Modulation Of Epithelial-mesenchymal Transition Research Articles

The miR-200 family regulates the epithelial-mesenchymal transition induced by EGF/EGFR in anaplastic thyroid cancer cells

The miR-200 family was recently identified as a suppressor of epithelial-mesenchymal transition (EMT). The loss or gain of miR-200 family members is associated with cancer invasion. The epidermal growth factor receptor (EGFR) is overexpressed in the majority of anaplastic thyroid cancers (ATCs). The activation of EGFR by its ligand, epidermal growth factor (EGF), activates a signaling cascade that results in the enhanced migration and invasiveness of thyroid cancer cells. However, little is known about the potential interrelationships between EGF/EGFR, miR-200s and the induction of EMT or mesenchymal-epithelial transition (MET) processes. This study aimed to investigate the regulatory role of miR-200s in EMT modulation by EGF/EGFR. Using transfection, real-time reverse transcription PCR and western blot analysis, we found that the EGF treatment of Nthy-ori 3-1 thyroid follicular cells resulted in the downregulation of E-cadherin and the upregulation of vimentin. By contrast, EGFR silencing in SW1736 human thyroid carcinoma cells led to the upregulation of E-cadherin and the downregulation of vimentin. In addition, EGF signaling correlated with the reduced expression of miR-200s and the re-expression of miR-200s abrogated the effects of EGF treatment and restored an epithelial phenotype to EGF-induced Nthy-ori 3-1 cells. Conversely, the silencing of miR-200s in SW1736 cells overcame siEGFR-induced changes in gene expression and phenotype. In addition, we demonstrate that miR-200s play a key role in in vitro EGF/EGFR-mediated thyroid cell invasion and in EMT in vivo. We, therefore, provide a mechanistic link between the miR-200 family and EGF/EGFR, which suggests that miR-200 upregulation may serve as a novel therapeutic strategy for highly invasive thyroid cancers.

Abstract 2308: MUC1 modulation of E-cadherin through miR-200c and ZEB1

Abstract MUC1, a cell-surface glycoprotein implicated in tumorigenesis and metastasis, has a cytoplasmic tail (MUC1.CT) that is phosphorylated by different receptor tyrosine kinases and conducts these signals by binding and altering the function or stability of transcription factors, thereby influencing gene transcription. MicroRNAs (miRNAs), short RNAs (19-22 nucleotides), regulate gene expression at the transcriptional level and are differentially regulated in cancer and metastasis. We discovered that MUC1.CT influences expression of miRNAs that contribute to metastasis, including miR-200c. MiR-200c is a known regulator of epithelial-to-mesenchymal transition (EMT) through regulation of ZEB1, a transcriptional repressor of E-cadherin. In addition, ZEB1 is a repressor of miR-200c in a feedback loop. Overexpression of MUC1 results in a dramatic decrease in miR-200c levels, resulting in increased levels of ZEB1. Chromatin immunoprecipitation (ChIP) was used to demonstrate that MUC1.CT occupies the ZEB1 binding motif upstream of the miR-200c start site. In addition, we found that ZEB1 occupancy of the miR-200c promoter region was enhanced 3.5-fold in MUC1 overexpressing cells, indicating that MUC1.CT may influence the expression and stability of ZEB1 at the miR-200c promoter. Co-immunoprecipitation experiments determined that MUC1.CT directly interacts with ZEB1. We evaluated levels of proteins affected by the miR-200c pathway in MUC1 overexpressing cells and observed an increase in ZEB1 levels and a decrease in E-cadherin levels, consistent with induction of EMT. We also confirmed an increase of ZEB1 occupancy at the E-cadherin promoter in MUC1 overexpressing cells, further confirming our hypothesis. In addition, MUC1.CT is differentially phosphorylated by several receptor tyrosine kinases. Antibodies against different phospho-isoforms were used with ChIP to investigate the signaling cascades that influence localization of the MUC1.CT to the miR-200c promoter. The phospho-YEKV form of MUC1.CT, which is representative of signaling through epidermal growth factor/receptor (EGF/EGFR), was the only phospho-MUC1.CT detected at the ZEB1 binding motif of the miR-200c promoter. EGF signaling is linked to a loss of E-cadherin, and our work is the first to link EGF signaling through the MUC1.CT as a direct regulator of miR-200c expression. These data portray a novel pathway of EMT modulation. EGF signaling via MUC1 regulates levels of miR-200c, altering regulation E-cadherin. Grant support was provided by grants from the NCI (Training Grant CA09476, R01CA57362, U01 CA111294) and student assistantships from UNMC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2308. doi:1538-7445.AM2012-2308

Notch: a key regulator of tumor angiogenesis and metastasis.

The Notch signaling pathway is critical for many developmental processes including physiologic angiogenesis. Notch is also implicated in having a key role in tumor angiogenesis. Preclinical and clinical experience with anti-angiogenic strategies indicates that they may be limited by tumor resistance and recurrence, which has led to the search for alternative angiogenic treatment strategies. Significant progress has been made in shedding light on the complex mechanisms by which Notch signaling can influence tumor growth by disrupting vasculature in an array of tumor models (Ridgway et al., 2006). These results have led to the consideration of Notch as an attractive target to block tumor angiogenesis and inhibit growth. However, studies of inhibition of Notch signaling in different tumor models have uncovered similarly variable results, and some unexpected adverse effects. The ability of Notch to function in a context-dependent manner as a determinant of cell fate, a tumor suppressor, and an oncogene may partially explain the complexity in interpreted the role of Notch signaling inhibitors in preclinical tumor studies. In addition, Notch may also play an important role in metastasis via its direct effects on the vasculature and by modulation of epithelial-mesenchymal transition in tumor cells. Here we present a current understanding of Notch signaling in tumor angiogenesis, and discuss recent work on the role of Notch in tumor metastatic progression.

P1-02-08: Modulation of EMT by Targeting E-Cadherin Restores Radiation Sensitivity in Human Breast Cancer Cells.

Abstract E-cadherin is the major adhesion protein associated with epithelial cells and loss of its expression is diagnostic of the epithelial to mesenchymal transition (EMT). Findings from our laboratory, suggest that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which typically are relatively radiosensitive. To definitively test this hypothesis we compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. We observed that many ER-a negative lines, were also E-cadherin negative, and the ER-a positive lines were positive for E-cadherin expression. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) and had high expression of E-cadherin, were also more sensitive to increasing doses of radiation. Treatment of MCF-7 cells with 100nM siRNA to E-cadherin for 72 hrs made the cells more resistant to radiation with the surviving fraction changing from 42.3% in control cells to 56.4% in siRNA treated cells. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 and Hs578t cells with a CDH1 -expression vector and isolated stable clones, which were then tested for radiosensitivity. Cells transfected with the backbone vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized both, MDA-MB-231 and Hs578t cells, compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. These observations suggest that ER-a signaling might regulate E-cadherin and implied that this regulation might influence EMT and radioresistance. Overall, our results suggest that the EMT status of breast cancer cells governs their response to radiation. A detailed investigation of the mechanism is ongoing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-08.

Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3B1, a novel epithelial-mesenchymal transition inducer in pancreatic cancer

Epithelial-mesenchymal transition (EMT) is a critical early event in tumorigenesis. The contribution of heparan sulfate (HS) to EMT has not been fully elucidated. HS D-glucosaminyl 3-O-sulfotransferase-3B1 (3-OST-3B1) participates in the final step of HS fine structure biosynthesis, whose involvement in cancer has yet to be determined. This study demonstrated that following treatment with trichostatin-A, a histone deacetylase inhibitor, 3-OST-3B1 gene expression was activated in the pancreatic cancer cell line, PANC-1. By chromatin immunoprecipitation analysis, permissive histone modifications including an increase in histone H3 lysine 9 monoactylation (H3 ac K9) but a decrease in methylated histone H3 (H3 me K9) were observed accompanying transcriptional activation of 3-OST-3B1. Functional, results revealed that increased 3-OST-3B1 levels were involved in the promotion of EMT processes. In vitro studies demonstrated that overexpression of 3-OST-3B1 in both pancreatic cancer cells and vascular endothelial cells could trigger an EMT-like phenotype as evidenced by the up-regulation of Snail at the mRNA and protein level, and its nuclear translocation. And 3-OST-3B1 appeared to be sufficient for the development of a more mesenchymal phenotype in vivo. Together, the results from this study unveiled a distinct function for 3-OST-3B1 as an EMT inducer in cancer and provided a link between histone modification and EMT modulation.

Abstract 1684: 2-Methoxyestradiol reverses the epithelial-mesenchymal transition in Barrett's adenocarcinoma cells

Abstract 2-Methoxyestradiol (2-ME2) is a novel anti-cancer agent because of its ability to potentiate apoptotic cell death and inhibit cancer cell growth and angiogenesis. In our study, we have investigated whether 2-ME2 is able to modulate epithelial-mesenchymal transition (EMT) in Barrett's esophageal adenocarcinoma cell (OE33), which has been postulated as an absolute requirement for tumor invasion and metastasis. Expression of epithelial (E-cadherin, β-catenin, Keratin-19) and mesenchymal markers (Oct-4, Vimentin) and stem cell markers (CD44, CD24) were studied at mRNA and protein levels in OE33 cells that were treated with 5µmolar/L of 2-ME2 for 24 h and 48 h. Our results indicate expression of β-catenin and E-cadherin is activated in a 2-ME2 dependent fashion in OE33 cells. Expression of Vimentin and Oct-4 is significantly down-regulated in 2-ME2 treated OE33 cells. Altogether, these results provide new insights into the role of 2-ME2 in EMT modulation, which may facilitate development of new therapeutic strategies to prevent the tumor invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1684. doi:10.1158/1538-7445.AM2011-1684

3D Models of Epithelial-Mesenchymal Transition in Breast Cancer Metastasis: High-Throughput Screening Assay Development, Validation, and Pilot Screen

Despite advancements in therapies developed for the treatment of cancer, patient prognosis and mortality rates have improved minimally, and metastasis remains the primary cause of cancer mortality worldwide. An underlying mechanism promoting metastasis in many types of cancer is epithelial-mesenchymal transition (EMT). Here the authors report a novel 3D model of EMT and metastatic breast cancer suitable for high-throughput screening (HTS) drug discovery. The primary assay incorporates the expression of the prognostic biomarker vimentin, as a luciferase reporter of EMT, in basil-like/triple-negative MDA-MB-231 breast carcinoma spheroids. Using this model, the authors developed a number of known antitumor agents as control modulators of EMT. U0126, PKC412, PF2341066, dasatinib, and axitinib downregulated vimentin expression by 70% to 90% as compared to untreated spheroids. Counterassays were developed to measure spheroid viability and the invasive potential of MDA-MB-231 spheroids after small-molecule treatment and used to confirm hits from primary screening. Finally, the authors conducted a pilot screen to validate this model for HTS using a purified library of marine secondary metabolites. From 230 compounds screened, they obtained a Z' score of 0.64, indicative of an excellent assay, and confirmed 4 hits, including isonaamidine B, papuamine, mycalolide E, and jaspamide. This HTS model demonstrates the potential to identify small-molecule modulators of EMT that could be used to discover novel antimetastatic agents for the treatment of cancer.

Abstract 2303: The redox-active protein thioredoxin: A modulator of epithelial-mesenchymal transition

Abstract Human thioredoxin is a ubiquitous redox-active protein that induces the activation of many redox-sensitive transcription factors, inhibits the activity of the apoptosis-signal regulating kinase, and activates the AKT signaling pathway. Thioredoxin is thus associated with many biological processes, including the induction of cellular proliferation leading to cancer progression and metastasis. The morphogenetic changes occurring during the initial stage of metastasis is referred to as epithelial-mesenchymal transition (EMT). EMT results in the transition of cancer cells from an epithelial to a fibroblastic or mesenchymal phenotype, accompanied by a large number of changes in gene expression, particularly down regulation in the expression of the epithelial marker E-cadherin and induction of expression and activation of the mesenchymal marker vimentin, N-cadherin, or fibronectin. Transforming growth factor (TGF)-β is regarded as the prototype cytokine for induction of EMT both in vitro and in vivo. Using a high throughput functional genomic screen, we previously showed that members of the TGF-β signaling pathway induce the expression of thioredoxin in both pancreatic and breast cancer cells. In addition, we showed that TGF-β1 stimulation in these cells resulted in a two-fold induction of thioredoxin expression. Therefore, we hypothesized that thioredoxin is a modulator of EMT. We based this hypothesis on the finding that increased expression of thioredoxin is associated with metastasis and that TGF-β1 induces thioredoxin expression. Herein we present evidence in both breast cancer cells and human biopsy samples of breast tumors that support our hypothesis. In vitro three-dimensional cell cultures of the MDA-MB-231 breast cancer cell line undergo phenotypic changes similar to the morphogenetic changes associated with EMT. Treating MDA-MB-231 cells with PX12, a potent inhibitor of thioredoxin, blocked the onset of these morphogenetic changes. We show a correlation between the expression of thioredoxin and the EMT markers utilizing Reverse-Phase Protein Array of 250 fine-needle aspirates from breast tumor patients and the Pearson correlation coefficients. There was a positive correlation between thioredoxin and N-cadherin (p<0.0001), and a negative correlation between thioredoxin and E-cadherin (p<0.0001) as well as between thioredoxin and β-catenin (p<0.0001). High expression of thioredoxin correlate with decreased expression of E-cadherin and β-catenin and an increased expression of N-cadherin. Our data demonstrated that thioredoxin might be an important regulator of EMT in breast cancer. Based on our findings we propose a model indicating that thioredoxin modulates TGF-β-induced EMT by functioning to maintain EMT once it is initiated. Ongoing studies by our group will enable us to confirm this model. 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 2303.

Epithelial mesenchymal transition during the neoplastic transformation of human breast epithelial cells by estrogen

Epithelial-mesenchymal transition (EMT) in epithelial cells has been indicated as an important component of neoplastic transformation although, the genetic mechanism involved in this process has not been defined. The aim of this study was to evaluate the expression of different genes related to EMT such as E-cadherin, TGFbeta1, TGFbeta2, h-RAS, TWIST1, SNAIL2, SMAD5, FN1, CEACAM1 and JAG1 using the in vitro-in vivo model of the estrogen induced cell transformation developed in our laboratory. The E2-transformed MCF-10F (E2 70) cells and the tumorigenic cell line C5-A8-T8 (C5-T8) exhibit progressive loss of ductulogenesis as demonstrated by growth in collagen matrix. MCF-10F cells form ductal structures while E2 70 cells form solid spherical masses that in histological sections exhibit a pattern of growth resembling ductal hyperplasia or carcinoma in situ. The tumorigenic cells C5-T8 did not form structures on collagen acquiring an invasive pattern with spindle like features. We have observed a reduction in E-cadherin expression in E2 70 cells and a complete loss in C5-T8 cells. TGFbeta1, TGFbeta2, CEACAM1 and JAG1 were down-regulated in E2 70 and C5-T8 cells. SMAD5 and h-RAS were up-regulated in the tumorigenic C5-T8 cells whereas FN1, Twist1 and Snail2 were up-regulated in C5-T8 and down-regulated in E2 70. We conclude that the loss of expression of TGFbeta1, TGFbeta2, CEACAM1 and JAG1 are related to ductulogenesis and branching and the overexpression of h-RAS with loss of E-cadherin expression and up-modulation of TWIST1, SNAIL2 and SMAD5 expressions are involved in the EMT modulation.