ZEB1 mRNA Research Articles

Correlation of BMI1 and ZEB1 expression with epithelial–mesenchymal transition in gingiva squamous cell carcinoma

ObjectiveOral squamous cell carcinoma is characterized by diverse histopathological features, tumorigenesis, and invasion. Gingiva squamous cell carcinoma (GSCC) is characterized by easily invading the jawbone. Epithelial–mesenchymal transition (EMT) is a crucial event for invasion of cancer cells and progression of carcinogenesis, inducing stem-like properties in epithelial cells. Here, expression of BMI1, which controls stem cell self-renewal, and ZEB1, a transcription factor that regulates EMT, was evaluated for its role in EMT and invasion of GSCC. Materials and methodsWe analyzed 36 gingival specimens (24 carcinomas and 12 dysplasias) by measuring protein and mRNA expression levels of BMI1, ZEB1, vimentin, and E-cadherin, and examining associations between their expression and clinicopathological parameters. ResultsCompared with adjacent normal squamous epithelium, E-cadherin expression was decreased significantly (P<0.05) and vimentin expression levels were increased significantly (P<0.05) at the invasive front, which are indicative of EMT in GSCC. Vimentin expression was significantly increased in poorly differentiated carcinoma (P=0.027), and vimentin and BMI1 expression was significantly increased in grade 4 mode of invasion (P<0.05). Furthermore, elevated levels of BMI1 were accompanied by downregulation of E-cadherin. A significant correlation was found between E-cadherin and BMI1 expression levels (P=0.014) and between BMI1 and ZEB1 mRNA expression (P=0.034). ConclusionThe present study shows that the expression levels of several biomarkers of gingiva carcinogenesis have unique characteristics that differ from those in tongue carcinogenesis. BMI1 and ZEB1 may be important factors associated with EMT in the invasion and progression of GSCC.

Differential Regulation of ZEB1 and EMT by MAPK-Interacting Protein Kinases (MNK) and eIF4E in Pancreatic Cancer.

Human pancreatic ductal adenocarcinoma (PDAC) tumors are associated with dysregulation of mRNA translation. In this report, it is demonstrated that PDAC cells grown in collagen exhibit increased activation of the MAPK-interacting protein kinases (MNK) that mediate eIF4E phosphorylation. Pharmacologic and genetic targeting of MNKs reverse epithelial-mesenchymal transition (EMT), decrease cell migration, and reduce protein expression of the EMT-regulator ZEB1 without affecting ZEB1 mRNA levels. Paradoxically, targeting eIF4E, the best-characterized effector of MNKs, increases ZEB1 mRNA expression through repression of ZEB1-targeting miRNAs, miR-200c and miR-141. In contrast, targeting the MNK effector hnRNPA1, which can function as a translational repressor, increases ZEB1 protein without increasing ZEB1 mRNA levels. Importantly, treatment with MNK inhibitors blocks growth of chemoresistant PDAC cells in collagen and decreases the number of aldehyde dehydrogenase activity-positive (Aldefluor+) cells. Significantly, MNK inhibitors increase E-cadherin mRNA levels and decrease vimentin mRNA levels in human PDAC organoids without affecting ZEB1 mRNA levels. Importantly, MNK inhibitors also decrease growth of human PDAC organoids. These results demonstrate differential regulation of ZEB1 and EMT by MNKs and eIF4E, and identify MNKs as potential targets in pancreatic cancer.

Abstract B03: HURP and ZEB1: Novel prognostic biomarkers in colorectal carcinomas

Abstract The current clinicopathologic diagnosis of colorectal cancer (CRC) is based on tumor-node-metastasis staging. This classification system has weaknesses due to the different genetic and epigenetic backgrounds not currently incorporated into CRC staging which can ultimately lead to treatment failure. Biological markers would improve early detection and guide clinicians in subsequent treatment decisions. Recently, we and others have identified “stem-like” molecules with the potential to identify patients at high risk of developing aggressive carcinomas. Herein, Hepatoma Up-Regulate Protein (HURP) and Zinc finger E-box binding homeobox 1 (ZEB1) are assessed as potential prognostic biomarkers in CRC. Tumors (N=21) obtained from colectomies and 5 µm formalin-fixed paraffin-embedded blocks were sectioned and immunostained for HURP and ZEB1. For certain cases (N=8), flash frozen tissues from tumor and adjacent normal colonic tissue were used to extract total RNA, synthesize cDNA, and perform RT-PCR. Presence of tumor cells in analyzed tissue sections was assessed by a pathologist. HURP mRNA expression was 4-fold higher (p=0.0005) in tumor tissue relative to adjacent normal tissue. Furthermore, an increase in HURP expression was evident in all tumor samples relative to their respective normal tissue control with an average 3-fold increase (p=0.001). Immunohistochemical analysis (IHC) of HURP revealed expression in all analyzed specimens. Although the stroma was weakly stained for HURP in some cases, all tumors were immunostained for this protein (average intensity score = ++, p&amp;lt;0.0001). These results suggest HURP’s potential as a tumor marker in CRC. In contrast to those results, ZEB1 mRNA expression in tumors did not differ from that observed in normal adjacent tissue; moreover, a tendency of mRNA to a decrease (n.s.) was observed. IHC for ZEB1 showed immunostaining in all analyzed cases. Weak staining was observed in both tumor and stroma (average intensity score = ++, n.s.). Further stratification of patients by race revealed that in Caucasian-American (CA) patients, staining of ZEB1 was less likely to be associated with disease progression or death after at least 2 years of follow-up. Interestingly, samples obtained from African-American (AA) individuals, known to have worse CRC outcomes, had an association between ZEB1 presence and poor survival regardless of the immunostaining intensity. These findings suggest that IHC localization of ZEB1 may represent a marker of a poor CRC prognosis, particularly in AA patients. However, one must be careful not to over-interpret these results as a Fischer’s Exact test revealed a non-significant p value = 0.387. Any conclusions about population-based distribution of poor prognosis with regards to ZEB1 positive CRC will require an adequately powered sample size. Overall, we have identified two stem cell-like molecules, HURP and ZEB1, with potential as markers of aggressiveness in CRC. Our findings also provide evidence of a possible biological basis for ethnicity-related differences with regards to stemness and regulatory factors of CRC aggressiveness. Citation Format: Logan Fair, Ingrid Espinoza, Xu Zhang, Abdelouahid Elkhattouti, Tangeng Ma, Joy King, Elizabeth Tarsi, Richard Whitlock, Vijay Kannuthurai, Ryan Jimenez, Tara Craft, Mary Graichen, Sharon Lobert, Roy Duhe, Charulochana Subramony, Christian R. Gomez, Christopher Lahr. HURP and ZEB1: Novel prognostic biomarkers in colorectal carcinomas. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B03.

Placental Growth Factor Promotes Ovarian Cancer Cell Invasion via ZEB2

Background/Aims: The aggressive manner of ovarian cancer (OVC) cells accounts for the majority of its lethality. Recently, we have shown that placental growth factor (PLGF) promotes metastases of OVC cells through miR-543-regulated MMP7. In the current study, we analyzed the effects of PLGF on another cell invasion associated protein, ZEB2, in OVC cells. Methods: The PLGF and ZEB2 levels in OVC tissues were compared to the paired adjacent non-tumor ovary tissue. We modified ZEB2 levels in OVC cells, and examined its effects on PLGF mRNA and protein levels by RT-qPCR and by Western blot, respectively. We also modified PLGF levels in OVC cells, and examined its effects on ZEB2 mRNA and protein levels by RT-qPCR and by Western blot, respectively. Then, we examined the cell invasiveness in PLGF-modified OVC cells in a transwell cell invasion assay. Finally, we used specific signal pathway inhibitors to treat PLGF-modified OVC cells and examined the effects on ZEB2 activation. Results: PLGF and ZEB2 levels were both significantly increased in OVC tissues, compared to the paired adjacent non-tumor ovary tissue. The PLGF and ZEB2 levels were strongly correlated. ZEB2 modification did not alter PLGF levels. Overexpression of PLGF in OVC cells significantly increased ZEB2 levels and cell invasiveness, while PLGF depletion in OVC cells significantly decreased ZEB2 levels and cell invasiveness. Application of a specific MAPK-p38 inhibitor, but not application of specific inhibitors for MAPK-p42/p44, PI3k/Akt, or JNK signaling pathways, to PLGF-overexpressing OVC cells substantially abolished the PLGF-induced ZEB2 activation. Conclusion: PLGF enhances OVC cell invasion through MAPK-p38-dependent activation of ZEB2.

Ginkgolide B Inhibits Human Bladder Cancer Cell Migration and Invasion Through MicroRNA-223-3p

Background/Aims: Ginkgolide B (GB) is currently used as an anticancer drug for treatment of some malignant cancers. However, whether it may have therapeutic effects on bladder cancer remains unknown. Here, we studied the effects of GB on bladder cancer cells. Methods: Bladder cells were treated with different doses of GB, and the effects on ZEB1 and microRNA-223-3p (miR-223-3p) were analyzed by RT-qPCR and/or Western blot. Prediction of a regulatory relationship between miR-93 and 3'-UTR of Beclin-1 mRNA was performed by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. Results: We found that GB dose-dependently decreased ZEB1 protein, but not mRNA, in bladder cancer cells, resulting in suppression of cell invasion. Moreover, in bladder cancer cells, GB dose-dependently decreased the levels of miR-223-3p, which suppressed the protein translation of ZEB1 through binding to 3'-UTR of ZEB1 mRNA. Overexpression of miR-223-3p decreased ZEB1 protein, while depletion of miR-223-3p increased ZEB1 protein in bladder cancer cells. Conclusion: GB inhibits bladder cancer cell invasiveness through suppressing ZEB1 protein translation via upregulating miR-223-3p.

The transcription factors ZEB2 and T-bet cooperate to program cytotoxic T cell terminal differentiation in response to LCMV viral infection.

The transcription factor T-bet is critical for cytotoxic T lymphocyte (CTL) differentiation, but it is unclear how it operates in a graded manner in the formation of both terminal effector and memory precursor cells during viral infection. We find that, at high concentrations, T-bet induced expression of Zeb2 mRNA, which then triggered CTLs to adopt terminally differentiated states. ZEB2 and T-bet cooperate to switch on a terminal CTL differentiation program, while simultaneously repressing genes necessary for central memory CTL development. Chromatin immunoprecipitation sequencing showed that a large proportion of these genes were bound by T-bet, and this binding was altered by ZEB2 deficiency. Furthermore, T-bet overexpression could not fully bypass ZEB2 function. Thus, the coordinated actions of T-bet and ZEB2 outline a novel genetic pathway that forces commitment of CTLs to terminal differentiation, thereby restricting their memory cell potential.

Role of miR-138 in the regulation of larynx carcinoma cell metastases.

The cases of larynx carcinoma (LC) with poor prognosis largely result from the distal metastases of the primary tumor. Since microRNAs (miRNAs) play critical roles during cancer metastases, determination of the involved miRNAs in the regulation of the LC metastases may provide novel therapeutic targets for LC treatment. Here, we studied the LC specimens from the patients and found that the levels of miR-138 were significantly decreased and the levels of ZEB2, a critical factor that regulates cancer cell invasiveness, were significantly increased in LC, compared to the paired normal larynx tissue. Metastatic LC appeared to contained lower levels of miR-138. Moreover, miR-138 and ZEB2 inversely correlated in LC specimens. Bioinformatics analyses showed that miR-138 targeted the 3'-untranslated region (3'-UTR) of ZEB2 mRNA to inhibit its translation, which was confirmed in a luciferase reporter assay. Further, miR-138 overexpression inhibited ZEB2-mediated cell invasiveness, while miR-138 depletion increased ZEB2-mediated cell invasiveness in LC cells. Together, our data suggest that miR-138 suppression in LC cells may promote ZEB2-mediated cancer metastases. Thus, miR-138 appears to be an intriguing therapeutic target to prevent metastases of LC.

MiR-429 regulates gastric cancer cell invasiveness through ZEB proteins.

MicroRNAs (miRNAs) play an essential role in the tumorigenesis of gastric carcinoma (GC). MiR-429 has been recently reported to inhibit GC growth; however, whether miR-429 may also regulate the invasion of GC cells is unknown. Here, we showed that miR-429 levels were significantly decreased and ZEB1 and ZEB2 levels were significantly increased in GC specimens, compared to the paired adjacent non-tumor gastric tissue. Moreover, the levels of miR-429 and ZEB1 or ZEB2 inversely correlated in GC specimens. Bioinformatics analyses showed that miR-429 targeted the 3'-untranslated region of both ZEB1 and ZEB2 mRNA to inhibit their translation, which was confirmed by luciferase reporter assay. Overexpression of miR-429 inhibited ZEB1/2-mediated cell invasiveness, while depletion of miR-429 augmented it. Together, our data suggest that miR-429 suppression in GC promotes ZEB1/2-mediated cancer cell invasion.

MiR-154 targeting ZEB2 in hepatocellular carcinoma functions as a potential tumor suppressor.

MicroRNA-154 (miR-154) has been identified as a tumor suppressor in several types of human cancers; however, its clinical significance and function in human hepatocellular carcinoma (HCC) remain unclear. The aim of the present study was to analyze the clinical significance and cellular function of miR-154 in HCC patients. The data showed that miR-154 expression was consistently lower in HCC tissues and cell lines compared to that in matched tumor-adjacent tissues and a normal hepatic cell line, and its expression was negatively correlated with tumor differentiation (P<0.01), TNM stage (P<0.01) and lymph node metastasis (P<0.01). Restoration of miR-154 expression in HepG2 cells inhibited cell proliferation, migration and invasion, and induced apoptosis and cell arrest at the G1 phase in vitro, as well as suppressed tumor growth in a nude mouse model. Using a luciferase assay, we identified that miR-154 was able to target the 3'-untranslated region (3'UTR) of ZEB2 mRNA. Then, we revealed that miR-154 was able to reduce ZEB2 expression at the levels of mRNA and protein using qRT-PCR and western blot analysis. Notably, restoration of expression of ZEB2 weakened miR-154-mediated suppression of tumor progression. In conclusion, these results indicate that miR-154 functions as a tumor suppressor in HCC by suppressing ZEB2, suggesting that miR-154 may serve as a potential target for HCC.

MicroRNA-33b inhibits lung adenocarcinoma cell growth, invasion, and epithelial-mesenchymal transition by suppressing Wnt/β-catenin/ZEB1 signaling.

Altered expression of microRNA (miRNA) is associated with lung carcinogenesis and metastasis. Our previous study of lung cancer miRNAs using the gene chip assay demonstrated altered miR-33b expression in lung adenocarcinoma. The present study further investigated miR-33b expression, function, and gene regulation in lung cancer cells in vitro and in nude mouse xenografts. Our data showed that the level of miR-33b expression was dramatically decreased in lung adenocarcinoma cell lines and tissues and that the reduced miR-33b expression was associated with tumor lymph node metastasis. Furthermore, restoration of miR-33b expression inhibited lung adenocarcinoma cell proliferation, migration, and invasion and tumor cell epithelial-mesenchymal transition (EMT) in vitro. Luciferase assay revealed that miR-33b bound to ZEB1 3'-UTR region and inhibited ZEB1 expression, while expression of ZEB1 mRNA and miR-33b was inversely associated with lung adenocarcinoma cell lines and tissues. Subsequently, we found that miR-33b suppressed the activity of WNT/β-catenin signaling in lung adenocarcinoma cells and in turn suppressed tumor cell growth and EMT in vitro and in vivo nude mouse xenografts. In conclusion, the present study provided novel insight into the molecular mechanism of lung adenocarcinoma progression. MicroRNA-33b should be further investigated as a potential therapeutic target in human lung adenocarcinoma.

MiR-205 promotes motility of ovarian cancer cells via targeting ZEB1

AimTo investigate the clinical significance of microRNA-205 (miR-205) and zinc finger E-box binding homeobox 1 (ZEB1) in epithelial ovarian cancer (EOC) and the underlying mechanisms by which they are involved into tumorigenesis. MethodsExpression levels of miR-205 and ZEB1 mRNA in EOC tissues were detected by quantitative real-time PCR. Their associations with clinicopathological features of EOC patients were statistically analyzed. Luciferase reporter assay was used to confirm target associations between miR-205 and ZEB1. After that, the functions of miR-205–ZEB1 axis on cell migration and invasion were further determined by transwell assay in vitro. ResultsExpression levels of miR-205 (P=0.0001) and ZEB1 mRNA (P<0.0001) in clinical EOC tissues were significantly higher and lower than those in normal tissues, respectively. Interestingly, there was a negative correlation between miR-205 and ZEB1 mRNA expression in EOC tissues (P=0.01). Additionally, miR-205-upregulation and/or ZEB1-downregulation were significantly associated with high pathological grade and advanced clinical stage of EOC patients (all P<0.05). Meantime, luciferase reporter assays identified ZEB1 as a direct target of miR-205 in EOC cells. Moreover, miR-205 blockage inhibited, whereas miR-205 mimics promoted the motility of EOC cells in vitro. Importantly, all the alterations of the above cellular phenotypes by blocking or enhancing of miR-205 could be alleviated by subsequent suppression or re-introduction of its target ZEB1, respectively. ConclusionMiR-205, acting as an oncogenic miRNA, may promote the clinical progression of EOC patients and enhance the cellular motility in vitro by directly and negatively regulating ZEB1, providing a potential therapeutic strategy for suppression of EOC metastasis.

MiR-200 suppresses metastases of colorectal cancer through ZEB1.

Poor prognosis of some colorectal cancer (CRC) cases largely results from early metastases of CRC to the distal organs. Thus, suppression of the invasion of CRC appears to be crucial therapy. Since microRNAs (miRNAs) play critical roles in the regulation of cancer metastases, identification of the involved miRNAs may provide novel therapeutic targets for CRC treatment. Here, we showed that the levels of miR-200 were significantly decreased and the levels of ZEB1 were significantly increased in the CRC specimens from patients, compared to the paired non-tumor tissue. Moreover, the levels of miR-200 and ZEB1 are inversely correlated. Bioinformatics analyses showed that miR-200 targeted the 3'-UTR of ZEB1 mRNA to inhibit its translation, which was confirmed by luciferase reporter assay. Moreover, miR-200 overexpression inhibited ZEB1-mediated cell invasiveness, while miR-200 depletion increased ZEB1-mediated cell invasiveness in CRC cells. Together, our data suggest that miR-200 suppression in CRC cells may promote ZEB1-mediated cancer metastasis. Our work thus highlights a novel molecular regulatory machinery that regulates metastases of CRC.

Abstract 4101: Clinical significance of ZEB1 mRNA levels in peritoneal washing for gastric cancer

Abstract Backgrounds. ZEB1 affects cancer progression by regulating Epithelial to mesenchymal transition (EMT) in some cancers. In gastric cancer, ZEB1 expression in cancer tissue has been identified as an independent prognostic factor. We also reported on a significant correlation between ZEB1 expression and diffuse phenotype in gastric cancer, and that patients with high ZEB1 expression had significantly poorer survival than those with low expression. It was also reported that ZEB1 was an independent predictor of peritoneal dissemination in gastric cancer patients, and was expressed in disseminated cancer cells in the peritoneum in the same pattern as that seen in primary lesions. Therefore, we hypothesized that ZEB1 mRNA levels in peritoneal washing (pZEB1) in conjunction with peritoneal washing cytology might predict intraperitoneal recurrence and prognosis. Objectives. The association between pZEB1 and clinicopathological parameters, and prognosis was investigated, and the possibility of using pZEB1 as a predictive marker was explored in this study. To our knowledge, this is the first study to examine the clinical implication of pZEB1 in gastric cancer. Methods. One hundred and seven consecutive patients with gastric cancer, who underwent surgical procedures that included the collection of peritoneal washing samples at the left subphrenic area at the beginning of surgery, were enrolled. RT-PCR was performed to quantify ZEB1 mRNA levels in peritoneal washing (pZEB1). The correlation between pZEB1 and clinicopathological variables and recurrence were statistically analyzed. Results. Patients were classified into the pZEB1High group (n = 27) or the pZEB1Low group (n = 80) based on their pZEB1 expression. pZEB1 was statistically correlated with pathological T category (P = 0.03) and vessel involvement (P = 0.03). 5-year disease-specific survival was 36.4% for the pZEB1High group and 64.7% for the pZEB1Low group (P = 0.02), and disease-free survival with the exception of StageIV disease was 46.9% for the pZEB1High group and 83.0% for the pZEB1Low group (P = 0.03). When subclassified into 4 categories based on washing cytology and pZEB1, the survival was worse in the pZEB1High group than in the pZEB1Low group (CY0 group: P = 0.01; CY1 group: P = 0.13). The recurrence rate for the pZEB1High group (8/27) was significantly higher than that of the pZEB1Low group (10/80; P = 0.03). Of these 18 patients, 6 developed to lymph node metastases, 6 peritoneal metastases, 5 liver metastases, and 1 lung metastasis. However, our results failed to show the statistical correlation between pZEB1 and peritoneal dissemination and peritoneal recurrence. Conclusions. pZEB1 could be a predictive marker for poor prognosis or tumor aggressiveness in gastric cancer in the same way as ZEB1 expression does so in primary lesions. It could add valuable information to conventional peritoneal washing cytology, and thus help select candidates for more powerful chemotherapies. Citation Format: Norimitsu Yabusaki, Suguru Yamada, Toshifumi Murai, Mitsuro Kanda, Daisuke Kobayashi, Chie Tanaka, Tsutomu Fujii, Goro Nakayama, Hiroyuki Sugimoto, Masahiko Koike, Shuji Nomoto, Michitaka Fujiwara, Yasuhiro Kodera. Clinical significance of ZEB1 mRNA levels in peritoneal washing for gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4101. doi:10.1158/1538-7445.AM2015-4101

Loss of MicroRNA‐101 Promotes Epithelial to Mesenchymal Transition in Hepatocytes

Epithelial-to-mesenchymal transition (EMT) has been implicated in embryonic development and various pathological events. However, the involvement of microRNA in the process of EMT remains to be fully defined in hepatocyte. ZEB1 is a well-known transcriptional repressor of E-cadherin and plays a major role in triggering EMT during organ fibrosis and cancer cell metastasis. Computational microRNA target predictions detect a conserved sequence matching to miR-101 in the 3'UTR of ZEB1 mRNA. Our results confirm that miR-101 suppresses ZEB1 expression by targeting the predicted site of ZEB1 3'UTR. Subsequent investigations show that miR-101 is significantly downregulated in the cultured hepatocytes undergoing EMT and in the hepatocytes isolated from fibrotic liver. Along with the loss of miR-101, the ZEB1 expression increases simultaneously in hepatocytes. In addition, miR-101 levels in HCC cell lines are negatively associated with the ZEB1 productions and the metastatic potentials of tumor cells. Mechanistically, we demonstrate that miR-101 significantly inhibits the TGF-β1-induced EMT in hepatocytes, whereas inhibition of miR-101 promotes the EMT process as indicated by the changes of morphology, cell migration, and the expression profiles of EMT markers. In the fibrotic liver, ectopic expression of miR-101 can significantly downregulate ZEB1 in the hepatocyte and thereby reduces the mesenchymal marker expression. Moreover, miR-101 significantly inhibits the proliferation and migration of HCC cell. Our results demonstrate that miR-101 regulates HCC cell phenotype by upregulating the epithelial marker genes and suppressing the mesenchymal ones.

Role and prognostic significance of the epithelial-mesenchymal transition factor ZEB2 in ovarian cancer.

ZEB2 is a key factor in epithelial-mesenchymal transition (EMT), a program controlling cell migration in embryonic development and adult tissue homeostasis. We demonstrated a role of ZEB2 in migration and anchorage-independent cell growth in ovarian cancer, as shown by ZEB2 silencing. We found that the RNA-binding protein HuR bound the 3'UTR of ZEB2 mRNA, acting as a positive regulator of ZEB2 protein expression. In Hey ovarian cell line, HuR silencing decreased ZEB2 and ZEB1 nuclear expression and impaired migration. In hypoglycemic conditions ZEB2 expression decreased, along with ZEB1, vimentin and cytoplasmic HuR, and a reduced cellular migration ability was observed. Analysis of ZEB2 and HuR expression in ovarian cancers revealed that nuclear ZEB2 is localized in tumor leading edge and co-localizes with cytoplasmic HuR. In a series of 143 ovarian cancer patients high expression of ZEB2 mRNA significantly correlated with a poor prognosis in term of both overall survival and progression- free survival. Moreover, at immunohistochemical evaluation, we found that prognostic significance of ZEB2 protein relies on its nuclear expression and co-localization with cytoplasmic HuR. In conclusion our findings indicated that nuclear ZEB2 may enhance progression of EMT transition and acquisition of an aggressive phenotype in ovarian cancer.

MicroRNA-138 Regulates Metastatic Potential of Bladder Cancer Through ZEB2

Background/Aims: The cases of bladder cancer (BC) with poor prognosis largely result from the distal metastases of the primary tumor. Since microRNAs (miRNAs) play critical roles during cancer metastases, determination of the involved miRNAs in the regulation of the metastases of BC may provide novel therapeutic targets for BC treatment. Here, we aimed to study the role of miR-138 in regulation of BC cell invasion and metastases. Methods: We analyzed the levels of miR-138 and ZEB2, a key factor that regulates cancer cell invasion, in the BC specimens from the patients. We also studied the correlation between miR-138 and ZEB2. We performed bioinformatics analyses on the binding of miR-138 to the 3'-UTR of ZEB2 mRNA, and verified the biological effects of this binding through promoter luciferase reporter assay. The effects of miR-138-modification on BC cell invasion were evaluated in a transwell cell invasion assay and a scratch would healing assay. Results: We found that the levels of miR-138 were significantly decreased and the levels of ZEB2 were significantly increased in BC specimens, compared to the paired normal bladder tissue. Metastatic BC appeared to contained lower levels of miR-138. Moreover, miR-138 and ZEB2 inversely correlated in BC specimens. Bioinformatics analyses showed that miR-138 targeted the 3'-UTR of ZEB2 mRNA to inhibit its translation. Furthermore, miR-138 overexpression inhibited ZEB2-mediated cell invasion and metastases, while miR-138 depletion increased ZEB2-mediated cell invasion and metastases in BC cells. Conclusion: Suppression of miR-138 in BC cells may promote ZEB2-mediated cancer invasion and metastases. Thus, miR-138 appears to be an intriguing therapeutic target to prevent metastases of BC.

Coordinative modulation of human zinc transporter 2 gene expression through active and suppressive regulators

Zinc transporter 2 (ZnT2) is one of the cellular factors responsible for Zn homeostasis. Upon Zn overload, ZnT2 reduces cellular Zn by transporting it into excretory vesicles. We investigated the molecular mechanism that regulates human ZnT2 (hZnT2) gene expression. Zn induces hZnT2 expression in dose- and time-dependent manners. Overexpression of metal-responsive transcription factor 1 (MTF-1) increases hZnT2 transcription, whereas depletion of MTF-1 reduces hZnT2 expression. There are five putative metal response elements (MREs) within 1kb upstream of the hZnT2 gene. A serial deletion of the hZnT2 promoter region (from 5′ to 3′) shows that the two MREs proximal to the gene are essential for Zn-induced promoter activity. Further mutation analysis concludes that the penultimate MRE (MREb) supports the metal-induced promoter activity. The hZnT2 promoter has also a zinc finger E-box binding homeobox (ZEB) binding element. Mutation or deletion of this ZEB binding element elevates the basal and Zn-induced hZnT2 promoter activities. Knockdown of ZEB1 mRNA enhances the hZnT2 transcript level in HEK-293 cells. In MCF-7 (ZEB-deficient) cells, expression of ZEB proteins attenuates the Zn-induced hZnT2 expression. However, expressions of MTF-1 target genes such as human ZnT1 and metallothionein IIA were not affected. Our study shows the expression of the hZnT2 gene is coordinately regulated via active and suppressive modulators.

The glycosyltransferase LARGE2 is repressed by Snail and ZEB1 in prostate cancer

Reductions in both expression of the dystroglycan core protein and functional glycosylation of the α-dystroglycan (αDG) subunit have been reported in a number of cancers and may contribute to disease progression. In the case of prostate cancer, one mechanism that contributes to αDG hypoglycosylation is transcriptional down-regulation of LARGE2 (GYLTY1B), a glycosyltransferase that produces the functional (laminin-binding) glycan on αDG, but the mechanism(s) underlying reduction of LARGE2 mRNA remain unclear. Here, we show that αDG hypoglycosylation is associated with epithelial-to-mesenchymal transition (EMT)-like status. We examined immunoreactivity for both functionally-glycosylated αDG and E-cadherin by flow cytometry and the relative expression of ZEB1 mRNA and the αDG glycosyltransferase LARGE2 mRNA in prostate and other cancer cell lines by quantitative RT-PCR. To study the role of ZEB1 and other transcription factors in the regulation of LARGE2, we employed overexpression and knockdown approaches. Snail- or ZEB1-driven EMT caused αDG hypoglycosylation by repressing expression of the LARGE2 mRNA, with both ZEB1-dependent and -independent mechanisms contributing to Snail-mediated LARGE2 repression. To examine the direct regulation of LARGE2 by Snail and ZEB1 we employed luciferase reporter and chromatin immunoprecipitation assays. Snail and ZEB1 were found to bind directly to the LARGE2 promoter, specifically to E/Z-box clusters. Furthermore, analysis of gene expression profiles of clinical samples in The Cancer Genome Atlas reveals negative correlation of LARGE2 and ZEB1 expression in various cancers. Collectively, our results suggest that LARGE2 is negatively regulated by Snail and/or ZEB1, revealing a mechanistic basis for αDG hypoglycosylation during prostate cancer progression and metastasis.

154 Drug response database with PDX tumor models in biomarker-driven multi-drug multi-arm clinical trial settings

also been analyzed. All samples used for histopathological examination were fixed in neutral-buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin-eosin. The immunohistochemical study was performed on paraffin-embedded sections using the avidin–biotin peroxidase method. To evaluate EGFR gene status, dual-color FISH analysis was performed on paraffin tissue arrays from 30 samples. Epigenetic analysis study of miRNAs and mRNA in 30 samples was performed using miRNA and mRNA Genechip Array (Affymetrix, Santa Clara, CA, USA). Expression levels of the selected miRNA (mir-200c) and mRNAs (CDH1, EGFR and ZEB1) were quantified using real-time reverse transcription-PCR (RT-PCR) analysis. MirRNA-200c showed a very significant difference between tumors having or not EGFR amplification. With respect to EGFR status our cases were categorized into three groups: high level EGFR amplification, low level EGFR amplification, and no EGFR amplification. Our results showed that microRNA-200c and E-cadherin expression are down-regulated, while ZEB1 is up-regulated, when tumors showed a high level of EGFR amplification. Conversely, ZEB1 mRNA expression levels were significantly lower in the group of tumors without EGFR amplification. Tumors with a low level of EGFR amplification showed ZEB1 expression levels comparable to those detected in the group with a high level of amplification. In this study we provide what is to our knowledge the first report of association between mirRNA-200c and EGFR amplification in glioblastomas. The mirRNA-200c plays an important role in epithelial–mesenchymal transition, but its implication in the behavior of glioblastoma is largely unknown, and we suggest that microRNA-200c may act as a potential regulator of glioblastoma migration and invasion by targeting ZEB1 mRNA.

MicroRNA-141 and MicroRNA-146b-5p Inhibit the Prometastatic Mesenchymal Characteristics through the RNA-binding Protein AUF1 Targeting the Transcription Factor ZEB1 and the Protein Kinase AKT

miR-141 and miR-146b-5p are two important tumor suppressor microRNAs, which control several cancer-related genes and processes. In the present report, we have shown that these microRNAs bind specific sites at the 3'-untranslated region (UTR) of the mRNA-binding protein AUF1, leading to its down-regulation. This inverse correlation between the levels of these microRNAs and AUF1 has been identified in various osteosarcoma cell lines. Additionally, we present clear evidence that AUF1 promotes mesenchymal features in osteosarcoma cells and that miR-141 and miR-146b-5p suppress this prometastatic process through AUF1 repression. Indeed, both microRNAs suppressed the invasion/migration and proliferation abilities of osteosarcoma cells through inhibiting the AKT protein kinase in an AUF1-dependent manner. We have also shown that AUF1 binds to and stabilizes the mRNA of the AKT activator phosphoinositide-dependent kinase-1 (PDK1). Furthermore, miR-141 and miR-146b-5p positively regulate the epithelial markers (E-cadherin and Epcam) and repress the mesenchymal markers (N-cadherin, Vimentin, Twist2, and ZEB1). These effects were mediated via the repression of the epithelial-to-mesenchymal inducer ZEB1 through targeting AUF1, which binds the 3'-UTR of the ZEB1 mRNA and reduces its turnover. These results indicate that at least some tumor suppressor functions of miR-141 and miR-146b-5p are mediated through the repression of the oncogenic potentials of AUF1. Therefore, these 3'-UTR-directed post-transcriptional gene expression regulators constitute promising new targets for diagnostic and/or therapeutic interventions.