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• W1996164472 abstract "The progression of colorectal carcinoma (CRC) to invasive and metastatic disease may involve localized occurrences of epithelial-mesenchymal transition (EMT). However, mechanisms of the EMT process in CRC progression are not fully understood. We previously showed that knockdown of signal transducer and activator of transcription 3 (STAT3) up-regulated E-cadherin (a key component in EMT progression) in CRC. In this study, we examined the roles of STAT3 in CRC EMT and ZEB1, an EMT inducer, in STAT3-induced down-regulation of E-cadherin. Knockdown of STAT3 significantly increased E-cadherin and decreased N-cadherin and vimentin expressions in highly invasive LoVo CRC cells. Meanwhile, overexpression of STAT3 significantly reduced E-cadherin and enhanced N-cadherin and vimentin expressions in weakly invasive SW1116 CRC cells. Activation of STAT3 significantly increased CRC cell invasiveness and resistance to apoptosis. Knockdown of STAT3 dramatically enhanced chemosensitivity of CRC cells to fluorouracil. STAT3 regulated ZEB1 expression in CRC cells, and the STAT3-induced decrease in E-cadherin and cell invasion depended on activation of ZEB1 in CRC cells. Additionally, pSTAT3Tyr-705 and ZEB1 expressions were significantly correlated with TNM (tumor, lymph node, and metastasis stages) (p < 0.01). In conclusion, STAT3 may directly mediate EMT progression and regulate ZEB1 expression in CRC. ZEB1 may participate in STAT3-induced cell invasion and E-cadherin down-regulation in CRC cells. The expressions of pSTAT3Tyr-705 and ZEB1 may be positively associated with CRC metastasis. Our data may provide potential targets to prevent and/or treat CRC invasion and metastasis. The progression of colorectal carcinoma (CRC) to invasive and metastatic disease may involve localized occurrences of epithelial-mesenchymal transition (EMT). However, mechanisms of the EMT process in CRC progression are not fully understood. We previously showed that knockdown of signal transducer and activator of transcription 3 (STAT3) up-regulated E-cadherin (a key component in EMT progression) in CRC. In this study, we examined the roles of STAT3 in CRC EMT and ZEB1, an EMT inducer, in STAT3-induced down-regulation of E-cadherin. Knockdown of STAT3 significantly increased E-cadherin and decreased N-cadherin and vimentin expressions in highly invasive LoVo CRC cells. Meanwhile, overexpression of STAT3 significantly reduced E-cadherin and enhanced N-cadherin and vimentin expressions in weakly invasive SW1116 CRC cells. Activation of STAT3 significantly increased CRC cell invasiveness and resistance to apoptosis. Knockdown of STAT3 dramatically enhanced chemosensitivity of CRC cells to fluorouracil. STAT3 regulated ZEB1 expression in CRC cells, and the STAT3-induced decrease in E-cadherin and cell invasion depended on activation of ZEB1 in CRC cells. Additionally, pSTAT3Tyr-705 and ZEB1 expressions were significantly correlated with TNM (tumor, lymph node, and metastasis stages) (p < 0.01). In conclusion, STAT3 may directly mediate EMT progression and regulate ZEB1 expression in CRC. ZEB1 may participate in STAT3-induced cell invasion and E-cadherin down-regulation in CRC cells. The expressions of pSTAT3Tyr-705 and ZEB1 may be positively associated with CRC metastasis. Our data may provide potential targets to prevent and/or treat CRC invasion and metastasis. Despite welcome declines in mortality rates over the past decade, colorectal cancer (CRC) 4The abbreviations used are: CRCcolorectal cancerEMTepithelial-mesenchymal transitionANOVAanalysis of variancentnucleotide(s)TNMtumor, lymph node, and metastasis. remains a common malignancy and one of the leading causes of morbidity and death in the world (1.Bates R.C. Mercurio A.M. The epithelial-mesenchymal transition (EMT) and colorectal cancer progression.Cancer Biol. Ther. 2005; 4: 365-370Crossref PubMed Google Scholar, 2.Markowitz S.D. Dawson D.M. Willis J. Willson J.K. Focus on colon cancer.Cancer Cell. 2002; 1: 233-236Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Epithelial-mesenchymal transition (EMT) is a crucial process in the initiation of the metastatic spread of tumor cells to distal organs (3.Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7557) Google Scholar). EMT may promote epithelial cells to escape from the rigid structural constraints provided by the tissue architecture and adopt a phenotype more amenable to cell migration and movement (4.Hay E.D. An overview of epithelio-mesenchymal transformation.Acta Anat. 1995; 154: 8-20Crossref PubMed Scopus (1238) Google Scholar, 5.Savagner P. Leaving the neighborhood. Molecular mechanisms involved during epithelial-mesenchymal transition.BioEssays. 2001; 23: 912-923Crossref PubMed Scopus (607) Google Scholar, 6.Thiery J.P. Epithelial-mesenchymal transitions in development and pathologies.Curr. Opin. Cell Biol. 2003; 15: 740-746Crossref PubMed Scopus (1435) Google Scholar). In this progression, epithelial cells may lose adhesion and cell-to-cell contacts (7.Arima Y. Inoue Y. Shibata T. Hayashi H. Nagano O. Saya H. Taya Y. Rb depletion results in deregulation of E-cadherin and induction of cellular phenotypic changes that are characteristic of the epithelial-to-mesenchymal transition.Cancer Res. 2008; 68: 5104-5112Crossref PubMed Scopus (133) Google Scholar, 8.Papageorgis P. Lambert A.W. Ozturk S. Gao F. Pan H. Manne U. Alekseyev Y.O. Thiagalingam A. Abdolmaleky H.M. Lenburg M. Thiagalingam S. Smad signaling is required to maintain epigenetic silencing during breast cancer progression.Cancer Res. 2010; 70: 968-978Crossref PubMed Scopus (145) Google Scholar). Therefore, EMT can be regarded as a pathological process that contributes to cancer progression, particularly in tumor cell invasion and metastasis (9.Arias A.M. Epithelial mesenchymal interactions in cancer and development.Cell. 2001; 105: 425-431Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). colorectal cancer epithelial-mesenchymal transition analysis of variance nucleotide(s) tumor, lymph node, and metastasis. The initiation and progression of EMT require transduction of cell signals. The transforming growth factor-β (TGF-β) signaling and activated Ras pathways have been implicated as key EMT inducers in CRC cancer (10.Bhowmick N.A. Ghiassi M. Bakin A. Aakre M. Lundquist C.A. Engel M.E. Arteaga C.L. Moses H.L. Transforming growth factor-β1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism.Mol. Biol. Cell. 2001; 12: 27-36Crossref PubMed Scopus (853) Google Scholar, 11.Ellenrieder V. Hendler S.F. Boeck W. Seufferlein T. Menke A. Ruhland C. Adler G. Gress T.M. Transforming growth factor beta1 treatment leads to an epithelial-mesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation.Cancer Res. 2001; 61: 4222-4228PubMed Google Scholar). The Wnt, PI3K/AKT, and other signaling pathways may also play an important role in the EMT process in CRC progression (12.Joyce T. Cantarella D. Isella C. Medico E. Pintzas A. A molecular signature for Epithelial to Mesenchymal transition in a human colon cancer cell system is revealed by large-scale microarray analysis.Clin. Exp. Metastasis. 2009; 26: 569-587Crossref PubMed Scopus (42) Google Scholar, 13.Gulhati P. Bowen K.A. Liu J. Stevens P.D. Rychahou P.G. Chen M. Lee E.Y. Weiss H.L. O'Connor K.L. Gao T. Evers B.M. mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways.Cancer Res. 2011; 71: 3246-3256Crossref PubMed Scopus (447) Google Scholar, 14.Cottonham C.L. Kaneko S. Xu L. miR-21 and miR-31 converge on TIAM1 to regulate migration and invasion of colon carcinoma cells.J. Biol. Chem. 2010; 285: 35293-35302Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 15.Kalogeropoulou M. Voulgari A. Kostourou V. Sandaltzopoulos R. Dikstein R. Davidson I. Tora L. Pintzas A. TAF4b and Jun/activating protein-1 collaborate to regulate the expression of integrin α6 and cancer cell migration properties.Mol. Cancer Res. 2010; 8: 554-568Crossref PubMed Scopus (11) Google Scholar). Recently, accumulating evidence has indicated that abnormalities in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway are involved in CRC oncogenesis (16.Spano J.P. Milano G. Rixe C. Fagard R. JAK/STAT signaling pathway in colorectal cancer. A new biological target with therapeutic implications.Eur. J. Cancer. 2006; 42: 2668-2670Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 17.Lassmann S. Schuster I. Walch A. Göbel H. Jütting U. Makowiec F. Hopt U. Werner M. STAT3 mRNA and protein expression in colorectal cancer. Effects on STAT3-inducible targets linked to cell survival and proliferation.J. Clin. Pathol. 2007; 60: 173-179Crossref PubMed Scopus (95) Google Scholar). As a key component of the JAK/STAT pathway, STAT3 is constitutively activated in CRC (18.Xiong H. Zhang Z.G. Tian X.Q. Sun D.F. Liang Q.C. Zhang Y.J. Lu R. Chen Y.X. Fang J.Y. Inhibition of JAK1,2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells.Neoplasia. 2008; 10: 287-297Crossref PubMed Scopus (329) Google Scholar), and several lines of evidence have supported its role in mediating cell motility and migration. STAT3 is important for the migration of sheets of cells in zebrafish embryo development (19.Yamashita S. Miyagi C. Carmany-Rampey A. Shimizu T. Fujii R. Schier A.F. Hirano T. Stat3 controls cell movements during zebrafish gastrulation.Dev. Cell. 2002; 2: 363-375Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), and conditional depletion of this molecule blocks wound healing in mouse keratinocytes (20.Sano S. Itami S. Takeda K. Tarutani M. Yamaguchi Y. Miura H. Yoshikawa K. Akira S. Takeda J. Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis.EMBO J. 1999; 18: 4657-4668Crossref PubMed Scopus (433) Google Scholar). Additionally, gastrin may induce EMT in CRC through the JAK2/STAT3 pathway (21.Ferrand A. Kowalski-Chauvel A. Bertrand C. Pradayrol L. Fourmy D. Dufresne M. Seva C. Involvement of JAK2 upstream of the PI 3-kinase in cell-cell adhesion regulation by gastrin.Exp. Cell Res. 2004; 301: 128-138Crossref PubMed Scopus (39) Google Scholar). EGF receptor is overexpressed in ovarian carcinoma, whereas EGF-induced EMT in ovarian cancer cells has been shown to depend on IL-6R and the JAK2/STAT3 pathway (22.Colomiere M. Ward A.C. Riley C. Trenerry M.K. Cameron-Smith D. Findlay J. Ackland L. Ahmed N. Cross-talk of signals between EGFR and IL-6R through JAK2/STAT3 mediate epithelial-mesenchymal transition in ovarian carcinomas.Br. J. Cancer. 2009; 100: 134-144Crossref PubMed Scopus (262) Google Scholar). However, the role of STAT3 in the EMT process of CRC progression is not fully understood. Loss of E-cadherin expression is a crucial step and fundamental event of EMT in cancer progression (3.Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7557) Google Scholar). As a key component of adherens junctions, E-cadherin plays a crucial role in the maintenance of epithelial integrity (23.Perez-Moreno M. Jamora C. Fuchs E. Sticky business. Orchestrating cellular signals at adherens junctions.Cell. 2003; 112: 535-548Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). Many studies have reported on the regulation of E-cadherin during cancer progression (3.Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7557) Google Scholar, 24.Qiao Y. Jiang X. Lee S.T. Karuturi R.K. Hooi S.C. Yu Q. FOXQ1 regulates epithelial-mesenchymal transition in human cancers.Cancer Res. 2011; 71: 3076-3086Crossref PubMed Scopus (138) Google Scholar, 25.Ren D. Minami Y. Nishita M. Critical role of Wnt5a-Ror2 signaling in motility and invasiveness of carcinoma cells following Snail-mediated epithelial-mesenchymal transition.Genes Cells. 2011; 16: 304-315Crossref PubMed Scopus (82) Google Scholar), and several proteins, including Snail, ZEB1, and ZEB2, have been identified that may down-regulate E-cadherin in various cancers (3.Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7557) Google Scholar). In our previous studies, we found that knockdown of STAT3 by RNA interference (RNAi) significantly increased E-cadherin expression in CRC cells (18.Xiong H. Zhang Z.G. Tian X.Q. Sun D.F. Liang Q.C. Zhang Y.J. Lu R. Chen Y.X. Fang J.Y. Inhibition of JAK1,2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells.Neoplasia. 2008; 10: 287-297Crossref PubMed Scopus (329) Google Scholar). Whether STAT3 contributes to the EMT process of CRC progression and the mechanisms of STAT3-induced E-cadherin down-regulation are not known. We now show that STAT3 may directly induce cell invasion and participate in resistance to chemotherapy drugs and apoptosis during EMT of CRC progression. To our knowledge, this is the first study to report that STAT3 may directly mediate the EMT process and ZEB1 expression of CRC progression. STAT3-induced cell invasion and decrease in E-cadherin expression depend on activation of ZEB1 in CRC cells. The combination of pSTAT3Tyr-705/ZEB1 may be a novel predictor of CRC metastasis and a potential therapeutic target. Two human CRC cell lines SW1116 and LoVo (ATCC, Manassas, VA) were cultured in RPMI 1640 medium (Invitrogen), supplemented with 10% fetal bovine serum (FBS) at 37 °C in humidified 5% CO2 atmosphere. For AG490 (pharmacological JAK2 inhibitor; Sigma) treatment, CRC cells were incubated with 100 μm AG490 for 24 h (18.Xiong H. Zhang Z.G. Tian X.Q. Sun D.F. Liang Q.C. Zhang Y.J. Lu R. Chen Y.X. Fang J.Y. Inhibition of JAK1,2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells.Neoplasia. 2008; 10: 287-297Crossref PubMed Scopus (329) Google Scholar) before harvesting for measurements. The DNA fragment encoding the STAT3 gene (GenBank® accession number NM_003150) was amplified from human cDNA with the primers STAT3-F (5′-GCTAAGCTTTATGGCCCAATGGAATCAGCTACAG-3′ and STAT3-R (5′-GCTCTCGAGTCATGGGGGAGGTAGCGCACTCCG-3′), which introduced the cloning sites HindIII and XhoI (underlined), respectively. The cDNA fragment obtained above was verified by sequencing and finally cloned into pCDNA3.1 between the HindIII and XhoI sites to obtain pCDNA3.1-STAT3. The wild type DNA fragment containing part of the promoter region (−520 to +70 from transcriptional initiation site) of the E-cadherin gene (GenBank® accession number NM_004360) and the wild type DNA fragment containing part of the promoter region (−500 to +100 from the transcriptional initiation site) of the ZEB1 gene (GenBank® accession number NM_001174094) were amplified from human genomic DNA with the following primers, respectively: E-cadherinP-F (5′-GGGGTACCTGTCTCTCTACAAAAAGGCA-3′) and E-cadherinP-R (5′-GGAAGATCTGGGCTGGAGCGGGCTGGAGT-3′); ZEB1 P-F (5′- GGGGTACCAAAGACGTTTCCTTATTCGA-3′) and ZEB1 P-R (5′- GAAGATCTAGAAAGGCGACGGGCTGACC-3′), which introduced the cloning sites KpnI and BglII (underlined), respectively. The DNA fragment obtained above was directly cloned into pGL3-basic (Promega, Madison, WI) between the KpnI and BglII sites to obtain pGL3-E-cadherinPWT and pGL3-ZEB1PWT. The mutant DNA sequences of the ZEB1 promoter region encompassing both of the two putative binding sites of STAT3 (−500 to +100 from the transcriptional initiation site) or the mutant DNA sequences of the E-cadherin promoter region encompassing both of the two putative binding sites of STAT3 and four putative binding sites of ZEB1 (−520 to +70 from transcriptional initiation site) were synthesized and inserted into pGL3-basic vector. The mutant type constructs were designated as pGL3-basic-ZEB1P MT, pGL3-basic-E-cadherinP STAT3B MT, pGL3-basic-E-cadherinP ZEB1B MT, and pGL3-basic-E-cadherinP STAT3B and ZEB1B MT, respectively. T was replaced with G in each STAT3 binding site of pGL3-basic-ZEB1P MT, pGL3-basic-E-cadherinP STAT3BMT, and pGL3-basic-E-cadherinP STAT3B and ZEB1B MT constructs. CT and CTG was replaced with AA and AAA in each ZEB1 binding site of pGL3-basic-E-cadherinP ZEB1B MT and pGL3-basic-E-cadherinP STAT3B and ZEB1B MT constructs, respectively. The siRNA against ZEB1 (TCF8; catalog no. L-006564-01-0005), the siRNA against STAT3 (catalog no. L-003544-00-0005), and the control siRNA were purchased from Dharmacon RNA Technology (Lafayette, CO). Twenty-four h before transfection at 30–40% confluence, CRC cells were transferred to 6-well plates. Transfection of siRNAs was carried out with DharmaFECT 1 siRNA transfection reagent (Dharmacon) according to the manufacturer's instructions. Cells were collected for analysis 48 h after transfection. For plasmid transfections, CRC cells (70% confluence, ∼5 × 106 cells) were transfected with 2 μg of pCDNA3.1-STAT3 or pCDNA3.1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The cells were collected for measurements 48 h after transfection. To stably knock down ZEB1, we infected SW1116 and LoVo cells with MISSION shRNA lentivirus particles (with the puromycin resistance gene) containing a U6 promoter driving shRNA targeting human ZEB1 or scramble negative control (Sigma-Aldrich). Methods used for lentivirus production and infection were performed as described by Gire et al. (26.Gire V. Roux P. Wynford-Thomas D. Brondello J.M. Dulic V. DNA damage checkpoint kinase Chk2 triggers replicative senescence.EMBO J. 2004; 23: 2554-2563Crossref PubMed Scopus (147) Google Scholar). Total RNA was extracted by TRIzol reagent (Invitrogen), according to the protocol of the manufacturer, and 1.5 μg of total RNA from cultured cells was reverse transcribed using the PrimeScriptPTMP RT reagent kit (Perfect Real Time) for RT-PCR (Takara, Shiga, Japan). Quantitative real-time PCR was carried out on an Applied Biosystems 7900 quantitative PCR system. The primers used were as follows: ZEB1-F (5′-GCCAATAAGCAAACGATTCTG-3′), ZEB1-R (5′-TTTGGCTGGATCACTTTCAAG-3′), ZEB2-F (5′-CGGTGCAAGAGGCGCAAACA-3′), ZEB2-R (5′-GGAGGACTCATGGTTGGGCA-3′), Snail1-F (5′-CACTATGCCGCGCTCTTTC-3′), Snail1-R (5′-GGTCGTAGGGCTGCTGGAA-3′), Snail2-F (5′-AAACTACAGCGAACTGGACACA-3′), Snail2-R (5′-GCCCCAAAGATGAGGAGTATC-3′), Twist1-F (5′-AGTCCGCAGTCTTACGAGGA-3′), Twist1-R (5′- GCCAGCTTGAGGGTCTGAAT-3′), Twist2-F (5′-CAAGCTGAGCAAGATCCAGAC-3′), Twist2-R (5′-GGTCATCTTATTGTCCATCTCG-3′), E12/E47-F (5′- TCAAGCAATAACTTCTCGTCCA-3′), E12/E47-R (5′-CGTCCAGGTGGTCTTCTATCTT-3′)18S-F (5′-CGGACAGGATTGACAGATTGATAGC-3′), and 18S-R (5′-TGCCAGAGTCTCGTTCGTTATCG-3′). All reactions were performed in triplicate in a 10-μl total volume containing Brilliant® SYBR® Green QPCR Master Mix (Takara, Shiga, Japan). The amplified transcript level of each specific gene was normalized to that of 18S. Western blot analysis was performed using standard techniques as described previously (27.Lu R. Wang X. Chen Z.F. Sun D.F. Tian X.Q. Fang J.Y. Inhibition of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway decreases DNA methylation in colon cancer cells.J. Biol. Chem. 2007; 282: 12249-12259Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Kangchen, Shanghai, China) was detected as a loading control. Antibodies used in this study were purchased from Cell Signaling Technology Inc. (Beverly, MA). All primary antibodies were used at a 1:1000 dilution. Cell proliferation assay was assessed by a tetrazolium salt (WST-8)–based colorimetric assay in Cell Counting Kit 8 (Dojindo, Kumamoto, Japan) (28.Morita Y. Naka T. Kawazoe Y. Fujimoto M. Narazaki M. Nakagawa R. Fukuyama H. Nagata S. Kishimoto T. Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor α-induced cell death in fibroblasts.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5405-5410Crossref PubMed Scopus (169) Google Scholar, 29.Wang Y.Y. Zhou G.B. Yin T. Chen B. Shi J.Y. Liang W.X. Jin X.L. You J.H. Yang G. Shen Z.X. Chen J. Xiong S.M. Chen G.Q. Xu F. Liu Y.W. Chen Z. Chen S.J. AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia. Implication in stepwise leukemogenesis and response to Gleevec.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 1104-1109Crossref PubMed Scopus (268) Google Scholar). Briefly, control and CRC cells treated with different doses of fluorouracil were seeded onto 96-well plates at an initial density of 5 × 103 cells/well. At specified time points, 10 μl of Cell Counting Kit 8 solution were added to each well of the plate, which was then incubated for 2 h. Cell viability was determined by scanning with a microplate reader at 450 nm. Data are expressed as the percentage of viable cells calculated as follows: cell survival rate (%) = (A450(treated) − A450(blank))/(A450(control) − A450(blank)) × 100%. For flow cytometric analysis, an annexin-V fluorescein isothiocyanate/PI double stain assay was performed in accordance with the manufacturer's protocol (BioVision, Mountain View, CA). Analysis was performed using a flow cytometer. Cell invasion assays were performed as described by Hecht et al. (30.Hecht M. Papoutsi M. Tran H.D. Wilting J. Schweigerer L. Hepatocyte growth factor/c-Met signaling promotes the progression of experimental human neuroblastomas.Cancer Res. 2004; 64: 6109-6118Crossref PubMed Scopus (69) Google Scholar). In brief, chambers with 8-μm pore polycarbonate membranes, coated with Matrigel on the upper side, were used (BD Biosciences). CRC cells or stable CRC cell lines with ZEB1 gene knockdown were transfected with STAT3 siRNA, control siRNA, pCDNA3.1-STAT3, or pCDNA3.1 for 24 h. Transfected cells were then harvested, and 1 × 105 cells were seeded in serum-free medium into the upper chamber, whereas medium supplemented with 15% FBS was applied to the lower chamber as a chemoattractant to induce invasion. After incubation for 48 h, migrated cells on the bottom surface of the filter were fixed, stained, and counted. Designated combinations of pGL3-E-cadherinPWT, pGL3-ZEB1PWT, and other mutant constructs with other siRNA or plasmids at 1.0 μg and 100 ng of phRL (Renilla luciferase) TK plasmid (Promega) for monitoring transfection efficiency were transiently transfected in triplicate with Lipofectamine 2000 (Invitrogen) or DharmaFECT 1 siRNA transfection reagent (Dharmacon) according to the manufacturer's directions. Twenty-four h after transfection, the cells were collected to detect luciferase activity using the Dual-Luciferase reporter assay system (Promega). Luciferase activity was measured by using a BD Monolight 3010 luminometer (BD Biosciences). Variation in transfection efficiency was normalized by dividing the luciferase activity of the construct by the corresponding Renilla luciferase activity. Promoter activity is reported as the mean ± S.E. Chromatin immunoprecipitation assays were performed using the ChIP assay kit (Upstate, Charlottesville, VA) following the manufacturer's protocol. ChIP analysis was performed as described previously (31.Fu X. Beer D.G. Behar J. Wands J. Lambeth D. Cao W. cAMP-response element-binding protein mediates acid-induced NADPH oxidase NOX5-S expression in Barrett esophageal adenocarcinoma cells.J. Biol. Chem. 2006; 281: 20368-20382Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Antibodies against ZEB1 (Cell Signal Technology), STAT3 (Cell Signal Technology), and normal rabbit IgG (Upstate) were used. Real-time PCR was performed in triplicate using an Applied Biosystems 7900 quantitative PCR system. Each PCR was carried out in a 10-μl reaction volume by using 3 μl of the eluted immunoprecipitated DNA. The amount of genomic DNA co-precipitated with the specific antibody was calculated in comparison with the total input DNA used for each immunoprecipitation as follows: CBTB = CBTB(genomic input) − CBTB(specific antibody), where CBTB(genomic input) and CBTB(specific antibody) are the mean threshold cycles of PCR performed in triplicate on DNA samples from the genomic input samples and the specific antibody samples, respectively. All specimens were from patients (35 primary colorectal adenocarcinomas) who underwent surgery in Shanghai Renji Hospital from July 2009 to December 2010. The protocol was approved by the ethics committee of Shanghai Jiao-Tong University School of Medicine Renji Hospital, and the research was carried out according to the provisions of the Helsinki Declaration of 1975. None of the patients received preoperative treatments, such as radiotherapy or chemotherapy. Meanwhile, 21 specimens of normal colonic epithelium, taken from patients without colorectal cancer, were used as negative controls. The expressions of STAT3, pSTAT3Tyr-705, ZEB1, and E-cadherin were examined with primary antibodies (STAT3, pSTAT3Tyr-705, ZEB1, and E-cadherin; dilution 1:100) in consecutive tissue sections using the LSAB+ kit (DakoCytomation, Copenhagen, Denmark) according to the manufacturer's instructions. The slides were examined independently by two investigators blinded to both clinical and pathologic data. Protein expression was quantified using a visual grading system based on the extent of staining (percentage of positive tumor cells graded on a scale of 0–4: 0, none; 1, 1–25%; 2, 26–50%; 3, 51–75%; 4, 475%) and the intensity of staining (graded on a scale of 0–3: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining). For further analysis, an index value was calculated as a product of grades of the extent and intensity of staining to define the cut-off value for high expression of the proteins, and the protein expression was classified into two categories: high (grades 4–12) and low (grades 0–3). Statistical analysis was performed with SPSS 13.0 software. Data are expressed as means ± S.E. Statistical differences between two groups were determined by Student's t test. Differences between multiple groups were tested using analysis of variance (ANOVA) and checked for significance using Fisher's protected least significant difference test. Analyses comparing the expressions of STAT3, pSTAT3Tyr-705, ZEB1, and E-cadherin were performed using χ2 analysis and Fisher's exact test. Results were considered significant if the p value was less than 0.05. Correlation analysis was performed between pSTAT3Tyr-705 and ZEB1. We previously showed that activated STAT3 is constitutively expressed in CRC and mediates cell proliferation, whereas knockdown of STAT3 significantly restores E-cadherin expression (18.Xiong H. Zhang Z.G. Tian X.Q. Sun D.F. Liang Q.C. Zhang Y.J. Lu R. Chen Y.X. Fang J.Y. Inhibition of JAK1,2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells.Neoplasia. 2008; 10: 287-297Crossref PubMed Scopus (329) Google Scholar). Down-regulation of E-cadherin is one of the EMT phenotypes in cancer progression (3.Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7557) Google Scholar). To determine whether STAT3 mediates EMT initiation in CRC cells, the effect of STAT3 siRNA was evaluated in highly invasive LoVo CRC cells. Western blot analysis showed that STAT3 siRNA significantly decreased STAT3 expression and phosphorylation in these cells (Fig. 1, A and B), indicating that the STAT3 was knocked down effectively. Knockdown of STAT3 significantly increased E-cadherin and decreased N-cadherin and vimentin expressions (Fig. 1, A and B), suggesting that STAT3 may contribute to EMT progression in LoVo cells. To further confirm the role of STAT3 in down-regulation of E-cadherin and up-regulation of N-cadherin and vimentin in CRC cells, we constructed and transfected the recombinant pCDNA3.1-STAT3 plasmid into low invasion SW1116 cells. Transfection of the pCDNA3.1-STAT3 plasmid significantly increased STAT3 expression and phosphorylation in SW1116 cells, when compared with the pCDNA3.1 control (Fig. 1, C and D), indicating that STAT3 was successfully overexpressed. Overexpression of STAT3 significantly reduced E-cadherin and enhanced N-cadherin and vimentin expressions in SW1116 cells, compared with pCDNA3.1 transfection. These results further indicate that STAT3 may mediate EMT initiation and progression in CRC cells. It has been reported that EMT may induce cell migration, alter invasion properties, promote chemotherapy drug resistance, and prevent apoptosis (3.Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7557) Google Scholar). Aberrant cell survival and resistance to apoptosis are also hallmarks of tumor EMT progression in epithelial carcinoma (32.Jäättelä M. Escaping cell death. Survival proteins in cancer.Exp. Cell Res. 1999; 248: 30-43Crossref PubMed Scopus (582) Google Scholar). Therefore, we examined whether STAT3 participates in these aspects of EMT progression in CRC cells. Transwell cell invasion assays showed that knockdown of STAT3 expression significantly reduced the invasion ability of LoVo cells normal" @default.
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• W1996164472 cites W1964081206 @default.
• W1996164472 cites W1964212105 @default.
• W1996164472 cites W1978559606 @default.
• W1996164472 cites W1981840337 @default.
• W1996164472 cites W1986834354 @default.
• W1996164472 cites W1992623236 @default.
• W1996164472 cites W1993817279 @default.
• W1996164472 cites W2001158436 @default.
• W1996164472 cites W2008555822 @default.
• W1996164472 cites W2009410973 @default.
• W1996164472 cites W2013480667 @default.
• W1996164472 cites W2019031019 @default.
• W1996164472 cites W2025647419 @default.
• W1996164472 cites W2032471037 @default.
• W1996164472 cites W2036699146 @default.
• W1996164472 cites W2038569449 @default.
• W1996164472 cites W2039298921 @default.
• W1996164472 cites W2041553436 @default.
• W1996164472 cites W2042502745 @default.
• W1996164472 cites W2043911925 @default.
• W1996164472 cites W2053271495 @default.
• W1996164472 cites W2053668040 @default.
• W1996164472 cites W2056956799 @default.
• W1996164472 cites W2060796494 @default.
• W1996164472 cites W2061023870 @default.
• W1996164472 cites W2066678136 @default.
• W1996164472 cites W2071749269 @default.
• W1996164472 cites W2076118448 @default.
• W1996164472 cites W2077494791 @default.
• W1996164472 cites W2079016331 @default.
• W1996164472 cites W2084487847 @default.
• W1996164472 cites W2084813619 @default.
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• W1996164472 cites W2118650242 @default.
• W1996164472 cites W2127357183 @default.
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