4089

SMAD4

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.

OBJECTIVE: The purpose of this study was to evaluate the prognostic relevance of SMAD4 expression in pancreatic cancer. METHODS: We analyzed the correlations between SMAD4 expression and clinicopathological parameters and outcome in 174 patients with pancreatic cancer. Specimens were also classified into subtypes reflecting epithelial-to-mesenchymal transition, based on E-cadherin and vimentin. RESULTS: We found that 59.8% (104/174) of patients were SMAD4 and 40.2% (70/174) were SMAD4. disease-specific survival in patients with SMAD4 was significantly better than that in SMAD4. SMAD4 status was significantly correlated with portal vein invasion, lymph vessel invasion, and perineural invasion and was an independent prognostic factor. SMAD4 was significantly associated with mesenchymal phenotype. The loss of SMAD4 expression was found in 49.4% of patients with no vascular invasion, 61.9% with portal vein invasion, 76.5% with common hepatic artery invasion, and 80.8% with superior mesenteric artery invasion. In addition, the specimens from 59.0% of patients with local recurrence, 66.7% of those with both local and distant recurrence, and 73.7% of those with distant recurrence were SMAD4. CONCLUSIONS: The loss of SMAD4 expression is an independent prognostic factor and seems to be associated with tumor progression, pattern of failure, and epithelial-to-mesenchymal transition status. Preoperative stratification based on SMAD4 could lead to appropriate treatment strategy.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and cetuximab, a monoclonal antibody targeting this receptor, is widely used to treat these patients. In the following investigation, we examined the role of SMAD4 down-regulation in mediating epithelial-to-mesenchymal transition (EMT) and cetuximab resistance in HNSCC. We determined that SMAD4 downregulation was significantly associated with increased cell motility, increased expression of vimentin, and cetuximab resistance in HNSCC cell lines. In the HNSCC genomic dataset obtained from The cancer Genome Atlas, SMAD4 was altered in 20/279 (7%) of HNSCC via homozygous deletion, and nonsense, missense, and silent mutations. When SMAD4 expression was compared with respect to human papillomavirus (HPV) status, HPV-positive tumors had higher expression compared to HPV-negative tumors. Furthermore, higher SMAD4 expression also correlated with higher CDKN2A (p16) expression. Our data suggest that SMAD4 down-regulation plays an important role in the induction of EMT and cetuximab resistance. Patients with higher SMAD4 expression may benefit from cetuximab use in the clinic.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Although AhR plays a crucial role in air toxicant-induced carcinogenesis, AhR expression was shown to negatively regulate tumorigenesis. Therefore, in the present study, we investigated the effect of AhR without ligand treatment on cancer invasion in lung cancer cell lines. Lung cancer cells expressing lower levels of AhR showed higher invasion ability (H1299 cells) compared with cells expressing higher levels of AhR (A549 cells). Overexpression of AhR in H1299 cells inhibited the invasion ability. We found that vimentin expression was inhibited in AhR-overexpressing H1299 cells. Additionally, the expression of EMT-related transcriptional factors Snail and ID-1 decreased. Interestingly, we found that Smad4 degradation was induced in AhR-overexpressing H1299 cells. Our data showed that AhR could interact with Jun-activation domain binding protein (Jab1) and Smad4, which may cause degradation of Smad4 by the proteasome. Our data suggest that AhR affects the transforming growth factor-beta signaling pathway by inducing Smad4 degradation by the proteasome and suppressing tumor metastasis via epithelial to mesenchymal transition reduction in lung cancer cells.CI - Copyright (c) 2016 Elsevier Ireland Ltd. All rights reserved.

The TGF-beta/Smad signaling pathway plays important roles in cancer cell proliferation, apoptosis, differentiation, angiogenesis and epithelial-mesenchymal transition (EMT), which is the key event in the early stages of cancer metastasis and enhances the capability of cell migration and invasion. Smad4 acts as the only Co-Smad of TGF/Smad signaling pathway and plays the key role in TGF-beta-mediated EMT. Nevertheless, the mRNA regulation mechanisms of Smad4 in human non-small cell lung cancer (NSCLC) remains largely unclear. Computational algorithms predicted that the 3'-UTR of Smad4 is a target of miR205. Here, we validated that miR205 could directly bind to 3'-UTR of Smad4 by luciferase assays. Moreover, we investigated the functional roles of miR205 and its molecular link to Smad4 in lung cancer cells. In this study, we confirmed that overexpression of miR205 suppressed the expression of Smad4, in turn, weakened the TGF-beta/Smad signaling pathway and inhibited TGF-beta/Smad4-induced EMT, invasion and migration ultimately. Furthermore, this study shows that miR205 can serve as a promising therapeutic target of highly aggressive NSCLC.

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.

Sec8 is one of the subunits of the exocyst, which is an evolutionarily conserved complex of eight proteins, comprising Sec3 (EXOC1), Sec5 (EXOC2), Sec6 (EXOC3), Sec8 (EXOC4), Sec10 (EXOC5), Sec15 (EXOC6), Exo70 (EXOC7), and Exo84 (EXOC8) subunits. Sec8 knockout mice embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly. During embryonic development, the first epithelial-mesenchymal transition (EMT) event occurs at gastrulation. Sec8 may be involved in the early embryonic development through EMT. However, the function of Sec8 in EMT remains unclear. In the present study, it was found that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression at the basal transcriptional level, thereby modulating cell migration and adhesion. Furthermore, Sec8 knockdown decreased CREB binding protein (CBP) expression at mRNA and protein levels. However, CBP knockdown did not affect Sec8 expression. These results indicated that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression through CBP, thereby mediating the EMT.CI - Copyright A(c) 2016 Elsevier Inc. All rights reserved.

In advanced prostate cancer, small ubiquitin-like modifier (SUMO)-specific cysteine protease 1 (SENP1) is up-regulated. However, the role of SENP1 in regulating deSUMOylation of TGF-beta/SMADs signaling is unknown. In this study, we developed a lentiviral vector, PLKO.1-shSENP1, to silence SENP1 in prostate cancer cells with high metastatic characteristics (PC3M). Likewise, we also created an adenovirus vector, Ad5/F11p-SENP1 to over-express SENP1 in prostate cancer cells with low metastatic potential (LNCaP). We showed that silencing of SENP1 promoted cellular apoptosis, and inhibited proliferation and migration of PC3M cells. Moreover, SENP1 silencing increased the SMAD4 expression at protein level, up-regulated E-cadherin and down-regulated Vimentin expression, indicating the inhibition of epithelial mesenchymal transition (EMT). Furthermore, SMAD4 interference abolished SENP1-mediated up-regulation of E-cadherin, suggesting that SENP1 regulated E-cadherin expression via SMAD4. SENP1 over-expression in LNCaP cells reduced SMAD4 protein, and promoted EMT via decreasing E-cadherin and increasing Vimentin. Moreover, down-regulation of SMAD4 and E-cadherin were blocked, after transfection with two SUMOylation sites mutated SMAD4, suggesting that SENP1 might reduce SMAD4 levels to regulate E-cadherin expression via deSUMOylation of SMAD4. In conclusion, SENP1 deSUMOylated SMAD4 to promote EMT via up-regulating E-cadherin in prostate cancer cells. Therefore, SENP1 is a potential target for treatment of advanced prostate cancer.

cancer stem cells (CSCs) are undifferentiated cancer cells with a high tumorigenic activity, the ability to undergo self-renewal, and a multilineage differentiation potential. Clinical evidence suggests that CSCs in a tumor mass are the cellular determinants to promote cancer invasion and metastasis. MicroRNAs (miRNAs) have emerged as important modulators of cancer stem cell characteristics. Unveiling the candidate miRNAs that regulate CSCs may provide novel therapeutic targets against cancer. We analyzed the miRNA expression profiles regulating the cancer stem-like cell characteristics in gastric cancer. Gastric cancer stem cells (GCSCs) were sorted using the stem cell marker CD44 by fluorescence-activated cell sorting. Functional studies revealed that CD44(+) cells formed more sphere colonies and showed higher invasiveness than CD44(-) cells. miRNA microarray analysis revealed that miR196a5p was significantly upregulated in CD44(+) cells than CD44(-) cells. Suppression of miR196a5p led to decreased colony formation and invasion of GCSCs. miR196a5p decreased the expression of Smad4 by targeting 3'-UTR of the mRNA. The expression of Smad4 in gastric cancer tissues was correlated with differentiation state of tumors, TNM stage and depth of invasion. The stimulation of epithelial-mesenchymal transition (EMT) by miR196a5p in cancer stem-like cells was abolished by overexpression of Smad4. Collectively, these data demonstrate that miR196a5p has a key role in EMT and invasion by targeting Smad4 in GCSCs. miR196a5p may serve as a potential target for gastric cancer therapy.

USP17 is upregulated in several cancers, indicating that USP17 might play essential functions in tumor development. However, the function of USP17 in osteosarcoma is still unknown. Our work aimed to investigate the function of USP17 in osteosarcoma. We found that the expression of USP17 was upregulated in osteosarcoma tissues and cell lines, including MG-63 and U2OS. Several functional experiments, such as colony formation analysis, Cell Counting Kit-8 assay, wound healing analysis, and transwell assay, showed that USP17 promoted cell proliferation, migration, and invasion. Moreover, we found that USP17 facilitated migration and invasion through promoting epithelial-mesenchymal transition. SMAD4 has been found to regulate epithelial-mesenchymal transition, co-immunopurification, and glutathione S-transferase pull-down analysis demonstrated that USP17 interacted with SMAD4. Furthermore, USP17 stabilized SMAD4 through its deubiquitinase activity. In conclusion, this study shows that USP17 enhances osteosarcoma cell proliferation and invasion through stabilizing SMAD4.

epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. During EMT, tumor cells acquire the capacity to migrate and invade the stroma. Activation of the transforming growth factor-b (TGF-b) signaling pathway is of major importance for the initiation of EMT. Smad4, an essential protein of this pathway, is known to complex with multiple transcription factors (e.g. Snail-1, Slug, Twist-1), in various types of cancer, promoting the repression or activation of target genes. The role of Smad4 in colorectal cancer (CRC) is not straightforward so far. In the present study forty eight resected CRC tumor specimens were immunohistochemically examined in order to assess the expression of Smad4 and its association with E-cadherin, Snail-1, Slug, Twist-1 protein expression and with various pathological parameters. Smad4 was found to be positively correlated with Snail-1, Slug and Twist-1 expression (p < 0.001). On the other hand it was negatively correlated with the expression of E-cadherin (p < 0.001). Furthermore, lymphatic invasion could be clearly associated with Smad4 expression, a finding complying with the metastatic ability of EMT cells. In conclusion, Smad4 could be considered as a central component of EMT transition in human colorectal cancer that combines with transcriptional factors to reduce E-cadherin and alter the expression of the epithelial phenotype.

Long noncoding RNAs (lncRNAs) are associated with various diseases including cataracts. The role of lncRNA potassium voltagegated channel subfamily Q member 1 opposite strand/antisense transcript 1 (KCNQ1OT1) on lens epithelial cell (LEC) proliferation and epithelialmesenchymal transition (EMT) in cataracts disease remains unclear. In the present study, KCNQ1OT1 and mothers against decapentaplegic homolog (SMAD)4 expression levels were upregulated in human cataract lens posterior capsular samples and in transforming growth factor (TGF)beta2treated SRA01/04 cells, as demonstrated by reverse transcriptionquantitative polymerase chain reaction, immunohistochemical staining and western blot analyses. A further loss of function test revealed that suppression of KCNQ1OT1 inhibited the proliferation and EMT of TGFbeta2treated SRA01/04 cells. Additionally, the present study reported that increase and decrease of KCNQ1OT1 regulated SMAD4 expression, which indicated that SMAD4 may be a downstream gene of KCNQ1OT1. Finally, a constructed SMAD4 RNA interference experiment confirmed that the function of KCNQ1OT1 was to act on LEC proliferation and EMT, and this was achieved via the SMAD4 signaling pathway. The findings of the present study may provide a novel target for molecular therapy of cataracts disease.

epithelial-mesenchymal transition (EMT) is considered a prerequisite for tumor invasion and metastasis in many cancers. However, the mechanisms underlying EMT in nasopharyngeal carcinoma (NPC) is largely unknown. In this study, we found that transforming growth factor-beta (TGF-beta), which reportedly promotes EMT in multiple cancers, can trigger EMT and increase the invasive and migratory capacities of NPC cells. Conversely, the downregulation of SMAD4, a vital member of the canonical TGF-beta pathway, reversed the TGF-beta-induced EMT, invasion, and migration. Further experiments revealed that SMAD4 was the target of miRNA-34a, which was downregulated in NPC tissues and suppressed NPC cell metastasis in vivo. miRNA-34a overexpression also antagonized the TGF-beta-induced EMT progression, invasion, and migration through SMAD4 inhibition. However, the restoration of SMAD4 expression rescued the inhibitory effects of miRNA-34a on tumorigenesis. All these results confirmed that miRNA-34a suppressed the TGF-beta-induced EMT, invasion, and migration of NPC cells by directly targeting SMAD4, which indicated the potential of miR-34a as a therapeutic target against NPC.CI - Copyright (c) 2018 Elsevier Masson SAS. All rights reserved.