TGFβ-induced Epithelial-mesenchymal Transition Research Articles

Wnt5a/β-catenin-mediated epithelial-mesenchymal transition: a key driver of subretinal fibrosis in neovascular age-related macular degeneration

BackgroundNeovascular age-related macular degeneration (nAMD), accounts for up to 90% of AMD-associated vision loss, ultimately resulting in the formation of fibrotic scar in the macular region. The pathogenesis of subretinal fibrosis in nAMD involves the process of epithelial–mesenchymal transition (EMT) occurring in retinal pigment epithelium (RPE). Here, we aim to investigate the underlying mechanisms involved in the Wnt signaling during the EMT of RPE cells and in the pathological process of subretinal fibrosis secondary to nAMD.MethodsIn vivo, the induction of subretinal fibrosis was performed in male C57BL/6J mice through laser photocoagulation. Either FH535 (a β-catenin inhibitor) or Box5 (a Wnt5a inhibitor) was intravitreally administered on the same day or 14 days following laser induction. The RPE-Bruch's membrane-choriocapillaris complex (RBCC) tissues were collected and subjected to Western blot analysis and immunofluorescence to examine fibrovascular and Wnt-related markers. In vitro, transforming growth factor beta 1 (TGFβ1)-treated ARPE-19 cells were co-incubated with or without FH535, Foxy-5 (a Wnt5a-mimicking peptide), Box5, or Wnt5a shRNA, respectively. The changes in EMT- and Wnt-related signaling molecules, as well as cell functions were assessed using qRT-PCR, nuclear-cytoplasmic fractionation assay, Western blot, immunofluorescence, scratch assay or transwell migration assay. The cell viability of ARPE-19 cells was determined using Cell Counting Kit (CCK)-8.ResultsThe in vivo analysis demonstrated Wnt5a/ROR1, but not Wnt3a, was upregulated in the RBCCs of the laser-induced CNV mice compared to the normal control group. Intravitreal injection of FH535 effectively reduced Wnt5a protein expression. Both FH535 and Box5 effectively attenuated subretinal fibrosis and EMT, as well as the activation of β-catenin in laser-induced CNV mice, as evidenced by the significant reduction in areas positive for fibronectin, alpha-smooth muscle actin (α-SMA), collagen I, and active β-catenin labeling. In vitro, Wnt5a/ROR1, active β-catenin, and some other Wnt signaling molecules were upregulated in the TGFβ1-induced EMT cell model using ARPE-19 cells. Co-treatment with FH535, Box5, or Wnt5a shRNA markedly suppressed the activation of Wnt5a, nuclear translocation of active β-catenin, as well as the EMT in TGFβ1-treated ARPE-19 cells. Conversely, treatment with Foxy-5 independently resulted in the activation of abovementioned molecules and subsequent induction of EMT in ARPE-19 cells.ConclusionsOur study reveals a reciprocal activation between Wnt5a and β-catenin to mediate EMT as a pivotal driver of subretinal fibrosis in nAMD. This positive feedback loop provides valuable insights into potential therapeutic strategies to treat subretinal fibrosis in nAMD patients.

MicroRNA-141-3p Suppressed the Progression of the Clear Cell Renal Cell Carcinoma by Targeting Transforming Growth Factor Beta 2 Gene Expression.

Clear cell renal cell carcinoma (ccRCC) is a malignant tumor of kidney epithelial cells, one of the most common tumors in the world. Transforming growth factor beta (TGFβ)1 is a crucial factor that induces epithelial-mesenchymal transition (EMT) in cancer cells. microRNA-141-3p (miR-141-3p) is a microRNA that is considered a tumor suppressor. However, the role and mechanism of miR-141-3p in TGFβ1-induced ccRCC cells are not fully understood. This study investigated the roles of miR-141-3p and its target gene in regulating EMT in ccRCC development. 786-0 and Caki-1cells were treated with TGFβ1 to induce EMT. The levels of miR-141-3p and TGFβ2 were determined by quantitative real-time polymerase chain reaction and Western blotting. The progression of EMT was evaluated by E-cadherin detection by immunofluorescence, and E-cadherin, N-cadherin, and vimentin detection by Western blotting. Furthermore, migration and invasion capacities were assessed using a Transwell system. The direct binding of miR-141-3p with the target gene TGFβ2 was confirmed by dual luciferase reporter gene assay. Results indicated that TGFβ1 treatment decreased the protein abundance of E-cadherin while increasing the protein expression of N-cadherin and vimentin, indicating TGFβ1-induced EMT was constructed successfully. Moreover, TGFβ1 treatment repressed the expression of miR-141-3p. miR-141-3p mimics reversed the effect of TGFβ1 on the migration, invasion, and expression of E-cadherin, N-cadherin, and vimentin. The miR-141-3p directly binds with the 3' untranslated region of TGFβ2 mRNA and suppresses its expression. Furthermore, TGFβ2 overexpression abrogated the above changes regulated by miR-141-3p mimics. Taken together, miR-141-3p inhibited TGFβ1-induced EMT by suppressing the migration and invasion of ccRCC cells via directly targeting TGFβ2 gene expression.

Modelling bronchial epithelial-fibroblast cross-talk in idiopathic pulmonary fibrosis (IPF) using a human-derived in vitro air liquid interface (ALI) culture

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3–4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air–liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFβ-2, ZO-1 and DN12. When stimulated with exogenous TGFβ, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFβ-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1β, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFβ isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFβ signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

Extracellular matrix viscoelasticity regulates TGFβ1-induced epithelial-mesenchymal transition and apoptosis via integrin linked kinase.

Transforming growth factor (TGF)-β1 is a multifunctional cytokine that plays important roles in health and disease. Previous studies have revealed that TGFβ1 activation, signaling, and downstream cell responses including epithelial-mesenchymal transition (EMT) and apoptosis are regulated by the elasticity or stiffness of the extracellular matrix. However, tissues within the body are not purely elastic, rather they are viscoelastic. How matrix viscoelasticity impacts cell fate decisions downstream of TGFβ1 remains unknown. Here, we synthesized polyacrylamide hydrogels that mimic the viscoelastic properties of breast tumor tissue. We found that increasing matrix viscous dissipation reduces TGFβ1-induced cell spreading, F-actin stress fiber formation, and EMT-associated gene expression changes, and promotes TGFβ1-induced apoptosis in mammary epithelial cells. Furthermore, TGFβ1-induced expression of integrin linked kinase (ILK) and colocalization of ILK with vinculin at cell adhesions is attenuated in mammary epithelial cells cultured on viscoelastic substrata in comparison to cells cultured on nearly elastic substrata. Overexpression of ILK promotes TGFβ1-induced EMT and reduces apoptosis in cells cultured on viscoelastic substrata, suggesting that ILK plays an important role in regulating cell fate downstream of TGFβ1 in response to matrix viscoelasticity.

Silibinin Prevents TGFβ-Induced EMT of RPE in Proliferative Vitreoretinopathy by Inhibiting Stat3 and Smad3 Phosphorylation.

The purpose of this study was to investigate the effects of silibinin on epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) and proliferative vitreoretinopathy (PVR) formation, as well as its underlying molecular mechanism. Cellular morphological change and EMT molecular markers were evaluated by using phase contrast imaging, qPCR, and Western blot (WB) to investigate the impact of silibinin on the EMT of ARPE-19 cells. Scratch assay and transwell assay were used to study the effect of silibinin on cell migration. An intravitreally injected RPE-induced rat PVR model was used to assess the effect of silibinin on PVR in vivo. RNA-seq was applied to study the molecular mechanism of silibinin-mediated PVR prevention. Silibinin inhibited TGFβ1-induced EMT and migration of RPE in a dose-dependent manner in vitro. Moreover, silibinin prevented proliferative membrane formation in an intravitreal injected RPE-induced rat PVR model. In line with these findings, RNA-seq revealed a global suppression of TGFβ1-induced EMT and migration-related genes by silibinin in RPEs. Mechanistically, silibinin reduced TGFβ1-induced phosphorylation levels of Smad3 and Stat3, and Smad3 nuclear translocation in RPE. Silibinin inhibits the EMT of RPE cells in vitro and prevents the formation of PVR membranes in vivo. Mechanistically, silibinin inhibits Smad3 phosphorylation and suppresses Smad3 nuclear translocation through the inhibition of Stat3 phosphorylation. These findings suggest that silibinin may serve as a potential treatment for PVR.

A circular RNA activated by TGFβ promotes tumor metastasis through enhancing IGF2BP3-mediated PDPN mRNA stability

Metastasis is the leading cause of cancer-related death, where TGFβ-induced epithelial-mesenchymal transition (EMT) process confers on cancer cells increased metastatic potential. However, the involvement of circRNAs in this process is still obscure. Here, we identify a TGFβ-induced circRNA called circITGB6 as an indispensable factor during the TGFβ-mediated EMT process. circITGB6 is significantly upregulated in metastatic cancer samples and its higher abundance is closely correlated to worse prognosis of colorectal cancer (CRC) patients. Through gain- and loss-of-function assays, circITGB6 is found to potently promote EMT process and tumor metastasis in various models in vitro and in vivo. Mechanistically, circITGB6 enhances the mRNA stability of PDPN, an EMT-promoting gene, by directly interacting with IGF2BP3. Notably, interfering circITGB6 with PEI-coated specific siRNA effectively represses liver metastasis. Therefore, our study reveals the function of a TGFβ-regulated circRNA in tumor metastasis and suggests that targeting circITGB6 is a promising strategy for cancer therapy.

Chlorogenic Acid Inhibited Epithelial-Mesenchymal Transition to Treat Pulmonary Fibrosis through Modulating Autophagy.

Chlorogenic acid (CGA), derived from dicotyledons and ferns, has been demonstrated with anti-inflammatory, anti-bacterial, and free radical-scavenging effects and can be used to treat pulmonary fibrosis (PF). However, the specific mechanism by which CGA treats PF needs to be further investigated. In this study, in vivo experiment was firstly performed to evaluate the effects of CGA on epithelial-mesenchymal transition (EMT) and autophagy in bleomycin (BLM)-induced PF mice. Then, the effects of CGA on EMT and autophagy was assessed using transforming growth factor beta (TGF-β) 1-induced EMT model in vitro. Furthermore, autophagy inhibitor (3-methyladenine) was used to verify that the inhibitory mechanism of CGA on EMT was associated with activating autophagy. Our results found that 60 mg/kg of CGA treatment significantly ameliorated lung inflammation and fibrosis in mice with BLM-induced PF. Besides, CGA inhibited EMT and promoted autophagy in mice with PF. In vitro studies also demonstrated that 50 µM of CGA treatment inhibited EMT and induced autophagy related factors in TGF-β1-induced EMT cell model. Moreover, the inhibitory effect of CGA on autophagy and EMT in vitro was abolished after using autophagy inhibitor. In conclusion, CGA could inhibit EMT to treat BLM-induced PF in mice through, activating autophagy.

Let-7c-3p suppresses lens epithelial-mesenchymal transition by inhibiting cadherin-11 expression in fibrotic cataract.

Fibrotic cataract, including anterior subcapsular cataract (ASC) and posterior capsule opacification, always lead to visual impairment. Epithelial-mesenchymal transition (EMT) is a well-known event that causes phenotypic alterations in lens epithelial cells (LECs) during lens fibrosis. Accumulating studies have demonstrated that microRNAs are important regulators of EMT and fibrosis. However, the evidence explaining how microRNAs modulate the behavior and alter the cellular phenotypes of the lens epithelium in fibrotic cataract is insufficient. In this study, we found that hsa-let-7c-3p is downregulated in LECs in human ASC in vivo as well as in TGFβ2-induced EMT in vitro, indicating that hsa-let-7c-3p may participate in modulating the profibrotic processes in the lens. We then demonstrated that overexpression of hsa-let-7c-3p markedly suppressed human LEC proliferation and migration and attenuated TGFβ2-induced EMT and injury-induced ASC in a mouse model. In addition, hsa-let-7c-3p mediated lens fibrosis by directly targeting the CDH11 gene, which encodes cadherin-11 protein, an important mediator in the EMT signaling pathway. It decreased cadherin-11 protein expression at the posttranscriptional level but not at the transcriptional level by binding to a specific site in the 3-untranslated region (3'-UTR) of CDH11 mRNA. Moreover, blockade of cadherin-11 expression with a specific short hairpin RNA reversed TGFβ2-induced EMT in LECs in vitro. Collectively, these data demonstrated that hsa-let-7c-3p plays a clear role in attenuating ASC development and may be a novel candidate therapeutic for halting fibrosis and maintaining vision.

Approaches in Hydroxytyrosol Supplementation on Epithelial-Mesenchymal Transition in TGFβ1-Induced Human Respiratory Epithelial Cells.

Hydroxytyrosol (HT) is an olive polyphenol with anti-inflammatory and antioxidant properties. This study aimed to investigate the effect of HT treatment on epithelial-mesenchymal transition (EMT) in primary human respiratory epithelial cells (RECs) isolated from human nasal turbinate. HT dose-response study and growth kinetic study on RECs was performed. Several approaches on HT treatment and TGFβ1 induction with varying durations and methods was studied. RECs morphology and migration ability were evaluated. Vimentin and E-cadherin immunofluorescence staining and Western blotting [E-cadherin, vimentin, SNAIL/SLUG, AKT, phosphorylated (p)AKT, SMAD2/3 and pSMAD2/3] were performed after 72-h treatment. In silico analysis (molecular docking) of HT was performed to evaluate the potential of HT to bind with the TGFβ receptor. The viability of the HT-treated RECs was concentration-dependent, where the median effective concentration (EC50) was 19.04 μg/mL. Testing of the effects of 1 and 10 µg/mL HT revealed that HT suppressed expression of the protein markers vimentin and SNAIL/SLUG while preserving E-cadherin protein expression. Supplementation with HT protected against SMAD and AKT pathway activation in the TGFβ1-induced RECs. Furthermore, HT demonstrated the potential to bind with ALK5 (a TGFβ receptor component) in comparison to oleuropein. TGFβ1-induced EMT in RECs and HT exerted a positive effect in modulating the effects of EMT.

Combination Treatment of a Phytochemical and a Histone Demethylase Inhibitor-A Novel Approach towards Targeting TGFβ-Induced EMT, Invasion, and Migration in Prostate Cancer.

Minimizing side effects, overcoming cancer drug resistance, and preventing metastasis of cancer cells are of growing interest in current cancer therapeutics. Phytochemicals are being researched in depth as they are protective to normal cells and have fewer side effects. Hesperetin is a citrus bioflavonoid known to inhibit TGFβ-induced epithelial-to-mesenchymal transition (EMT), migration, and invasion of prostate cancer cells. Targeting epigenetic modifications that cause cancer is another class of upcoming therapeutics, as these changes are reversible. Global H3K27me3 levels have been found to be reduced in invasive prostate adenocarcinomas. Combining a demethylase inhibitor and a known anti-cancer phytochemical is a unique approach to targeting cancer to attain the aforementioned objectives. In the current study, we used an H3K27 demethylase (JMJD3/KDM6B) inhibitor to study its effects on TGFβ-induced EMT in prostate cancer cells. We then gave a combined hesperetin and GSK-J4 treatment to the PC-3 and LNCaP cells. There was a dose-dependent increase in cytotoxicity and inhibition of TGFβ-induced migration and invasion of prostate cancer cells after GSK-J4 treatment. GSK-J4 not only induced trimethylation of H3K27 but also induced the trimethylation of H3K4. Surprisingly, there was a reduction in the H3K9me3 levels. GSK-J4 alone and a combination of hesperetin and GSK-J4 treatment effectively inhibit the important hallmarks of cancer, such as cell proliferation, migration, and invasion, by altering the epigenetic landscape of cancer cells.

TGFβ1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation.

Epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, the main cause of cancer-related mortality. Growing evidence that SNAIL and ZEB transcription factors, typically portrayed as master regulators of EMT, may be dispensable for this process, led us to re-investigate its mechanistic underpinnings. For this, we used an unbiased computational approach that integrated time-resolved analyses of chromatin structure and differential gene expression, to predict transcriptional regulators of TGFβ1-inducible EMT in the MCF10A mammary epithelial cell line model. Bioinformatic analyses indicated comparatively minor contributions of SNAIL proteins and ZEB1 to TGFβ1-induced EMT, whereas the AP-1 subunit JUNB was anticipated to have a much larger impact. CRISPR/Cas9-mediated loss-of-function studies confirmed that TGFβ1-induced EMT proceeded independently of SNAIL proteins and ZEB1. In contrast, JUNB was necessary and sufficient for EMT in MCF10A cells, but not in A549 lung cancer cells, indicating cell-type-specificity of JUNB EMT-regulatory capacity. Nonetheless, the JUNB-dependence of EMT-associated transcriptional reprogramming in MCF10A cells allowed to define a gene expression signature which was regulated by TGFβ1 in diverse cellular backgrounds, showed positively correlated expression with TGFβ signaling in multiple cancer transcriptomes, and was predictive of patient survival in several cancer types. Altogether, our findings provide novel mechanistic insights into the context-dependent control of TGFβ1-driven EMT and thereby may lead to improved diagnostic and therapeutic options.

Nintedanib-αVβ6 Integrin Ligand Conjugates Reduce TGFβ-Induced EMT in Human Non-Small Cell Lung Cancer.

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGFβ is one of the most powerful inducers of this transition, as it induces overexpression of the fibronectin receptor, αvβ6 integrin, in cancer cells which, in turn, is strongly associated with EMT. Thus, αvβ6 integrin receptors may be exploited as a target for the selective delivery of anti-tumor agents. We introduce three novel synthesized conjugates, in which a selective αvβ6 receptor ligand is linked to nintedanib, a potent kinase inhibitor used to treat advanced adenocarcinoma lung cancer in clinics. The αvβ6 integrin ligand directs nintedanib activity to the target cells of the tumor microenvironment, avoiding the onset of negative side effects in normal cells. We found that the three conjugates inhibit the adhesion of cancer cells to fibronectin in a concentration-dependent manner and that αvβ6-expressing cells internalized the conjugated compounds, thus permitting nintedanib to inhibit 2D and 3D cancer cell growth and suppress the clonogenic ability of the EMT phenotype as well as intervening in other aspects associated with the EMT transition. These results highlight αvβ6 receptors as privileged access points for dual-targeting molecular conjugates engaged in an efficient and precise strategy against non-small cell lung cancer.

Expression of dual-specificity phosphatases in TGFß1-induced EMT in SKOV3 cells.

The study aims to profile the dual-specificity phosphatases (DUSP) expression in response to Transforming growth factor β1 (TGFβ1)-induced epithelial- mesenchymal transition (EMT) in ovarian adenocarcinoma cells. The ovarian adenocarcinoma cell line SKOV3 was used as a TGFβ1-induced EMT model. Cells were incubated with 5 ng/mL TGFβ1 to induce EMT. EMT was confirmed with real-time qPCR, western blot, and immunofluorescence analyses of various EMT markers. Western blot was used to analyze phospho- and total MAPK protein levels. Typical and atypical DUSPs mRNA expression profile was determined by real-time qPCR. The epithelial marker E-cadherin expressions were decreased and mesenchymal EMT markers Snail and Slug expression levelswere increased after TGFβ1 induction. Phosphorylation of ERK1/2 and p38 MAPK were enhanced in response to TGFβ1 treatment. The expression of DUSP2, DUSP6, DUSP8, DUSP10, and DUSP13 were decreased while DUSP7, DUSP16, DUSP18, DUSP21, and DUSP27 were increased by TGFβ1. TGFβ1 induced EMT which was accompanied by increased activity of MAPKs, and led to marked changes in expressions of several DUSPs in SKOV3 cells.

Limb expression 1-like protein promotes epithelial-mesenchymal transition and epidermal growth factor receptor-tyrosine kinase inhibitor resistance via nucleolin-mediated ribosomal RNA synthesis in non-small cell lung cancer.

Limb expression 1-like protein (LIX1L) might be an RNA-binding protein involved in post-transcriptional regulation. However, little is known regarding the biological function and mechanism of LIX1L in cancer cells. Here we demonstrate a clear correlation between LIX1L expression and epithelial-mesenchymal transition (EMT) markers in 81 non-small cell lung cancer (NSCLC) tissues and The Cancer Genome Atlas database, suggesting that LIX1L is a mesenchymal marker. Besides, LIX1L expression is obviously elevated in TGFβ1-induced EMT NSCLC cells and enhances cell migration, invasion, anoikis resistance, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance, and proliferation. Interestingly, the increased LIX1L expression prominently localizes to the nucleoli, where it physically interacts with the key ribosome biogenesis regulator NCL protein, inducing ribosomal RNA (rRNA) synthesis in EMT NSCLC cells. NCL knockdown or inhibition of rRNA synthesis reverses the enhanced EMT functions and proliferation ability caused by LIX1L overexpression in NSCLC cells, indicating that NCL expression and rRNA synthesis participates in LIX1L-mediated biological functions during EMT. Collectively, our findings suggest that the LIX1L-NCL-rRNA synthesis axis is a novel EMT-activated mechanism. Targeting the pathway might be a therapeutic option for EMT and EGFR-TKI resistance in NSCLC.

Hesperetin modulates TGFβ induced metastatic potential of prostate cancer cells by altering histone methylation marks

Prostate cancer is the most prevalent cancer in males, and usually, death occurs due to bone metastasis. TGFβ has been shown to play an essential role in the metastasis of prostate cancer cells by promoting epithelial-to-mesenchymal transition (EMT). Hesperetin is known to possess anti-microbial, anti-fungal, antioxidant, and anti-cancer properties and good bioavailability. Therefore, this study investigated the effect of hesperetin on TGFβ-induced cell proliferation and EMT in prostate cancer cells (PC3). Interestingly, we found that hesperetin can significantly inhibit the cell proliferation of PC3 cells and arrest the cells in the S and G2M phases of the cell cycle. The invasion and migration assay results decipher the inhibitory effect of hesperetin on TGFβ-induced invasion and migration of prostate cancer cells. Our results confirmed that hesperetin also acts through the canonical signaling pathway, as we observed a significant decrease in the expression of pSmad3. Hesperetin can inhibit the TGFβ induced EMT by increasing E-cadherin expression and decreasing N-cadherin expression. Hesperetin could modulate the TGFβ induced histone methylation marks. Further investigation is required to understand the role of hesperetin in modulating these marks and thus inhibiting TGFβ-induced EMT. Hence, the results of our study identified the potential of hesperetin to modulate TGFβ-induced cell proliferation and invasion and migration of prostate cancer cells which may help inhibit the metastatic growth of prostate cancer cells.

Hepatoprotective Effects of a Natural Flavanol 3,3'-Diindolylmethane against CCl4-Induced Chronic Liver Injury in Mice and TGFβ1-Induced EMT in Mouse Hepatocytes via Activation of Nrf2 Cascade.

Hepatic fibrosis is a form of irregular wound-healing response with acute and chronic injury triggered by the deposition of excessive extracellular matrix. Epithelial–mesenchymal transition (EMT) is a dynamic process that plays a crucial role in the fibrogenic response and pathogenesis of liver fibrosis. In the present study, we postulated a protective role of 3,3′-diindolylmethane (DIM) against TGF-β1 mediated epithelial–mesenchymal transition (EMT) in vitro and carbon tetrachloride (CCl4)-induced liver fibrosis in mice. TGF-β1-induced AML-12 hepatocyte injury was evaluated by monitoring cell morphology, measuring reactive oxygen species (ROS) and mitochondrial membrane potential, and quantifying apoptosis, inflammatory, and EMT-related proteins. Furthermore, CCl4-induced liver fibrosis in mice was evaluated by performing liver function tests, including serum ALT and AST, total bilirubin, and albumin to assess liver injury and by performing H&E and Sirius red staining to determine the degree of liver fibrosis. Immunoblotting was performed to determine the expression levels of inflammation, apoptosis, and Nrf2/HO-1 signaling-related proteins. DIM treatment significantly restored TGF-β1-induced morphological changes, inhibited the expression of mesenchymal markers by activating E-cadherin, decreased mitochondrial membrane potential, reduced ROS intensity, and upregulated levels of Nrf2-responsive antioxidant genes. In the mouse model of CCl4-induced liver fibrosis, DIM remarkably attenuated liver injury and liver fibrosis, as reflected by the reduced ALT and AST parameters with increased serum Alb activity and fewer lesions in H&E staining. It also mitigated the fibrosis area in Sirius red and Masson staining. Taken together, our results suggest a possible molecular mechanism of DIM by suppressing TGF-β1-induced EMT in mouse hepatocytes and CCl4-induced liver fibrosis in mice.

The TFEB-TGIF1 axis regulates EMT in mouse epicardial cells

Epithelial-mesenchymal transition (EMT) is a complex and pivotal process involved in organogenesis and is related to several pathological processes, including cancer and fibrosis. During heart development, EMT mediates the conversion of epicardial cells into vascular smooth muscle cells and cardiac interstitial fibroblasts. Here, we show that the oncogenic transcription factor EB (TFEB) is a key regulator of EMT in epicardial cells and that its genetic overexpression in mouse epicardium is lethal due to heart defects linked to impaired EMT. TFEB specifically orchestrates the EMT-promoting function of transforming growth factor (TGF) β, and this effect results from activated transcription of thymine-guanine-interacting factor (TGIF)1, a TGFβ/Smad pathway repressor. The Tgif1 promoter is activated by TFEB, and in vitro and in vivo findings demonstrate its increased expression when Tfeb is overexpressed. Furthermore, Tfeb overexpression in vitro prevents TGFβ-induced EMT, and this effect is abolished by Tgif1 silencing. Tfeb loss of function, similar to that of Tgif1, sensitizes cells to TGFβ, inducing an EMT response to low doses of TGFβ. Together, our findings reveal an unexpected function of TFEB in regulating EMT, which might provide insights into injured heart repair and control of cancer progression.

Upregulation of dual-specificity phosphatase-26 is required for transforming growth factor β1(TGFβ1)-induced Epithelial-mesenchymal transition in A549 and PANC1 cells.

Transforming Growth Factor β (TGFβ) proteins are potent inducers of the epithelial-mesenchymal transition (EMT) in tumor cells. Although mitogen-activated protein kinase (MAPK) family has been shown to be involved in TGFβ-induced EMT, role of Dual Specificity Phosphatases (DUSP), key regulators of MAPK activity, in TGFβ-induced EMT is largely unkonwn. Real-time qPCR analyses were performed to determine the effect of TGFβ1 on expression of EMT genes and DUSP proteins in the non-small cell lung cancer model A549 and pancreatic adenocarcinoma model PANC1 cells. Western blot analyses were conducted to study the changes in protein levels of EMT proteins and select DUSP proteins, as well as phosphorylations of MAPK proteins upon TGFβ1 stimulation. Small interfering RNA (siRNA) was utilized to reduce expressions of DUSP genes. We observed that the EMT phenotype coincided with increases in phosphorylations of the MAPK proteins ERK1/2, p38MAPK, and JNK upon TGFβ1 stimulation. Real-time qPCR analysis showed increases in DUSP15 and DUSP26 mRNA levels and Western blot analysis confirmed the increase in DUSP26 protein levels in both A549 and PANC1 cells treated with TGFβ1 relative to control. Silencing of DUSP26 expression by siRNA markedly suppressed the effect of TGFβ1 on E-cadherin and mesenchymal genes in the cells. Data provided suggest that TGFβ1 modulates the expression of DUSP genes and that upregulation of DUSP26 may be required for TGFβ1-promoted EMT in A549 and PANC1 cells. Further studies should be carried out to elucidate the requirement of individual DUSPs in TGFβ1-associated EMT in tumor cells.

PGC1α Cooperates with FOXA1 to Regulate Epithelial Mesenchymal Transition through the TCF4-TWIST1

The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) is a critical transcriptional coactivator that maintains metabolic homeostasis and energy expenditure by cooperating with various transcription factors. Recent studies have shown that PGC1α deficiency promotes lung cancer metastasis to the bone through activation of TCF4 and TWIST1-mediated epithelial–mesenchymal transition (EMT), which is suppressed by the inhibitor of DNA binding 1 (ID1); however, it is not clear which transcription factor participates in PGC1α-mediated EMT and lung cancer metastasis. Here, we identified forkhead box A1 (FOXA1) as a potential transcription factor that coordinates with PGC1α and ID1 for EMT gene expression using transcriptome analysis. Cooperation between FOXA1 and PGC1α inhibits promoter occupancy of TCF4 and TWIST1 on CDH1 and CDH2 proximal promoter regions due to increased ID1, consequently regulating the expression of EMT-related genes such as CDH1, CDH2, VIM, and PTHLH. Transforming growth factor beta 1 (TGFβ1), a major EMT-promoting factor, was found to decrease ID1 due to the suppression of FOXA1 and PGC1α. In addition, ectopic expression of ID1, FOXA1, and PGC1α reversed TGFβ1-induced EMT gene expression. Our findings suggest that FOXA1- and PGC1α-mediated ID1 expression involves EMT by suppressing TCF4 and TWIST1 in response to TGFβ1. Taken together, this transcriptional framework is a promising molecular target for the development of therapeutic strategies for lung cancer metastasis.

Long Non-Coding RNA MIR31HG Promotes the Transforming Growth Factor β-Induced Epithelial-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma Cells.

The epithelial-to-mesenchymal transition (EMT) describes a biological process in which polarized epithelial cells are converted into highly motile mesenchymal cells. It promotes cancer cell dissemination, allowing them to form distal metastases, and also involves drug resistance in metastatic cancers. Transforming growth factor β (TGFβ) is a multifunctional cytokine that plays essential roles in development and carcinogenesis. It is a major inducer of the EMT. The MIR31 host gene (MIR31HG) is a newly identified long non-coding (lnc)RNA that exhibits ambiguous roles in cancer. In this study, a cancer genomics analysis predicted that MIR31HG overexpression was positively correlated with poorer disease-free survival of pancreatic ductal adenocarcinoma (PDAC) patients, which was associated with upregulation of genes related to TGFβ signaling and the EMT. In vitro evidence demonstrated that TGFβ induced MIR31HG expression in PDAC cells, and knockdown of MIR31HG expression reversed TGFβ-induced EMT phenotypes and cancer cell migration. Therefore, MIR31HG has an oncogenic role in PDAC by promoting the EMT.