Activation Of Epithelial-mesenchymal Transition Research Articles

Targeting MDK Abrogates IFN-γ-Elicited Metastasis inCancers of Various Origins.

IFN-γ is a pleiotropic cytokine with immunomodulatory and tumoricidal functions. It has been used as an anti-tumor agent in adjuvant therapies for various cancers. Paradoxically, recent advances have also demonstrated pro-tumorigenic effects of IFN-γ, especially in promoting cancer metastasis, with the mechanism remains unclear. This will undoubtedly hinder the application of IFN-γ in cancer treatment. Here, we verified that IFN-γ treatment led to activation of the epithelial-to-mesenchymal transition (EMT) programme and metastasis in cell lines of various cancers, including the kidney cancer cell line Caki-1, the lung cancer cell line A549, the cervical carcinoma cell line CaSki, the breast cancer cell line BT549 and the colon cancer cell line HCT116. We further disclosed that midkine (MDK), an emerging oncoprotein and EMT inducer, is a common responsive target of IFN-γ in these cell lines. Mechanistically, IFN-γ upregulated MDK via STAT1, a principle downstream effector in the IFN-γ signalling. MDK is elevated in the majority of cancer types in the TCGA database, and its overexpression drove EMT activation and cancer metastasis in all examined cell lines. Targeting MDK using a specific MDK inhibitor (iMDK) broadly reversed IFN-γ-activated EMT, and subsequently abrogated IFN-γ-triggered metastasis. Collectively, our data uncover a MDK-dependent EMT inducing mechanism underlying IFN-γ-driven metastasis across cancers which could be attenuated by pharmacological inhibition of MDK. Based on these findings, we propose that MDK may be used as a potential therapeutic target to eliminate IFN-γ-elicited pro-metastatic adverse effect, and that combined MDK utilization may expand the application of IFN-γ in cancer and improve the clinical benefits from IFN-γ-based therapies.

Fangchinoline targets epithelial–mesenchymal transition process by modulating activation of multiple cell‐signaling pathways

Epithelial-mesenchymal transition (EMT) is a key process, which can promote the transition of tumor cells into other organs by weakening the cell-cell junctions. Tumor cell invasion and metastasis arising because of EMT can determine the prognosis of cancer. EMT can be induced by several growth factors including transforming growth factor-β (TGF-β), which can exert their effects by affecting several cell-signaling pathways. Fangchinoline (FCN), a kind of bisbenzylisoquinoline, belongs to the family Menispermaceae. FCN can display substantial antitumor effects against various malignant cell lines but its possible impact on EMT has not been explored. We examined the potential impact of FCN in affecting the activation of EMT in human colon cancer cells. We evaluated the influence of FCN on EMT in colon cancer cells by using Western blot analysis and reverse transcription-polymerase chain reactionassays. The cellular invasion and migration were observed by Boyden chamber and wound healing assays. Thereafter, the effect of the drug on proliferation and invasion was also evaluated by real-time cell analysis. FCN suppressed the levels of TGF-β-induced mesenchymal markers, such as fibronectin, vimentin, MMP-9, MMP-2, N-cadherin, Twist, and Snail. However, FCN markedly enhanced the expression of epithelial markers such as occludin and E-cadherin. These results imply that FCN can potentially inhibit tumor metastasis through abrogating EMT. In addition, FCN downregulated c-Met/PI3K/Akt/mTOR and Wnt/β-catenin cell signaling pathways and mitigated tumor migration as well as invasion. Overall, our study suggests a potential novel role of FCN as an antimetastatic agent against human colon cancer cells.

NR4A1 inhibits the epithelial-mesenchymal transition of hepatic stellate cells: Involvement of TGF-β-Smad2/3/4-ZEB signaling.

This study aimed to examine whether nuclear receptor 4a1 (NR4A1) is involved in inhibiting hepatic stellate cell (HSC) activation and liver fibrosis through the epithelial–mesenchymal transition (EMT). HSC-T6 cells were divided into the control group, the acetaldehyde (200 μM, an EMT activator) group, and the NR4A1 activation group (Cytosporone B; 1 μM). The expression levels of the epithelial marker E-cadherin, the mesenchymal markers fibronectin (FN), vimentin, smooth muscle alpha-actin (α-SMA), and fibroblast-specific protein 1 (FSP-1), and the components of the transforming growth factor (TGF)-β pathway were detected by real-time polymerase chain reaction and western blotting. Compared with the control group, E-cadherin in the acetaldehyde group was downregulated, whereas FN, FSP-1, vimentin, α-SMA, and COL1A1/COL1A2 were upregulated (P < 0.05). Compared with the acetaldehyde group, NR4A1 agonist upregulated E-cadherin and downregulated FN, FSP-1, vimentin, α-SMA, and COL1A1/COL1A2 (P < 0.05). After acetaldehyde stimulation, TGF-β, Smad2/3/4, and zinc finger E-box-binding homeobox (ZEB) were upregulated, while Smad7 mRNA levels were downregulated (all P < 0.05). Compared with acetaldehyde alone, NR4A1 agonist increased Smad7 mRNA levels and reduced TGF-β, Smad2/3/4, and ZEB mRNA levels (all P < 0.05). NR4A1 activation suppresses acetaldehyde-induced EMT, as shown by epithelial and mesenchymal marker expression. The inhibition of the TGF-β–Smad2/3/4–ZEB signaling during HSC activation might be involved.

Aryl hydrocarbon Receptor (AhR) Promotes Cell Growth, Induces Stemness Like Characteristics and Metastasis in Ovarian Cancer Cells via Activation of Akt, β‐Catenin and EMT

BackgroundOvarian cancer is the most lethal form of all gynecological malignancies with highpitched tumor relapse and chemoresistance. Cancer stem cells (CSCs), a small sub population of cancer cells driving tumor initiation, progression, and metastasis, are known to be a major contributor of therapy resistance. Recent studies have depicted a link between CSC development and exposure to environmental pollutants like 2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD). Reports suggest that one of the major pathways that mediate the metabolism of these environmental chemicals is the aryl hydrocarbon receptor (AhR) pathway, whose role in several CSCs is lately demonstrated. However, its role in ovarian cancer development and chemoresistance remains uninvestigated. We hypothesize that AhR and its regulatory genes CYP1A1 and CYP1B1 are involved in the ovarian cancer progression and chemoresistance. The objectives of the present study were a) to examine the role and involvement of AhR in ovarian cancer progression and chemoresistance and b) to explore the underlying mechanisms.MethodsTo test our hypothesis, seven ovarian cancer cell lines; A2780, A2780cis (cisplatin resistant), OVCAR‐3, SKOV‐3, CAOV‐3, and SW‐626 were utilized in this study. Ovarian cancer cells were treated for 24 h with a potent AhR activator, tetrachlorodibenzo‐p‐dioxin (TCDD, 10 nM), thereafter protein expression of AhR, CYP1A1, CYP1B1, pro‐ and anti‐apoptotic, and stemness markers were determined by immunoblotting.Resultsour study showed that SKOV‐3 cells showed highest basal expression of AhR, whereas A2780cis and A2780 expressed the highest basal expression of CYP1A1 and CYP1B1 proteins, respectively. At the inducible levels, treatment of selected cells (OVCAR‐3, SKOV‐3 and A2780) with TCDD significantly induced the protein level of AhR, CYP1A1 in OVCAR‐3 and SKOV‐3, and CYP1B1 in only SKOV‐3 and A2780 cells. To investigate the mechanism involved, we utilized A2780 cells as a model to explore the effect of TCDD on pro‐ and anti‐apototic, Epithelial to Mesenchymal Transition (EMT), and stemness markers. Our results showed that activation of AhR by TCDD in A2780 cells induced phosphorylation of Akt protein with a concomitant increase in the expression of anti‐apoptotic proteins BCL‐2, BCL‐xl and MCL‐1. In addition, a significant increase of aldehyde dehydrogenase (ALDH1), a functional CSC‐specific marker exhibiting important role in cancer progression and chemoresistance was also observed. This effect was also associated with an accumulation of βCatenin, a Wnt transcription factor. Moreover, we found a decrease in E‐cadherin, an epithelial marker, followed by a concurrent increase in Vimentin, a mesenchymal marker, upon AhR activation, confirming the induction of EMT.ConclusionThe results from our study indicate that AhR regulates ovarian cancer progression, exerts stemness characteristics and induces metastasis via activation of Akt, Wnt/β‐Catenin and EMT. This study provides a better understanding of the regulation of ovarian cancer cells in relation to AhR that could help in devising novel therapeutics for better management of ovarian cancer and prevention.

Epithelial-mesenchymal transition and cancer stem cells: A route to acquired cisplatin resistance through epigenetics in HNSCC.

Chemoresistance is associated with tumor recurrence, metastases, and short survival. Cisplatin is one of the most used chemotherapies in cancer treatment, including head and neck squamous cell carcinoma (HNSCC), and many patients develop resistance. Here, we established cell lines resistant to cisplatin to better understand epigenetics and biological differences driving the progression of HNSCC after treatment. Cisplatin resistance was established in CAL-27 and SCC-9 cell lines. Gene expression of HDAC1, HDAC2, SIRT1, MTA1, KAT2B, KAT6A, KAT6B, and BRD4 indicated the cisplatin activates the epigenetic machinery. Increases in tumor aggressiveness were detected by BMI-1 and KI-67 in more resistant cell lines. Changes in cellular shape and epithelial-mesenchymal transition (EMT) activation were also observed. HDAC1 and ZEB1 presented an opposite distribution with down-regulation of HDAC1 and up-regulation of ZEB1 in the course of chemoresistance. Up-regulation of ZEB1 and BMI-1 in patients with HNSCC is also associated with a poor response to therapy. Cancer stem cells (CSC) population increased significantly with chemoresistance. Down-regulation of HDAC1, HDAC2, and SIRT1 and accumulation of Vimentin and ZEB1 were observed in the CSC population. Our results suggest that in the route to cisplatin chemoresistance, epigenetic modifications can be associated with EMT activation and CSC accumulation which originate more aggressive tumors.

Hydrogen Sulfide Inhibits Bronchial Epithelial Cell Epithelial Mesenchymal Transition Through Regulating Endoplasm Reticulum Stress.

Epithelial mesenchymal transition (EMT) is a contributing factor in remodeling events of chronic obstructive pulmonary disease (COPD). Hydrogen sulfide (H2S) has been implicated in the pathogenesis of COPD, but the effect of H2S in regulating EMT and the underlying mechanisms is not clear. In this study, we assessed endoplasmic reticulum (ER) stress markers, EMT markers and associated signal molecules in rat lungs, bronchial epithelial cells, and human peripheral lung tissues to investigate the effect of H2S in regulating EMT and the underlying mechanisms. We found that EMT and ER stress occurred in lung epithelial cells, especially in the bronchial epithelial cells of smokers and COPD patients. In cigarette smoke (CS)-exposed rats, intraperitoneal injection of NaHS significantly alleviated CS-induced lung tissue damage, small airway fibrosis, ER stress, and EMT, while intraperitoneal injection of propargylglycine (cystathionine-gamma-lyase inhibitor) aggravated these effects induced by CS. In the nicotine-exposed 16HBE cells, an appropriate concentration of H2S donor not only inhibited nicotine-induced ER stress, but also inhibited nicotine-induced enhancement of cell migration ability and EMT. ER stress nonspecific inhibitors taurine and 4-phenyl butyric acid also inhibited nicotine-induced enhancement of cell migration ability and EMT. Both H2S and inositol-requiring enzyme 1 (IRE1) activation inhibitor 4μ8C inhibited nicotine-induced activation of IRE1, Smad2/3 and EMT. These results suggest that H2S inhibits CS- or nicotine-induced ER stress and EMT in bronchial epithelial cells and alleviates CS-induced lung tissue damage and small airway fibrosis. The IRE1 signal pathway and Smad2/3 may be responsible for the inhibitory effect of H2S.

The clinical relevance of epithelial-mesenchymal transition and its correlations with tumorigenic immune infiltrates in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a cancer with extremely high mortality. Epithelial-mesenchymal transition (EMT) may play an important role in the occurrence, invasion and prognosis of HCC; however, its relationship with immunity in HCC has not yet been studied. Therefore, we investigated the diagnostic and prognostic values of EMT and explored its potential connections with tumorigenic immune infiltrates in HCC. We first proposed a quantitative metric of EMT activity, the EMT score. After applying this metric to 20 datasets from the Integrative Molecular Database of Hepatocellular Carcinoma, the Cancer Genome Atlas, and the Gene Expression Omnibus, we explored the ability of the EMT score to stratify across sample types. We then applied the EMT score for survival analysis and to differentiate patients with/without vascular invasion to test its prognostic value. We also collected and calculated data on the abundance of immune cells and immune cell markers in HCC and investigated their correlations with EMT scores. Finally, we synthesized and analyzed 20 datasets and constructed an EMT-gene-immune linkage network. The results showed higher EMT scores in HCC samples than in cirrhotic and normal livers. The cases with higher EMT scores also showed poorer performance in terms of prognostic factors such as vascular invasion and overall survival time. Our research demonstrated a broad correlation between EMT and the tumor immune microenvironment, and we uncovered multiple potential linkers associated with both EMT and immunity. Studying EMT has clinical relevance and high diagnostic and prognostic value for HCC.

In Silico Analysis of Ion Channels and Their Correlation with Epithelial to Mesenchymal Transition in Breast Cancer.

Simple SummaryBreast cancer involves changes in the healthy cells of the breast resulting in rapid and abnormal division of cells that later spread to other parts of the body through the process of metastasis, which involves epithelial mesenchymal transition (EMT). Ion channels play a significant role in the switch from epithelial to mesenchymal transition through their contributions to cellular motility, cell volume regulation and cell cycle progression. Comprehensive computational analyses were performed to understand the role of ion channels in tumor/metastatic samples of breast cancer and their correlation with EMT.Uncontrolled growth of breast cells due to altered gene expression is a key feature of breast cancer. Alterations in the expression of ion channels lead to variations in cellular activities, thus contributing to attributes of cancer hallmarks. Changes in the expression levels of ion channels were observed as a consequence of EMT. Additionally, ion channels were reported in the activation of EMT and maintenance of a mesenchymal phenotype. Here, to identify altered ion channels in breast cancer patients, differential gene expression and weighted gene co-expression network analyses were performed using transcriptomic data. Protein–protein interactions network analysis was carried out to determine the ion channels interacting with hub EMT-related genes in breast cancer. Thirty-two ion channels were found interacting with twenty-six hub EMT-related genes. The identified ion channels were further correlated with EMT scores, indicating mesenchymal phenotype. Further, the pathway map was generated to represent a snapshot of deregulated cellular processes by altered ion channels and EMT-related genes. Kaplan–Meier five-year survival analysis and Cox regressions indicated the expression of CACNA1B, ANO6, TRPV3, VDAC1 and VDAC2 to be potentially associated with poor survival. Deregulated ion channels correlate with EMT-related genes and have a crucial role in breast cancer-associated tumorigenesis. Most likely, they are potential candidates for the determination of prognosis in patients with breast cancer.

An integrative gene expression signature analysis identifies CMS4 KRAS-mutated colorectal cancers sensitive to combined MEK and SRC targeted therapy

BackgroundOver half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s).MethodsWe classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi “bypass” resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154).ResultsHere we report a central role of SRC in mediating “bypass”-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P < 10–320), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs.ConclusionsLarge human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node–-an Achilles’ heel–-in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC.

Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGF\u03b2R2 signaling pathway

BRCA1 deficient breast cancers are aggressive and chemoresistant due, in part, to their enrichment of cancer stem cells that can be generated from carcinoma cells by an epithelial-mesenchymal transition (EMT). We previously discovered that BRCA1 deficiency activates EMT in mammary tumorigenesis. How BRCA1 controls EMT and how to effectively target BRCA1-deficient cancers remain elusive. We analyzed murine and human tumors and identified a role for Tgfβr2 in governing the molecular aspects of EMT that occur with Brca1 loss. We utilized CRISPR to delete Tgfβr2 and specific inhibitors to block Tgfβr2 activity and followed up with the molecular analysis of assays for tumor growth and metastasis. We discovered that heterozygous germline deletion, or epithelia-specific deletion of Brca1 in mice, activates Tgfβr2 signaling pathways in mammary tumors. BRCA1 depletion promotes TGFβ-mediated EMT activation in cancer cells. BRCA1 binds to the TGFβR2 locus to repress its transcription. Targeted deletion or pharmaceutical inhibition of Tgfβr2 in Brca1-deficient tumor cells reduces EMT and suppresses tumorigenesis and metastasis. BRCA1 and TGFβR2 expression levels are inversely related in human breast cancers. This study reveals for the first time that a targetable TGFβR signaling pathway is directly activated by BRCA1-deficiency in the induction of EMT in breast cancer progression.

Artesunate inhibits proliferation and migration of RPE cells and TGF-β2 mediated epithelial mesenchymal transition by suppressing PI3K/AKT pathway

To study the braking effectiveness of artesunate on transforming growth factor (TGF)-β2 mediated epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) in vitro. The fostered ARPE-19 cells were processed with artesunate alone or combined with the TGF-β2. The CCK-8 examination was utilized to test the cell propagation. Cell migration was detected by scratch as well as the Transwell examination. The EMT characters and activation of PI3K/Akt signal channel were estimated by Western blotting and immunofluorescence. The Western blotting was utilized in order to confirm the vitreous of controls as well as patients with proliferative vitreoretinopathy (PVR) were collected and the levels of PI3K, phospho-PI3K, Akt. Disposal of ARPE-19 cells with artesunate (50-150 µmol/L) obviously suppressed their propagation and immigration, which dependent on the concentration and time. Artesunate suppressed the EMT which was induced by TGF-β2 in ARPE-19 cells through sustaining the expression of vimentin and α-SMA through the suppression of PI3K, phospho-PI3K, phospho-Akt and Akt. Levels of PI3K, phospho-PI3K, AKT and phospho-Akt was increased in the vitreous in PVR (P<0.05). Such findings indicate that PI3K/Akt signal channel is highly activated in vitreous of PVR. Artesuante is an operative depressor of the propagation, immigration and TGF-β2-mediated EMT of ARPE-19 cells by reduced the expression of PI3K/Akt channel.

Nifuroxazide ameliorates pulmonary fibrosis by blocking myofibroblast genesis: a drug repurposing study

BackgroundIdiopathic pulmonary fibrosis (IPF) is a serious interstitial lung disease with a complex pathogenesis and high mortality. The development of new drugs is time-consuming and laborious; therefore, research on the new use of old drugs can save time and clinical costs and even avoid serious side effects. Nifuroxazide (NIF) was originally used to treat diarrhoea, but more recently, it has been found to have additional pharmacological effects, such as anti-tumour effects and inhibition of inflammatory diseases related to diabetic nephropathy. However, there are no reports regarding its role in pulmonary fibrosis.MethodsThe therapeutic effect of NIF on pulmonary fibrosis in vivo was measured by ELISA, hydroxyproline content, H&E and Masson staining, immunohistochemistry (IHC) and western blot. Immune cell content in lung tissue was also analysed by flow cytometry. NIF cytotoxicity was evaluated in NIH/3T3 cells, human pulmonary fibroblasts (HPFs), A549 cells and rat primary lung fibroblasts (RPLFs) using the MTT assay. Finally, an in vitro cell model created by transforming growth factor-β1 (TGF-β1) stimulation was assessed using different experiments (immunofluorescence, western blot and wound migration assay) to evaluate the effects of NIF on the activation of NIH/3T3 and HPF cells and the epithelial-mesenchymal transition (EMT) and migration of A549 cells.ResultsIn vivo, intraperitoneal injection of NIF relieved and reversed pulmonary fibrosis caused by bleomycin (BLM) bronchial instillation. In addition, NIF inhibited the expression of a variety of cellular inflammatory factors and immune cells. Furthermore, NIF suppressed the activation of fibroblasts and EMT of epithelial cells induced by TGF-β1. Most importantly, we used an analytical docking experiment and thermal shift assay to further verify that NIF functions in conjunction with signal transducer and activator of transcription 3 (Stat3). Moreover, NIF inhibited the TGF-β/Smad pathway in vitro and decreased the expression of phosphorylated Stat3 in vitro and in vivo.ConclusionTaken together, we conclude that NIF inhibits and reverses pulmonary fibrosis, and these results support NIF as a viable therapeutic option for IPF treatment.Graphic

Quantifying the Patterns of Metabolic Plasticity and Heterogeneity along the Epithelial-Hybrid-Mesenchymal Spectrum in Cancer.

Cancer metastasis is the leading cause of cancer-related mortality and the process of the epithelial-to-mesenchymal transition (EMT) is crucial for cancer metastasis. Both partial and complete EMT have been reported to influence the metabolic plasticity of cancer cells in terms of switching among the oxidative phosphorylation, fatty acid oxidation and glycolysis pathways. However, a comprehensive analysis of these major metabolic pathways and their associations with EMT across different cancers is lacking. Here, we analyse more than 180 cancer cell datasets and show the diverse associations of these metabolic pathways with the EMT status of cancer cells. Our bulk data analysis shows that EMT generally positively correlates with glycolysis but negatively with oxidative phosphorylation and fatty acid metabolism. These correlations are also consistent at the level of their molecular master regulators, namely AMPK and HIF1α. Yet, these associations are shown to not be universal. The analysis of single-cell data for EMT induction shows dynamic changes along the different axes of metabolic pathways, consistent with general trends seen in bulk samples. Further, assessing the association of EMT and metabolic activity with patient survival shows that a higher extent of EMT and glycolysis predicts a worse prognosis in many cancers. Together, our results reveal the underlying patterns of metabolic plasticity and heterogeneity as cancer cells traverse through the epithelial–hybrid–mesenchymal spectrum of states.

Targeting hypoxia regulated sodium driven bicarbonate transporters reduces triple negative breast cancer metastasis.

Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple negative breast cancer subtype (TNBC). Metastasis is the cause of 90% of breast cancer patient deaths. Regions of tumour hypoxia tend to be more acidic and both hypoxia and acidosis increase tumour metastasis. In line with this the metastatic process is dependent on pH regulatory mechanisms. We and others have previously identified increased hypoxic expression of Na+ driven bicarbonate transporters (NDBTs) as a major mechanism of tumour pH regulation. Hypoxia induced the expression of NDBTs in TNBC, most frequently SLC4A4 and SLC4A5. NDBT inhibition (S0859) and shRNA knockdown suppressed migration (40% reduction) and invasion (70% reduction) in vitro. Tumour xenograft metastasis in vivo was significantly reduced by NDBT knockdown. To investigate the mechanism by which NDBTs support metastasis, we investigated their role in regulation of phospho-signalling, epithelial-to-mesenchymal transition (EMT) and metabolism. NDBT knockdown resulted in an attenuation in hypoxic phospho-signalling activation; most notably LYN (Y397) reduced by 75%, and LCK (Y394) by 72%. The metastatic process is associated with EMT. We showed that NDBT knockdown inhibited EMT, modulating the expression of key EMT transcription factors and ablating the expression of vimentin whilst increasing the expression of E-cadherin. NDBT knockdown also altered metabolic activity reducing overall ATP and extracellular lactate levels. These results demonstrate that targeting hypoxia-induced NDBT can be used as an approach to modulate phospho-signalling, EMT, and metabolic activity and reduce tumour migration, invasion, and metastasis in vivo.

Silencing CDCA8 inhibits the proliferation and invasion of gastric cancer cells and induces apoptosis by blocking the Akt pathway

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To investigate the effect of cell division cycle associated 8 (CDCA8) on malignant progression of gastric cancer (GC) cells.&#x0D; Methods: Short hairpin RNAs (shRNA) were transfected into two gastric cell lines to knock down expression of CDCA8. Transfection efficiency was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Then, Cell Counting Kit 8 and colony formation and Transwell assays were utilized to explore the effect of CDCA8 knockdown on the proliferation, invasion, and migration of GC cells. Flow cytometry was conducted to analyze the effect of CDCA8 knockdown on cell cycle progression and apoptosis. The relationship between CDCA8, epithelial-mesenchymal transition (EMT), and Akt pathway activity was determined by western blot analysis.&#x0D; Results: Proliferation, invasion, and migration of GC cells were significantly inhibited by CDCA8 knockdown. Knockdown of CDCA8 induced cell cycle arrest in G1 phase and apoptosis, and inhibited EMT and Akt pathway activity.&#x0D; Conclusion: Knockdown of CDCA8 inhibits GC growth and metastasis in vitro by reducing Akt pathway activity. Thus, this molecule presents a potential strategy for the management of GC.&#x0D; &#x0D; &#x0D; &#x0D;

KCNIP3 silence promotes proliferation and epithelial-mesenchymal transition of papillary thyroid carcinoma through activating Wnt/β-catenin pathway

PurposePapillary thyroid carcinoma (PTC) is the common endocrine malignancy. Kv channel interacting protein 3 (KCNIP3) has been investigated in a variety of diseases, but its role and underlying mechanism in PTC are not fully delineated. Based on this, this study mainly explored the possible mechanism of KCNIP3 in PTC. MethodsKCNIP3 expression in PTC tissues was analyzed by ENCORI and validated by quantitative real-time PCR (qRT-PCR). KCNIP3 overexpression (oe-KCNIP3) or KCNIP3 silence (si-KCNIP3) was transfected into IHH4 and FTC-133 cells, respectively. Then cell biological behaviors were detected by cell function assays. The expressions of epithelial-mesenchymal transition (EMT)- and Wnt/β-catenin pathway-related proteins were quantified by qRT-PCR and western blot. Lastly, IHH4 cells were treated with LiCl and the above assays were performed again. ResultsThe expression of KCNIP3 was decreased in PTC. After transfection, oe-KCNIP3 inhibited the PTC cell viability, cloning, migration and invasion but promoted apoptosis, and meanwhile, oe-KCNIP3 reduced the EMT and Wnt pathway activation. In contrast, si-KCNIP3 had the opposite effect. Moreover, LiCl, a Wnt signaling pathway activator, could reverse the above effects of oe-KCNIP3. ConclusionKCNIP3 might play an anticarcinogenic role in PTC via inhibiting the activation of Wnt/β-catenin signaling pathway.

B7‑H3 promotes the epithelial‑mesenchymal transition of NSCLC by targeting SIRT1 through the PI3K/AKT pathway.

Epithelial-mesenchymal transition (EMT) is a key step in cancer metastasis. B7-H3, a co-signaling molecule associated with poor prognosis of non-small cell lung cancer (NSCLC), promotes the metastasis of NSCLC by activating the EMT process. However, its underlying mechanism remains poorly understood. In the present study, it was shown that CRISPR/Cas9-mediated B7-H3 deletion downregulated the expression of the class III histone deacetylase, sirtuin-1 (SIRT1), in NSCLC A549 cells. Accordingly, SIRT1 silencing resulted in markedly decreased migration and invasion of A549 cells. Both B7-H3 gene-edited and SIRT1-silenced cells were typically characterized by an increased expression of the epithelial marker E-cadherin, and downregulation of the mesenchymal markers N-cadherin and vimentin, as compared with mock-edited and scrambled negative small interfering RNA control, respectively. It was further demonstrated that B7-H3 ablation significantly downregulated phosphorylated AKT/protein kinase B expression, and SIRT1 expression was substantially suppressed by the PI3K-specific inhibitor, LY294002. Taken together, the findings of the present study revealed that B7-H3-induced signaling upregulates SIRT1 expression via the PI3K/AKT pathway to promote EMT activation that is associated with metastasis in NSCLC.

SNF5, a core subunit of SWI/SNF complex, regulates melanoma cancer cell growth, metastasis, and immune escape in response to matrix stiffness

Increased stiffness of the extracellular matrix is an important hallmark of melanoma development and progression, but its regulatory role and related mechanisms remain unclear. We adapted polydimethylsiloxane (PDMS)-micropillar-based matrix platform and investigated the effect of matrix stiffness on the proliferation, epithelial-mesenchymal transition (EMT), and immune escape of melanoma cells. We observed a stiff matrix enhanced cell proliferation, EMT, and immune escape of A375 cells. Furthermore, the expression of SNF5 on the stiffer matrix was higher than that on the softer matrix. Next, we investigated whether SNF5 is an important transducer in response to matrix stiffness. Our results revealed that knockdown of SNF5 significantly decreased stiff matrix-induced activation of cell proliferation, EMT and immune escape. Meanwhile, the overexpression of SNF5 showed its ability to increase cell proliferation, invasion and immune escape by activating the STAT-3 pathway in vitro. Furthermore, SNF5 deficiency elevated the level of tumor-infiltrating CD8+T cells and decreased the number of PD-L1 positive cells in vivo. Together, our findings suggested that stiffer substrate enhanced melanoma development by upregulating SNF5 expression, and SNF5 is a key mediator of stiffer matrix-induced immune evasion of melanoma cancer cells.

Single Nucleotide Polymorphisms of IL-33 Gene Correlated with Renal Allograft Fibrosis in Kidney Transplant Recipients.

Background Nowadays, renal allograft survival is confined by the development of allograft fibrosis. Previous studies have reported interleukin-33 (IL-33) upregulated significantly in patients with chronic renal allograft dysfunction, and it could induce renal tubular epithelial to mesenchymal transition (EMT), which eventually contributed to renal allograft fibrosis. Our study intended to detect the underlying association between single nucleotide polymorphisms (SNPs) of IL-33 gene and renal allograft fibrosis in kidney transplant recipients. Methods We collected blood samples from 200 renal transplant recipients for the identification of SNPs and transplanted kidney tissue samples for identifying differentially expressed genes (DEGs). Intersection of SNP-related genes and DEGs was conducted for further analysis. Relationships between these SNPs and renal allograft fibrosis were evaluated by the inheritance models. Immunohistochemical (IHC) staining and western blotting (WB) were used to detect the expression of IL-33 and the markers of EMT in human kidney tissues obtained from control and chronic renal allograft dysfunction (CAD) patients. In vitro, we detected the progressions of EMT-related markers and the levels of MAPK signaling pathway mediators after transfecting IL-33 mutant plasmids in HK2 cells. Results Three intersected genes including IL-33 genes were significantly expressed. IL-33 expression was validated in kidney tissues by IHC and WB. Thirty-nine IL-33-related SNPs were identified in targeted sequencing, in which 26 tagger SNPs were found by linkage disequilibrium analysis for further analysis. General linear models indicated sirolimus administration significantly influenced renal allograft fibrosis (P < 0.05), adjustment of which was conducted in the following analysis. By multiple inheritance model analyses, SNP rs10975519 of IL-33 gene was found closely related to renal allograft fibrosis (P < 0.005). Furthermore, HK2 cells transfected with mutated plasmid of rs10975519 showed stronger mobility and migration ability. Moreover, IL-33 mutant plasmids could promote the IL-33-induced EMT through the sustained activation of p38 MAPK signaling pathway in HK2 cells. Conclusion In our study, rs10975519 on the IL-33 gene was found to be statistically associated with the development of renal allograft fibrosis in kidney transplant recipients. This process may be related to the IL-33-induced EMT and sustained activation of p38 MAPK signaling pathway.

Ferritinophagic Flux Was a Driving Force in Determination of Status of EMT, Ferroptosis, and NDRG1 Activation in Action of Mechanism of 2-Pyridylhydrazone Dithiocarbamate S-Acetic Acid.

Ferritinophagy is a process of ferritin degradation in lysosomes; however, how its effect on other cellular events, such as epithelial-mesenchymal transition (EMT) and ferroptosis remains elusive. In this study, we determined how ferritinophagic flux influence the status of EMT and ferroptosis in HepG2 cell. Our data revealed that 2-pyridylhydrazone dithiocarbamate s-acetic acid (PdtaA) induced EMT inhibition involved ferritinophagy-mediated ROS production, but addition of ferrostatin-1 could attenuate the effect of PdtaA on the regulation of EMT-related proteins, suggesting that ferroptosis might involve in the EMT regulation. Next, downregulation of Gpx4 and xCT as well as enhanced lipid peroxidation further supported that PdtaA was able to induce ferroptosis. Knockdown of NCOA4 significantly attenuated the regulatory effect of PdtaA on related proteins which highlighted that the strength of ferritinophagic flux (NCOA4/ferritin) was a driving force in determination of the status of EMT and ferroptosis. Furthermore, NDRG1 activation was also observed, and knockdown of NDRG1 similarly influenced the expressions of ferroptosis-related proteins, suggesting that NDRG1 also involved ferroptosis induction, which was first reported. Taken together, PdtaA-induced EMT inhibition, ferroptosis, and NDRG1 activation all depended on the strength of ferritinophagic flux.