Nintedanib reduces ventilation‐augmented bleomycin‐induced epithelial–mesenchymal transition and lung fibrosis through suppression of the Src pathway

Mechanical ventilation (MV) used in patients with acute respiratory distress syndrome (ARDS) can increase lung inflammation and pulmonary fibrogenesis. Src is crucial in mediating the transforming growth factor (TGF)‐β1‐induced epithelial–mesenchymal transition (EMT) during the fibroproliferative phase of ARDS. Nintedanib, a multitargeted tyrosine kinase inhibitor that directly blocks Src, has been approved for the treatment of idiopathic pulmonary fibrosis. The mechanisms regulating interactions among MV, EMT and Src remain unclear. In this study, we suggested hypothesized that nintedanib can suppress MV‐augmented bleomycin‐induced EMT and pulmonary fibrosis by inhibiting the Src pathway. Five days after administrating bleomycin to mimic acute lung injury (ALI), C57BL/6 mice, either wild‐type or Src‐deficient were exposed to low tidal volume (V_T) (6 ml/kg) or high V_T (30 ml/kg) MV with room air for 5 hrs. Oral nintedanib was administered once daily in doses of 30, 60 and 100 mg/kg for 5 days before MV. Non‐ventilated mice were used as control groups. Following bleomycin exposure in wild‐type mice, high V_T MV induced substantial increases in microvascular permeability, TGF‐β1, malondialdehyde, Masson's trichrome staining, collagen 1a1 gene expression, EMT (identified by colocalization of increased staining of α‐smooth muscle actin and decreased staining of E‐cadherin) and alveolar epithelial apoptosis (P < 0.05). Oral nintedanib, which simulated genetic downregulation of Src signalling using Src‐deficient mice, dampened the MV‐augmented profibrotic mediators, EMT profile, epithelial apoptotic cell death and pathologic fibrotic scores (P < 0.05). Our data indicate that nintedanib reduces high V_T MV‐augmented EMT and pulmonary fibrosis after bleomycin‐induced ALI, partly by inhibiting the Src pathway.     

Exosomes mediate an epithelial‐mesenchymal transition cascade in retinal pigment epithelial cells: Implications for proliferative vitreoretinopathy

Exosomes have recently emerged as a pivotal mediator of many physiological and pathological processes. However, the role of exosomes in proliferative vitreoretinopathy (PVR) has not been reported. In this study, we aimed to investigate the role of exosomes in PVR. Transforming growth factor beta 2 (TGFß‐2) was used to induce epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, as an in vitro model of PVR. Exosomes from normal and EMTed RPE cells were extracted and identified. We incubated extracted exosomes with recipient RPE cells, and co‐cultured EMTed RPE cells and recipient RPE cells in the presence of the exosome inhibitor GW4869. Both experiments suggested that there are further EMT‐promoting effects of exosomes from EMTed RPE cells. MicroRNA sequencing was also performed to identify the miRNA profiles in exosomes from both groups. We identified 34 differentially expressed exosomal miRNAs (P <. 05). Importantly, miR‐543 was found in exosomes from EMTed RPE cells, and miR‐543‐enriched exosomes significantly induced the EMT of recipient RPE cells. Our study demonstrates that exosomal miRNA is differentially expressed in RPE cells during EMT and that these exosomal miRNAs may play pivotal roles in EMT induction. Our results highlight the importance of exosomes as cellular communicators within the microenvironment of PVR.     

Activin A regulates growth of gastro‐intestinal epithelial cells by mediating epithelial‐mesenchymal interaction

The importance of epithelial–mesenchymal interaction on the development of gastro‐intestinal (GI) organs has been repeatedly reported, but its molecular mechanism has not been fully understood though several factors including hepatocyte growth factor and endothelin‐3 have been shown to mediate it. Activins have been demonstrated to play important roles in the regulation of organogenesis in vertebrates, but their roles in the regulation of growth and differentiation of GI organs remain to be solved. In the present study, we examined expression of activins in developing rat GI tract, and found that inhibin bA encoding activin A was specifically expressed by GI mesenchymes, while inhibin bB encoding activin B was expressed by both epithelial and mesenchymal components. We then examined the effect of activin A on the growth of fetal rat GI epithelial cells in primary culture. We found that activin A inhibited the growth of forestomach and glandular stomach epithelial cells while it stimulated the growth of colonic epithelial cells. These results suggest that activin A secreted from GI mesenchymes region‐specifically regulates the growth of attaching epithelial cells. We thus conclude that activin A mediates epithelial‐mesenchymal interaction in the developing GI tract.     

Aldolase A as a prognostic factor and mediator of progression via inducing epithelial–mesenchymal transition in gastric cancer

Glycolysis is regarded as the hallmark of cancer development and progression, which involves a multistep enzymatic reaction. This study aimed to explore the clinicopathological significance and potential role of glycolytic enzyme aldolase A (ALDOA) in the carcinogenesis and progression of gastric cancer (GC). ALDOA was screened from three paired liver metastasis tissues and primary GC tissues and further explored with clinical samples and in vitro studies. The ALDOA protein level significantly correlated with a larger tumor diameter (P = .004), advanced T stage (P < .001), N stage (P < .001) and lymphovascular invasion (P = .001). Moreover, the expression of ALDOA was an independent prognostic factor for the 5‐year overall survival and disease‐free survival of patients with GC in both univariate and multivariate survival analyses (P < .05). Silencing the expression of ALDOA in GC cell lines significantly impaired cell growth, proliferation and invasion ability (P < .05). Knockdown of the expression of ALDOA reversed the epithelial–mesenchymal transition process. Mechanically, ALDOA could affect the hypoxia‐inducible factor (HIF)‐1α activity as demonstrated by the HIF‐1α response element–luciferase activity in GC cells. Collectively, this study revealed that ALDOA was a potential biomarker of GC prognosis and was important in the carcinogenesis and progression of human GC.     

miR‐200c/Bmi1 axis and epithelial–mesenchymal transition contribute to acquired resistance to BRAF inhibitor treatment

Resistance to BRAF inhibitors (BRAFi) is one of the major challenges for targeted therapies for BRAF‐mutant melanomas. However, little is known about the role of microRNAs in conferring BRAFi resistance. Herein, we demonstrate that miR‐200c expression is significantly reduced whereas miR‐200c target genes including Bmi1, Zeb2, Tubb3, ABCG5, and MDR1 are significantly increased in melanomas that acquired BRAFi resistance compared to pretreatment tumor biopsies. Similar changes were observed in BRAFi‐resistant melanoma cell lines. Overexpression of miR‐200c or knock‐down of Bmi1 in resistant melanoma cells restores their sensitivities to BRAFi, leading to deactivation of the PI3K/AKT and MAPK signaling cascades, and acquisition of epithelial–mesenchymal transition‐like phenotypes, including upregulation of E‐cadherin, downregulation of N‐cadherin, and ABCG5 and MDR1 expression. Conversely, knock‐down of miR‐200c or overexpression of Bmi1 in BRAFi‐sensitive melanoma cells activates the PI3K/AKT and MAPK pathways, upregulates N‐cadherin, ABCG5, and MDR1 expression, and downregulates E‐cadherin expression, leading to BRAFi resistance. Together, our data identify miR‐200c as a critical signaling node in BRAFi‐resistant melanomas impacting the MAPK and PI3K/AKT pathways, suggesting miR‐200c as a potential therapeutic target for overcoming acquired BRAFi resistance.     

Polydatin alleviated radiation‐induced lung injury through activation of Sirt3 and inhibition of epithelial–mesenchymal transition

Radiation‐induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy. It is characterized with two main features including early radiation pneumonitis and fibrosis in later phase. This study was to investigate the potential radioprotective effects of polydatin (PD), which was shown to exert anti‐inflammation and anti‐oxidative capacities in other diseases. In this study, we demonstrated that PD‐mitigated acute inflammation and late fibrosis caused by irradiation. PD treatment inhibited TGF‐β1‐Smad3 signalling pathway and epithelial–mesenchymal transition. Moreover, radiation‐induced imbalance of Th1/Th2 was also alleviated by PD treatment. Besides its free radical scavenging capacity, PD induced a huge increase of Sirt3 in culture cells and lung tissues. The level of Nrf2 and PGC1α in lung tissues was also elevated. In conclusion, our data showed that PD attenuated radiation‐induced lung injury through inhibiting epithelial–mesenchymal transition and increased the expression of Sirt3, suggesting PD as a novel potential radioprotector for RILI.     

FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

Transforming growth factor (TGF) β2 and fibroblast growth factor (FGF) 2 are involved in regulation of posterior capsule opacification (PCO) and other processes of epithelial–mesenchymal transition (EMT) such as cancer progression, wound healing and tissue fibrosis as well as normal embryonic development. We previously used an in vivo rodent PCO model to show the expression of tropomyosin (Tpm) 1/2 was aberrantly up‐regulated in remodelling the actin cytoskeleton during EMT. In this in vitro study, we show the Tpms family of cytoskeleton proteins are involved in regulating and stabilizing actin microfilaments (F‐actin) and are induced by TGFβ2 during EMT in lens epithelial cells (LECs). Importantly, we found TGFβ2 and FGF2 played contrasting roles. Stress fibre formation and up‐regulation of α‐smooth muscle actin (αSMA) induced by TGFβ2 could be reversed by Tpm1/2 knock‐down by siRNA. Expression of Tpm1/2 and stress fibre formation induced by TGFβ2 could be reversed by FGF2. Furthermore, FGF2 delivery to TGFβ‐treated LECs perturbed EMT by reactivating the mitogen‐activated protein kinase (MAPK)/ extracellular signal‐regulated kinase (ERK) pathway and subsequently enhanced EMT. Conversely, MEK inhibitor (PD98059) abated the FGF2‐mediated Tpm1/2 and αSMA suppression. However, we found that normal LECs which underwent EMT showed enhanced migration in response to combined TGFβ and FGF2 stimulation. These findings may help clarify the mechanism reprogramming the actin cytoskeleton during morphogenetic EMT cell proliferation and fibre regeneration in PCO. We propose that understanding the physiological link between levels of FGF2, Tpm1/2 expression and TGFβs‐driven EMT orchestration may provide clue(s) to develop therapeutic strategies to treat PCO based on Tpm1/2.     

Trichostatin A,a histone deacetylase inhibitor,suppresses proliferation and epithelial–mesenchymal transition in retinal pigment epithelium cells

The proliferation and epithelial–mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells are the major pathological changes in development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. Histone deacetylases (HDACs)‐mediated epigenetic mechanisms play important roles in controlling various physiological and pathological events. However, whether HDACs are involved in the regulation of proliferation and EMT in PRE cells remains unidentified. In this study, we evaluated the expression profile of HDAC family (18 genes) and found that some of class I and class II HDACs were up‐regulated in transforming growth factor‐β2 (TGF‐β2)/TGF‐β1‐stimulated RPE cells. Tricostatin A (TSA), a class I and II HDAC inhibitor, suppressed the proliferation of RPE cells by G1 phase cell cycle arrest through inhibition of cyclin/CDK/p‐Rb and induction of p21 and p27. In the meantime, TSA strongly prevented TGF‐β2–induced morphological changes and the up‐regulation of α‐SMA, collagen type I, collagen type IV, fibronectin, Snail and Slug. We also demonstrated that TSA affected not only the canonical Smad signalling pathway but also the non‐canonical TGF‐β/Akt, MAPK and ERK1/2 pathways. Finally, we found that the underlying mechanism of TSA affects EMT in RPE cells also through down‐regulating the Jagged/Notch signalling pathway. Therefore, this study may provide a new insight into the pathogenesis of PVR, and suggests that epigenetic treatment with HDAC inhibitors may have therapeutic value in the prevention and treatment of PVR.     

Exosomal miR‐663b targets Ets2‐repressor factor to promote proliferation and the epithelial–mesenchymal transition of bladder cancer cells

Exosomes circulating in biological fluids have the potential to be utilized as cancer biomarkers and are associated with cancer progression and metastasis. MicroRNA (miR)‐663b has been found to be elevated in plasma from patients with bladder cancer (BC). However, the functional role of exosomal miR‐663b in BC processes remains unknown. Here, we isolated exosomes from plasma and found that the miR‐663b level was elevated in exosomes from plasma of patients with BC compared with healthy controls. Exosomal miR‐663b from BC cells promoted cell proliferation and epithelial–mesenchymal transition. Moreover, exosomal miR‐663b targeted Ets2‐repressor factor and acted as a tumor promoter in BC cells. Taken together, our findings suggested that exosomal miR‐663b is a promising potential biomarker and target for clinical detection and therapy in BC.     

Silencing of Prrx1b suppresses cellular proliferation,migration, invasion and epithelial–mesenchymal transition in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired‐related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial–mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up‐regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal‐like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/β‐catenin signaling pathway and induced the mesenchymal‐epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.     

lncRNA‐POIR promotes epithelial–mesenchymal transition and suppresses sorafenib sensitivity simultaneously in hepatocellular carcinoma by sponging miR‐182‐5p

Sorafenib (SOR) resistance remains a major obstacle in the effective treatment of hepatocellular carcinoma (HCC). A number of long noncoding RNAs (lncRNAs) are responsible for this chemoresistance. This study aimed to reveal the essential function of a recently defined lncRNA, lncRNA‐POIR, in the epithelial–mesenchymal transition (EMT) and SOR sensitivity of HCC cells. SOR‐induced cytotoxicity was analyzed via cell counting kit‐8 and ethynyl‐2'‐deoxyuridine incorporation assays, whereas immunoblotting and confocal immunofluorescence were used to determine the expression levels of EMT markers. Furthermore, loss‐ or gain‐of‐function approaches were used to demonstrate the role of lncRNA‐POIR/miR‐182‐5p on EMT and SOR sensitivity in HCC. The direct interaction between lncRNA‐POIR and miR‐182‐5p was verified using a luciferase reporter assay. We found that knockdown of lncRNA‐POIR sensitized HCC cells to SOR and simultaneously reversed EMT. As expected, miR‐182‐5p was confirmed as the downstream target of lncRNA‐POIR. Moreover, miR‐182‐5p overexpression clearly reversed EMT and promoted SOR‐induced cytotoxicity in representative HCC cells, whereas miR‐182‐5p downregulation played a contrasting role; miR‐182‐5p knockdown abolished the modulatory effects of lncRNA‐POIR siRNA on EMT and SOR sensitivity. Together, these pieces of data suggest that lncRNA‐POIR promotes EMT progression and suppresses SOR sensitivity simultaneously by sponging miR‐182‐5p. Thus, we proposed a compelling rationale for the use of lncRNA‐POIR as a promising predictor of SOR response and as a potential therapeutic target for HCC treatment in the future.     

Analysis of epithelial–mesenchymal transition markers in psoriatic epidermal keratinocytes

Psoriasis is similar to endpoints of epithelial–mesenchymal transition (EMT), a process of epithelial cells transformed into fibroblast-like cells. The molecular epithelial and mesenchymal markers were analysed in psoriatic keratinocytes. No obvious alteration of epithelial markers E-cadherin (E-cad), keratin 10 (K10), K14 and K16 was detected in psoriatic keratinocytes. However, significantly increased expression of Vim, FN, plasminogen activator inhibitor 1 (PAI-1) and Slug was seen. IL-17A and IL-13 at 50 ng ml⁻¹ strongly decreased expression of K10, Vim and FN. TGF-β1 at 50 ng ml⁻¹ promoted the production of N-cad, Vim, FN and PAI-1. Slug was decreased by dexamethasone (Dex), but E-cad was upregulated by Dex. Silencing of ERK partially increased E-cad and K16, but remarkably inhibited K14, FN, Vim, β-catenin, Slug and α5 integrin. Moreover, inhibition of Rho and GSK3 by their inhibitors Y27632 and SB216763, respectively, strongly raised E-cad, β-catenin and Slug. Dex decreased Y27632-mediated increase of β-catenin. Dex at 2.0 µM inhibited SB216763-regulated E-cad, β-catenin and slug. In conclusion, EMT in psoriatic keratinocytes may be defined as an intermediate phenotype of type 2 EMT. ERK, Rho and GSK3 play active roles in the process of EMT in psoriatic keratinocytes.