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Hepatoprotective versus Oncogenic Functions of STAT3 in Liver Tumorigenesis

2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2011.05.005

1525-2191

Hua Wang, Fouad Lafdil, Lei Wang, Ogyi Park, Shi Yin, Junyang Niu, Andrew M. Miller, Zhaoli Sun, Bin Gao,

PI3K/AKT/mTOR signaling in cancer

Aberrantly hyperactivated STAT3 has been found in human liver cancers as an oncogene; however, STAT3 has also been shown to exert hepatoprotective effects during liver injury. The balancing act that STAT3 plays between hepatoprotection and liver tumorigenesis remains poorly defined. In this study, the diethylnitrosamine (DEN)-induced liver tumor model and the chronic carbon tetrachloride (CCl4)–induced liver fibrosis model were both used to investigate the role of STAT3 in liver tumorigenesis. Hepatocyte-specific STAT3 knockout mice were resistant to liver tumorigenesis induced by a single DEN injection, whose tumorigenesis was associated with minimal chronic liver inflammation, injury, and fibrosis. In contrast, long-term CCl4 treatment resulted in severe hepatic oxidative damage, inflammation, and fibrosis but rarely induced liver tumor formation in wild-type mice. Despite the oncogenic function of STAT3 in DEN-induced liver tumor, hepatocyte-specific STAT3 knockout mice were more susceptible to liver tumorigenesis after 16 weeks of CCl4 injection, which was associated with higher levels of liver injury, inflammation, fibrosis, and oxidative DNA damage compared with wild-type mice. These findings suggest that the hepatoprotective feature of STAT3 prevents hepatic damage and fibrosis under the condition of persistent inflammatory stress, consequently suppressing injury-driven liver tumor initiation. Once liver tumor cells have developed, STAT3 likely acts as an oncogenic factor to promote tumor growth. Aberrantly hyperactivated STAT3 has been found in human liver cancers as an oncogene; however, STAT3 has also been shown to exert hepatoprotective effects during liver injury. The balancing act that STAT3 plays between hepatoprotection and liver tumorigenesis remains poorly defined. In this study, the diethylnitrosamine (DEN)-induced liver tumor model and the chronic carbon tetrachloride (CCl4)–induced liver fibrosis model were both used to investigate the role of STAT3 in liver tumorigenesis. Hepatocyte-specific STAT3 knockout mice were resistant to liver tumorigenesis induced by a single DEN injection, whose tumorigenesis was associated with minimal chronic liver inflammation, injury, and fibrosis. In contrast, long-term CCl4 treatment resulted in severe hepatic oxidative damage, inflammation, and fibrosis but rarely induced liver tumor formation in wild-type mice. Despite the oncogenic function of STAT3 in DEN-induced liver tumor, hepatocyte-specific STAT3 knockout mice were more susceptible to liver tumorigenesis after 16 weeks of CCl4 injection, which was associated with higher levels of liver injury, inflammation, fibrosis, and oxidative DNA damage compared with wild-type mice. These findings suggest that the hepatoprotective feature of STAT3 prevents hepatic damage and fibrosis under the condition of persistent inflammatory stress, consequently suppressing injury-driven liver tumor initiation. Once liver tumor cells have developed, STAT3 likely acts as an oncogenic factor to promote tumor growth. Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and one of the fastest growing cancers in terms of incidence in developed countries.1Ferenci P. Fried M. Labrecque D. Bruix J. Sherman M. Omata M. Heathcote J. Piratsivuth T. Kew M. Otegbayo J.A. Zheng S.S. Sarin S. Hamid S.S. Modawi S.B. Fleig W. Fedail S. Thomson A. Khan A. Malfertheiner P. Lau G. Carillo F.J. Krabshuis J. Le Mair A. Hepatocellular carcinoma (HCC): a global perspective.J Clin Gastroenterol. 2010; 44: 239-245Crossref PubMed Scopus (225) Google Scholar, 2El-Serag H.B. Rudolph K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.Gastroenterology. 2007; 132: 2557-2576Abstract Full Text Full Text PDF PubMed Scopus (4442) Google Scholar Despite recent progress in understanding its pathogenic mechanisms,2El-Serag H.B. Rudolph K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.Gastroenterology. 2007; 132: 2557-2576Abstract Full Text Full Text PDF PubMed Scopus (4442) Google Scholar, 3Aravalli R.N. Steer C.J. Cressman E.N. Molecular mechanisms of hepatocellular carcinoma.Hepatology. 2008; 48: 2047-2063Crossref PubMed Scopus (562) Google Scholar, 4Mishra L. Banker T. Murray J. Byers S. Thenappan A. He A.R. Shetty K. Johnson L. Reddy E.P. Liver stem cells and hepatocellular carcinoma.Hepatology. 2009; 49: 318-329Crossref PubMed Scopus (272) Google Scholar HCC is still considered one of the deadliest cancers worldwide with a poor prognosis, as very few patients are eligible for potential curative treatment, such as surgical resection and liver transplantation.5El-Serag H.B. Marrero J.A. Rudolph L. Reddy K.R. Diagnosis and treatment of hepatocellular carcinoma.Gastroenterology. 2008; 134: 1752-1763Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 6Llovet J.M. Bruix J. Molecular targeted therapies in hepatocellular carcinoma.Hepatology. 2008; 48: 1312-1327Crossref PubMed Scopus (898) Google Scholar, 7Bruix J. Llovet J.M. Major achievements in hepatocellular carcinoma.Lancet. 2009; 373: 614-616Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 8Villanueva A. Llovet J.M. Targeted therapies for hepatocellular carcinoma.Gastroenterology. 2011; 140: 1410-1426Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar Various etiologies, including hepatotropic viruses (eg, hepatitis B and C viruses), chronic alcohol consumption, nonalcoholic steatohepatitis, and toxins (eg, aflatoxin B), can lead to chronic liver injury, inflammation, or fibrosis/cirrhosis, which can then culminate in liver cancer.1Ferenci P. Fried M. Labrecque D. Bruix J. Sherman M. Omata M. Heathcote J. Piratsivuth T. Kew M. Otegbayo J.A. Zheng S.S. Sarin S. Hamid S.S. Modawi S.B. Fleig W. Fedail S. Thomson A. Khan A. Malfertheiner P. Lau G. Carillo F.J. Krabshuis J. Le Mair A. Hepatocellular carcinoma (HCC): a global perspective.J Clin Gastroenterol. 2010; 44: 239-245Crossref PubMed Scopus (225) Google Scholar, 2El-Serag H.B. Rudolph K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.Gastroenterology. 2007; 132: 2557-2576Abstract Full Text Full Text PDF PubMed Scopus (4442) Google Scholar, 3Aravalli R.N. Steer C.J. Cressman E.N. Molecular mechanisms of hepatocellular carcinoma.Hepatology. 2008; 48: 2047-2063Crossref PubMed Scopus (562) Google Scholar Although the close contributory relationship between chronic liver inflammation and carcinogenesis has been well documented,2El-Serag H.B. Rudolph K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.Gastroenterology. 2007; 132: 2557-2576Abstract Full Text Full Text PDF PubMed Scopus (4442) Google Scholar, 3Aravalli R.N. Steer C.J. Cressman E.N. Molecular mechanisms of hepatocellular carcinoma.Hepatology. 2008; 48: 2047-2063Crossref PubMed Scopus (562) Google Scholar the exact mechanisms that direct inflammation-induced liver cancer remain elusive. Recent evidence suggests that liver cancer formation can be generally divided into three stages: initiation, promotion, and progression. Liver tumor initiation can be induced by many initiators, including hepatitis B virus, cirrhosis, and aflatoxin B. These factors can induce irreversible genetic mutations that subsequently cause proto-oncogene activation and/or loss of tumor suppression. For tumor development to take place, initiation must be followed by a committed tumor promotion process that requires interactions between initiated cells and their microenvironment,9Albini A. Sporn M.B. The tumour microenvironment as a target for chemoprevention.Nat Rev Cancer. 2007; 7: 139-147Crossref PubMed Scopus (663) Google Scholar of which inflammation is an important factor.10Grivennikov S.I. Greten F.R. Karin M. Immunity, inflammation, and cancer.Cell. 2010; 140: 883-899Abstract Full Text Full Text PDF PubMed Scopus (7427) Google Scholar Although HCC usually appears after exposure to carcinogens such as aflatoxin B, viral hepatitis, and alcohol intake, it may take many years, even decades, to develop HCC from liver cirrhosis after chronic liver injury and fibrosis.2El-Serag H.B. Rudolph K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.Gastroenterology. 2007; 132: 2557-2576Abstract Full Text Full Text PDF PubMed Scopus (4442) Google Scholar, 3Aravalli R.N. Steer C.J. Cressman E.N. Molecular mechanisms of hepatocellular carcinoma.Hepatology. 2008; 48: 2047-2063Crossref PubMed Scopus (562) Google Scholar Although the early etiologic events involved in liver tumorigenesis have been well documented, little is known about mechanisms underlying the progression from premalignant lesions to overt carcinomas. Without greater knowledge of these mechanisms, it is impossible to improve current unsatisfactory and suboptimal therapeutic options for HCC. Recent results of a phase 3 clinical trial showed that sorafenib, a multikinase inhibitor that blunts multiple signaling pathways, markedly improved survival in patients with advanced HCC tumors.11Llovet J.M. Ricci S. Mazzaferro V. Hilgard P. Gane E. Blanc J.F. de Oliveira A.C. Santoro A. Raoul J.L. Forner A. Schwartz M. Porta C. Zeuzem S. Bolondi L. Greten T.F. Galle P.R. Seitz J.F. Borbath I. Haussinger D. Giannaris T. Shan M. Moscovici M. Voliotis D. Bruix J. Sorafenib in advanced hepatocellular carcinoma.N Engl J Med. 2008; 359: 378-390Crossref PubMed Scopus (8967) Google Scholar, 12Siegel A.B. Olsen S.K. Magun A. Brown Jr, R.S. Sorafenib: where do we go from here?.Hepatology. 2010; 52: 360-369Crossref PubMed Scopus (86) Google Scholar This pivotal study has stimulated scientific research on novel molecular therapies targeting specific signaling pathways to treat this malignancy.6Llovet J.M. Bruix J. Molecular targeted therapies in hepatocellular carcinoma.Hepatology. 2008; 48: 1312-1327Crossref PubMed Scopus (898) Google Scholar A growing body of evidence suggests that persistent activation of STAT3 is oncogenic and is prevalent in a variety of human cancers, including liver cancer.13Yu H. Pardoll D. Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3.Nat Rev Cancer. 2009; 9: 798-809Crossref PubMed Scopus (3085) Google Scholar Hepatic activation of STAT3 occurs through various cytokines, growth factors, hormones, and hepatitis viral proteins.14Wang H. Lafdil F. Kong X. Gao B. Signal transducer and activator of transcription 3 in liver diseases: a novel therapeutic target.Int J Biol Sci. 2011; 7: 536-550Crossref PubMed Scopus (197) Google Scholar Among them, IL-6 and IL-22 are two potent inducers of STAT3 activation in hepatocytes.14Wang H. Lafdil F. Kong X. Gao B. Signal transducer and activator of transcription 3 in liver diseases: a novel therapeutic target.Int J Biol Sci. 2011; 7: 536-550Crossref PubMed Scopus (197) Google Scholar Activation of STAT3 begins on binding of IL-6 to its corresponding receptor on hepatocytes, resulting in dimerization of the signal transducer protein, gp130. The gp130-associated Janus kinases then combine to form dimers and phosphorylate each other, the gp130 protein, and cytoplasmic STAT3 monomers. IL-22 binds to IL-22R and IL-10R2 on hepatocytes, followed by inducing Janus kinase phosphorylation and subsequently STAT3 phosphorylation. Consequently, phosphorylated STAT3 (pSTAT3) monomers interact with each other and form dimers that translocate into the nuclei to induce transcription of genes involved in cell survival and proliferation.15Kishimoto T. IL-6: from its discovery to clinical applications.Int Immunol. 2010; 22: 347-352Crossref PubMed Scopus (593) Google Scholar Activation of STAT3 by IL-6 or IL-22 has been shown to contribute to the tumorigenesis of a variety of tumors, including HCC.13Yu H. Pardoll D. Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3.Nat Rev Cancer. 2009; 9: 798-809Crossref PubMed Scopus (3085) Google Scholar, 16Bromberg J. Wang T.C. Inflammation and cancer: IL-6 and STAT3 complete the link.Cancer Cell. 2009; 15: 79-80Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 17Naugler W.E. Sakurai T. Kim S. Maeda S. Kim K. Elsharkawy A.M. Karin M. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production.Science. 2007; 317: 121-124Crossref PubMed Scopus (1472) Google Scholar, 18Radaeva S. Sun R. Pan H.N. Hong F. Gao B. Interleukin 22 (IL-22) plays a protective role in T cell-mediated murine hepatitis: IL-22 is a survival factor for hepatocytes via STAT3 activation.Hepatology. 2004; 39: 1332-1342Crossref PubMed Scopus (482) Google Scholar, 19Park O. Wang H. Weng H. Feigenbaum L. Li H. Yin S. Ki S. Yoo S.H. Dooley S. Wang F.S. Young H.A. Gao B. In vivo consequences of liver-specific interleukin-22 expression: implications for human liver disease progression.Hepatology. 2011; ([Epub ahead of print])https://doi.org/10.1002/hep.24339Crossref Scopus (183) Google Scholar Thus, intense efforts have been made to identify suitable anti-STAT3 agents to treat human cancers such as HCC.20Yue P. Turkson J. Targeting STAT3 in cancer: how successful are we?.Expert Opin Investig Drugs. 2009; 18: 45-56Crossref PubMed Scopus (346) Google Scholar However, historically, evidence from a wealth of previous studies has confirmed the hepatoprotective function of STAT3 in numerous models of liver injury.14Wang H. Lafdil F. Kong X. Gao B. Signal transducer and activator of transcription 3 in liver diseases: a novel therapeutic target.Int J Biol Sci. 2011; 7: 536-550Crossref PubMed Scopus (197) Google Scholar Thus, an understanding of the pathologic/oncogenic versus protective functions of STAT3 in liver tumorigenesis is needed to carefully assess the risks and benefits of anti-STAT3 treatment for HCC in the clinical setting. A previous study reported that conditional deletion of STAT3 in hepatocytes prevents liver tumor development induced by a single diethylnitrosamine (DEN) injection at age 15 days,21He G. Yu G.Y. Temkin V. Ogata H. Kuntzen C. Sakurai T. Sieghart W. Peck-Radosavljevic M. Leffert H.L. Karin M. Hepatocyte IKKβ/NF-κB inhibits tumor promotion and progression by preventing oxidative stress-driven STAT3 activation.Cancer Cell. 2010; 17: 286-297Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar which was also confirmed in the present study. However, this DEN model is not associated with significant chronic liver injury, inflammation, and fibrosis, which is different from most human liver cancers that develop after chronic hepatocellular damage, inflammation, and fibrosis/cirrhosis.2El-Serag H.B. Rudolph K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.Gastroenterology. 2007; 132: 2557-2576Abstract Full Text Full Text PDF PubMed Scopus (4442) Google Scholar, 3Aravalli R.N. Steer C.J. Cressman E.N. Molecular mechanisms of hepatocellular carcinoma.Hepatology. 2008; 48: 2047-2063Crossref PubMed Scopus (562) Google Scholar Thus, by using liver-specific STAT3 knockout mice, we also explored the role of hepatic STAT3 in a model of liver tumor induced by chronic carbon tetrachloride (CCl4) treatment that is associated with strong chronic liver injury, inflammation, and fibrosis. The present results suggest that hepatic STAT3 protects against CCl4-induced chronic liver injury and consequently against tumorigenesis. Hepatocyte-specific STAT3 knockout mice (STAT3Hep−/−) (AlbCre+/− STAT3flox/flox) and littermate wild-type controls (AlbCre−STAT3flox/flox) were described previously.22Horiguchi N. Wang L. Mukhopadhyay P. Park O. Jeong W.I. Lafdil F. Osei-Hyiaman D. Moh A. Fu X.Y. Pacher P. Kunos G. Gao B. Cell type-dependent pro- and anti-inflammatory role of signal transducer and activator of transcription 3 in alcoholic liver injury.Gastroenterology. 2008; 134: 1148-1158Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar STAT1−/− and STAT1−/−STAT3Hep−/− mice were generated as described previously.23Wang H. Park O. Lafdil F. Shen K. Horiguchi N. Yin S. Fu X.Y. Kunos G. Gao B. Interplay of hepatic and myeloid signal transducer and activator of transcription 3 in facilitating liver regeneration via tempering innate immunity.Hepatology. 2010; 51: 1354-1362Crossref PubMed Scopus (34) Google Scholar All the animal experiments were approved by the Institutional Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism. The DEN-induced liver tumor model was established as described previously.24Vesselinovitch S.D. Mihailovich N. Kinetics of diethylnitrosamine hepatocarcinogenesis in the infant mouse.Cancer Res. 1983; 43: 4253-4259PubMed Google Scholar, 25Fan Y. Boivin G.P. Knudsen E.S. Nebert D.W. Xia Y. Puga A. The aryl hydrocarbon receptor functions as a tumor suppressor of liver carcinogenesis.Cancer Res. 2010; 70: 212-220Crossref PubMed Scopus (139) Google Scholar Briefly, 15-day-old STAT3Hep−/− mice and their littermates were injected with 5 or 20 μg/g of DEN (Sigma-Aldrich, St. Louis, MO). After 4 or 9 months on normal chow, the mice were sacrificed. Their livers were then removed, separated into individual lobes, analyzed for the presence of HCCs, and subjected to analysis of histologic and immunochemical parameters. Eight- to 10-week-old male STAT3Hep−/− mice and their wild-type littermates were injected i.p. with 2 mL/kg body weight of 10% (v/v) CCl4 (Sigma-Aldrich) dissolved in olive oil three times a week for up to 16 weeks. Control groups were treated with vehicle (10% olive oil, 2 mL/kg). The mice were sacrificed at different time points after the last injection of chronic CCl4 treatment, and liver tissues were harvested for experiments. No mortality was observed in these mice after chronic CCl4 treatment. Ascites were reported in the mice after chronic CCl4 treatment plus phenobarbital in drinking water, especially after the inhalation protocol.26Domenicali M. Caraceni P. Giannone F. Baldassarre M. Lucchetti G. Quarta C. Patti C. Catani L. Nanni C. Lemoli R.M. Bernardi M. A novel model of CCl4-induced cirrhosis with ascites in the mouse.J Hepatol. 2009; 51: 991-999Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Chronic i.p. administration of CCl4 plus phenobarbital in drinking water also induced ascites but to a lesser extent.26Domenicali M. Caraceni P. Giannone F. Baldassarre M. Lucchetti G. Quarta C. Patti C. Catani L. Nanni C. Lemoli R.M. Bernardi M. A novel model of CCl4-induced cirrhosis with ascites in the mouse.J Hepatol. 2009; 51: 991-999Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar No obvious ascites were observed in the mice treated with i.p. injection of CCl4 alone in the present protocol. The whole liver was carefully removed from the euthanized animal, washed, and placed in cold PBS. The number of surface liver tumor nodules was counted for all the liver lobes in a blinded manner. Some reasonably sized tumor nodules (>2 mm in diameter) were carefully removed from the liver lobes using fine forceps and were placed in fresh cold PBS. These separated nodules were then halved using a sterile razor blade and were split into samples for RNA and protein extraction. Histologic sections, including larger tumor nodules (>2 mm in diameter), were collected and fixed in 10% formalin. Tissue section slides were stained with H&E using standard protocols. Liver nodules typically presented as basophilic foci with crowded nuclei and were classified as atypical foci (HCC) or hepatocellular adenomas. Adenomas were distinguished from atypical foci based on the presence of clearly defined margins and compression of surrounding parenchyma. Formalin-fixed liver samples were processed, and paraffin sections were stained with H&E. The score of liver injury (necrosis and inflammation) was evaluated blindly by two pathologists as described previously.27Goodman Z.D. Grading and staging systems for inflammation and fibrosis in chronic liver diseases.J Hepatol. 2007; 47: 598-607Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar Liver fibrosis was determined by Sirius red staining for collagens or by immunohistochemical (IHC) staining for activated hepatic stellate cells with anti–α-smooth muscle actin (Dako, Carpinteria, CA) and were quantified by digital imaging using NIH Scion Image and Adobe Photoshop (Adobe Systems Inc., San Jose, CA).28Radaeva S. Sun R. Jaruga B. Nguyen V.T. Tian Z. Gao B. Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners.Gastroenterology. 2006; 130: 435-452Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 29Paik Y.H. Kim J.K. Lee J.I. Kang S.H. Kim D.Y. An S.H. Lee S.J. Lee D.K. Han K.H. Chon C.Y. Lee S.I. Lee K.S. Brenner D.A. Celecoxib induces hepatic stellate cell apoptosis through inhibition of Akt activation and suppresses hepatic fibrosis in rats.Gut. 2009; 58: 1517-1527Crossref PubMed Scopus (88) Google Scholar Serum alanine transaminase (ALT), aspartate aminotransferase (AST), and albumin levels were determined using a chemistry analyzer (Prochem V; Drew Scientific Co., Barrow-in-Furness, UK). Serum levels of C-reactive protein were analyzed by immunoperoxidase assay (Immunology Consultants Laboratory, Newberg, OR). Serum cytokine levels were measured by Cytometric Bead Array (BD Biosciences, San Diego, CA). Hepatocyte apoptosis was detected by using the ApopTag TUNEL apoptosis detection kit (Chemicon International, Temecula, CA). Hepatocyte proliferation in vivo was determined using the bromodeoxyuridine (BrdU) incorporation assay. Briefly, mice were injected (i.p) with BrdU (50 μg/g body weight) and euthanized 2 hours later, and the livers were harvested for IHC staining of BrdU using a kit (BD Biosciences). BrdU-labeled hepatocytes in each slide were counted in five low-power (×100) fields. Real-time PCR was performed with the indicated primers as described previously.22Horiguchi N. Wang L. Mukhopadhyay P. Park O. Jeong W.I. Lafdil F. Osei-Hyiaman D. Moh A. Fu X.Y. Pacher P. Kunos G. Gao B. Cell type-dependent pro- and anti-inflammatory role of signal transducer and activator of transcription 3 in alcoholic liver injury.Gastroenterology. 2008; 134: 1148-1158Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar Western blot analyses were performed as described previously22Horiguchi N. Wang L. Mukhopadhyay P. Park O. Jeong W.I. Lafdil F. Osei-Hyiaman D. Moh A. Fu X.Y. Pacher P. Kunos G. Gao B. Cell type-dependent pro- and anti-inflammatory role of signal transducer and activator of transcription 3 in alcoholic liver injury.Gastroenterology. 2008; 134: 1148-1158Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar using STAT3, STAT1, glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology Inc., Danvers, MA), and anti-CYP2E1 (Chemicon International, Billerica, MA) antibodies. Malondialdehyde (MDA) was measured using the thiobarbituric acid method and is expressed as nanomoles of MDA per milligram of protein.30Gavino V.C. Miller J.S. Ikharebha S.O. Milo G.E. Cornwell D.G. Effect of polyunsaturated fatty acids and antioxidants on lipid peroxidation in tissue cultures.J Lipid Res. 1981; 22: 763-769Abstract Full Text PDF PubMed Google Scholar, 31Ohkawa H. Ohishi N. Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Anal Biochem. 1979; 95: 351-358Crossref PubMed Scopus (23164) Google Scholar Glutathione (GSH) was measured as described previously.32Baker M.A. Cerniglia G.J. Zaman A. Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples.Anal Biochem. 1990; 190: 360-365Crossref PubMed Scopus (803) Google Scholar The hepatic GSH concentrations were adjusted according to the original wet weight of liver tissue (nanomoles per milligram of liver tissue). Hepatic 8-hydroxy-2-deoxyguanosine (8-OHdG) was measured using an enzyme-linked immunosorbent assay kit (OXIS Health Products Inc., Portland, OR) according to the manufacturer's manual. Data are expressed as mean ± SD (n = 5 to 12 in each group). Student's t-test was performed to compare values from two groups. To compare values obtained from three or more groups, one-factor analysis of variance was used, followed by Tukey's post hoc test. Statistical significance was taken at the P < 0.05 level. To address the function of hepatic STAT3 in the development of DEN-induced HCC, STAT3Hep−/− mice and their wild-type littermates were injected with DEN at age 15 days. Figure 1A shows hepatic expression of STAT3 that was significantly lower in STAT3Hep−/− mice than in wild-type mice, confirming hepatic STAT3 deletion in STAT3Hep−/− mice at age 15 days. In the initial experimental design, we were unsure of the susceptibility of the strains of wild-type and STAT3Hep−/− mice to DEN-induced HCC development, so we injected mice with 5 or 20 μg/g of DEN. As illustrated in Figure 1B, injection of both doses induced significant liver tumor formation 9 months after injection. Injection of 20 μg/g of DEN induced a slightly higher number and larger size of tumors than did injection of 5 μg/g of DEN, but this difference did not reach statistical significance. The number and size of tumors were much smaller in STAT3Hep−/− mice than in wild-type mice after injection of either 5 or 20 μg/g of DEN. Tumor and nontumor tissues from wild-type and STAT3Hep−/− mice were collected and subjected to real-time PCR (Figure 1C) and Western blot analyses (Figure 1D). Figure 1C shows that expression of the typical cell cycle protein cyclin D was reduced and that expression of cell cycle regulatory protein p21 was higher in tumor and nontumor samples from STAT3Hep−/− mice than from wild-type mice. Hepatic expression of p27 was detected at low levels and was comparable in wild-type and STAT3Hep−/− mice (data not shown). Western blot analyses in Figure 1D show that pSTAT3 expression was elevated in nontumor and tumor tissues in DEN-treated wild-type mice. Such expression, as expected, was barely detected in STAT3Hep−/− mice. Expression of pSTAT1 was elevated in nontumor tissues and, to a lesser extent, in tumor tissues in DEN-treated wild-type mice compared with that in nontreated control mice. In addition, expression of pSTAT1 and STAT1 was comparable between STAT3Hep−/− and WT mice. Figure 1E shows representative images of liver, H&E staining, Ki-67 immunostaining, and TUNEL staining from DEN-treated wild-type and knockout mice. Microscopically, the tumors in both groups formed discrete nodes surrounded by almost normal liver tissues. Histologic analyses show that the tumors from both groups had a similarly moderate degree of differentiation, with an increased nuclear-to-cytoplasmic index, enlarged and hyperchromatic nuclei, and expansive growth. In the area of tumor tissues, the normal liver architecture, such as bile duct and portal tract formation, was lost. In addition, wild-type mice had greater numbers of Ki-67+ and TUNEL+ hepatocytes than did STAT3Hep−/− mice 9 months after DEN exposure. To explore the role of STAT1 in DEN-induced liver tumor formation, wild-type, STAT1−/−, STAT3Hep−/−, and STAT1−/−STAT3Hep−/− double knockout mice were treated with a single dose of DEN at age 15 days. Despite the well-documented antioncogenic effect of STAT1, STAT1−/− mice had a similar number and size of tumors as wild-type mice 9 months after DEN injection (Figure 1F). An additional deletion of STAT1 did not further increase liver tumor number, size, and incidence compared with STAT3Hep−/− mice (Figure 1F). These results indicate that STAT1 signaling is not enhanced and is not responsible for the reduced liver tumorigenesis in STAT3Hep−/− mice. Because a causal relationship between chronic injury and inflammation and liver carcinogenesis in most patients with HCC is well established, we wondered whether the DEN-induced liver cancer mouse model is associated with chronic hepatocyte injury, inflammation, and fibrosis. As illustrated in Figure 2A, elevation of serum ALT and liver oxidative stress MDA and 8-OHdG levels and down-regulation of hepatic GSH levels were observed only 24 hours after a single dose of acute DEN injection. Such changes were comparable between wild-type and STAT3Hep−/− mice. H&E staining shows that no obvious hepatocyte necrosis and inflammatory foci were detected in the livers 4 or 9 months after DEN injection (Figure 2B). Sirius red staining revealed no obvious liver fibrosis in these DEN-treated mice (Figure 2B). Only in the very late stage of HCC, when the liver tumor grows too fast, were necrotic tumor tissue and inflammation seen in the large tumor tissues (data not shown). Real-time PCR analyses in Figure 2C show that expression of CCR2, F4/80, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and transforming growth factor (TGF)-β was up-regulated in liver tumor tissues but not in nontumor tissues from DEN-treated wild-type mice compared with nontreated mice. Such induction was not observed in tumor tissues from DEN-treated STAT3Hep−/− mice, suggesting that hepatic STAT3 plays a role in stimulating inflammation in tumor tissues (Figure 2C). The previous data show that the DEN model is not associated with significant liver injury and inflammation; however, most human liver cancers develop after chronic liver injury, inflammation, and fibrosis.2El-Serag H.B. Rudolph K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.Gastroenterology. 2007; 132: 2557-2576Abstract Full Text Full Text PDF PubMed Scopus (4442) Google Scholar, 3Aravalli R.N. Steer C.J. Cressman E.N. Molecular mechanisms of hepatocellular carcinoma.Hepatology. 2008; 48: 2047-2063Crossref PubMed Scopus (562) Google Scholar To further explore the role of STAT3 in liver tumorigenesis that is ass