ZEB1 Protein Research Articles

ZEB1 is Estrogen Responsive In Vitro in Human Foreskin Cells and is Over Expressed in Penile Skin in Patients With Severe Hypospadias

We determined the effect of estrogen on ZEB1 in vitro and tested the hypothesis that ZEB1 is over expressed in the penile skin of subjects with hypospadias. Hs68 cells, a fibroblast cell line derived from human foreskin, were exposed to 0, 1, 10 and 100 nM estrogen, and the expression level of ZEB1 was assessed using reverse transcription real-time polymerase chain reaction, Western blot and immunocytochemical analysis. Next, preputial skin was prospectively collected from case and control subjects at hypospadias repair (37 cases) and circumcision (11). Hypospadias was classified as severe (13 cases) or mild (24) based on the position of the urethral meatus. ZEB1 expression was quantified using reverse transcription real-time polymerase chain reaction, Western blot and immunohistochemical analysis. Estrogen increased ZEB1 expression at the mRNA and protein levels in Hs68 cells in a concentration dependent fashion (p <0.01). Subjects with severe hypospadias had significantly higher ZEB1 mRNA levels and protein expression compared to controls or subjects with mild hypospadias (both p <0.01). Subjects with severe hypospadias had increased expression of ZEB1 in the basal layers of the preputial epidermis. Estrogen increases ZEB1 expression in a human foreskin fibroblast cell line in vitro. Furthermore, ZEB1 is significantly over expressed in the penile skin of subjects with severe hypospadias. We propose that ZEB1 overexpression may contribute to development of hypospadias and may mediate the effect of estrogen on developing external male genitalia.

249 ZEB1 IS ESTROGEN RESPONSIVE IN VITRO IN HUMAN FORESKIN CELLS AND IS OVEREXPRESSED IN PENILE SKIN IN PATIENTS WITH SEVERE HYPOSPADIAS

You have accessJournal of UrologyPediatrics: Basic Research1 Apr 2011249 ZEB1 IS ESTROGEN RESPONSIVE IN VITRO IN HUMAN FORESKIN CELLS AND IS OVEREXPRESSED IN PENILE SKIN IN PATIENTS WITH SEVERE HYPOSPADIAS Liang Qiao, Gregory Tasian, Zhang Haiyang, Gerald Cunha, and Laurence Baskin Liang QiaoLiang Qiao San Francisco, CA More articles by this author , Gregory TasianGregory Tasian San Francisco, CA More articles by this author , Zhang HaiyangZhang Haiyang San Francisco, CA More articles by this author , Gerald CunhaGerald Cunha San Francisco, CA More articles by this author , and Laurence BaskinLaurence Baskin San Francisco, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.340AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES ZEB1, which acts as a transcriptional repressor and down-regulates cellular adhesion molecules, is one of many genes upregulated in patients with isolated hypospadias. This study determined the effect of estrogen on ZEB1 expression in vitro and tested the hypothesis that ZEB1 expression in penile skin is overexpressed in subjects with hypospadias. METHODS Hs68 cells, a fibroblast cell line derived from human foreskin, were exposed to 0, 1, 10, and 100nm of estrogen, after which the expression level of ZEB1 was assessed using reverse transcription real-time PCR, Western blot, and immunocytochemistry. Secondly, preputial skin was prospectively collected from case and control human subjects at the time of hypospadias repair (n = 37) and circumcision (n = 11), respectively. Hypospadias was classified as severe (n = 13) or mild (n = 24) based on position of the urethral meatus. ZEB1 expression was quantified using reverse transcription real-time PCR, Western blot and immunohistochemistry. RESULTS Estrogen increased ZEB1 expression at the mRNA and protein level in Hs68 cells in a concentration-dependent fashion (p < 0.01). Subjects with severe hypospadias had significantly higher ZEB1 mRNA levels and protein expression compared to control subjects and subjects with mild hypospadias (all p < 0.01). Subjects with severe hypospadias have increased expression of ZEB1 in the basal layers of the preputial epidermis. CONCLUSIONS Estrogen increases ZEB1 expression in a human foreskin fibroblast cell line in vitro. Furthermore, zeb1 is significantly over-expressed in the penile skin of subjects with severe hypospadias. We propose that ZEB1 overexpression may contribute to the development of hypospadias and may mediate the effect of estrogen on developing external male genitalia. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e100-e101 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Liang Qiao San Francisco, CA More articles by this author Gregory Tasian San Francisco, CA More articles by this author Zhang Haiyang San Francisco, CA More articles by this author Gerald Cunha San Francisco, CA More articles by this author Laurence Baskin San Francisco, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Nuclear Snail1 and nuclear ZEB1 protein expression in invasive and intraductal human breast carcinomas

Snail1 and ZEB1 are transcriptional repressors that drive tumor initiation and metastasis in animal models. Snail1 and ZEB1 are frequently coexpressed in tumor cell lines, suggesting that these factors may cooperate to promote tumor progression. However, coexpression of these transcriptional repressors in primary human cancer specimens has not been investigated. Previous studies assessed expression in primary breast cancers of Snail1 messenger RNA, which does not reflect Snail1 activity because Snail1 is subject to posttranslational modifications that inhibit its nuclear localization/activity. In the current study, using breast tumor cell lines of known Snail1 and ZEB1 expression status, we developed immunohistochemistry protocols for detecting nuclear Snail1 and nuclear ZEB1 proteins. Using these protocols, we assessed nuclear Snail1 and nuclear ZEB1 expressions in primary human breast cancers of varying subtypes (n = 78). Nuclear Snail1 and estrogen receptor α expressions were inversely associated in primary breast cancers, and nuclear Snail1 was expressed in approximately 80% of triple-negative breast cancers (lacking estrogen receptor α, progesterone receptor, and human epidermal growth factor receptor 2 overexpression). In contrast, nuclear ZEB1 was expressed at a significantly lower frequency in these breast cancers. Notably, nuclear Snail1 protein was detected in 45% of ductal carcinoma in situ specimens (n = 29), raising the important possibility that nuclear Snail1 expression in early stage breast lesions may predict future development of invasive breast cancer. Collectively, our studies demonstrate frequent expression of nuclear Snail1, but not nuclear ZEB1, in invasive, triple-negative breast cancers as well as in intraductal carcinomas.

Abstract P3-04-03: Combined Epigenetic Targeting Reverses Epithelial-Mesencymal Transition (EMT) and Reduces Invasiveness of Human Breast Cancer Cells

Abstract Background: Epithelial-Mesenchymal-transition (EMT) is a highly conserved biologic process that is involved in organ development, tissue regeneration, tumor invasion and metastasis. EMT is phenotypically associated with the loss E-cadherin and a gain of mesenchymal markers (e.g., vimentin). Both promoter methylation by DNMT1 and histone H3- K27 trimethylation by the histone methyl transferase EZH2 have been shown to cause E-cadherin expression. Several transcription factors, including ZEB1, Snail, Slug and Twist, directly bind to E-cadherin promoter, recruit histone deacetylase (HDAC) activity and repress E-cadherin. We have previously shown that the pan-HDAC inhibitor panobinostat (PS, Novartis Pharmaceuticals) depletes EZH2 and DNMT1 levels, and co-treatment with PS and DNMT1 inhibitor decitabine (DAC) synergistically depletes EZH2 and DNMT1. Methods: Here, we determined the effects of PS (20 to 100 nM) and/or DAC (0.5 to 5.0 uM) on E-cadherin levels and other EMT-associated markers, as well as on invasiveness of human breast cancer MB-231 and SUM159PT cells, which have undetectable levels of E-cadherin. Results: Treatment with PS dose dependently inhibited the migration and invasion of the breast cancer cells (by Boyden chamber assay). This was associated with induction of mRNA and protein levels of E-cadherin and decline in vimentin levels. PS treatment inhibited HDAC activity and reduced DNMT1 and EZH2 levels. Additionally PS repressed ZEB1 mRNA and protein levels. Co-treatment with bortezomib partially restored ZEB1 levels, indicating that PS treatment promotes proteasomal degradation of ZEB1. Chromatin immunoprecipitation (ChIP) analyses demonstrated that PS treatment inhibited H3K27 chromatin repressive and increased H3K4 chromatin permissive mark associated with the E-cadherin promoter. PS treatment also diminished ZEB1 occupancy on the E-cadherin promoter. Pre-treatment of MB-231 and SUM 159PT cells with DAC or 5-azacytidine (AC, also a DNMT1 inhibitor) (0.5 to 5.0 uM) further enhanced PS-mediated induction of E-cadherin, which was associated with further decline in ZEB1 levels. Additionally, co-treatment with PS and DAC caused greater inhibition of in vitro invasion by the breast cancer cells. Discussion: Collectively, these findings indicate that combined epigenetic targeting with PS and DAC depletes ZEB1, induces E-cadherin and reverses EMT, thereby, attenuating invasiveness of human breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-03.

MiR-200a Regulates Epithelial-Mesenchymal to Stem-like Transition via ZEB2 and β-Catenin Signaling

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133(+) side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.

Repression of E-cadherin by SNAIL, ZEB1, and TWIST in invasive ductal carcinomas of the breast: a cooperative effort?

It has been suggested that down-regulation of E-cadherin in invasive breast ductal carcinomas is mediated by the aberrant expression of several of its transcriptional repressors, but their inhibitory role and clinical importance are not yet well established. We investigated gene and protein expression patterns of the E-cadherin repressors SNAIL, ZEB1, and TWIST in relation to clinicopathologic parameters, in a series of 88 patients with invasive breast ductal carcinomas. Up-regulation of SNAIL messenger RNA (P = .008) and down-regulation of TWIST (P = .022) were associated with triple-negative tumors, whereas ZEB1 gene expression was more frequent in hormone-positive tumors (P = .004). Loss of E-cadherin was found in 19% of the tumors, but it did not correlate with aberrant expression of any of the repressors investigated herein. Nonetheless, we found that ZEB-1 protein overexpression inversely correlated with high tumor grade (P = .018), nuclear grade (P = .002), and presence of lymph nodes (P = .001), and these data were consistent with the gene expression data for ZEB1. Clinically, down-regulation of ZEB1 messenger RNA was associated with poor overall survival (P = .011) and disease-free survival (P = .053), whereas patients with TWIST negative tumors had a worse overall survival (P = .008) and disease-free survival (P = .006). Our data indicate that deregulation of TWIST is somehow important in the aggressiveness of triple-negative carcinomas and poor patient outcome, whereas down-regulation of ZEB1 seems to play a role in tumor spread, metastases, and poor survival.

SPROUTY-2 and E-cadherin regulate reciprocally and dictate colon cancer cell tumourigenicity

SPROUTY-2 (SPRY2) regulates receptor tyrosine kinase signalling and therefore cell growth and differentiation. In this study, we show that SPRY2 expression in colon cancer cells is inhibited by the active vitamin D metabolite 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) through E-cadherin-dependent and -independent mechanisms. In turn, SPRY2 represses both basal and 1,25(OH)(2)D(3)-induced E-cadherin expression. In line with this, SPRY2 induces ZEB1 RNA and protein, but not that of other epithelial-to-mesenchymal transition inducers that repress the CDH1/E-cadherin promoter. Consistently, SPRY2 and E-cadherin protein levels inversely correlate in colon cancer cell lines and xenografted tumours. Moreover, SPRY2 knockdown by small hairpin RNA increases CDH1/E-cadherin expression and, reciprocally, CDH1/E-cadherin knockdown increases that of SPRY2. In colon cancer patients, SPRY2 is upregulated in undifferentiated high-grade tumours and at the invasive front of low-grade carcinomas. Quantification of protein expression in 34 tumours confirmed an inverse correlation between SPRY2 and E-cadherin. Our data demonstrate a tumourigenic action of SPRY2 that is based on the repression of E-cadherin, probably by the induction of ZEB1, and a reciprocal regulation of SPRY2 and E-cadherin that dictates cell phenotype. We propose SPRY2 as a candidate novel marker for high-grade tumours and a target of therapeutic intervention in colon cancer.

Mir-200c Regulates Induction of Apoptosis through CD95 by Targeting FAP-1

Tumor progression shares many characteristics with the process of epithelial-to-mesenchymal transition (EMT). Cells that have undergone an EMT are known to have an increased resistance to apoptosis. CD95/Fas is an apoptosis-inducing receptor expressed on many tissues and tumor cells. During tumor progression CD95 is frequently downregulated, and tumor cells lose apoptosis sensitivity. miR-200 microRNAs repress both the EMT-inducing ZEB1 and ZEB2 transcription factors. We now demonstrate that miR-200c sensitizes cells to apoptosis mediated by CD95. We have identified the apoptosis inhibitor FAP-1 as a target for miR-200c. FAP-1 was demonstrated to be responsible for the reduced sensitivity to CD95-mediated apoptosis in cells with inhibited miR-200. The identification of FAP-1 as an miR-200c target provides a molecular mechanism to explain both the downregulation of CD95 expression and the reduction in sensitivity of cells to CD95-mediated apoptosis that is observed in the context of reduced miR-200 expression during tumor progression.

Abstract 3172: ZEB1 regulation in breast epithelial cells: Role of IGF-1 in the CCN6 modulation pathway

Abstract Background: During cancer progression, the transcription factor ZEB1 has been implicated in the regulation of key genes triggering the EMT (Epithelial to Mesenchymal Transition) program. We previously reported that the matricellular protein CCN6 is lost or reduced in highly aggressive breast carcinomas. Furthermore, we have identified CCN6 as a tumor suppressor in breast cancer whose down-regulation enhances IGF-1 signaling on breast epithelial cells. Recently, our lab observed that stable inhibition of CCN6 in benign mammary epithelial cells causes EMT and invasion with marked up-regulation of ZEB1. Since IGF-1 is shown to up-regulate ZEB1 expression in epithelial prostate cancer cells we hypothesize that CCN6 may regulate EMT in breast cancers by modulating IGF-1 signaling and thereby regulating ZEB1 expression. Methods: Stable CCN6-deficient HME (Human Mammary Epithelial cells)(HME-CCN6 KD) were incubated with 500 ng/mL rhCCN6 protein. Western blots (WB) for ZEB1 and vimentin, inmunofluorescence, invasion and proliferation assays were performed. ZEB1 mRNA levels were evaluated by RT-qPCR (Taqman). IGF-1 expression was analyzed by RT-qPCR and WB on MDA-MB-231 and HME-CCN6 KD (pre- and post-rhCCN6 treatment) while IGF-1R, phospho-IGF-1R, IRS-1 and phospho-IRS-1 expression were investigated by WB. Lentivirus-mediated (pLK.O1) short hairpin RNA targeting IGF1-R was performed in HME control and HME-CCN6 KD cells. WBs were done to assess expression of proteins of interest. mRNA content was evaluated by RT-qPCR. Results: CCN6 incubation of HME-CCN6 KD cells induced down-regulation of ZEB1 mRNA and protein levels as well as subcellular localization changes of the mesenchymal marker vimentin (“cap” pattern) evidenced as a decrease in vimentin expression by WB. hrCCN6 protein treatment also reduced proliferation and invasion abilities of HME-CCN6 KD cells compared to controls. In Addition, cell lines expressing low levels of CCN6 such us HME-CCN6 KD and MDA-MB-231 secreted IGF-1 in the conditioned media and showed activation of the IGF-1 signaling pathway. This phenomenon was reverted by CCN6 treatment, with reduction of both, IGF-1 protein and mRNA levels. IGF-1R down-regulation in HME-CCN6 KD cells decreased ZEB1 expression and induced the re-expression of cytokeratin-18 (epithelial marker). Conclusions: Our results suggest that CCN6 regulates ZEB1 expression and EMT in breast epithelial cells by modulating the IGF-1 signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3172.

Abstract 2362: Defining the role of ZEB1 in the molecular pathogenesis of lung cancer

Abstract Background: Epithelial-to-mesenchymal transition (EMT) is an important biologic process in both early development and cancer whereby an epithelial cell transforms to a mesenchymal phenotype resulting in enhanced migration, invasiveness, and resistance to apoptosis. The transcription factor ZEB1 promotes EMT by repressing E-cadherin, and has been shown to promote tumor metastasis and increase resistance to standard chemotherapies in several human cancers. Its role in the tumorigenic progression of lung cancer however is less understood. Methods and Results: We analyzed genome-wide mRNA expression profiles across a cohort of 77 non small cell lung cancer (NSCLC) lines representing all common NSCLC histologies and oncogenotypes using Affymetrix GeneChip U133 Plus 2.0 arrays. Stratification of NSCLC lines by high/low ZEB1 expression identified 640 genes with significantly (p&amp;lt;0.001) different expression between the two groups. E-cadherin (CDH1) was significantly higher in cell lines with low ZEB1 expression, as was family member CDH3 and ERBB3. Mesenchymal markers vimentin, N-cadherin, (CDH2) as well as neuroendocrine markers (RTN1, NCAM1 and NEUROD1) were significantly up-regulated in cell lines with high ZEB1 expression. E-cadherin (r=-0.75) and Claudin 7 (r=-0.74) demonstrated the most significant indirect correlation with ZEB1 expression. To investigate the ability of ZEB1 to transform ‘normal’ epithelial cells we analyzed ZEB1 expression in an isogenic series of human bronchial epithelial cells (HBECs) - immortalized without viral oncoproteins - genetically manipulated to have single and multiple oncogenic changes common in lung cancer such as those for KRAS, p53, EGFR, and c-MYC. ZEB1 mRNA and protein expression was significantly increased in HBECs with combined oncogenic KRASV12 and p53 knockdown that had also been clonally selected for growth in soft agar. These transformed HBEC(KRASP53), which are tumorigenic in NOD/SCID mice in vivo, exhibit a decrease in epithelial markers and an increase in mesenchymal markers when compared to parental cells, indicating the cells have undergone EMT. To determine whether the observed increase in ZEB1 expression was causal to tumorigenicity or a bystander effect, ZEB1 expression was knocked down in HBEC(KRASP53) with RNA interference resulting in a significant decrease in colony formation, invasiveness and cell motility (p&amp;lt;0.005). Conclusion: These findings indicate that in NSCLC, ZEB1 expression is highly variable, is associated with an mRNA gene expression signature and is inversely correlated with E-cadherin expression, a known negative target of ZEB1. analyzing an isogenic series of progressively transformed HBECs we found ZEB1 increased with tumorigenic progression and an invasive phenotype. This phenotype could in turn be reversed by knockdown of ZEB1 suggesting ZEB1 plays an important role in tumorigenic transformation of human bronchial epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2362.

Abstract 2295: Knockdown of ZEB1, a master EMT gene, suppresses anchorage-independent cell growth of lung cancer cells

Abstract Background: Epithelial-to-mesenchymal transition (EMT), an essential process during embryonic development and in wound healing, has been shown to be a key event in tumor migration, invasion and metastasis. EMT program is controlled by several master regulators including Twist, ZEB1, SIP1, Snail, Slug, and Goosecoid which are called master EMT genes1),2). ZEB1 protein binds E-boxes within the promoter region of E-cadherin gene, leading to its transcriptional repression. Also, it directly represses many other genes encoding components of the epithelial junctional complex and cell polarity factors. ZEB1 was shown to promote metastasis in colorectal cancer and breast cancer3), and the association between ZEB1 and tumor progression has been studied in several human cancers. Purpose: To evaluate the association between ZEB1 expression and mesenchymal phenotype in lung cancer, and to test the effect of ZEB1 knockdown with RNA interference on the growth of lung cancer cells. Methods: We analyzed the expression of E-cadherin (epithelial marker), Vimentin (mesenchymal marker), and four master EMT genes (Snail, Slug, ZEB1, SIP1) in 19 NSCLC cell lines and 32 NSCLC tumor tissues. Transient knockdown of ZEB1 with RNA interference was done in three NSCLC cell lines with high expression of ZEB1: H157, H1299, and H460. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin and Vimentin were done. Cellular proliferation was measured by WST-1 assay and clonogenic growth was measured by liquid (anchorage-dependent) and soft agar (anchorage-independent) colony formation assays. Apoptosis was evaluated by FACS and western blot of cleaved caspase-3. Results: In cell lines, of four master EMT genes, only ZEB1 expression significantly correlated with both E-cadherin and Vimentin expression. Most EGFR mutant lines showed epithelial phenotype (ratio of Vimentin to E-cadherin (RVE) &amp;lt; 1.0). ZEB1 expression was also inversely correlated with miR-200c and miR-205. In tumor tissues, ZEB1 and Snail expression correlated with the ratio of Vimentin to E-cadherin, and ZEB1 expression highly significantly correlated with Vimentin expression. After ZEB1 knockdown E-cadherin protein was reexpressed in H460 but not in H1299 and H157 cells. ZEB1 knockdown suppressed cell proliferation and liquid colony formation significantly in H1299 and modestly in H157 and H460. Notably, ZEB1 knockdown dramatically suppressed growth of soft agar in the all three cell lines studied. FACS and western blot of cleaved caspase-3 showed apoptosis induction by ZEB1 knockdown in H460, suggesting that growth inhibitory effect of ZEB1 knockdown in lung cancer was caused in part by apoptosis. Conclusions: ZEB1 has the dominant role in maintaining mesencymal phenotype in NSCLC and removal of ZEB1 in lung cancer cell lines induces inhibition of soft agar growth. These results suggest that ZEB1 could be a therapeutic target for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2295.

Abstract 707: Anti-SSTR antibodies can reverse neuroendocrine tumor cellular dedifferentiation induced by neutralizing EMT regulator miR-200c in pancreatic neuroendocrine tumor BON cells while increase cell death

Abstract Somatostatin receptors (SSTR) are antiproliferative and antisecretory receptors that belong to the Class A of the seven-transmembrane G-protein coupled receptors. SSTR is comprised of five major subtypes namely subtype 1, 2 (A and B), 3, 4 and 5. Recently, we reported that SST signaling serves as a determinant in neuroendocrine-mesenchymal transition (NMT), a process that is dependent on the TGF-beta - SST signaling axis. NMT is similar to the epithelial-mesenchymal transition (EMT), but it has the opposite cellular differentiation outcome in response to the TGF-beta treatment. For instance, TGF-beta promotes cell dedifferentiation in EMT but promotes cell differentiation in NMT. In epithelial cancers, the micro RNA 200c (miR-200c) has been shown to negatively regulate the zinc finger homeodomain enhancer-binding protein (ZEB1) (an E-cadherin's transcriptional repressor) and thus able to keep EMT invasiveness in check. In this study, we found that upon transient transfection of the miR-200c repressor (X-miR-200c made by Oligoengine Inc., designed by Todd Hauser) into BON cells, the X-miR-200c was able to drive NMT forward. In general, the X-miR-200c triggers the expression of tumor metastasis markers to change, such as ZEB1 protein increase coupled with E-cadherin protein decrease, these changes altogether lead to both Vimentin and Twist protein increase. Interestingly, our anti-SSTR 2, 3, and 5 antibodies derived from the extracellular loop 2 ectodomain region were able to reverse BON cells from the mesenchymal state back to the neuroendocrine state. Additionally, the wound gap healing assay demonstrated that the gap closes at a much slower rate (∼2.5x slower) in the presence of either anti-SSTR2 antibody or anti-SSTR5 antibody treatment than the isotype IgG treatment. Our results suggest that similar to EMT, the miR-200c is also involved in controlling the process of NMT and it is reversible in the presence of anti-SSTR2, 3 and 5 antibodies. Furthermore, we also found that these anti-SSTR antibodies examined can increase neuroendocrine tumor cell death that may or may not be dependent on the NMT reversal. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 707.

Abstract B74: A microRNA expression signature of response to EGFR inhibition targets EMT in both lung and pancreatic cell lines

Abstract Purpose: The intent of the study described herein is to develop and characterize a microRNA gene expression signature of response to EGFR inhibition portable to solid tumors for which treatment with an EGFR inhibitor is indicated. Experimental Design: Treatment with inhibitors of the epidermal growth factor receptor (EGFR) can result in clinical response in both non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC), but only in a minority of unselected patients. Identification of biomarkers that can direct treatment to the responsive population is essential and here we describe generation of a microRNA gene signature of response to EGFR inhibition. Using a Taqman MicroRNA Array of 381 probes, we demonstrate that a 20-gene microRNA signature derived from NSCLC cell lines can predict sensitivity of both lung and pancreatic cancer cell lines to erlotinib, a small molecule inhibitor of EGFR. Overlap of independently generated microRNA expression signatures of NSCLC and pancreatic cell lines produces a fourgene signature. We characterize a target of the reduced signature, ZEB1, in NSCLC and pancreatic cancer cell lines by evaluation of mRNA and protein expression. We evaluated ZEB1 protein and the ZEB1 target, E-cadherin, after transfection of mir-200c into inhibitor-resistant NSCLC cell line, A549. Results: We have identified a 20-gene microRNA signature of response to EGFR inhibition that predicts both inhibitor-sensitive NSCLC and pancreatic cell lines. By overlapping independent signatures of response in NSCLC and pancreatic cancer cell lines, a four gene microRNA signature is identified that targets proteins responsible for epithelial to mesenchymal transition (EMT). Of those proteins, ZEB1 is a transcription factor responsible for repression of E-cadherin expression. Both ZEB1 mRNA and protein expression are down-regulated in erlotinib-sensitive NSCLC in which the microRNA genes 200b, 200c, and 141 are up-regulated. Ectopic expression of mir-200c in inhibitor-resistant lines induces cell death, suggesting that down-regulation of this microRNA is essential for viability in these NSCLC lines. Conclusions: The 20-gene miRNA signature of sensitivity provides a means for defining response that can be generated from FFPE samples of tumor rather than fresh tumor samples. Whereas mRNA is difficult to isolate from FFPE samples due to degradation, microRNA may survive the fixation process. Thus, response to EGFR inhibition in the second- or third-line can be predicted from fixed tumor samples collected early in the treatment of an individual patient, eliminating the need for acquisition of fresh tumor. Further, the microRNA signature identifies the well-studied EMT pathway indicative of EGFR-inhibitor sensitivity and provides biological significance for the members of the signature. Citation Information: Cancer Res 2009;69(23 Suppl):B74.

Reciprocal regulation of ZEB1 and AR in triple negative breast cancer cells

Zinc-finger enhancer binding protein (ZEB1) is a transcription factor involved in the progression of cancer primarily through promoting epithelial to mesenchymal transition (EMT). ZEB1 represses the expression of E-cadherin by binding to E-box sequences in the promoter, thus decreasing epithelial differentiation. We show that ZEB1 and androgen receptor (AR) cross-talk in triple negative breast cancer cell lines. Chromatin immunoprecipitation analysis demonstrates that ZEB1 binds directly to the E-box located in the AR promoter. ZEB1 suppression by stably transfecting shRNA in a triple negative breast cancer cell line resulted in a decrease of AR mRNA, protein, and AR downstream targets. ZEB1 knockdown in triple negative breast cancer cells sensitized the cells to bicalutamide by reducing migration compared to the control cells. Conversely, blockade of AR signaling with bicalutamide resulted in a suppression of ZEB1 protein expression in two triple negative breast cancer cell lines. Furthermore, using a breast cancer tissue microarray, a majority of triple negative breast cancers exhibit positive staining for both ZEB1 and AR. Taken together, these results indicate that ZEB1 and AR regulate each other to promote cell migration in triple negative breast cancer cells.

Abstract 4911: miR-200c is Up-regulated by Oxidative Stress and Induces Endothelial Cell Apoptosis and Senescence

Background. microRNAs (miR) are small non-coding RNAs that inhibit translation of messenger RNAs (mRNA) into proteins and play a key role in the modulation of cell function. The objective of this work was to examine the effect of oxidative stress on human umbilical vein endothelial cells (HUVEC) miR expression. Methods and results. HUVEC were exposed to 200 μ M H 2 O 2 for 8 to 24 hours; out of 254 miRNAs examined only 2 increased &gt; 8-fold, miR-200c (28.30 ± 8.8 fold vs control; p &lt; 0.01) and the co-transcribed miR-141(26.72 ± 10.23 fold vs control; p &lt; 0.02); peak expression was achieved after 16 hours exposure to oxidative stress. All other members of the miR-8 gene family to which miR-200c and miR-141 belong, i.e. miR429, miR200a and miR200b were induced by H 2 O 2 , albeit to a lower level: i.e. 4 – 8 fold increase. This response to oxidative stress was not limited to HUVEC since a similar increase in miR-200c was also found in C2C12 cells growing or differentiated in myotubes exposed to H 2 O 2 . Further, oxidative stress induced with 0.25 mM BCNU also up-regulated miR-200c (3.18 ± 0.96 fold; p &lt; 0.02) and this response was prevented by pre-incubation with the free radical scavenger NAC (10 mM). Over-expression of miR-200c in HUVEC induced cell growth arrest, enhanced apoptosis (3.36 ± 1.05 fold vs control; p &lt; 0.01) and induced cellular senescence as indicated by positive beta-galactosidase ( β -gal)staining (scramble miR:12.66% ± 2.27 β -gal positive cells; miR-200c: 87.36%± 4.85 β -gal positive cells); all these effects on cell proliferation, apoptosis and senescence are known to be caused by oxidative stress. ZEB1 is a well-known target of miR-200c and lack of ZEB1 has been linked to an enhanced cellular senescence. It was found that under our experimental conditions in HUVEC exposed to H 2 O 2 both ZEB1 mRNA and protein decreased and the over-expression of miR-200c in HUVEC down-regulates ZEB1 mRNA and protein. Conclusions. Oxidative stress markedly increases miR-200c and its family members and down-regulates ZEB1, which is a known miR-200c target. Further, the over-expression of miR-200c induces cell growth arrest, enhances apoptosis and induces cell senescence. Thus, miR-200c may play a key role in mediating the effect of oxidative stress on cell function and survival.

SIP1 protein protects cells from DNA damage-induced apoptosis and has independent prognostic value in bladder cancer

The epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Two ZEB family members, ZEB1 and ZEB2(SIP1), inhibit transcription of the E-cadherin gene and induce EMT in vitro. However, their relevance to human cancer is insufficiently studied. Here, we performed a comparative study of SIP1 and ZEB1 proteins in cancer cell lines and in one form of human malignancy, carcinoma of the bladder. Whereas ZEB1 protein was expressed in all E-cadherin-negative carcinoma cell lines, being in part responsible for the high motility of bladder cancer cells, SIP1 was hardly ever detectable in carcinoma cells in culture. However, SIP1 represented an independent factor of poor prognosis (P = 0.005) in a series of bladder cancer specimens obtained from patients treated with radiotherapy. In contrast, ZEB1 was rarely expressed in tumor tissues; and E-cadherin status did not correlate with the patients' survival. SIP1 protected cells from UV- and cisplatin-induced apoptosis in vitro but had no effect on the level of DNA damage. The anti-apoptotic effect of SIP1 was independent of either cell cycle arrest or loss of cell-cell adhesion and was associated with reduced phosphorylation of ATM/ATR targets in UV-treated cells. The prognostic value of SIP1 and its role in DNA damage response establish a link between genetic instability and metastasis and suggest a potential importance for this protein as a therapeutic target. In addition, we conclude that the nature of an EMT pathway rather than the deregulation of E-cadherin per se is critical for the progression of the disease and patients' survival.

Dithiolethione modified valproate and diclofenac increase E-cadherin expression and decrease proliferation of non-small cell lung cancer cells

The effects of dithiolethione modified valproate, diclofenac and sulindac on non-small cell lung cancer (NSCLC) cells were investigated. Sulfur(S)-valproate and S-diclofenac at 1 μg/ml concentrations significantly reduced prostaglandin (PG)E 2 levels in NSCLC cell lines A549 and NCI-H1299 as did the COX-2 inhibitor DuP-697. In vitro, S-valproate, S-diclofenac and S-sulindac half-maximally inhibited the clonal growth of NCI-H1299 cells at 6, 6 and 15 μg/ml, respectively. Using the MTT assay, 10 μg/ml S-valproate, NO-aspirin and Cay10404, a selective COX-2 inhibitor, but not SC-560, a selective COX-1 inhibitor, inhibited the growth of A549 cells. In vivo, 18 mg/kg i.p. of S-valproate and S-diclofenac, but not S-sulindac, significantly inhibited A549 or NCI-H1299 xenograft proliferation in nude mice, but had no effect on the nude mouse body weight. The mechanism by which S-valproate and S-diclofenac inhibited the growth of NSCLC cells was investigated. Nitric oxide-aspirin but not S-valproate caused apoptosis of NSCLC cells. By Western blot, S-valproate and S-diclofenac increased E-cadherin but reduced vimentin and ZEB1 (a transcriptional suppressor of E-cadherin) protein expression in NSCLC cells. Because S-valproate and S-diclofenac inhibit the growth of NSCLC cells and reduce PGE 2 levels, they may prove beneficial in the chemoprevention and/or therapy of NSCLC.

ZEB1 Links p63 and p73 in a Novel Neuronal Survival Pathway Rapidly Induced in Response to Cortical Ischemia

BackgroundAcute hypoxic/ischemic insults to the forebrain, often resulting in significant cellular loss of the cortical parenchyma, are a major cause of debilitating injury in the industrialized world. A clearer understanding of the pro-death/pro-survival signaling pathways and their downstream targets is critical to the development of therapeutic interventions to mitigate permanent neurological damage.Methodology/Principal FindingsWe demonstrate here that the transcriptional repressor ZEB1, thought to be involved in regulating the timing and spatial boundaries of basic-Helix-Loop-Helix transactivator-mediated neurogenic determination/differentiation programs, functions to link a pro-survival transcriptional cascade rapidly induced in cortical neurons in response to experimentally induced ischemia. Employing histological, tissue culture, and molecular biological read-outs, we show that this novel pro-survival response, initiated through the rapid induction of p63, is mediated ultimately by the transcriptional repression of a pro-apoptotic isoform of p73 by ZEB1. We show further that this phylogenetically conserved pathway is induced as well in the human cortex subjected to episodes of clinically relevant stroke.Conclusions/SignificanceThe data presented here provide the first evidence that ZEB1 induction is part of a protective response by neurons to ischemia. The stroke-induced increase in ZEB1 mRNA and protein levels in cortical neurons is both developmentally and phylogenetically conserved and may therefore be part of a fundamental cellular response to this insult. Beyond the context of stroke, the finding that ZEB1 is regulated by a member of the p53 family has implications for cell survival in other tissue and cellular environments subjected to ischemia, such as the myocardium and, in particular, tumor masses.

Inhibition of E-cadherin is associated with down-regulation of members of the miR-200 family but not with promoter methylation in human breast cancer cell lines.

Abstract Abstract #6017 Background: Increasing evidence indicates the fundamental role of epithelial to mesenchymal transition (EMT) in breast cancer progression and this is viewed as an essential early step in tumor cell invasion and metastasis. One of the pivotal events of EMT is down-regulation of E-cadherin. Recent findings have demonstrated the importance of the miR-200 microRNA family in the regulation of E-cadherin and EMT. In view of these findings, we examined the relationship between expression of members of the miR-200 family and status of E-cadherin levels in various human breast cancer cell lines.&amp;#x2028; Materials and Methods: Using quantitative real-time PCR analysis, we analyzed the expression of miR-200b, miR-200c, and miR-141 in 15 human breast cancer cell lines. Additionally, we measured the level of ZEB1, ZEB2, and E-cadherin proteins using Western blot analysis and E-cadherin promoter methylation by methylation-specific PCR.&amp;#x2028; Results: We found a substantial down-regulation of miR-200b, miR-200c, and miR-141 in the breast cancer cell lines. The extent of down-regulation was correlated with metastatic potential of cells. The down-regulation of members of the miR-200 family was associated with up-regulation of ZEB1 and ZEB2, transcriptional repressors of E-cadherin, and a subsequent decrease in the E-cadherin levels. More importantly, inhibition of E-cadherin expression was not due to its promoter hypermethylation. Treatment of breast cancer cells with demethylating agent 5-aza-2'-deoxycytidine resulted in over-expression of miR-200b and miR-200c, increase of the level of E-cadherin, and diminished metastatic properties of cancer cells.Discussion: Previous studies have revealed the important role of promoter methylation-related inhibition of E-cadherin in EMT in human breast cancer. However, the transcriptional silencing of E-cadherin is not always related to promoter hypermethylation, which indicates the existence of other mechanisms in E-cadherin down-regulation. In the present study, we demonstrated the crucial role of members of the miR-200 family in up-regulation of ZEB1 and ZEB2 proteins leading to subsequent E-cadherin down-regulation in human breast cancer cell lines. The extent of down-regulation of the miR-200 family was dependent of cancer stage and was associated with metastatic potential of cancer cell lines. This suggests that expression of miR-200 may be a powerful diagnostic marker determining breast cancer stage and cancer invasive phenotype. More importantly, the results indicate the great potential for reversing the invasive phenotype of cancer cells by up-regulation of miR-200 microRNAs. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6017.

Reciprocal regulation of ZEB1 and AR in triple negative breast cancer cells.

Abstract Abstract #3054 Introduction: Approximately 60-80% of breast tumors are positive for androgen receptor (AR) and negative for estrogen receptor (ER) and progesterone (PR) (Isola et al. J Pathol. 1993). This suggests that AR may function independently of ER and PR to cross-talk with other factors such as Zinc-finger enhancer binding protein (ZEB1). ZEB1 is a transcription factor involved in the progression of breast cancer primarily through promoting epithelial to mesenchymal transition (EMT). ZEB1 represses the expression of E-cadherin by binding to E-box sequences in the promoter, thus decreasing epithelial differentiation and promotes cell migration.&amp;#x2028; Methods: We established a stable control and ZEB1 knockdown using shRNA in MDA-MB-231 cells, a highly metastatic triple negative breast cancer cell line with mesechymal morphology. The stable cell lines were characterized by immunoblot and RT-PCR analysis and were stimulated with synthetic androgens or an androgen antagonist and cell growth and migration were monitored.&amp;#x2028; Results: ZEB1 regulated the expression of AR in triple breast cancer cell lines. AR promoter contains two E-boxes, which may be critical to the transcriptional regulation of this gene. ZEB1 suppression by stably transfecting shRNA in MDA-MB-231 cells resulted in a decrease of AR mRNA, protein and AR downstream targets. In addition, ZEB1 suppression reduced the protein expression of Cyclin D1 and decreased cell proliferation. Conversely, blockade of AR signaling with bicalutamide resulted in suppression of ZEB1 protein expression in two triple negative breast cancer cell lines. ZEB1 knockdown in MDA-MB-231 cells sensitized the cells to bicalutamide by inducing a greater amount of cell death and a reduction in migration compared to the control cells.&amp;#x2028; Conclusions: Taken together, these results indicate that ZEB1 and AR regulate each other to promote cell growth and cell migration in triple negative breast cancer cells. Inhibition of AR signaling could decrease cell proliferation via ZEB1 in a subset of triple negative breast cancers. The growth of triple negative breast cancers that express ZEB1 and AR could be inhibited by antiandrogen treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3054.