1. Volume 142, Issue 7, Pages 1547-1558.e14 (June 2012)
p28GANK Prevents Degradation of Oct4 and Promotes Expansion of Tumor-Initiating Cells in Hepatocarcinogenesis 
You–Wen Qian, Yao Chen, Wen Yang, Jing Fu, Jie Cao, Yi–Bin Ren, Jun–Jie Zhu, Bo Su, Tao Luo, Xiao–Fang Zhao, Rong–Yang Dai, Juan–Juan Li, Wen Sun, Meng–Chao Wu, Gen–Sheng Feng, Hong–Yang Wang 
Gastroenterology 
Volume 142, Issue 7, Pages e14 (June 2012)
DOI: /j.gastro
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2. Figure 1
p28GANK is preferentially expressed in liver T-ICs. (A) HCC TMA sections were used for immunohistochemical study on expression of p28GANK and OV6 expression. Representative staining of p28GANK and OV6 is shown (upper panel; original magnification 200×). The percentage of patients with high OV6 staining was higher in the p28GANK-high group (left lower panel). Correlation of expression levels of p28GANK and OV6 in HCCs (right lower panel). Spearman ρ = , P= (B) The disease-free and overall survival rates of 130 patients with HCC were compared among different groups. (C) qRT-PCR analysis of magnetic sorted OV6+ or CD133+ subpopulation was performed to evaluate mRNA expression level of p28GANK in SMMC-7721 and HCC-LM3 cells. Data are shown as mean ± SD. *P < .05; **P < .01. (D) Tumor cells isolated from freshly resected human HCC specimens were capable of forming spheroids in nonadherent conditions. Representative cases are shown (scale bar = 200 μm). qRT-PCR analysis was performed for p28GANK and stem cell-associated genes (CD133, EpCAM) in mechanically dissociated cancer spheroids vs primary adherent HCC cells (n = 45). The correlation between p28GANK and CD133 or EpCAM is shown (Spearman ρ = 0.52, P = .03; Spearman ρ = 0.49, P = .0006, respectively).
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3. Figure 2
Overexpression of p28GANK enhances cancer stem cell properties of T-ICs. (A) qRT-PCR analysis was performed for the indicated genes in magnetic sorted SMCC-7721 LV-p28GANK OV6+ or SMCC-7721 LV-luc OV6+ cells. Data represent mean ± SD. *P < .05; **P < .01 (left and middle panels). Flow cytometric analysis revealed that the percentage of OV6+ cells was significantly increased in LV-p28GANK–treated SMCC-7721 cells compared with LV-luc–treated cells. Representative results from 3 experiments are shown (right panel). (B) SMMC-7721 LV-p28GANK OV6+ cells were able to generate an increased number of primary and secondary passaged spheroids and an increased size of primary passaged spheroids as compared with SMMC-7721 LV-luc OV6+ cells (middle and right panels). Representative phase-contrast image of HCC spheroids derived from OV6+ cells (scale bar = 500 μm) (left panel). Experiments were performed in triplicate, and data are shown as mean ± SD. *P < .05; **P < .01. (C) The same amount of magnetic sorted LV-GFP OV6+ and LV-p28GANK OV6+ SMMC-7721 cells was mixed and cultured. One week later, the percentage of OV6+ subpopulation was determined by flow cytometric analysis. (D) Flow cytometric analysis showed standard chemotherapy with ADR (1 μg/mL) or cis-platinum (1.5 μg/mL) enriched the OV6+ subpopulation in SMMC-7721 cells after 4 days. Representative results from 3 experiments are shown. (E) p28GANK overexpression enhanced the colony formation capability of SMMC-7721 OV6+ cells in the presence of ADR (0.1 μg/mL) or cis-platinum (0.6 μg/mL) for 10 days; n = 3000 cells in 3 trials; **P < .01 (upper panel). Cell viability was also determined by cyclocystokinin-8 assay (lower panel). (F) Overexpression of p28GANK increased the percentage of side population cells in HCC-LM3 cells (left panel). Representative results from 3 experiments are shown. SMMC-7721 LV-p28GANK or LV-luc cells were treated with ADR (1 μg/mL) or cis-platinum (1.5 μg/mL) for 4 days, and qRT-PCR was performed for the indicated genes (right panel). Data represented mean ± SD. *P < .05; **P < .01.
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4. Figure 3
Overexpression of p28GANK enhances the tumorigenic and metastasis potential of OV6+ HCC cells in vivo. (A) Tumor incidence in mouse xenografts injected with the indicated number of LV-p28GANK– or LV-luc–infected SMMC-7721 OV6+ cells after 25 days is shown (left upper panel). Representative subcutaneous tumors from mice injected with 2 × 104 lentivirus-infected T-ICs after 6 weeks of transplantation are shown (left lower panel). Western blot shows the enhanced expression of exogenous p28GANK in subcutaneous tumors (right upper panel). Tumor volumes from each group (n = 6) were also measured (right lower panel). (B) Representative lung tissue sections of NOD/SCID mice killed at 11 weeks from each group are shown (H&E; original magnification 100×). Black arrows indicate lung metastatic tumors (upper panel). The number of lung metastatic foci in each group (n = 5) was calculated (lower panel). Results display the mean ± SD. **P < .01 vs control. (C) Immunostaining of p28GANK and OV6 were performed on subcutaneous tumors inoculated after 11 weeks (original magnification 200×).
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5. Figure 4
Knockdown of p28GANK attenuates cancer stem cell properties of T-ICs. (A) qRT-PCR analysis was performed for p28GANK, CD133, CD90, and EpCAM in magnetic sorted OV6+ or CD133+ SMCC-7721 LV-miNon and SMMC-7721 LV- mip28GANK 1# and 2# cells. Data represent mean ± SD. *P < .05; **P < .01. (B) Flow cytometric analysis revealed that the percentage of OV6+ and double staining of CD133 and EpCAM antigen cells was significantly decreased in LV-mip28GANK 2# treated–Huh7 cells compared with LV-miNon–treated cells. Representative results from 3 experiments are shown. (C) The same amounts of magnetic sorted LV-miNon OV6+ and LV-mip28GANK (which expressed green fluorescent protein) OV6+ SMMC-7721 cells were mixed and cultured. One week later, the percentage of the OV6+ subpopulation was determined by flow cytometric analysis. (D) Tumor incidence in mouse xenografts injected with the indicated number of LV-miNon or LV-mip28GANK 2# infected SMMC-7721 OV6+ cells after 25 days is shown (left upper panel). Representative subcutaneous tumors from 2 × 104 lentivirus-infected T-ICs after 6 weeks of transplantation are shown (left lower panel). Western blot analyses show efficient knockdown of p28GANK in subcutaneous tumors (right upper panel). Tumor volumes from each group (n = 6) were also measured (right lower panel). (E) Representative lung tissue sections of NOD/SCID mice killed at 11 weeks from each group are shown (H&E; original magnification 100×). Black arrows indicate lung metastatic tumors (upper panel). The number of lung metastatic foci in each group (n = 5) was calculated (lower panel). Results show the mean ± SD. *P < .05 vs control. (F) Immunostaining of p28GANK and OV6 was performed on subcutaneous tumors inoculated after 11 weeks (original magnification 200×).
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6. Figure 5
p28GANK causes elevated Oct4 expression at the protein level. (A) Expression of Oct4 in SMMC-7721 cells infected with Ad-Laz or Ad-p28GANK and in HCC-LM3 cells infected with Ad-GFP or Ad-sip28GANK was detected by Western blot. (B) Tumors generated from LV-luc–, LV- p28GANK–, LV-miNon–, or LV-mip28GANK–infected SMMC-7721 OV6+ cells were immunostained for Oct4. Black arrows indicate nuclear expression of Oct4. Representative pictures are shown. (C) Western blot analysis showed that p28GANK expression was associated with Oct4 in tumor tissue from 134 patients with HCC. Representative samples are shown (left panel). Correlation of expression levels of p28GANK and Oct4 is also shown (Spearman ρ = , P < .0001) (right panel). The protein expression values were analyzed by Quantity One software (Bio-Rad Laboratories, Hercules, CA). (D) Representative p28GANK and Oct4 expression in HCC TMA sections of 130 patients (original magnification 200×). Black arrows indicate nuclear location of Oct4 (upper panel). The immunostaining levels of p28GANK and Oct4 were also scored from 0 to 3+. Correlation of expression levels of p28GANK and Oct4 is shown (lower panel; Spearman ρ = , P < .0001). (E) The disease-free and overall survival rates were compared between the high p28GANK expression/low Oct4 expression group (n = 16) and the high p28GANK expression/high Oct4 expression group (n = 25).
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7. Figure 6
p28GANK activates liver T-ICs by impeding Oct4 degradation. (A) p28GANK attenuates Oct4 degradation by WWP2. 293T cells were transfected with 0.5 μg of Flag-Oct4 along with 1 μg/2 μg of HA-WWP2 or 0.1 μg of Myc- p28GANK. At 36 hours after transfection, cells were treated with cycloheximide (CHX) at a concentration of 15 mg/mL for 16 hours before harvesting. (B) Coimmunoprecipitation of Oct4, WWP2, and p28GANK in 293T cells. Flag-Oct4 (0.5 μg), HA-WWP2 (2 μg), and Myc-p28GANK (3.75 or 7.5 μg) were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated with anti-Flag antibody or control immunoglobulin G and analyzed by Western blotting with anti-HA or anti-ubiquitin antibody (upper and middle panels). Cell lysates were also subjected to immunoblotting (lower panel). (C and D) p28GANK interacts with exogenous and endogenous WWP2. (C) Myc-p28GANK (6 μg) and HA-WWP2 (4 μg) were cotransfected into 293T cells. Whole cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (upper panel). Cell lysates were also subjected to immunoblotting (lower panel). (D) SMMC-7721 cells were treated with Ad-Laz or Ad-p28GANK. Whole cell lysates were immunoprecipitated with anti-p28GANK antibody or control immunoglobulin G and analyzed by Western blotting (upper panel). The whole cell lysates were also analyzed by Western blotting (lower panel). (E) Interaction of HA-WWP2 with Myc-tagged p28GANK mutants. 293T cells cotransfected with HA-WWP2 (4 μg) and Myc-tagged deletion mutants of p28GANK (6 μg) (mutants with each of the ankyrin repeats [dA1–dA5], the N terminus [dN, depleted of amino acids 1–38], or the C terminus [dC, depleted of amino acids 204–226] deleted) were immunoprecipitated and immunoblotted with the indicated antibodies. (F) Representative images of spheroid from SMMC-7721-OV6+ LV-GFP and LV-mip28GANK cells transfected with pCDNA3.1 or pCDNA3.1-Oct4 (scale bar = 500 μm) (left panel). Experiments were performed in triplicate, and data are shown as mean ± SD. **P < .01 (right panel).
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8. Figure 7
A model for the regulation of Oct4 by p28GANK in liver T-ICs. p28GANK overexpression promotes Oct4 expression via blockage of WWP2-mediated Oct4 ubiquitination and degradation by 26S proteasome, promoting self-renewal of T-ICs and progression of HCC.
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9. Supplementary Figure 1
(A) qRT-PCR analysis of the magnetic sorted OV6+ subpopulation was performed to evaluate mRNA expression level of p28GANK in Huh7 and HepG2 cells. Data are shown as mean ± SD from 3 independent experiments. **P < .01. (B) qRT-PCR analysis of the magnetic sorted CD133+ subpopulation was performed to evaluate sorting efficiency in SMMC-7721 and HCC-LM3 cells. Data are shown as mean ± SD from 3 independent experiments. *P < .05, **P < .01. (C) qRT-PCR analysis of the magnetic sorted OV6+ or CD133+ subpopulation was performed to evaluate mRNA expression level of p28GANK in SMMC-7721 and HCC-LM3 spheroids. Data are shown as mean ± SD from 3 independent experiments. *P < .05, **P < .01. (D) qRT-PCR analysis of p28GANK expression of SMMC-7721 and HCC-LM3 cells in different culture conditions. Data are shown as mean ± SD from 3 independent experiments. **P < .01.
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10. Supplementary Figure 2
(A) Expression of exogenous p28GANK in SMMC-7721 cells infected with LV-luc or LV-p28GANK and in HCC-LM3 cells infected with LV-con or LV- p28GANK was detected by Western blot. (B) qRT-PCR analysis was performed for p28GANK and hepatic stem cell markers in magnetic sorted CD133+ SMCC-7721 cells treated with LV-p28GANK or LV-luc (upper panel) and in OV6+/CD133+ HCC-LM3 cells treated with LV-p28GANK or LV-con (middle and lower panels). Data represent mean ± SD from 3 independent experiments. *P < .05; **P < .01. (C) Flow cytometric analysis revealed that the percentage of OV6+ cells was significantly increased in LV-p28GANK–treated SMCC-7721 cells compared with LV-luc–treated cells. The experiments were performed in triplicate, and data are presented as mean ± SD. **P < .01. The related representative result is shown in Figure 2A (right panel). (D) Flow cytometric analysis revealed that the percentage of double-stained OV6+ and CD133+ cells was significantly increased in LV-p28GANK–treated Huh7 cells compared with LV-luc–treated cells. The representative result is shown (left panel). Data from 3 independent experiments are also presented as mean ± SD (right panel). *P < .01. (E) qRT-PCR analysis of hepatic cell differentiation markers, such as G6P, ALB, AFP, and CK-18, in OV6+ SMMC-7721 cells treated with LV-p28GANK or LV-luc. Data represent mean ± SD from 3 independent experiments. **P < .01. G6P, glucose-6-phosphate; ALB, albumin; AFP, α-fetoprotein; CK-18, cytokeratin 18.
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11. Supplementary Figure 3
(A) Results from flow cytometric analysis showed that the proportion of cells in S phase was increased in LV-p28GANK SMMC-7721 cells as compared with their control cells. A representative result is shown. (B) Flow cytometric analysis showed 5-ethynyl-2′-deoxyuridine (EdU)-based S-phase detection offers sufficient evidence that p28GANK promotes S-phase DNA synthesis in OV6+ T-ICs as well as in total SMMC-7721 cells. A representative result is shown in the left panel. Data from 3 independent experiments are also presented as mean ± SD (right panel). *P < .05. (C) Flow cytometric analysis showed no marked effect on cell death. A representative result is shown in the left panel. Data from 3 independent experiments are also presented as mean ± SD (right panel). PI, propidium iodide.
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12. Supplementary Figure 4
(A) Flow cytometric analysis showed that standard chemotherapy with ADR (1 μg/mL) or cis-platinum (DDP; 1.5 μg/mL) enriched the OV6+ subpopulation in SMMC-7721 cells after 4 days. The experiments were performed in triplicate, and data are presented as mean ± SD. **P < .01. The related representative results are shown in Figure 2D. (B) Flow cytometric analysis showed that standard chemotherapy with ADR (1 μg/mL) or DDP (1.5 μg/mL) enriched the OV6+ subpopulation in HCC-LM3 cells after 4 days. Representative results are shown (upper panel). Data from 3 independent experiments are also presented as mean ± SD (lower panel). **P < .01. (C) p28GANK overexpression enhanced the colony formation capability of HCC-LM3 OV6+ cells in the presence of ADR (0.1 μg/mL) or DDP (0.6 μg/mL) for 10 days. n = 3000 cells in 3 trials; *P < .05, **P < .01 (upper panel). Cell viability was also determined by cholecystokinin-8 assay (lower panel). (D) Overexpression of p28GANK increased the percentage of side population cells in HCC-LM3 cells. The experiments were performed in triplicate, and data are presented as mean ± SD. **P < .01. The related representative result is shown in Figure 2F (left panel).
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13. Supplementary Figure 5
(A) Expression of endogenous p28GANK in SMMC-7721 cells infected with LV-GFP, LV-mip28GANK 1#, and LV-mip28GANK 2# was detected by Western blot at indicated time points. (B) Flow cytometric analysis revealed that the percentage of OV6+ and double staining of CD133 and EpCAM antigen cells was significantly decreased in LV-mip28GANK 2#–treated Huh7 cells compared with LV-miNon–treated cells. The experiments were performed in triplicate, and data are presented as mean ± SD. **P < .01. The related representative results are shown in Figure 4B. (C) qRT-PCR analysis of hepatic cell differentiation markers, such as G6P, ALB, AFP, and CK-18, in OV6+ SMMC-7721 cells treated with LV-mip28GANK 1#, LV-mip28GANK 2#, or LV-miNon. Data represent mean ± SD from 3 independent experiments; *P < .05; **P < .01. G6P, glucose-6-phosphate; ALB, albumin; AFP, α-fetoprotein; CK-18, cytokeratin 18.
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14. Supplementary Figure 6
(A) Expression of Oct4 in HepG2, Huh7, PLC/PRF/5, and Hep3B cells infected with Ad-Laz or Ad-p28GANK was detected by Western blot. (B) Coimmunoprecipitation of p28GANK and Oct4 in 293T cells. Plasmids encoding 2.5 μg of Flag-OCT4 and 7.5 μg of Myc- p28GANK were cotransfected into 293T cells with or without MG132. Whole cell lysates were immunoprecipitated with anti-Myc, anti-Flag antibody, or control immunoglobulin G (IgG) and analyzed by Western blotting with anti-Flag and anti-Myc antibody (upper panel). Cell lysates were also subjected to immunoblotting with anti-Flag, anti-Myc, and anti-GAPDH antibodies (lower panel).
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15. Supplementary Figure 7
Western blot analysis of p28GANK and Oct4 expression in 134 HCC tissues.
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16. Supplementary Figure 8
(A) The elevated spheroid numbers of SMMC-7721 LV-p28GANK OV6+ cells as compared with SMMC-7721 LV-luc OV6+ cells were partly reversed in the presence of a PI3K-AKT inhibitor (LY294002). Representative phase-contrast images are shown (scale bar = 500 μm) (upper panel). Experiments were performed in triplicate, and data are shown as mean ± SD. **P < .01 (lower panel). (B) The reduced spheroid numbers of SMMC-7721 LV-mip28GANK 2# OV6+ cells as compared with SMMC-7721 LV-miNon OV6+ cells were partly reversed with ectopic AKT expression. A representative phase-contrast image is shown (scale bar = 500 μm) (upper panel). Experiments were performed in triplicate, and data are shown as mean ± SD. **P < .01 (lower panel). (C) Chromatin immunoprecipitation was performed with chromatin from SMMC-7721/HCC-LM3–derived cell pools using β-catenin–specific antibody for precipitation. Analysis was performed using a specific primer for the promoter region of c-Myc containing T-cell factor 4–binding elements. As controls, 1/50th of the starting chromatin (Input) was used.
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17. Supplementary Figure 9
(A) Flow cytometric analysis showed that the percentage of OV6+ cells was significantly increased in Ad-p28GANK-infected Hep3B cells compared with Ad-Laz–infected cells. The representative result is shown (upper panel). Experiments were performed in triplicate, and data are shown as mean ± SD. **P < .01 (lower panel). (B) qRT-PCR analysis was performed for p28GANK, CD133, CD90, EpCAM, and Oct4 in p28GANK overexpression and knockdown adenovirus-treated Hep3B cells. Data represent mean ± SD from 3 independent experiments, *P < .05; **P < .01.
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