Transcriptional Repressor ZEB1 Research Articles

MiR-200 can repress breast cancer metastasis through ZEB1-independent but moesin-dependent pathways

The microRNA-200 (miR-200) family has a critical role in regulating epithelial-mesenchymal transition and cancer cell invasion through inhibition of the E-cadherin transcriptional repressors ZEB1 and ZEB2. Recent studies have indicated that the miR-200 family may exert their effects at distinct stages in the metastatic process, with an overall effect of enhancing metastasis in a syngeneic mouse breast cancer model. We find in a xenograft orthotopic model of breast cancer metastasis that ectopic expression of members of the miR-200b/200c/429, but not the miR-141/200a, functional groups limits tumour cell invasion and metastasis. Despite modulation of the ZEB1-E-cadherin axis, restoration of ZEB1 in miR-200b-expressing cells was not able to alter metastatic potential suggesting that other targets contribute to this process. Instead, we found that miR-200b repressed several actin-associated genes, with the knockdown of the ezrin-radixin-moesin family member moesin alone phenocopying the repression of cell invasion by miR-200b. Moesin was verified to be directly targeted by miR-200b, and restoration of moesin in miR-200b-expressing cells was sufficient to alleviate metastatic repression. In breast cancer cell lines and patient samples, the expression of moesin significantly inversely correlated with miR-200 expression, and high levels of moesin were associated with poor relapse-free survival. These findings highlight the context-dependent effects of miR-200 in breast cancer metastasis and demonstrate the existence of a moesin-dependent pathway, distinct from the ZEB1-E-cadherin axis, through which miR-200 can regulate tumour cell plasticity and metastasis.

Acquired Expression of NFATc1 Downregulates E-Cadherin and Promotes Cancer Cell Invasion

NFATc1 is a transcription factor that regulates T-cell development, osteoclastogenesis, and macrophage function. Given that T cells, osteoclasts, and macrophages in the tumor microenvironment are thought to modulate tumor progression, tumor cells may acquire NFATc1 expression through fusion with these NFATc1-expressing normal cells. We here revealed that a small proportion of tumor cells in human carcinoma specimens expressed NFATc1. To investigate the consequences of NFATc1 acquisition by tumor cells, we established A549 and MCF7 cell lines expressing a constitutively active form of NFATc1 (NFATc1CA) in an inducible manner. The expression of NFATc1CA promoted cancer cell invasion in association with changes in cell morphology. Analysis of gene expression and RNA interference experiments revealed that NFATc1CA suppressed E-cadherin expression by upregulating the transcriptional repressors Snail and Zeb1 in a manner independent of TGF-β signaling. Induced expression of NFATc1CA also downregulated E-cadherin expression and increased invasive activity in tumor xenografts in vivo. Our results thus suggest that the acquisition of NFATc1 expression contributes to tumor progression.

Activation of miR200 by c-Myb depends on ZEB1 expression and miR200 promoter methylation

Tumor progression to metastasis is a complex, sequential process that requires proliferation, resistance to apoptosis, motility and invasion to colonize at distant sites. The acquisition of these features implies a phenotypic plasticity by tumor cells that must adapt to different conditions by modulating several signaling pathways1 during the journey to the final site of metastasis. Several transcription factors and microRNA play a role in tumor progression, but less is known about the control of their expression during this process. Here, we demonstrate by ectopic expression and gene silencing that the proto-oncogene c-Myb activates the expression of the 5 members of miR200 family (miR200b, miR200a, miR429, miR200c and miR141) that are involved in the control of epithelial-mesenchymal transition (EMT) and metastasis in many types of cancers. Transcriptional activation of miR200 by c-Myb occurs through binding to myb binding sites located in the promoter regions of miR200 genes on human chromosomes 1 and 12. Furthermore, when c-Myb and the transcriptional repressor ZEB1 are co-expressed, as at the onset EMT, the repression by ZEB1 prevails over the activation by c-Myb, and the expression of miR200 is inhibited. We also demonstrate that during EMT induced by TGF-β, the promoters of miR200 genes are methylated, and their transcription is repressed regardless of the presence of repressors such as ZEB1 and activators such as c-Myb. Finally, we find a correlation between the expression of c-Myb and that of four out of 5 miR200 in a data set of 207 breast cancer patients.

Gain-of-function mutant p53 downregulates miR-223 contributing to chemoresistance of cultured tumor cells

Mutant p53 proteins are expressed at high frequency in human tumors and are associated with poor clinical prognosis and resistance to chemotherapeutic treatments. Here we show that mutant p53 proteins downregulate micro-RNA (miR)-223 expression in breast and colon cancer cell lines. Mutant p53 binds the miR-223 promoter and reduces its transcriptional activity. This requires the transcriptional repressor ZEB-1. We found that miR-223 exogenous expression sensitizes breast and colon cancer cell lines expressing mutant p53 to treatment with DNA-damaging drugs. Among the putative miR-223 targets, we focused on stathmin-1 (STMN-1), an oncoprotein known to confer resistance to chemotherapeutic drugs associated with poor clinical prognosis. Mutant p53 silencing or miR-223 exogenous expression lowers the levels of STMN-1 and knockdown of STMN-1 by small interfering RNA increases cell death of mutant p53-expressing cell lines. On the basis of these findings, we propose that one of the pathways affected by mutant p53 to increase cellular resistance to chemotherapeutic agents involves miR-223 downregulation and the consequent upregulation of STMN-1.

Salt‐inducible kinase 1 regulates E‐cadherin expression and intercellular junction stability

The protein kinase liver kinase B1 (LKB1) regulates cell polarity and intercellular junction stability. Also, LKB1 controls the activity of salt-inducible kinase 1 (SIK1). The role and relevance of SIK1 and its downstream effectors in linking the LKB1 signals within these processes are partially understood. We hypothesize that SIK1 may link LKB1 signals to the maintenance of epithelial junction stability by regulating E-cadherin expression. Results from our studies using a mouse lung alveolar epithelial (MLE-12) cell line or human renal proximal tubule (HK2) cell line transiently or stably lacking the expression of SIK1 (using SIK1 siRNAs or shRNAs), or with its expression abrogated (sik1(+/+) vs. sik1(-/-) mice), indicate that suppression of SIK1 (∼40%) increases the expression of the transcriptional repressors Snail2 (∼12-fold), Zeb1 (∼100%), Zeb2 (∼50%), and TWIST (∼20-fold) by activating cAMP-response element binding protein. The lack of SIK1 and activation of transcriptional repressors decreases the availability of E-cadherin (mRNA and protein expression by ∼100 and 80%, respectively) and the stability of intercellular junctions in epithelia (decreases in transepithelial resistance). Furthermore, LKB1-mediated increases in E-cadherin expression are impaired in cells where SIK1 has been disabled. We conclude that SIK1 is a key regulator of E-cadherin expression, and thereby contributes to the stability of intercellular junctions.

P1-02-04: Estrogen Receptor beta Inhibits Breast Cancer EMT by Regulating the Expression of miR-200.

Abstract Estrogen receptor beta (ERβ) mediates the effects of estrogens in a variety of human tissues and regulates cellular processes involved in initiation and progression of breast cancer such as cell proliferation and migration. Clinical studies produced contradictory data regarding the role for ERβ in prognosis of metastatic breast cancer and the molecular mechanism through which ERβ influences cell migration and invasion has not been fully elucidated. Here we show that induction of ERβ expression inhibits epithelial to mesenchymal transition (EMT) in metastatic breast cancer cells. This correlates with an ERβ-mediated induction in the expression of the epithelial marker E-cadherin and downregulation of its transcriptional repressors ZEB1 and SIP1. ERβ alters the expression of ZEB1 and SIP1 by inducing the expression of the miR-200a, miR200b and miR-429. Downregulation of these miRNAs in ERβ-expressing cells resulted in decreased cell-cell contact and decline of E-cadherin levels. In addition, ERβ was found to inhibit the invasiveness of metastatic breast cancer cells in a zebrafish xenotransplantation model. We are now examining breast cancer specimens derived from ductal carcinomas and metaplastic breast cancers to see whether ERβ levels decline in the mesenchymal regions and ERβ expression is correlated with epithelial markers. These data propose a crucial role for ERβ in the regulation of EMT and in prognosis of invasive and metastatic breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-04.

Recruitment of histone deacetylases HDAC1 and HDAC2 by the transcriptional repressor ZEB1 downregulates E-cadherin expression in pancreatic cancer

ObjectivePancreatic cancer is characterised by invasive tumour spread and early metastasis formation. During epithelial–mesenchymal transition, loss of the cell adhesion molecule E-cadherin is frequent and can be caused by genetic...

NF-κB mediates the 12(S)-HETE-induced endothelial to mesenchymal transition of lymphendothelial cells during the intravasation of breast carcinoma cells.

Background:The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts.Methods:To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs.Results:We show that tumour spheroids generate ‘circular chemorepellent-induced defects’ (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 μM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general.Interpretation:These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell–cell contacts, migration, and the generation of CCID.

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.

Tissue Transglutaminase Promotes Drug Resistance and Invasion by Inducing Mesenchymal Transition in Mammary Epithelial Cells

Recent observations that aberrant expression of tissue transglutaminase (TG2) promotes growth, survival, and metastasis of multiple tumor types is of great significance and could yield novel therapeutic targets for improved patient outcomes. To accomplish this, a clear understanding of how TG2 contributes to these phenotypes is essential. Using mammary epithelial cell lines (MCF10A, MCF12A, MCF7 and MCF7/RT) as a model system, we determined the impact of TG2 expression on cell growth, cell survival, invasion, and differentiation. Our results show that TG2 expression promotes drug resistance and invasive functions by inducing epithelial-mesenchymal transition (EMT). Thus, TG2 expression supported anchorage-independent growth of mammary epithelial cells in soft-agar, disrupted the apical-basal polarity, and resulted in disorganized acini structures when grown in 3D-culture. At molecular level, TG2 expression resulted in loss of E-cadherin and increased the expression of various transcriptional repressors (Snail1, Zeb1, Zeb2 and Twist1). Tumor growth factor-beta (TGF-β) failed to induce EMT in cells lacking TG2 expression, suggesting that TG2 is a downstream effector of TGF-β-induced EMT. Moreover, TG2 expression induced stem cell-like phenotype in mammary epithelial cells as revealed by enrichment of CD44+/CD24-/low cell populations. Overall, our studies show that aberrant expression of TG2 is sufficient for inducing EMT in epithelial cells and establish a strong link between TG2 expression and progression of metastatic breast disease.

BRCA1-Associated Epigenetic Regulation of p73 Mediates an Effector Pathway for Chemosensitivity in Ovarian Carcinoma

The majority of tumors arising in BRCA1 mutation carriers exhibit inactivation of p53, a key effector of cell death after DNA damage. Despite the loss of p53, BRCA1-deficient tumor cells exhibit increased sensitivity to cisplatin, and patients with BRCA1-associated ovarian carcinomas experience improved outcomes with platinum-based chemotherapy compared with sporadic cases. Although it is known that chemosensitivity in BRCA1-associated cancers is associated with unrepaired DNA damage, the specific effector pathway mediating the cellular response to platinum-induced damage in these tumors is poorly understood. Here, we show that the p53-related gene p73, encoding a proapoptotic protein that is linked to chemosensitivity in many settings, is upregulated through a novel epigenetic mechanism in both human and murine models of BRCA1-associated ovarian carcinoma. BRCA1-deficient ovarian carcinoma cells exhibit hypermethylation within a p73 regulatory region, which includes the binding site for the p73 transcriptional repressor ZEB1, leading to the abrogation of ZEB1 binding and increased expression of transactivating p73 isoforms (TAp73). Cisplatin chemotherapy induces TAp73 target genes specifically in BRCA1-deficient cells, and knockdown of TAp73 in these cells causes chemoresistance while having little or no effect on BRCA1-expressing tumor cells. In primary ovarian carcinomas, ZEB1 binding site methylation and TAp73 expression correlate with BRCA1 status and with clinical response. Together, these findings uncover a novel regulatory mechanism that supports the contribution of TAp73 as an important mediator of the response to platinum chemotherapy in a subset of ovarian carcinomas. TAp73 might represent a response predictor and potential therapeutic target for enhancing chemosensitivity in this disease.

The role of ZEB1 in the inflammation-induced promotion of EMT in HNSCC

Objectives To determine the role of ZEB1 in the inflammation-induced promotion of the epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC). Study Design A molecular biology study. Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemical staining of human HNSCC tissue sections were used to determine how inflammation affects the transcriptional repressor, ZEB1. Setting An academic hospital laboratory. Subjects and Methods Relative ZEB1 RNA levels were determined by RT-PCR, and protein expression was evaluated in situ by immunohistochemical staining of human HNSCC tissue sections. Results IL-1β-treated HNSCC cell lines demonstrated a significant decrease in E-cadherin mRNA and an increase in the mRNA expression of the transcriptional repressor ZEB1. IL-1β exposure led to enhanced ZEB1 binding at the chromatin level, as determined by chromatin immunoprecipitation assays (ChIP). An inverse relationship between ZEB1 and E-cadherin was demonstrated in situ by immunohistochemical staining of human HNSCC tissue sections. Conclusions Our recent investigations indicate that inflammatory mediators are potent regulators of EMT in HNSCC. This is the first report indicating the role of ZEB1 in the inflammation-induced promotion of EMT in HNSCC. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy.

Abstract C45: Somatostatin receptor activation by agonist-like antibodies can reverse neuroendocrine tumor BON cell dedifferentiation and increase cell death

Abstract The somatostatin receptors (SSTR) are effective therapeutic targets for treating neuroendocrine tumors, primarily achieved through the inhibition of both cellular proliferation and hormone secretion. SSTR are comprised of five major subtypes, namely subtypes 1, 2 (A and B), 3, 4 and 5. The somatostatin signaling also works as a determinant in the TGF-β dependent neuroendocrine-mesenchymal transition (NMT) process. NMT is similar to the epithelial-mesenchymal transition (EMT), but has the opposite cellular differentiation response to TGF-β. For instance, TGF-β promotes cell dedifferentiation in EMT but promotes cell differentiation in NMT. The process of EMT is a critical morphogenic event during the progression of epithelial tumors. Recently, in epithelial tumors, the micro RNA 200c (miR-200c) has been found to negatively regulate E-cadherin's transcriptional repressor ZEB1 and capable of keeping the EMT invasiveness in check. In our study, the miR-200c repressor (Oligoengine Inc. designed by Todd Hauser) has been successful in up-regulating ZEB1 and ultimately driving the neuroendocrine tumor BON cells into the dedifferentiated mesenchymal state. The miR-200c repressor induces changes in the tumor metastasis markers, such as the ZEB1 increase, E-cadherin decrease, and ultimately with both Vimentin and Twist increases. Our anti-SSTR antibodies raised against receptor's ectodomain region (anti-SSTR ECL2 antibodies) were able to reverse the BON cell from its mesenchymal state back to the differentiated neuroendocrine state. Our results suggest that similar to EMT, both miR-200c and ZEB1 are also involved in the NMT dedifferentiation programming. In summary, we conclude that miR-200c/ZEB1 signaling can be regulated by the SSTR activation. This is evident because anti-SSTR agonist-like SSTR2, 3 and 5 antibodies are capable of overcoming neuroendocrine tumor's miR-200c commitment to metastasis. Furthermore, we also found that these anti-SSTR antibodies can sensitize tumor cells to death. Citation Information: Cancer Res 2009;69(23 Suppl):C45.

Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

BackgroundEpithelial to Mesenchymal Transition (EMT) induced by Transforming Growth Factor-β (TGF-β) is an important cellular event in organogenesis, cancer, and organ fibrosis. The process to reverse EMT is not well established. Our purpose is to define signaling pathways and transcription factors that maintain the TGF-β-induced mesenchymal state.ResultsInhibitors of five kinases implicated in EMT, TGF-β Type I receptor kinase (TβRI), p38 mitogen-activated protein kinase (p38 MAPK), MAP kinase kinase/extracellular signal-regulated kinase activator kinase (MEK1), c-Jun NH-terminal kinase (JNK), and Rho kinase (ROCK), were evaluated for reversal of the mesenchymal state induced in renal tubular epithelial cells. Single agents did not fully reverse EMT as determined by cellular morphology and gene expression. However, exposure to the TβRI inhibitor SB431542, combined with the ROCK inhibitor Y27632, eliminated detectable actin stress fibers and mesenchymal gene expression while restoring epithelial E-cadherin and Kidney-specific cadherin (Ksp-cadherin) expression. A second combination, the TβRI inhibitor SB431542 together with the p38 MAPK inhibitor SB203580, was partially effective in reversing EMT. Furthermore, JNK inhibitor SP600125 inhibits the effectiveness of the TβRI inhibitor SB431542 to reverse EMT. To explore the molecular basis underlying EMT reversal, we also targeted the transcriptional repressors ZEB1 and ZEB2/SIP1. Decreasing ZEB1 and ZEB2 expression in mouse mammary gland cells with shRNAs was sufficient to up-regulate expression of epithelial proteins such as E-cadherin and to re-establish epithelial features. However, complete restoration of cortical F-actin required incubation with the ROCK inhibitor Y27632 in combination with ZEB1/2 knockdown.ConclusionsWe demonstrate that reversal of EMT requires re-establishing both epithelial transcription and structural components by sustained and independent signaling through TβRI and ROCK. These findings indicate that combination small molecule therapy targeting multiple kinases may be necessary to reverse disease conditions.

43 Different Candidate Genes and Their Role During Tumor Progression, Invasion and Metastasis

Introduction: Invasion and metastasis are the key characteristics of tumor progression. Epithelial-mesenchymal-transition (EMT) is a developmental program that is often activated during cancer invasion and metastasis, especially at the invasive front. In our cellular model of human oral-esophageal carcinogenesis we were able to recapitulate tumor development in a stepwise fashion. Cyclin D1 overexpression and p53 inactivation (OKF6D1/d.n.p53) led to immortalization, additional EGFR overexpression (OKF6D1/d.n.p53/EGFR) induced an In Vitro transformed phenotype, whereas additional c-myc overexpression (OKF6D1/ d.n.p53/EGFR/c-myc) resulted in invasive cancer cells. To investigate the role of a master regulator of EMT and metastasis we overexpressed the transcriptional repressor ZEB1 in the different cell types. Additionally, to study the ability of these different cells to migrate we analyzed the expression and localization of different candidates involved in tumor invasion and metastasis: 1. collagen XVII a hemidesmosomal epithelial adhesion protein and 2. cathepsin B a lysosomal cystein protease. Methods: The expression of collagen XVII, cathepsin B and E-cadherin as a ZEB1 target gene was assayed in the distinct cell types by RT-PCR and western blot analysis. The cellular distribution was studied by immunofluorescence. The proteolytic activity of cathepsin B on the cell surface was determined by DQ-Collagen IV. Functional relevance of ZEB1 overexpression was analyzed in several cell types using a matrigel assay. Results: RT-PCR revealed a downregulation of collagen XVII expression in In Vitro transformed cells followed by an upregulation in cancer cells. Immunofluorescence demonstrated that the cellular localization of collagen XVII and cathepsin B is influenced by malignant transformation, i.e. collagen XVII is first translocated to the cytoplasm and relocated to the cell membrane in cancer cells. Cathepsin B is recruited from the lysosomes to the membrane during malignant transformation. Initial results showed a downregulation of the epithelial marker E-cadherin and an upregulation of the mesenchymal marker vimentin in ZEB1 overexpressing cells. Conclusions: In our human cellular model of oral-esophageal carcinogenesis we demonstrated that collagen XVII is differentially expressed during carcinogenesis. Cellular localization of collagen XVII and cathepsin B is defined by the distinct steps of tumor development. The recruitment to the membrane and secretion of cathepsin B leads to an increased degradation of the ECM. In the next step ZEB1 overexpression or at least E-cadherin downregultaion seems to be necessary for a invasive and metastatic phenotype.

44 High Efficacy of Gene Expression Profiling and Regularized Discriminant Analysis in the Prediction of Neuroendocrine Tumor Subtypes and Their Metastases

Introduction: Invasion and metastasis are the key characteristics of tumor progression. Epithelial-mesenchymal-transition (EMT) is a developmental program that is often activated during cancer invasion and metastasis, especially at the invasive front. In our cellular model of human oral-esophageal carcinogenesis we were able to recapitulate tumor development in a stepwise fashion. Cyclin D1 overexpression and p53 inactivation (OKF6D1/d.n.p53) led to immortalization, additional EGFR overexpression (OKF6D1/d.n.p53/EGFR) induced an In Vitro transformed phenotype, whereas additional c-myc overexpression (OKF6D1/ d.n.p53/EGFR/c-myc) resulted in invasive cancer cells. To investigate the role of a master regulator of EMT and metastasis we overexpressed the transcriptional repressor ZEB1 in the different cell types. Additionally, to study the ability of these different cells to migrate we analyzed the expression and localization of different candidates involved in tumor invasion and metastasis: 1. collagen XVII a hemidesmosomal epithelial adhesion protein and 2. cathepsin B a lysosomal cystein protease. Methods: The expression of collagen XVII, cathepsin B and E-cadherin as a ZEB1 target gene was assayed in the distinct cell types by RT-PCR and western blot analysis. The cellular distribution was studied by immunofluorescence. The proteolytic activity of cathepsin B on the cell surface was determined by DQ-Collagen IV. Functional relevance of ZEB1 overexpression was analyzed in several cell types using a matrigel assay. Results: RT-PCR revealed a downregulation of collagen XVII expression in In Vitro transformed cells followed by an upregulation in cancer cells. Immunofluorescence demonstrated that the cellular localization of collagen XVII and cathepsin B is influenced by malignant transformation, i.e. collagen XVII is first translocated to the cytoplasm and relocated to the cell membrane in cancer cells. Cathepsin B is recruited from the lysosomes to the membrane during malignant transformation. Initial results showed a downregulation of the epithelial marker E-cadherin and an upregulation of the mesenchymal marker vimentin in ZEB1 overexpressing cells. Conclusions: In our human cellular model of oral-esophageal carcinogenesis we demonstrated that collagen XVII is differentially expressed during carcinogenesis. Cellular localization of collagen XVII and cathepsin B is defined by the distinct steps of tumor development. The recruitment to the membrane and secretion of cathepsin B leads to an increased degradation of the ECM. In the next step ZEB1 overexpression or at least E-cadherin downregultaion seems to be necessary for a invasive and metastatic phenotype.

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

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.

Understanding EMT in HNSCC

Problem Down regulation of E-cadherin is the hallmark of epithelial mesenchymal transition (EMT). The loss of E-cadherin confers a more invasive phenotype and is one of the steps leading to metastasis. Our recent investigations indicate that inflammatory mediators are potent regulators of EMT in HNSCC. Methods Immunohistochemical analysis of HNSCC tumor tissue specimens, in vitro analysis of E-cadherin regulation in HNSCC cells, and in vivo analysis of tumor growth and metastasis ina murine model. Results An inverse relationship between COX-2 and E-cadherin was demonstrated in situ by double immunohistochemical staining of human HNSCC tissue sections. Treatment of HNSCC cells with exogenous IL-1B, also caused the down regulation of E-cadherin expression and up regulation of Cox-2 expression in HNSCC cell lines in a concentration- and time-dependent manner. IL-1B -treated HNSCC cell lines demonstrated a significant decrease in E-cadherin mRNA and an increase in the mRNA of the transcriptional repressors SNAIL and ZEB1. IL-1B exposure led to enhanced ZEB1 and Snail binding at the chromatin level as determined by chromatin immunoprecipitation assays (ChIP). An inverse relationship between E-cadherin and ZEB1 and a direct relationship between COX-2 and ZEB1 were demonstrated by immunohistochemical staining of human HNSCC tissue sections. When HNSCC cell lines that over express Snail were injected into SCID mice, the primary tumor and metastatic burdens were significantly greater than HNSCC vector controls. Conclusion These findings indicate that 1L-1B modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in HNSCC. This is the first report indicating the role of E-cadherin transcriptional repressors in the inflammation-induced promotion of EMT in HNSCC. Significance This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy. Support AA0–HNS Surgeon Scientist CDA.

The miR-200 Family Inhibits Epithelial-Mesenchymal Transition and Cancer Cell Migration by Direct Targeting of E-cadherin Transcriptional Repressors ZEB1 and ZEB2

MicroRNAs are small non-coding RNA molecules that can regulate gene expression by interacting with multiple mRNAs and inducing either translation suppression or degradation of mRNA. Recently, several miRNAs were identified as either promoters or suppressors of metastasis. However, it is unclear in which step(s) of the multistep metastatic cascade these miRNAs play a defined functional role. To study the functional importance of miRNAs in epithelial-mesenchymal transition (EMT), a process thought to initiate metastasis by enhancing the motility of tumor cells, we used a well established in vitro EMT assay: transforming growth factor-beta-induced EMT in NMuMG murine mammary epithelial cells. We found that members of the miR-200 family, organized as two clusters in the genome, were repressed during EMT. Overexpression of each miRNA individually or as clusters in NMuMG cells hindered EMT by enhancing E-cadherin expression through direct targeting of ZEB1 and ZEB2, which encode transcriptional repressors of E-cadherin. In the 4TO7 mouse carcinoma cell line, which expresses low levels of endogenous E-cadherin and displays a mesenchymal phenotype, ectopic expression of the miR-200 family miRNAs significantly increased E-cadherin expression and altered cell morphology to an epithelial phenotype. Furthermore, ectopic expression of each miR-200 miRNA cluster significantly reduced the in vitro motility of 4TO7 cells in migration assays. These results suggested that loss of expression of the miR-200 family members may play a critical role in the repression of E-cadherin by ZEB1 and ZEB2 during EMT, thereby enhancing migration and invasion during cancer progression.