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Roles of STAT3 and ZEB1 Proteins in E-cadherin Down-regulation and Human Colorectal Cancer Epithelial-Mesenchymal Transition *

Roles of STAT3 and ZEB1 Proteins in E-cadherin Down-regulation and Human Colorectal Cancer... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 8, pp. 5819 –5832, February 17, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Roles of STAT3 and ZEB1 Proteins in E-cadherin Down-regulation and Human Colorectal Cancer Epithelial-Mesenchymal Transition Received for publication, August 19, 2011, and in revised form, December 21, 2011 Published, JBC Papers in Press, December 28, 2011, DOI 10.1074/jbc.M111.295964 1 1,2 Hua Xiong , Jie Hong , Wan Du, Yan-wei Lin, Lin-lin Ren, Ying-chao Wang, Wen-yu Su, Ji-lin Wang, Yun Cui, Zhen-hua Wang, and Jing-Yuan Fang From the Gastrointestinal Division, Shanghai Jiao-Tong University School of Medicine Renji Hospital, Shanghai Institution of Digestive Disease, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health (Shanghai Jiao-Tong University), State Key Laboratory of Oncogenes and Related Genes, 145 Middle Shandong Road, Shanghai 200001, China Background: Colorectal cancer (CRC) to metastatic disease may involve the epithelial-mesenchymal transition (EMT). Results: STAT3 may regulate N-cadherin, vimentin, and ZEB1 expressions. STAT3-induced cell invasion and down-regulation of E-cadherin may depend on ZEB1. Conclusion: STAT3 may mediate CRC EMT progression and ZEB1 expression. Activation of STAT3 and ZEB1 proteins may contribute to worse prognosis in CRC patients. Significance: Our data may provide potential targets to prevent and/or treat CRC invasion. The progression of colorectal carcinoma (CRC) to invasive Despite welcome declines in mortality rates over the past and metastatic disease may involve localized occurrences of epi- decade, colorectal cancer (CRC) remains a common malig- thelial-mesenchymal transition (EMT). However, mechanisms nancy and one of the leading causes of morbidity and death in of the EMT process in CRC progression are not fully under- the world (1, 2). Epithelial-mesenchymal transition (EMT) is a stood. We previously showed that knockdown of signal trans- crucial process in the initiation of the metastatic spread of ducer and activator of transcription 3 (STAT3) up-regulated tumor cells to distal organs (3). EMT may promote epithelial E-cadherin (a key component in EMT progression) in CRC. In cells to escape from the rigid structural constraints provided by this study, we examined the roles of STAT3 in CRC EMT and the tissue architecture and adopt a phenotype more amenable ZEB1, an EMT inducer, in STAT3-induced down-regulation of to cell migration and movement (4–6). In this progression, E-cadherin. Knockdown of STAT3 significantly increased E-cad- epithelial cells may lose adhesion and cell-to-cell contacts (7, 8). herin and decreased N-cadherin and vimentin expressions in Therefore, EMT can be regarded as a pathological process that highly invasive LoVo CRC cells. Meanwhile, overexpression of contributes to cancer progression, particularly in tumor cell STAT3 significantly reduced E-cadherin and enhanced N-cad- invasion and metastasis (9). herin and vimentin expressions in weakly invasive SW1116 CRC The initiation and progression of EMT require transduction cells. Activation of STAT3 significantly increased CRC cell inva- of cell signals. The transforming growth factor- (TGF-) sig- siveness and resistance to apoptosis. Knockdown of STAT3 dra- naling and activated Ras pathways have been implicated as key matically enhanced chemosensitivity of CRC cells to fluorouracil. EMT inducers in CRC cancer (10–11). The Wnt, PI3K/AKT, STAT3 regulated ZEB1 expression in CRC cells, and the STAT3- and other signaling pathways may also play an important role in induceddecreaseinE-cadherinandcellinvasiondependedonacti- the EMT process in CRC progression (12–15). Recently, accu- Tyr-705 vation of ZEB1 in CRC cells. Additionally, pSTAT3 and mulating evidence has indicated that abnormalities in the Janus ZEB1 expressions were significantly correlated with TNM (tumor, kinase/signal transducer and activator of transcription (JAK/ lymph node, and metastasis stages) (p < 0.01). In conclusion, STAT) pathway are involved in CRC oncogenesis (16, 17). As a STAT3 may directly mediate EMT progression and regulate ZEB1 key component of the JAK/STAT pathway, STAT3 is constitu- expression in CRC. ZEB1 may participate in STAT3-induced cell tively activated in CRC (18), and several lines of evidence have invasion and E-cadherin down-regulation in CRC cells. The supported its role in mediating cell motility and migration. Tyr-705 expressions of pSTAT3 and ZEB1 may be positively associ- STAT3 is important for the migration of sheets of cells in ated with CRC metastasis. Our data may provide potential targets zebrafish embryo development (19), and conditional depletion to prevent and/or treat CRC invasion and metastasis. of this molecule blocks wound healing in mouse keratinocytes (20). Additionally, gastrin may induce EMT in CRC through the JAK2/STAT3 pathway (21). EGF receptor is overexpressed in * This work was supported by National Natural Science Foundation of China ovarian carcinoma, whereas EGF-induced EMT in ovarian can- Grant 30900757 (to F. J. Y.), National Natural Science Foundation of China Grant 91129724 (to J. H.), and “Chen Guang” Project Grant 09CG13 (to cer cells has been shown to depend on IL-6R and the JAK2/ J. H.). Both authors contributed equally to this work. To whom correspondence may be addressed. Tel.: 86-21-63200874; Fax: 86-21-63266027; E-mail: [email protected]. The abbreviations used are: CRC, colorectal cancer; EMT, epithelial-mesen- To whom correspondence may be addressed. Tel.: 86-21-63200874; Fax: chymal transition; ANOVA, analysis of variance; nt, nucleotide(s); TNM, 86-21-63266027; E-mail: [email protected]. tumor, lymph node, and metastasis. FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5819 This is an Open Access article under the CC BY license. STAT3 and ZEB1 Signaling in CRC EMT Progression STAT3 pathway (22). However, the role of STAT3 in the EMT mega, Madison, WI) between the KpnI and BglII sites to obtain process of CRC progression is not fully understood. pGL3-E-cadherinPWT and pGL3-ZEB1PWT. Loss of E-cadherin expression is a crucial step and funda- The mutant DNA sequences of the ZEB1 promoter region mental event of EMT in cancer progression (3). As a key com- encompassing both of the two putative binding sites of STAT3 ponent of adherens junctions, E-cadherin plays a crucial role in (500 to 100 from the transcriptional initiation site) or the the maintenance of epithelial integrity (23). Many studies have mutant DNA sequences of the E-cadherin promoter region reported on the regulation of E-cadherin during cancer pro- encompassing both of the two putative binding sites of STAT3 gression (3, 24, 25), and several proteins, including Snail, ZEB1, and four putative binding sites of ZEB1 (520 to 70 from and ZEB2, have been identified that may down-regulate E-cad- transcriptional initiation site) were synthesized and inserted herin in various cancers (3). In our previous studies, we found into pGL3-basic vector. The mutant type constructs were des- that knockdown of STAT3 by RNA interference (RNAi) signif- ignated as pGL3-basic-ZEB1P MT, pGL3-basic-E-cadherinP icantly increased E-cadherin expression in CRC cells (18). STAT3B MT, pGL3-basic-E-cadherinP ZEB1B MT, and pGL3- Whether STAT3 contributes to the EMT process of CRC pro- basic-E-cadherinP STAT3B and ZEB1B MT, respectively. T gression and the mechanisms of STAT3-induced E-cadherin was replaced with G in each STAT3 binding site of pGL3-basic- down-regulation are not known. We now show that STAT3 ZEB1P MT, pGL3-basic-E-cadherinP STAT3BMT, and pGL3- may directly induce cell invasion and participate in resistance basic-E-cadherinP STAT3B and ZEB1B MT constructs. CT to chemotherapy drugs and apoptosis during EMT of CRC pro- and CTG was replaced with AA and AAA in each ZEB1 binding gression. To our knowledge, this is the first study to report that site of pGL3-basic-E-cadherinP ZEB1B MT and pGL3- STAT3 may directly mediate the EMT process and ZEB1 basic-E-cadherinP STAT3B and ZEB1B MT constructs, expression of CRC progression. STAT3-induced cell invasion respectively. and decrease in E-cadherin expression depend on activation of Small Interfering RNA (siRNA) Plasmid Transfections and Tyr-705 ZEB1 in CRC cells. The combination of pSTAT3 Lentiviral Transduction—The siRNA against ZEB1 (TCF8; cat- /ZEB1 may be a novel predictor of CRC metastasis and a potential alog no. L-006564-01-0005), the siRNA against STAT3 (catalog therapeutic target. no. L-003544-00-0005), and the control siRNA were purchased from Dharmacon RNA Technology (Lafayette, CO). Twenty- EXPERIMENTAL PROCEDURES four h before transfection at 30–40% confluence, CRC cells Cell Culture and AG490 Treatment—Two human CRC cell were transferred to 6-well plates. Transfection of siRNAs was lines SW1116 and LoVo (ATCC, Manassas, VA) were cultured carried out with DharmaFECT 1 siRNA transfection reagent in RPMI 1640 medium (Invitrogen), supplemented with 10% (Dharmacon) according to the manufacturer’s instructions. fetal bovine serum (FBS) at 37 °C in humidified 5% CO Cells were collected for analysis 48 h after transfection. atmo- sphere. For AG490 (pharmacological JAK2 inhibitor; Sigma) For plasmid transfections, CRC cells (70% confluence, 5 treatment, CRC cells were incubated with 100 M AG490 for 10 cells) were transfected with 2 g of pCDNA3.1-STAT3 or 24 h (18) before harvesting for measurements. pCDNA3.1 using Lipofectamine 2000 (Invitrogen) according to Construction of Plasmids—The DNA fragment encoding the the manufacturer’s instructions. The cells were collected for STAT3 gene (GenBank accession number NM_003150) was measurements 48 h after transfection. amplified from human cDNA with the primers STAT3-F (5- To stably knock down ZEB1, we infected SW1116 and LoVo GCTAAGCTTTATGGCCCAATGGAATCAGCTACAG-3 cells with MISSION shRNA lentivirus particles (with the puro- and STAT3-R (5-GCTCTCGAGTCATGGGGGAGGTAGC- mycin resistance gene) containing a U6 promoter driving GCACTCCG-3), which introduced the cloning sites HindIII shRNA targeting human ZEB1 or scramble negative control and XhoI (underlined), respectively. The cDNA fragment (Sigma-Aldrich). Methods used for lentivirus production and obtained above was verified by sequencing and finally cloned infection were performed as described by Gire et al. (26). into pCDNA3.1 between the HindIII and XhoI sites to obtain Reverse Transcription-PCR (RT-PCR)—Total RNA was pCDNA3.1-STAT3. extracted by TRIzol reagent (Invitrogen), according to the pro- The wild type DNA fragment containing part of the pro- tocol of the manufacturer, and 1.5 g of total RNA from cul- TMP moter region (520 to70 from transcriptional initiation site) tured cells was reverse transcribed using the PrimeScriptP of the E-cadherin gene (GenBank accession number RT reagent kit (Perfect Real Time) for RT-PCR (Takara, Shiga, NM_004360) and the wild type DNA fragment containing part Japan). of the promoter region (500 to100 from the transcriptional Quantitative Real-time PCR—Quantitative real-time PCR initiation site) of the ZEB1 gene (GenBank accession number was carried out on an Applied Biosystems 7900 quantitative NM_001174094) were amplified from human genomic DNA PCR system. The primers used were as follows: ZEB1-F (5- with the following primers, respectively: E-cadherinP-F GCCAATAAGCAAACGATTCTG-3), ZEB1-R (5-TTTGG- (5-GGGGTACCTGTCTCTCTACAAAAAGGCA-3) and CTGGATCACTTTCAAG-3), ZEB2-F (5-CGGTGCAAGA- E-cadherinP-R (5-GGAAGATCTGGGCTGGAGCGGGCT- GGCGCAAACA-3), ZEB2-R (5-GGAGGACTCATGGTTG- GGAGT-3); ZEB1 P-F (5- GGGGTACCAAAGACGTTTCC- GGCA-3), Snail1-F (5-CACTATGCCGCGCTCTTTC-3), TTATTCGA-3) and ZEB1 P-R (5- GAAGATCTAGAAAG- Snail1-R (5-GGTCGTAGGGCTGCTGGAA-3), Snail2-F (5- GCGACGGGCTGACC-3), which introduced the cloning AAACTACAGCGAACTGGACACA-3), Snail2-R (5-GCCC- sites KpnI and BglII (underlined), respectively. The DNA frag- CAAAGATGAGGAGTATC-3), Twist1-F (5-AGTCCGCA- ment obtained above was directly cloned into pGL3-basic (Pro- GTCTTACGAGGA-3), Twist1-R (5- GCCAGCTTGAGGG- 5820 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 1. Effect of STAT3 on expression of epithelial and mesenchymal markers in LoVo and SW1116 cells. A representative Western blot (A) and the summarized data (B) show that knockdown of STAT3 expression significantly decreased the phosphorylation of STAT3 and the expressions of STAT3, N-cad- herin, and vimentin in LoVo cells, whereas E-cadherin expression was dramatically increased, indicating that STAT3 may participate in the regulation of epithelial and mesenchymal markers in colon cancer. Another representative Western blot (C) and the summarized data (D) show that STAT3 overexpression significantly increased the phosphorylation of STAT3 and expressions of STAT3, N-cadherin, and vimentin in SW1116 cells, whereas E-cadherin expression was dramatically reduced. Seventy-five pmol of siRNA duplexes of STAT3, control siRNA, or plasmids complexed with liposomes were applied to each well. After 72 h of transfection, the cells were collected for analysis. n 3, t test; *, p 0.01; **, p 0.05, compared with the pCDNA3.1 or control siRNA groups. Error bars, S.E. TCTGAAT-3), Twist2-F (5-CAAGCTGAGCAAGATCCA- determined by scanning with a microplate reader at 450 nm. GAC-3), Twist2-R (5-GGTCATCTTATTGTCCATCTCG- Data are expressed as the percentage of viable cells calculated as 3), E12/E47-F (5- TCAAGCAATAACTTCTCGTCCA-3), follows: cell survival rate (%)  (A (treated)  A (blank))/ 450 450 E12/E47-R (5-CGTCCAGGTGGTCTTCTATCTT-3)18S-F (A (control)  A (blank))  100%. 450 450 (5-CGGACAGGATTGACAGATTGATAGC-3), and 18S-R Detection of Apoptosis—For flow cytometric analysis, an (5-TGCCAGAGTCTCGTTCGTTATCG-3). All reactions annexin-V fluorescein isothiocyanate/PI double stain assay was were performed in triplicate in a 10-l total volume containing performed in accordance with the manufacturer’s protocol Brilliant SYBR Green QPCR Master Mix (Takara, Shiga, (BioVision, Mountain View, CA). Analysis was performed Japan). The amplified transcript level of each specific gene was using a flow cytometer. normalized to that of 18S. In Vitro Invasion Assay—Cell invasion assays were per- Western Blot and Antibodies—Western blot analysis was per- formed as described by Hecht et al. (30). In brief, chambers with formed using standard techniques as described previously 8-m pore polycarbonate membranes, coated with Matrigel on (27). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) the upper side, were used (BD Biosciences). CRC cells or stable (Kangchen, Shanghai, China) was detected as a loading control. CRC cell lines with ZEB1 gene knockdown were transfected Antibodies used in this study were purchased from Cell Signal- with STAT3 siRNA, control siRNA, pCDNA3.1-STAT3, or ing Technology Inc. (Beverly, MA). All primary antibodies were pCDNA3.1 for 24 h. Transfected cells were then harvested, and used at a 1:1000 dilution. 1 10 cells were seeded in serum-free medium into the upper Drug Sensitivity Assays to Fluorouracil—Cell proliferation chamber, whereas medium supplemented with 15% FBS was assay was assessed by a tetrazolium salt (WST-8)–based color- applied to the lower chamber as a chemoattractant to induce imetric assay in Cell Counting Kit 8 (Dojindo, Kumamoto, invasion. After incubation for 48 h, migrated cells on the bot- Japan) (28, 29). Briefly, control and CRC cells treated with dif- tom surface of the filter were fixed, stained, and counted. ferent doses of fluorouracil were seeded onto 96-well plates at Luciferase Assay—Designated combinations of pGL3-E-cad- an initial density of 5  10 cells/well. At specified time points, herinPWT, pGL3-ZEB1PWT, and other mutant constructs 10 l of Cell Counting Kit 8 solution were added to each well of with other siRNA or plasmids at 1.0 g and 100 ng of phRL the plate, which was then incubated for 2 h. Cell viability was (Renilla luciferase) TK plasmid (Promega) for monitoring FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5821 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 2. Role of STAT3 in CRC cell EMT phenotypes. Transwell Matrigel invasion assays were performed in LoVo cells transfected with control siRNA or STAT3 siRNA (A) and in SW1116 cells transfected with pCDNA3.1 or pCDNA3.1-STAT3 (B). Twenty-four h after transfection, the cells were plated on transwell inserts, and invasion was assessed after incubation for 72 h. Cells were observed under a microscope and photographed. Cells were counted from five random microscopic fields (200) per insert in triplicate. The migrated cell numbers were normalized to that of the control group. Data are shown as mean S.D. (error bars) from three separate experiments (*, p  0.05 compared with control siRNA group; **, p  0.01 compared with the pCDNA3.1 group). Representative colony-forming assays and relative quantitations show inhibition of growth in LoVo cells transfected with STAT3 siRNA compared with control siRNA (C) and increase of growth of SW1116 cells transfected with pCDNA3.1-STAT3 compared with pCDNA3.1 (D). Colony numbers following transfection with STAT3 siRNA or pCDNA3.1-STAT3 are expressed as the relative percentages of colonies compared with the corresponding control groups. Data are means S.E. (error bars) of five randomly selected microscopic fields from three independent wells in each group. *, p  0.05 compared with the control siRNA group; **, p  0.01 compared with the pCDNA3.1 group. E, dose-response curve of a representative experiment showing relative fluorouracil sensitivity determined by Cell Counting Kit 8 cell proliferation. LoVo cells were treated with fluorouracil after transfection with control or STAT3 siRNA. n 3, t test; *, p 0.05, compared with control siRNA group. F, apoptosis of SW116 cells were analyzed by flow cytometric analysis after transfection with pCDNA3.1-STAT3 or pCDNA3.1 following treatment with different doses of etopside. n  3, t test; Œ, p  0.01, compared with the pCDNA3.1 group. transfection efficiency were transiently transfected in triplicate STAT3 (Cell Signal Technology), and normal rabbit IgG with Lipofectamine 2000 (Invitrogen) or DharmaFECT 1 (Upstate) were used. siRNA transfection reagent (Dharmacon) according to the Real-time PCR Quantification of Genomic DNA ChIP—Real- manufacturer’s directions. Twenty-four h after transfection, time PCR was performed in triplicate using an Applied Biosys- the cells were collected to detect luciferase activity using the tems 7900 quantitative PCR system. Each PCR was carried out Dual-Luciferase reporter assay system (Promega). Luciferase in a 10-l reaction volume by using 3 l of the eluted immuno- activity was measured by using a BD Monolight 3010 luminom- precipitated DNA. The amount of genomic DNA co-precipi- eter (BD Biosciences). Variation in transfection efficiency was tated with the specific antibody was calculated in comparison normalized by dividing the luciferase activity of the construct with the total input DNA used for each immunoprecipitation as by the corresponding Renilla luciferase activity. Promoter follows: CB  CB (genomic input)  CB (specific anti- TB TB TB activity is reported as the mean  S.E. body), where CB (genomic input) and CB (specific anti- TB TB Chromatin Immunoprecipitation (ChIP) Assay—Chromatin body) are the mean threshold cycles of PCR performed in trip- immunoprecipitation assays were performed using the ChIP licate on DNA samples from the genomic input samples and the assay kit (Upstate, Charlottesville, VA) following the manufac- specific antibody samples, respectively. turer’s protocol. ChIP analysis was performed as described pre- Immunohistochemical Staining—All specimens were from viously (31). Antibodies against ZEB1 (Cell Signal Technology), patients (35 primary colorectal adenocarcinomas) who under- 5822 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 3. Role of JAK/STAT3 pathway in ZEB1 expression in CRC cells. Real-time RT-PCR showed that the JAK2 inhibitor AG490 significantly decreased the expression of ZEB1 in SW1116 (A) and LoVo (B) cells, when compared with the control cells. AG490 treatment did not dramatically affect the expression of ZEB2, Snail1, Snail2, Twist1, Twist2, or E12/E47. C, knockdown of STAT3 significantly decreased the ZEB1 mRNA level in SW1116 cells. D, knockdown of STAT3 significantly decreased the ZEB1 mRNA level in LoVo cells. Cells were collected for analysis after treatment with 100 M AG490 for 24 h. Seventy-five pmol of siRNA duplex of STAT3 or control siRNA complexed with liposomes were applied in each well. After 48 h of transfection, the cells were collected for analysis. n 3, ANOVA; *, p  0.05, compared with control; n  3, t test; **, p  0.05; Œ, p  0.01, compared with control siRNA. Error bars, S.E. went surgery in Shanghai Renji Hospital from July 2009 to Statistical Analysis—Statistical analysis was performed with December 2010. The protocol was approved by the ethics com- SPSS 13.0 software. Data are expressed as means  S.E. Statis- mittee of Shanghai Jiao-Tong University School of Medicine tical differences between two groups were determined by Stu- Renji Hospital, and the research was carried out according to dent’s t test. Differences between multiple groups were tested the provisions of the Helsinki Declaration of 1975. None of the using analysis of variance (ANOVA) and checked for signifi- patients received preoperative treatments, such as radiotherapy cance using Fisher’s protected least significant difference test. Tyr-705 or chemotherapy. Meanwhile, 21 specimens of normal colonic Analyses comparing the expressions of STAT3, pSTAT3 , epithelium, taken from patients without colorectal cancer, were ZEB1, and E-cadherin were performed using  analysis and used as negative controls. The expressions of STAT3, Fisher’s exact test. Results were considered significant if the p Tyr-705 pSTAT3 , ZEB1, and E-cadherin were examined with pri- value was less than 0.05. Correlation analysis was performed Tyr-705 Tyr-705 mary antibodies (STAT3, pSTAT3 , ZEB1, and E-cad- between pSTAT3 and ZEB1. herin; dilution 1:100) in consecutive tissue sections using the RESULTS LSAB kit (DakoCytomation, Copenhagen, Denmark) accord- ing to the manufacturer’s instructions. Effect of STAT3 on E-cadherin, N-cadherin, and Vimentin The slides were examined independently by two investiga- Expressions in CRC Cells—We previously showed that acti- tors blinded to both clinical and pathologic data. Protein vated STAT3 is constitutively expressed in CRC and mediates expression was quantified using a visual grading system based cell proliferation, whereas knockdown of STAT3 significantly on the extent of staining (percentage of positive tumor cells restores E-cadherin expression (18). Down-regulation of graded on a scale of 0–4: 0, none; 1, 1–25%; 2, 26–50%; 3, E-cadherin is one of the EMT phenotypes in cancer progression 51–75%; 4, 475%) and the intensity of staining (graded on a (3). To determine whether STAT3 mediates EMT initiation in scale of 0–3: 0, no staining; 1, weak staining; 2, moderate stain- CRC cells, the effect of STAT3 siRNA was evaluated in highly ing; 3, strong staining). For further analysis, an index value was invasive LoVo CRC cells. Western blot analysis showed that calculated as a product of grades of the extent and intensity of STAT3 siRNA significantly decreased STAT3 expression and staining to define the cut-off value for high expression of the phosphorylation in these cells (Fig. 1, A and B), indicating that proteins, and the protein expression was classified into two cat- the STAT3 was knocked down effectively. Knockdown of egories: high (grades 4–12) and low (grades 0–3). STAT3 significantly increased E-cadherin and decreased FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5823 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 4. Effect of STAT3 on ZEB1 expression and function in CRC cells. A representative Western blot (A) and the summarized data (B) show that transfection with STAT3 siRNA significantly decreased STAT3 phosphorylation and expressions of STAT3 and ZEB1 in LoVo cells. Another Western blot (C) and the summarized data (D) show that STAT3 phosphorylation and expressions of STAT3 and ZEB1 were boosted in STAT3-overexpressing SW1116 cells. E, stable knockdown of ZEB1 dramatically decreased cell invasion in basal condition and blocked STAT3-induced cell invasion in LoVo cells. n  3, t test; *, p  0.05, compared with control siRNA. n  3, t test; **, p  0.05, compared with pCDNA3.1. n  3, ANOVA; Œ, p  0.01, compared with pCDNA3.1 control shRNA lentivirus; ŒŒ, p  0.05, compared with pCDNA3.1-STAT3 control shRNA lentivirus. Error bars, S.E. N-cadherin and vimentin expressions (Fig. 1, A and B), suggest- apoptosis are also hallmarks of tumor EMT progression in epi- ing that STAT3 may contribute to EMT progression in LoVo thelial carcinoma (32). Therefore, we examined whether cells. STAT3 participates in these aspects of EMT progression in To further confirm the role of STAT3 in down-regulation of CRC cells. Transwell cell invasion assays showed that knock- E-cadherin and up-regulation of N-cadherin and vimentin in down of STAT3 expression significantly reduced the invasion CRC cells, we constructed and transfected the recombinant ability of LoVo cells normally characterized as highly invasive pCDNA3.1-STAT3 plasmid into low invasion SW1116 cells. (Fig. 2A). Meanwhile, overexpression of STAT3 dramatically Transfection of the pCDNA3.1-STAT3 plasmid significantly increased the invasiveness of SW1116 cells (Fig. 2B), indicating increased STAT3 expression and phosphorylation in SW1116 that STAT3 may have significant effects on cell migration and cells, when compared with the pCDNA3.1 control (Fig. 1, C and invasion in CRC cells. In cell colony-forming assays, the num- D), indicating that STAT3 was successfully overexpressed. ber of colonies of LoVo cells was reduced by nearly 80% when Overexpression of STAT3 significantly reduced E-cadherin transfected with STAT3 siRNA as compared with control and enhanced N-cadherin and vimentin expressions in siRNA (Fig. 2C). Up-regulation of STAT3 significantly SW1116 cells, compared with pCDNA3.1 transfection. These increased the number of SW1116 cell colonies, compared with results further indicate that STAT3 may mediate EMT initia- transfection of pCDNA3.1 (Fig. 2D). The data suggest that tion and progression in CRC cells. STAT3 may mediate cell colony formation in CRC cells. In Role of STAT3 in Cell Invasion, Cell Colony Formation, Che- addition, the CRC cell survival rate was lower in the STAT3 mosensitivity, and Resistance to Apoptosis in CRC Cells—It has siRNA-transfected group than in the control siRNA group after been reported that EMT may induce cell migration, alter inva- treatment with different doses of fluorouracil (Fig. 2E), indicat- sion properties, promote chemotherapy drug resistance, and ing that knockdown of STAT3 significantly enhanced chemo- prevent apoptosis (3). Aberrant cell survival and resistance to sensitivity of CRC to this drug. Furthermore, overexpression of 5824 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 5. Role of STAT3 in the regulation of ZEB1 expression in CRC cells. A, bioinformatic analysis of STAT3 transcriptional factor binding site in part of the ZEB1 gene promoter region. The numbers on the left side indicate the locations upstream of the first base of the initial transcription site. STAT3-binding sites are highlighted, and the DNA sequence encompassed by two arrows was amplified in the ChIP assay. B, representative results from three experiments showed that ZEB1 DNA was detectable in the chromatin sample immunoprecipitated from SW1116 cells using an antibody against STAT3, suggesting that STAT3 binds to the ZEB1 promoter. Input DNA was used as a positive control; rabbit IgG and cell lysates without antibody were used as negative controls. C, overexpression of STAT3 caused a nearly 40% increase in ZEB1 promoter luciferase activity in SW1116 cells. D, knockdown of STAT3 significantly decreased ZEB1 promoter luciferase activity in SW1116 cells. E, two putative STAT3 binding sites were located between nt 500 and 100. White and black rhombuses indicate wild type and mutant sequences for STAT3 binding sites, respectively. WT, wild type; MT, mutant type. Mutation of STAT3 binding sites significantly decreased transcriptional activity of the ZEB1 promoter in a luciferase assay. n  3, t test; **, p  0.05, compared with pCDNA3.1pGL3-basic-ZEB1PWT. n  3, t test; *, p  0.05, compared with control siRNApGL3-basic-ZEB1PWT; Œ, p  0.01, compared with pGL3-basic-ZEB1PWT. Error bars, S.E. STAT3 significantly decreased cell apoptosis in response to dif- SW1116 and LoVo cells. The data further indicate that STAT3 ferent doses of etopside (Fig. 2F), indicating that STAT3 may may participate in regulating ZEB1 expression in CRC cells. Next, we examined whether ZEB1 expression depended on play an important role in resistance to fluorouracil treatment and etopside-induced apoptosis during the EMT process of STAT3 in CRC cells. After transfection of STAT3 siRNA into CRC progression. LoVo cells for 48 h, Western blot analysis showed that knock- JAK/STAT3 Pathway May Regulate ZEB1 Expression in CRC down of STAT3 significantly down-regulated ZEB1 expression Cells—It has been confirmed that ZEB1, ZEB2, Snail1, Snail2, compared with the control (Fig. 4, A and B), indicating that Twist1, Twist2, and E12/E47 are critical transcription factors STAT3 may mediate ZEB1 expression in CRC cells. By con- that repress E-cadherin expression in EMT of cancer progres- trast, when pCDNA3.1-STAT3 was transfected into SW1116 sion (3). After having found that STAT3 may regulate E-cad- cells for 48 h, the overexpression of STAT3 significantly increased ZEB1 expression, compared with pCDNA3.1 (Fig. 4, herin expression, we next detected the expressions of ZEB1, ZEB2, Snail1, Snail2, Twist1, Twist2, and E12/E47 in CRC cells. C and D). These results further indicate that STAT3 may par- Although treatment with the JAK/STAT3 pathway inhibitor ticipate in regulation of ZEB1 expression in CRC cells. More- AG490 significantly down-regulated ZEB1 expression in over, stable knockdown of ZEB1 dramatically decreased cell SW1116 (Fig. 3A) and LoVo (Fig. 3B) cells, it did not dramati- invasion in basal conditions. STAT3-induced CRC cell invasion cally affect the expressions of any of the other molecules was significantly decreased by stable knockdown of ZEB1 detected at the mRNA level in CRC cells, indicating that the expression in CRC cells (Fig. 4E), indicating that STAT3-in- JAK/STAT3 pathway may specifically mediate ZEB1 expres- duced CRC cell invasion may depend on ZEB1. sion in CRC cells. As shown in Fig. 3, C and D, knockdown of Furthermore, DNA sequence analysis of the ZEB1 promoter STAT3 significantly down-regulated ZEB1 expression in both regions (nt 500 to 100) revealed two putative STAT3 binding FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5825 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 6. Role of ZEB1 in STAT3-regulated E-cadherin expression in CRC cells. A, a representative Western blot analysis showed that transfection of ZEB1 siRNA significantly reduced the expression of ZEB1. A representative Western blot (B) and the summarized data (C) showed that STAT3 overexpression did not down-regulate E-cadherin expression after ZEB1 knockdown. D, knockdown of STAT3 or ZEB1 expression caused a nearly 2-fold increase in E-cadherin promoter luciferase activity in SW1116 cells. E, overexpression of STAT3 significantly decreased E-cadherin promoter luciferase activity in SW1116 cells. ZEB1 siRNA significantly blocked STAT3-induced down-regulation of E-cadherin promoter luciferase activity in SW1116 cells; n 3, ANOVA; Œ, p 0.05, compared with pCDNA3.1control siRNA; ŒŒ, p  0.05, compared with pCDNA3.1-STAT3control siRNA; n  3, ANOVA; *, p  0.05, compared with control siRNApGL3-basic-E-cadherinPWT; **, p  0.05, compared with pCDNA3.1control siRNApGL3-basic-E-cadherinPWT; ***, p  0.05, compared with pCDNA3.1-STAT3control siRNApGL3-basic-E-cadherinPWT. Error bars, S.E. sites (Fig. 5A). Thus, in the ChIP assay, we designed one 48 h later. Western blot analysis showed that knockdown of primer set (nt 310 to 130, containing two putative ZEB1 expression significantly increased the expression of STAT3 binding sites), with product sizes of 200 bp, to E-cadherin in CRC cells. Furthermore, STAT3-induced E-cad- amplify part of the ZEB1 promoter regions. As shown in Fig. herin down-regulation was dramatically blocked by down-reg- 5B, this primer set showed amplifiable products. In contrast, ulation of ZEB1 (Fig. 6, B and C), indicating that ZEB1 may no detectable amplification was observed in cell lysates incu- participate in STAT3-induced E-cadherin down-regulation in bated with non-relevant rabbit IgG or cell lysate without CRC cells. antibody incubation (negative controls). In the dual lucifer- Luciferase assays were then performed to further confirm ase assay, mutation of STAT3 binding sites of ZEB1 pro- whether STAT3 repressed E-cadherin expression via altering moter exhibited almost a 60% decrease in relative luciferase ZEB1 regulation of E-cadherin transcription. We found that activity compared with that of wild type construct (Fig. 5E), knockdown of STAT3 or ZEB1 significantly up-regulated suggesting that STAT3 may directly bind to the ZEB1 pro- E-cadherin promoter activity in CRC cells (Fig. 6D). Knock- moter and regulate the transcriptional activity of ZEB1.We down of ZEB1 significantly abolished the STAT3-induced further found that overexpression of STAT3 significantly decrease in E-cadherin promoter activity (Fig. 6E). Further- up-regulated transcriptional activity of the ZEB1 promoter more, DNA sequence analysis of the E-cadherin promoter in CRC cells (Fig. 5C). Knockdown of STAT3 significantly regions (nt520 to70) revealed two putative STAT3 binding down-regulated transcriptional activity of the ZEB1 pro- sites and four putative ZEB1 binding sites (Fig. 7A). Thus, in the moter (Fig. 5D). The data suggest that STAT3-induced up- ChIP assay, we designed one primer set (E-cadherin-a, nt206 regulation of ZEB1 may depend on activation of ZEB1 tran- to 40, containing three putative ZEB1 binding sites), with prod- scription in CRC cells. uct sizes of250 bp, to amplify part of the E-cadherin promoter Effects of ZEB1 on STAT3-induced E-cadherin Down- regions. As shown in Fig. 7, B and C, this primer set showed regulation—In order to determine whether ZEB1 mediates amplifiable products. In contrast, no detectable amplification STAT3-induced E-cadherin down-regulation in CRC cells, was observed in cell lysates incubated with non-relevant rabbit pCDNA3.1-STAT3 and control plasmid were introduced into IgG or cell lysate without antibody incubation (negative con- CRC cells after ZEB1 siRNA and control siRNA transfection trols). These results indicate that ZEB1 may directly bind to the 5826 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 7. A, bioinformatic analysis of STAT3 and ZEB1 transcriptional factor binding site in part of the E-cadherin gene promoter region. The numbers on the left indicate the locations upstream of the first base of the initial transcription site. STAT3-binding sites and ZEB1-binding sites are highlighted, and the DNA sequence surrounded by two arrows was for ChIP. Representative results from three experiments show that E-cadherin DNA was detected in the chromatin sample immunoprecipitated from SW1116 (B) and LoVo (C) cells using an antibody against ZEB1, respectively, suggesting that ZEB1 binds to the E-cadherin promoter. Input DNA was used as a positive control; rabbit IgG and cell lysates without antibody were used as negative controls. Real-time PCR of the ChIP samples showed that overexpression of STAT3 dramatically increased the binding efficiency of ZEB1 to the E-cadherin promoter in SW1116 (D) and LoVo (E) cells. Knockdown of STAT3 significantly decreased the binding efficiency of ZEB1 to the E-cadherin promoter in SW1116 (F) and LoVo (G) cells. n  3, t test; *, p  0.01, compared with pCDNA3.1. n  3, t test; **, p  0.05, compared with control siRNA. Error bars, S.E. E-cadherin promoter. Furthermore, results of a ChIP assay and dual luciferase assay showed that the transcriptional activity of real-time PCR analysis demonstrated that overexpression of E-cadherin promoter was not dramatically changed after muta- STAT3 increased ZEB1 recruitment to the E-cadherin pro- tion of STAT3 binding sites in E-cadherin promoter (Fig. 8). moter (Fig. 7, D and E), whereas it was decreased by knockdown The data suggest that STAT3 may not directly bind to the of STAT3 (Fig. 7, F and G). The data suggest that STAT3 may E-cadherin promoter. play an important role in ZEB1 binding to the E-cadherin pro- Expressions of pSTAT3, STAT3, ZEB1, and E-cadherin in moter in SW1116 and LoVo cells. Moreover, the transcrip- CRC Tissues—Table 1 showed the frequencies of STAT3, Tyr-705 tional activity of the E-cadherin promoter was significantly pSTAT3 , ZEB1, and E-cadherin expression in normal increased after mutation of ZEB1 binding sites or dual mutation and tumor colon tissues by immunohistochemical staining. Tyr-705 of STAT3 and ZEB1 binding sites in the E-cadherin promoter In consecutive tissue sections, pSTAT3 , mostly pres- (Fig. 8). The data further suggests that ZEB1 may directly bind ent in the nucleus, was found with higher expression in to the E-cadherin promoter and regulate the transcriptional 16.7% of the normal colon epithelium samples and 65.7% of activity of E-cadherin. the colon adenocarcinoma samples (Fig. 9, B and F). ZEB1 In addition, in order to examine whether STAT3 may showed predominantly nuclear localization, with occasional directly bind to the E-cadherin promoter, we designed two cytoplasmic staining (Fig. 9, A and E). Immunostaining with an primer sets (E-cadherin-b, nt 416 to 224, containing one anti-ZEB1 antibody showed higher expression in 19 and 60% of putative STAT3 binding site; E-cadherin-c, nt 83 to 56, con- normal colon epithelium samples and colon adenocarcinoma taining one putative STAT3 binding site), with product sizes of samples, respectively. E-cadherin was observed at higher expres- 200 bp to amplify parts of the E-cadherin promoter regions. sion in 100% of the normal colon epithelium samples and in 20% of However, no E-cadherin DNA was detectable in the immuno- the adenocarcinoma samples (Fig. 9, D and H). Our data suggest Tyr-705 precipitated chromatin sample of SW1116 or LoVo cell lysates that the expressions of pSTAT3 and ZEB1 were significantly by using an antibody against STAT3 (data not shown). Further up-regulated ( test, p 0.001), whereas the expression of E-cad- FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5827 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 8. Two putative STAT3 binding sites and four putative ZEB1 binding sites were located between nt520 and70 of the E-cadherin 5-flanking region. White and black rhombuses indicate a wild or mutant sequence for STAT3 binding sites, respectively. White and black triangles indicate a wild type or mutant sequence for ZEB1 binding sites, respectively. WT, wild type; STAT3B MT, mutant type of each STAT3 mutation binding site; ZEB1B MT, mutant type of each ZEB1 mutation binding site; ZEB1B and STAT3B MT, mutant type of each ZEB1 and STAT3 mutation binding site. Mutation of ZEB1 binding sites or dual mutation of STAT3 and ZEB1 binding sites significantly increased the transcriptional activity of E-cadherin promoter in the luciferase assay. Mutation of STAT3 binding sites had no obvious effect on the transcriptional activity of the E-cadherin promoter. n  3, ANOVA; *, p  0.05, compared with pGL3-basic-E- cadherinPWT. Error bars, S.E. TABLE 1 Frequencies of STAT3, pSTAT3, ZEB1, and E-cadherin expression in normal and tumor colon samples Tyr-705 2 pSTAT3 and ZEB1 were mostly present in adenocarcinoma, whereas the expression of E-cadherin was significantly decreased in adenocarcinoma ( test). There was no significant difference in STAT3 expression between normal epithelia and adenocarcinoma. STAT3 pSTAT3 ZEB1 E-cadherin n High Low p value High Low p value High Low p value High Low p value a a a Normal epithelium 21 19 2 0.14 3 18 0.001 417 0.01 21 0 0.001 Adenocarcinoma 35 26 9 23 12 21 14 7 28 p  0.001. 2 Tyr-705 herin was significantly decreased ( test, p  0.001) in colorectal pSTAT3 and ZEB1 may play important roles in CRC adenocarcinoma. metastasis. Moreover, Table 2 showed that the high expressions of Tyr-705 pSTAT3 and ZEB1 were significantly more frequent in DISCUSSION poorly differentiated CRC tissue than in well differentiated The JAK/STAT signaling pathway plays a significant role in CRC tissue (Fisher’s exact test, p  0.001). Furthermore, high Tyr-705 immune function, cell growth, and differentiation (33). Accu- expression of pSTAT3 was significantly correlated with mulating evidence has indicated that STAT3 correlates with high expression of ZEB1 (r  0.7385, p  0.001). There was no cell proliferation in breast carcinoma (34) and non-small cell significant difference in high STAT3 expressions between the lung cancer (35). In a previous study, we found that activation of CRC tissues of different differentiation levels. In addition, other STAT3 may mediate human CRC tumorigenesis and progres- clinical characteristics, including age and gender, were not Tyr-705 sion, and knockdown of STAT3 significantly increases the directly related to the expressions of STAT3, pSTAT3 , and ZEB1. The results indicate that the high expressions of expression of E-cadherin (18). 5828 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression Tyr-705 FIGURE 9. Expressions of STAT3, ZEB1, and E-cadherin in CRC. Shown is immunohistochemical analysis of consecutive tissue sections for ZEB1, pSTAT3 , Tyr-705 STAT3, and E-cadherin in normal colorectal mucosa and high grade CRC. A and B, nuclear staining of ZEB1 and pSTAT3 were mostly presented in adenocarci- Tyr-705 noma. Absent expression of ZEB1 (E) or pSTAT3 (F) was evident in the stroma or epithelium of the normal colorectal mucosa. C and G, cytoplasmic and nuclear staining of STAT3 was frequently detected in normal mucosa and adenocarcinoma. H, cytoplasmic staining of E-cadherin was predominantly detected in normal colorectal mucosa. D, staining of E-cadherin was dramatically decreased in adenocarcinoma, compared with normal colorectal epithelium. A–H (original magnifica- tion,200), representative areas from A1–H1 (original magnification,40), respectively. TABLE 2 STAT3, pSTAT3, and ZEB1 expressions in CRC tissues with different levels of differentiation Tyr-705 Correlation analysis of expressions of STAT3, pSTAT3 , and ZEB1 and clinicopathologic characteristics in CRC patients. Statistical analysis was conducted with Tyr-705 Fisher’s exact test. p values less than 0.05 were considered statistically significant. pSTAT3 and ZEB1 expressions were significantly related to TNM stage (Fisher exact test), suggesting that constitutive activation of STAT3 and elevation of ZEB1 may contribute to poor prognosis in CRC patients. STAT3 pSTAT3 ZEB1 High Low p value High Low p value High Low p value Sex Male 16 5 1 13 8 0.72 12 9 0.68 Female 10 4 10 4 7 7 Gender 65 16 3 0.25 14 5 0.3 10 9 0.83 65 10 6 9 7 9 7 TNM stage a a I/II 10 6 0.25 4 12 0.001 511 0.001 III/IV 16 3 19 0 16 3 p  0.001. As a calcium-dependent cell adhesion molecule, E-cadherin loss of E-cadherin expression is a crucial step in the initiation of is expressed predominantly in epithelial tissues (36). It is an tumor metastasis and a fundamental event in EMT (3). EMT important tumor suppressor gene, which may lose expression plays important roles in tumor formation and progression and function in tumor progression and invasion (37–40). The through enhancing cell proliferation, migration, and drug FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5829 STAT3 and ZEB1 Signaling in CRC EMT Progression resistance and preventing apoptosis (3). It has been reported examined whether STAT3 may directly bind to the E-cadherin that the TGF, Wnt, Notch, and EGF signaling pathways may promoter. Although we found two putative STAT3 binding induce EMT in the progression of different cancers (41–44). sites in the E-cadherin promoter region, no E-cadherin DNA Although IL-6 may induce an EMT phenotype in human breast was detected in the immunoprecipitated chromatin sample of cancer cells (45) and TGF-1 induces EMT in mouse hepato- SW1116 or LoVo cell lysates by using an antibody against cytes (46) via activation of STAT3, the role of STAT3 is not yet STAT3 (data not shown). In addition, the transcriptional activ- well understood in human CRC cancer EMT progression. ity of E-cadherin promoter was not dramatically changed after In our present study, we found that activation of STAT3 may mutation of putative STAT3 binding sites in the E-cadherin induce changes in epithelial cells resembling that of EMT pro- promoter (Fig. 8). The data suggest that STAT3 may not gression for the following reasons. 1) Knockdown of STAT3 directly bind to the E-cadherin promoter. Based on the data significantly decreased N-cadherin and vimentin expressions described above, we hypothesized that ZEB1 may mediate (Fig. 1, A and B). 2) Overexpression of STAT3 significantly STAT3-induced E-cadherin down-regulation in CRC cells. reduced E-cadherin and enhanced N-cadherin and vimentin This hypothesis was further supported by our finding that expressions (Fig. 1, C and D). 3) Down-regulation of STAT3 STAT3-induced decrease of E-cadherin expression (Fig. 6, B significantly reduced cell invasion ability (Fig. 2A) and cell col- and C) and promoter transcription activity (Fig. 6E) was signif- ony numbers (Fig. 2C) of CRC cells. 4) Overexpression of icantly blocked by knockdown of ZEB1 in CRC cells. In the STAT3 dramatically increased the invasion ability (Fig. 2B) and real-time ChIP PCR assay, overexpression of STAT3 increased cell colony numbers (Fig. 2D) in CRC cells. Furthermore, ZEB1 recruitment to the E-cadherin promoter (Fig. 7, D and E), Thiery et al. (3) have demonstrated that EMT may be involved whereas it was decreased by knockdown of STAT3 (Fig. 7, F and in more than invasion in cancer; it may be involved as well in G). The transcriptional activity of the E-cadherin promoter was resistance to chemotherapy and cell death. Therefore, we also significantly increased after mutation of ZEB1 binding sites or explored whether STAT3 participates in the resistance to fluo- dual mutation of STAT3 and ZEB1 binding sites in the E-cad- rouracil (a chemotherapy drug) treatment and promotes CRC herin promoter in the dual luciferase assay (Fig. 8). The data cell resistance to etopside-induced cell apoptosis. Our data (Fig. suggest that ZEB1 may contribute to the STAT3-induced 2, E and F) indicate that STAT3-induced EMT may cause resis- decrease in E-cadherin expression via increasing its binding tance to conventional chemotherapy and cell death in colon efficiency to the E-cadherin promoter region. Tyr-705 cancer. Our detection of pSTAT3 and ZEB1 expressions by Because down-regulation of E-cadherin expression is a key immunohistochemistry further showed that they were remark- initiating event in EMT, transcription factors that repress ably increased in colon adenocarcinoma, compared with nor- E-cadherin have been defined as inducers of EMT. The major mal colon epithelium (Fig. 9). However, E-cadherin expression transcriptional repressors that are known to down-regulate was significantly decreased in high grade colon adenocarci- E-cadherin expression include those in the Snail and ZEB fam- noma (Fig. 9). The data are consistent with other reports showing Tyr-705 ilies (6, 47). In our study, ZEB1 expression was significantly that pSTAT3 is constitutively activated and correlated with reduced after treatment with the JAK/STAT pathway inhibitor CRC dedifferentiation (49). ZEB1 expression is absent in normal AG490 and STAT3 siRNA transfection (Fig. 3, A–D). However, colon and breast tissues, whereas ZEB1-positive tumor tissues ZEB2, Snail1, Snail2, Twist1, Twist2, and E12/E47 expressions express low levels of cytokeratin (50). Furthermore, our study also were not obviously altered in response to AG490. The data demonstrates that high tumor, lymph node, and metastasis suggest that ZEB1 but not ZEB2, Snail1, Snail2, Twist1, Twist2, (TNM) staging of CRC was significantly more frequent in patients Tyr-705 or E12/E47 may mediate STAT3-induced colon cancer pro- with high expressions of pSTAT3 and ZEB1 than in those gression. Results of the cell invasion assays further supported with low expression (Tables 1 and 2). Moreover, a very strong Tyr-705 this conclusion. Stable knockdown of ZEB1 significantly positive correlation between pSTAT3 and ZEB1 expression reduced cell invasion at the basal conditions and blocked was identified in cancerous tissues. These observations imply that Tyr-705 STAT3-induced cell invasion in CRC cells (Fig. 4E). This result pSTAT3 and ZEB1 expression may be powerful indicators is consistent with another report showing that ZEB1 expression of CRC metastasis. is important during colon cancer progression (48). In addition, In conclusion, STAT3 may play an important role in the we further found that ZEB1 expression was significantly EMT process of CRC progression. As an EMT inducer, ZEB1 decreased by knockdown of STAT3 (Fig. 4, A and B), whereas it expression may be regulated by STAT3. ZEB1 may participate was dramatically increased by overexpression of STAT3 (Fig. 4, in STAT3-induced cell invasion in CRC cells. Moreover, ZEB1 C and D). These results further indicate that the activation of may mediate STAT3-induced down-regulation of E-cadherin STAT3 is highly important for ZEB1 expression in colon carci- via directly inhibiting transcriptional activity of the E-cadherin noma. Further analysis revealed two putative STAT3 binding promoter in CRC. 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Aigner, K., Dampier, B., Descovich, L., Mikula, M., Sultan, A., Schreiber, 943–950 M., Mikulits, W., Brabletz, T., Strand, D., Obrist, P., Sommergruber, W., 45. Sullivan, N. J., Sasser, A. K., Axel, A. E., Vesuna, F., Raman, V., Ramirez, N., Schweifer, N., Wernitznig, A., Beug, H., Foisner, R., and Eger, A. (2007) Oberyszyn, T. M., and Hall, B. M. (2009) Interleukin-6 induces an epithe- The transcription factor ZEB1 (EF1) promotes tumor cell dedifferentia- lial-mesenchymal transition phenotype in human breast cancer cells. On- tion by repressing master regulators of epithelial polarity. Oncogene 26, cogene 28, 2940–2947 6979–6988 5832 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Roles of STAT3 and ZEB1 Proteins in E-cadherin Down-regulation and Human Colorectal Cancer Epithelial-Mesenchymal Transition *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2012 Elsevier Inc.
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0021-9258
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10.1074/jbc.m111.295964
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 8, pp. 5819 –5832, February 17, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Roles of STAT3 and ZEB1 Proteins in E-cadherin Down-regulation and Human Colorectal Cancer Epithelial-Mesenchymal Transition Received for publication, August 19, 2011, and in revised form, December 21, 2011 Published, JBC Papers in Press, December 28, 2011, DOI 10.1074/jbc.M111.295964 1 1,2 Hua Xiong , Jie Hong , Wan Du, Yan-wei Lin, Lin-lin Ren, Ying-chao Wang, Wen-yu Su, Ji-lin Wang, Yun Cui, Zhen-hua Wang, and Jing-Yuan Fang From the Gastrointestinal Division, Shanghai Jiao-Tong University School of Medicine Renji Hospital, Shanghai Institution of Digestive Disease, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health (Shanghai Jiao-Tong University), State Key Laboratory of Oncogenes and Related Genes, 145 Middle Shandong Road, Shanghai 200001, China Background: Colorectal cancer (CRC) to metastatic disease may involve the epithelial-mesenchymal transition (EMT). Results: STAT3 may regulate N-cadherin, vimentin, and ZEB1 expressions. STAT3-induced cell invasion and down-regulation of E-cadherin may depend on ZEB1. Conclusion: STAT3 may mediate CRC EMT progression and ZEB1 expression. Activation of STAT3 and ZEB1 proteins may contribute to worse prognosis in CRC patients. Significance: Our data may provide potential targets to prevent and/or treat CRC invasion. The progression of colorectal carcinoma (CRC) to invasive Despite welcome declines in mortality rates over the past and metastatic disease may involve localized occurrences of epi- decade, colorectal cancer (CRC) remains a common malig- thelial-mesenchymal transition (EMT). However, mechanisms nancy and one of the leading causes of morbidity and death in of the EMT process in CRC progression are not fully under- the world (1, 2). Epithelial-mesenchymal transition (EMT) is a stood. We previously showed that knockdown of signal trans- crucial process in the initiation of the metastatic spread of ducer and activator of transcription 3 (STAT3) up-regulated tumor cells to distal organs (3). EMT may promote epithelial E-cadherin (a key component in EMT progression) in CRC. In cells to escape from the rigid structural constraints provided by this study, we examined the roles of STAT3 in CRC EMT and the tissue architecture and adopt a phenotype more amenable ZEB1, an EMT inducer, in STAT3-induced down-regulation of to cell migration and movement (4–6). In this progression, E-cadherin. Knockdown of STAT3 significantly increased E-cad- epithelial cells may lose adhesion and cell-to-cell contacts (7, 8). herin and decreased N-cadherin and vimentin expressions in Therefore, EMT can be regarded as a pathological process that highly invasive LoVo CRC cells. Meanwhile, overexpression of contributes to cancer progression, particularly in tumor cell STAT3 significantly reduced E-cadherin and enhanced N-cad- invasion and metastasis (9). herin and vimentin expressions in weakly invasive SW1116 CRC The initiation and progression of EMT require transduction cells. Activation of STAT3 significantly increased CRC cell inva- of cell signals. The transforming growth factor- (TGF-) sig- siveness and resistance to apoptosis. Knockdown of STAT3 dra- naling and activated Ras pathways have been implicated as key matically enhanced chemosensitivity of CRC cells to fluorouracil. EMT inducers in CRC cancer (10–11). The Wnt, PI3K/AKT, STAT3 regulated ZEB1 expression in CRC cells, and the STAT3- and other signaling pathways may also play an important role in induceddecreaseinE-cadherinandcellinvasiondependedonacti- the EMT process in CRC progression (12–15). Recently, accu- Tyr-705 vation of ZEB1 in CRC cells. Additionally, pSTAT3 and mulating evidence has indicated that abnormalities in the Janus ZEB1 expressions were significantly correlated with TNM (tumor, kinase/signal transducer and activator of transcription (JAK/ lymph node, and metastasis stages) (p < 0.01). In conclusion, STAT) pathway are involved in CRC oncogenesis (16, 17). As a STAT3 may directly mediate EMT progression and regulate ZEB1 key component of the JAK/STAT pathway, STAT3 is constitu- expression in CRC. ZEB1 may participate in STAT3-induced cell tively activated in CRC (18), and several lines of evidence have invasion and E-cadherin down-regulation in CRC cells. The supported its role in mediating cell motility and migration. Tyr-705 expressions of pSTAT3 and ZEB1 may be positively associ- STAT3 is important for the migration of sheets of cells in ated with CRC metastasis. Our data may provide potential targets zebrafish embryo development (19), and conditional depletion to prevent and/or treat CRC invasion and metastasis. of this molecule blocks wound healing in mouse keratinocytes (20). Additionally, gastrin may induce EMT in CRC through the JAK2/STAT3 pathway (21). EGF receptor is overexpressed in * This work was supported by National Natural Science Foundation of China ovarian carcinoma, whereas EGF-induced EMT in ovarian can- Grant 30900757 (to F. J. Y.), National Natural Science Foundation of China Grant 91129724 (to J. H.), and “Chen Guang” Project Grant 09CG13 (to cer cells has been shown to depend on IL-6R and the JAK2/ J. H.). Both authors contributed equally to this work. To whom correspondence may be addressed. Tel.: 86-21-63200874; Fax: 86-21-63266027; E-mail: [email protected]. The abbreviations used are: CRC, colorectal cancer; EMT, epithelial-mesen- To whom correspondence may be addressed. Tel.: 86-21-63200874; Fax: chymal transition; ANOVA, analysis of variance; nt, nucleotide(s); TNM, 86-21-63266027; E-mail: [email protected]. tumor, lymph node, and metastasis. FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5819 This is an Open Access article under the CC BY license. STAT3 and ZEB1 Signaling in CRC EMT Progression STAT3 pathway (22). However, the role of STAT3 in the EMT mega, Madison, WI) between the KpnI and BglII sites to obtain process of CRC progression is not fully understood. pGL3-E-cadherinPWT and pGL3-ZEB1PWT. Loss of E-cadherin expression is a crucial step and funda- The mutant DNA sequences of the ZEB1 promoter region mental event of EMT in cancer progression (3). As a key com- encompassing both of the two putative binding sites of STAT3 ponent of adherens junctions, E-cadherin plays a crucial role in (500 to 100 from the transcriptional initiation site) or the the maintenance of epithelial integrity (23). Many studies have mutant DNA sequences of the E-cadherin promoter region reported on the regulation of E-cadherin during cancer pro- encompassing both of the two putative binding sites of STAT3 gression (3, 24, 25), and several proteins, including Snail, ZEB1, and four putative binding sites of ZEB1 (520 to 70 from and ZEB2, have been identified that may down-regulate E-cad- transcriptional initiation site) were synthesized and inserted herin in various cancers (3). In our previous studies, we found into pGL3-basic vector. The mutant type constructs were des- that knockdown of STAT3 by RNA interference (RNAi) signif- ignated as pGL3-basic-ZEB1P MT, pGL3-basic-E-cadherinP icantly increased E-cadherin expression in CRC cells (18). STAT3B MT, pGL3-basic-E-cadherinP ZEB1B MT, and pGL3- Whether STAT3 contributes to the EMT process of CRC pro- basic-E-cadherinP STAT3B and ZEB1B MT, respectively. T gression and the mechanisms of STAT3-induced E-cadherin was replaced with G in each STAT3 binding site of pGL3-basic- down-regulation are not known. We now show that STAT3 ZEB1P MT, pGL3-basic-E-cadherinP STAT3BMT, and pGL3- may directly induce cell invasion and participate in resistance basic-E-cadherinP STAT3B and ZEB1B MT constructs. CT to chemotherapy drugs and apoptosis during EMT of CRC pro- and CTG was replaced with AA and AAA in each ZEB1 binding gression. To our knowledge, this is the first study to report that site of pGL3-basic-E-cadherinP ZEB1B MT and pGL3- STAT3 may directly mediate the EMT process and ZEB1 basic-E-cadherinP STAT3B and ZEB1B MT constructs, expression of CRC progression. STAT3-induced cell invasion respectively. and decrease in E-cadherin expression depend on activation of Small Interfering RNA (siRNA) Plasmid Transfections and Tyr-705 ZEB1 in CRC cells. The combination of pSTAT3 Lentiviral Transduction—The siRNA against ZEB1 (TCF8; cat- /ZEB1 may be a novel predictor of CRC metastasis and a potential alog no. L-006564-01-0005), the siRNA against STAT3 (catalog therapeutic target. no. L-003544-00-0005), and the control siRNA were purchased from Dharmacon RNA Technology (Lafayette, CO). Twenty- EXPERIMENTAL PROCEDURES four h before transfection at 30–40% confluence, CRC cells Cell Culture and AG490 Treatment—Two human CRC cell were transferred to 6-well plates. Transfection of siRNAs was lines SW1116 and LoVo (ATCC, Manassas, VA) were cultured carried out with DharmaFECT 1 siRNA transfection reagent in RPMI 1640 medium (Invitrogen), supplemented with 10% (Dharmacon) according to the manufacturer’s instructions. fetal bovine serum (FBS) at 37 °C in humidified 5% CO Cells were collected for analysis 48 h after transfection. atmo- sphere. For AG490 (pharmacological JAK2 inhibitor; Sigma) For plasmid transfections, CRC cells (70% confluence, 5 treatment, CRC cells were incubated with 100 M AG490 for 10 cells) were transfected with 2 g of pCDNA3.1-STAT3 or 24 h (18) before harvesting for measurements. pCDNA3.1 using Lipofectamine 2000 (Invitrogen) according to Construction of Plasmids—The DNA fragment encoding the the manufacturer’s instructions. The cells were collected for STAT3 gene (GenBank accession number NM_003150) was measurements 48 h after transfection. amplified from human cDNA with the primers STAT3-F (5- To stably knock down ZEB1, we infected SW1116 and LoVo GCTAAGCTTTATGGCCCAATGGAATCAGCTACAG-3 cells with MISSION shRNA lentivirus particles (with the puro- and STAT3-R (5-GCTCTCGAGTCATGGGGGAGGTAGC- mycin resistance gene) containing a U6 promoter driving GCACTCCG-3), which introduced the cloning sites HindIII shRNA targeting human ZEB1 or scramble negative control and XhoI (underlined), respectively. The cDNA fragment (Sigma-Aldrich). Methods used for lentivirus production and obtained above was verified by sequencing and finally cloned infection were performed as described by Gire et al. (26). into pCDNA3.1 between the HindIII and XhoI sites to obtain Reverse Transcription-PCR (RT-PCR)—Total RNA was pCDNA3.1-STAT3. extracted by TRIzol reagent (Invitrogen), according to the pro- The wild type DNA fragment containing part of the pro- tocol of the manufacturer, and 1.5 g of total RNA from cul- TMP moter region (520 to70 from transcriptional initiation site) tured cells was reverse transcribed using the PrimeScriptP of the E-cadherin gene (GenBank accession number RT reagent kit (Perfect Real Time) for RT-PCR (Takara, Shiga, NM_004360) and the wild type DNA fragment containing part Japan). of the promoter region (500 to100 from the transcriptional Quantitative Real-time PCR—Quantitative real-time PCR initiation site) of the ZEB1 gene (GenBank accession number was carried out on an Applied Biosystems 7900 quantitative NM_001174094) were amplified from human genomic DNA PCR system. The primers used were as follows: ZEB1-F (5- with the following primers, respectively: E-cadherinP-F GCCAATAAGCAAACGATTCTG-3), ZEB1-R (5-TTTGG- (5-GGGGTACCTGTCTCTCTACAAAAAGGCA-3) and CTGGATCACTTTCAAG-3), ZEB2-F (5-CGGTGCAAGA- E-cadherinP-R (5-GGAAGATCTGGGCTGGAGCGGGCT- GGCGCAAACA-3), ZEB2-R (5-GGAGGACTCATGGTTG- GGAGT-3); ZEB1 P-F (5- GGGGTACCAAAGACGTTTCC- GGCA-3), Snail1-F (5-CACTATGCCGCGCTCTTTC-3), TTATTCGA-3) and ZEB1 P-R (5- GAAGATCTAGAAAG- Snail1-R (5-GGTCGTAGGGCTGCTGGAA-3), Snail2-F (5- GCGACGGGCTGACC-3), which introduced the cloning AAACTACAGCGAACTGGACACA-3), Snail2-R (5-GCCC- sites KpnI and BglII (underlined), respectively. The DNA frag- CAAAGATGAGGAGTATC-3), Twist1-F (5-AGTCCGCA- ment obtained above was directly cloned into pGL3-basic (Pro- GTCTTACGAGGA-3), Twist1-R (5- GCCAGCTTGAGGG- 5820 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 1. Effect of STAT3 on expression of epithelial and mesenchymal markers in LoVo and SW1116 cells. A representative Western blot (A) and the summarized data (B) show that knockdown of STAT3 expression significantly decreased the phosphorylation of STAT3 and the expressions of STAT3, N-cad- herin, and vimentin in LoVo cells, whereas E-cadherin expression was dramatically increased, indicating that STAT3 may participate in the regulation of epithelial and mesenchymal markers in colon cancer. Another representative Western blot (C) and the summarized data (D) show that STAT3 overexpression significantly increased the phosphorylation of STAT3 and expressions of STAT3, N-cadherin, and vimentin in SW1116 cells, whereas E-cadherin expression was dramatically reduced. Seventy-five pmol of siRNA duplexes of STAT3, control siRNA, or plasmids complexed with liposomes were applied to each well. After 72 h of transfection, the cells were collected for analysis. n 3, t test; *, p 0.01; **, p 0.05, compared with the pCDNA3.1 or control siRNA groups. Error bars, S.E. TCTGAAT-3), Twist2-F (5-CAAGCTGAGCAAGATCCA- determined by scanning with a microplate reader at 450 nm. GAC-3), Twist2-R (5-GGTCATCTTATTGTCCATCTCG- Data are expressed as the percentage of viable cells calculated as 3), E12/E47-F (5- TCAAGCAATAACTTCTCGTCCA-3), follows: cell survival rate (%)  (A (treated)  A (blank))/ 450 450 E12/E47-R (5-CGTCCAGGTGGTCTTCTATCTT-3)18S-F (A (control)  A (blank))  100%. 450 450 (5-CGGACAGGATTGACAGATTGATAGC-3), and 18S-R Detection of Apoptosis—For flow cytometric analysis, an (5-TGCCAGAGTCTCGTTCGTTATCG-3). All reactions annexin-V fluorescein isothiocyanate/PI double stain assay was were performed in triplicate in a 10-l total volume containing performed in accordance with the manufacturer’s protocol Brilliant SYBR Green QPCR Master Mix (Takara, Shiga, (BioVision, Mountain View, CA). Analysis was performed Japan). The amplified transcript level of each specific gene was using a flow cytometer. normalized to that of 18S. In Vitro Invasion Assay—Cell invasion assays were per- Western Blot and Antibodies—Western blot analysis was per- formed as described by Hecht et al. (30). In brief, chambers with formed using standard techniques as described previously 8-m pore polycarbonate membranes, coated with Matrigel on (27). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) the upper side, were used (BD Biosciences). CRC cells or stable (Kangchen, Shanghai, China) was detected as a loading control. CRC cell lines with ZEB1 gene knockdown were transfected Antibodies used in this study were purchased from Cell Signal- with STAT3 siRNA, control siRNA, pCDNA3.1-STAT3, or ing Technology Inc. (Beverly, MA). All primary antibodies were pCDNA3.1 for 24 h. Transfected cells were then harvested, and used at a 1:1000 dilution. 1 10 cells were seeded in serum-free medium into the upper Drug Sensitivity Assays to Fluorouracil—Cell proliferation chamber, whereas medium supplemented with 15% FBS was assay was assessed by a tetrazolium salt (WST-8)–based color- applied to the lower chamber as a chemoattractant to induce imetric assay in Cell Counting Kit 8 (Dojindo, Kumamoto, invasion. After incubation for 48 h, migrated cells on the bot- Japan) (28, 29). Briefly, control and CRC cells treated with dif- tom surface of the filter were fixed, stained, and counted. ferent doses of fluorouracil were seeded onto 96-well plates at Luciferase Assay—Designated combinations of pGL3-E-cad- an initial density of 5  10 cells/well. At specified time points, herinPWT, pGL3-ZEB1PWT, and other mutant constructs 10 l of Cell Counting Kit 8 solution were added to each well of with other siRNA or plasmids at 1.0 g and 100 ng of phRL the plate, which was then incubated for 2 h. Cell viability was (Renilla luciferase) TK plasmid (Promega) for monitoring FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5821 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 2. Role of STAT3 in CRC cell EMT phenotypes. Transwell Matrigel invasion assays were performed in LoVo cells transfected with control siRNA or STAT3 siRNA (A) and in SW1116 cells transfected with pCDNA3.1 or pCDNA3.1-STAT3 (B). Twenty-four h after transfection, the cells were plated on transwell inserts, and invasion was assessed after incubation for 72 h. Cells were observed under a microscope and photographed. Cells were counted from five random microscopic fields (200) per insert in triplicate. The migrated cell numbers were normalized to that of the control group. Data are shown as mean S.D. (error bars) from three separate experiments (*, p  0.05 compared with control siRNA group; **, p  0.01 compared with the pCDNA3.1 group). Representative colony-forming assays and relative quantitations show inhibition of growth in LoVo cells transfected with STAT3 siRNA compared with control siRNA (C) and increase of growth of SW1116 cells transfected with pCDNA3.1-STAT3 compared with pCDNA3.1 (D). Colony numbers following transfection with STAT3 siRNA or pCDNA3.1-STAT3 are expressed as the relative percentages of colonies compared with the corresponding control groups. Data are means S.E. (error bars) of five randomly selected microscopic fields from three independent wells in each group. *, p  0.05 compared with the control siRNA group; **, p  0.01 compared with the pCDNA3.1 group. E, dose-response curve of a representative experiment showing relative fluorouracil sensitivity determined by Cell Counting Kit 8 cell proliferation. LoVo cells were treated with fluorouracil after transfection with control or STAT3 siRNA. n 3, t test; *, p 0.05, compared with control siRNA group. F, apoptosis of SW116 cells were analyzed by flow cytometric analysis after transfection with pCDNA3.1-STAT3 or pCDNA3.1 following treatment with different doses of etopside. n  3, t test; Œ, p  0.01, compared with the pCDNA3.1 group. transfection efficiency were transiently transfected in triplicate STAT3 (Cell Signal Technology), and normal rabbit IgG with Lipofectamine 2000 (Invitrogen) or DharmaFECT 1 (Upstate) were used. siRNA transfection reagent (Dharmacon) according to the Real-time PCR Quantification of Genomic DNA ChIP—Real- manufacturer’s directions. Twenty-four h after transfection, time PCR was performed in triplicate using an Applied Biosys- the cells were collected to detect luciferase activity using the tems 7900 quantitative PCR system. Each PCR was carried out Dual-Luciferase reporter assay system (Promega). Luciferase in a 10-l reaction volume by using 3 l of the eluted immuno- activity was measured by using a BD Monolight 3010 luminom- precipitated DNA. The amount of genomic DNA co-precipi- eter (BD Biosciences). Variation in transfection efficiency was tated with the specific antibody was calculated in comparison normalized by dividing the luciferase activity of the construct with the total input DNA used for each immunoprecipitation as by the corresponding Renilla luciferase activity. Promoter follows: CB  CB (genomic input)  CB (specific anti- TB TB TB activity is reported as the mean  S.E. body), where CB (genomic input) and CB (specific anti- TB TB Chromatin Immunoprecipitation (ChIP) Assay—Chromatin body) are the mean threshold cycles of PCR performed in trip- immunoprecipitation assays were performed using the ChIP licate on DNA samples from the genomic input samples and the assay kit (Upstate, Charlottesville, VA) following the manufac- specific antibody samples, respectively. turer’s protocol. ChIP analysis was performed as described pre- Immunohistochemical Staining—All specimens were from viously (31). Antibodies against ZEB1 (Cell Signal Technology), patients (35 primary colorectal adenocarcinomas) who under- 5822 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 3. Role of JAK/STAT3 pathway in ZEB1 expression in CRC cells. Real-time RT-PCR showed that the JAK2 inhibitor AG490 significantly decreased the expression of ZEB1 in SW1116 (A) and LoVo (B) cells, when compared with the control cells. AG490 treatment did not dramatically affect the expression of ZEB2, Snail1, Snail2, Twist1, Twist2, or E12/E47. C, knockdown of STAT3 significantly decreased the ZEB1 mRNA level in SW1116 cells. D, knockdown of STAT3 significantly decreased the ZEB1 mRNA level in LoVo cells. Cells were collected for analysis after treatment with 100 M AG490 for 24 h. Seventy-five pmol of siRNA duplex of STAT3 or control siRNA complexed with liposomes were applied in each well. After 48 h of transfection, the cells were collected for analysis. n 3, ANOVA; *, p  0.05, compared with control; n  3, t test; **, p  0.05; Œ, p  0.01, compared with control siRNA. Error bars, S.E. went surgery in Shanghai Renji Hospital from July 2009 to Statistical Analysis—Statistical analysis was performed with December 2010. The protocol was approved by the ethics com- SPSS 13.0 software. Data are expressed as means  S.E. Statis- mittee of Shanghai Jiao-Tong University School of Medicine tical differences between two groups were determined by Stu- Renji Hospital, and the research was carried out according to dent’s t test. Differences between multiple groups were tested the provisions of the Helsinki Declaration of 1975. None of the using analysis of variance (ANOVA) and checked for signifi- patients received preoperative treatments, such as radiotherapy cance using Fisher’s protected least significant difference test. Tyr-705 or chemotherapy. Meanwhile, 21 specimens of normal colonic Analyses comparing the expressions of STAT3, pSTAT3 , epithelium, taken from patients without colorectal cancer, were ZEB1, and E-cadherin were performed using  analysis and used as negative controls. The expressions of STAT3, Fisher’s exact test. Results were considered significant if the p Tyr-705 pSTAT3 , ZEB1, and E-cadherin were examined with pri- value was less than 0.05. Correlation analysis was performed Tyr-705 Tyr-705 mary antibodies (STAT3, pSTAT3 , ZEB1, and E-cad- between pSTAT3 and ZEB1. herin; dilution 1:100) in consecutive tissue sections using the RESULTS LSAB kit (DakoCytomation, Copenhagen, Denmark) accord- ing to the manufacturer’s instructions. Effect of STAT3 on E-cadherin, N-cadherin, and Vimentin The slides were examined independently by two investiga- Expressions in CRC Cells—We previously showed that acti- tors blinded to both clinical and pathologic data. Protein vated STAT3 is constitutively expressed in CRC and mediates expression was quantified using a visual grading system based cell proliferation, whereas knockdown of STAT3 significantly on the extent of staining (percentage of positive tumor cells restores E-cadherin expression (18). Down-regulation of graded on a scale of 0–4: 0, none; 1, 1–25%; 2, 26–50%; 3, E-cadherin is one of the EMT phenotypes in cancer progression 51–75%; 4, 475%) and the intensity of staining (graded on a (3). To determine whether STAT3 mediates EMT initiation in scale of 0–3: 0, no staining; 1, weak staining; 2, moderate stain- CRC cells, the effect of STAT3 siRNA was evaluated in highly ing; 3, strong staining). For further analysis, an index value was invasive LoVo CRC cells. Western blot analysis showed that calculated as a product of grades of the extent and intensity of STAT3 siRNA significantly decreased STAT3 expression and staining to define the cut-off value for high expression of the phosphorylation in these cells (Fig. 1, A and B), indicating that proteins, and the protein expression was classified into two cat- the STAT3 was knocked down effectively. Knockdown of egories: high (grades 4–12) and low (grades 0–3). STAT3 significantly increased E-cadherin and decreased FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5823 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 4. Effect of STAT3 on ZEB1 expression and function in CRC cells. A representative Western blot (A) and the summarized data (B) show that transfection with STAT3 siRNA significantly decreased STAT3 phosphorylation and expressions of STAT3 and ZEB1 in LoVo cells. Another Western blot (C) and the summarized data (D) show that STAT3 phosphorylation and expressions of STAT3 and ZEB1 were boosted in STAT3-overexpressing SW1116 cells. E, stable knockdown of ZEB1 dramatically decreased cell invasion in basal condition and blocked STAT3-induced cell invasion in LoVo cells. n  3, t test; *, p  0.05, compared with control siRNA. n  3, t test; **, p  0.05, compared with pCDNA3.1. n  3, ANOVA; Œ, p  0.01, compared with pCDNA3.1 control shRNA lentivirus; ŒŒ, p  0.05, compared with pCDNA3.1-STAT3 control shRNA lentivirus. Error bars, S.E. N-cadherin and vimentin expressions (Fig. 1, A and B), suggest- apoptosis are also hallmarks of tumor EMT progression in epi- ing that STAT3 may contribute to EMT progression in LoVo thelial carcinoma (32). Therefore, we examined whether cells. STAT3 participates in these aspects of EMT progression in To further confirm the role of STAT3 in down-regulation of CRC cells. Transwell cell invasion assays showed that knock- E-cadherin and up-regulation of N-cadherin and vimentin in down of STAT3 expression significantly reduced the invasion CRC cells, we constructed and transfected the recombinant ability of LoVo cells normally characterized as highly invasive pCDNA3.1-STAT3 plasmid into low invasion SW1116 cells. (Fig. 2A). Meanwhile, overexpression of STAT3 dramatically Transfection of the pCDNA3.1-STAT3 plasmid significantly increased the invasiveness of SW1116 cells (Fig. 2B), indicating increased STAT3 expression and phosphorylation in SW1116 that STAT3 may have significant effects on cell migration and cells, when compared with the pCDNA3.1 control (Fig. 1, C and invasion in CRC cells. In cell colony-forming assays, the num- D), indicating that STAT3 was successfully overexpressed. ber of colonies of LoVo cells was reduced by nearly 80% when Overexpression of STAT3 significantly reduced E-cadherin transfected with STAT3 siRNA as compared with control and enhanced N-cadherin and vimentin expressions in siRNA (Fig. 2C). Up-regulation of STAT3 significantly SW1116 cells, compared with pCDNA3.1 transfection. These increased the number of SW1116 cell colonies, compared with results further indicate that STAT3 may mediate EMT initia- transfection of pCDNA3.1 (Fig. 2D). The data suggest that tion and progression in CRC cells. STAT3 may mediate cell colony formation in CRC cells. In Role of STAT3 in Cell Invasion, Cell Colony Formation, Che- addition, the CRC cell survival rate was lower in the STAT3 mosensitivity, and Resistance to Apoptosis in CRC Cells—It has siRNA-transfected group than in the control siRNA group after been reported that EMT may induce cell migration, alter inva- treatment with different doses of fluorouracil (Fig. 2E), indicat- sion properties, promote chemotherapy drug resistance, and ing that knockdown of STAT3 significantly enhanced chemo- prevent apoptosis (3). Aberrant cell survival and resistance to sensitivity of CRC to this drug. Furthermore, overexpression of 5824 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 5. Role of STAT3 in the regulation of ZEB1 expression in CRC cells. A, bioinformatic analysis of STAT3 transcriptional factor binding site in part of the ZEB1 gene promoter region. The numbers on the left side indicate the locations upstream of the first base of the initial transcription site. STAT3-binding sites are highlighted, and the DNA sequence encompassed by two arrows was amplified in the ChIP assay. B, representative results from three experiments showed that ZEB1 DNA was detectable in the chromatin sample immunoprecipitated from SW1116 cells using an antibody against STAT3, suggesting that STAT3 binds to the ZEB1 promoter. Input DNA was used as a positive control; rabbit IgG and cell lysates without antibody were used as negative controls. C, overexpression of STAT3 caused a nearly 40% increase in ZEB1 promoter luciferase activity in SW1116 cells. D, knockdown of STAT3 significantly decreased ZEB1 promoter luciferase activity in SW1116 cells. E, two putative STAT3 binding sites were located between nt 500 and 100. White and black rhombuses indicate wild type and mutant sequences for STAT3 binding sites, respectively. WT, wild type; MT, mutant type. Mutation of STAT3 binding sites significantly decreased transcriptional activity of the ZEB1 promoter in a luciferase assay. n  3, t test; **, p  0.05, compared with pCDNA3.1pGL3-basic-ZEB1PWT. n  3, t test; *, p  0.05, compared with control siRNApGL3-basic-ZEB1PWT; Œ, p  0.01, compared with pGL3-basic-ZEB1PWT. Error bars, S.E. STAT3 significantly decreased cell apoptosis in response to dif- SW1116 and LoVo cells. The data further indicate that STAT3 ferent doses of etopside (Fig. 2F), indicating that STAT3 may may participate in regulating ZEB1 expression in CRC cells. Next, we examined whether ZEB1 expression depended on play an important role in resistance to fluorouracil treatment and etopside-induced apoptosis during the EMT process of STAT3 in CRC cells. After transfection of STAT3 siRNA into CRC progression. LoVo cells for 48 h, Western blot analysis showed that knock- JAK/STAT3 Pathway May Regulate ZEB1 Expression in CRC down of STAT3 significantly down-regulated ZEB1 expression Cells—It has been confirmed that ZEB1, ZEB2, Snail1, Snail2, compared with the control (Fig. 4, A and B), indicating that Twist1, Twist2, and E12/E47 are critical transcription factors STAT3 may mediate ZEB1 expression in CRC cells. By con- that repress E-cadherin expression in EMT of cancer progres- trast, when pCDNA3.1-STAT3 was transfected into SW1116 sion (3). After having found that STAT3 may regulate E-cad- cells for 48 h, the overexpression of STAT3 significantly increased ZEB1 expression, compared with pCDNA3.1 (Fig. 4, herin expression, we next detected the expressions of ZEB1, ZEB2, Snail1, Snail2, Twist1, Twist2, and E12/E47 in CRC cells. C and D). These results further indicate that STAT3 may par- Although treatment with the JAK/STAT3 pathway inhibitor ticipate in regulation of ZEB1 expression in CRC cells. More- AG490 significantly down-regulated ZEB1 expression in over, stable knockdown of ZEB1 dramatically decreased cell SW1116 (Fig. 3A) and LoVo (Fig. 3B) cells, it did not dramati- invasion in basal conditions. STAT3-induced CRC cell invasion cally affect the expressions of any of the other molecules was significantly decreased by stable knockdown of ZEB1 detected at the mRNA level in CRC cells, indicating that the expression in CRC cells (Fig. 4E), indicating that STAT3-in- JAK/STAT3 pathway may specifically mediate ZEB1 expres- duced CRC cell invasion may depend on ZEB1. sion in CRC cells. As shown in Fig. 3, C and D, knockdown of Furthermore, DNA sequence analysis of the ZEB1 promoter STAT3 significantly down-regulated ZEB1 expression in both regions (nt 500 to 100) revealed two putative STAT3 binding FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5825 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 6. Role of ZEB1 in STAT3-regulated E-cadherin expression in CRC cells. A, a representative Western blot analysis showed that transfection of ZEB1 siRNA significantly reduced the expression of ZEB1. A representative Western blot (B) and the summarized data (C) showed that STAT3 overexpression did not down-regulate E-cadherin expression after ZEB1 knockdown. D, knockdown of STAT3 or ZEB1 expression caused a nearly 2-fold increase in E-cadherin promoter luciferase activity in SW1116 cells. E, overexpression of STAT3 significantly decreased E-cadherin promoter luciferase activity in SW1116 cells. ZEB1 siRNA significantly blocked STAT3-induced down-regulation of E-cadherin promoter luciferase activity in SW1116 cells; n 3, ANOVA; Œ, p 0.05, compared with pCDNA3.1control siRNA; ŒŒ, p  0.05, compared with pCDNA3.1-STAT3control siRNA; n  3, ANOVA; *, p  0.05, compared with control siRNApGL3-basic-E-cadherinPWT; **, p  0.05, compared with pCDNA3.1control siRNApGL3-basic-E-cadherinPWT; ***, p  0.05, compared with pCDNA3.1-STAT3control siRNApGL3-basic-E-cadherinPWT. Error bars, S.E. sites (Fig. 5A). Thus, in the ChIP assay, we designed one 48 h later. Western blot analysis showed that knockdown of primer set (nt 310 to 130, containing two putative ZEB1 expression significantly increased the expression of STAT3 binding sites), with product sizes of 200 bp, to E-cadherin in CRC cells. Furthermore, STAT3-induced E-cad- amplify part of the ZEB1 promoter regions. As shown in Fig. herin down-regulation was dramatically blocked by down-reg- 5B, this primer set showed amplifiable products. In contrast, ulation of ZEB1 (Fig. 6, B and C), indicating that ZEB1 may no detectable amplification was observed in cell lysates incu- participate in STAT3-induced E-cadherin down-regulation in bated with non-relevant rabbit IgG or cell lysate without CRC cells. antibody incubation (negative controls). In the dual lucifer- Luciferase assays were then performed to further confirm ase assay, mutation of STAT3 binding sites of ZEB1 pro- whether STAT3 repressed E-cadherin expression via altering moter exhibited almost a 60% decrease in relative luciferase ZEB1 regulation of E-cadherin transcription. We found that activity compared with that of wild type construct (Fig. 5E), knockdown of STAT3 or ZEB1 significantly up-regulated suggesting that STAT3 may directly bind to the ZEB1 pro- E-cadherin promoter activity in CRC cells (Fig. 6D). Knock- moter and regulate the transcriptional activity of ZEB1.We down of ZEB1 significantly abolished the STAT3-induced further found that overexpression of STAT3 significantly decrease in E-cadherin promoter activity (Fig. 6E). Further- up-regulated transcriptional activity of the ZEB1 promoter more, DNA sequence analysis of the E-cadherin promoter in CRC cells (Fig. 5C). Knockdown of STAT3 significantly regions (nt520 to70) revealed two putative STAT3 binding down-regulated transcriptional activity of the ZEB1 pro- sites and four putative ZEB1 binding sites (Fig. 7A). Thus, in the moter (Fig. 5D). The data suggest that STAT3-induced up- ChIP assay, we designed one primer set (E-cadherin-a, nt206 regulation of ZEB1 may depend on activation of ZEB1 tran- to 40, containing three putative ZEB1 binding sites), with prod- scription in CRC cells. uct sizes of250 bp, to amplify part of the E-cadherin promoter Effects of ZEB1 on STAT3-induced E-cadherin Down- regions. As shown in Fig. 7, B and C, this primer set showed regulation—In order to determine whether ZEB1 mediates amplifiable products. In contrast, no detectable amplification STAT3-induced E-cadherin down-regulation in CRC cells, was observed in cell lysates incubated with non-relevant rabbit pCDNA3.1-STAT3 and control plasmid were introduced into IgG or cell lysate without antibody incubation (negative con- CRC cells after ZEB1 siRNA and control siRNA transfection trols). These results indicate that ZEB1 may directly bind to the 5826 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 7. A, bioinformatic analysis of STAT3 and ZEB1 transcriptional factor binding site in part of the E-cadherin gene promoter region. The numbers on the left indicate the locations upstream of the first base of the initial transcription site. STAT3-binding sites and ZEB1-binding sites are highlighted, and the DNA sequence surrounded by two arrows was for ChIP. Representative results from three experiments show that E-cadherin DNA was detected in the chromatin sample immunoprecipitated from SW1116 (B) and LoVo (C) cells using an antibody against ZEB1, respectively, suggesting that ZEB1 binds to the E-cadherin promoter. Input DNA was used as a positive control; rabbit IgG and cell lysates without antibody were used as negative controls. Real-time PCR of the ChIP samples showed that overexpression of STAT3 dramatically increased the binding efficiency of ZEB1 to the E-cadherin promoter in SW1116 (D) and LoVo (E) cells. Knockdown of STAT3 significantly decreased the binding efficiency of ZEB1 to the E-cadherin promoter in SW1116 (F) and LoVo (G) cells. n  3, t test; *, p  0.01, compared with pCDNA3.1. n  3, t test; **, p  0.05, compared with control siRNA. Error bars, S.E. E-cadherin promoter. Furthermore, results of a ChIP assay and dual luciferase assay showed that the transcriptional activity of real-time PCR analysis demonstrated that overexpression of E-cadherin promoter was not dramatically changed after muta- STAT3 increased ZEB1 recruitment to the E-cadherin pro- tion of STAT3 binding sites in E-cadherin promoter (Fig. 8). moter (Fig. 7, D and E), whereas it was decreased by knockdown The data suggest that STAT3 may not directly bind to the of STAT3 (Fig. 7, F and G). The data suggest that STAT3 may E-cadherin promoter. play an important role in ZEB1 binding to the E-cadherin pro- Expressions of pSTAT3, STAT3, ZEB1, and E-cadherin in moter in SW1116 and LoVo cells. Moreover, the transcrip- CRC Tissues—Table 1 showed the frequencies of STAT3, Tyr-705 tional activity of the E-cadherin promoter was significantly pSTAT3 , ZEB1, and E-cadherin expression in normal increased after mutation of ZEB1 binding sites or dual mutation and tumor colon tissues by immunohistochemical staining. Tyr-705 of STAT3 and ZEB1 binding sites in the E-cadherin promoter In consecutive tissue sections, pSTAT3 , mostly pres- (Fig. 8). The data further suggests that ZEB1 may directly bind ent in the nucleus, was found with higher expression in to the E-cadherin promoter and regulate the transcriptional 16.7% of the normal colon epithelium samples and 65.7% of activity of E-cadherin. the colon adenocarcinoma samples (Fig. 9, B and F). ZEB1 In addition, in order to examine whether STAT3 may showed predominantly nuclear localization, with occasional directly bind to the E-cadherin promoter, we designed two cytoplasmic staining (Fig. 9, A and E). Immunostaining with an primer sets (E-cadherin-b, nt 416 to 224, containing one anti-ZEB1 antibody showed higher expression in 19 and 60% of putative STAT3 binding site; E-cadherin-c, nt 83 to 56, con- normal colon epithelium samples and colon adenocarcinoma taining one putative STAT3 binding site), with product sizes of samples, respectively. E-cadherin was observed at higher expres- 200 bp to amplify parts of the E-cadherin promoter regions. sion in 100% of the normal colon epithelium samples and in 20% of However, no E-cadherin DNA was detectable in the immuno- the adenocarcinoma samples (Fig. 9, D and H). Our data suggest Tyr-705 precipitated chromatin sample of SW1116 or LoVo cell lysates that the expressions of pSTAT3 and ZEB1 were significantly by using an antibody against STAT3 (data not shown). Further up-regulated ( test, p 0.001), whereas the expression of E-cad- FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5827 STAT3 and ZEB1 Signaling in CRC EMT Progression FIGURE 8. Two putative STAT3 binding sites and four putative ZEB1 binding sites were located between nt520 and70 of the E-cadherin 5-flanking region. White and black rhombuses indicate a wild or mutant sequence for STAT3 binding sites, respectively. White and black triangles indicate a wild type or mutant sequence for ZEB1 binding sites, respectively. WT, wild type; STAT3B MT, mutant type of each STAT3 mutation binding site; ZEB1B MT, mutant type of each ZEB1 mutation binding site; ZEB1B and STAT3B MT, mutant type of each ZEB1 and STAT3 mutation binding site. Mutation of ZEB1 binding sites or dual mutation of STAT3 and ZEB1 binding sites significantly increased the transcriptional activity of E-cadherin promoter in the luciferase assay. Mutation of STAT3 binding sites had no obvious effect on the transcriptional activity of the E-cadherin promoter. n  3, ANOVA; *, p  0.05, compared with pGL3-basic-E- cadherinPWT. Error bars, S.E. TABLE 1 Frequencies of STAT3, pSTAT3, ZEB1, and E-cadherin expression in normal and tumor colon samples Tyr-705 2 pSTAT3 and ZEB1 were mostly present in adenocarcinoma, whereas the expression of E-cadherin was significantly decreased in adenocarcinoma ( test). There was no significant difference in STAT3 expression between normal epithelia and adenocarcinoma. STAT3 pSTAT3 ZEB1 E-cadherin n High Low p value High Low p value High Low p value High Low p value a a a Normal epithelium 21 19 2 0.14 3 18 0.001 417 0.01 21 0 0.001 Adenocarcinoma 35 26 9 23 12 21 14 7 28 p  0.001. 2 Tyr-705 herin was significantly decreased ( test, p  0.001) in colorectal pSTAT3 and ZEB1 may play important roles in CRC adenocarcinoma. metastasis. Moreover, Table 2 showed that the high expressions of Tyr-705 pSTAT3 and ZEB1 were significantly more frequent in DISCUSSION poorly differentiated CRC tissue than in well differentiated The JAK/STAT signaling pathway plays a significant role in CRC tissue (Fisher’s exact test, p  0.001). Furthermore, high Tyr-705 immune function, cell growth, and differentiation (33). Accu- expression of pSTAT3 was significantly correlated with mulating evidence has indicated that STAT3 correlates with high expression of ZEB1 (r  0.7385, p  0.001). There was no cell proliferation in breast carcinoma (34) and non-small cell significant difference in high STAT3 expressions between the lung cancer (35). In a previous study, we found that activation of CRC tissues of different differentiation levels. In addition, other STAT3 may mediate human CRC tumorigenesis and progres- clinical characteristics, including age and gender, were not Tyr-705 sion, and knockdown of STAT3 significantly increases the directly related to the expressions of STAT3, pSTAT3 , and ZEB1. The results indicate that the high expressions of expression of E-cadherin (18). 5828 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 8 •FEBRUARY 17, 2012 STAT3 and ZEB1 Signaling in CRC EMT Progression Tyr-705 FIGURE 9. Expressions of STAT3, ZEB1, and E-cadherin in CRC. Shown is immunohistochemical analysis of consecutive tissue sections for ZEB1, pSTAT3 , Tyr-705 STAT3, and E-cadherin in normal colorectal mucosa and high grade CRC. A and B, nuclear staining of ZEB1 and pSTAT3 were mostly presented in adenocarci- Tyr-705 noma. Absent expression of ZEB1 (E) or pSTAT3 (F) was evident in the stroma or epithelium of the normal colorectal mucosa. C and G, cytoplasmic and nuclear staining of STAT3 was frequently detected in normal mucosa and adenocarcinoma. H, cytoplasmic staining of E-cadherin was predominantly detected in normal colorectal mucosa. D, staining of E-cadherin was dramatically decreased in adenocarcinoma, compared with normal colorectal epithelium. A–H (original magnifica- tion,200), representative areas from A1–H1 (original magnification,40), respectively. TABLE 2 STAT3, pSTAT3, and ZEB1 expressions in CRC tissues with different levels of differentiation Tyr-705 Correlation analysis of expressions of STAT3, pSTAT3 , and ZEB1 and clinicopathologic characteristics in CRC patients. Statistical analysis was conducted with Tyr-705 Fisher’s exact test. p values less than 0.05 were considered statistically significant. pSTAT3 and ZEB1 expressions were significantly related to TNM stage (Fisher exact test), suggesting that constitutive activation of STAT3 and elevation of ZEB1 may contribute to poor prognosis in CRC patients. STAT3 pSTAT3 ZEB1 High Low p value High Low p value High Low p value Sex Male 16 5 1 13 8 0.72 12 9 0.68 Female 10 4 10 4 7 7 Gender 65 16 3 0.25 14 5 0.3 10 9 0.83 65 10 6 9 7 9 7 TNM stage a a I/II 10 6 0.25 4 12 0.001 511 0.001 III/IV 16 3 19 0 16 3 p  0.001. As a calcium-dependent cell adhesion molecule, E-cadherin loss of E-cadherin expression is a crucial step in the initiation of is expressed predominantly in epithelial tissues (36). It is an tumor metastasis and a fundamental event in EMT (3). EMT important tumor suppressor gene, which may lose expression plays important roles in tumor formation and progression and function in tumor progression and invasion (37–40). The through enhancing cell proliferation, migration, and drug FEBRUARY 17, 2012• VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 5829 STAT3 and ZEB1 Signaling in CRC EMT Progression resistance and preventing apoptosis (3). It has been reported examined whether STAT3 may directly bind to the E-cadherin that the TGF, Wnt, Notch, and EGF signaling pathways may promoter. Although we found two putative STAT3 binding induce EMT in the progression of different cancers (41–44). sites in the E-cadherin promoter region, no E-cadherin DNA Although IL-6 may induce an EMT phenotype in human breast was detected in the immunoprecipitated chromatin sample of cancer cells (45) and TGF-1 induces EMT in mouse hepato- SW1116 or LoVo cell lysates by using an antibody against cytes (46) via activation of STAT3, the role of STAT3 is not yet STAT3 (data not shown). In addition, the transcriptional activ- well understood in human CRC cancer EMT progression. ity of E-cadherin promoter was not dramatically changed after In our present study, we found that activation of STAT3 may mutation of putative STAT3 binding sites in the E-cadherin induce changes in epithelial cells resembling that of EMT pro- promoter (Fig. 8). The data suggest that STAT3 may not gression for the following reasons. 1) Knockdown of STAT3 directly bind to the E-cadherin promoter. Based on the data significantly decreased N-cadherin and vimentin expressions described above, we hypothesized that ZEB1 may mediate (Fig. 1, A and B). 2) Overexpression of STAT3 significantly STAT3-induced E-cadherin down-regulation in CRC cells. reduced E-cadherin and enhanced N-cadherin and vimentin This hypothesis was further supported by our finding that expressions (Fig. 1, C and D). 3) Down-regulation of STAT3 STAT3-induced decrease of E-cadherin expression (Fig. 6, B significantly reduced cell invasion ability (Fig. 2A) and cell col- and C) and promoter transcription activity (Fig. 6E) was signif- ony numbers (Fig. 2C) of CRC cells. 4) Overexpression of icantly blocked by knockdown of ZEB1 in CRC cells. In the STAT3 dramatically increased the invasion ability (Fig. 2B) and real-time ChIP PCR assay, overexpression of STAT3 increased cell colony numbers (Fig. 2D) in CRC cells. Furthermore, ZEB1 recruitment to the E-cadherin promoter (Fig. 7, D and E), Thiery et al. (3) have demonstrated that EMT may be involved whereas it was decreased by knockdown of STAT3 (Fig. 7, F and in more than invasion in cancer; it may be involved as well in G). The transcriptional activity of the E-cadherin promoter was resistance to chemotherapy and cell death. Therefore, we also significantly increased after mutation of ZEB1 binding sites or explored whether STAT3 participates in the resistance to fluo- dual mutation of STAT3 and ZEB1 binding sites in the E-cad- rouracil (a chemotherapy drug) treatment and promotes CRC herin promoter in the dual luciferase assay (Fig. 8). The data cell resistance to etopside-induced cell apoptosis. Our data (Fig. suggest that ZEB1 may contribute to the STAT3-induced 2, E and F) indicate that STAT3-induced EMT may cause resis- decrease in E-cadherin expression via increasing its binding tance to conventional chemotherapy and cell death in colon efficiency to the E-cadherin promoter region. Tyr-705 cancer. Our detection of pSTAT3 and ZEB1 expressions by Because down-regulation of E-cadherin expression is a key immunohistochemistry further showed that they were remark- initiating event in EMT, transcription factors that repress ably increased in colon adenocarcinoma, compared with nor- E-cadherin have been defined as inducers of EMT. The major mal colon epithelium (Fig. 9). However, E-cadherin expression transcriptional repressors that are known to down-regulate was significantly decreased in high grade colon adenocarci- E-cadherin expression include those in the Snail and ZEB fam- noma (Fig. 9). The data are consistent with other reports showing Tyr-705 ilies (6, 47). In our study, ZEB1 expression was significantly that pSTAT3 is constitutively activated and correlated with reduced after treatment with the JAK/STAT pathway inhibitor CRC dedifferentiation (49). ZEB1 expression is absent in normal AG490 and STAT3 siRNA transfection (Fig. 3, A–D). However, colon and breast tissues, whereas ZEB1-positive tumor tissues ZEB2, Snail1, Snail2, Twist1, Twist2, and E12/E47 expressions express low levels of cytokeratin (50). Furthermore, our study also were not obviously altered in response to AG490. The data demonstrates that high tumor, lymph node, and metastasis suggest that ZEB1 but not ZEB2, Snail1, Snail2, Twist1, Twist2, (TNM) staging of CRC was significantly more frequent in patients Tyr-705 or E12/E47 may mediate STAT3-induced colon cancer pro- with high expressions of pSTAT3 and ZEB1 than in those gression. Results of the cell invasion assays further supported with low expression (Tables 1 and 2). Moreover, a very strong Tyr-705 this conclusion. Stable knockdown of ZEB1 significantly positive correlation between pSTAT3 and ZEB1 expression reduced cell invasion at the basal conditions and blocked was identified in cancerous tissues. These observations imply that Tyr-705 STAT3-induced cell invasion in CRC cells (Fig. 4E). This result pSTAT3 and ZEB1 expression may be powerful indicators is consistent with another report showing that ZEB1 expression of CRC metastasis. is important during colon cancer progression (48). In addition, In conclusion, STAT3 may play an important role in the we further found that ZEB1 expression was significantly EMT process of CRC progression. As an EMT inducer, ZEB1 decreased by knockdown of STAT3 (Fig. 4, A and B), whereas it expression may be regulated by STAT3. ZEB1 may participate was dramatically increased by overexpression of STAT3 (Fig. 4, in STAT3-induced cell invasion in CRC cells. Moreover, ZEB1 C and D). These results further indicate that the activation of may mediate STAT3-induced down-regulation of E-cadherin STAT3 is highly important for ZEB1 expression in colon carci- via directly inhibiting transcriptional activity of the E-cadherin noma. Further analysis revealed two putative STAT3 binding promoter in CRC. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Feb 17, 2012

Keywords: Cell Signaling; Metastasis; STAT3; Tumor; Tumor Therapy; ZEB1; Colorectal Cancer; Epithelial-Mesenchymal Transition

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