Volume 139, Issue 6, Pages 2146-2157.e12 (December 2010) Up-regulation of Krüppel-Like Factor 8 Promotes Tumor Invasion and Indicates Poor Prognosis for Hepatocellular Carcinoma  Jia–Chu Li, Xin–Rong Yang, Hai–Xiang Sun, Yang Xu, Jian Zhou, Shuang–Jian Qiu, Ai–Wu Ke, Yue–Hong Cui, Zhi–Jun Wang, Wei– Min Wang, Kang–Da Liu, Jia Fan  Gastroenterology  Volume 139, Issue 6, Pages 2146-2157.e12 (December 2010) DOI: 10.1053/j.gastro.2010.08.004 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Expression of KLF8 in HCC cell lines and tissue samples. (A) Relative KLF8 mRNA levels in different cell lines by qRT-PCR analysis (a: P < .05 compared with L-02; b: P < .05 compared with HepG2, SMMC7721, and SF/SMMC7721; c: P < .05 compared with MHCC97L; d: P < .05 compared with MHCC97H); Western blot and immunocytochemical analysis of KLF8 protein expression in liver cell lines L-02, HepG2, MHCC97L, and HCCLM3. (B) qRT-PCR results showed HCC cell lines with high KLF8 expression had high vimentin expression and low E-cadherin expression. (C) The patients with high expression of KLF8 have low E-cadherin expresion (r = −0.476, P = .0005) and no relation with KLF1 or KLF3 (P > .05). (D) Patients suffering HCC recurrence exhibited higher KLF8 mRNA expression levels than those without recurrence (P = .001). (E) Protein levels of KLF8 in tumor samples of HCC patients with or without recurrence by Western blot analysis. KLF, Krüppel-like factor; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; mRNA, messenger RNA. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Function analysis of KLF8 by siRNA. (A) KLF8 expression in HCCLM3 cells treated by siRNA for 72 hours in Western blot analysis; Bcl-xL and Cyclin D1 were down-regulated after KLF8 siRNA; gelatinase activity assay showed that secretion of matrix metalloproteinase 2 in the KLF8 siRNA group was lower compared with control group, and the activity of matrix metalloproteinase 9 was not affected. (B) Transwell Matrigel Invasion assays showed that the number of invasive cells in the KLF8 siRNA-treated group decreased significantly compared with those in the control group. (C) In MTT assay, the cell proliferation was significantly suppressed 72 hours after KLF8 siRNA transfection (P < .001). (D) Cell cycle analysis showed that KLF8 siRNA could increase tumor cell apoptosis (sub-G1: 16.8 ± 1.3 vs 7.4 ± 0.9, respectively) and cause S phase arrest (S phase: 18.7 ± 1.6 vs 30.3 ± 2.0, respectively). (E) The E-cadherin was up-regulated in immunofluorescence analysis after HCCLM3 KLF8 siRNA. (F) The EMT biomarkers including E-cadherin, N-cadherin, vimentin, and fibronectin were detected by RT-PCR. Loss of mesenchymal markers and gain of the epithelial markers after KLF8 siRNA in HCCLM3 are shown. KLF8, Krüppel-like factor 8; siRNA, small interference RNA; RT-PCR, reverse transcription polymerase chain reaction. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Effects of down-regulation of KLF8 on tumor growth and metastasis in vivo. (A) The morphologic characteristics of tumor in KLF8 shRNA group and control group. (B) The characteristic pictures of tumor specimen (a and d), lung metastases (b and e) by H&E, and KLF8 expression by immunohistochemistry (c and f) in KLF8 shRNA group and control shRNA group. (C) There are significant differences in tumor weight (1.0 ± 0.8 g vs 3.7 ± 0.8 g, respectively, P < .001) and lung metastasis rate (16.7% vs 100%, respectively, P = .015) between the KLF8 shRNA group and the control shRNA group. (D and E) KLF8 shRNA group showed increase in the number of apoptosis index (47.3% ± 5.9% vs 10.0% ± 1.7%, respectively, P < .01), substantial decrease in proliferation rate (14.1% ± 2.6% vs 56.1% ± 4.0%, respectively, P < .01), and a significant decrease of tumor vessels density (64.3 ± 7.0 vs 21.0 ± 3.2 Vessels/5HP, respectively, P < .01). Scale bars, 100 μm. KLF8, Krüppel-like factor 8; siRNA, small interference RNA; shRNA, small hairpin RNA. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Typical expressions of KLF8 in TMAs by immunochemistry analysis (A and B: negative; C and D: weak positive; E and F: strong positive; Scale bars, 50 μm). KLF8, Krüppel-like factor 8; TMAs, tissue microarrays. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Kaplan-Meier analysis of TTR and OS in 314 cases based on KLF8 expressions. Compared with KLF8 positive group, the TTR (A) and OS (B) were significantly higher in KLF8 negative group. The prognostic function of KLF8 is especially useful for patients with BCLC stage 0+A (C), TNM stage I (D), Edmondson stages I-II (E), and normal AFP levels (F). KLF8, Krüppel-like factor 8; BCLC, Barcelona Clinic Liver Cancer; AFP, α-fetoprotein; OS, overall survival; TTR, time to recurrence. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 The prognostic significance of KLF8 in early recurrence and late recurrence by Kaplan-Meier analysis. The prognostic significance of KLF8 existed in the early recurrence group (P = .001, A) but not in the later recurrence group (P = .623, B). KLF8, Krüppel-like factor 8. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 The apoptosis analysis and active caspase-3, -8, and -9 detection; chromatin immunoprecipitation (ChIP) analysis; and promoter activity assay. (A) The apoptosis was detected by Annexin V/propidium iodide (PI) staining, and we found that the apoptosis ratio was higher in the Krüppel-like factor (KLF) 8 small interfering RNA (siRNA) group; numbers in the respective quadrant profiles indicate the percentage of the cells present in this area. LL, living cells (Annexin V negative/PI negative); LR, early apoptotic/primary apoptotic cells (Annexin V positive/PI negative); UR, late apoptotic/secondary apoptotic cells (Annexin V positive/PI positive); UL, necrotic cells (Annexin V negative/PI positive). (B) The activity of caspase-3, -8, and -9 in HCCLM3 after KLF8 siRNA for 72 hours. The ratio of OD 405 nm of transfected cells/OD 405 nm of parental cells was calculated, and the value of caspase-3 and -9 were significantly higher in the KLF8 siRNA group, whereas caspase-8 level had no significant change after KLF8 siRNA. (C) KLF8 can physically bind to human Bcl-xL and Cyclin D1 promoter, whereas there was no significantly different affinity with MMP2. (D) In promoter analysis, KLF8 induced about a 2-fold increase of luciferase activity for Cyclin D1 and more than a 1.5-fold increase of Bcl-xL, whereas it induced no changes of MMP2 (*P < .01 vs control). Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Overexpression of Krüppel-like factor (KLF) 8 promotes SMMC7721 proliferation and invasion in vitro and in vivo. (A) KLF8 expressions in SMMC7721-KLF8 and SMMC7721-Mock group were detected by Western blot analysis. (B) Compared with the SMMC7721-Mock group, the cell proliferation of SMMC7721-KLF8 was significantly increased. (C) For trans-well Matrigel Invasion Assays, the invasion potential of SMMC7721-KLF8 cells were significantly higher than those of SMMC7721-Mock cells. (D) Overexpression of KLF8 in SMMC7721 resulted in a shift in epithelial-to-mesenchymal transition (EMT) marker expression and up-regulation of cyclin D1 and Bcl-xL. (E) The tumor weights in SMMC7721-KLF8 group were significantly greater than those in the SMMC7721-Mock group (2.6 ± 0.3 g vs 0.2 ± 0.1 g, P < .001); The characteristic pictures of tumor and intrahepatic metastasis; the metastatic ratio of intrahepatic metastasis in SMMC7721-KLF8 group was obviously increased compared with the SMMC7721-Mock group (100% vs 0%, respectively). White arrow, metastatic nodules. (F) The characteristic pictures of tumor specimen of H&E staining and KLF8 immunohistochemistry staining in pcDNA 3.1 and pcDNA3.1 KLF8 groups. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Validation gene changes after Krüppel-like factor (KLF) 8 small interfering RNA (siRNA). (A) The fold change of KLF1, 3, 4, 5, 6, and 8 expressions after siRNA was detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). (B) Several differentially expressed genes (IRF7, Rsu-1, cyclin G2, TGF-α, MMP1, MMP7, cyclin D1, and FAK) by microarray experiments were validated by qRT-PCR. (C) FAK protein expression was detected by Western blot. (D) Microarray-based gene expression profiles. Two hundred twenty-nine genes were up-regulated (ratio, >2.0), and 224 genes were down-regulated (ratio, <0.5) between siRNA/KLF8 and scrambled siRNA-treated HCCLM3, which were mainly categorized into 17 groups. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 The protein expression in Western blot analysis was quantitated by densitometry (using Image J 1.38 software; National Institutes of Health, Bethesda, MD). Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5 The schematic diagram for the role of Krüppel-like factor (KLF) 8 in HCC. KLF8 expression causes epithelial-to-mesenchymal transition (EMT) and promotes tumor cell proliferation status by up-regulating cyclin D1 expression, enhances antiapoptosis potential by up-regulating Bcl-xL expression, and induces invasive and metastatic ability by matrix metalloproteinase (MMP) 2 secretion. The roles of KLF8, both in proliferation and survival at the tumor origin and metastasis, as well as invasion function during hematogenous dissemination of cancer cells, were responsible for the high recurrence rate and poor prognosis observed in KLF8-positive HCC patients. Gastroenterology 2010 139, 2146-2157.e12DOI: (10.1053/j.gastro.2010.08.004) Copyright © 2010 AGA Institute Terms and Conditions