Downregulation Of Smad3 Research Articles

The Role of MicroRNA-21 in Venous Neointimal Hyperplasia: Implications for Targeting miR-21 for VNH Treatment.

The molecular mechanism of hemodialysis access arteriovenous fistula (AVF) failure due to venous neointimal hyperplasia (VNH) is not known. The role of microRNA-21 (miR-21) in VNH associated with AVF failure was investigated by performing in vivo and in vitro experiments. In situ hybridization results revealed that miR-21 expression increased and was associated with fibroblasts in failed AVFs from patients. In a murine AVF model, qRT-PCR gene expression results showed a significant increase in miR-21 and a decrease in miR-21 target genes in graft veins (GVs) compared to contralateral veins in mouse AVF. miR-21 knockdown in GVs was performed using a lentivirus-mediated small hairpin RNA (shRNA), and this improved AVF patency with a decrease in neointima compared to control GVs. Moreover, loss of miR-21 in GVs significantly decreased the Tgfβ1, Col-Ia, and Col-Iva genes. Immunohistochemistry demonstrated a significant decrease in myofibroblasts and proliferation with an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in miR-21-knockdown vessels, along with a decrease in hypoxia-inducible factor-1 alpha (HIF-1α) and phospho-SMAD2 (pSMAD-2) and phospho-SMAD3 (pSMAD-3) and an increase in phosphatase and tensin homolog (PTEN) staining. Hypoxic fibroblast knockdown for miR-21 showed a significant decrease in Tgfβ-1 expression and pSMAD-2 and -3 levels and a decrease in myofibroblasts. These results indicate that miR-21 upregulation causes VNH formation by fibroblast-to-myofibroblast differentiation.

Abstract 5314: Smad4 loss upregulates chemokine expression and promotes tumor inflammation and growth in head and neck cancer

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide. Of the patients diagnosed with HNSCC, previous findings show that SMAD4 loss is more common in recurrent HNSCC patients compared with newly diagnosed cases and is strongly associated with poor patient survival and metastases. SMAD4 is a tumor suppressor gene and is a central mediator of the transforming growth factor β (TGF-β) signaling pathway and acts as a co-SMAD which binds receptor-regulated SMADs such as SMAD2 and 3. SMAD2/3/4 complex is important for regulating downstream gene transcription that is involved in tumor invasion and metastasis. Purpose: Since the TGF-β pathway is highly dysregulated in HNSCC, we investigate in this study how SMAD4 downregulation alters TGF-β pathway signaling and the tumor microenvironment. Results: Using isogenic HNSCC cell lines, we visualized using immunofluorescence staining that levels of activated phospho-SMAD2 in the nucleus are correlated with expression of SMAD4. Genome-wide screening using Gene Set Enrichment Analysis (GSEA) further revealed changes in gene expression during SMAD4 knockdown in HNSCC cells. From GSEA results, the 20 most significantly altered Gene Ontology (GO) pathways were determined. It was shown that majority of these GO pathways are associated with regulating neutrophil migration and chemotaxis with an upregulation of CXC chemokine gene expression, specifically with CXCL1. In contrast, anti-tumor chemokines, such as CXCL9 and CXCL11, were downregulated during loss of SMAD4 expression. Using an orthotopic mouse model we show that in vivo, decreased expression of SMAD4 promotes tumor take and growth. Conclusion: Collectively, these results suggest that HNSCC tumors with SMAD4 loss may have poor prognosis attributed to immunosuppressive functions of chemokine expression that acts on tumor-associated neutrophils. Citation Format: Jude Masannat, Xuefeng Wang, Biwei Cao, FeiFei Song, Ritu Chaudhary, Janis de la Iglasia, Ciaara Gorgoglione, Robbert Slebos, Christine Chung. Smad4 loss upregulates chemokine expression and promotes tumor inflammation and growth in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5314.

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-β Signaling and Upregulating CDK Inhibitors P16 and P27.

PurposeKrüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) cell fate while suppressing mesenchymal properties. TGF-β plays a crucial role in cell differentiation and development, and if dysregulated, it induces epithelial-mesenchymal transition (EMT). As KLF4 and TGF-β regulate each other in a context-dependent manner, we evaluated the role of the crosstalk between KLF4 and TGF-β-signaling in CE homeostasis.MethodsWe used spatiotemporally regulated ablation of Klf4 within the adult mouse CE in ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/ Krt12rtTA/rtTA/ Tet-O-Cre) mice and short hairpin RNA (shRNA)-mediated knockdown or lentiviral vector-mediated overexpression of KLF4 in human corneal limbal epithelial (HCLE) cells to evaluate the crosstalk between KLF4 and TGF-β-signaling components. Expression of TGF-β signaling components and cyclin-dependent kinase (CDK) inhibitors was quantified by quantitative PCR, immunoblots, and/or immunofluorescent staining.ResultsCE-specific ablation of Klf4 resulted in (1) upregulation of TGF-β1, -β2, -βR1, and -βR2; (2) downregulation of inhibitory Smad7; (3) hyperphosphorylation of Smad2/3; (4) elevated nuclear localization of phospho-Smad2/3 and Smad4; and (5) downregulation of CDK inhibitors p16 and p27. Consistently, shRNA-mediated knockdown of KLF4 in HCLE cells resulted in upregulation of TGF-β1 and -β2, hyperphosphorylation and nuclear localization of SMAD2/3, downregulation of SMAD7, and elevated SMAD4 nuclear localization. Furthermore, overexpression of KLF4 in HCLE cells resulted in downregulation of TGF-β1, -βR1, and -βR2 and upregulation of SMAD7, p16, and p27.ConclusionsCollectively, these results demonstrate that KLF4 regulates CE cell cycle progression by suppressing canonical TGF-β signaling and overcomes the undesirable concomitant decrease in TGF-β–dependent CDK inhibitors p16 and p27 expression by directly upregulating them.

Weaning-induced cell cycle arrest through up-regulation of p21 and p27 in the jejunum of piglets

This study was aimed at investigating the effects of weaning on jejunal redox status, morphology and expression of cell cycle regulators in piglets. A total of 120 new-born piglets from 12 litters were divided into two groups: the control (suckled normally) and weaning group (weaned at day 21). Six piglets from each group were slaughtered at day 22, day 25 and day 28. Results showed that weaning increased the serum hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA), decreased inhibitory hydroxyl radical (IHR) concentrations and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities at day 25. Furthermore, the jejunal nitric oxide (NO) level was increased at days 22 and 28, while H 2 O 2 concentration was increased at days 22, 25 and 28. Results of jejunum morphology showed weaning decreased villous height and width, and increased crypt depth at day 25. Relative mRNA and protein expression showed that Smad4 was decreased at day 25 and then increased at day 28. The expression of p21 and p27 were significantly increased by weaning at days 25 and 28, but cyclin D and cyclin E showed no significant differences compared with control group. In conclusion, weaning increased the serum and jejunal reactive oxygen species (ROS) production, inhibited jejunum development and cell cycle progression through down-regulation of Smad4 and up-regulation of p21 and p27 of piglets, which further explained weaning-induced stress syndrome. Keywords : Proliferation, intestine, reactive oxygen species, weaning

INTS8 accelerates the epithelial-to-mesenchymal transition in hepatocellular carcinoma by upregulating the TGF-β signaling pathway.

BackgroundHepatocellular carcinoma (HCC) is the third leading cause of death by malignancy worldwide. HCC has a poor prognosis due to tumor invasiveness and metastasis. There is substantial evidence that the epithelial-to-mesenchymal transition (EMT) plays a central role in cancer metastasis. In a previous study, a possible association between integrator complex 8 (INTS8) and the progression and development of HCC was discovered. However, its role and the molecular mechanisms in HCC are poorly understood.MethodsThe PROGgeneV2 platform database and Kaplan–Meier plotter analysis were used to analyze the potential effects of INTS8 in HCC. Moreover, we performed migration, transwell, and metastasis assays to investigate the effects of INTS8 on HCC cells. In addition, relevant signaling pathways were examined by western blot and RT-qPCR assays.ResultsWe used the PROGgeneV2 platform database and Kaplan–Meier plotter analysis, which indicated that increased expression of INTS8 is associated with poor overall survival of HCC. Moreover, INTS8 expression was higher in HCC tissues than in adjacent noncancerous tissues. INTS8 depletion reduced the invasion and migration of HCC cell lines. Downregulation of INTS8 in vivo resulted in fewer observed metastatic nodules in lungs. Moreover, INTS8 knockdown also increased the expression of epithelial markers (E-cadherin) and decreased the expression of mesenchymal markers (N-cadherin and vimentin) following the downregulation of SMAD4. In addition, pretreatment with TGF-β1 could partly prevent the decrease in the expression of SMAD4 and EMT markers induced by INTS8 knockdown.ConclusionOverall, these findings suggest that INTS8 accelerates the EMT in HCC by upregulating the TGF-β signaling pathway.

Nuclear import of transcriptional corepressor BCOR occurs through interaction with karyopherin α expressed in human periodontal ligament

Mutations in the gene encoding BCL-6 corepressor (BCOR) are responsible for oculofaciocardiodental (OFCD) syndrome, which is a rare X-linked dominant disorder characterized by radiculomegaly of permanent teeth as the most typical symptom. To function as a transcriptional corepressor, BCOR needs to enter the nucleus; however, the molecular pathway for its nuclear translocation during dental root formation remains unclear. The purpose of this study was to determine the mechanism underlying BCOR transport into the nucleus. Our results showed that human periodontal ligament (PDL) cells expressed karyopherin α (KPNA)2, KPNA4, and KPNA6 belonging to a family of nuclear import proteins, which interacted with BCOR in the immunoprecipitation assay. Site-directed mutagenesis targeting the two nuclear localization signals (NLSs) within BCOR reduced its nuclear translocation; however, co-expression of KPNA2, KPNA4, or KPNA6 with BCOR carrying a previously described mutation which eliminated one of the two NLSs significantly increased nuclear accumulation of the mutant BCOR, indicating participation of KPNA in BCOR nuclear translocation. Comparative expression profiling of PDL cells isolated from normal and OFCD patients revealed significant downregulation of SMAD4, GLI1, and nuclear factor 1-C (NFIC) mRNA expression, suggesting that BCOR mutations cause hyperactive root formation in OFCD syndrome by inhibiting SMAD4-Hedgehog-NFIC signaling implicated in dental root development. Our study contributes to understanding of the mechanisms providing nuclear import of BCOR during root formation.

Influence of nourishing yin and tonifying yang sequential therapy combined with Western medicine on TGF-β1/Smads signaling pathway in anovulatory infertility rats with diminished ovarian reserve

To explore the influence for combination of nourishing yin and tonifying yang sequential therapy (NYTYST) with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve (DOR) based on TGF-β1/Smads signaling pathway. Methods: A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group (n=8 in each group). The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy (HRT) and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor β1 receptor (TGF-β1R) in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot. Results: Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group (P<0.01); the levels of TGF-β1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly (P<0.01). Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group (P<0.05 or P<0.01). The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group (P<0.05). Compared with the Western medicine group, the levels of TGF-β1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group (P<0.05 or P<0.01). Conclusion: NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-β1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.

Upregulated microRNA-146a expression induced by granulocyte colony-stimulating factor enhanced low-dosage chemotherapy response in aged acute myeloid leukemia patients

The selection of chemotherapy regimen for elderly patients with acute myeloid leukemia (AML) remains challenging. Here, we report that granulocyte colony-stimulating factor (G-CSF) upregulates the expression of microRNA (miR)-146a in a nuclear factor kappaB-dependent manner, leading to direct decreases in the expression of the target proteins CXCR4 and Smad4 in AML cells in vitro. The reduction in CXCR4 expression suppressed the migration abilities of leukemia cells. Downregulation of Smad4 promoted cell cycle entry in leukemia cells. Furthermore, an increase in apoptosis was observed when leukemia cells were treated sequentially with G-CSF and cytosine arabinoside in vitro. These findings suggest that G-CSF treatment may disrupt the protection of bone marrow niches from leukemia cells. In a review of data from 78 cases of primary AML, we found that a high miR-146a expression and/or upregulation of this miRNA during G-CSF priming chemotherapy was predictive of better clinical outcomes. Our findings suggest that miR-146a may be a novel biomarker for evaluating the clinical prognosis and treatment effects of a G-CSF priming protocol in elderly patients with AML.

MiRNA-34a reversed TGF-β-induced epithelial-mesenchymal transition via suppression of SMAD4 in NPC cells

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-β (TGF-β), 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-β pathway, reversed the TGF-β-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-β-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-β-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.

Effect of histone deacetylase inhibitor on epithelial-mesenchymal transition of liver fibrosis.

Liver fibrosis is an excessively reversible wound healing process and the fibrotic disorder is the activation of hepatic stellate cell that requires extensive alterations in gene expression. As reversible deacetylation of histone proteins modulate gene expression, we examined the effect of valproic acid (VPA) as selective histone deacetylase inhibitor on CCl-4 induced liver fibrosis. Thirty rats were divided into three equal groups; control group, fibrotic group and VPA-treated group. The rats were sacrificed after 6 weeks of liver fibrosis induction. The histopathological effect on liver tissue was examined. The expression of α-SMA and Smad-4 mRNA and serum levels of TGF-β1, alanine aminotransferase, and aspartate aminotransferase were determined. Treatment of rats with VPA attenuated carbon tetrachloride-induced liver fibrosis. Moreover, α-SMA and Smad-4 expression was repressed under VPA treatment and both serum TGF-β1 and liver enzymes were significantly decreased. The histone deacetylase inhibitor-1 VPA inhibits the epithelial-mesenchymal transition and affects hepatic stellate cell activation during liver fibrosis through downregulation of Smad4 and α-SMA expression which may serve as a promising agent in liver fibrosis treatment. © 2018 IUBMB Life, 70(6):511-518, 2018.

MiR-181b-induced SMAD7 downregulation controls granulosa cell apoptosis through TGF-β signaling by interacting with the TGFBR1 promoter.

SMAD7 disrupts the TGF-β signaling pathway by influencing TGFBR1 stability and by blocking the binding of TGFBR1 to SMAD2/3. In this study, we showed that SMAD7 attenuated the TGF-β signaling pathway in ovarian granulosa cells (GCs) by regulating TGFBR1 transcriptional activity. To function as a transcription factor, SMAD7 downregulated the mRNA levels of TGFBR1 via direct binding to the SMAD-binding elements (SBEs) within the promoter region of pig TGFBR1. We also showed that SMAD7 enhanced porcine GC apoptosis by interrupting TGFBR1 and the TGF-β signaling pathway. Interestingly, miR-181b, a microRNA that is downregulated during porcine follicular atresia, was identified to be directly targeting SMAD7 at its 3'-UTR. By inhibiting SMAD7, miR-181b could inhibit GC apoptosis by activating the TGF-β signaling pathway. Our findings provide new insights into the mechanisms underlying the regulation of the TGF-β signaling pathway by SMAD7 and miR-181b.

Novel RAS inhibitor 25-O-methylalisol F attenuates epithelial-to-mesenchymal transition and tubulo-interstitial fibrosis by selectively inhibiting TGF-β-mediated Smad3 phosphorylation

BackgroundTubulo-interstitial fibrosis (TIF) is the common pathway in the chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) is a major contributor to the TIF by the increased myofibroblasts. Renin-angiotensin system (RAS) is critical mediator on EMT in progressive CKD. Angiotensin II (ANG) mediates EMT and causes TIF by stimulating transforming growth factor-β1 (TGF-β1). RAS activation could further activate TGF-β1. Inhibition of the RAS is one of the most powerful therapies for progressive CKD. 25-O-methylalisol F (MAF) is a new tetracyclic triterpenoid compound isolated from the Alismatis rhizoma, which is extensively used for anti-hypertensive, diuretic and anti-hyperlipidemic effects. MethodsInhibitory effect of MAF on EMT is investigated in both TGF-β1- and ANG-induced tubular epithelial cells (NRK-52E) and fibroblasts (NRK-49F). Western blot analysis, qRT-PCR, siRNA, immunofluorescence staining and co-immunoprecipitation techniques were used to evaluate the inhibition of MAF on EMT and further revealed the intervention effects on RAS, TGF-β/Smad and Wnt/β-catenin pathways. ResultsMAF treatment significantly inhibited TGF-β1 and ANG-induced expressions of collagen I, fibronectin, α-SMA, vimentin and E-cadherin at both mRNA and protein levels in the NRK-52E and NRK-49F cells. The action mechanism revealed that MAF significantly ameliorated upregulation of angiotensinogen, renin, ACE and AT1R expressions. Further, MAF attenuated upregulation of Smad3 phosphorylation and downregulation of Smad7, but did not affect the phosphorylation of Smad2, PI3K, ERK1/2 and p38 expressions and Smad4 expression in NRK-52E cells. Co-immunoprecipitation analysis indicated that MAF selectively blocked the combination of Smad3 with TGFβRI and Smad3 with SARA without interfering with the Smad2, TGFβRI and SARA interaction. Additionally, MAF suppressed the expressions of Wnt1 and β-catenin as well as its downstream target Snail1, Twist, MMP-7, PAI-1 and FSP1 expressions in NRK-52E cells. ConclusionsMAF simultaneously targeted multiple RAS components and it was a novel RAS inhibitor. MAF inhibited EMT by Smad3-specific signaling in the TGF-β/Smad-dependent pathway and Wnt/β-catenin pathway. MAF has an important effect on crosstalk between the TGF-β/Smad and Wnt/β-catenin pathway in EMT process by activation of RAS.

Syringic acid, a phenolic acid, promotes osteoblast differentiation by stimulation of Runx2 expression and targeting of Smad7 by miR-21 in mouse mesenchymal stem cells.

Syringic acid (SA), a phenolic acid, has been used in Chinese and Indian medicine for treating diabetes but its role in osteogenesis has not yet been investigated. In the present study, at the molecular and cellular levels, we evaluated the effects of SA on osteoblast differentiation. At the cellular level, there was increased alkaline phosphatase (ALP) activity and calcium deposition by SA treatment in mouse mesenchymal stem cells (mMSCs). At the molecular level, SA treatment of these cells stimulated expression of Runx2, a bone transcription factor, and of osteoblast differentiation marker genes such as ALP, type I collagen, and osteocalcin. It is known that Smad7 is an antagonist of TGF-β/Smad signaling and is a negative regulator of Runx2. microRNAs (miRNAs) play a key role in the regulation of osteogenesis genes at the post-transcriptional level and studies have reported that Smad7 is one of the target genes of miR-21. We found that there was down regulation of Smad7 and up regulation of miR-21 in SA-treated mMSCs. We further identified that the 3'-untranslated region (UTR) of Smad7 was directly targeted by miR-21 in these cells. Thus, our results suggested that SA promotes osteoblast differentiation via increased expression of Runx2 by miR-21-mediated down regulation of Smad7. Hence, SA may have potential in orthopedic applications.

Dysregulation of the miR-146a-Smad4 axis impairs osteogenesis of bone mesenchymal stem cells under inflammation

Osteoporosis is a common disease that affects patient quality of life, especially among the elderly population. Although inflammation contributes significantly to osteoporosis, the underlying mechanism is unclear. In this study, we found that tumor necrosis factor (TNF)-α, an inflammatory environment mimic, inhibits osteogenesis of bone mesenchymal stem cells (BMSCs), induces miR-146a and decreases Smad4. Moreover, overexpression of miR-146a inhibited the osteogenic ability of BMSCs, whereas blocking miR-146a partially rescued the osteogenesis deficiency under TNF-α treatment. Molecularly, miR-146a decreased Smad4 expression at the protein level by binding to an element located in the Smad4 3′-untranslated region, and restoration of Smad4 reversed the inhibitory effects of miR-146a on osteogenesis. Together, our results showed that the inflammatory environment mimic TNF-α inhibits osteogenesis via upregulation of miR-146a and subsequent downregulation of Smad4, thus suggesting that therapeutic manipulation of miR-146a maybe a potential strategy to improve osteogenesis in the context of osteoporosis.

Nanocurcumin-Mediated Down-Regulation of Telomerase Via Stimulating TGFβ1 Signaling Pathway in Hepatocellular Carcinoma Cells

Background:Curcumin, extracted from turmeric, represents enormous potential to serve as an anticancer agent. Telomerase is viewed as a prominent molecular target of curcumin, and transforming growth factor-β1 (TGFβ1) has proven to be a major inhibitory signaling pathway for telomerase activity. In the current study, we aimed to explore suppressive effects of nanocurcumin on telomerase expression through TGFβ1 pathway in a hepatocellular carcinoma cell line (Huh7).Methods:MTT assay was used to determine the effect of nonocurcumin on viability of Huh7 cells. RT-PCR was used to analyze the gene expression patterns.Results:MTT assay revealed that nanocurcumin acts in a dose- and time-dependent manner to diminish the cell viability. RT-PCR analysis indicated that nanocurcumin results in augmentation of TGFβ1 72 hours post treatment and leads to the reduction of telomerase expression 48 and 72 hours post exposure. Also, up-regulation of Smad3 and E2F1 and down-regulation of Smad7 confirmed the effect of nanocurcumin on intermediate components of TGFβ1 pathway. Furthermore, transfection of the proximal promoter of telomerase triggered a significant reduction in luciferase activity.Conclusion:The data from the present study lead us to develop a deeper understanding of the mechanisms underlying nanocurcumin-mediated regulation of telomerase expression, thereby presenting a new perspective to the landscape of using nanocurcumin as a cancer-oriented therapeutic agent.

MicroRNA-124 inhibits the proliferation of C6 glioma cells by targeting Smad4.

MicroRNA-124 (miR-124) has been shown to be downregulated in glioma; however, its biological functions in glioma are not yet fully understood. The aim of this study was to examine the Smad4‑dependent effects of miR‑124 on C6glioma cell proliferation. In this study, the level of miR‑124 was found to be enhanced in C6cells upon transfection with miR‑124 mimics, and the mechanisms of action of miR‑124 in C6cells were investigated by reverse transcriptase-quantitative polymerase chain reaction, MTT assay, western blot analysis and luciferase reporter assays invitro. The results revealed that miR‑124 expression was significantly lower in the C6cells than in either normal rat brain tissue or astrocytes. Upon the overexpression of miR‑124, the proliferation of the C6cells decreased and Smad4 expression was significantly suppressed. Smad4 was identified as a direct target of miR‑124 through luciferase reporter assays. Furthermore, miR‑124 was found to modulate signal transducer and activator of transcription3(Stat3) by downregulating Smad4 expression. Using small interfering RNA targeting Smad4 mRNA, we also confirmed that miR‑124 downregulated c‑Myc by modulating Smad4 expression. In addition, caspase‑3 expression was induced by miR‑124 overexpression, but not via Smad4 downregulation. On the whole, our results demonstrate that miR‑124 upregulation inhibits the growth of C6glioma cells by targeting Smad4 directly. These findings may be clinically useful for the development of therapeutic strategies directed toward miR‑124 function in patients with glioma.

Abstract 2835: Potential opposing roles of AKT isoforms in indolent versus aggressive prostate cancer

Abstract Prostate cancer (CaP) is the most commonly diagnosed non-cutaneous cancer and the second highest contributor to cancer-related deaths in U.S. men. Localized CaP has a 5-year survival rate of approximately 100%, but disseminated, metastatic disease (mCaP) dramatically reduces 5-year survival to &amp;lt;30%. A known driving force in the progression of CaP is activation of the PI3K/Akt signaling pathway through loss of PTEN, with widespread effects on cell proliferation and migration. Additional genomic changes, such as the loss of TP53 or RB1, or the upregulation of MYC, correlate with increased incidence and aggressiveness of mCaP. However, only a fraction of men with high levels of circulating and disseminated CaP cells progress to overt, macro-metastatic disease - the lethal phenotype in CaP. Importantly, little is known regarding the drivers of indolent vs. aggressive mCaP. We used two transgenic (Tg) mouse models of mCaP with clinically relevant genetic alterations which exhibit Akt activation and Rb1 loss, yet one produces indolent mCaP and the other, systemic aggressive mCaP. Specifically, mice lacking Rb1 and the metastasis-suppressor, SSeCKS/Akap12, develop high-grade prostatic intraepithelial neoplasia (HG-PIN) and disseminated disease in the lymph nodes whereas those lacking Pten and Rb1 develop CaP and aggressive mCaP. The metastasis-suppressing functions of Akap12 relates to its ability to scaffold Src and PKC, thereby attenuating their oncogenic signaling pathways. We will show data suggesting that the pathological differences between these two Tg models is based on the differential use of PI3K-p110 and Akt isoforms, with aggressiveness correlating with the downregulation of Smad4, a known metastasis-suppressor in Tg CaP models. Additionally, differences in Akt isoform usage likely controls the expression of neurogenesis genes that are especially upregulated in castration-recurrent CaP that fails second-line androgen receptor antagonist inhibitors such as enzalutamide. By comparing the differential transcriptomes and signaling networks in primary-site tumors, lymph node metastases, and CaP cell lines derived from these two Tg models, several new potential therapeutic targets have been identified that could be exploited to prevent or treat aggressive mCaP. Citation Format: Karina A. Miller, Hyun-Kyung Ko, Irwin H. Gelman. Potential opposing roles of AKT isoforms in indolent versus aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2835. doi:10.1158/1538-7445.AM2017-2835

Abstract 4628: Selection of Smad2 for TGF-β to suppress dendritic cells by STAT3/c-Ski-induced repression of Smad3

Abstract The mechanisms how Smad-mediated transforming growth factor-β (TGF-β) signalling regulates dendritic cells (DCs) remain largely unknown, despite its crucial regulatory roles in the differentiation and activation of DCs. Here, we show that STAT3/c-Ski-induced downregulation of Smad3 during differentiation selects Smad2 as the specific TGF-β receptor-regulated Smad (R-Smad) to suppress the immunogenicity of DCs. Smad3 transcribed by unphosphorylated Smad2/3 prevented the progenitor cells from DC differentiation with repression of Flt3, IRF4 and STAT3. Repression of Smad3 by synergy of phosphorylated STAT3 and c-Ski was essential for DC differentiation. TGF-β suppressed DC activation via Smad2, hence DC-specific Smad2 deletion enhanced anti-lymphoma effector functions of cytotoxic T lymphocytes (CTLs) and type 1 helper T (Th1) cells in a mouse EL4 lymphoma model. Our findings reveal stepwise regulatory roles of R-Smads in DC regulation: Smad3 inhibits differentiation and Smad2 suppresses immunogenicity. Citation Format: Jeong-Hwan Yoon, Eunjin Bae, Katsuko Sudo, Masakatsu Takanashi, Jin Soo Han, Seok Hee Park, Michael Weinstein, Susumu Nakae, Tadashi Yamashita, In-Kyu Lee, Ji Hyeon Ju, Takayuki Sumida, Masahiko Kuroda, Keiji Miyazawa, Mitsuyasu Kato, Mizuko Mamura. Selection of Smad2 for TGF-β to suppress dendritic cells by STAT3/c-Ski-induced repression of Smad3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4628. doi:10.1158/1538-7445.AM2017-4628

SUMO-Specific Cysteine Protease 1 Promotes Epithelial Mesenchymal Transition of Prostate Cancer Cells via Regulating SMAD4 deSUMOylation.

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-β/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.

Effect of TGF-β/smad signaling pathway blocking on expression profiles of miR-335, miR-150, miR-194, miR-27a, and miR-199a of hepatic stellate cells (HSCs).

The aim of this study was to determine the effect of inhibition of TGF-β/smad signaling on the expression profiles of miR-335, miR-150, miR-194, miR-27a, miR-199a of hepatic stellate cells (HSCs). Liver fibrosis is excessive deposition of extracellular matrix proteins due to ongoing inflammation and HSC activation that occurs in most types of chronic liver diseases. Recent studies have shown the importance of microRNAs in the pathogenesis of chronic liver diseases. In this study, for inhibition of TGF-β smad-signaling pathway, expressing Smad4 shRNA plasmids were transfected into HSCs. Subsequently, using Real Time-PCR, we measured the expression levels of miR-335, miR-150, miR-194, miR-27a and miR-199a. Gene expression analysis showed that downregulation of Smad4 by vector Smad4shRNA significantly increased the expression levels of miR-335 (P<0.01) and miR-150 (P<0.001) and decreased the expression level of miR-27a (P<0.05). The results of this study suggest that blocking TGF-β smad-signaling can also differentially modulate microRNA expression in support of activation and fibrogenesis of HSCs.