Volume 142, Issue 3, Pages 521-530.e3 (March 2012) Reciprocal Activation Between PLK1 and Stat3 Contributes to Survival and Proliferation of Esophageal Cancer Cells  Yu Zhang, Xiao–Li Du, Cheng–Ji Wang, De–Chen Lin, Xia Ruan, Yan–Bin Feng, Yan–Qiu Huo, Haiyong Peng, Jing–Lu Cui, Tong–Tong Zhang, Yong–Quan Wang, Hongbing Zhang, Qi–Min Zhan, Ming–Rong Wang  Gastroenterology  Volume 142, Issue 3, Pages 521-530.e3 (March 2012) DOI: 10.1053/j.gastro.2011.11.023 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Stat3 is constitutively activated in ESCC and positively regulates PLK1 expression. (A) The expression level of p-Stat3 (Tyr705) in ESCC tissues was detected by immunoblotting (left panel) or immunohistochemistry staining (right panel). T, tumor; N, the corresponding normal epithelia. (B) ESCC cells were serum-starved for 24 hours and then treated with or without 100 μmol/L AG490 for 24 hours. Apoptosis was determined by Annexin V–FITC/PI staining (left panel). Data are mean ± SD. **P < .01. (C) KYSE510 cells were transfected with Stat3-siRNA for 24 hours and then synchronized with or without 400 ng/mL nocodazole for 24 hours. (D) KYSE510 cells were transfected with EGFP-tagged Stat3β (Stat3-DN) or empty vector for 48 hours. PLK1 expression was detected by immunofluorescence staining. Representative images are shown. Original magnification, ×630 (left panel). The percentage of PLK1 down-regulated cells among the transfected cells in 25 fields chosen randomly from 2 independent experiments were analyzed and plotted (right panel). Data are mean ± SD. ***P < .001. (E) pBabe-Stat3C or empty vector stably transfected NIH3T3 cells were synchronized with nocodazole for 24 hours. (B, C, and E) The cell lysates were immunoblotted for the indicated proteins. (F) Quantitative reverse-transcription PCR analysis for ESCC cells transfected with Stat3-siRNA (left panel) or NIH3T3 cells stably expressed Stat3C (right panel). Data are mean ± SEM. *P < .05; **P < .01. Gastroenterology 2012 142, 521-530.e3DOI: (10.1053/j.gastro.2011.11.023) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Stat3 activates PLK1 transcription. (A) The schematic diagram depicts 4 putative Stat3 binding sites in the human PLK1 promoter and primers used in the ChIP assay. (B) The ChIP assay was performed using the chromatin prepared from KYSE510 cells. (C) EMSAs with PLK1-SIE probes and nuclear extracts from Stat3C (3C) or empty vector (pB) stably transfected NIH3T3 cells synchronized with nocodazole, as well as ESCC cells treated with or without JSI-124. (D–F) PLK1 transcriptional activity was analyzed using luciferase reporter assay. (D) Schematic diagram of PLK1 reporter constructs. In the pGL3-PLK1-DM construct, the PLK1-SIE core sequence was deleted and is shown as a dashed line. (E) NIH3T3 fibroblasts were cotransfected with Stat3C, Stat3Y705F (YF), and empty vector pBabe along with pGL3-PLK1. The PLK1 promoter activity was measured at 48 hours after transfection. (F) ESCC cells were transfected with pGL3-PLK1 or pGL3-PLK1-DM. At 24 hours after transfection, the cells were synchronized with 100 μmol/L mimosine (Mimo) or 400 ng/mL nocodazole (Noco) for 24 hours, and then the PLK1 promoter activity was measured. Data are mean ± SD. ***P < .001. nsd, no significant difference. Gastroenterology 2012 142, 521-530.e3DOI: (10.1053/j.gastro.2011.11.023) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 PLK1 positively regulates Stat3 expression involving β-catenin. (A and B) ESCC cells were transfected with PLK1-siRNA for 48 hours. (A) Immunoblotting (left panel) and quantitative reverse-transcription PCR analysis (right panel). Data are mean ± SEM. **P < .01. (B) Stat3 transcriptional activity was measured using reporter assay. Data are mean ± SD. ***P < .001. (C) PLK1-EGFP or empty vector transiently transfected NIH3T3 cells were synchronized with nocodazole for 24 hours, and then subjected to immunoblotting (left panel) or quantitative reverse-transcription PCR (right panel). Data are mean ± SEM. **P < .01. (D-F) KYSE510 cells were transfected with (D) PLK1 siRNA, (E) β-catenin siRNA, or (F) co-transfected with β-catenin S37A mutant or empty vector along with PLK1 siRNA, and then the cell lysates were immunoblotted for the indicated proteins. Gastroenterology 2012 142, 521-530.e3DOI: (10.1053/j.gastro.2011.11.023) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 The reciprocal regulation of Stat3 and PLK1 is critical for resistance to apoptosis. (A) KYSE510 cells were transfected with siRNA against Stat3 or PLK1 for 48 hours. (B) KYSE510 cells were transiently transfected with pEGFP-PLK1 or empty vector, and treated with 10 μmol/L JSI-124 for 4 hours at 48 hours after transfection. (C) KYSE510 cells stably expressing Stat3C or empty vector were treated with 50 nmol/L BI 2536 for 24 hours. Apoptosis was determined by Annexin V–FITC/PI or Annexin V–PE/7-AAD double staining, and representative results are shown. Data are mean ± SEM. **P < .01. Stat3 and PLK1 expression were determined by Western blot. Gastroenterology 2012 142, 521-530.e3DOI: (10.1053/j.gastro.2011.11.023) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Disruption of PLK1 activity suppresses the growth of ESCC xenografts. (A) Immunostaining analysis for ESCC cells treated with or without 50 nmol/L BI 2536 for 24 hours. (B) Colony formation assays with KYSE510 cells exposed to 50 nmol/L BI 2536 or vehicle for only one time. Representative results are shown. (C and D) Nude mice bearing established KYSE510 xenograft tumors (average volume, ∼50 mm3) were treated with 50 mg/kg BI 2536 or vehicle control twice weekly on 2 consecutive days for 4 cycles (n = 5 per group). (C) Tumor volumes were measured weekly and data are mean ± SEM. **P < .01; ***P < .001. (D) Tumor weight was quantified at 4 weeks after drug administration. (E and F) KYSE510 xenograft tumors were excised from nude mice treated with 50 mg/kg BI 25636 or vehicle control for 48 hours. (E) Tumor lysates were immunoblotted for the indicated proteins. (F) Immunohistochemistry staining of PLK1 and Stat3 in SC tumor tissues sections Original magnification, ×400. Degree of intratumoral proliferation was determined by Ki67 staining, and apoptosis was measured by TUNEL assay Original magnification, ×200. Gastroenterology 2012 142, 521-530.e3DOI: (10.1053/j.gastro.2011.11.023) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 The functional interaction of Stat3 and PLK1 contributes to tumorigenicity of KYSE510 cells. KYSE510 cells stably expressing Stat3-shRNA (sh-Stat3) were infected with pLXIN-PLK1-hyg (sh-Stat3-PLK1) or empty vector pLXIN-hyg (sh-Stat3-Vec), and KYSE510 cells stably expressing scrambled shRNA (Ctrl) infected with empty vector pLXIN-hyg (Ctrl-Vec) were used as control. (A) Immunoblotting analysis for cells treated with 100 μmol/L mimosine for 24 hours. (B) Colony formation assays. Representative results are shown and colony number was plotted. Data are mean ± SD. nsd, no significant difference. (C–E) The stable clone cells were implanted SC into athymic mice (Nu/Nu) (n = 5 per group), and SC tumors were excised at 4 weeks after inoculation. (C) Photograghs of SC tumors are shown and tumor weight was quantified. (D) Tumor lysates were immunoblotted for the indicated proteins. (E) Immunohistochemistry staining of Stat3 and PLK1 in SC tumor tissue sections Original magnification, ×400. Degree of intratumoral proliferation was determined by Ki67 staining, and apoptosis was detected by TUNEL assay Original magnification, ×200. Gastroenterology 2012 142, 521-530.e3DOI: (10.1053/j.gastro.2011.11.023) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Blockade of JAK/Stat3 signaling induces apoptosis accompanied with PLK1 down-regulation in ESCC cells. (A) KYSE150 and KYSE510 cells were serum-starved for 24 hours and then treated with or without 100 μmol/L AG490 for 24 hours. Apoptosis was determined by Annexin V–FITC/PI staining and representative results are shown (left panel). (B) KYSE510 cells were treated with 10 μmol/L JSI-124 for 4 hours. (C) KYSE150 cells were transfected with Stat3-siRNA for 48 hours. (D) KYSE150 cells were transiently transfected with pEGFP-Stat3-DN (Stat3β) or empty vector. At 24 hours after transfection, the cells were synchronized with nocodazole for 24 hours. (A–D) Stat3, p-Stat3 (Tyr705), and PLK1 protein levels were detected by immunoblotting. Gastroenterology 2012 142, 521-530.e3DOI: (10.1053/j.gastro.2011.11.023) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Stat3 phosphorylation positively correlates with PLK1 expression in ESCC tissues. Representative results of p-Stat3 (Tyr705) and PLK1 immunostaining in sequential sections from the same tissue microarray. Original magnification, ×200. T35 (negative), T20 (moderate), and T87 (strong) are the case numbers. Gastroenterology 2012 142, 521-530.e3DOI: (10.1053/j.gastro.2011.11.023) Copyright © 2012 AGA Institute Terms and Conditions