Published OnlineFirst February 18, 2016; DOI: 10.1158/0008-5472.CAN-15-1548 Cancer Tumor and Stem Cell Biology Research Inflammation-Dependent IL18 Signaling Restricts Hepatocellular Carcinoma Growth by Enhancing the Accumulation and Activity of Tumor- Infiltrating Lymphocytes Geoffrey J. Markowitz1, Pengyuan Yang1,2,3, Jing Fu3, Gregory A. Michelotti4, Rui Chen1, Jianhua Sui5, Bin Yang2, Wen-Hao Qin3, Zheng Zhang6, Fu-Sheng Wang6, Anna Mae Diehl4, Qi-Jing Li7, Hongyang Wang3, and Xiao-Fan Wang1 Abstract Chronic inflammation in liver tissue is an underlying cause of IL18R1 deletion increased tumor burden. Mechanistically, we hepatocellular carcinoma. High levels of inflammatory cytokine foundthatIL18exertedinflammation-dependent tumor-sup- IL18 in the circulation of patients with hepatocellular carcinoma pressive effects largely by promoting the differentiation, activ- correlates with poor prognosis. However, conflicting results have ity, and survival of tumor-infiltrating T cells. Finally, differences been reported for IL18 in hepatocellular carcinoma development in the expression of IL18 in tumor tissue versus nontumor and progression. In this study, we used tissue specimens from tissueweremorepredictiveofpatientoutcomethanoverall hepatocellular carcinoma patients and clinically relevant mouse tissue expression. Taken together, our findings resolve a long- models of hepatocellular carcinoma to evaluate IL18 expression standing contradiction regarding a tumor-suppressive role for and function. In a mouse model of liver fibrosis that recapitulates IL18 in established hepatocellular carcinoma and provide a a tumor-promoting microenvironment, global deletion of the mechanistic explanation for the complex relationship between IL18 receptor IL18R1 enhanced tumor growth and burden. Sim- its expression pattern and hepatocellular carcinoma prognosis. ilarly, in a carcinogen-induced model of liver tumorigenesis, Cancer Res; 76(8); 1–12. Ó2016 AACR. Introduction Because of the well-known intimate relationship of hepatocel- lular carcinoma with inflammation, previous studies have dem- Liver cancer is the third most deadly cancer worldwide, and onstrated both pro and antitumorigenic roles of a variety of unlike many other cancers, the incidence of this disease and its immune cell types and mediators. Important effector cells, such most common type, hepatocellular carcinoma, is rising despite as macrophages, natural killer (NK) cells, and T cells, have been advances in surveillance and therapeutics (1, 2). Hepatocellular shown to play varying and diverse roles in regulating the multistep carcinoma derives from multiple etiologic factors, including tumorigenic process, often in a stage- and even etiology-depen- infection by hepatitis viruses, alcohol abuse, and metabolic dent fashion (6–14). Cytokines, including TNFa, IL6, IL1a, IL1b, syndrome (1). These etiologic factors induce a state of chronic TGFb, IFNg, and CCL22, have been reported to display robust and inflammation, fibrosis, and cirrhosis, which in turn leads to disparate functionalities in modulating hepatocellular carcinoma malignant transformation of hepatocytes, hepatocellular carcino- initiation and progression (7, 13, 15–20). Examination of hepa- ma development, and progression (1, 3–5). tocellular carcinoma patient samples has also indicated a tight correlation among inflammation, disease progression, and prog- 1 nosis, although these correlations may vary based on the etiology Department of Pharmacology and Cancer Biology, Duke University – Medical Center, Durham, North Carolina. 2Key Laboratory of Infection (4, 14, 21 24). and Immunity, Institute of Biophysics, Chinese Academy of Sciences, IL18 is a member of the IL1 family of cytokines (25). Originally Beijing, China. 3National Center for Liver Cancer, Second Military described as IFNg-inducing factor, its canonical function is to Medical University, Shanghai, China. 4Department of Gastroenterolo- 5 promote the production of IFNg from a variety of immune cells, gy, Duke University Medical Center, Durham, North Carolina. Biolo- þ gics Research Center, National Institute of Biological Sciences, Beijing, primarily Th1 CD4 T cells and NK cells, frequently in conjunc- China. 6Research Center for Biological Therapy, Beijing 302 Hospital, tion with IL12 (25). IL18 is produced by a variety of cells, 7 Beijing, China. Department of Immunology, Duke University Medical including but not limited to dendritic cells, macrophages, and Center, Durham, North Carolina. epithelial cells (25). Its functional production is regulated both Note: Supplementary data for this article are available at Cancer Research transcriptionally and via proteolytic processing of its precursor Online (http://cancerres.aacrjournals.org/). peptide by the inflammasome (25). Clinical data have shown that Corresponding Author: Xiao-Fan Wang, Duke University Medical Center, Box IL18 levels in serum are elevated in patients with chronic hepatitis 3813, Durham, NC 27710. Phone: 919-681-4861; Fax: 919-681-7152; E-mail: B, hepatitis C, and hepatocellular carcinoma, and higher levels of [email protected] circulating IL18 are correlated with worse prognosis of hepato- doi: 10.1158/0008-5472.CAN-15-1548 cellular carcinoma (26–28). However, conflicting experimental Ó2016 American Association for Cancer Research. results have been reported showing either pro or antitumorigenic www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 18, 2016; DOI: 10.1158/0008-5472.CAN-15-1548 Markowitz et al. functions of IL18. Multiple carcinogen-induced and spontaneous Fibrosis induction tumorigenesis studies have demonstrated a protumorigenic role Carbon tetrachloride–induced liver fibrosis. Six- to 7-week-old À À of IL18, whereas investigations utilizing the implantation of male WT or IL18R1 / mice were treated with either olive oil or tumor cells in combination with methods to enhance IL18 carbon tetrachloride (CCl4) diluted 1:20 in olive oil at 600 mL/kg. expression revealed potential IL-18–induced tumor suppression Mice received biweekly intraperitoneal injections for 6 weeks and (10, 29, 30). Thus, despite the established connection between its were sacrificed 2 days following the 12th injection. presence in the circulation of patients and correlated poorer prognosis, a pathologic feature frequently associated with tumor Bile duct ligation–induced liver fibrosis. Twelve-week-old male WT À À promoters, the role of IL18 in hepatocellular carcinoma devel- or IL18R1 / mice were anesthetized and the common bile duct opment and progression is still contentious. To address this isolated and transected between two ligations. Mice were sacri- critical question, we conducted comprehensive analyses of two ficed 14 days postsurgery. sets of patient samples in conjunction with clinically relevant mouse models to examine the role of IL-18 in hepatocellular Orthotopic implantation carcinoma. We reveal a tumor-suppressive role for IL18 that is Implantation occurred either 2 days after the 8th injection of consistent with the complex expression patterns of this cytokine in CCl4 or olive oil or immediately following bile duct ligation. Mice our patient cohorts. Differing from previous reports, we demon- were anesthetized, the abdomen opened, and the liver exposed. strate a powerful effect of endogenous IL18 signaling modulating  6 þ Hepa1-6-GFP-Luc cells (3 10 ) were suspended in 30 mL growth the accumulation and activity of both CD8 and multiple subsets – þ factor reduced Matrigel and injected into the left lobe of the liver. of CD4 T cells to effect antitumor activity. We further demon- The peritoneum and skin were subsequently sutured shut. The 9th strate that IL18 signaling serves as a key mediator of complemen- dose of CCl or olive oil occurred 2 days after surgery, and the 12th þ – 4 tary CD8 T-cell- and NK cell dependent tumor suppression. dose occurred 12 days after surgery. Mice were monitored for Finally, we show that the differences in the level of IL18 expression health and sacrificed 14 days postimplantation for both fibrotic between matched tumor and nontumor tissue provided more allograft models. reliable prognostic value than the overall levels of IL18. Taken together, our findings establish that, contrary to an expected DEN-induced carcinogenesis protumorigenic activity due to its elevated levels in circulation, À À Post-natal-day-15 WT or IL18R1 / mice received a single IL18 actually exerts a stage-dependent tumor-suppressive effect intraperitoneal injection of N-nitrosodiethylamine (DEN) at 25 on hepatocellular carcinoma progression, primarily through the mg/kg. Mice were sacrificed 6 or 12 months postadministration. antitumor activities of tumor-infiltrating lymphocytes. Alternatively, starting one week post-DEN treatment, mice also received weekly doses of 500 mL/kg CCl4 via intraperitoneal fi Materials and Methods injection. These mice received 22 doses of CCl4 and were sacri ced Patient samples 2 days after the last injection. Patient samples were obtained following informed consent according to established protocols approved by the Ethics Com- Sample harvest and preparation Livers and spleens were harvested and either postfixed in 10% mittees of the Eastern Hepatobiliary Surgery Hospital (Shanghai, China) and the 302 Military Hospital (Beijing, China). Details on phosphate-buffered formalin at 4 C on an orbital shaker for 1 day fi patient collection, characterization, and survival