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Hepatocellular carcinoma (HCC) occurs in a significant number of patients with hepatitis C virus (HCV) infection. HCV causes double-strand DNA breaks and enhances the mutation frequency of proto-oncogenes and tumor suppressors. However, the underlying mechanisms for these oncogenic events are still elusive. Here we study the role of c-Jun, STAT3, and nitric oxide (NO) in spontaneous and diethylnitrosamine (DEN)-initiated and/or phenobarbital (Pb)-promoted HCC development using HCV core transgenic (Tg) mice. The viral core protein induces hepatocarcinogenesis induction as a tumor initiator under promotion by Pb treatment alone. Conditional knockout of c-jun and stat3 in hepatocytes achieves a nearly complete, additive effect on prevention of core-induced spontaneous HCC or core-enhanced HCC incidence caused by DEN/Pb. Core induces hepatocyte proliferation and the expression of inflammatory cytokines (IL-6, TNFα, IL-1) and iNOS; the former is dependent on c-Jun and STAT3, and the latter on c-Jun. Oxidative DNA damage repair activity is impaired by the HCV core protein due to reduced DNA glycosylase activity for the excision of 8-oxodG. This impairment is abrogated by iNOS inhibition or c-Jun deficiency, but aggravated by the NO donor or iNOS inducing cytokines. The core protein also suppresses apoptosis mediated by Fas ligand because of c-Jun-dependent Fas down-regulation. These results indicate that the HCV core protein potentiates chemically induced HCC through c-Jun and STAT3 activation, which in turn, enhances cell proliferation, suppresses apoptosis, and impairs oxidative DNA damage repair, leading to hepatocellular transformation.
Hepatitis C virus (HCV) causes chronic hepatitis and liver cirrhosis and greatly increases the risk for HCC (1–3). In both HCC and chronic hepatitis, the transcription factor AP-1 is activated and implicated (4). The ectopic expression of HCV core protein in cell cultures also activates AP-1 (c-Jun) (5) via the activation of c-Jun N-terminal kinase (JNK) and MAPKK (6, 7), and HCV core transgenic (Tg) mice develop liver tumors (8), suggesting the role of c-Jun in core-induced oncogenesis.
Transcription activator c-Jun is required for cell proliferation in postnatal hepatocytes (9). Mice deficient in c-Jun die between embryonic days E12.5 and E13.5 from massive apoptosis of hepatoblasts, erythroblasts, and other cell types, indicating the requirement of c-Jun in normal liver development and hematopoiesis (10, 11). To rescue embryonic lethality, a “floxed” c-jun allele is deleted in a designated cell type upon expression of the Cre recombinase under the control of a cell-type specific promoter. Using this conditional gene disruption, the requirement for c-jun is also shown for chemically-induced HCC in mice where c-Jun deficiency in hepatocytes reduces both the number and size of HCC after tumor initiation with diethylnitrosamine (DEN), while increasing apoptosis (12).
HCV core protein induces reactive oxygen species (ROS), and HCV core Tg mice have higher hepatic levels of 8-oxodeoxyguanosine, indicative of DNA damage by ROS (13). In fact, HCV core Tg mice show increased mutation frequencies of tumor suppressor and proto-oncogenes (13, 14). ROS also activates c-Jun and STAT3 (15). Therefore, the core protein may increase the growth and survival of initiated tumor cells via activation of c-Jun and STAT3. However, the mechanisms by which c-Jun and STAT3 specifically contribute to liver oncogenesis induced by interactions of HCV core and environmental carcinogens, remain to be elucidated. Further, whether HCV core protein works as a tumor initiator or promoter, has not been determined (16). The present study demonstrates that the mitogenic and anti-apoptotic effects mediated by c-Jun/AP-1 and STAT3 are both required for hepatocytes’ susceptibility to HCV core-initiated hepatocellular transformation, and that this is caused by fixation of genetic mutations induced by oxidative stress and impaired DNA repair, resulting from activation of c-Jun and nitric oxide.
For animal studies, mice expressing the HCV core gene genotype 1b under control of the human elongation factor (EF) 1a promoter, were generated and bred at the USC transgenic mouse facility (8–13 and 8–20 lines). The c-junflox/flox mice are a generous gift from Dr. Carter in Vanderbilt University. The stat3 flox/flox mice are generated by standard procedures.
The adenovirus which expresses cre recombinase under the albumin promoter, was used to disrupt the c-jun gene. The removal of the neo gene was confirmed by PCR or Southern blotting, demonstrating that the targeted c-jun allele contains the protein-coding sequence flanked by loxP sites (17).
Statistical comparisons of the groups were made by one-way analysis of variance, and when they were statistically significant, each group was compared with others by Fisher’s PLSD test (Statview 4.0 Abacus Concept, Inc., Berkeley, CA).
To determine whether HCV core promotes carcinogen-induced liver tumorigenesis, we injected HCV core Tg mice with the genotoxic carcinogen diethylnitrosamine (DEN) as a tumor initiator at six weeks of age and administered the tumor promoter phenobarbital (Pb) in drinking water starting from 10 weeks of age, until 22 months of age (Fig. 1A). Mortality of core Tg mice given DEN and Pb became evident at eight months old and continued to increase with time. By 20 months, the DEN/Pb treatment caused 42% mortality among core Tg mice as compared to 12% among wild type (WT) mice (p<0.05) (Fig. 1B). Autopsy results confirmed that the lethality of the Tg mice was associated with primary liver tumors. Without DEN tumor induction, Tg mice developed spontaneous HCC at the rate of 14%, 28%, and 38% at 14, 18, 22 months old, respectively, while none of the WT littermates developed the tumor (Fig. 1C); the results are consistent with the previously reported finding (8). The DEN/Pb treatment resulted in 22% liver tumor incidence in WT mice but 62% incidence with enhanced dysplastic changes in core Tg mice at 14 months of age (p<0.05, Fig. 1C and 1D–f). At 18 and 22 months, the magnitude of the difference in the liver tumor incidence between WT and Tg mice diminished, although the latter animals still showed higher incidence (Fig. 1C). Histological analysis revealed that the tumors were mainly adenoma and HCC with occasional angiosarcoma (Fig. 1D-c). In most cases, multiple hepatocellular neoplasms of various sizes were present in the liver of these mice (Fig. 1C). The treatment of the core Tg mice with DEN and Pb induced more severe steatosis and dysplasia (Fig. 1D-a and 1D–f). Administration of DEN resulted in comparable increases in the serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), the markers for liver damage, in both WT and core Tg mice (data not shown) suggesting that the hepatotoxin induced comparable liver necrosis in both groups of mice.
The extent of hepatocellular proliferation, as assessed by PCNA staining and liver weight, was nearly two-fold higher in the HCV core Tg mice than in WT mice under DEN/Pb treatment (Fig. 1E), indicating that dysregulated hepatocyte proliferation may be the cause of increased hepatocellular transformation in Tg mice. This difference was not apparent in carcinogen-untreated mice (Fig. 1E). To test if the livers in core Tg mice had JNK and STAT3 activation, we stained tissue sections for phospho-STAT3 and phospho-JNK. The staining and phospho-STAT3 protein levels as determined by immunoblotting were clearly increased in DEN/Pb-treated Tg mice as compared to WT mice (Fig. 1F). These results indicate that increased liver tumor development in HCV core Tg mice is associated with enhanced hepatocyte proliferation and activation of JNK and STAT3.
To determine the possible role of c-jun in core-induced or -enhanced liver oncogenesis, we bred core Tg mice with c-jun conditional knockout (c-jun flox/flox) mice. The c-jun gene in this mouse line is flanked by the lox site, which will recombine to delete the c-jun gene in the presence of the Cre recombinase. We injected mice with a recombinant adenovirus that expresses Cre (A5CMVCre) to induce the deletion of the c-jun gene primarily in the liver. As a control, adenovirus expressing lacZ (Ad.LAcZ) was injected (see the experimental design in Fig. 2A). Immunoblot and qRT-PCR of c-Jun demonstrated effective c-Jun deficiency in animals which received Ad5CMVCre (Fig. 2A, lower blots and graph). The mortality associated with DEN/Pb treatment in core Tg mice was significantly attenuated by c-Jun deficiency (Fig. 2B). Spontaneous HCC development (without DEN/Pb) in core Tg mice was largely abrogated by c-Jun deficiency (Fig. 2C). The enhanced tumor incidence in the core-Tg mice treated with DEN/Pb was also reduced by 60% (p < 0.001) due to c-Jun deficiency (Fig. 2C and 2E) while a smaller 30% reduction was observed in c-Jun deficient WT mice given DEN/Pb (Fig. 2C). The number of cells double-positive for CD133 and CD49f, markers for cancer stem cells, clearly increased in core Tg mice treated with DEN/Pb but not in c-Jun deficient core Tg mice or WT mice treated with the carcinogens (Fig. 2F). Thus, our results demonstrate that HCV core protein not only serves as an independent tumor inducer, but also accentuates carcinogen-induced HCC development in a manner largely dependent on c-Jun.
HCV core enhanced DEN/Pb-induced hepatocarcinogenesis. However, we do not know whether this effect is due to the core’s role as a tumor initiator or promoter. To address this question, WT and Tg mice were treated with Pb (a tumor promoter) or DEN (a tumor initiator) alone and compared for the effect of core on the number and size of liver tumors induced. As shown in Fig. 3B, even with Pb treatment alone, HCV core Tg mice developed more than threefold larger and numerous liver tumors than WT mice, the increments which resembled those seen with DEN/Pb treatment (Fig. 3A). Further, c-Jun deficiency markedly abrogated these oncogenic effects in core Tg mice treated with DEN+Pb or PB alone. In contrast, HCV core Tg mice treated with DEN alone, developed liver tumors with much smaller mass and fewer numbers than those treated with DEN+Pb or PB alone (Fig. 3A–C). These results indicate that HCV core initiates not promotes hepatocarcinogenesis.
Our previous in vitro data indicate that the HCV core protein induces DNA mutations via an increase in the production of ROS and reactive nitrogen species (RNS) (18, 19). Thus, we investigated next whether the administration of an antioxidant reduces core-enhanced liver tumor development under DEN+Pb treatment. Butylated hydroxyanisole (BHA), an anti-oxidant that scavenges ROS and RNS, was administered via drinking water for 12 months (Fig. 3D). The treatment of BHA significantly reduced HCV core-induced enhancements of liver tumor size and number, indicating that ROS or RNS-mediated oncogenic mutation is important for the enhanced liver oncogenesis in core Tg mice given DEN/Pb (Fig. 3D). To make a mechanistic connection of hepatocarcinogenesis and DNA repair, DNA mutation frequency was determined by plasmid-based sequencing from genomic DNA using p53 gene as a marker of HCV core transgenic mice in the presence or absence of antioxidant treatment (BHA). The data showed that core transgenic mice have a significantly higher frequency of mutation, which is abrogated by BHA treatment (Fig. 3D Table, P < 0.01). These results indicate that HCV core-induced ROS/RNS enhances DNA mutation frequency of major tumor suppressor gene p53, which is abrogated by blocking ROS/RNS, in livers of HCV core transgenic mice.
Next, we tested whether suppressed liver tumor formation with BHA is associated with inhibition of hepatocellular proliferation. For this analysis, we examined 5-bromo-2′deoxyuridine (BrdU) incorporation in the livers at various time points (2.5~26 months) of DEN/Pb treatment in WT and core Tg mice, with or without BHA treatment (Fig. 3E). In parallel, we also analyzed the effect of c-Jun deficiency. At the young age of 2.5 months, the proliferative activity is high, particularly in core Tg mice treated with DEN/Pb, and this is reduced 50~60% by c-Jun deficiency and 30% by BHA treatment. BrdU index was lower in all groups at the older ages, but the effects of c-Jun deficiency and BHA were still evident (p<0.05, Fig. 3E). Suppressed proliferation in c-Jun deficient core Tg mice also corroborated reduced PCNA and Ki-67 mRNA levels detected by qRT-PCR (Fig. 3F). These results demonstrate that the contribution of core-enhanced cellular proliferation takes place during the early stage of DEN/Pb-induced carcinogenesis, consistent with the notion that core serves as a tumor initiator. The BHA effect indicates a role of oxidative stress in hepatocellular proliferation (Fig. 3E). Interestingly, p-STAT3 levels were also reduced by c-jun disruption in core Tg mice, suggesting that core-induced STAT3 activation is dependent on c-Jun (Fig. 3F). These data demonstrate that HCV core promotes hepatocellular proliferation via oxidative stress and c-Jun. Since p-STAT3 has known mitogenic effects (20), c-Jun dependent STAT3 phosphorylation suggests the contribution of this mitogenic factor as a downstream effector of c-Jun for core-induced hepatocyte proliferation.
Our results demonstrate the importance of c-Jun in the HCV core’s ability to synergistically enhance DEN/Pb-induced hepatocarconogenesis as a tumor initiator. We asked next what c-Jun/AP-1 target genes are upregulated and implicated in our synergism model. c-Jun/AP-1 activates the promoter of matrix metalloproteinases (MMPs) (21), which play a critical role in acute, fulminant hepatitis by degrading the extracellular matrix and allowing massive leukocyte influx in the liver (22) and is involved in cancer migration, growth and vasculogenesis (26). Indeed, our qRT-PCR analysis revealed increased expression of MMP-9 and MMP-13, but not MMP-2 in the livers of core Tg mice given DEN/Pb, as compared to carcinogen-treated WT mice, and abrogation of this induction by c-Jun deficiency (Fig. 4A). Induction of MMP-9 in core Tg mice is also confirmed by zymography (Fig. 4B). Concomitantly, pro-inflammatory cytokines known to induce MMPs, such as IL-1α and TNF-α (23), are upregulated in core Tg mice and similarly repressed by c-Jun deficiency (Fig. 4A). IL-6 which is a known agonist for STAT3 activation and implicated in carciongenesis (20, 24, 25), is also induced in core Tg mice in a c-Jun dependent manner (Fig. 4A). HCV core induces iNOS, RNS/ROS generation, and DNA hypermutation in vitro (19), and these changes are implicated in enhanced double-strand DNA breaks and increased levels of oxidatively damaged DNA (8-oxodG) in the livers of core Tg mice (18). Indeed, our analysis shows upregulation of iNOS in core Tg mice and its abrogation by c-Jun deficiency, suggesting that the core protein is upstream of c-Jun, which contributes to DNA damage via iNOS induction.
To test direct activation of AP-1 by the core protein, we next performed a transient transfection experiment using an AP-1 reporter construct and primary hepatocytes from WT (c-jun+/+) and c-Jun deficient (c-jun−/−) mice. Core expression increased the AP-1 promoter activity in c-jun+/+ but not in c-jun−/− hepatocytes (Fig. 4C). In addition, BHA significantly reduced the core-induced AP-1 promoter activity (Fig. 4C), suggesting the role of ROS and RNS in the activation of the AP-1 promoter induced by the core protein.
STAT3 plays an important role in DEN-induced hepatocarcinogenesis (31). HCV core protein induces generation of ROS (13) and the expression of IL-6 (Fig 4A), both of which are known agonists for STAT3 activation (13, 14). Indeed, our results demonstrate enhanced activation of STAT in core Tg mice (Fig. 1F) and the potential role of p-STAT3 in c-Jun dependent pro-oncogenic effects of the core (Fig. 5F). To test the importance of STAT3 in core-induced or -promoted hepatocarcinogenesis, we examined the effects of hepatocyte specific deletion of stat3 (stat3flox/flox mice crossed with mice expressing albumin promoter-Cre) on liver oncogenesis induced by DEN/Pb treatment (Fig. 5A). Our results in c-junflox/flox mice injected with the adenoviral vector expressing Cre, supported the role of c-Jun in core-mediated and -enhanced liver tumor formation. However, this technique inevitably deletes c-jun in both parenchymal and non-parenchymal liver cells. To further test hepatocyte-specific deletion of the c-jun, the compound mice harboring a cre gene under albumin promoter, c-junflox/flox, and a core transgene, were generated and also tested for DEN/Pb-induced hepatocarcinogenesis. The mice were divided into eight groups (n = 35 to 48 in each group) based on the presence or absence of c-jun, stat3, and the viral core protein, and the use of DEN and Phenobarbital (Fig. 5A). Conditional knockout of c-jun or stat3 reduced both spontaneous and DEN-induced tumor incidence (Fig. 5B). Furthermore, dual knockout of c-jun and stat3 showed an additive effect, resulting in a remarkable 80% reduction in the incidence (Fig. 5A–C). To determine the role of STAT3 in core-enhanced hepatocellualr proliferation, Ki-67 mRNA levels were measured in WT and Stat3−/− mice treated with DEN/Pb. Core-induced Ki-67 expression was significantly reduced in STAT3 deficient mice (Fig. 5E). This result and the c-Jun-dependent mitogenic effect (Fig. 3F) suggest that both c-Jun and STAT3 mediate core-induced hepatocellular proliferation. Furthermore, the number of apoptotic cells was significantly increased by c-Jun or STAT3 deficiency in tumor-bearing liver tissues of core Tg mice (Fig. 5F). Interestingly, double knockout of c-jun and stat3 had a synergistic effect on the frequency of apoptotic cells in core Tg mice (Fig. 5F). In no-tumor bearing tissues of core Tg mice or tumor-bearing tissues of WT mice, c-Jun deficiency, but not STAT3, deficiency significantly increased apoptosis (Fig. 5F). HCV infection is associated with Fas-dependent apoptosis of infected hepatocytes via cytotoxic T lympocytes (26). For this reason, we tested the effects of agonistic anti-Fas antibody (Jo2) on hepatocytes isolated from WT and core Tg mice treated with DEN/Pb. Hepatocytes from WT mice released significantly higher levels of AST into the medium and showed frequent TUNEL staining in response to Jo2. In contrast, the cells from core Tg mice had significantly reduced release of AST and almost complete absence of TUNEL staining (Supl. Fig. 1A, B, D). Furthermore, hepatocytes from c-Jun deficient core Tg mice, restored Jo2-induced cell death response (Suppl. Fig. 1). These differential apoptotic effects between core and WT hepatocytes were closely associated with c-Jun-dependent reduction of Fas expression in core hepatocytes (Suppl. Fig. 1C).
HCV core serves as a tumor initiator (Fig. 3B) through genetic damage caused by core-stimulated generation of ROS or RNS (19). Furthermore, DNA repair mechanisms may be inhibited by core-generated nitric oxide (NO) (27–29). As the antioxidant butylated hydroxyanisole (BHA) inhibits nitrite release (30) and HCV core-induced oncogenesis (Fig. 3D), we hypothesized that core-stimulated generation of NO inhibits DNA-damage repair, especially oxidative DNA damage repair. To test this notion, cell lysates from WT and core Tg mouse hepatocytes with or without a prior treatment with NOS inhibitors, were examined for their ability to promote in vitro incorporation of a radio-labeled nucleotide (32P-dGTP) into a damaged DNA substrate. If dGTP is efficiently incorporated into the substrate with a lysate, this means that the lysate contained fully functional repair mechanisms to excise damaged bases and to incorporate new dGTP. Our results showed that dGTP was incorporated into the damaged DNA when the lysate from WT hepatocytes was used, while no dGTP incorporation was evident using the core Tg hepatocyte lysate (lanes 1 vs. 4 of Fig. 6A). Pre-treatment with a specific iNOS inhibitor (1400W) or a general NOS inhibitor (N ω-nitro-L-arginine methyl ester: L-NMMA) nearly normalized the dGTP incorporation activity with the lysate from Tg hepatocytes (lanes 5 and 6, Fig. 6A). Similarly, the lysate from core Tg hepatocytes treated with BHA, also had normal dGTP incorporation as seen in the WT lyaste (lane 4, Fig. 6B). Furthermore, the treatment of WT hepatocytes with a mixture of NO-inducing cytokines (IFN-γ, TNF-α, IL-1β) or a NO donor (SNAP), caused a complete failure in dGTP incorporation (lanes 7 and 8 of Fig. 6B)
Next, we tested the role of c-Jun in core-induced inhibition of dGTP incorporation. The lysate from core Tg mouse hepatocytes deficient in c-Jun (albumin-cre:c-junflox/flox: c-jun−/−) showed the normal level of dGTP incorporation as opposed to severely impaired activity with the lysate from core Tg/c-jun+/+ mice (lane 4 vs. 10, Fig. 6C). These results support the obligatory role of c-Jun in mediating core-induced inhibition of DNA repair via NO. To extend this conclusion to natural HCV infection, we tested the lysate from Huh7.5.1 cells infected or uninfected with HCV for the dGTP incorporation analysis. Indeed, HCV-infected cell lysate (HCV+) showed impaired incorporation activity, which was again normalized by treatment with 1400W or L-NMMA (Fig. 6D). In addition, the treatment of HCV-uninfected cells (HCV−) with the NO donor SNAP or the NO-inducing cytokine mixture, obliterated the incorporation activity, and the latter effect was prevented with 1400W (Fig. 6D). Thus, these results confirm HCV-mediated inhibition of oxidative DNA damage repair via NO generation in the setting of HCV infection. HCV expresses several other structural and non-structural proteins besides core. Thus we next tested these viral proteins for their effects on DNA repair. For this analysis, the 32P-dGTP incorporation assay was performed on Huh7 cells expressing individual viral proteins (Fig. 6E). Among seven viral proteins examined, core and NS3 proteins equally impaired the incorporation activity (Fig. 6E), which was restored by treatment with NO inhibitors (Fig. 6F). Similar results were obtained using the lysate from Huh7 cells containing an HCV replicon, which included NS3 (Fig. 6F). The control cell line containing a neomycin-resistant gene exhibited normal dGTP-incorporation activity, which was not affected by the NO inhibitors. These results indicate that NO induced by core and NS3 proteins is responsible for inhibition of DNA repair associated with HCV infection (Fig. 6A–F).
HCV infection or core protein inhibits dGTP-incorporation activity in a c-Jun and NO-dependent manner, which is mainly facilitated by base excision repair (BER). BER removes a variety of DNA lesions such as spontaneous hydrolytic depurination, deamination of cytosine and 5-methylcytosine, products of reactions with hydroxyl radical, and covalent DNA adducts (31). The BER components include Polβ, polδ, polε, APE1 (AP-endonuclease), and Ogg1 (8-oxoguanine-DNA glycosylase) (32). To determine whether HCV core protein affects the BER, we performed immunoblot analysis to determine the expression of the components of the BER in HepG2 cells with and without stable core protein expression. We also performed co-immunoprecipitation analysis to assess the interactions between the BER components and the HCV core protein. Neither alteration of protein or mRNA levels of the BER components (Fig. 7A and 7B), nor the interaction of the core protein with the components (the data not shown), was observed.
We next analyzed whether the accumulation of 8-oxodG in HCV-infected Huh7.5.1 cells, core-transduced HepG2 cells, and primary hepatocytes from core Tg mice, is accompanied by alterations in DNA glycosylase activity for the repair of oxidative damage. For this assessment, we measured the activity which specifically removes 8-oxodG using a duplex oligo containing a radio-labeled 8-oxodG residue (33). As predicted, HCV infection or core expression either by stable transduction or as a transgene, increased the content of 8-xodG in a manner dependent on iNOS or oxidant stress (Fig. 7C). Accumulation of this oxidative DNA modification was also shown to be dependent on c-Jun in primary hepatocytes from core Tg vs. Tg:c-jun−/− mice (Fig. 7E, the last panel). As shown in Fig. 7D, these increases in the 8-oxodG content are closely associated with concomitant reductions in the release of 8-oxodG by DNA glycosylase activity of respective cell lysate. Furthermore, the protective effects of the iNOS inhibitor (1400W), antioxidant (BHA), or c-Jun deficiency (c-jun−/−), tightly correlated with enhanced DNA glycosylase activity (Fig. 7D).
We demonstrated that dual ablation of c-jun and stat3 results in an additive and nearly complete prevention of both spontaneous and DEN-induced HCC in HCV core Tg mice, highlighting the critical role of both c-Jun and STAT3 in HCV hepatocarcinogenesis. The core-induced proliferative effects on hepatocytes required activation of c-Jun/AP-1 and STAT3 particularly during tumor initiation and early progression. Furthermore, our data suggest that c-Jun is upstream of STAT3 activation (Fig. 3F), probably via c-Jun-mediated IL-6 induction (Fig. 4A). The BHA’s antioxidant effect is most likely upstream, scavenging ROS, which in turn suppresses c-Jun activation (34) and oxidative DNA damage.
These results demonstrate that HCV core protein induces specific signaling via c-Jun and STAT3 that culminate to the multiple levels of mutagenic and pro-oncogenic effects as a tumor initiator to induce spontaneous HCC and to enhance carcinogen/promoter-induced hepatic carcinogenesis. Based on this conclusion, c-Jun and STAT3 inhibitors (35) may be particularly useful during pre-cancerous stages such as cirrhosis or chronic viral infection, as chemopreventive agents.
We thank Dr. Carter in Vanderbilt University for c-jun flox/flox mice and Mr. Sean Vorah, Ms. Ling Zhou, Ms. Minyi Helene Liu, Ms. Claudine Kashiwabara, and Mr. Jeffery Hwang from University of Southern California for technical assistance, Dr. Francis Chisari for Huh7.5.1 cells, Dr. Takaji Wakita for JFH-1 strain, and Dr. Hua Yu from City of Hope for the breeding of STAT3flox/flox mice. This project was supported by NIH research grants AI40038, 1R01 AA018857-01, P50AA11999, P01CA123328, Zumberge Fellowship, Wright Foundation, American Cancer Society pilot funding, pilot funding from Research Center for Liver Diseases, and CA108302.