Zinc Finger E-box-binding Homeobox 1 Expression Research Articles

CAFs-derived TIAM1 Promotes OSCC Cell Growth and Metastasis by Regulating ZEB2.

Previous studies have suggested that cancer-associated fibroblasts (CAFs) within the tumor microenvironment are a critical factor in tumorigenesis and tumor development. However, the regulatory mechanisms of CAFs on oral squamous cell carcinoma (OSCC) are poorly defined. A CAF-conditioned medium (CAF-CM) was collected and applied to culture OSCC cells. Then, cell viability, proliferation, migration, and invasion were evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and scratch healing assays. T-Lymphoma Invasion and Metastasis 1 (TIAM1), zinc finger E-box-binding homeobox 2 (ZEB2), E-cadherin, and increased N-cadherin protein levels were determined using western blot. TIAM1 and ZEB2 mRNA levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR). Their interaction was analyzed using Co-immunoprecipitation (Co-IP) assay. SCC25 cells with or without (TIAM1-silencing) CAFs were subcutaneously inoculated in nude mice to assess the effect of TIAM1 in CAFs on OSCC tumor growth in vivo. CAFs expedited OSCC cell proliferation, migration, invasion, and EMT. TIAM1 and ZEB2 expression were upregulated in OSCC patients and OSCC cells, and the TIAM1 level was much higher in CAFs than in OSCC cells. Furthermore, TIAM1 knockdown in CAFs might partly abolish the promotion of CAFs on OSCC cell development, implying that TIAM1 might be secreted by CAFs into the culture medium to exert its effects inside OSCCs. TIAM1 might increase ZEB2 expression, and ZEB2 upregulation might partly reverse the repression of TIAM1 silencing in CAFs on OSCC cell malignant behaviors. In vivo studies confirmed that CAFs accelerated OSCC tumor growth, these effects were partially counteracted by TIAM1 downregulation. Overall, TIAM1 secreted by CAFs could expedite OSCC cell growth and metastasis by regulating ZEB2, providing a promising therapeutic target for OSCC treatment.

Utilising deep learning networks to classify ZEB2 expression images in cervical cancer.

Aims/Background Cervical cancer continues to be a significant cause of cancer-related deaths among women, especially in low-resource settings where screening and follow-up care are lacking. The transcription factor zinc finger E-box-binding homeobox 2 (ZEB2) has been identified as a potential marker for tumour aggressiveness and cancer progression in cervical cancer tissues. Methods This study presents a hybrid deep learning system developed to classify cervical cancer images based on ZEB2 expression. The system integrates multiple convolutional neural network models-EfficientNet, DenseNet, and InceptionNet-using ensemble voting. We utilised the gradient-weighted class activation mapping (Grad-CAM) visualisation technique to improve the interpretability of the decisions made by the convolutional neural networks. The dataset consisted of 649 annotated images, which were divided into training, validation, and testing sets. Results The hybrid model exhibited a high classification accuracy of 94.4% on the test set. The Grad-CAM visualisations offered insights into the model's decision-making process, emphasising the image regions crucial for classifying ZEB2 expression levels. Conclusion The proposed hybrid deep learning model presents an effective and interpretable method for the classification of cervical cancer based on ZEB2 expression. This approach holds the potential to substantially aid in early diagnosis, thereby potentially enhancing patient outcomes and mitigating healthcare costs. Future endeavours will concentrate on enhancing the model's accuracy and investigating its applicability to other cancer types.

Circ-10720 as a ceRNA adsorbs microRNA-1238 and modulates ZEB2 to boost NSCLC development by activating EMT

BackgroundCircular RNAs (circRNAs) are critical regulators in the progression of tumors. This experimental design aimed to explore the mechanism of circ-10720 in non-small cell lung cancer (NSCLC).MethodsWe used RT-qPCR to measure circ-10720 expression in clinical samples and analyzed its relationship with the clinicopathological characteristics of NSCLC patients. The expression levels of microRNA-1238 (miR-1238) and Zinc Finger E-box-binding Homeobox 2 (ZEB2) in clinical samples were detected by RT-qPCR. NSCLC cells were transfected with relevant plasmids or sequences. Circ-10720, miR-1238, and ZEB2 expressions in cells were analyzed via RT-qPCR or western blot. Cell proliferation, apoptosis, migration, and invasion were assessed with CCK-8, flow cytometry, and transwell assay, respectively. The protein expression of ZEB2 and epithelial–mesenchymal transition (EMT)-related markers (E-cadherin, Vimentin, N-cadherin) were detected via western blot. Xenograft assay was used to determine the effect of circ-10720 on NSCLC in vivo. Circ-10720 and ZEB2 expressions in tumors were detected using RT-qPCR or Western blot. Immunohistochemistry was used to evaluate E-cadherin and N-cadherin expression in tumors. Finally, the binding relationship between miR-1238 with circ-10720 or ZEB2 was verified by the bioinformatics website, dual luciferase reporter assay, RNA pull-down assay, and RIP assay.ResultsCirc-10720 was upregulated in NSCLC and correlated with TNM stage of NSCLC patients. MiR-1238 was lowly expressed but ZEB2 was highly expressed in NSCLC. Circ-10720 silencing suppressed the proliferation, metastasis, and EMT of NSCLC cells. Mechanically, circ-10720 was a competitive endogenous RNA (ceRNA) for miR-1238, and ZEB2 was a target of miR-1238. circ-10720-modulated ZEB2 via competitively binding with miR-1238 to control NSCLC progression. In addition, circ-10720 knockdown suppressed tumor growth in vivo.ConclusionsCirc-10720 acts as a ceRNA to adsorb miR-1238 and modulate ZEB2 to facilitate the proliferation, migration, invasion, and EMT of NSCLC cells.Graphical

Cisplatin Disrupts Proteasome Bounce-Back Effect through Suppressing ZEB1/Nfe2l1 in Cholangiocarcinoma.

Bortezomib (BTZ) is a powerful proteasome inhibitor that has been approved for the treatment of haematologic malignancies. Its effectiveness has been assessed against different types of solid tumours. BTZ is ineffective in most solid tumours because of drug resistance, including cholangiocarcinoma, which is associated with a proteasome bounce-back effect. However, the mechanism through which proteasome inhibitors induce the proteasome bounce-back effect remains largely unknown. Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry. Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells. Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.

The mechanism of PFK-1 in the occurrence and development of bladder cancer by regulating ZEB1 lactylation

BackgroundBladder cancer (BC) is one of the most common malignancies of the genitourinary system. Phosphofructokinase 1 (PFK-1) is one of member of PFK, which plays an important role in reprogramming cancer metabolism, such as lactylation modification. Zinc finger E-box-binding homeobox 1 (ZEB1) has been demonstrated to be a oncogene in many cancers. Therefore, this study was performed to explore the effects of PFK-1 on the lactylation of ZEB1 in BC development.MethodsCell viability was measured using the CCK-8 kit. The glucose assay kit and lactate assay kit were used to detect glucose utilization and lactate production. The DNA was purified and quantified by qRT-PCR.ResultsIn the present study, we found that ZEB1 expression levels were significantly elevated in bladder cancer cells. Impaired PFK-1 expression inhibits proliferation, migration, and invasion of BC cells and suppresses tumour growth in vivo. We subsequently found that knockdown of PFK-1 decreases glycolysis, including reduced glucose consumption, lactate production and total extracellular acidification rate (ECAR). Mechanistically, PFK-1 inhibits histone lactylation of bladder cancer cells, and thus inhibits the transcription activity of ZEB1.ConclusionOur results suggest that PFK-1 can inhibit the malignant phenotype of bladder cancer cells by mediating the lactylation of ZEB1. These findings suggested PFK-1 to be a new potential target for bladder cancer therapy.

Long Non-coding RNA X-Inactive Specific Transcript Promotes Esophageal Squamous Cell Carcinoma Progression via the MicroRNA 34a/Zinc Finger E-box–Binding Homeobox 1 Pathway

BackgroundThe long non-coding RNA X-inactive specific transcript (XIST) plays a crucial role in transcriptional silencing of the X chromosome. Zinc finger E-box-binding homeobox 1 (ZEB1) is a transcription factor involved in epithelial-mesenchymal transition (EMT) regulation.AimsThis study aimed to investigate the impact of XIST on esophageal squamous cell carcinoma (ESCC) progression and its underlying mechanism involving the miR-34a/ZEB1/E-cadherin/EMT pathway.MethodsXIST and ZEB1 expression were analyzed using quantitative PCR and immunohistochemistry. XIST knockdown was achieved in KYSE150 ESCC cells using siRNA or shRNA lentivirus transfection. Proliferation, migration, and invasion abilities were assessed, and luciferase reporter assays were performed to confirm XIST-miR-34a-ZEB1 interactions. In vivo ESCC growth was evaluated using a xenograft mouse model.ResultsXIST and ZEB1 were upregulated in tumor tissues, correlating with metastasis and reduced survival. XIST knockdown inhibited proliferation, migration, and invasion of KYSE150 cells. It decreased ZEB1 expression, increased E-cadherin and miR-34a levels. Luciferase reporter assays confirmed miR-34a binding to XIST and ZEB1. XIST knockdown suppressed xenograft tumor growth.ConclusionXIST promotes ESCC progression via the miR-34a/ZEB1/E-cadherin/EMT pathway. Targeting the XIST/miR-34a/ZEB1 axis holds therapeutic potential and serves as a prognostic biomarker in ESCC.Graphical This study has discovered an XIST-associated pathway involved in the regulation of ZEB1 expression, thereby influencing the progression of squamous cell carcinoma (ESCC). The specific mechanism is as follows: XIST can regulates ZEB1 expression by sponging miR-34a, ultimately promoting the metastasis of tumor by down-regulating the expression of E-cadherin.

IKBKE promotes the ZEB2-mediated EMT process by phosphorylating HMGA1a in glioblastoma

IKBKE (Inhibitor of Nuclear Factor Kappa-B Kinase Subunit Epsilon) is an important oncogenic protein in a variety of tumors, which can promote tumor growth, proliferation, invasion and drug resistance, and plays a critical regulatory role in the occurrence and progression of malignant tumors. HMGA1a (High Mobility Group AT-hook 1a) functions as a cofactor for proper transcriptional regulation and is highly expressed in multiple types of tumors. ZEB2 (Zinc finger E-box Binding homeobox 2) exerts active functions in epithelial mesenchymal transformation (EMT). In our current study, we confirmed that IKBKE can increase the proliferation, invasion and migration of glioblastoma cells. We then found that IKBKE can phosphorylate HMGA1a at Ser 36 and/or Ser 44 sites and inhibit the degradation process of HMGA1a, and regulate the nuclear translocation of HMGA1a. Crucially, we observed that HMGA1a can regulate ZEB2 gene expression by interacting with ZEB2 promoter region. Hence, HMGA1a was found to promote the ZEB2-related metastasis. Consequently, we demonstrated that IKBKE can exert its oncogenic functions via the IKBKE/HMGA1a/ZEB2 signalling axis, and IKBKE may be a prominent biomarker for the treatment of glioblastoma in the future.

MiR-34b-5p suppresses the epithelial-mesenchymal transition and metastasis in endometrial cancer AN3CA cells by targeting ZEB1.

Tumor metastasis is a primary cause of recurrence and mortality in endometrial cancer. miR-34b-5p is abnormally expressed in various cancers and participates in tumor cell progression and metastasis. The objective of this study was to elucidate the biological functions and molecular mechanisms of miR-34b-5p in regulating the epithelial-mesenchymal transition (EMT) and metastasis in AN3CA endometrial cancer cells. The expression levels of miR-34b-5p and zinc finger E-box-binding homeobox 1 (ZEB1) in endometrial cancer cells were analyzed by qRT-PCR, and ZEB1 expression in endometrial cancer tissues was examined by immunohistochemistry. Proliferation, migration, and invasion of endometrial cancer AN3CA cells were evaluated using CCK8, scratch, and transwell assays, respectively. Bioinformatic analysis and dual-luciferase reporter gene assays were used to validate the targeting relationship between miR-34b-5p and ZEB1. Western blotting was performed to analyze the expression levels of ZEB1 and EMT-related proteins. miR-34b-5p was significantly downregulated in endometrial cancer AN3CA cells. Overexpression of miR-34b-5p significantly inhibited proliferation, invasion, migration, and the EMT of endometrial cancer AN3CA cells. ZEB1, which was identified as a direct target gene of miR-34b-5p, exhibited high expression in endometrial cancer cells and tissues. Additionally, ZEB1 upregulation partially reversed the inhibitory effects of miR-34b-5p on proliferation, migration, invasion, and the EMT of endometrial cancer AN3CA cells. miR-34b-5p suppresses the EMT and metastasis in endometrial cancer AN3CA cells by targeting ZEB1, indicating that the miR-34b-5p-ZEB1-EMT axis may be a therapeutic target for endometrial cancer.

Clinicopathologic Significance of Quaking Expression in Hepatocellular Carcinoma.

The RNA binding protein quaking (QKI) is associated with the development and progression of tumor suppressors in various cancers. However, the clinical implications of QKI expression have not yet been fully elucidated. In this study, we aimed to investigate the clinicopathological and prognostic significance of QKI expression in hepatocellular carcinoma (HCC). We performed QKI, Zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin, and glutathione peroxidase 4 (GPX4) immunohistochemical staining on 166 HCC patient tissue samples. X-tile bioinformatics software was used to set the cut-off value for high QKI expression. Correlations between QKI expression and various clinicopathological parameters were assessed. The best cut-off value for high QKI expression was 12.5. High QKI expression was observed in 28 of 166 patients (16.9%) and was an independent prognostic factor for inferior recurrence-free survival (RFS). In addition, high ZEB1 and GPX4 expression correlated with high QKI expression, but not with the loss of E-cadherin expression. High QKI expression was identified in HCCs and associated with poor RFS. QKI might be a prognostic biomarker of HCCs associated with epithelial-to-mesenchymal transition and a potential candidate therapeutic target.

Astragaloside IV inhibits epithelial-mesenchymal transition and pulmonary fibrosis via lncRNA-ATB/miR-200c/ZEB1 signaling pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease involving multiple factors and genes. Astragaloside IV (ASV) is one of the main bioactive ingredients extracted from the root of Astragalus membranaceus, which plays an important role in anti-inflammatory, antioxidant and improve cardiopulmonary function. Epithelial-mesenchymal transition (EMT) is a key driver of the process of pulmonary fibrosis, and Zinc finger E-box-binding homeobox 1 (ZEB1) can promote pulmonary fibrosis in an EMT-dependent manner. Here, we found that ASV effectively inhibited the ZEB1 and EMT in both bleomycin (BLM)-induced rat pulmonary fibrosis and TGF-β1-treated A549 cells. To further elucidate the molecular mechanisms underlying effects of ASV in IPF, we explored the truth using bioinformatics, plasmid construction, immunofluorescence staining, western blotting and other experiments. Dual luciferase reporter assay and bioinformatics proved that miR-200c not only acts as an upstream regulatory miRNA of ZEB1 but also has binding sites for the lncRNA-ATB. In A549 cell-based EMT models, ASV reduced the expression of lncRNA-ATB and upregulated miR-200c. Furthermore, overexpression of lncRNA-ATB and silencing of miR-200c reversed the down-regulation of ZEB1 and the inhibition of EMT processes by ASV. In addition, the intervention of ASV prevented lncRNA-ATB as a ceRNA from regulating the expression of ZEB1 through sponging miR-200c. Taken together, the results showed that ASV inhibited the EMT process through the lncRNA-ATB/miR-200c/ZEB1 signaling pathway, which provides a novel approach to the treatment of IPF.

Protein-protein interactions between RUNX3 and ZEB1 in chronic lung injury induced by methamphetamine abuse.

Methamphetamine (MA) is the most common and highly addictive substance abuse drug. Runt-related transcription factor 3 (RUNX3) and Zinc finger E-box-binding homeobox 1 (ZEB1) are associated with lung inflammation and fibrosis. However, the protein-protein interactions (PPIs) between RUNX3 and ZEB1 and its involvement in MA-induced chronic lung injury is still unclear. In this study, we evaluated lung injury using echocardiography, hematoxylin and eosin staining, and western blot analysis. The viability of alveolar epithelial cells (AECs) was assessed using cell counting kit-8. Molecular Operating Environment software, Search Tool for the Retrieval of Interacting Genes/Proteins database, co-immunoprecipitation, assay and confocal immunofluorescence assay were used to predict and identify the PPIs between RUNX3 and ZEB1. The expression of RUNX3 and ZEB1 were knockdown in AECs using siRNA. The results revealed that MA exposure increased the peak blood flow velocity of the pulmonary artery and the acceleration time of pulmonary artery blood flow. Further, exposure to MA also causes adhesion and fusion of the alveolar walls and altered AEC activity. A decrease in the expression of RUNX3 and an increase in the expression of ZEB1 and its downstream signaling molecules were observed on MA exposure. The PPIs between RUNX3 and ZEB1 were identified. Further, an increase in the protein binding rate of RUNX3-ZEB1 was observed in MA-induced lung injury. These results show interactions between RUNX3 and ZEB1. RUNX3 protects against lung injury; however, ZEB1 expression and the PPIs between ZEB1 and RUNX3 has deleterious effects on chronic lung injury induced by MA exposure. Our results provide a new therapeutic approach for the treatment of chronic lung injury due to MA exposure.

Hsa_circ_0005529 promotes ZEB1 expression by regulating miR-873-5p and enhancing proliferation, invasion, and migration in gastric cancer cell lines.

Gastric cancer is a relatively common tumor. As circular RNAs (circRNAs) are documented to modulate proliferation and metastasis in various cancers, we evaluated the functions of circRNAs, in particular, hsa_circ_0005529, in gastric cancer cells. Levels of hsa_circ_0005529 and miR-873-5p were examined by qRT-PCR, and the presence of hsa_circ_0005529 was confirmed by RNase R treatment. CCK-8, wound-healing, and Transwell assays were used to assess proliferation, migration, and invasion, respectively, while Western blotting was used to determine levels of zinc finger E-box-binding homeobox 1 (ZEB1) and dual-luciferase reporter assays to examine relationships between hsa_circ_0005529 and miR-873-5p. hsa_circ_0005529 was strongly expressed in gastric cancer where it stimulated tumorigenic behavior. Furthermore, hsa_circ_0005529 was shown to promote ZEB1 expression by sponging miR-873-5p, an inhibitor of ZEB1 expression. Our research showed that hsa_circ_0005529 promoted tumorigenic behavior in gastric cancer cells by adsorbing miR-873-5p to modulate ZEB1 levels. This suggests that hsa_circ_0005529 may be useful as a biomarker and target for diagnosing and treating gastric cancer.

Study on inhibition of Britannin on triple-negative breast carcinoma through degrading ZEB1 proteins

BackgroundTriple-negative breast carcinomas (TNBCs) are a breast carcinoma with the most aggressive form, which is demonstrated as enhanced invasion and recurrence. Britannin is extracted mainly from the traditional Chinese herb Inula japonica Thunb, and few studies have focused on its effect on TNBC. Moreover, there is still no report concerning the role of Britannin in degrading the transcripts of Zinc finger E-box-binding homeobox 1 (ZEB1) proteins. PurposeTo explore the potential effect of Britannin on invasion and stemness of TNBCs and its underlying mechanism. MethodsCellular activity was measured using MTT, and cell cycle was measured using flow cytometry (FCM). The effect of Britannin on the migrating and invading abilities of MDA-MB-231 and 4T1 cells were measured using the wound healing and transwell assays. The sizes and number of breast carcinoma cells were measured by tumor formation assay and in vitro limiting-dilution assay. CD44 expression in tumor spheroids was tested by immunofluorescence assay. Nextly, the expressions of epithelial-mesenchymal transition (EMT) markers and ZEB1 protein expressional level were detected by western blot . ZEB1 mRNA expressional level was analyzed using RT-qPCR. Drug affinity-responsive target stability (DARTS) method was used to detect the binding activity between Britannin and ZEB1. Co-immunoprecipitation (Co-IP) analysis was applied to test the ubiquitination of ZEB1. The mouse models for experimental lung metastasis of 4T1 cells were established to detect the anti-metastasis effect of Britannin in vivo, and the expressional levels of EMT markers in lung metastases were detected by immunohistochemistry. ResultsBritannin could inhibit cell growth and G2/M arrest in TNBC cells. Britannin could inhibit the migrating and invading ability without inducing severe apoptosis of MDA-MB-231 and 4T1 cells. Meanwhile, Britannin reduced the size and number of spheroids formed in these two cells, and decreased the expressional level of stem cells biomarker CD44 in tumor spheroids. Mechanism research showed that Britannin specifically bound to ZEB1 and induced its ubiquitination in MDA-MB-231 cells. Afterwards, Britannin disturbed protein stability and promoted ZEB1 protein degradation. Importantly, Britannin could not inhibit cell invasion and spheroid formation after ZEB1 expression was knocked down. Finally, Britannin inhibition of 4T1 cell metastasis was confirmed through establishing mouse models for the experimental lung metastasis. It was proved that both Britannin and paclitaxel could decrease the lung metastases, and Britannin could also down-regulate the protein expressional levels of ZEB1, MMP9 and CD44. ConclusionThis study reveals that Britannin suppresses the invasion and metastasis of TNBC cells through degrading ZEB1, which suggests that Britannin can be used to prevent tumor metastasis and recurrence via degrading ZEB1proteins.

Abstract 5721: c-Myc expression coupled with loss of cytoplasmic Miz-1 expression and its re-localization to a nuclear expression in high grade cholangio and hepatocholangio carcinomas

Abstract Miz-1 (Myc-interacting zinc finger) activates target genes through transcription factors that are regulated by specific antimitogenic signals. Association with Myc converts Miz-1 from being a transcriptional activator to a repressor. This binding and the formation of Myc/Miz-1 complex induces a change in the biophysical properties of the Miz-1 protein, rendering the complex insoluble and causing to relocalize inside the nucleus. In addition, this association stabilizes Myc and as a result represses transcription. With uncontrolled tumor progress, metastatic tumor expansion can occur through epithelial-to-mesenchymal transition (EMT) and the induction of ZEB-1 (Zinc Finger E-Box Binding Homeobox-1). Zeb-1 expression is highly associated with aggressive behavior tumor types. A cohort of 98 human cholangiocarcinoma carcinoma (CC) including 14 with mixed (CC)/(HCC) resected cases whose formalin fixed paraffin embedded (FFPE) tissue sections containing adjacent benign and malignant tumor lesions were examined by immunohistochemistry (IHC) using Miz-1 (ZBTB17), ZEB-1 (OT13G6), c-Myc (Y69), and Ki-67 (Mib-1) monoclonal antibodies. The clinicopathologic variables, including viral hepatitis, cirrhosis, tumor size, grade and number, vascular invasion, tumor-node-metastasis (TNM) stage, were obtained from each patient’s medical records. The variables were assessed for correlation with the immunohistochemical results. Compared to the adjacent benign liver section, reduction of Miz-1 expression was observed in 98/98 (100%) (P<0.0001) cases, while positive expression of c-Myc, Ki-67 (> 5%), and ZEB-1 was observed in 18/98 (18.36%) (P<0.0001), 27/98 (27.55%) (P=0.000003), and 15/98 (15.30%) (P<0.00001) cases respectively. Of note, complete loss of Miz-1 expression was noted in 20/36 (55.55%) (P=0.106) high grade tumors (III/IV) associated with positive c-Myc, Ki-67 (>5%), ZEB-1, and nuclear Miz-1 expression in 12/20 (60%) (P=0.12) cases (4 CC and 8 mixed (CC)/(HCC) cases). All 12 cases with positive ZEB-1 had confirmed positive lymph node involvement. These results support the role of Miz-1 as one of the intermediary checkpoints required for the induction of cell cycle arrest and apoptosis. Positive c-Myc/ZEB-1 coupled with Miz-1 reduced cytoplasmic and its re-localization to a nuclear expression confirms the transcriptional repressor role of the Myc/Miz-1 complex and the activation of the ZEB-1 gene as a predictor of increased metastatic expansion, therapy resistance, and poor survival in high grade CC, and CC/HCC cases. These findings correlate with a cohort of an adequate number of differently graded lesions from 147 HCC cases that showed similar expression pattern of Miz-1, c-Myc and Zeb-1 to better confirm our hypothesis in epithelial derived human malignancies with frequently amplified 8q24 chromosome. Citation Format: Joeffrey J. Chahine, Sumeyye Culfaci, DongHyang Kwon, Pamela Tuma, Bhaskar V.S. Kallakury. c-Myc expression coupled with loss of cytoplasmic Miz-1 expression and its re-localization to a nuclear expression in high grade cholangio and hepatocholangio carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5721.

SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvβ3 and the Transcription Factor ZEB1 in Prostate Cancer Cells

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, is a matricellular protein that modulates interactions between cells and their microenvironment. SPARC is expressed during extracellular matrix remodeling and is abundant in bone marrow and high-grade prostate cancer (PCa). In PCa, SPARC induces changes associated with epithelial–mesenchymal transition (EMT), enhancing migration and invasion and increasing the expression of EMT transcriptional factor Zinc finger E-box-binding homeobox 1 (ZEB1), but not Zinc finger protein SNAI1 (Snail) or Zinc finger protein SNAI2 (Slug). It is unknown whether the SPARC-induced downregulation of E-cadherin in PCa cells depends on ZEB1. Several integrins are mediators of SPARC effects in cancer cells. Because integrin signaling can induce EMT programs, we hypothesize that SPARC induces E-cadherin repression through the activation of integrins and ZEB1. Through stable knockdown and the overexpression of SPARC in PCa cells, we demonstrate that SPARC downregulates E-cadherin and increases vimentin, ZEB1, and integrin β3 expression. Knocking down SPARC in PCa cells decreases the tyrosine-925 phosphorylation of FAK and impairs focal adhesion formation. Blocking integrin αvβ3 and silencing ZEB1 revert both the SPARC-induced downregulation of E-cadherin and cell migration enhancement. We conclude that SPARC induces E-cadherin repression and enhances PCa cell migration through the integrin αvβ3/ZEB1 signaling pathway.

The transcription factor ZEB1 mediates the progression of epithelial ovarian cancer by promoting the transcription of CircANKRD17

Zinc finger E-box-binding homeobox 1 (ZEB1) is a key transcription factor that regulates the process of epithelial-mesenchymal transition (EMT) in various tumors. However, its role in epithelial ovarian cancer (EOC) is far from understood. The present study aimed to explore the role and potential mechanism of action of ZEB1 in EOC. A quantitativereverse transcription-polymerase chain reaction was used to detect ZEB1 expression levels. The Cell Counting Kit-8 assay, transwell assays, and flow cytometry were used to verify the effects of ZEB1 on the proliferation, invasion, migration, apoptosis, and EMT of EOC, respectively. RNAsequencing identified the effect of knocking down ZEB1 on circular RNAs in EOC cells. Dual-luciferase activity assay and chromatin immunoprecipitation experiments were used to verify the regulatory effect of ZEB1 on the circular RNA ANKRD17 (CircANKRD17; ID: hsa_circ_0007883) at the transcriptional level. Higher ZEB1 expression was found in EOC tissues and cells and was closely related to tumor metastasis, advanced stages, and lower survival rates. Furthermore, silencing ZEB1 inhibited the proliferation, invasion, migration, and EMT of EOC cells but enhanced cell apoptosis. Mechanistically, knockdown of ZEB1 resulted in the greatest downregulation of CircANKRD17 in EOC cells, and ZEB1 significantly promoted the expression of CircANKRD17 and had no significant effect on ANKRD17 messenger RNA expression. Further experiments verified that ZEB1 mediates the regulation of EOC processes by CircANKRD17. Highly expressed ZEB1 promoted proliferation, invasion, migration, and EMT while it inhibited cell apoptosis in EOC by promoting the transcription of CircANKRD17, providing a potential target for the treatment of EOC.

High expression of ZEB1 is associated with EMAST & metastasis in colorectal cancer patients.

Transforming growth factor-beta (TGF-β) signalling pathway has been reported to be involved in metastasis and at the same time has been considered compellingly an important mediator of epithelial-to-mesenchymal transition (EMT). Besides, EMT process is maintained by zinc-finger E-box-binding homeobox 1 (ZEB1) gene which is induced by TGF-β pathway. TGF-β has been shown to be associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) phenomenon, which is one of the prognostic biomarkers of colorectal cancer (CRC). This study was conducted to determine the link among ZEB1-induced TGF-β, EMAST status and metastasis. The expression level of ZEB1 was evaluated using quantitative reverse transcription (qRT) real-time PCR in 122 formalin fixed paraffin-embedded tissues of CRC sample with known EMAST status and TGF-β/Smad-dependent pathways. The association among ZEB1 expression, TGF-β signalling pathway, EMAST status and metastatic behaviour was examined. ZEB1 gene expression level was higher in tumour tissues as compared to normal samples (P<0.045). In addition, ZEB1 positive expression level was associated significantly with metastasis (P=0.05), EMAST+ status (P=0.052) and activated TGF-β signalling pathway (P=0.002). Our results validated significant association between activated TGF-β signalling pathway and EMAST+ phenotype with higher expression of ZEB1 and higher level of metastasis.

Tumor-associated macrophages induced spheroid formation by CCL18-ZEB1-M-CSF feedback loop to promote transcoelomic metastasis of ovarian cancer

BackgroundOvarian cancer (OvCa)-tumor-associated macrophages (TAMs) spheroids are abundantly present within ascites of high malignant patients. This study investigated the mutual interaction of OvCa cells and TAMs in the spheroids.MethodsThree-dimensional coculture...

Overexpression of Cystine/Glutamate Antiporter xCT Correlates with Nutrient Flexibility and ZEB1 Expression in Highly Clonogenic Glioblastoma Stem-like Cells (GSCs)

Simple SummaryGlioblastoma (GBM) is the most aggressive form of glioma (WHO grade IV), and mounting evidence suggests that glioblastoma stem-like cells (GSCs) play an important role in tumor growth and response to therapy. In the current study, we sought to understand the metabolic dependencies of GSCs using high-resolution proton magnetic resonance spectroscopy (1H-NMR). In a defined experimental setting, we stratified in vitro GSC models into two subtypes (Gln/GluHigh, Gln/GluLow) and used diverse molecular approaches to perform comprehensive analyses in GSC neurosphere cultures and primary GBM samples.Cancer stem-like cells mediate tumor initiation, progression, and therapy resistance; however, their identification and selective eradication remain challenging. Herein, we analyze the metabolic dependencies of glioblastoma stem-like cells (GSCs) with high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. We stratify our in vitro GSC models into two subtypes primarily based on their relative amount of glutamine in relationship to glutamate (Gln/Glu). Gln/GluHigh GSCs were found to be resistant to glutamine deprivation, whereas Gln/GluLow GSCs respond with significantly decreased in vitro clonogenicity and impaired cell growth. The starvation resistance appeared to be mediated by an increased expression of the glutamate/cystine antiporter SLC7A11/xCT and efficient cellular clearance of reactive oxygen species (ROS). Moreover, we were able to directly correlate xCT-dependent starvation resistance and high Gln/Glu ratios with in vitro clonogenicity, since targeted differentiation of GSCs with bone morphogenic protein 4 (BMP4) impaired xCT expression, decreased the Gln/Glu ratio, and restored the sensitivity to glutamine starvation. Moreover, significantly reduced levels of the oncometabolites lactate (Lac), phosphocholine (PC), total choline (tCho), myo-inositol (Myo-I), and glycine (Gly) were observed in differentiated GSCs. Furthermore, we found a strong association between high Gln/Glu ratios and increased expression of Zinc finger E-box-binding homeobox 1 (ZEB1) and xCT in primary GBM tumor tissues. Our analyses suggest that the inhibition of xCT represents a potential therapeutic target in glioblastoma; thus, we could further extend its importance in GSC biology and stress responses. We also propose that monitoring of the intracellular Gln/Glu ratio can be used to predict nutrient stress resistance.

Positive Reciprocal Feedback of lncRNA ZEB1-AS1 and HIF-1α Contributes to Hypoxia-Promoted Tumorigenesis and Metastasis of Pancreatic Cancer.

BackgroundMany studies have reported the roles of the extracellular hypoxia microenvironment in the tumorigenesis and metastasis of multiple cancers. However, long noncoding RNAs (lncRNAs) that induce cancer oncogenicity and metastasis of pancreatic cancer (PC) under hypoxia conditions remain unclear.MethodsIn PC cells, the expression levels of lncRNAs in different conditions (normoxia or hypoxia) were compared through RNA sequencing (RNA-seq). The effects of the zinc finger E-box-binding homeobox 1 (ZEB1-AS1) antisense lncRNA on PC cells cultured in normoxia/hypoxia medium were measured through gain and loss-of-function experiments. Fluorescence in situ hybridization and luciferase reporter assays in addition to in vivo studies were utilized to explore the adaptive mechanisms of ZEB1-AS1 in the hypoxia-promoted proliferation, migration, and invasion ability of PC cells. Moreover, the level of ZEB1-AS1 and its associated targets or pathways were investigated in both PC and pancreatic normal tissues.ResultsRNA-seq revealed that ZEB1-AS1 was significantly upregulated in PC cells under hypoxia conditions. The ZEB1-AS1 expression level was closely associated with poor prognosis of PC patients. Knockdown of ZEB1-AS1 suppressed the proliferation, migration, and invasion of PC cells in vitro as well as PC xenograft tumor growth in vivo. In PC cells, RNAi-mediated reduction of ZEB1-AS1 inhibited zinc finger E-box-binding homeobox 1 (ZEB1), while ZEB1-AS1 overexpression rescued ZEB1 expression, indicating that ZEB1-AS1 promotes ZEB1 expression. Moreover, hypoxia-inducible factor-1α (HIF-1α)induced the expression of ZEB1-AS1 by binding to the ZEB1-AS1 promoter, which contains a putative hypoxia response element (HRE). Mechanistically, ZEB1-AS1 scaffolded the interaction among HIF-1α, ZEB1, and histone deacetylase 1 (HDAC1), leading to deacetylation-mediated stabilization of HIF-1α. We further revealed that ZEB1 induced the deacetylase capacity of HDAC1 to suppress the acetylation or degradation of HIF-1α, improving HIF-1α assembly. Thus, hypoxia-induced ZEB1-AS1 facilitated ZEB1 transcription and the stability of HIF-1α, which promoted the metastasis of PC cells. Clinically, dysregulated ZEB1 and HIF-1α expression was significantly correlated with histological grade, lymphatic metastasis, and distant metastasis in PC patients.ConclusionsOur results emphasized that the positive reciprocal loop of HIF-1α/ZEB1-AS1/ZEB1/HDAC1 contributes to hypoxia-promoted oncogenicity and PC metastasis, indicating that it might be a novel therapeutic target for PC.