1. Volume 143, Issue 6, Pages 1641-1649.e5 (December 2012)
Sorafenib Down-regulates Expression of HTATIP2 to Promote Invasiveness and Metastasis of Orthotopic Hepatocellular Carcinoma Tumors in Mice 
Wei Zhang, Hui–Chuan Sun, Wen–Quan Wang, Qiang–Bo Zhang, Peng–Yuan Zhuang, Yu–Quan Xiong, Xiao–Dong Zhu, Hua–Xiang Xu, Ling–Qun Kong, Wei–Zhong Wu, Lu Wang, Tian–Qiang Song, Qiang Li, Zhao–You Tang 
Gastroenterology 
Volume 143, Issue 6, Pages e5 (December 2012)
DOI: /j.gastro
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2. Figure 1
Sorafenib increased invasiveness and metastatic potential in LM3-RFP and SMMC7721, but not HepG2, orthotopic HCC models. The LM3-RFP model was first used to explore the effect of sorafenib on invasiveness and metastatic potential of HCC. (A) Tumor-bearing mice treated with sorafenib survived 88 days, compared with 70 days for controls (P < .01, left panel). Tumor size was 3.78 ± 0.25 cm3 in controls and 1.23 ± 0.16 cm3 in the group treated with sorafenib (P < .001, right panel). (B) Sorafenib reduced the number of lung metastasis (191.0 ± 22.8 vs 97.5 ± 18.0; P < .05) but did not reduce the rate of lung metastasis, which was 6 of 6 in the sorafenib group and 9 of 10 in controls (left panel). The standardized number of lung metastasis was greater in the sorafenib group than in controls (45.3 ± 6.8 vs 87.5 ± 19.5; P < .05; right panel). (C) The percentage of circulating tumor cells in sorafenib-treated mice was 5.23% ± 1.23%, which was higher than that in controls (0.44% ± 0.22%; P < .05). (D) Sorafenib induced more IHMs. (Left panel) Three tumors from controls and 3 sorafenib-treated tumors were compared by both bright field (b) and fluorescence (f) (arrows indicates IHM). (Center panel) Comparison of IHM (P < .05; Mann-Whitney U test). (Right panel) Confirmation of the presence of IHM by H&E staining. (E) A group of EMT markers detected by real-time PCR and Western blotting showed that E-cadherin was down-regulated and N-cadherin and vimentin were up-regulated. (F) Further evidence of the effect of sorafenib on invasiveness of HCC was found in HCC-LM3, SMMC7721, and HepG2 models (left panel). (Right panel) Tumor volumes in controls versus the sorafenib group were 1.43 ± 0.21 versus 0.57 ± 0.11 cm3 in the SMMC7721 model (P = .0072), 1.25 ± 0.25 versus 0.76 ± 0.11 cm3 in the HepG2 model (P = .1438), and 3.57 ± 0.38 versus 1.01 ± 0.11 cm3 in the HCC-LM3 model (P < .0001). Sorafenib significantly promoted IHM in the HCC-LM3 (P = .0196) and SMMC7721 (P = .0193) models but not in the HepG2 model (P = .1626).
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3. Figure 2
Sorafenib down-regulated HTATIP2 at the transcript and protein level. (A) Seven significantly down-regulated genes (>2-fold) were antioncogenes. (B) Expression of the 7 antioncogenes. (C) Gene network analysis by Ariadne software shows the changes in 84 genes. (D and E) Down-regulation of HTATIP2 by sorafenib was confirmed by PCR and Western blotting assays.
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4. Figure 3
Down-regulation of HTATIP2 by sorafenib contributed to increased invasiveness of HCC. (A) Expression of HTATIP2 was evaluated at the messenger RNA and protein level. (B) Tumor volumes in controls versus the sorafenib group were 3.57 ± 0.38 versus 1.01 ± 0.11 cm3 in the LM3-wt model (P < .0001) and 1.67 ± 0.45 versus 0.32 ± 0.09 cm3 in the LM3-LV-shHTATIP2 model (P = .0098). The number of IHMs in the controls versus sorafenib-treated group was 1.6 ± 0.4 versus 3.7 ± 0.6 in the LM3-wt model (P = .017; Mann-Whitney U test) and 3.2 ± 1 versus 3.8 ± 0.8 in the LM3-LV-shHTAITP2 model (P = .69; Mann-Whitney U test). (C–F) The proinvasive effect of sorafenib was HTATIP2 dependent and dose dependent. (C) Migration and invasion of HCC cell lines with different expression levels of HTATIP2. (D) Quantification of invasion assay. *LM3-wt compared with LM3-sh-HTATIP2; **HepG2-wt compared with HepG2-LV-HTATIP2; #LM3-wt compared with HepG2-wt and SMMC7721; *,**,#P < .05. (E) At 10 μmol/L, sorafenib significantly inhibited invasion in all 7 cell lines. (F) At 5 μmol/L, sorafenib inhibited invasion of cell lines with low expression of HTATIP2 but promoted invasiveness in cell lines with high expression of HTATIP2.
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5. Figure 4
Examination of HTATIP2 and EMT markers by Western blotting in 7 cell lines with sorafenib treatment in vitro. (A) Expression of HTATIP2 and EMT markers. (B) Sorafenib up-regulated N-cadherin and vimentin and down-regulated E-cadherin in SMMC7721 cells. (C) In LM3-wt and LM3-LV-shNon cells, 1 μmol/L and 5 μmol/L sorafenib down-regulated HTATIP2; N-cadherin and vimentin were up-regulated, while E-cadherin was not significantly different. In LM3-LV-shHTATIP2 cells, sorafenib did not change the expression of vimentin and N-cadherin. (D) In HepG2-wt and HepG2-LV-Non cells, 1 μmol/L and 5 μmol/L sorafenib did not change the expression of HTATIP2, N-cadherin, or vimentin. In HepG2-LV-HTATIP2 cells, sorafenib down-regulated HTATIP2 and E-cadherin and up-regulated N-cadherin and vimentin.
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6. Figure 5
HTATIP2 was regulated by sorafenib through the JAK/STAT3 pathway. (A) After 12-hour treatment, a down-regulation of pSTAT3 corresponded with a decrease of HTATIP2. (B) The down-regulation of HTATIP2 messenger RNA was confirmed by real-time PCR. (C and D) The down-regulation of HTATIP2 at (C) 12 hours and (D) 24 hours after administration of sorafenib was dose dependent. (E) Inhibition of pSTAT3 by AG490 at 100 μmol/L. (F) The dose-dependent inhibition of HTATIP2 by sorafenib became less significant when HCC-LM3 cells were pretreated with 100 μmol/L AG490 for 4 hours.
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7. Supplementary Figure 1
Proliferation assay. Sorafenib did not inhibit proliferation of 7 cell lines at a concentration of 5 μmol/L at 48 hours. At a concentration of 10 μmol/L, sorafenib inhibited proliferation at 48 hours in almost all cell lines.
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8. Supplementary Figure 2
Immunofluorescence assay for HTATIP2 and EMT markers. Changes in HTATIP2 protein and EMT markers were similar to those detected by Western blotting.
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9. Supplementary Figure 3
The proinvasive effect of sorafenib in immunocompetent mouse models. (A) HTAIP2 expression was lower in H22 than in Hepa1-6. When treated with sorafenib, Hepa1-6, but not H22, had decreased expression of HATIP2. (B) Intrahepatic metastasis was not increased with sorafenib treatment in H22 tumors. (C) Intrahepatic metastasis increased with sorafenib treatment in Hepa1-6 tumors.
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10. Supplementary Figure 4
Sorafenib tended to prolong recurrence-free survival (RFS) in patients with low expression of HTATIP2. (A) HTATIP2 expression was quantified by immunohistochemistry, and the median value was set as the cutoff point. Tumor samples with high expression and low expression of HTATIP2 are shown. (B) RFS curves for sorafenib-treated patients and controls. (C) Sorafenib did not prolong RFS in patients with high tumor expression of HTATIP2. (D) Sorafenib prolonged RFS in patients with low tumor expression of HTATIP2.
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