6935

ZEB1

zinc finger E-box binding homeobox 1;ZEB1;zinc finger E-box binding homeobox 1

delta-crystallin enhancer binding factor 1|negative regulator of IL2|posterior polymorphous corneal dystrophy 3|transcription factor 8 (represses interleukin 2 expression)|zinc finger E-box-binding homeobox 1|zinc finger homeodomain enhancer-binding prote

10p11.2

protein-coding

The ubiquitin ligase CUL4A has been implicated in tumorigenesis, but its contributions to progression and metastasis have not been evaluated. Here, we show that CUL4A is elevated in breast cancer as well as in ovarian, gastric, and colorectal tumors in which its expression level correlates positively with distant metastasis. CUL4A overexpression in normal or malignant human mammary epithelial cells increased their neoplastic properties in vitro and in vivo, markedly increasing epithelial-mesenchymal transition (EMT) and the metastatic capacity of malignant cells. In contrast, silencing CUL4A in aggressive breast cancer cells inhibited these processes. Mechanistically, we found that CUL4A modulated histone H3K4me3 at the promoter of the EMT regulatory gene ZEB1 in a manner associated with its transcription. ZEB1 silencing blocked CUL4A-driven proliferation, EMT, tumorigenesis, and metastasis. Furthermore, in human breast cancers, ZEB1 expression correlated positively with CUL4A expression and distant metastasis. Taken together, our findings reveal a pivotal role of CUL4A in regulating the metastatic behavior of breast cancer cells.

Resistance to trastuzumab and concomitantly distal metastasis are leading causes of mortality in HER2-positive breast cancers, the molecular basis of which remains largely unknown. Here, we generated trastuzumab-resistant breast cancer cells with increased tumorigenicity and invasiveness compared with parental cells, and observed robust epithelial-mesenchymal transition (EMT) and consistently elevated TGF-beta signaling in these cells. MiR-200c, which was the most significantly downregulated miRNA in trastuzumab-resistant cells, restored trastuzumab sensitivity and suppressed invasion of breast cancer cells by concurrently targeting ZNF217, a transcriptional activator of TGF-beta, and ZEB1, a known mediator of TGF-beta signaling. Given the reported backward inhibition of miR-200c by ZEB1, ZNF217 also exerts a feedback suppression of miR-200c via TGF-beta/ZEB1 signaling. Restoration of miR-200c, silencing of ZEB1 or ZNF217 or blockade of TGF-beta signaling increased trastuzumab sensitivity and suppressed invasiveness of breast cancer cells. Therefore, our study unraveled nested regulatory circuits of miR-200c/ZEB1 and miR-200c/ZNF217/TGF-beta/ZEB1 in synergistically promoting trastuzumab resistance and metastasis of breast cancer cells. These findings provide novel insights into the common role of EMT and related molecular machinery in mediating the malignant phenotypes of breast cancers.

Growing evidence illustrates that aberrant activation of the epithelial-mesenchymal transition plays a key role in tumor cell invasion and metastasis. Transcription factors SNAI1 and ZEB1 regulate the epithelial-mesenchymal transition. To determine if SNAI1 and ZEB1 are involved in the metastasis of cervical cancer, we used immunohistochemistry to evaluate the expression of SNAI1, ZEB1, and vimentin in tumor and stromal compartments for a large set of cervical carcinoma samples. Results were evaluated using an H score (percentage x intensity). Of 70 samples, 64 cases (91.43%) were positive for SNAI1 expression. The median SNAI1 H score was 174.00 (range, 5-285). Sixty-seven cases (95.71%) were positive for ZEB1, with a median H score of 165.77 (range, 5-260). Nuclear expression of SNAI1 and ZEB1 in tumor cells was positively associated with International Federation of Gynecology and Obstetrics (FIGO) stages (P = .015 and P = .008, respectively) and lymph node metastasis (P = .007 and P = .007, respectively); meanwhile, expression of vimentin in tumor cells was positively associated with lymph node metastasis (P = .019). According to negative vimentin expression, nuclear expression of ZEB1 in tumor cells was positively associated with FIGO stages (P = .04). According to positive vimentin expression, nuclear expression of SNAI1 in tumor cells was positively associated with FIGO stages (P = .018) and pN (P = .029). In light of these findings, we propose that SNAI1 and ZEB1 have the potential to be used as a novel predictor of pelvic lymph node metastasis and represent promising therapeutic targets in patients with cervical cancer.

Undifferentiated endometrial carcinomas are very aggressive high-grade endometrial carcinomas that are frequently under-recognized. This study aimed to analyze the molecular alterations underlying the development of these endometrial carcinomas, focusing on those related to dedifferentiation. We assessed a series of 120 tumors: 57 grade 1 and 2 endometrioid endometrial carcinomas, 15 grade 3 endometrioid endometrial carcinomas, 27 endometrial serous carcinomas, and 21 undifferentiated endometrial carcinomas. We found a high frequency of DNA mismatch repair deficiency (38%) and moderate rate of p53 overexpression ( approximately 33%) in undifferentiated carcinomas. In contrast to the characteristic endometrioid phenotype, there was a dramatic downregulation of E-cadherin expression in the undifferentiated subtype. Quantitative methylation studies dismissed CDH1 promoter hypermethylation as the mechanism responsible for this change in gene expression, while immunohistochemistry revealed that the E-cadherin repressor ZEB1 was frequently overexpressed (62%) in undifferentiated endometrial carcinomas. This finding was accompanied by a sharp downregulation in the expression of the miR-200 family of microRNAs, well-known targets of ZEB1. Furthermore, there was enhanced expression of epithelial-to-mesenchymal transition markers in undifferentiated endometrial carcinomas, such as N-cadherin, cytoplasmic p120, and osteonectin. In addition, HMGA2, a regulator of epithelial-to-mesenchymal transition that is expressed in aggressive endometrial tumors, such as endometrial serous carcinomas and carcinosarcomas, was expressed in >20% of undifferentiated carcinomas. These results suggest that ZEB1 overexpression, associated with E-cadherin and miR-200s downregulation, and the expression of mesenchymal markers might enhance the metastatic potential of undifferentiated endometrial carcinomas, leading to a poor prognosis. In addition, our observations suggest that the immnohistochemical analysis of E-cadherin and ZEB1 can aid in the differential diagnosis of the more agressive undifferentiated endometrial carcinomas from grade 3 endometrioid carcinomas.

The aim of this study was to investigate the expression of ZEB1 in gastric carcinoma, its correlation with the clinicopathology of gastric carcinoma, and the role of ZEB1 in invasion and metastasis in gastric carcinoma. ZEB1 expression was analyzed by immunohistochemistry and Western blot in 45 gastric carcinoma tissue samples that contained the adjacent gastric mucosa. The correlation between ZEB1 expression, the occurrence and development of gastric cancer, and clinical pathology was investigated. ZEB1 expression in the human gastric carcinoma cell line AGS was downregulated by RNA interference, and changes in ZEB1 expression corresponded with changes in the invasive and metastatic ability of AGS cells. Immunohistochemistry revealed that ZEB1 protein expression in gastric carcinoma tissues was significantly higher than in normal gastric mucosa tissues (p < 0.001). A lower degree of differentiation of gastric cancer (p = 0.009), a higher TNM (tumor, node, and metastasis) stage (p = 0.010), and a larger scope of invasion were correlated with higher expression of ZEB1 (p = 0.041, 0.002). However, the expression of ZEB1 in gastric carcinoma tissue was independent of gender, age, and tumor size (p > 0.05). Western blot results also showed that ZEB1 protein expression was significantly higher in gastric carcinoma tissue than in the adjacent normal gastric mucosa tissue (p = 0.008). A lower degree of differentiation of the gastric carcinoma correlated with a higher TNM stage, and a larger scope of invasion correlated with increased ZEB1 expression (p = 0.023). Transfection of ZEB1 siRNA in AGS cells significantly decreased the expression level of ZEB1 protein (p = 0.035). Furthermore, the number of cells that could pass through the Transwell chamber was significantly lower in the transfected group than in the non-transfected control group (p = 0.039), indicating that the suppression of ZEB1 expression could significantly reduce the invasive and metastatic ability of AGS cells (p = 0.005). Concluding, in gastric carcinoma tissue, overexpression of ZEB1 may be related to the occurrence and development as well as invasion and metastasis of gastric carcinoma.

OBJECTIVES: 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. METHODS: tumor initiating capability of HNC-CH133(+) cells with ZEB1/2 knockdown or co-overexpression was presented in vitro and in vivo. RESULTS: 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. CONCLUSION: Therapies targeting ZEB1/ZEB2 in HNC-CD133(+) cells may provide a new approach for HNC therapy in the future.

Epithelial to mesenchymal transition (EMT) facilitates tissue remodelling during embryonic development and is viewed as an essential early step in tumour metastasis. We found that all five members of the microRNA-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) and miR-205 were markedly downregulated in cells that had undergone EMT in response to transforming growth factor (TGF)-beta or to ectopic expression of the protein tyrosine phosphatase Pez. Enforced expression of the miR-200 family alone was sufficient to prevent TGF-beta-induced EMT. Together, these microRNAs cooperatively regulate expression of the E-cadherin transcriptional repressors ZEB1 (also known as deltaEF1) and SIP1 (also known as ZEB2), factors previously implicated in EMT and tumour metastasis. Inhibition of the microRNAs was sufficient to induce EMT in a process requiring upregulation of ZEB1 and/or SIP1. Conversely, ectopic expression of these microRNAs in mesenchymal cells initiated mesenchymal to epithelial transition (MET). Consistent with their role in regulating EMT, expression of these microRNAs was found to be lost in invasive breast cancer cell lines with mesenchymal phenotype. expression of the miR-200 family was also lost in regions of metaplastic breast cancer specimens lacking E-cadherin. These data suggest that downregulation of the microRNAs may be an important step in tumour progression.

The tumor suppressor gene p53 has been implicated in the regulation of epithelial-mesenchymal transition (EMT) and tumor metastasis by regulating microRNA (miRNA) expression. Here, we report that mutant p53 exerts oncogenic functions and promotes EMT in endometrial cancer (EC) by directly binding to the promoter of miR-130b (a negative regulator of ZEB1) and inhibiting its transcription. We transduced p53 mutants into p53-null EC cells, profiled the miRNA expression by miRNA microarray and identified miR-130b as a potential target of mutant p53. Ectopic expression of p53 mutants repressed the expression of miR-130b and triggered ZEB1-dependent EMT and cancer cell invasion. Loss of an endogenous p53 mutation increased the expression of miR-130b, which resulted in reduced ZEB1 expression and attenuation of the EMT phenotype. Furthermore, re-expression of miR-130b suppressed mutant p53-induced EMT and ZEB1 expression. Importantly, the expression of miR-130 was significantly reduced in EC tissues, and patients with higher expression levels of miR-130b survived longer. These data provide a novel understanding of the roles of p53 gain-of-function mutations in accelerating tumor progression and metastasis through modulation of the miR-130b-ZEB1 axis.

We examined 56 malignant mesotheliomas, 117 lung adenocarcinomas, and 34 metastatic lung adenocarcinomas with antibodies to transcription factors involved in epitheliomesenchymal transition. The tumors were stained immunohistochemically with antibodies zeb1, twist, and snail. Malignant mesotheliomas exhibited a stronger expression of zeb1 and twist than lung adenocarcinomas (p < 0.001 for both). metastatic adenocarcinomas displayed a more frequent expression of zeb1 and twist (p < 0.001 for both) than lung adenocarcinomas. Patients with snail positive mesotheliomas experienced a better survival (p = 0.046), whereas in lung adenocarcinomas, this trait predicted worse survival (p = 0.024). Biphasic mesotheliomas had a more frequent expression of snail or zeb1 than epithelioid and sarcomatoid mesotheliomas as a group (p = 0.034 and p = 0.005, respectively). E-cadherin was more commonly present in epithelioid mesotheliomas (p = 0.002). Cases with snail positivity showed a significantly lower apoptotic index (p = 0.039). Malignant mesotheliomas display strong twist and zeb1 expression, which may be an indication of transdifferentiation between the epithelioid and sarcomatoid cell types with biphasic mesotheliomas representing tumors in active transition. metastatic adenocarcinomas show a higher expression of zeb1 and twist than primary lung tumors, confirming our previous findings and highlighting their significance in the metastatic process. Snail expression is associated with poor prognosis in lung adenocarcinomas, but an opposite effect was seen in mesothelioma, a fact which may be related to the different cellular origin of epithelial and mesothelial cells.

BACKGROUND: This study aimed to investigate the expression of ZEB1 in osteosarcoma tissues and to discuss the relationship between ZEB1 expression and osteosarcoma metastasis. METHODS: Using RT-PCR and Western blotting, the mRNA and protein expressions of ZEB1 in the osteosarcoma and normal bone tissues were detected. Using the RNA interference technique, the expression of ZEB1 in the human osteosarcoma MG-63 cell line was downregulated, and the changes in the invasion of MG-63 cells were examined. RESULTS: The positive mRNA expression rate of ZEB1 in the osteosarcoma tissues was significantly higher than that in normal bone tissue (P < 0.05). The protein expression level of ZEB1 in the sarcoma tissues from patients with positive lung metastasis was significantly higher than that from patients without lung metastasis (P < 0.05). After the transfection of ZEB1 siRNA into the MG-63 cells, the protein expression of ZEB1 was significantly reduced (P < 0.05), and the number of cells that passed through the Transwell chamber was significantly lower than that in the non-transfected control group as well as the transfected control group (P < 0.05). CONCLUSIONS: The overexpression of ZEB1 in osteosarcoma may be related to the carcinogenesis and development as well as metastasis and invasion of osteosarcoma.

BACKGROUND: Progress has been made against early events of malignant transformation and drug resistance associated with epithelial ovarian cancer; uncontrolled metastases, however, still accounts for most patient deaths. The molecular mechanism that regulates the process of epithelial ovarian cancer metastases is not yet clearly understood. The purpose of this study was to investigate the effect of down-regulating the transcriptional repressor zinc-finger E-box-binding homeobox 1 (ZEB1) on an epithelial-mesenchymal transition (EMT) of human ovarian cancer SKOV3 cell line in vitro and in vivo. METHODS: The human ovarian cancer cells SKOV3 and HO8910 were transfected with an expression vector-based small hairpin RNA (shRNA) targeting ZEB1 (shZEB1), and the stably transfected cells were selected. Colony-forming, wound-healing, and cellular migration assays were respectively used. The tumorigenicity of shZEB1-SKOV3 was also evaluated in mice. RESULTS: The shZEB1-SKOV3 and shZEB1-HO8910 cells showed a lower level of ZEB1 expression and weaker cell migration than the control cells. Moreover, down-regulating ZEB1 expression with shRNA in the cells enhanced the expression of miR-200c that acted as a tumor suppressor to inhibit the epithelial-mesenchymal transition of shZEB1-SKOV3 cells and to block shZEB1-SKOV3 cell metastasis in vivo. The shRNA-mediated down-regulation ZEB1 in SKOV3 cells significantly decreased the tumor growth in the xenograft mice. CONCLUSION: The shZEB1-mediated down-regulation of the ZEB1 expression in the SKOV3 cells may be considered for future clinical trials.

Ovarian cancer, particularly high-grade serous ovarian carcinoma (HG-SOC), is still the main cause of death among gynecological malignancies. However, the molecular mechanisms related to its malignant biological behavior are still unclear. Recent studies indicate that microRNAs (miRNAs) play an important role in tumor metastasis. Here, we report that miR-1236-3p expression was downregulated in HG-SOC when compared to that in normal fallopian tube tissue. Manipulation of miR-1236-3p significantly influenced the morphology, migration and invasion of ovarian cancer cell lines (A2780 and SKOV3). With dual-luciferase reporter assay, we demonstrated that miR1236-3p binds to the 3UTR of zinc-finger E-box binding homeobox 1 (ZEB1) mRNA, and functions as a negative regulator of ZEB1. Furthermore, we revealed that manipulation of miR-1236-3p modulates ZEB1 expression and influences expression of its downstream genes E-cadherin and N-cadherin at both the mRNA and protein levels. We also found an inverse relationship between miR1236-3p and ZEB1 expression in the HG-SOC tissue samples. Taken together, our results indicate that miR-1236-3p regulates ovarian cancer metastasis by directly targeting ZEB1, and it may play an important role in the diagnosis and treatment of ovarian cancer.

BACKGROUND AND OBJECTIES: Phenotypic plasticity of cancer cells via epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) is essential for tumor progression and metastasis. METHODS: Tissue samples were obtained from 76 pancreatic head cancers. We assessed the expression of E-cadherin, vimentin, ZEB-1, and ZEB-2 by immunohistochemical and immunofluorescence staining. Next, 147 metastatic lymph nodes from 45 pancreatic cancers with low expression of E-cadherin were obtained and divided into two categories according to the maximum diameter of the metastases: 2 mm or more and less than 2 mm. RESULTS: High expressions of ZEB-1 and ZEB-2 in the primary tumors were significantly associated with repression of E-cadherin (P = 0.0007), and poorer prognosis (P = 0.0322). Forty-three (29.3%) of the 147 metastatic tumors from pancreatic cancers with low expression of E-cadherin showed high E-cadherin expression. cancer cells in the larger metastases showed high expression of E-cadherin (P = 0.0061) and low expression of ZEB-1 (P = 0.0170) and ZEB-2 (P = 0.0036) compared with those in the smaller metastases. CONCLUSIONS: In primary pancreatic tumors and metastatic lymph nodes, high and low expression of ZEB-1 and ZEB-2 was associated with mesenchymal and epithelial phenotype of cancer cells, respectively.

metastatic colonization involves cancer cell lodgment or adherence in the microvasculature and subsequent migration of those cells across the endothelium into a secondary organ site. To study this process further, we analyzed transendothelial migration of human PC-3 prostate cancer cells in vitro. We isolated a subpopulation of cells, TEM4-18, that crossed an endothelial barrier more efficiently, but surprisingly, were less invasive than parental PC-3 cells in other contexts in vitro. Importantly, TEM4-18 cells were more aggressive than PC-3 cells in a murine metastatic colonization model. Microarray and FACS analysis of these cells showed that the expression of many genes previously associated with leukocyte trafficking and cancer cell extravasation were either unchanged or down-regulated. Instead, TEM4-18 cells exhibited characteristic molecular markers of an epithelial-to-mesenchymal transition (EMT), including frank loss of E-cadherin expression and up-regulation of the E-cadherin repressor ZEB1. Silencing ZEB1 in TEM4-18 cells resulted in increased E-cadherin and reduced transendothelial migration. TEM4-18 cells also express N-cadherin, which was found to be necessary, but not sufficient for increased transendothelial migration. Our results extend the role of EMT in metastasis to transendothelial migration and implicate ZEB1 and N-cadherin in this process in prostate cancer cells.

MicroRNAs (miRNAs) represent a class of small, non-coding RNAs that control gene expression by targeting mRNA and triggering either translational repression or RNA degradation. The objective of our study was to evaluate the involvement of miRNAs in human medullary thyroid carcinoma (MTC) and to identify the markers of metastatic cells and aggressive tumour behaviour. Using matched primary and metastatic tumour samples, we identified a subset of miRNAs aberrantly regulated in metastatic MTC. Deregulated miRNAs were confirmed by quantitative real-time PCR and validated by in situ hybridisation on a large independent set of primary and metastatic MTC samples. Our results uncovered ten miRNAs that were significantly expressed and deregulated in metastatic tumours: miR-10a, miR-200b/-200c, miR-7 and miR-29c were down-regulated and miR-130a, miR-138, miR-193a-3p, miR-373 and miR-498 were up-regulated. Bioinformatic approaches revealed potential miRNA targets and signals involved in metastatic MTC pathways. Migration, proliferation and invasion assays were performed in cell lines treated with miR-200 antagomirs to ascertain a direct role for this miRNA in MTC tumourigenesis. We show that the members of miR-200 family regulate the expression of E-cadherin by directly targeting ZEB1 and ZEB2 mRNA and through the enhanced expression of tumour growth factor beta (TGFbeta)-2 and TGFbeta-1. Overall, the treated cells shifted to a mesenchymal phenotype, thereby acquiring an aggressive phenotype with increased motility and invasion. Our data identify a robust miRNA signature associated with metastatic MTC and distinct biological processes, e.g., TGFbeta signalling pathway, providing new potential insights into the mechanisms of MTC metastasis.

The process by which epithelial features are lost in favor of a mesenchymal phenotype is referred to as epithelial-mesenchymal transition (EMT). Most carcinomas use this mechanism to evade into neighboring tissues. Reduction or a loss of E-cadherin expression is a well-established hallmark of EMT. As a potent suppressor of E-cadherin, transcription factor ZEB1 is one of the key inducers of EMT, whose expression promotes tumorigenesis and metastasis of carcinomas. Bcl-2-associated athanogene 3 (BAG3) affects multifaceted cellular functions, including proliferation, apoptosis, cell adhesion and invasion, viral infection, and autophagy. Recently, we have reported a novel role of BAG3 implicated in EMT, while the mechanisms are poorly elucidated. The current study demonstrated that knockdown of BAG3 induced EMT, and increased cell migratory and invasiveness in thyroid cancer cells via transcriptional activation of ZEB1. We also found that BAG3 knockdown led to nuclear accumulation of beta-catenin, which was responsible for the transcriptional activation of ZEB1. These results indicate BAG3 as a regulator of ZEB1 expression in EMT and as a regulator of metastasis in thyroid cancer cells, providing potential targets to prevent and/or treat thyroid cancer cell invasion and metastasis.