Regulation Of ZEB1 Research Articles

Retraction Note: HOXD9 promotes epithelial–mesenchymal transition and cancer metastasis by ZEB1 regulation in hepatocellular carcinoma

Retraction Note: HOXD9 promotes epithelial–mesenchymal transition and cancer metastasis by ZEB1 regulation in hepatocellular carcinoma

Conditional deletion of Zeb1 in Csf1r+ cells reduces inflammatory response of the cornea to alkali burn

ZEB1 is an essential factor in embryonic development. In adults, it is often highly expressed in malignant tumors with low expression in normal tissues. The major biological function of ZEB1 in developing embryos and progressing cancers is to transdifferentiate cells from an epithelial to mesenchymal phenotype; but what roles ZEB1 plays in normal adult tissues are largely unknown. We previously reported that the reduction of Zeb1 in monoallelic global knockout (Zeb1+/-) mice reduced corneal inflammation-associated neovascularization following alkali burn. To uncover the cellular mechanism underlying the Zeb1 regulation of corneal inflammation, we functionally deleted Zeb1 alleles in Csf1r+ myeloid cells using a conditional knockout (cKO) strategy and found that Zeb1 cKO reduced leukocytes in the cornea after alkali burn. The reduction of immune cells was due to their increased apoptotic rate and linked to a Zeb1-downregulated apoptotic pathway. We conclude that Zeb1 facilitates corneal inflammatory response by maintaining Csf1r+ cell viability.

Abstract 5422: Identification of ZEB1-regulated circular RNAs in lung cancer cells

Abstract ZEB1 is a master transcription factor playing a crucial role in promoting epithelial-mesenchymal transition (EMT), as well as the progression and metastasis of lung cancer. The progression of lung cancer is regulated by non-coding RNAs, including microRNAs, long non-coding RNAs, and circular RNAs (circRNAs). In this study, we aimed to identify circRNAs under the regulation of ZEB1 and elucidate their functions within lung cancer cells. Through RNA sequencing and subsequent CIRI-full analysis in ZEB1-overexpressing lung cancer cells, we identified 25 circRNAs displaying differential expression compared to control cells. Among these circRNAs, we targeted circDennd1b, which showed down-regulation in ZEB1-overexpressing cells. We used siRNAs to target the back-splicing junction of circDennd1b, resulting in its knockdown and consequently promoting the invasion of lung cancer cells. Now, we are studying how circDennd1b affects the progression and metastasis of lung cancer. In summary, the circRNAs modulated by ZEB1 are anticipated to play a key role in the invasion, progression, and metastasis of lung cancer. Citation Format: Seongran Cho, Jin-A Choi, Young-Ho Ahn. Identification of ZEB1-regulated circular RNAs in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5422.

CD73 mitigates ZEB1 expression in papillary thyroid carcinoma

BackgroundZEB1, a core transcription factor involved in epithelial-mesenchymal transition (EMT), is associated with aggressive cancer cell behavior, treatment resistance, and poor prognosis across various tumor types. Similarly, the expression and activity of CD73, an ectonucleotidase implicated in adenosine generation, is an important marker of tumor malignancy. Growing evidence suggests that EMT and the adenosinergic pathway are intricately linked and play a pivotal role in cancer development. Therefore, this study focuses on exploring the correlations between CD73 and ZEB1, considering their impact on tumor progression.MethodsWe employed CRISPR/Cas9 technology to silence CD73 expression in cell lines derived from papillary thyroid carcinoma. These same cells underwent lentiviral transduction of a reporter of ZEB1 non-coding RNA regulation. We conducted studies on cell migration using scratch assays and analyses of cellular speed and polarity. Additionally, we examined ZEB1 reporter expression through flow cytometry and immunocytochemistry, complemented by Western blot analysis for protein quantification. For further insights, we applied gene signatures representing different EMT states in an RNA-seq expression analysis of papillary thyroid carcinoma samples from The Cancer Genome Atlas.ResultsSilencing CD73 expression led to a reduction in ZEB1 non-coding RNA regulation reporter expression in a papillary thyroid carcinoma-derived cell line. Additionally, it also mitigated ZEB1 protein expression. Moreover, the expression of CD73 and ZEB1 was correlated with alterations in cell morphology characteristics crucial for cell migration, promoting an increase in cell polarity index and cell migration speed. RNA-seq analysis revealed higher expression of NT5E (CD73) in samples with BRAF mutations, accompanied by a prevalence of partial-EMT/hybrid state signature expression.ConclusionsCollectively, our findings suggest an association between CD73 expression and/or activity and the post-transcriptional regulation of ZEB1 by non-coding RNA, indicating a reduction in its absence. Further investigations are warranted to elucidate the relationship between CD73 and ZEB1, with the potential for targeting them as therapeutic alternatives for cancer treatment in the near future.

Vitamin D deficiency contributes to the diabetic kidney disease progression via increase ZEB1/ZEB2 expressions

BackgroundDiabetic kidney disease (DKD) remains one of the main causes of end-stage renal disease (ESRD) and mortality in diabetic patients worldwide. Vitamin D deficiency (VitDD) is one of the main consequences of different chronic kidney disease (CKD) types and is associated with rapid progression to ESRD. Nevertheless, the mechanisms that lead to this process are poorly understood. This study aimed to characterize a model of diabetic nephropathy progression in VitDD and the epithelial-mesenchymal-transition (EMT) role in these processes.MethodsWistar Hannover rats received a diet with or without VitD before type 1 diabetes (T1D) induction. After this procedure, the rats were accompanied for 12 and 24 weeks after T1D induction and the renal function, structure, cell transdifferentiating markers and zinc finger e-box binding homeobox 1/2 (ZEB1/ZEB2) contribution to kidney damage were evaluated during the DKD progression.ResultsThe results showed an increase in glomerular tuft, mesangial and interstitial relative areas and renal function impairment in VitD-deficient diabetic rats compared to diabetic rats that received a VitD-containing diet. These alterations can be associated with increased expression of EMT markers, ZEB1 gene expression, ZEB2 protein expression and TGF-β1 urinary excretion. Decreased miR-200b expression, an important post-transcriptional regulator of ZEB1 and ZEB2 was also observed.ConclusionOur data demonstrated that VitD deficiency contributes to the rapid development and progression of DKD in diabetic rats induced by increase ZEB1/ZEB2 expressions and miR-200b downregulation.

Senescent neutrophils-derived exosomal piRNA-17560 promotes chemoresistance and EMT of breast cancer via FTO-mediated m6A demethylation

Cellular senescence is characterized by a tumor-suppressive program as well as a pro-inflammatory secretome. Neutrophils constitute significant compositions of malignancies and play key roles in tumor development. However, the role of senescent neutrophils in cancer progression is presently unexplored. Here, we demonstrate that neutrophils display enhanced senescence in breast cancer patients receiving chemotherapy. The senescent neutrophils produce increased number of exosomes, which confer drug resistance to tumor cells in vitro and in vivo. Mechanistically, senescent neutrophils-derived exosomal piRNA-17560 enhances the expression of fat mass and obesity-associated protein (FTO) in breast cancer cells. The upregulation of FTO further strengthens ZEB1 transcripts stability and expression by decreasing N6-methyladenosine (m6A) RNA methylation, leading to chemoresistance and epithelial-mesenchymal transition (EMT) of tumor cells. Clinically, the level of exosomal piR-17560 correlates with poor chemotherapy response in patients with breast cancer. In addition, YTHDF2 is essential for the posttranscriptional regulation of ZEB1 by piRNA-17560/FTO signaling. Senescent neutrophils secret exosomal piR-17560 in a STAT3-dependent manner. Altogether, this study suggests that senescent neutrophils-derived exosomal piR-17560 confers chemoresistance to tumor cells and senescent neutrophils may serve as a potential therapeutic target in breast cancer.

Regulation of ZEB1 Function and Molecular Associations in Tumor Progression and Metastasis.

Simple SummaryGiven the importance of the epithelial-to-mesenchymal transition in tumor progression and the pivotal role of ZEB1 as a transcriptional repressor during this process, the regulation of ZEB1-targeted gene expression is an active area of investigation. Diverse signaling pathways converge to induce ZEB1 activity, but few studies have focused on the enzyme-dependent modifications of ZEB1 that occur after its translation from mRNA (i.e., post-translational modifications, PTM). In addition to outlining the current knowledge in the field, we outline several questions regarding the PTM-mediated regulation of ZEB1 that remain unanswered. Thus, the areas of research covered in this review paper will provide a noteworthy conceptual advancement in our understanding of ZEB1′s biological function, as well as its post-transcriptional and post-translational regulation. Furthermore, the review will enhance the development of treatment strategies by identifying knowledge gaps in ZEB1′s regulatory mechanisms that could potentially be targeted to prevent and treat metastasis in cancer patients.Zinc finger E-box binding homeobox 1 (ZEB1) is a pleiotropic transcription factor frequently expressed in carcinomas. ZEB1 orchestrates the transcription of genes in the control of several key developmental processes and tumor metastasis via the epithelial-to-mesenchymal transition (EMT). The biological function of ZEB1 is regulated through pathways that influence its transcription and post-transcriptional mechanisms. Diverse signaling pathways converge to induce ZEB1 activity; however, only a few studies have focused on the molecular associations or functional changes of ZEB1 by post-translational modifications (PTMs). Due to the robust effect of ZEB1 as a transcription repressor of epithelial genes during EMT, the contribution of PTMs in the regulation of ZEB1-targeted gene expression is an active area of investigation. Herein, we review the pivotal roles that phosphorylation, acetylation, ubiquitination, sumoylation, and other modifications have in regulating the molecular associations and behavior of ZEB1. We also outline several questions regarding the PTM-mediated regulation of ZEB1 that remain unanswered. The areas of research covered in this review are contributing to new treatment strategies for cancer by improving our mechanistic understanding of ZEB1-mediated EMT.

Zeb1 regulation of wound-healing-induced inflammation in alkali-damaged corneas

SummaryThe cornea is an avascular tissue for vision clarity. Alkali burn could cause severe traumatic damage on the cornea with inflammation and neovascularization (NV), leading to vision reduction and blindness. Mechanisms underlying corneal inflammation and NV are not as clear. We previously reported that Zeb1 is an important factor in corneal NV, and we sought to clarify whether it is also involved in regulation of corneal inflammation. We analyzed the alkali burn-induced corneal inflammation and wound healing in both Zeb1+/+ and Zeb1−/+ littermates through a multidisciplinary approach. We provide evidence that Zeb1 forms a positive regulatory loop with Tgfb to regulate early corneal inflammation by maintenance of immune cell viability and mobility and later wound healing by activation of both Nf-κb and Tgfb-related Stat3 signaling pathways. We believe that ZEB1 is a potential therapeutic target, and inactivation of ZEB1 could be a strategy to treat severe corneal inflammation condition.

Functional mechanism of hsa-miR-128-3p in epithelial-mesenchymal transition of pancreatic cancer cells via ZEB1 regulation.

Pancreatic cancer (PC) often correlates with high mortality due to late diagnosis, rapid metastasis, and resistance to chemotherapy. miR-128-3p has been validated as a tumor suppressor in PC. This study explored the functional mechanism of miR-128-3p in epithelial-mesenchymal transition (EMT) of PC cells. Four PC cancer cell lines with different degrees of malignancy and normal pancreatic cells were selected to detect expressions of hsa-miR-128-3p and ZEB1 by RT-qPCR and Western blot. miR-128-3p mimic or si-ZEB1 was delivered into PANC-1 cells and miR-128-3p inhibitor or oe-ZEB1 was delivered into AsPC-1 cells. Expressions of epithelial and mesenchymal markers were analyzed by Western blot and cell fluorescence staining. The binding relationship between miR-128-3p and ZEB1 was examined by bioinformatics analysis and dual-luciferase assay, and verified by RT-qPCR and Western blot. PC cell invasion and migration were assessed by Transwell assays. Generally, hsa-miR-128-3p was poorly-expressed in PC cells. However, it was relatively more expressed in AsPC-1 cells with epithelial phenotypes relative to PANC-1 cells with mesenchymal phenotype, whereas ZEB1 expression showed opposite tendencies. PANC-1 cells transfected with miR-128-3p mimic or si-ZEB1 showed upregulated E-cadherin and downregulated N-cadherin, and transformed from mesenchymal phenotypes to epithelial phenotypes, with decreased invasion and migration, while opposite results occurred in AsPC-1 cells transfected with miR-128-3p inhibitor or oe-ZEB1. miR-128-3p targeted ZEB1. oe-ZEB1 antagonized the inhibition of miR-128-3p mimic on PANC-1 cell EMT, invasion, and migration, while si-ZEB1 reversed the facilitation of miR-128-3p inhibitor in AsPC-1 cells. In conclusion, miR-128-3p inhibited PC cell EMT, invasion, and migration by targeting ZEB1.

MicroRNA-200c/429 mediated regulation of Zeb1 augments N-Cadherin in mouse cardiac mesenchymal cells.

Cardiac mesenchymal cells (CMCs) are a promising cell type that showed therapeutic potential in heart failure models. The analysis of the underlying mechanisms by which the CMCs improve cardiac function ison track. This study aimed to investigate the expression of N-Cadherin, a transmembrane protein that enhances cell adhesion, and recently gained attention for differentiation and augmentation of stem cell function. The mouse CMCs were isolated and analyzed for the mesenchymal markers using flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were used to assess the expression of N-Cadherin along with its counteracting molecule E-Cadherin and their regulator Zeb1 in CMCs and dermal fibroblast. The expression level of miR-200c and miR-429 was analyzed using miRNA assays. Transient transfection of miR-200c followed by qRT-PCR, western blot analysis, and immunostaining was done in CMCs to analyze the expression of Zeb1, N-Cadherin, and E-Cadherin. Flow cytometry analysis showed that CMCs possess mesenchymal markers and absence for hematopoietic and immune cell markers. Increased expression of N-Cadherin and Zeb1 in CMCs was observed in CMCs at both RNA and protein levels compared to fibroblast. We found significant downregulation of miR-200c and miR-429 in CMCs. The ectopic expression of miR-200c in CMCs significantly downregulated Zeb1 and N-Cadherin expression. Our findings suggest that the significant downregulation of miR-200c/429 in CMCs maintains the expression of N-Cadherin, which may be important for its functional integrity.

Perfluorooctane sulfonate (PFOS) triggers migration and invasion of esophageal squamous cell carcinoma cells via regulation of Zeb1

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent and deadly cancers worldwide, especially in Eastern Asia. As a potential endocrine-disrupting chemical (EDC), perfluorooctane sulfonate (PFOS) can mimic estrogen, disturb the estrogen signals, and then cause various diseases. Although ESCC can be directly exposed to PFOS during food digestion, the effects and mechanisms of PFOS on the development of ESCC are still not well illustrated. This study showed that PFOS can promote the migration and invasion of ESCC cells. Further, PFOS treatment can increase the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, while decreasing the expression of E-Cadherin (E-Cad). Zeb1, an important transcription factor for cell motility, was essential for PFOS induced migration and invasion of ESCC cells. PFOS can increase the expression of Zeb1 via upregulation of its transcription and proteins stability. A-kinase interacting protein 1 (AKIP1) and ataxia-telangiectasia mutated (ATM) were responsible for PFOS induced transcription and proteins stability of Zeb1 in ESCC cells, respectively. Collectively, our data indicated that environmental exposure and body accumulation of PFOS might be an important risk factor for ESCC progression.

Zinc-Dependent Regulation of ZEB1 and YAP1 Coactivation Promotes Epithelial-Mesenchymal Transition Plasticity and Metastasis in Pancreatic Cancer

Pancreatic cancer is characterized by extensive metastasis. Epithelial-mesenchymal transition (EMT) plasticity plays a critical role in tumor progression and metastasis by maintaining the transition between EMT and mesenchymal-epithelial transition states. Our aim is to understand the molecular events regulating metastasis and EMT plasticity in pancreatic cancer. The interactions between a cancer-promoting zinc transporter ZIP4, a zinc-dependent EMT transcriptional factor ZEB1, a coactivator YAP1, and integrin α3 (ITGA3) were examined in human pancreatic cancer cells, clinical specimens, spontaneous mouse models (KPC and KPCZ) and orthotopic xenografts, and 3-dimensional spheroid and organoid models. Correlations between ZIP4, miR-373, and its downstream targets were assessed by RNA in situ hybridization and immunohistochemical staining. The transcriptional regulation of ZEB1, YAP1, and ITGA3 by ZIP4 wasdetermined by chromatin immunoprecipitation, co-immunoprecipitation, and luciferase reporter assays. The Hippo pathway effector YAP1 is a potent transcriptional coactivator and forms a complex with ZEB1 to activate ITGA3 transcription through the YAP1/transcriptional enhanced associate domain (TEAD) binding sites in human pancreatic cancer cells and KPC-derived mouse cells. ZIP4 upregulated YAP1 expression via activation of miR-373 and inhibition of the YAP1 repressor large tumor suppressor 2 kinase (LATS2). Furthermore, upregulation of ZIP4 promoted EMT plasticity, cell adhesion, spheroid formation, and organogenesis both in human pancreatic cancer cells, 3-dimensional spheroid model, xenograft model, and spontaneous mouse models (KPC andKPCZ) through ZEB1/YAP1-ITGA3 signaling axis. We demonstrated that ZIP4 activates ZEB1 and YAP1 through distinct mechanisms. The ZIP4-miR-373-LATS2-ZEB1/YAP1-ITGA3 signaling axis has a significant impact on pancreatic cancer metastasis and EMT plasticity.

NCSTN promotes hepatocellular carcinoma cell growth and metastasis via β-catenin activation in a Notch1/AKT dependent manner

BackgroundHepatocellular carcinoma is the third top cause of cancer-related mortalities worldwide. The prognosis of HCC patients remains poor due to rapid progression and high incidence of tumor recurrence. Nicastrin (NCSTN), a core subunit of γ-Secretase, has been reported to play a vital role in tumor progression. However, no study till now has revealed its role in HCC.MethodsThe expression of NCSTN was evaluated by immunohistochemical staining, Western blot, and quantitative real-time PCR. Cell counting kit-8, colony formation and cell cycle assays were used for evaluating cell growth in vitro. Transwell and wound-healing assays were used for evaluating cell migration and invasion capacity. Immunofluorescence, subcellular protein fractionation and co-immunoprecipitation were used for location analysis of β-catenin. The in vivo functions of NCSTN were illustrated by xenograft tumor models.ResultsNCSTN was dramatically overexpressed in HCC compared to normal liver tissues. Elevated NCSTN expression level was significantly correlated to worse overall and recurrence-free survival of HCC patients. Enhanced NCSTN expression promoted HCC cell growth, migration and invasion in vitro and in vivo. Mechanistic investigations showed that NCSTN induced epithelial-mesenchymal transition (EMT) process via upregulation of Zeb1. Subsequently, we revealed that NCSTN facilitated nuclear translocation of β-catenin, a positive transcriptional regulator of Zeb1. Using Notch and AKT inhibitors, we revealed that NCSTN promoted β-catenin activation through Notch1 and AKT signaling pathway. NCSTN increased AKT and GSK-3β phosphorylation by cleavage of Notch1, which decreased GSK-3β/β-catenin complex. The inactivation of GSK-3β inhibited the β-catenin degradation and promoted nuclear translocation of β-catenin to initiate transcription of Zeb1, resulting in malignant phenotype.ConclusionsOur results demonstrated that NCSTN promoted HCC cell growth and metastasis via β-catenin-mediated upregulation of Zeb1 in a Notch1/AKT dependent manner, suggesting that NCSTN might serve as a potential prognostic marker and therapeutic target for HCC.

Regulation of cell growth and migration by miR-96 and miR-183 in a breast cancer model of epithelial-mesenchymal transition

Breast cancer is the most commonly diagnosed malignancy in women, and has the second highest mortality rate. Over 90% of all cancer-related deaths are due to metastasis, which is the spread of malignant cells from the primary tumor to a secondary site in the body. It is hypothesized that one cause of metastasis involves epithelial-mesenchymal transition (EMT). When epithelial cells undergo EMT and transition into mesenchymal cells, they display increased levels of cell proliferation and invasion, resulting in a more aggressive phenotype. While many factors regulate EMT, microRNAs have been implicated in driving this process. MicroRNAs are short noncoding RNAs that suppress protein production, therefore loss of microRNAs may promote the overexpression of specific target proteins important for EMT. The goal of this study was to investigate the role of miR-96 and miR-183 in EMT in breast cancer. Both miR-96 and miR-183 were found to be downregulated in post-EMT breast cancer cells. When microRNA mimics were transfected into these cells, there was a significant decrease in cell viability and migration, and a shift from a mesenchymal to an epithelial morphology (mesenchymal-epithelial transition or MET). These MET-related changes may be facilitated in part by the regulation of ZEB1 and vimentin, as both of these proteins were downregulated when miR-96 and miR-183 were overexpressed in post-EMT cells. These findings indicate that the loss of miR-96 and miR-183 may help facilitate EMT and contribute to the maintenance of a mesenchymal phenotype. Understanding the role of microRNAs in regulating EMT is significant in order to not only further elucidate the pathways that facilitate metastasis, but also identify potential therapeutic options for preventing or reversing this process.

ASPP1 deficiency promotes epithelial-mesenchymal transition, invasion and metastasis in colorectal cancer

The apoptosis-stimulating protein of p53 (ASPP) family of proteins can regulate apoptosis by interacting with the p53 family and have been identified to play an important role in cancer progression. Previously, we have demonstrated that ASPP2 downregulation can promote invasion and migration by controlling β-catenin-dependent regulation of ZEB1, however, the role of ASPP1 in colorectal cancer (CRC) remains unclear. We analyzed data from The Cancer Genome Atlas (TCGA) and coupled this to in vitro experiments in CRC cell lines as well as to experimental pulmonary metastasis in vivo. Tissue microarrays of CRC patients with information of clinical-pathological parameters were also used to investigate the expression and function of ASPP1 in CRC. Here, we report that loss of ASPP1 is capable of enhancing migration and invasion in CRC, both in vivo and in vitro. We demonstrate that depletion of ASPP1 could activate expression of Snail2 via the NF-κB pathway and in turn, induce EMT; and this process is further exacerbated in RAS-mutated CRC. ASPP1 could be a prognostic factor in CRC, and the use of NF-κB inhibitors may provide new strategies for therapy against metastasis in ASPP1-depleted CRC patients.

Schlafen12 Reduces the Aggressiveness of Triple Negative Breast Cancer through Post-Transcriptional Regulation of ZEB1 That Drives Stem Cell Differentiation.

Schlafen12 (SLFN12) promotes human intestinal and prostatic epithelial differentiation. We sought to determine whether SLFN12 reduces triple-negative breast cancer (TNBC) aggressiveness. We validated bioinformatics analyses of publicly available databases by staining human TNBC. After virally overexpressing or siRNA-reducing SLFN12 in TNBC cell lines, we measured proliferation by CCK-8 assay, invasion into basement-membrane-coated pores, mRNA by q-RT-PCR and protein by Western blotting. Flow cytometry assessed proliferation and stem cell marker expression, and sorted CD44+/CD24- cells. Stemness was also assessed by mammosphere formation, and translation by click-it-AHA chemistry. SLFN12 expression was lower in TNBC tumors and correlated with survival. SLFN12 overexpression reduced TNBC MDA-MB-231, BT549, and Hs578T proliferation. In MDA-MB-231 cells, AdSLFN12 reduced invasion, promoted cell cycle arrest, increased E-cadherin promoter activity, mRNA, and protein, and reduced vimentin expression and protein. SLFN12 knockdown increased vimentin. AdSLFN12 reduced the proportion of MDA-MB-231 CD44+CD24- cells, with parallel differentiation changes. SLFN12 overexpression reduced MDA-MB-231 mammosphere formation. SLFN12 overexpression decreased ZEB1 and Slug protein despite increased ZEB1 and Slug mRNA in all three lines. SLFN12 overexpression accelerated MDA-MB-231 ZEB1 proteasomal degradation and slowed ZEB1 translation. SLFN12 knockdown increased ZEB1 protein. Coexpressing ZEB1 attenuated the SLFN12 effect on E-cadherin mRNA and proliferation in all three lines. SLFN12 may reduce TNBC aggressiveness and improve survival in part by a post-transcriptional decrease in ZEB1 that promotes TNBC cancer stem cell differentiation.

PLAGL2 promotes epithelial\u2013mesenchymal transition and mediates colorectal cancer metastasis via \u03b2-catenin-dependent regulation of ZEB1

BackgroundWe previously demonstrated that the pleomorphic adenoma gene like-2 (PLAGL2) is involved in the pathogenesis of Hirschsprung disease. Enhanced PLAGL2 expression was observed in several malignant tumours. However, the exact function of PLAGL2 and its underlying mechanism in colorectal cancer (CRC) remain largely unknown.MethodsImmunohistochemical analysis of PLAGL2 was performed. A series of in vitro and in vivo experiments were conducted to reveal the role of PLAGL2 in the progression of CRC.ResultsEnhanced PLAGL2 expression was significantly associated with EMT-related proteins in CRC. The data revealed that PLAGL2 promotes CRC cell proliferation, migration, invasion and EMT both in vitro and in vivo. Mechanistically, PLAGL2 promoted the expression of ZEB1. PLAGL2 enhanced the expression and nuclear translocation of β-catenin by decreasing its phosphorylation. The depletion of β-catenin neutralised the regulation of ZEB1 that was caused by enhanced PLAGL2 expression. The small-molecule inhibitor PNU-74654, also impaired the enhancement of ZEB1 that resulted from the modified PLAGL2 expression. The depletion of ZEB1 could block the biological function of PLAGL2 in CRC cells.ConclusionsCollectively, our findings suggest that PLAGL2 mediates EMT to promote colorectal cancer metastasis via β-catenin-dependent regulation of ZEB1.

E-cadherin, Snail, ZEB-1, DNMT1, DNMT3A and DNMT3B expression in normal and breast cancer tissues.

Breast cancer is known as one of very important cancers among females, given that a variety of external (i.e., environmental risk factors) and internal factors (i.e., genetics, and epigenetics) are related to the emergence and progression of breast cancer. Among genetic and epigenetic factors, DNA methyltransferase and EMT related genes have critical roles in breast cancer pathogenesis. In the study presented here, we investigated expression of DNA methyltransferases (e.g., DNMT1, DNMT3A and DNMT3B) and EMT related genes (e.g., E-cadherin, Snail, ZEB-1). Tissue samples were collected from 18 cancer and 24 normal breast tissues. We evaluated the expression levels of DNA methyltransferases and EMT related genes using Quantitative real-time PCR (qRT-PCR). Our results indicated that the expression levels of ZEB-1, Snail, and DNMT3B were increased in breast cancer subjects in comparison to the control group. On the other hand, there was a significant decrease in E-cadherin expression in breast cancer tissues in comparison to the normal tissues. Moreover, there were no significant changes for DNMT1 and DNMT3A expression in breast cancer tissues when compared to the normal tissues. Taken together, our finding show that up regulation of ZEB-1 and Snail could be associated with down regulation of E-cadherin and results in promotion of cancer cell invasion. Moreover, down regulation of E-cadherin may be related to deterioration of DNMT3B inpatients with breast cancer.

A Novel Calcium-Mediated EMT Pathway Controlled by Lipids: An Opportunity for Prostate Cancer Adjuvant Therapy.

The composition of periprostatic adipose tissue (PPAT) has been shown to play a role in prostate cancer (PCa) progression. We recently reported an inverse association between PCa aggressiveness and elevated PPAT linoleic acid (LA) and eicosapentaenoic acid (EPA) content. In the present study, we identified a new signaling pathway with a positive feedback loop between the epithelial-to-mesenchymal transition (EMT) transcription factor Zeb1 and the Ca2+-activated K+ channel SK3, which leads to an amplification of Ca2+ entry and cellular migration. Using in vitro experiments and ex vivo cultures of human PCa slices, we demonstrated that LA and EPA exert anticancer effects, by modulating Ca2+ entry, which was involved in Zeb1 regulation and cancer cellular migration. This functional approach using human prostate tumors highlights the clinical relevance of our observations, and may allow us to consider the possibility of targeting cancer spread by altering the lipid microenvironment.

ZIP4 Increases Expression of Transcription Factor ZEB1 to Promote Integrin α3β1 Signaling and Inhibit Expression of the Gemcitabine Transporter ENT1 in Pancreatic Cancer Cells

Pancreatic tumors undergo rapid growth and progression, become resistant to chemotherapy, and recur after surgery. We studied the functions of the solute carrier family 39 member 4 (SLC39A4, also called ZIP4), which regulates concentrations of intracellular zinc and is increased in pancreatic cancer cells, in cell lines and mice. We obtained 93 pancreatic cancer specimens (tumor and adjacent nontumor tissues) from patients who underwent surgery and gemcitabine chemotherapy and analyzed them by immunohistochemistry. ZIP4 and/or ITGA3 or ITGB1 were overexpressed or knocked down with short hairpin RNAs in AsPC-1 and MIA PaCa-2 pancreatic cancer cells lines, and in pancreatic cells from KPC and KPC-ZEB1-knockout mice, and pancreatic spheroids were established; cells and spheroids were analyzed by immunoblots, reverse transcription polymerase chain reaction, and liquid chromatography tandem mass spectrometry. We studied transcriptional regulation of ZEB1, ITGA3, ITGB1, JNK, and ENT1 by ZIP4 using chromatin precipitation and luciferase reporter assays. Nude mice were given injections of genetically manipulated AsPC-1 and MIA PaCa-2 cells, and growth of xenograft tumors and metastases was measured. In pancreatic cancer specimens from patients, increased levels of ZIP4 were associated with shorter survival times. MIA PaCa-2 cells that overexpressed ZIP4 had increased resistance to gemcitabine, 5-fluorouracil, and cisplatin, whereas AsPC-1 cells with ZIP4 knockdown had increased sensitivity to these drugs. In mice, xenograft tumors grown from AsPC-1 cells with ZIP4 knockdown were smaller and more sensitive to gemcitabine. ZIP4 overexpression significantly reduced accumulation of gemcitabine in pancreatic cancer cells, increased growth of xenograft tumors in mice, and increased expression of the integrin subunits ITGA3 and ITGB1; expression levels of ITGA3 and ITGB1 were reduced in cells with ZIP4 knockdown. Pancreatic cancer cells with ITGA3 or ITGB1 knockdown had reduced proliferation and formed smaller tumors in mice, despite overexpression of ZIP4; spheroids established from these cells had increased sensitivity to gemcitabine. We found ZIP4 to activate STAT3 to induce expression of ZEB1, which induced expression of ITGA3 and ITGB1 in KPC cells. Increased ITGA3 and ITGB1 expression and subsequent integrin α3β1 signaling, via c-Jun-N-terminal kinase (JNK), inhibited expression of the gemcitabine transporter ENT1, which reduced gemcitabine uptake by pancreatic cancer cells. ZEB1-knockdown cells had increased sensitivity to gemcitabine. In studies of pancreatic cancer cell lines and mice, we found that ZIP4 increases expression of the transcription factor ZEB1, which activates expression of ITGA3 and ITGB1. The subsequent increase in integrin α3β1 signaling, via JNK, inhibits expression of the gemcitabine transporter ENT1, so that cells take up smaller amounts of the drug. Activation of this pathway might help mediate resistance of pancreatic tumors to chemotherapeutic agents.