that TGFbeta/SMAD pathway, specifically SMAD2, may play a critical role for CLDN6 downregulation through DNA methyltransferase 1 (DNMT1) mediated DNA methylation. To test this hypothesis, we blocked the SMAD2 pathway with SB431542 in two human breast cancer cell lines (MCF-7 and SKBR-3). Our results showed that treatment with SB431542 led to a decrease of DNMT1 expression and the binding activity for CLDN6 promoter. The methylation level of CLDN6 promoter was decreased, and simultaneously CLDN6 protein expression increased. Upregulation of CLDN6 inhibited epithelial to mesenchymal transition (EMT) and reduced the migration and invasion ability of both MCF-7 and SKBR-3 cells. Furthermore, knocked down of CLDN6 abolished SB431542 effects on suppression of EMT associated gene expression and inhibition of migration and invasion. Thus, we demonstrated that the downregulation of CLDN6 is regulated through promoter methylation by DNMT1, which depends on the SMAD2 pathway, and that CLDN6 is a key regulator in the SMAD2/DNMT1/CLDN6 pathway to inhibit EMT, migration and invasion of breast cancer cells.

Idiopathic pulmonary fibrosis (IPF) is a chronic and usually progressive lung disease and the epithelial-mesenchymal transition (EMT) may play an important role in the pathogenesis of pulmonary fibrosis. IL-17 is a proinflammatory cytokine which promotes EMT profiles in lung inflammatory diseases. In this study, we investigated the effect of IL-17 on EMT in alveolar epithelial cell line A549 and the role of TGFbeta1-Smad and ERK signaling pathways in the process. Morphological observation on the cells was performed under inverted microscope. The mRNA and protein expressions of E-cad and alpha-SMA were detected by quantitative RT-PCR and western blotting. The mRNA and protein expressions of TGF-beta1 were analyzed via quantitative RT-PCR and ELISA. expressions of Smad2/3, p-Smad2/3, ERK1/2, p-ERK1/2 and p-JNK were examined by western blotting. The results indicated that IL-17 can induce A549 cells to undergo morphological changes and phenotypic markers changes, such as down-regulated E-cad expression and up-regulated alpha-SMA expression. Additionally, IL-17 enhanced TGF-beta1 expression and stimulated Smad2/3 and ERK1/2 phosphorylation in A549 cells. However, there were no significant differences in the expression of phosphorylated JNK in A549 cells with or without IL-17 treatment. SB431542 or U0126 treated cells showed inhibited morphological changes and phenotypic markers expression, such as up-regulated E-cad expression and down-regulated alpha-SMA expression. In summary, our results suggest that IL-17 can induce A549 alveolar epithelial cells to undergo EMT via the TGF-beta1 mediated Smad2/3 and ERK1/2 activation.

Thymoquinone, a major ingredient of black seed oil (Nigella sativa), has been shown to exhibit anticancer capacity in various types of cancers. However, there are few studies concerning the correlation between thymoquinone and epithelial-to-mesenchymal transition (EMT) in prostate cancer. In the present study, we firstly found that thymoquinone showed antimetastatic capacity in prostate cancer DU145 and PC3 cells. Additionally, thymoquinone reversed EMT by increasing E-cadherin expression and decreasing vimentin and Slug expression in a concentration-dependent manner. Recent studies have shown that the transforming growth factor-beta (TGF-beta) signaling pathway may be associated with EMT. Intriguingly, the expression of TGF-beta, Smad2 and Smad3 at the mRNA and protein levels was notably reduced upon thymoquinone treatment in prostate cancer DU145 and PC3 cells. Subsequently, we confirmed that thymoquinone repressed metastasis and EMT of prostate cancer through downregulation of the TGF-beta/Smad2/3 signaling pathway, which may be partially reversed by TGF-beta overexpression. In summary, our findings demonstrated that thymoquinone suppressed the metastatic phenotype and reversed EMT of prostate cancer cells by negatively regulating the TGF-beta/Smad2/3 signaling pathway. These findings suggest that thymoquinone is a potential therapeutic agent against prostate cancer which functions by targeting TGF-beta.

The pathological development of lens epithelial cells (LECs) leads to posterior capsular opacification (PCO). This study was undertaken to investigate the effects of microRNA-486-5p (miR-486-5p) on TGF-beta2-induced proliferation, invasion and epithelial-mesenchymal transition (EMT) in the lens epithelial cell line SRA01/04, and to explore the underlying molecular mechanisms. The expression of miR-486-5p in TGF-beta2-induced SRA01/04 cells was down-regulated, and the expression of Smad2, p-Smad2 and p-Smad3 was up-regulated. A dual-luciferase reporter assay revealed that miR-486-5p directly targets the 30'-UTR of Smad2. MiR-486-5p mimic transfection markedly down-regulated the expression levels of Smad2, thus inhibiting the expression of p-Smad2 and p-Smad3. MiR-486-5p overexpression in SRA01/04 cells markedly suppressed TGF-beta2-induced proliferation and invasion, inhibited protein expression of CDK2 and CDK4, down-regulated fibronectin, alpha-SMA and vimentin and up-regulated E-cadherin; these effects were partly reversed by Smad2 overexpression. In short, these data show that miR-486-5p overexpression can inhibit TGF-beta2-induced proliferation, invasion and EMT in SRA01/04 cells by repressing Smad2/Smad3 signalling, implying that miR-486-5p may be an effective target to interfere in the progression of PCO.

The transforming growth factor-beta1 (TGF-beta1) signaling pathways contribute to cell metastasis and epithelial-mesenchymal transition (EMT). Golgi protein 73 (GP73), a type II transmembrane protein in the Golgi apparatus, was initially regarded as a potential biomarker for the diagnosis of hepatocellular carcinoma (HCC). Recently, it was reported that GP73 acts as a key oncogene by promoting HCC growth and metastasis. However, the role of GP73 in metastasis, especially when involving signaling pathways, is uncertain. Here, we report that GP73, which is upregulated in HCC tissues and cell lines, is associated with tumor size, tumor node metastasis stage, distant metastasis and vascular invasion. The ectopic overexpression of GP73 increased HCC cell invasion, EMT and metastasis both in vitro and in vivo. Conversely, GP73 knockdown inhibited invasion and EMT. Moreover, GP73 enhanced p-Smad2 and p-Smad3 levels by mediating TGF-beta1, thus leading to the promotion of EMT and invasion in HCC cells. In contrast, we used SB431542 (SB) to repress p-Smad2 and p-Smad3 expression, which resulted in a reversion of EMT. Furthermore, when the TGF-beta1/Smad pathway was blocked, upregulation of GP73 still caused an enhanced EMT and invasion, and downregulation of GP73 resulted in a decreased in EMT and invasion. In clinical HCC samples, GP73 positively correlated with TGF-beta1/Smad2, which was upregulated in HCC. Taken together, our findings highlight the important role of GP73 in regulating EMT and metastasis in HCC partly by targeting TGF-beta1/Smad2 signaling, suggesting that GP73 may represent a novel potential therapeutic target and prognostic marker for the treatment and diagnosis of HCC.

Pancreatic cancer is the fourth leading cause of cancer-associated mortality worldwide, with an overall 5-year survival rate <8%. We studied the therapeutic effect of itraconazole (ITZ), a commonly used broad-spectrum anti-fungal agent, in the treatment of pancreatic cancer, and to reveal the underlying anticancer mechanisms. Effects of ITZ on cell proliferation, apoptosis, invasion and migration were observed by MTT assays and colony formation assays, flow cytometry, wound scratch assays and transwell assays, respectively. Western blotting and immunofluorescence were performed to investigate the effect of ITZ on the epithelial to mesenchymal transition (EMT) of pancreatic cancer cells. Recombinant transforming growth factor-beta (TGF-beta) and TGF-beta neutralizing antibody were used to study the effect of ITZ on the TGF-beta/SMAD2/3 signaling. Transgenic engineered mice which harboring the spontaneous pancreatic cancer was applied to investigate the therapeutic role of ITZ in vivo. We report that ITZ inhibited the viability and induced apoptosis of pancreatic cancer cells. Furthermore, ITZ suppressed the invasion and migration of pancreatic cancer cells. We found that ITZ treatment was efficient in suppressing EMT and that the effect of ITZ was partially mediated by impaired TGF-beta/SMAD2/3 signaling. The role of TGF-beta/SMAD2/3 signaling in mediating the effect of ITZ was confirmed based on the results that recombinant TGF-beta induced, but the TGF-beta neutralizing antibody inhibited EMT as well as the invasion and migration of pancreatic cancer cells. Also, the anticancer effect of ITZ could be partially reversed by recombinant TGF-beta. Furthermore, treatment with ITZ suppressed growth of tumor in vivo. Taken together, we suggest that ITZ may potentially serve as a new chemotherapeutic agent for the treatment of pancreatic cancer.

AIM: Our previous study found S100A11 was significantly raised in intrahepatic cholangiocarcinoma cells, but the relationship between S100A11 and intrahepatic cholangiocarcinoma remains unclear. METHODS: We investigated the effect of silencing S100A11 on TGF-beta1-induced epithelial-mesenchymal transition (EMT), cell migration and invasion. RESULTS: Our results demonstrated silencing S100A11 inhibited TGF-beta1-induced cell migration, invasion and EMT, expression of EMT markers E-cadherin, N-cadherin, beta-catenin, vimentin, Slug and Snail was reversed. Furthermore, TGF-beta1-induced p-SMAD2 and 3 were also inhibited due to low S100A11 expression. CONCLUSION: Our present study indicated that S100A11 promotes EMT through accumulation of TGF-beta1 expression, and TGF-beta1-induced upregulation of p-SMAD2 and 3.

Activation of metastatic reprogramming is critical for tumour metastasis. However, more detailed knowledge of the underlying mechanism is needed to enable targeted intervention. Here, we show that paraspeckle component 1 (PSPC1), identified in an aberrant 13q12.11 locus, is upregulated and associated with poor survival in patients with cancer. PSPC1 promotes tumorigenesis, epithelial-to-mesenchymal transition (EMT), stemness and metastasis in multiple cell types and in spontaneous mouse cancer models. PSPC1 is the master activator for transcription factors of EMT and stemness and accompanies c-Myc activation to facilitate tumour growth. PSPC1 increases transforming growth factor-beta1 (TGF-beta1) secretion through an interaction with phosphorylated and nuclear Smad2/3 to potentiate TGF-beta1 autocrine signalling. Moreover, PSPC1 acts as a contextual determinant of the TGF-beta1 pro-metastatic switch to alter Smad2/3 binding preference from tumour-suppressor to pro-metastatic genes. Having validated the PSPC1-Smads-TGF-beta1 axis in various cancers, we conclude that PSPC1 is a master activator of pro-metastatic switches and a potential target for anti-metastasis drugs.

We previously reported that osteopontin (OPN), a matrix structural glycophosphoprotein, is upregulated in the injured mouse lens prior to the epithelial-mesenchymal transition (EMT). Here, we investigated the role of this protein in EMT of the lens epithelium during wound healing. The crystalline lens was injured by needle puncture in OPN-null (KO, n=40) and wild-type (WT, n=40) mice. The animals were killed at day 1, 2, 5, and 10 postinjury. Immunohistochemistry was employed to detect alpha-smooth muscle action (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and phospho-Smad2/3. Cell proliferation was assayed by examining uptake of bromodeoxyuridine (BrdU). The results showed that injury-induced EMT of mouse lens epithelium, as evaluated by histology, expression pattern of alphaSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFbeta1 and TGFbeta2 in the epithelium was also inhibited. Cell proliferation was more active in KO mice as compared with WT mice at day 1 and 2, but not at day 5 and 10. An in vitro experiment shows OPN facilitates cell adhesion of lens epithelial cell line. OPN is required for activation of Smad2/3 signal in an injured lens epithelium and lens cell EMT.

PURPOSE: Extracellular matrix (ECM) accumulates during the development of posterior capsule opacification (PCO). Vitronectin, an ECM component that is generally prominent in wound healing, has been detected in PCO specimens. Here we set out to investigate the distribution of vitronectin in the lens and determine how it, and other ECM components, influence the lens epithelial phenotype. METHODS: Rat lens epithelial explants were cultured on vitronectin, fibronectin, and laminin substrata. Explants were monitored for cell migration and the appearance of markers for epithelial mesenchymal transition (EMT), using phase contrast microscopy and immunohistochemistry, respectively. Explants were also monitored for evidence of Smad signaling. Vitronectin expression was analyzed in embryonic and postnatal rodent lens development by immunohistochemistry, western blotting, and in situ hybridization. RESULTS: Vitronectin, like fibronectin and laminin, provided a good substratum for cellular attachment and migration. However, in the case of vitronectin and fibronectin, this was accompanied by a major phenotypic change. On either vitronectin or fibronectin, but not laminin, most of the cells became elongated, spindle-shaped and were strongly reactive for filamentous alpha-smooth muscle actin. In these respects this transition was typical of the well known TGFbeta-induced EMT. In explants cultured on vitronectin and fibronectin, but not laminin, cell nuclei showed prominent reactivity for Smad 2/3. Vitronectin was also shown to be expressed during embryonic and postnatal development. Initially mRNA and protein were detected in all lens cells, however as development progressed, expression became restricted to cells of the epithelium and transition zone. CONCLUSIONS: The results clearly show that lens cell engagement with a vitronectin or a fibronectin, but not laminin, substratum has a potent EMT promoting effect and that Smad 2/3 signaling is involved. Thus when considering strategies to slow or prevent PCO, these results highlight the need to take into account ECM molecules such as vitronectin that have the capacity to promote EMT.

TGF-beta1 induces epithelial-mesenchymal transition (EMT) and autophagy in a variety of cells. However, the role of autophagy in TGF-beta1-induced EMT has not been clearly elucidated and the underlying mechanisms are unclear. In the present study, we found that TGF-beta1 induced both autophagy and EMT in mouse tubular epithelial C1.1 cells. Inhibition of autophagy by 3-methyladenine or siRNA knockdown of Beclin 1 reduced TGF-beta1-induced increase of vimentin and decreased E-cadherin expression. In contrast, rapamycin-associated enhancement of TGF-beta1-induced autophagy increased EMT of C1.1 cells. Serum rescue inhibited autophagy followed by reversal of EMT. Blocking of autophagosome-lysosomal but not proteosomal degradation reduced the decrease of E-cadherin, demonstrating a role for autophagy in degradation of E-cadherin during EMT. Autophagy promoted the activation of Src and Src-associated phosphorylation of beta-catenin at Y-654 leading to pY654-beta-catenin/p-Smad2 complex formation. Chromatin immunoprecipitation assay demonstrated binding by the pY654-beta-catenin/p-Smad2 complex to ILK promoter thus increasing ILK expression. Taken together, our results demonstrate that TGF-beta1-induced autophagy links beta-catenin and Smad signaling to promote EMT in C1.1 cells through a novel pY654-beta-catenin/p-Smad2/ILK pathway. The pathway delineated links disruption of E-cadherin/beta-catenin-mediated cell-cell contact to induction of EMT via upregulation of ILK.CI - Copyright (c) 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Extracellular matrix deposition during tubulointerstitial fibrosis (TIF), a central pathological process in patients with diabetic nephropathy (DN), is driven by locally activated, disease-relevant myofibroblasts. Myofibroblasts can arise from various cellular sources, e.g., tubular epithelial cells via a process named epithelial-to-mesenchymal transition (EMT). Transforming growth factor beta 1 (TGF-beta1) and its downstream Smad signaling play a critical role in both TIF and EMT. Whereas Smad3 is one central mediator, the role of the other prominently expressed variant, Smad2, is not completely understood. In this study, we sought to analyze the role of renal Smad2 in the development of TIF and EMT during streptozotocin-induced DN by using a fibroblast-specific protein 1 (FSP1)-promotor-driven SMAD2 knockout mouse model with decreased tubular, endothelial, and interstitial Smad2 expression. In contrast to wild-type diabetic mice, diabetic SMAD2 knockout mice showed the following features: (1) significantly reduced DN and TIF (shown by KIM1 expression; periodic acid Schiff staining; collagen I and III, fibronectin, and connective tissue growth factor deposition); (2) significantly reduced tubular EMT-like changes (e.g., altered Snail1, E-cadherin, matrix metalloproteinase 2, and vimentin deposition); and (3) significantly decreased expression of myofibroblast markers (alpha-smooth muscle actin, FSP1). As one mechanism for the protection against diabetes-induced TIF and EMT, decreased Smad3 protein levels and, as a possible consequence, reduced TGF-beta1 levels were observed in diabetic SMAD2 knockout mice. Our findings thus support the important role of Smad2 for pro-fibrotic TGF-beta/Smad3 signaling in experimental DN.

epithelial-mesenchymal transition (EMT) is a process associated with airway remodeling in chronic obstructive pulmonary disease (COPD), which leads to progressive pulmonary destruction. Panax ginseng is a traditional herbal medicine that has been shown to improve pulmonary function and exercise capacity in patients with COPD. Ginsenoside Rg1 is one of the main active components and was shown to inhibit oxidative stress and inflammation. The present study investigated the hypothesis that ginsenoside Rg1 attenuates EMT in COPD rats induced by cigarette smoke (CS) and human bronchial epithelial (HBE) cells exposed to cigarette smoke extract (CSE). Our data showed that CS or CSE exposure increased expression of the mesenchymal marker alpha-smooth muscle actin (alpha-SMA) and decreased expression of the epithelial marker epithelial cadherin (E-cad) in both lung tissues and HBE cells, which was markedly suppressed by ginsenoside Rg1. Importantly, CS-induced upregulation of TGF-beta1/Smad pathway components, including TGF-beta1, TGF-betaR1, phospho-Smad2, and phospho-Smad3, was also inhibited by ginsenoside Rg1. Additionally, ginsenoside Rg1 mimicked the effect of SB525334, a TGF-betaR1-Smad2/3 inhibitor, on suppression of EMT in CSE-induced HBE cells. Collectively, we concluded that ginsenoside Rg1 alleviates CS-induced pulmonary EMT, in both COPD rats and HBE cells, via inhibition of the TGF-beta1/Smad pathway.