Epithelial-mesenchymal Transition Related Genes Research Articles

Caffeic acid phenethyl ester activates pro-apoptotic and epithelial-mesenchymal transition-related genes in ovarian cancer cells A2780 and A2780cis.

Ovarian cancer is a highly aggressive pathology, displaying a poor prognosis and chemoresistance to classical therapy. The present study was conducted to evaluate the effect of caffeic acid phenethyl ester (CAPE) on survival of ovarian cancer cell lines, A2780 (sensitive to cisplatin) and A2780cis (resistant to cisplatin). MTT assay was used to evaluate cell viability, while the apoptotic processes were examined by flow cytometry and qRT-PCR. A reduction of cell proliferation and activation of the apoptosis was observed in both cell lines. qRT-PCR evaluation demonstrated the activation of the pro-apoptotic genes (BAD, CASP8, FAS, FADD, p53) in both cell lines. The limited therapeutic effect in A2780 cells is explained by the activation of epithelial-mesenchymal transition-related genes (ZEB1, ZEB2, or TGFBB1) as displayed by Ingenuity Network analysis. Overall data suggest that CAPE can be used as an alternative in sensitizing cells to chemotherapy.

Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma.

Epithelial-mesenchymal transition (EMT) is a pivotal event in the progression of cancer towards metastasis. Given that pigment epithelium-derived factor (PEDF) inhibits angiogenesis, the present study analyzed whether PEDF expression is associated with EMT and prognosis in invasive ductal breast cancer (IDC). Immunohistochemical analysis was used to examine the expression levels of PEDF, E-cadherin, vimentin, Snail and nuclear factor-κB (NF-κB) in 119 cases of IDC. Correlations between PEDF expression and EMT-related genes, and clinicopathological features and clinical prognosis were analyzed. E-cadherin, vimentin, Snail and NF-κB expression was correlated with tumor size, lymph node metastasis and clinicopathological stage. PEDF expression was closely associated with tumor size. Spearman's rank correlation analysis revealed a positive correlation between PEDF and E-cadherin, vimentin, Snail and NF-κB expression (P<0.05). Additionally, Kaplan-Meier survival analysis demonstrated that the five-year survival rate was higher for patients with PEDF- and E-cadherin-positive tumors, but was lower for those with vimentin-, Snail- and NF-κB-positive tumors. Vimentin, E-cadherin and NF-κB levels were dependent prognostic factors of favorable outcomes in IDC, as determined by Cox multivariate analysis. PEDF expression in breast cancer was significantly associated with EMT-related genes, suggesting that it may be an EMT suppressor. However, its potential as a prognostic indicator in breast cancer warrants further investigation.

Abstract 4114: Chemopreventive and chemotherapeutic effect of Withaferin-A on in vivo models of prostate cancer

Abstract Several groups have emphasized on the functional role of phytochemicals in chemoprevention and chemotherapy of prostate cancer (CaP). In this study we have focused on Withaferin A (WA) a bioactive compound derived from Withania somnifera, which has been extensively used in Asian and African traditional medicine system. In our in vitro studies, WA inhibits the growth of CRPC cells by targeting AKT mediated pro-survival signaling and simultaneously activating FOXO3a dependent apoptosis in CaP cell lines. The objective of the present study was to evaluate the in vivo efficacy of WA on both xenograft and transgenic adenocarcinoma of mouse prostrate (TRAMP) models. DU-145 and C4-2B CaP cells stably overexpressing AKT were xenografted in nude mice and WA (5mg/kg body weight) was orally administered along with control groups fed with oil alone for 4-5 weeks. AKT-overexpressing tumors showed 2-3-fold faster growth than pCMV-expressing tumors and oral administration of WA significantly reduced AKT-induced tumor growth. Immunohistochemistry of tumor sections revealed high expression of AKT, pAKT and down regulation of nuclear FOXO3a in tumor sections derived from AKT-overexpressing tumors. On the contrary, WA treated controls as well as AKT over expressed tumor sections clearly revealed that WA reverted FOXO3a mediated apoptotic signaling by down regulating AKT activation. In addition, activation of AKT has emerged as a central feature of epithelial-mesenchymal transition (EMT). Our results also revealed elevated expression of important EMT related transcription factors and genes such as snail, slug, β-catenin, vimentin, and MMPs and decrease in the expression of E-cadherin expression in AKT overexpressing tumors. Simultaneously, WA treatment of pCMV and AKT-overexpressing tumors showed decrease in the expression of these EMT markers and increase in the expression of E-cadherin. Similarly, TRAMP mice were randomized into controls and WA (oral gavage-3mg/kg body weight) for 31 weeks. Control and WA-treated mice were sacrificed periodically at 11, 20, 27 and 31 weeks. Histopathological examination revealed both the groups (control and WA-treated) showed prostatic hyperplasia with no signs of metastasis up to 20 weeks. However, 58.3% mice in the control group showed metastasis to lungs, liver or kidneys, whereas in the treatment group 16.6% mice showed metastasis in 31 weeks. Molecular analysis showed higher expression of phosphorylated AKT, FOXO3a, along with EMT markers like β-catenin, snail, vimentin and down regulation of E-cadherin expression in control-TRAMP tumors. On the other hand, WA-treated tumor tissues revealed low expression of pAKT and pFOXO3a in WA treated mice. Also, low expression of β-catenin; snail, vimentin and upregulation of E-cadherin in WA treated mice was observed. These results potentiate the chemopreventive and chemotherapeutic effects of WA in CaP and steers the clinical translation of this biomolecule. Citation Format: Suman Suman, Trinath P. Das, Murali K. Ankem, Chendil Damodaran. Chemopreventive and chemotherapeutic effect of Withaferin-A on in vivo models of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2015-4114

Snail-mediated Cripto-1 repression regulates the cell cycle and epithelial–mesenchymal transition-related gene expression

Transcription factor Snail mediates epithelial to mesenchymal transitions (EMT) by coordinate repression of epithelial markers, facilitating mass cell movement during germ layer formation. Aberrant reprogramming in its signaling pathways causes metastatic cancer. Snail’s involvement in “fate-changing” decisions is however not understood. Cripto-1 shares a common temporal expression pattern with Snail during development. While Cripto-1 is required for mammary morphogenesis and hematopoietic stem cell renewal, its unregulated expression causes metastatic cancers. Therefore, we suspected that Snail regulates the expression of Cripto-1 controlling decisions such as motility, transformation and differentiation. We demonstrate that Snail represses Cripto-1 gene by direct transcriptional interaction and propose a novel mechanism by which it co-ordinately regulates cell fate decisions during development and could be causal of cancers.

The effects of pseudomonasaeruginosa mannose sensitive hamemagglutination vaccine on epithelial-mesenchymal transition of hepatocellular carcinoma

Objective To investigate the effects of pseudomonasaeruginosa mannose sensitive hamemagglutination vaccine(PA-MSHA) on the epithelial-mesenchymal transition(EMT) of hepatocellular carcinoma(HCC). Methods MHCC97L and HepG2 cells were cultured and treated with different dose of PA - MSHA. After that, morphological changes of cells were observed under a light microscope. Immunofluorescence and Western blot analysis were used to evaluate the expression of EMT related genes and transcription factors. Immunohistochemistry and reverse transcription polymerase chain reaction(RT- PCR)analysis were used to evaluate the expression of E- cadherin,Snail and Slug in the tumor tissue after the mice were treated with PA- MSHA. Results Western blot and immunofluorescence analysis reveled that the expression of E-cadherin was significantly elevated while expression of Vimentin was significantly inhibited by PA- MSHA,in a dose dependent manner. Moreover, the expression of fibronectin, nuclear factor- kappa B(NF-κB),Snail and Slug were also inhibited by PA- MSHA, but theses effects of PA- MSHA were blocked by mannose. After PA- MSHA treatment, HCC cells have altered from irregular shape to epithelial cobblestone appearance.In vivo study, PA- MSHA also significantly increased the expression of E- cadherin and inhibited the expression of Snail mRNA and Slug mRNA. Conclusion PA-MSHA can significantly inhibit EMT of HCC in a mannose-mediated manner. Key words: Hepatocellular carcinoma; Pseudomonas aeruginosa; Mannose; Epithelialmesenchymal transition

Adipocyte hypoxia promotes epithelial-mesenchymal transition-related gene expression and estrogen receptor-negative phenotype in breast cancer cells.

The development of breast cancer is linked to the loss of estrogen receptor (ER) during the course of tumor progression, resulting in loss of responsiveness to hormonal treatment. The mechanisms underlying dynamic ERα gene expression change in breast cancer remain unclear. A range of physiological and biological changes, including increased adipose tissue hypoxia, accompanies obesity. Hypoxia in adipocytes can establish a pro-malignancy environment in breast tissues. Epidemiological studies have linked obesity with basal-like breast cancer risk and poor disease outcome, suggesting that obesity may affect the tumor phenotype by skewing the microenvironment toward support of more aggressive tumor phenotypes. In the present study, human SGBS adipocytes were co-cultured with ER-positive MCF7 cells for 24 h. After co-culture, HIF1α, TGF-β, and lectin-type oxidized LDL receptor 1 (LOX1) mRNA levels in the SGBS cells were increased. Expression levels of the epithelial-mesenchymal transition (EMT)-inducing transcription factors FOXC2 and TWIST1 were increased in the co-cultured MCF7 cells. In addition, the E-cadherin mRNA level was decreased, while the N-cadherin mRNA level was increased in the co-cultured MCF7 cells. ERα mRNA levels were significantly repressed in the co-cultured MCF7 cells. ERα gene expression in the MCF7 cells was decreased due to increased HIF1α in the SGBS cells. These results suggest that adipocytes can modify breast cancer cell ER gene expression through hypoxia and also can promote EMT processes in breast cancer cells, supporting an important role of obesity in aggressive breast cancer development.

Role of microRNA221 in regulating normal mammary epithelial hierarchy and breast cancer stem-like cells

Increasing evidence suggests that lineage specific subpopulations and stem-like cells exist in normal and malignant breast tissues. Epigenetic mechanisms maintaining this hierarchical homeostasis remain to be investigated. In this study, we found the level of microRNA221 (miR-221) was higher in stem-like and myoepithelial cells than in luminal cells isolated from normal and malignant breast tissue. In normal breast cells, over-expression of miR-221 generated more myoepithelial cells whereas knock-down of miR-221 increased luminal cells. Over-expression of miR-221 stimulated stem-like cells in luminal type of cancer and the miR-221 level was correlated with clinical outcome in breast cancer patients. Epithelial-mesenchymal transition (EMT) was induced by overexpression of miR-221 in normal and breast cancer cells. The EMT related gene ATXN1 was found to be a miR-221 target gene regulating breast cell hierarchy. In conclusion, we propose that miR-221 contributes to lineage homeostasis of normal and malignant breast epithelium.

Cell fusion between gastric epithelial cells and mesenchymal stem cells results in epithelial-to-mesenchymal transition and malignant transformation.

BackgroundThe discovery of cancer stem cells and tumor heterogeneity prompted the exploration of additional mechanisms aside from genetic mutations for carcinogenesis and cancer progression. The aim of the present study was to investigate the effect of cell fusion between mesenchymal stem cells and the gastric epithelial cells in tumorigenesis.MethodsCell fusion between cord blood mesenchymal stem cells and human gastric epithelial cells was performed in vitro. Cell scratch and transwell assays were performed to determine migration and invasion abilities of the hybrids. The expressions of epithelial-mesenchymal transition-related proteins and genes were analyzed by immunocytochemistry and real time quantitative PCR. Tumorigenesis of the hybrids was evaluated through in vivo inoculation in nude mice.ResultsHybrids expressed the phenotypes of both donor cells. Aneuploidy was observed in 84.1% of cells. The hybrids showed increased proliferation, migration and invasion abilities compared with the parental cells. In addition, the expression of N-cadherin and vimentin in the hybrids was significantly higher than that of the epithelial cells, and the mRNA expression of the epithelial-mesenchymal transition-related genes, Twist and Slug, in the hybrids was also increased compared with that of the parental epithelial cells. Furthermore, the hybrids formed masses of epithelial origin with glandular structures in BALB/c nude mice.ConclusionsThese findings suggest that cell fusion between gastric epithelial cells and mesenchymal stem cells may result in epithelial to mesenchymal transition and malignant transformation.

Abstract LB-48: DCLK1 targeted monoclonal antibodies demonstrate therapeutic potential against pancreatic ductal adenocarcinoma

Abstract Doublecortin-like kinase 1 (DCLK1) is overexpressed and marks a population of tumor-initiating cells in pancreatic ductal adenocarcinoma (PDAC). It regulates key oncogenes, pluripotency factors, angiogenic factors, and epithelial mesenchymal transition (EMT) related transcription factors. Therefore, DCLK1 may be a novel therapeutic target in PDAC. In this study we evaluated whether DCLK1-targeted monoclonal antibodies (mAbs) demonstrate therapeutic activity against pancreatic cancer. Human pancreatic cancer cells (AsPC-1) were treated with mAbs against the C-terminal domain of human DCLK1 to assess their effect on gene expression, cell proliferation, migration, invasion, and tumorigenicity. Gene expression levels were analyzed by quantitative real-time RT-PCR. The proliferative and invasive potential of cells with or without mAb treatment were compared using a MTT assay for proliferation, wound healing assay for migration, and Matrigel coated transwell assay for invasion. Tumor xenografts originated from AsPC-1 cells in nude mice were also treated with these mAbs. Here we demonstrate that treating AsPC-1 cells with mAb against DCLK1 resulted in a more than 50% decrease in DCLK1 mRNA expression. The expression levels of several key tumor suppressor microRNAs, let7a, miR144, and miR200, were upregulated in cells treated with these mAbs. The expression levels of pancreatic cancer related oncogenes KRAS, NOTCH, and REG4, angiogenic factors, VEGFR1 and VEGFR2, and EMT related genes, ZEB1, ZEB2, SNAIL, SLUG, and TWIST, were significantly downregulated (among 30-50% reduction) in the cells treated with mAb against DCLK1. Furthermore, AsPC-1 cells exhibited more than 20% decrease in migration and more than 40% decrease in invasion following anti-DCLK1 mAb treatment. Moreover, AsPC-1 xenograft tumor volumes were reduced approximately 20% following mAb treatment. These data implicate the C-terminal of DCLK1 as a potential signal transduction regulator of DCLK1 in cancer via tumor suppressor microRNA related mechanism similar to siRNA mediated knockdown of DCLK1. These results suggest that utilizing mAb targeting DCLK1 may represent a potential novel therapeutic strategy to reduce the tumor stem-like cell subpopulation in PDAC. Citation Format: Dongfeng Qu, Nathaniel Weygant, Randal May, Parthasarathy Chandrakesan, Daniel Owen, Sripathi Sureban, Courtney Houchen. DCLK1 targeted monoclonal antibodies demonstrate therapeutic potential against pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-48. doi:10.1158/1538-7445.AM2014-LB-48

Two cases of placental mesenchymal dysplasia: Case reports

Two cases of placental mesenchymal dysplasia: Case reports

PTEN Loss-Mediated Akt Activation Increases the Properties of Cancer Stem-Like Cell Populations in Prostate Cancer

Objective: To demonstrate that the PTEN/PI3K/Akt/NF-κB pathway plays an important role in regulating the prostate cancer stem-like cell population by upregulating ABCG2. Methods: Targeted PTEN knockdown in human prostate DU145 and 22Rv1 cells using a small interfering RNA were confirmed by immunoblot analysis using antibodies of PTEN, phospho-Akt, Akt, and a-tubulin. Knockdown PTEN DU145 and 22Rv1 cells were augmented, and the stem cell-like properties were examined by cell viability and tumor sphere formation and treated by Akt IV inhibitor to provide the signal transduction pathway. Luciferase activity assays were performed. Results: The knockdown of PTEN in prostate cancer cell lines increased the stem-like properties of the cells, including their sphere-forming ability, stem cell population number, epithelial-mesenchymal transition-related gene expression, and ABCG2 expression. Additionally, PTEN expression was highly associated with elevated expression of phospho-Akt. Treatment with an Akt inhibitor suppressed the PTEN-mediated effects on the properties of these stem-like cells as well as drug resistance, ABCG2 expression, and the NF-κB pathway. Conclusion: The loss of PTEN in prostate cancer cells resulted in an increased PI3K/Akt pathway. Due to the Akt activation, PTEN loss may play an important role in prostate cancer by promoting cancer stemness through a mechanism that involves enhanced NF-κB signaling.

Elucidation of pathogenic mechanisms for Fabry disease by transcriptome analysis (690.10)

Fabry disease is an X‐linked inborn error of glycosphingolipid metabolism associated with deficient alpha‐galactosidase A activity. The major clinical feature of Fabry disease, such as fibrosis in cardiomyopathy and renal failure has been observed with abnormal accumulation of globotriaosylceramide (Gb3) in biological fluids, vascular endothelium, heart and kidney. In addition to Gb3, increased concentration of deacylated Gb3 (lyso‐Gb3) in the plasma of symptomatic patients also has been suggested as a causative molecular event. However, the correlation between fibrogenesis and elevated levels of these sphingolipids is poorly understood. To elucidate the genetic mechanisms involved in renal fibrosis in fabry disease, we analyzed the changes of global gene expression before and after Gb3 or lyso‐Gb3 treatment on two types of kidney cell lines, human proximal renal tubular epithelial cells (HK‐2) and mouse renal glomerular mesangial cells (SV40MES13), using microarray. The results showed that Gb3 and lyso‐Gb3 regulate the expression of 199 and 328 genes in each cell type, with at least 2.0‐fold change considerations. To identify key biological functions, we classified all the genes into 4 groups based on the expression patterns (1) Gb3 up, (2) Gb3 down, (3)lyso‐Gb3 up, (4)lyso‐Gb3 down regulated genes and performed functional annotation analysis using the DAVID tool. Most biological functions were related to fibrogenesis or epithelial‐mesenchymal transition (EMT). Interestingly, the gene expression patterns were significantly different between treated with Gb3 and treated with lyso‐Gb3. The expressions of EMT related genes (F8, FOXP2, HOXA11, DLL1 and WT1) were confirmed by realtime‐PCR or Western blot. These findings suggested that Gb3 and lyso‐Gb3 lead to renal fibrosis in Fabry disease with different biochemical modulations.

Inhibition of mTOR pathway attenuates migration and invasion of gallbladder cancer via EMT inhibition

Gallbladder cancer (GBC) is an aggressive disease in which epithelial-mesenchymal transition (EMT) plays a critical role. Whether inhibition of mTOR effects via EMT reversal in GBC remains unclear. Using genetic and pharmacologic inhibitions of mTOR, we investigated the changes of EMT levels in GBC cells. Expressions of EMT related genes were also studied. Migration and invasion assays were carried out and in vivo tumour metastasis mouse models were established. Circulating tumour DNA was quantified. We used EMT index (ratio of Vimentin/Ecadherin expression) to profile EMT levels. We found that inhibition of mTOR using shRNAs and rapamycin inhibited EMT in GBC-SD gallbladder cancer cells. Inhibition of mTOR inhibited EMT in GBC-SD cells in TGF-β-dependent manner, which was contributed majorly by mTORC2 inhibition. Rapamycin decreased invasiveness and migration of GBC-SD cells in vitro and in vivo. We have in the current study shown that rapamycin diminishes the ability of invasion and migration of GBC via inhibition of TGF-β-dependent EMT. Our findings contribute to the understanding of the carcinogenesis of GBC.

The Effect of CXCR4 Silencing on Epithelial‐Mesenchymal Transition Related Genes in Glioma U87 Cells

The epithelial-mesenchymal transition (EMT) of tumor cells is deemed to be closely associated with tumor metastasis. CXCR4 has been proved to play an important role in the process of tumor metastasis. This study illustrates the function and expression of CXCR4 silencing and the EMT related genes in the human glioma cell line U87. The results showed that CXCR4 silencing could inhibit the cell invasive and adhesion potentials, expression of N-cadherin, vimentin, β-catenin, TGF-β1, p-Smad2, and p-Akt, and the activity of transcription factors NF-κB, AP-1, Snail, and twist. Meanwhile, CXCR4 silencing could also up-regulate the expression of E-cadherin, indicating that silencing of CXCR4 expression can inhibit the expression of EMT related genes in U87 cells. The study would provide a potential theoretical basis for the further exploration of the role of CXCR4 in human glioma.

Expression and Prognostic Significance of a Comprehensive Epithelial-Mesenchymal Transition Gene Set in Renal Cell Carcinoma

Epithelial-mesenchymal transition enhances tumor cell motility and has a critical role in invasion and metastasis in a number of carcinomas. A set of transcription factors acts as a master regulator of the epithelial-mesenchymal transition process. To our knowledge it is unknown whether epithelial-mesenchymal transition is important for clear cell renal cell carcinoma progression. Therefore, we comprehensively assessed mRNA levels of epithelial-mesenchymal transition associated genes in renal cell carcinoma as well as their prognostic relevance. We determined the expression of a set of 46 epithelial-mesenchymal transition related genes by oligonucleotide microarray and gene set enrichment analyses using RNA from 14 samples each of normal kidneys, and G1 and G3 primary renal cell carcinomas. Expression of select epithelial-mesenchymal transition genes was validated by real-time polymerase chain reaction in normal kidneys, primary renal cell carcinomas and metastases in an independent cohort of 112 patients. Results were combined with followup data for survival analysis. The epithelial-mesenchymal transition gene set was preferentially expressed in primary renal cell carcinoma compared to normal tissue (false discovery rate 0.01). No difference was found between G1 and G3 tumors. Quantitative reverse transcriptase-polymerase chain reaction revealed down-regulation of critical epithelial-mesenchymal transition genes such as CDH2 and ZEB1 in metastases, suggesting epithelial-mesenchymal transition reversal during metastasis. Kaplan-Meier analysis demonstrated a better outcome in patients with low CXCR4, vimentin, fibronectin and TWIST1 mRNA levels. Multivariate analyses revealed that CXCR4 and VIM up-regulation represents an independent prognostic marker for poor cancer specific survival in patients with renal cell carcinoma. Taken together, our data provide strong evidence that epithelial-mesenchymal transition occurs in renal cell carcinoma. Thus, interference with epithelial-mesenchymal transition in renal cell carcinoma might represent a future therapeutic option.

Abstract 960: Blocking the Notch pathway induces mesenchymal-to-epithelial transition in lung adenocarcinoma: A novel approach to overcoming chemoresistance.

Abstract Backgroung: The Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. EMT cells possess the characteristics of cell motility, invasiveness, and have stem cells properties. EMT expression profile correlates with resistance to chemotherapy and poor outcome in multiple tumors and there is evidence suggesting an integral role of Notch pathway in mediating an EMT status. In this study, we investigated Notch activity and EMT status and the correlation with chemoressitance in lung adenocarcinoma. Results: Transduction of multiple lung adenocarcinoma cell lines, NCI-H1299, NCI-H460, NCI-H358, NCI-H441 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-positive). We compared cell migration capacity of GFP-positive and -negative cells utilizing a transwell invasion assay. Cells with high Notch activity, as evidenced by GFP expression, had a significantly higher migration capability as compared to GFP-negative cells. To evaluate resistance to chemotherapy, we treated GFP-pos and GFP-neg cells with docetaxel or cisplatin and measured apoptosis by Annexin V expression with flow cytometry. GFP-pos cells showed major resistance to both agents as compared to GFP-neg cells. When GFP-pos cells were exposed to a Notch activity blocker, gamma secretase inhibitor (GSI), these cells were rendered sensitive to cisplatin or docetaxel exposure as evident by an increase in Annexin V expression. Using RNA microarray analysis and RT-PCR assays, there was an increased expression of EMT related genes (Vimentin, N-Cadherin, Snail-1,….) in GFP-pos cells as compared to GFP-neg cells. Furthermore, NOD/SCID mice with subcutaneous primary lung tumor xenografts treated with GSI showed decreased expression of downstream effectors of Notch as as well as vimentin. Mice treated with combination of GSI and docetaxel had better tumor control as compared to those treated with GSI and docetaxel as single agents. Conclusion: These studies suggest that the Notch pathway is an important regulator of EMT status and that its inhibition induces mesenchymal to epithelial transition overcoming chemoresistnace. Notch inhibitors in combination with standard chemotherapy present a potential effective approach to achieve better disease control in lung cancer. Citation Format: Khaled Hassan, Luo Wang, Hasan Korkaya, Paige O'Dowd, Sarah Dubaisi, Rayyan Kadi, Rawad Nasr, Binghui Zhang, April Davis, Gregory Kalemkerian, Max Wicha. Blocking the Notch pathway induces mesenchymal-to-epithelial transition in lung adenocarcinoma: A novel approach to overcoming chemoresistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 960. doi:10.1158/1538-7445.AM2013-960

456 IDENTIFICATION OF EMT-RELATED MIRNA-200 FAMILY REGULATED NOVEL CANCER PATHWAYS IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research (III)1 Apr 2013456 IDENTIFICATION OF EMT-RELATED MIRNA-200 FAMILY REGULATED NOVEL CANCER PATHWAYS IN RENAL CELL CARCINOMA Hirofumi Yoshino, Naohiko Seki, Hideo Hidaka, Takeshi Yamasaki, Toshihiko Idesako, Tomokazu Yonezawa, Hideki Enokida, and Masayuki Nakagawa Hirofumi YoshinoHirofumi Yoshino Kagoshima, Japan More articles by this author , Naohiko SekiNaohiko Seki Chiba, Japan More articles by this author , Hideo HidakaHideo Hidaka Kagoshima, Japan More articles by this author , Takeshi YamasakiTakeshi Yamasaki Kagoshima, Japan More articles by this author , Toshihiko IdesakoToshihiko Idesako Kagoshima, Japan More articles by this author , Tomokazu YonezawaTomokazu Yonezawa Kagoshima, Japan More articles by this author , Hideki EnokidaHideki Enokida Kagoshima, Japan More articles by this author , and Masayuki NakagawaMasayuki Nakagawa Kagoshima, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1847AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recently, miR-200 family was reported to play a major role to epithelial to mesenchymal transition (EMT). Our recent microRNA (miRNA) expression signatures of RCC revealed that miR-200-family (miR-200a/b/c, miR-141 and miR-429) was significantly reduced in RCC clinical specimens. The aim of this study was to investigate the functional significance of miR-200-family in cancer cells and to identify novel miR-200-family-mediated cancer pathways in RCC. METHODS Gain-of-function studies were performed to investigate cancer cell viability by mature miRNAs transfection into RCC cells. To identify the biological processes or pathways potentially regulated by the miRNAs, we applied genome-wide gene expression analysis and in silico study; TargetScan database [http://www.targetscan.org/] and GENECODIS software, which assigned a number of the putative miRNA targets to known pathways in KEGG [http://www.genome.jp/kegg/pathway.html]. Furthermore, we performed gene expression analyses of all candidate genes involved in each of the pathways by using microarray expression data containing 53 clinical RCCs and 23 normal kidneys, and constructed heat map diagrams. RESULTS Expression levels of miR-200-family were significantly reduced in RCC clinical specimens compared to adjacent non-cancerous tissues (P<0.0001). Restoration of each miR-200-family significantly inhibited cancer cell proliferation, migration, and invasion in the RCC cell lines. The mRNA expression levels of ZEB1 and ZEB2 (EMT related genes) were significantly decreased, whereas that of E-cadherin (epithelium marker) was increased by miR-200-family transfection into the RCC cells. Our in silico analysis showed that miR-200-family appeared to be an important modulator of tumor cell processes through suppression of many targets, particularly those involved in ‘Pathways in cancer' and ‘ErbB' signaling pathways (P<0.05). Furthermore, gene expression analysis showed that MYC and EGFR in those pathway were significantly up-regulated in clinical RCCs (P<0.05). CONCLUSIONS Our analysis data showed that tumor suppressive miR-200-family might contribute to EMT through regulating several oncogenic pathways, especially related to MYC and EGFR, suggesting that these genes might be key molecules for RCC treatment. Elucidation of tumor suppressive miR-200-family-mediated cancer pathways provides the novel therapeutic strategy to control local tumor recurrence or distant metastasis of RCC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e187 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hirofumi Yoshino Kagoshima, Japan More articles by this author Naohiko Seki Chiba, Japan More articles by this author Hideo Hidaka Kagoshima, Japan More articles by this author Takeshi Yamasaki Kagoshima, Japan More articles by this author Toshihiko Idesako Kagoshima, Japan More articles by this author Tomokazu Yonezawa Kagoshima, Japan More articles by this author Hideki Enokida Kagoshima, Japan More articles by this author Masayuki Nakagawa Kagoshima, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Loss of the Keratin Cytoskeleton Is Not Sufficient to Induce Epithelial Mesenchymal Transition in a Novel KRAS Driven Sporadic Lung Cancer Mouse Model

Epithelial-to-mesenchymal transition (EMT), the phenotypical change of cells from an epithelial to a mesenchymal type, is thought to be a key event in invasion and metastasis of adenocarcinomas. These changes involve loss of keratin expression as well as loss of cell polarity and adhesion. We here aimed to determine whether the loss of keratin expression itself drives increased invasion and metastasis in adenocarcinomas and whether keratin loss leads to the phenotypic changes associated with EMT. Therefore, we employed a recently described murine model in which conditional deletion of the Keratin cluster II by Cre-recombinase leads to the loss of the entire keratinmultiprotein family. These mice were crossed into a newly generated Cre-recombinase inducible KRAS-driven murine lung cancer model to examine the effect of keratin loss on morphology, invasion and metastasis as well as expression of EMT related genes in the resulting tumors. We here clearly show that loss of a functional keratin cytoskeleton did not significantly alter tumor morphology or biology in terms of invasion, metastasis, proliferation or tumor burden and did not lead to induction of EMT. Further, tumor cells did not induce synchronously expression of vimentin, which is often seen in EMT, to compensate for keratin loss. In summary, our data suggest that changes in cell shape and migration that underlie EMT are dependent on changes in signaling pathways that cause secondary changes in keratin expression and organization. Thus, we conclude that loss of the keratin cytoskeleton per se is not sufficient to causally drive EMT in this tumor model.

Abstract C90: Gross appearance of hepatocellular carcinoma reflects expression of cancer stem cells, epithelial-mesenchymal transition, and hypoxia-related genes

Abstract The molecular background for the various gross appearances of hepatocellular carcinoma (HCC) have not yet been clarified, although the prognostic value thereof has been previously reported. Accordingly, in the present study, differences in the expression of cancer stem cells (CSC), epithelial-mesenchymal transition (EMT), and hypoxia related genes, according to the gross type of HCC, were evaluated in two independent cohorts composed of surgically resected HCCs (Cohort 1, n=99; cohort 2, n=110). The gross appearances of HCC were evaluated according to the “General Rules for the Clinical and Pathological Study of Primary Liver Cancer” set by the Liver Cancer Study Group of Japan, and categorized into single nodular (SN) type, single nodular type with extranodular growth (SNEG) type, confluent multinodular (CM) type, and infiltrative (IF) type. Additionally, transcription levels of CSC related genes (EpCAM, Oct3/4, Nanog), EMT regulator (Snail, Twist), and hypoxia related genes (HIF1α, CA9) were evaluated in cohort 1, and the protein expressions of EpCAM and CA9 were evaluated in cohort 2. Clinicopathologic findings, including vascular invasion and capsule formation, as well as prognosis, were also compared according to the gross types of HCC. As observed in this study, IF type HCCs were associated with more frequent portal vein invasion, tumor multiplicity and lack of capsule formation, compared to the other types of HCC (P&amp;lt;0.05 for all), in cohort 1; a similar trend was also identified in cohort 2. In cohort 1, IF type HCCs demonstrated significantly higher transcription levels of EpCAM, Snail and HIF1α (P&amp;lt;0.05 for all) than SN type HCCs. Moreover, the transcription level of HIF1α was also higher in IF type HCCs than both SNEG and CM type HCCs. SN type HCCs exhibited lower transcription levels of EpCAM and Snail compared to CM type and IF type HCCs. In cohort 2, CA9 protein expression was more frequently noted in IF type HCCs than other types of HCC (P&amp;lt;0.05 at all). The expression levels of CSC, EMT and hypoxia related genes showed a tendency to increase in SN, SNEG, CM and IF type HCCs, in that order as like the invasive pathological properties of the HCCs. IF type HCCs showed significantly decreased disease free survival and overall survival in comparison to the other types of HCC in both cohorts 1 and 2. In conclusion, our study demonstrated that IF type HCCs are associated with an increased expression of CSCs, EMT, and hypoxia related genes, all of which are potentially related to their invasive nature and poor prognosis. Citation Format: Hyungjin Rhee, Na Deuk Chae, Park Young Nyun. Gross appearance of hepatocellular carcinoma reflects expression of cancer stem cells, epithelial-mesenchymal transition, and hypoxia-related genes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C90.

Epithelial-mesenchymal transition-related gene expression as a new prognostic marker for neuroblastoma

Neuroblastoma (NB) is a highly metastatic tumor in children. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. This study investigated the role of EMT in the progression of NB. Using EMT assays on samples from 11 tumors, we identified 14 genes that were either differentially expressed between tumors of different stages or highly upregulated in NB. Quantitative RT-PCR of these genes was conducted in 96 NB tumors and their expression levels were compared between stages and between tumors with the presence and absence of MYCN amplification. The association of survival rate with differential gene expression was investigated. Expression of KRT19 was significantly decreased in stage 3 or 4 NB as well as stage 4S NB compared with stage 1 or 2 NB. Expression levels of KRT19 and ERBB3 were significantly low, and expression levels of TWST1 and TCF3 were high in MYCN-amplified NB. The patients with low expression of KRT19 or ERBB3 showed significantly worse overall survival. Furthermore, the correlation between high invasive ability and low expression of KRT19 and ERBB3 was suggested in vitro using six NB cell lines. The authors conclude that downregulation of KRT19 is highly associated with tumor progression in NB and metastasis in localized primary NB and that low expression of ERBB3 is also associated with progression of NB.