Clinical Cancer Research CR Helping Patients Advances in Breast Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 14, 4045, July 1, 2008. doi: 10.1158/1078-0432.CCR-07-5040
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hsu, M.-C.
Right arrow Articles by Hung, W.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hsu, M.-C.
Right arrow Articles by Hung, W.-C.

Human Cancer Biology

Overexpression of Jab1 in Hepatocellular Carcinoma and Its Inhibition by Peroxisome Proliferator-Activated Receptor{gamma} Ligands In vitro and In vivo

Ming-Chuan Hsu1, Chao-Cheng Huang2, Hui-Chiu Chang1,6, Tsung-Hui Hu3 and Wen-Chun Hung4,5,6

Authors' Affiliations: 1 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University; 2 Department of Pathology, and 3 Division of Gastroenterology, Department of Internal Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center; 4 Institute of Biomedical Sciences, National Sun Yat-Sen University; 5 Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan; and 6 National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Taiwan, People's Republic of China

Requests for reprints: Wen-Chun Hung, Institute of Biomedical Sciences, National Sun Yat-Sen University, No. 70, Lien-Hai Road, Kaohsiung 804, Taiwan, People's Republic of China. Phone: 886-7-5252000, ext. 5817; Fax: 886-7-2259573; E-mail: hung1228{at}ms10.hinet.net.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Purpose: Jun activation domain-binding protein 1 (Jab1) is the fifth subunit of the COP9 signalosome and exhibits oncogenic activity. We investigated Jab1 expression in hepatocellular carcinoma (HCC) tissues and cell lines and tested the effect of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligands on Jab1 expression.

Experimental Design: Jab1 expression in HCC tissues and cell lines was studied by real-time reverse transcription-PCR, immunohistochemical staining, and Western blotting. Promoter activity and chromatin immunoprecipitation assays were done to address the inhibition of Jab1 promoter by PPAR{gamma} ligands. RNA interference was used to clarify PPAR{gamma} ligand-induced inhibition of Jab1. Anticancer and Jab1-suppressing activity of PPAR{gamma} ligands was tested in nude mice.

Results: Jab1 was detected in the nucleus and cytoplasm of HCC tissues and 37% (37 of 99) of tissues exhibited Jab1 overexpression. Jab1 expression correlated with sex and hepatitis C virus infection, whereas it was negatively associated with hepatitis B virus infection. Additionally, Jab1 was overexpressed in HCC cell lines. PPAR{gamma} ligands troglitazone and rosiglitazone down-regulated Jab1 expression in HCC cells, and troglitazone directly suppressed Jab1 promoter activity by inhibiting Sp1- and Tcf4-mediated transcription. This suppression was mediated via both PPAR{gamma}-dependent and PPAR{gamma}-independent mechanisms. Ectopic expression of Jab1 counteracted troglitazone-induced growth inhibition. Animal studies verified that intratumor or i.p. injection of troglitazone attenuated HCC growth and reduced Jab1 expression in tumor tissues.

Conclusions: Our results indicate that Jab1 is overexpressed in HCC and PPAR{gamma} ligands may suppress Jab1 to inhibit the proliferation of HCC cells.

Jun activation domain-binding protein 1 (Jab1) was originally identified as a coactivator of activator protein-1 transcription factor and was important for the specificity of activation of target genes by activator protein-1 (1). Subsequent studies indicated that Jab1 is also the fifth subunit of the 450K COP9/signalosome complex (2, 3). Recent studies suggested that Jab1 is a potential oncogene and may play critical roles in tumorigenesis. First, Jab1 may induce p27Kip1 nuclear exportation and degradation to promote cell proliferation (4). Second, Jab1 may trigger the proteolysis of p53 to enhance oncogenic activity (5). Third, Jab1 interacts directly with Smad4 and induces its degradation via the ubiquitin/proteasome pathway, which leads to inhibition of transforming growth factor-β–induced gene transcription (6). Because transforming growth factor-β exhibits anticancer activity in the early stages of tumorigenesis, inhibition of transforming growth factor-β signaling pathway by Jab1 may promote tumor formation. Fourth, Jab1 binds hypoxia-inducible factor-1, prevents its degradation, and enhances its transcriptional activity (7). Up-regulation of hypoxia-inducible factor-1 by Jab1 stimulates the expression of vascular endothelial growth factor to increase tumor angiogenesis. Pathologic investigations also showed that Jab1 was up-regulated in several types of human cancer including breast, colon, pancreatic, and lung carcinomas (8–11). These data suggest that Jab1 is a tumor promoter. However, the functional role of Jab1 in the development of hepatocellular carcinoma (HCC) is still unclear.

Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) are ligand-activated nuclear receptors involved in the control of diverse biological processes via activation of downstream target genes (12). Ligands for PPAR{gamma} are clinically used for the treatment of non-insulin-dependent diabetes mellitus (13). Recent studies indicated that these ligands exhibited potent anticancer activity (reviewed in ref. 14). Two previous studies showed that a PPAR{gamma} ligand troglitazone effectively inhibited proliferation of HCC cells in vitro and in vivo (15, 16). The authors also showed that the F-box protein p45Skp2 was a molecular target for troglitazone. In this study, we tested whether Jab1 may be another target for PPAR{gamma} ligands. In addition, the expression of Jab1 in HCC tissues and its clinical significance were also addressed.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Patients and tissues. Surgically excised HCC tissues and surrounding nontumor liver tissues were collected from the archives of the Department of Pathology at Chang Gung Memorial Hospital-Kaohsiung Medical Center between January 1986 and December 1996. Nineteen females and 80 males were included in this study. The mean age of these patients is 55.2 years. Among 99 patients, 65 were infected hepatitis B virus (HBV) and 27 were infected with hepatitis C virus (HCV). Sixty-two HCC cases were concurrent with cirrhosis and 66 cases were with grade 3 to 4 fibrosis. Tumors with size larger than 5 cm were detected in 55 cases, and 26 HCC tissues were poorly differentially. {alpha}-Fetoprotein was assayed by MEIA method by using Abbot AXSYM autoanalyzer system, and the cutoff value is 15 ng/mL. Seventy-three HCC cases were {alpha}-fetoprotein positive (range, 16-42,350 ng/mL). Written consent forms were obtained before tissue collection, and the study was approved by the Medical Ethics and the Human Clinical Trial Committee of Chang Gung Memorial Hospital-Kaohsiung Medical Center. All of the HCC slides were reviewed by a pathologist (C-C.H.) and representative paraffin-embedded blocks consisting of tumor and adjacent nontumor parts were retrieved for immunohistochemical staining.

Immunohistochemical staining and scoring. The paraffin-embedded tissue blocks were sectioned into 2-µm slices and placed on the slides coated with poly-L-lysine. After overnight incubation in a 37°C oven, the slides were dewaxed and blocked for 5 min with 3% hydrogen peroxide to deprive the endogenous peroxidase activity. After antigen retrieval with microwave, Jab1 antibody (1:100 dilution; Santa Cruz Biotechnology) was applied onto the specimens and incubated for 30 min at room temperature. The sections were then washed with PBS and incubated with horseradish peroxidase/F(ab) polymer conjugate (PicTure Polymer Detection kit; Zymed) for another 30 min. Finally, the slides were washed and incubated with 3,3'-diaminobenzidine for 5 min, counterstained with Mayer's hematoxylin, and observed under the microscope. The paraffin-embedded tissue of human colon cancer was used as the positive control. Negative control was done by omitting the primary antibody. The nuclear and cytoplasmic staining results of Jab1 were graded as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining) in HCC specimens and their adjacent normal tissues by comparing with the positive control, respectively.

Cell culture and reagents. HepG2, Hep3B, and SK-Hep-1 cells were provided by Dr. M.H. Tai (Kaohsiung Veterans Hospital). Jab1, PPAR{gamma}, β-catenin, Sp3, and Sp4 antibodies were obtained from Santa Cruz Biotechnology. Anti-Sp1 and anti-Tcf4 antibodies were obtained from Upstate. p27Kip1 antibody was purchased from BD Transduction Laboratories. Troglitazone and rosiglitazone were purchased from Cayman Chemical. GW9662 was obtained from Tocris. pcDNA-Myc-Jab1 expression vector was provided by Dr. S.J. Kim (National Cancer Institute). PPAR{gamma} short hairpin RNA (shRNA) was provided by Dr. M.D. Lai (National Cheng Kung University). jetPEI-Hepatocyte transfection reagent was from Polyplus transfection.

Real-time reverse transcription-PCR. Total RNA (1 µg) was reverse transcribed into cDNA. mRNA level of Jab1 was quantified by real-time reverse transcription-PCR (RT-PCR) using iQ SYBR Green Supermix (Bio-Rad). DNA-directed RNA polymerase II polypeptide A served as an internal control for total cDNA content. Samples were amplified by using the MJ Mini real-time PCR detection system (Bio-Rad). The PCR primers used are Jab1-forward 5'-CAAGAAACAGCAGCAAGAAATC-3', Jab1-reverse 5'-AGCATCAGACCCATCACTTCC-3', polymerase II polypeptide A-forward 5'-CCCACTTACTCGCCCACTTCC-3', and polymerase II polypeptide A-reverse 5'-TTCTCCTCGTCACTGTCATCCG-3'.

Western blot analysis. Cells were harvested in a lysis buffer and equal amount of proteins was subjected to SDS-PAGE as described previously (17). Proteins were transferred to nitrocellulose membranes and the blots were probed with primary antibodies. Enhanced chemiluminescence reagents were used to depict the protein bands on the blots.

Jab1 promoter-luciferase reporter plasmids and reporter activity assay. Construction of Jab1 promoter-luciferase reporter plasmids and promoter activity assay were done as described previously (18, 19).

Chromatin immunoprecipitation assay. Vehicle- or troglitazone-treated cells were fixed with 1% formaldehyde at 37°C for 10 min. Cells were harvested and chromatin immunoprecipitation assay was done as described previously (19). Antibodies against Sp1, Sp3, Sp4, Tcf4, or Myc (as a negative control) were used for precipitating the protein/DNA complex. DNA fragments were recovered and subjected to PCR amplification by using the primers specific for the detection of regions that contained the Sp1 binding site (-347/-335) and Tcf4 binding site (-380/-368) in human Jab1 gene promoter. The sequences for the primers are Sp1-forward 5'-CCTATACCCACACAAAAACACTTTC-3', Sp1-reverse 5'-ACCGGAAGAAGGGCATGTA-3', Tcf4-forward, 5'-CCGTTAGCTTTTACGCATGT-3', and Tcf4-reverse 5'-TGTGTGGGTATAGGTACCTTAAATGA-3'.

shRNA targeting. shRNA targeting experiments were done as described previously (20). HepG2 cells were transfected with control or PPAR{gamma} shRNA for 48 h. After transfection, cells were treated with vehicle or troglitazone (50 µmol/L) and expression of PPAR{gamma} and Jab1 was studied. The sequence used to target PPAR{gamma} mRNA is 5'-CGCGGAGATCTCCAGTGATATTCAAGAGATATCACTGGAGATCTCCGCTTTTTGGAAA-3' (sense).

Tumor xenografts in BALB/c nude mice. Animal studies were approved by the Animal Care and Ethics Committee of the National Sun Yat-Sen University. Five-week-old male athymic nude mice (BALB/c nu/nu) were obtained from the National Laboratory Animal Center. On day 1, each mouse was s.c. injected with HepG2 cells (1 x 107 cells in 0.1 mL PBS) on the right dorsal flank. When tumors reached 2 to 3 mm diameter (6 weeks after injection), 15 mice were randomly divided into three groups. The first group received intratumor injection of vehicle (0.5% DMSO) every 3 days. The second group received intratumor injection of troglitazone (final concentration 50 µmol/L) every 3 days. The final group received i.p. injection (0.6 mg/mouse) of troglitazone every 3 days. This dose reflected the similar dose used in human for the treatment of diabetes (800 mg/d). Tumor diameter was measured with a caliber ruler every 3 days. Tumor volume (mm3) was estimated by using the formula: volume = (shortest diameter)2 x (longest diameter) x 0.5. After continuous treatment for another 6 weeks, mice were sacrificed and the tumors were dissected. Weight of tumors and organs (including liver, heart, lung, kidney, and spleen) were measured. Total RNA and cellular proteins were extracted and the Jab1 mRNA and protein levels in tumor tissues were studied.

Statistical analysis. The associations between Jab1 and clinicopathologic variables were assessed by using {chi}2 test. The difference in tumor volume between control and troglitazone-treated groups was determined by Student's t test. Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Overexpression of Jab1 in HCC tissues. We compared Jab1 expression between HCC tumor parts and their adjacent nontumor parts. Jab1 was expressed in nontumor and tumor tissues and was detected in both the nucleus and the cytoplasm (Fig. 1A ). Jab1 was highly expressed in the nucleus of tumor part, although weak staining was also observed in the cytoplasm (Fig. 1B). Conversely, Jab1 expression was very low in the normal part (Fig. 1C). Some of the tumor tissues exhibited high cytoplasmic staining, whereas the nuclear staining was low (Fig. 1D and E). Similarly, the normal part had only slight staining (Fig. 1F). We scored the nuclear and cytoplasmic Jab1 intensities separately and compared the expression between normal and tumor parts. Overexpression of nuclear Jab1 was detected in 27% (27 of 99) of HCC tissues. In addition, Jab1 was overexpressed in the cytoplasm of 19% (19 of 99) of HCC tissues. When the scores of the nuclear and cytoplasmic staining were pooled together, we found that Jab1 was overexpressed in 37% (37 of 99) of HCC tissues. Jab1 expression was not associated with age, tumor size, grade, stage, cirrhosis, fibrosis, and {alpha}-fetoprotein (Table 1 ). Interestingly, Jab1 overexpression was higher in female (P = 0.0003) and was positively associated with HCV infection, whereas it was negatively correlated with HBV infection. No significant association was found between Jab1 overexpression and recurrence (P = 0.8696).


Figure 1
View larger version (136K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Nuclear and cytoplasmic staining of Jab1 in nontumor (NT) and tumor (T) parts in HCC specimens. A, representative HCC tissue section containing nontumor and tumor part (x100). B, strong Jab1 nuclear staining of the tumor part of A (x400). C, weak Jab1 nuclear staining of the nontumor part of A (x400). D, representative HCC tissue section containing nontumor and tumor part (x100). E, strong Jab1 cytoplasmic staining of the tumor part of Fig. 2A (x400). F, weak Jab1 cytoplasmic staining of the nontumor part of Fig. 2A (x400).

 

Figure 2
View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Overexpression of Jab1 in human hepatoma cell lines and its inhibition by troglitazone. A, Jab1 protein level of three nontumor liver tissues (N1-N3), HepG2 (G2), Hep3B (3B), and SK-Hep-1 (SK) HCC cells was investigated by Western blot analysis. Actin served as an internal control. The signal intensity of Jab1 was normalized to that of actin and the ratio was shown. B, total RNA was extracted from N1 to N3 tissues and HCC cell lines and real-time RT-PCR was done to investigate the Jab1 mRNA level. The ratios of Jab1 and DNA directed RNA polymerase II polypeptide A (POLR2A; internal control) of three independent studies were expressed as mean ± SD. C, HepG2 cells were treated with vehicle (C; 0.5% DMSO) and various concentrations (µmol/L) of troglitazone (T) or rosiglitazone (R) for 24 h. Western blot analysis was done to investigate the Jab1 protein level. The signal intensity of Jab1 was normalized to that of actin and the ratio was shown. D, cells were treated with 50 µmol/L troglitazone (T50) or rosiglitazone (R50) and Jab1 mRNA level was studied by real-time RT-PCR. *, P < 0.05, difference between control and troglitazone-treated groups determined by Student's t test.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Correlation between Jab1 and clinicopathologic variables in HCC patients

 
Jab1 is overexpressed in HCC cell lines and transcriptionally inhibited by PPAR{gamma}ligands troglitazone and rosiglitazone. We next studied Jab1 expression in human HCC cell lines. Jab1 protein was highly expressed in HepG2, Hep3B, and SK-Hep-1 cells, whereas normal liver tissues exhibited low level of Jab1 protein (Fig. 2A ). Real-time RT-PCR data showed that Jab1 mRNA level of HepG2, Hep3B, and Sk-Hep1 cells was 2.8-, 2.0-, and 3.3-fold higher than the average Jab1 level of three normal liver tissues respectively (Fig. 2B). We tested the effect of PPAR{gamma} ligands troglitazone and rosiglitazone on Jab1 expression. We found that PPAR{gamma} ligands down-regulated Jab1 protein (Fig. 2C) and mRNA (Fig. 2D) level in a dose-dependent manner in HCC cell lines. These results suggested that Jab1 is a target for PPAR{gamma} ligands. Because HepG2 cells were more sensitive to PPAR{gamma} ligands, we used this cell line to address the molecular mechanism in our subsequent study.

Troglitazone suppresses Jab1 promoter activity via Sp1 and Tcf4 binding sites. We constructed human Jab1 promoter-luciferase plasmids and tested the effect of troglitazone. As shown in Fig. 3A , troglitazone inhibited Jab1 promoter activity in a concentration-dependent fashion. By using deletion mutants, we found that troglitazone inhibited Jab1 promoter activity via the -405/-223 region (Fig. 3B). Computer prediction analysis indicated that Sp1, Tcf4, and STAT1 transcription factor binding sites were found in this region. Mutation of Sp1 and Tcf4 sites attenuated the inhibitory effect of troglitazone on Jab1 promoter, whereas mutation of STAT1 site had no effect (Fig. 3C). We next studied the protein factors that bound to Jab1 promoter. Chromatin immunoprecipitation assay showed that Sp1 constitutively bound to Jab1 promoter and its binding was attenuated by ~40% by troglitazone (Fig. 3D). Sp3 and Sp4 also bound to Jab1 promoter. However, their binding was very weak (when compared with the binding of Sp1) and was not affected by troglitazone. The attenuation of Sp1 binding to Jab1 promoter by troglitazone was not due to the reduction of Sp1 protein because only very minor alteration (<10%) of Sp1 protein level was found in troglitazone-treated cells (Fig. 3E). Chromatin immunoprecipitation data also showed that Tcf4 and β-catenin constitutively bound to the Tcf4 site of Jab1 promoter and their binding was repressed by 40% to 50% by troglitazone (Fig. 3D). Taken together, our promoter and chromatin immunoprecipitation data suggested that troglitazone might inhibit Jab1 expression via reduction of Sp1- and Tcf4-mediated gene transcription.


Figure 3
View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Troglitazone represses Jab1 promoter activity via Sp1 and Tcf4 sites. A, HepG2 cells were transfected with full-length pJab1965 promoter-luciferase construct and treated without (0.5% DMSO) or with 20 µmol/L troglitazone (T20) or 50 µmol/L troglitazone for 24 h. Luciferase activity was determined by using dual-luciferase assay system and normalized for Renilla luciferase activity. Mean ± SE of three independent experiments. *, P < 0.05, difference of promoter activity between control and troglitazone-treated groups determined by Student's t test. A diagram indicating full-length promoter-luciferase construct was shown. B, Jab1 promoter deletion mutants pJab1433, pJab405, and pJab223 were transfected into HepG2 cells. Cells were treated without or with 50 µmol/L troglitazone for 24 h and promoter activity was determined. *, P < 0.05. A diagram indicating various deletion promoter-luciferase constructs was shown. C, mutation constructs in which Sp1 (pJabmSp1), Tcf4 (pJabmTcf), and STAT1 (pJabmSTAT1) sites were mutated from pJab405 separately were transfected into HepG2 cells. Cells were treated without or with 50 µmol/L troglitazone for 24 h and promoter activity was determined. *, P < 0.05. A diagram indicating the mutation sites in the pJab405 construct was shown. D, HepG2 cells were incubated in 10% FCS containing 0.5% DMSO or 50 µmol/L troglitazone for 24 h. Cells were fixed with 1% formaldehyde and sonicated to shear DNA. Sp1, Sp3, Sp4, Tcf4, β-catenin, or Myc (as negative control) antibodies were added and incubated overnight at 4°C with rotation. DNA fragments were recovered and subjected to PCR amplification by using the primers specific for the detection of regions that contained the Sp1 binding site (-347/-335) and Tcf4 binding site (-380/-368) in human Jab1 gene promoter. The signal intensities were measured and normalized to input controls. The ratios were shown below each lane. E, HepG2 cells were incubated in 10% FCS containing 0.5% DMSO or 50 µmol/L troglitazone for 24 h. Western blot analysis was done to study the Sp1, Sp3, and Sp4 protein levels.

 
Suppression of Jab1 expression by troglitazone via PPAR{gamma}-dependent and PPAR{gamma}-independent mechanisms. PPAR{gamma} has been shown to interact with other transcription factors, including Sp1 and Tcf4, to regulate gene transcription (21–23). Therefore, we tested whether inhibition of Jab1 by troglitazone was dependent on PPAR{gamma}. As shown in Fig. 4A , treatment of PPAR{gamma} inhibitor GW9662 reversed the inhibition of Jab1 by troglitazone. In addition, GW9662 also reversed troglitazone- induced inhibition of Jab1 promoter activity (Fig. 4B). Chromatin immunoprecipitation assay showed that reduction of binding of Sp1 and Tcf4 to Jab1 promoter by troglitazone was reversed by GW9662 (Fig. 4C). We used shRNA to knockdown PPAR{gamma} and tested the effect of troglitazone. PPAR{gamma} shRNA effectively suppressed PPAR{gamma} expression (Fig. 4D) and partially reversed troglitazone-induced inhibition of Jab1 (Fig. 4E). These data indicated that PPAR{gamma} is required for the down-regulation of Jab1 by troglitazone. However, our results suggested that PPAR{gamma}-independent mechanisms were also involved in this process. First, the dose of troglitazone needed to repress Jab1 is higher than the dose routinely used to stimulate receptor activation. Second, PPAR{gamma} shRNA and chemical inhibitor GW9662 could not completely reversed the effect of troglitazone. Therefore, troglitazone acted via both PPAR{gamma}-dependent and PPAR{gamma}-independent pathways to repress Jab1. We next tested whether inhibition of Jab1 by troglitazone is important for its growth-inhibitory effect on HCC cells. Indeed, troglitazone inhibited proliferation of HepG2 cells and ectopic expression of Jab1 counteracted troglitazone-induced growth inhibition (Fig. 4F). However, ectopic expression of Jab1 could not fully rescue the growth-inhibitory effect of troglitazone. Therefore, Jab1 is one of the mediators for troglitazone to suppress HCC growth.


Figure 4
View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Troglitazone inhibition of Jab1 is PPAR{gamma} dependent. A, HepG2 cells were preincubated with PPAR{gamma} inhibitor GW9662 (G; 10 µmol/L) and then treated with vehicle or troglitazone (50 µmol/L) for 24 h. Real-time RT-PCR was done to study the Jab1 mRNA and the ratios of Jab1 to DNA directed RNA polymerase II polypeptide A (internal control) from three independent studies were expressed as mean ± SE. *, P < 0.05. B, cells were transfected with pJab405 promoter-luciferase construct and treated with drugs as described above. Luciferase activity was determined by using dual-luciferase assay system and normalized for Renilla luciferase activity. *, P < 0.05. C, HepG2 cells were preincubated with PPAR{gamma} inhibitor GW9662 (10 µmol/L) and then treated with vehicle or troglitazone (50 µmol/L) for 24 h. Cells were fixed with 1% formaldehyde and sonicated to shear DNA. Sp1, Tcf4, or Myc (negative control) antibodies were added to immunoprecipitate the DNA/transcription factor complex. DNA fragments were recovered and were subjected to PCR amplification by using the primers specific for the detection of regions that contained the Sp1 binding site (-347/-335) and Tcf4 binding site (-380/-368) in human Jab1 gene promoter. The signal intensities were measured and normalized to input controls. The ratios were shown below each lane. D, cells were transfected with control or PPAR{gamma} (P) shRNA expression vector for 48 h and RT-PCR was done to investigate the relative level of PPAR{gamma} mRNA. E, cells were transfected with control or PPARcnr75040 shRNA expression vector for 48 h. After transfection, cells were treated with vehicle (0.5% DMSO) or troglitazone (50 µmol/L) for another 24 h. Jab1 mRNA level was then investigated by real-time RT-PCR. *, P < 0.05. F, HepG2 cells were transfected with 3 µg/well control or Jab1 expression vector by using jetPEI-Hepatocyte transfection reagent for 24 h and incubated with vehicle (–) or troglitazone (+; 50 µmol/L) for another 48 h. Viable cell number was counted by trypan blue exclusion assay. *, P < 0.05.

 
Troglitazone suppresses Jab1 expression and HCC growth in vivo. We carried out animal study to verify that troglitazone also repressed Jab1 in vivo. HepG2 cells (1 x 107 per mouse) were injected s.c. on the right dorsal flank of BALB/c nude mice. After 6 weeks, when the tumor nodules were seen in the injection sites, mice were randomly subdivided into three groups. The first group received intratumor injection of vehicle (0.5% DMSO) every 3 days. The second group received intratumor injection of troglitazone (50 µmol/L) every 3 days. To mimic the treatment under physiologic condition, the final group received i.p. injection (0.6 mg/mouse) of troglitazone every 3 days. This dose reflected the similar dose used in human for the treatment of diabetes (800 mg/d). After continuous treatment for another 6 weeks, mice were sacrificed and the tumors were dissected. No significant differences in the weight of body, liver, heart, lung, kidney, and spleen were observed in these experimental groups (data not shown). Tumors of the control group were large in size and rich in blood vessels (Fig. 5A ). Intratumor injection of troglitazone potently reduced tumor volume by 90% and few blood vessels were observed around the tumors (Fig. 5A). I.p. injection of troglitazone also inhibited tumor growth by 60% to 70%. Real-time RT-PCR study showed that Jab1 expression in troglitazone-treated groups was down-regulated (Fig. 5B). Western blot analysis also showed that Jab1 protein was reduced in tumor tissues of both troglitazone-treated groups (Fig. 5C). Inhibition of Jab1 expression by troglitazone in vivo was of biological importance because protein level of the tumor suppressor p27Kip1 was significantly increased. Thus, troglitazone repressed Jab1 expression and growth of HCC in vitro and in vivo.


Figure 5
View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Inhibition of Jab1 expression by troglitazone in vivo. A, each mouse was s.c. injected with HepG2 cells (1 x 107 in 0.1 mL PBS) on the right dorsal flank. When tumors reached 2 to 3 mm diameter (6 wk after injection), 15 mice were randomly divided into three experimental groups. The first group received intratumor injection of vehicle (0.5% DMSO) every 3 d. The second group received intratumor injection of troglitazone (IT; 50 µmol/L) every 3 d. The final group received i.p. injection (IP; 0.6 mg/mouse) of troglitazone every 3 d. Tumor diameter was measured with a caliber ruler every 3 d. Tumor volume (mm3) was estimated by using the formula: volume = (shortest diameter)2 x (longest diameter) x 0.5. *, P < 0.05, when the tumor size of control and troglitazone-treated groups (IT and IP) were compared. B, total RNA was extracted from tumors and the Jab1 mRNA was studied by real-time RT-PCR. Mean ± SE of the each experimental group. *, P < 0.05, when the results of control and troglitazone-treated groups were compared. C, a typical Western blot showed the Jab1 and p27Kip1 protein level in tumors of three experimental groups.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In this study, we show that Jab1 is overexpressed in HCC and may play a critical role in the tumorigenesis of this cancer. By using array-based comparative genomic hybridization, Patil et al. showed that high expression of Jab1 in HCC significantly correlated with DNA copy number gain at chromosome 8q (24). In agreement with our current finding, the authors showed that Jab1 protein was detected in both the nucleus and the cytoplasm and knockdown of Jab1 by small interfering RNA inhibited the proliferation of Hep3B cells. However, they did not address the correlation between Jab1 and clinicopathologic variables. Interestingly, we found that Jab1 overexpression was higher in female (P = 0.0003) and is positively associated with HCV infection, whereas it was negatively correlated with HBV infection. A very recent study showed that HBV X protein directly interacted with Jab1 to activate activator protein-1–mediated transcription (25). Because HBV X protein binds directly to Jab1 via protein-protein interaction, it seems possible that HBV-infected HCC cells do not need to increase Jab1 expression to promote hepatocarcinogenesis. On the contrary, HBV-negative HCC may exhibit Jab1 gene amplification or overexpression more frequently. Another important finding is the positive correlation between HCV infection and Jab1 up-regulation. Whether HCV core protein may stimulate Jab1 expression to enhance the development of HCC warrants further investigation.

The association between sex and Jab1 overexpression is complex. Previous studies have shown that steroids and their cognate receptors (especially estrogen receptor) may play critical roles in the development of HCC (26). Recent reports showed that Jab1 may physically interact with estrogen and progesterone receptors (27, 28). In addition, Jab1 also directly binds with the steroid receptor coactivator SRC-1 (28). Jab1 is involved in ligand-dependent degradation of estrogen receptor by the proteasome, whereas it stabilizes the progesterone-SRC-1 complex and enhance transcriptional activity. Therefore, the protein level of these steroid receptors may modulate the Jab1 protein expression in HCC tissues.

Another critical finding of our study is the identification of Jab1 as a molecular target for PPAR{gamma}-induced growth arrest of HCC cells. The anticancer effect of troglitazone on HCC cells was originally reported by Koga et al. (15). These authors showed that troglitazone up-regulated the expression of p21Waf1, p27Kip1, and p18Ink4c and induced growth inhibition in various HCC cell lines. The authors also showed that troglitazone inhibited the expression of p45Skp2 to increase p27Kip1 protein stability and to inhibit cell growth. In this study, we provide the first evidence that Jab1 is another target for troglitazone. We find that troglitazone directly suppresses the transcription of Jab1 gene via Sp1 and Tcf4. More importantly, this inhibition caused significantly up-regulation of p27Kip1 and attenuated tumor growth in vitro and in vivo. PPAR{gamma} has been shown to attenuate the binding of Sp1 to the consensus site in the thromboxane receptor gene to repress transcription (21). Functional interplay between PPAR{gamma} and Tcf4-mediated gene expression has also been shown recently. Liu et al. showed that PPAR{gamma} target β-catenin to proteasome-mediated degradation and attenuate Tcf4-stimulated gene transcription (23). Our results were consistent with this hypothesis and we found that intracellular protein level of β-catenin was reduced after troglitazone treatment (data not shown). Results of this study suggest that PPAR{gamma} ligands can be developed as a novel class of drugs for inhibiting Jab1. Collectively, we conclude that Jab1 is overexpressed in HCC and may be a rational target for the treatment of HCC.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
We thank Drs. M.H. Tai, Y.C. Wu, M.D. Lai, and S.J. Kim for providing cell lines and experimental materials.


   Footnotes
 
Grant support: Center for Gene Regulation and Signal Transduction Research of Cheng Kung University and National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center (W-C. Hung).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/ 2/07; revised 1/21/08; accepted 2/12/08.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Claret FX, Hibi M, Dhut S, Toda T, Karin M. A new group of conserved coactivators that increase the specificity of AP-1 transcription factors. Nature 1996;383:453–7.[CrossRef][Medline]
  2. Seeger M, Kraft R, Ferrell K, Bech-Otschir D, Dumdey R, Schade R. A novel protein complex involved in signal transduction possessing similarities to 26S proteasome subunits. FASEB J 1998;12:469–78.[Abstract/Free Full Text]
  3. Wei N, Tsuge T, Serino G, et al. The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex. Curr Biol 1998;8:919–22.[CrossRef][Medline]
  4. Tomoda K, Kubota Y, Kato J. Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1. Nature 1999;398:160–5.[CrossRef][Medline]
  5. Bech-Otschir D, Kraft R, Huang X, et al. COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system. EMBO J 2001;20:1630–9.[CrossRef][Medline]
  6. Wan M, Cao X, Wu Y, et al. Jab1 antagonizes TGF-β signaling by inducing Smad4 degradation. EMBO Rep 2002;3:171–6.[CrossRef][Medline]
  7. Bae MK, Ahn MY, Jeong JW, et al. Jab1 interacts directly with HIF-1{alpha} and regulates its stability. J Biol Chem 2002;277:9–12.[Abstract/Free Full Text]
  8. Tomoda K, Kato JY, Tatsumi E, Takahashi T, Matsuo Y, Yoneda-Kato N. The Jab1/COP9 signalosome subcomplex is a downstream mediator of Bcr-Abl kinase activity and facilitates cell-cycle progression. Blood 2005;105:775–83.[Abstract/Free Full Text]
  9. Kouvaraki MA, Korapati AL, Rassidakis GZ, et al. Potential role of Jun activation domain-binding protein 1 as a negative regulator of p27kip1 in pancreatic adenocarcinoma. Cancer Res 2006;66:8581–9.[Abstract/Free Full Text]
  10. Fukomoto A, Tomoda K, Yoneda-Kato N, Nakajima Y, Kato JY. Depletion of Jab1 inhibits proliferation of pancreatic cancer cell lines. FEBS Lett 2006;580:5836–40.[CrossRef][Medline]
  11. Esteva FJ, Sahin AA, Rassidakis GZ, et al. Jun activation domain binding protein 1 expression is associated with low p27(Kip1) levels in node-negative breast cancer. Clin Cancer Res 2003;9:5652–9.[Abstract/Free Full Text]
  12. Green S. PPAR: a mediator of peroxisome proliferator action. Mutat Res 1995;333:101–9.[Medline]
  13. Compbell IW. The clinical significance of PPAR{gamma} agonism. Curr Mol Med 2005;5:349–63.[CrossRef][Medline]
  14. Murphy GJ, Holder JC. PPAR-{gamma} agonists: therapeutic role in diabetes, inflammation and cancer. Trends Pharmacol Sci 2000;21:469–74.[CrossRef][Medline]
  15. Koga H, Sakisaka S, Harada M, et al. Involvement of p21(WAF1/Cip1), p27(Kip1), and p18(INK4c) in troglitazone-induced cell-cycle arrest in human hepatoma cell lines. Hepatology 2001;33:1087–97.[CrossRef][Medline]
  16. Koga H, Harada M, Ohtsubo MS, et al. Troglitazone induces p27Kip1- associated cell-cycle arrest through down-regulating Skp2 in human hepatoma cells. Hepatology 2003;37:1086–96.[CrossRef][Medline]
  17. Hung WC, Chang HC, Pan MR, Lee TH, Chuang LY. Induction of p27(KIP1) as a mechanism underlying NS398-induced growth inhibition in human lung cancer cells. Mol Pharmacol 2000;58:1398–403.[Medline]
  18. Hsu MC, Chang HC, Hung WC. HER-2/neu transcriptionally activates Jab1 expression via the AKT/β-catenin pathway in breast cancer cells. Endocr Relat Cancer 2007;14:655–67.[Abstract/Free Full Text]
  19. Hsu MC, Chang HC, Hung WC. HER-2/neu represses the metastasis suppressor RECK via ERK and Sp transcription factors to promote cell invasion. J Biol Chem 2006;281:4718–25.[Abstract/Free Full Text]
  20. Chang HC, Cho CY, Hung WC. Silencing of the metastasis suppressor RECK by RAS oncogene is mediated by DNA methyltransferase 3b-induced promoter methylation. Cancer Res 2006;66:8413–20.[Abstract/Free Full Text]
  21. Sugawara A, Uruno A, Kudo M, et al. Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-{gamma} via an interaction with Sp1 in vascular smooth muscle cells. J Biol Chem 2002;277:9676–83.[Abstract/Free Full Text]
  22. Hong J, Samudio I, Liu S, Abdelrahim M, Safe S. Peroxisome proliferator- activated receptor {gamma}-dependent activation of p21 in Panc-28 pancreatic cancer cells involves Sp1 and Sp4 proteins. Endocrinology 2004;145:5774–85.[Abstract/Free Full Text]
  23. Liu J, Wang H, Zuo Y, Farmer SR. Functional interaction between peroxisome proliferator-activated receptor {gamma} and β-catenin. Mol Cell Biol 2006;26:5827–37.[Abstract/Free Full Text]
  24. Patil MA, Gutgemann I, Zhang J, et al. Array-based comparative genomic hybridization reveals recurrent chromosomal aberrations and Jab1 as a potential target for 8q gain in hepatocellular carcinoma. Carcinogenesis 2005;26:2050–7.[Abstract/Free Full Text]
  25. Tanaka Y, Kanai F, Ichimura T, et al. The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1. Oncogene 2006;25:633–42.[Medline]
  26. Giannitrapani L, Soresi M, La Spada E, Cervello M, D'Alessandro N, Montalto G. Sex hormones and risk of liver tumor. Ann N Y Acad Sci 2006;1089:228–36.[CrossRef][Medline]
  27. Callige M, Kieffer I, Richard-Foy H. CSN5/Jab1 is involved in ligand- dependent degradation of estrogen receptor {alpha} by the proteasome. Mol Cell Biol 2005;25:4349–58.[Abstract/Free Full Text]
  28. Chauchereau A, Georgiakaki M, Perrin-Wolff M, Milgrom E, Loosfelt H. JAB1 interacts with both the progesterone receptor and SRC-1. J Biol Chem 2000;275:8540–8.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
Y. Liu, S. V. Shah, X. Xiang, J. Wang, Z.-b. Deng, C. Liu, L. Zhang, J. Wu, T. Edmonds, C. Jambor, et al.
COP9-Associated CSN5 Regulates Exosomal Protein Deubiquitination and Sorting
Am. J. Pathol., April 1, 2009; 174(4): 1415 - 1425.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hsu, M.-C.
Right arrow Articles by Hung, W.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hsu, M.-C.
Right arrow Articles by Hung, W.-C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online