Epithelial-mesenchymal Transition Transcription Factors Zeb1 Research Articles

Zeb1 mediates EMT/plasticity-associated ferroptosis sensitivity in cancer cells by regulating lipogenic enzyme expression and phospholipid composition

Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial–mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGFβ stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very long-chain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1.

In vitro effect of 1-methyltryptophan isomers on epithelial-mesenchymal transition transcription factors in tubular epithelial cells after ischemia-reperfusion injury.

The compound 1-methyltryptophan (1-MT) has been shown to act protectively in renal ischemia-reperfusion injury. Toll-like receptor 4 signaling is also a regular process of epithelial-mesenchymal transition (EMT) that can after ischemia-reperfusion injury (IRI) result in an increase in renal fibrosis. EMT is associated with specific transcription factors: Snai1, Snai2, Zeb1, and Twist. 1-MT could regulate EMT and act as an antifibrotic agent. This study aimed to investigate the effect of 1-MT on EMT transcription factors in tubular epithelial cells that underwent 30 min. Renal tubular epithelial cells (TECs) were isolated from Lewis rats using a standard protocol with Fe2O3 magnetic separation and selective media as previously mentioned. Cells were cultivated and divided into 4 groups, namely C-TECs: control cells, IRI-TECs: IRI-induced TECs, D-IRI-TECs: IRI-induced TECs treated with 1-methyl-D-tryptophan, and L-IRI-TECs: IRI-induced TECs treated with 1-methyl-L-tryptophan. IRI was induced in all groups for 30 min by mineral oil (except for C-TECs) followed by 48-hour reperfusion. RNA and proteins were isolated from harvested cells. Using a semi-quantitative polymerase chain reaction, we assessed the relative mRNA expression of EMT transcription factors Snai1, Snai2, Zeb1, and Twist. Hereby, we showed that the treatment of ischemia-induced TECs with both 1-MT isomers lowered the expression of EMT transcription factors Snai1 and Zeb1 which were increased by ischemia and reperfusion of TECs. This could act favorably in renal IRI decreasing EMT and renal fibrosis, therefore showing the potential of 1-MT as a part of therapy in renal transplantation aimed at renal ischemia-reperfusion injury.

A novel antagonist of the CCL5/CCR5 axis suppresses the tumor growth and metastasis of triple-negative breast cancer by CCR5-YAP1 regulation

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC) with a high mortality rate, and few effective therapeutic strategies are available. CCL5/CCR5 is an appealing immunotherapeutic target for TNBC. However, its signaling mechanism is poorly understood and its direct antagonists have not been reported. Here, we developed a high-throughput screening (HTS) assay for discovering its antagonists. Verteporfin was identified as a more selective and potent antagonist than the known CCR5 antagonist maraviroc. Without photodynamic therapy, verteporfin demonstrated significant inhibition on TNBC tumor growth through immune regulation, remarkable suppression of lung metastasis by cell-intrinsic mechanism, and a significant extension of overall survival in vivo. Mechanistically, CCR5 was found to be essential for expression of the key hippo effector YAP1. It promoted YAP1 transcription via HIF-1α and exerted further control over the migration of CD8+ T, NK, and MDSC immune cells through chemokines CXCL16 and CXCL8 which were identified from RNA-seq. Moreover, the CCR5-YAP1 axis played a vital role in promoting metastasis by modulating β-catenin and core epithelial-mesenchymal transition transcription factors ZEB1 and ZEB2. It is noteworthy that the regulatory relationship between CCR5 and YAP1 was observed across various BC subtypes, TNBC patients, and showed potential relevance in fifteen additional cancer types. Overall, this study introduced an easy-to-use HTS assay that streamlines the discovery of CCL5/CCR5 axis antagonists. Verteporfin was identified as a specific molecular probe of this axis with great potentials as a therapeutic agent for treating sixteen malignant diseases characterized by heightened CCR5 and YAP1 levels.

MYL9 expressed in cancer-associated fibroblasts regulate the immune microenvironment of colorectal cancer and promotes tumor progression in an autocrine manner

BackgroundThe tumor microenvironment (TME) is an important factor that regulates the progression of colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs) are the main mesenchymal cells in the TME and play a vital role in tumor progression; however, the specific underlying mechanisms require further study.MethodsMultiple single-cell and transcriptome data were analyzed and validated. Primary CAFs isolation, CCK8 assay, co-culture assay, western blotting, multiple immunofluorescence, qRT-PCR, ELISA, immunoprecipitation, ChIP, double luciferase, and animal experiments were used to explore the potential mechanism of MYL9 regulation in CRC.ResultsOur findings revealed that MYL9 was predominantly localized and expressed in CAFs rather than in CRC cells, and bioinformatics analysis revealed that high MYL9 expression was strongly associated with poor overall and disease-free survival in various tumors. In addition, high MYL9 expression is closely associated with M2 macrophage infiltration, which can lead to an immunosuppressive microenvironment in CRC, making it insensitive to immunotherapy. Mechanically, MYL9 can regulate the secretion of CAFs on CCL2 and TGF-β1, thus affecting the immune microenvironment and progression of CRC. In addition, MYL9 bounded with IQGAP1 to regulate CCL2 and TGF-β1 secretion through the ERK 1/2 pathway, and CCL2 and TGF-β1 synergistically promoted CRC cells progression through the PI3K-AKT pathway. Furthermore, MYL9 promotes epithelial-mesenchymal transition (EMT) in CRC. During the upstream regulation of MYL9 in CAFs, we found that the EMT transcription factor ZEB1 could bind to the MYL9 promoter in CAFs, enhancing the activity and function of MYL9. Therefore, MYL9 is predominantly expressed in CAFs and can indirectly influence tumor biology and EMT by affecting CAFs protein expression in CRC.ConclusionsMYL9 regulates the secretion of cytokines and chemokines in CAFs, which can affect the immune microenvironment of CRC and promote CRC progression. The relationship between MYL9 expression and CRC clinical staging and immunotherapy is closer in CAFs than in tumor cells; therefore, studies using CAFs as a model deserve more attention when exploring tumor molecular targets in clinical research.

MP04-16 EPITHELIAL TO MESENCHYMAL TRANSITION PROMOTES PD-L1 EXPRESSION IN RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023MP04-16 EPITHELIAL TO MESENCHYMAL TRANSITION PROMOTES PD-L1 EXPRESSION IN RENAL CELL CARCINOMA Allison May, Jonathan Jiang, Tyler Robinson, and Evan Keller Allison MayAllison May More articles by this author , Jonathan JiangJonathan Jiang More articles by this author , Tyler RobinsonTyler Robinson More articles by this author , and Evan KellerEvan Keller More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Sarcomatoid dedifferentiation in renal cell carcinoma (RCC) is thought to occur when the parental cell type, such as clear cell RCC (ccRCC), undergoes an epithelial mesenchymal transition (EMT). The mesenchymal sarcomatoid cells have increased ability to invade and metastasize, and thus sarcomatoid RCC (sRCC) has a very poor prognosis. Interestingly however, there is encouraging evidence that sRCC responds at higher rates to PD-1/PD-L1 directed immunotherapy than ccRCC. We aim to test the hypothesis that EMT directly upregulates PD-L1 expression in sRCC and that PD-L1 may have positive feedback on induction of EMT. METHODS: Baseline expression of EMT markers, EMT transcription factors and PD-L1 were measured using Western Blot and qPCR in two clear cell RCC lines (Caki-1 and 786-O) and five sarcomatoid RCC lines (RCJ-41T1, RCJ-41T2, RCJ-41M, UOK-276, UOK-127). EMT was induced in the cells through treatment with multiple agents including TGFB, IFNg, HGF, SPP1, and CoCl2 and by over expression of Snail or Zeb1. Effect of EMT on PD-L1 expression was measured. To evaluate the role of PD-L1 on EMT, PD-L1 expression was decreased using siRNA and its effect on EMT related protein expression was measured using Western Blot. RESULTS: At baseline, sRCC cell lines generally had higher expression of EMT related markers and transcription factors than ccRCC lines. Baseline PD-L1 expression positively correlated with increasing EMT state in all cell lines. Treatment of cells with known EMT inducers, TGFB, IFNg, and hypoxia led to increased levels of mesenchymal marker N-cadherin, decreased epithelial marker E-cadherin, and increased levels of EMT transcription factors Zeb1, Snail, and Slug. Treatment of cells with HGF and SPP1, two molecules associated with sRCC based on our prior work, also induced EMT. Finally, over expression of known EMT transcription factors Snail and Zeb1 led to EMT as well. All methods of EMT induction concomitantly increased PD-L1 expression. Knockdown of PD-L1 was tested in UOK-276 and 786-O cells and led to decreased expression levels of EMT transcription factors and increased epithelial markers. CONCLUSIONS: These data support the theory that sRCC arises from EMT and that EMT directly leads to upregulation of PD-L1. Furthermore, these results suggest that PD-L1 maintains the mesenchymal state. These findings may help explain the enhanced response to PD-1/PD-L1 directed therapy in sRCC and suggest that markers of EMT state could represent a biomarker for immunotherapy response in RCC. Source of Funding: Clarke Family Fellowship for Kidney Cancer Research © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e39 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Allison May More articles by this author Jonathan Jiang More articles by this author Tyler Robinson More articles by this author Evan Keller More articles by this author Expand All Advertisement PDF downloadLoading ...

Opposing roles of ZEB1 in the cytoplasm and nucleus control cytoskeletal assembly and YAP1 activity.

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and is initiated by mesenchyme-driving transcription factors and actin cytoskeletal assembly. We show a cytoplasmic-to-nuclear transport gradient of the EMT transcription factor Zeb1 toward sites of invasion in lung adenocarcinoma (LUAD), driven by the EMT inducer Tgfb, which is expressed in M2 polarized macrophages. We show that Zeb1 binds free actin monomers and RhoA in the cytoplasm to inhibit actin polymerization, blocking cell migration and Yap1 nuclear transport. Tgfb causes turnover of the scaffold protein Rassf1a, which targets RhoA. Release of this RhoA inhibition in response to Tgfb overcomes Zeb1's block of cytoskeleton assembly and frees it for nuclear transport. A ZEB1 nuclear transport signature highlights EMT progression, identifies dedifferentiated invasive/metastatic human LUADs, and predicts survival. Blocking Zeb1 nuclear transport with a small molecule identified in this study inhibits cytoskeleton assembly, cell migration, Yap1 nuclear transport, EMT, and precancerous-to-malignant transition.

Molecular mechanism of CD163+ tumor-associated macrophage (TAM)-derived exosome-induced cisplatin resistance in ovarian cancer ascites

BackgroundStudies have found that tumor-associated macrophages (TAMs) in the malignant ascites of patients with serous ovarian cancer exhibit a mixed polarization phenotype and highly variable expression of the surface marker CD163. The exosomes secreted by mesenchymal cells can be taken up by tumor cells, affecting the malignant biological behavior of them.MethodsUsing reverse transcription-polymerase chain reaction (RT-PCR) to detect the genes expression of drug resistance related factors cancer stem cells (CSCs)/multidrug resistance (MDR)/epithelial-mesenchymal transition (EMT) after CD163+ TAMs exosomes in ovarian cancer ascites co-cultured with A2780 and A2780/cis-diamminedichloroplatinum (DDP). Differences of the level of miR-221-3p expression between CD163+ TAMs cells (M2) and peripheral blood mononuclear cells M0 detect by microarray screening, and bioinformatics analysis predicts that its target gene is ADAMTS6. Western blot (WB) and immunohistochemical detection of ADAMTS6 expression level in epithelial ovarian cancer (EOC) clinical sample tissues, and analysis of the ADAMTS6 expression in ovarian cancer tissues and its correlation with clinicopathological factors. WB was used to detect the expression of AKT pathway and downstream EMT-related molecules [SNAIL1, ZEB1, Vimentin (VIM)] after co-culture of CD163+ TAMs exosomes with ovarian cancer cell lines A2780, A2780/DDP. WB detects the expression of EGFR and TGF-β1 upstream molecules of the AKT signaling pathway (AKT-pAKT) and downstream EMT molecules (SNAIL1, ZEB1, VIM) after overexpression of ADAMTS6 in A2780 and A2780/DDP.ResultsWe demonstrated that after CD163+ TAM exosomes were taken up by EOC cells, the highly expressed miR-221-3p could downregulate the level of ADAMTS6, further activate the AKT signaling pathway, and increase the expression of EMT transcription factors SNAIL1 and ZEB1 and the mesothelial marker VIM. Decreased expression of the epithelial marker E-cadherin induced EMT, triggering a switch to a CSC-like phenotype and MDR, thereby promoting EOC cell proliferation, adhesion, migration, and resistance. Compared with benign ovarian tumors, ADAMTS6 expression was low in EOC tissues and was closely related to the clinical stage, age, and survival curve of ovarian cancer patients.ConclusionsOverexpression of ADAMTS6 reduced the IC50 of cisplatin in ovarian cancer cells. The mechanism may be related to the inhibition of EMT mediated by the EGFR/TGF-β/AKT pathway of EOC cells, which has potential value in the treatment of ovarian cancer.

Abstract LB095: A domain-specific antibody to NRP2 down-regulated epithelial-mesenchymal transition genes and enhanced efficacy of standard-of-care therapeutics for aggressive breast cancer

Abstract INTRODUCTION: A growing body of evidence suggests that increased expression of the vascular endothelial growth factor (VEGF) co-receptor Neuropilin-2 (NRP2) is associated with aggressive breast cancers and that VEGF/NRP2 signaling contributes to clinical resistance to chemotherapy and tumor recurrence, making NRP2 a promising therapeutic target. A major limitation that has hampered the development of such a therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that specifically block VEGF/NRP2 signaling. aTyr has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of diseases that involve NRP2 signaling. Among them, ATYR2810 has been characterized to bind to the b1 domain of NRP2 that encompasses the VEGF binding sites. It completely blocks the binding of VEGF to NRP2, and VEGF-induced NRP2/VEGFR dimerization. Importantly, ATYR2810 had no effect on Semaphorin 3F (Sema3F) induced NRP2/PlexinA1 dimerization, demonstrating its specificity for blocking the VEGF/NRP2 pathway. We have shown that ATYR2810, but not a Sema3F-blocking mAb has tumor-inhibitory effects on triple negative breast cancer (TNBC) cell lines or patient-derived organoids. RESULTS: We further evaluated the efficacy of ATYR2810 in combination with standard-of-care therapeutics including Cisplatin and Bevacizumab (anti-VEGF-A blocking antibody). In in vitro 3D methylcellulose colony formation assays, ATYR2810 sensitized TNBC cells to Cisplatin or Bevacizumab and considerably reduced colony formation in combination with these treatments. In an in vivo TNBC xenograft cancer model (MDA-MB-231), ATYR2810 augmented the anti-tumor effects of Cisplatin or Bevacizumab. To explore the underlying molecular mechanism, we performed gene expression profiling with samples treated by ATYR2810 alone and the combo therapy with Cisplatin or Bevacizumab. A number of gene markers of cancer stem cells (CSC) and/or epithelial-mesenchymal transition (EMT) were found to be down-regulated by ATYR2810 treatment in TNBC patient-derived organoids, including a key EMT transcription factor ZEB1. We also confirmed the reduction of ZEB1 protein expression by ATYR2810 treatment in TNBC cells. CONCLUSIONS: These results demonstrate the efficacy of ATYR2810 in combination with standard-of-care therapeutics in in vitro and in vivo TNBC models, and suggest its activity is mediated through inhibiting both EMT and cellular dedifferentiation that renders tumors more sensitive to the treatment regimes. The targeting of VEGF/NRP2 signaling by ATYR2810 may provide a new therapeutic option, and lead to the identification of new treatment biomarkers, which could offer improved efficacy and reduced toxicity in aggressive breast cancers. Citation Format: Zhiwen Xu, Christoph Burkart, Hira L. Goel, Justin Rahman, Clara Polizzi, Matt Seikkula, Luke Burman, Arthur M. Mercurio, Leslie A. Nangle. A domain-specific antibody to NRP2 down-regulated epithelial-mesenchymal transition genes and enhanced efficacy of standard-of-care therapeutics for aggressive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB095.

A Theoretical Approach to Coupling the Epithelial-Mesenchymal Transition (EMT) to Extracellular Matrix (ECM) Stiffness via LOXL2.

Simple SummaryEpithelial-mesenchymal transition (EMT) is a key process in cancer progression through which cells weaken their cell-cell adhesion and gain mobility and invasive traits. Besides chemical signaling, recent studies have established the connection of EMT to mechanical microenvironment, such as the stiffness of extracellular matrix (ECM). LOXL2 is representative of a family of enzymes that promotes fiber cross-linking in ECM. With increased cross-linking comes increased stiffness, which induces EMT that can, in turn, elevate LOXL2 levels. As such, a positive feedback loop among EMT, LOXL2, and ECM stiffness can be formed. We built a mathematical model on a core biochemical reaction network featuring this feedback loop, and showed how strongly it drives EMT. We also illustrated mechanistically how cross-linking connects with stiffness, using a mechanical model of collagen (a major component of ECM). Using this theoretical framework, we demonstrated the heterogeneity of LOXL2/stiffness and its implications on migrating cancer cells that could seed metastasis, the growth of secondary malignant tumors. This framework can inspire experimental studies of more fine-grained mechanotransduction and biomechanical heterogeneity in cancers.The epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression, being responsible in many cases for the onset of the metastatic cascade and being integral in the ability of cells to resist drug treatment. Most studies of EMT focus on its induction via chemical signals such as TGF-β or Notch ligands, but it has become increasingly clear that biomechanical features of the microenvironment such as extracellular matrix (ECM) stiffness can be equally important. Here, we introduce a coupled feedback loop connecting stiffness to the EMT transcription factor ZEB1, which acts via increasing the secretion of LOXL2 that leads to increased cross-linking of collagen fibers in the ECM. This increased cross-linking can effectively increase ECM stiffness and increase ZEB1 levels, thus setting a positive feedback loop between ZEB1 and ECM stiffness. To investigate the impact of this non-cell-autonomous effect, we introduce a computational approach capable of connecting LOXL2 concentration to increased stiffness and thereby to higher ZEB1 levels. Our results indicate that this positive feedback loop, once activated, can effectively lock the cells in a mesenchymal state. The spatial-temporal heterogeneity of the LOXL2 concentration and thus the mechanical stiffness also has direct implications for migrating cells that attempt to escape the primary tumor.

Cancer-associated fibroblasts induce epithelial\u2013mesenchymal transition of bladder cancer cells through paracrine IL-6 signalling

BackgroundCancer-associated fibroblasts (CAFs), activated by tumour cells, are the predominant type of stromal cells in cancer tissue and play an important role in interacting with neoplastic cells to promote cancer progression. Epithelial-mesenchymal transition (EMT) is a key feature of metastatic cells. However, the mechanism by which CAFs induce EMT program in bladder cancer cells remains unclear.MethodsTo investigate the role of CAFs in bladder cancer progression, healthy primary bladder fibroblasts (HFs) were induced into CAFs (iCAFs) by bladder cancer-derived exosomes. Effect of conditioned medium from iCAFs (CM iCAF) on EMT markers expression of non-invasive RT4 bladder cancer cell line was determined by qPCR and Western blot. IL6 expression in iCAFs was evaluated by ELISA and Western blot. RT4 cell proliferation, migration and invasion were assessed in CM iCAF +/− anti-IL6 neutralizing antibody using cyQUANT assay, scratch test and transwell chamber respectively. We investigated IL6 expression relevance for bladder cancer progression by querying gene expression datasets of human bladder cancer specimens from TCGA and GEO genomic data platforms.ResultsCancer exosome-treated HFs showed CAFs characteristics with high expression levels of αSMA and FAP. We showed that the CM iCAF induces the upregulation of mesenchymal markers, such as N-cadherin and vimentin, while repressing epithelial markers E-cadherin and p-ß-catenin expression in non-invasive RT4 cells. Moreover, EMT transcription factors SNAIL1, TWIST1 and ZEB1 were upregulated in CM iCAF-cultured RT4 cells compared to control. We also showed that the IL-6 cytokine was highly expressed by CAFs, and its receptor IL-6R was found on RT4 bladder cancer cells. The culture of RT4 bladder cancer cells with CM iCAF resulted in markedly promoted cell growth, migration and invasion. Importantly, inhibition of CAFs-secreted IL-6 by neutralizing antibody significantly reversed the IL-6-induced EMT phenotype, suggesting that this cytokine is necessary for CAF-induced EMT in the progression of human bladder cancer. Finally, we observed that IL6 expression is up-regulated in aggressive bladder cancer and correlate with CAF marker ACTA2.ConclusionsWe conclude that CAFs promote aggressive phenotypes of non-invasive bladder cancer cells through an EMT induced by the secretion of IL-6.

PBX3 Is Part of an EMT Regulatory Network and Indicates Poor Outcome in Colorectal Cancer.

Purpose: Colorectal cancers are composed of phenotypically different tumor cell subpopulations within the same core genetic background. Here, we identify high expression of the TALE transcription factor PBX3 in tumor cells undergoing epithelial-mesenchymal transition (EMT), analyze PBX3 regulation, and determine clinical associations in colorectal cancer.Experimental design: We used transcriptomic and in situ analyses to identify PBX3 expression in colorectal cancer and cell biology approaches to determine its regulation and function. Clinical associations were analyzed in independent tissue collections and gene expression datasets of colorectal cancers with recorded follow-up data.Results: PBX3 was expressed in tumor cells with high WNT activity undergoing EMT at the leading tumor edge of colorectal cancers, whereas stromal cells were PBX3 negative. PBX3 expression was induced by WNT activation and by the EMT transcription factors SNAIL and ZEB1, whereas these effects were mediated indirectly through microRNA miR-200. PBX3 was required for a full EMT phenotype in colon cancer cells. On the protein level, PBX3 expression indicated poor cancer-specific and disease-free survival in a cohort of 244 UICC stage II colorectal cancers, and was associated with metastasis in a case-control collection consisting of 90 cases with or without distant metastasis. On the mRNA level, high PBX3 expression was strongly linked to poor disease-free survival.Conclusions: PBX3 is a novel indicator of EMT in colorectal cancer, part of an EMT regulatory network, and a promising prognostic predictor that may aid in therapeutic decision making for patients with colorectal cancer. Clin Cancer Res; 24(8); 1974-86. ©2018 AACR.

SOX4 contributes to TGF-β-induced epithelial–mesenchymal transition and stem cell characteristics of gastric cancer cells

SOX4 is highly expressed in gastric cancer (GC) and is associated with tumor grade, metastasis and prognosis, however the mechanism is not clear. We report herein that SOX4 was upregulated and overexpression of SOX4 was associated with increased expression of the markers of Epithelial–mesenchymal transition (EMT) and stemness in clinic patient samples. In vitro, overexpression of SOX4 promoted the invasion as showed by Transwell assay and stemness of GC cells as assessed by sphere formation assay, which was suppressed by silencing SOX4 with shRNA. Further studies showed that SOX4 up-regulated the expression of EMT transcription factors Twist1, snail1 and zeb1 and stemness transcription factors SOX2 and OCT4, and promoted the nuclear translocation of β-catenin. Moreover, we revealed that TGF-β treatment significantly up-regulated the expression of SOX4 and silencing SOX4 reversed TGF-β induced invasion and sphere formation ability of GC cells. Finally, we showed that SOX4 promoted the lung metastasis and tumor formation ability of gastric cancer cells in nude mice. Our results suggest that SOX4 is a target TGF-β signaling and mediates TGF-β-induced EMT and stem cell characteristics of GC cells, revealing a novel role of TGF-β/SOX4 axis in the regulation of malignant behavior of GC.

MiR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis

Epithelial tumour cells can gain invasive and metastatic capabilities by undergoing an epithelial–mesenchymal transition. Transcriptional regulators and post-transcriptional effectors like microRNAs orchestrate this process of high cellular plasticity and its malignant consequences. Here, using microRNA sequencing in a time-resolved manner and functional validation, we have identified microRNAs that are critical for the regulation of an epithelial–mesenchymal transition and of mesenchymal tumour cell migration. We report that miR-1199-5p is downregulated in its expression during an epithelial–mesenchymal transition, while its forced expression prevents an epithelial–mesenchymal transition, tumour cell migration and invasion in vitro, and lung metastasis in vivo. Mechanistically, miR-1199-5p acts in a reciprocal double-negative feedback loop with the epithelial–mesenchymal transition transcription factor Zeb1. This function resembles the activities of miR-200 family members, guardians of an epithelial cell phenotype. However, miR-1199-5p and miR-200 family members share only six target genes, indicating that, besides regulating Zeb1 expression, they exert distinct functions during an epithelial–mesenchymal transition.

KLF4 Plays an Essential Role in Corneal Epithelial Homeostasis by Promoting Epithelial Cell Fate and Suppressing Epithelial-Mesenchymal Transition.

PurposeThe purpose of this study was to test the hypothesis that KLF4 promotes corneal epithelial (CE) cell fate by suppressing the epithelial–mesenchymal transition (EMT), using spatiotemporally regulated CE-specific ablation of Klf4 in Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mice.MethodsCE-specific ablation of Klf4 was achieved by feeding Klf4Δ/ΔCE mice with doxycycline chow. The wild-type (WT; normal chow-fed littermates) and the Klf4Δ/ΔCE histology was compared by hematoxylin and eosin–stained sections; EMT marker expression was quantified by quantitative PCR, immunoblots, and immunofluorescent staining; and wound healing rate was measured by CE debridement using Algerbrush. KLF4 and EMT markers were quantified in human corneal limbal epithelial (HCLE) cells undergoing TGF-β1–induced EMT by quantitative PCR, immunoblots, and immunofluorescent staining.ResultsThe epithelial markers E-cadherin, Krt12, claudin-3, and claudin-4 were down-regulated, whereas the mesenchymal markers vimentin, β-catenin, survivin, and cyclin-D1 and the EMT transcription factors Snail, Slug, Twist1, Twist2, Zeb1, and Zeb2 were up-regulated in the Klf4Δ/ΔCE corneas. The Klf4Δ/ΔCE cells migrated faster, filling 93% of the debrided area within 16 hours compared with 61% in the WT. After 7 days of wounding, the Klf4Δ/ΔCE cells that filled the gap failed to regain epithelial characteristics, as they displayed abnormal stratification; down-regulation of E-cadherin and Krt12; up-regulation of β-catenin, survivin, and cyclin-D1; and a 2.5-fold increase in the number of proliferative Ki67+ cells. WT CE cells at the migrating edge and the HCLE cells undergoing TGF-β1–induced EMT displayed significant down-regulation of KLF4.ConclusionsCollectively, these results reveal that KLF4 plays an essential role in CE homeostasis by promoting epithelial cell fate and suppressing EMT.

Highlights of This Issue

Metabolomics captures the interactions between genetic alterations, enzymatic activity, and metabolic reactions. Here, comparative mass spectrometry-based metabolomics was performed using matched frozen and fixed isogenic prostate cancer cells and clinical specimens. A sufficient number of metabolites were retained in formalin-fixed, paraffin-embedded (FFPE) tissue to allow unsupervised hierarchical clustering of cell populations. This metabolomic profiling approach creates the opportunity, for research and diagnostic purposes, to interrogate annotated tumor banks retrospectively and link metabolomics with clinicopathologic endpoints. Combined, metabolic profiling to discover and validate metabolism-based diagnostic, prognostic or predictive biomarkers in archival diagnostic tissue adds a new, powerful technology for precision medicine.A critical aspect of metastatic epithelial ovarian cancer (EOC) is tumor cell dormancy, rendering these cells resistant to chemotherapeutics. Employing a systematic approach to analyze the role of negative growth factors, MacDonald and colleagues demonstrate that disruption of the DP, RB family, E2F, and MuvB complex (DREAM) results in death of EOC cells. Specifically, loss of the DREAM assembly factor Dyrk1A, or the DREAM component p130, impairs repression of DREAM target genes, leads to continued DNA synthesis, and is coincident with increased cell death. Importantly, chemical inhibition of Dyrk1A synergizes with conventional chemotherapies to kill EOC cells under dormant conditions.The basis of resistance or sensitivity to irinotecan in colorectal cancer has still not been clearly established. Strategies to identify susceptible patients and targetable pathways to reverse resistance are urgently needed. Cabal-Hierro and O’Dwyer highlight a role for RIP1 kinase in SN38 sensitivity by mediating cell death/DNA damage signaling in colon adenocarcinoma model systems through the TNF/TNFR pathway. Downregulation of RIP1 protects from SN-38 in vitro and in vivo, suggesting that RIP1 has potential as a biomarker. Activation of TNF/TNFR signaling potentiates SN38 activity in a RIP1-dependent manner; thus, suggesting a re-evaluation of TNF-based interventions to enhance treatment efficacy.Early metastasis and therapeutic resistance are huge setbacks to patients diagnosed with pancreatic ductal adenocarcinoma (PDAC). Here, Smigiel and colleagues report a unique function of the tumor microenvironment (TME) cytokine oncostatin M (OSM) in inducing epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties in pancreatic cancer cells. Elevated levels of OSM in PDAC induce EMT transcription factors ZEB1 and Snail, and induce CD44+ CSC that are more migratory, tumorigenic, highly metastatic and resistant to gemcitabine. As such, targeting OSM or OSM-signaling in PDAC is a potential therapeutic strategy to prevent de novo acquisition of CSC properties such as drug resistance and metastasis.

Plasma DCLK1 is a marker of hepatocellular carcinoma (HCC): Targeting DCLK1 prevents HCC tumor xenograft growth via a microRNA-dependent mechanism.

Tumor stem cell marker Doublecortin-like kinase1 (DCLK1) is upregulated in several solid tumors. The role of DCLK1 in hepatocellular carcinoma (HCC) is unclear. We immunostained tissues from human livers with HCC, cirrhosis controls (CC), and non-cirrhosis controls (NCC) for DCLK1. Western blot and ELISA analyses for DCLK1 were performed with stored plasma samples. We observed increased immunoreactive DCLK1 in epithelia and stroma in HCC and CCs compared with NCCs, and observed a marked increase in plasma DCLK1 from patients with HCC compared with CC and NCC. Analysis of the Cancer Genome Atlas' HCC dataset revealed that DCLK1 is overexpressed in HCC tumors relative to adjacent normal tissues. High DCLK1-expressing cells had more epithelial-mesenchymal transition (EMT). Various tumor suppressor miRNAs were also downregulated in HCC tumors. We evaluated the effects of DCLK1 knockdown on Huh7.5-derived tumor xenograft growth. This was associated with growth arrest and a marked downregulation of cMYC, and EMT transcription factors ZEB1, ZEB2, SNAIL, and SLUG via let-7a and miR-200 miRNA-dependent mechanisms. Furthermore, upregulation of miR-143/145, a corresponding decrease in pluripotency factors OCT4, NANOG, KLF4, and LIN28, and a reduction of let-7a, miR-143/145, and miR-200-specific luciferase activity was observed. These findings suggest that the detection of elevated plasma DCLK1 may provide a cost-effective, less invasive tool for confirmation of clinical signs of cirrhosis, and a potential companion diagnostic marker for patients with cirrhosis and HCC. Our results support evaluating DCLK1 as a biomarker for detection and as a therapeutic target for eradicating HCC.

Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors.MethodsiNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models.ResultsHigh endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.ConclusionsConsidering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0527-x) contains supplementary material, which is available to authorized users.

IL-22 contributes to TGF-β1-mediated epithelial-mesenchymal transition in asthmatic bronchial epithelial cells

BackgroundAllergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. Epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-β1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects.MethodsPrimary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-β1, IL-22 and TGF-β1+IL-22.ResultsPrimary bronchial epithelial cells stimulated with TGF-β1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-β1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics.ConclusionThe impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-β1 cooperativity in driving EMT in primary human bronchial epithelial cells.

A novel function of HPV16-E6/E7 in epithelial–mesenchymal transition

Human papillomavirus (HPV) 16 is among the most important etiological factors in many human cancers, including head and neck squamous cell carcinomas (HNSCCs) not associated with alcohol or tobacco use. HPV16-E6 and E7 oncoproteins target intracellular signaling networks, altering key molecular and cellular events during tumor progression. The present study investigates the role of HPV16-E6 and E7 oncogenes on the epithelial–mesenchymal transition (EMT), a cellular process thought to be critical for tumor cell invasion and metastasis. Using the epithelial MDCK cell line as an in vitro model, we show that the stable expression of HPV16-E6 or E7 induces morphological conversion from cobblestone-shaped epithelium to spindle-shaped mesenchyme-like phenotype. Consistent with these morphological changes, both E6 and E7 induce expression of the EMT-activating transcriptional factors Slug, Twist, ZEB1 and ZEB2, especially ZEBs, accompanied with switch from epithelial to mesenchymal markers. Importantly, E6 and E7 expression results in induction of the migratory and invasive potential, a functional hallmark of EMT. When we examined the association between HPV16 and the EMT signature in HNSCC cell lines derived from head and neck cancer patients, we found a correlation between HPV16 positivity and the expression of EMT transcription factor ZEB1. Taken together, our findings suggest HPV16 induces EMT-like processes via induction of the EMT transcription factors which may contribute to tumor progression and metastasis.

Epithelial–mesenchymal transition transcription factor ZEB1/ZEB2 co-expression predicts poor prognosis and maintains tumor-initiating properties in head and neck cancer

Both epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties may be involved in metastasis, which contributes to the high mortality rate of patients with head and neck cancers (HNCs). However, the mechanisms through which the EMT transcription factors ZEB1 and ZEB2 regulate HNC are still unclear. Tumor initiating capability of HNC-CH133(+) cells with ZEB1/2 knockdown or co-overexpression was presented in vitro and in vivo. In the present study, we demonstrated that ZEB1/ZEB2 expression was significantly increased in HNC-CD133(+) CSC-like cells compared with HNC-CD133(-) cells. The small interfering RNA (siRNA)-mediated co-knockdown of ZEB1 and ZEB2 (siZEB1/2) in HNC-CH133(+) cells suppressed their CSC-like properties, including self-renewal ability, the expression of stemness markers, and drug resistance. In contrast, the co-overexpression of ZEB1/ZEB2 in HNC-CD133(-) cells enhanced their sphere-forming ability and increased the percentage of CD44-positive cells and side population cells. In vivo studies showed that the delivery of siZEB1/2 to xenograft tumors in nude mice reduced tumor growth and the rate of distant metastasis. In clinical samples, the levels of ZEB1/ZEB2 expression were low in local lesions but high in metastatic lymph nodes in HNC tissues. Patients with tumors that co-expressed ZEB1(high) and ZEB2(high) had especially poor survival rates. Therapies targeting ZEB1/ZEB2 in HNC-CD133(+) cells may provide a new approach for HNC therapy in the future.