Published OnlineFirst June 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2885 IGFBP7 Deletion Promotes Hepatocellular Carcinoma Maaged Akiel1, Chunqing Guo1, Xia Li1, Devaraja Rajasekaran1, Rachel G. Mendoza1, Chadia L. Robertson1, Nidhi Jariwala1, Fang Yuan1, Mark A. Subler1, Jolene Windle1, Dawn K. Garcia2, Zhao Lai2, Hung-I Harry Chen2, Yidong Chen2,3, Shah Giashuddin4, Paul B. Fisher1,5,6, Xiang-Yang Wang1,5,6, Devanand Sarkar1,5,6,7 Departments of 1Human and Molecular Genetics, 5VCU Massey Cancer Center; 6VCU Institute of Molecular Medicine (VIMM), Virginia Commonwealth University, Richmond, VA 23298, USA; 2Greehey Children’s Cancer Research Institute, 3Department of Epidemiology and Biostatistics, University of Texas Health Science Center San Antonio, San Antonio, TX 78229; 4Department of Pathology, New York Presbyterian Health System at Weill Cornell Medical College, New York, NY. Running title: Promotion of HCC in Igfbp7ko mice Key-words: IGF signaling, antigen presentation, tumor suppression, inflammation, mouse model Financial support The present study was supported in part by National Cancer Institute Grant R21 CA183954, National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK107451 and VCU Massey Cancer Center (MCC) Pilot Project Grant (D. Sarkar), and R01 CA175033 and R01 CA154708 (X-Y. Wang). C.L. Robertson is supported by a National Institute of Diabetes And Digestive And Kidney Diseases Grant T32DK007150. Services in support of this project were provided by the VCU Massey Cancer Center 1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2885 Transgenic/Knock-out Mouse Facility and Flow Cytometry Facility, supported in part with funding from NIH-NCI Cancer Center Support Grant P30 CA016059. 7Corresponding author: Devanand Sarkar 1220 East Broad St, Box 980035 Richmond, VA 23298 Tel: 804-827-2339 Fax: 804-628-1176 e-mail: [email protected] Disclosure: All authors have no potential conflicts. Abbreviations: IGFBP7: Insulin-like growth factor binding protein-7 HCC: Hepatocellular carcinoma MHC: Major histocompatibility complex PI3K: Phosphatidylinositol-3-Kinase ERK: Extracellular Signal-Regulated Kinase GSK3β: Glycogen synthase kinase 3β PCNA: Proliferating cell nuclear antigen AFP: α-feto protein MTT: 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide BrdU: 5-bromo-2’-deoxyuridine MDSC: Myeloid-derived suppressor cells BMDC: Bone marrow-derived dendritic cells 2 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2885 NK: Natural killer cells NSG: NOD scid Gamma CM: Conditioned media 3 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2885 Abstract Activation of IGF signaling is a major oncogenic event in diverse cancers, including hepatocellular carcinoma (HCC). In this setting, the insulin-like growth factor binding protein IGFBP7 inhibits IGF signaling by binding the IGF-1 receptor (IGF-1R), functioning as a candidate tumor suppressor. IGFBP7 abrogates tumors by inhibiting angiogenesis and inducing cancer-specific senescence and apoptosis. Here we report that Igfbp7-deficient mice exhibit constitutively active IGF signaling, presenting with pro- inflammatory and immunosuppressive microenvironments and spontaneous liver and lung tumors occurring with increased incidence in carcinogen-treated subjects. Igfbp7 deletion increased proliferation and decreased senescence of hepatocytes and mouse embryonic fibroblasts, effects that were blocked by treatment with IGF-1 receptor inhibitor. Significant inhibition of genes regulating immune surveillance was observed in Igfbp7-/- murine livers, which was associated with a marked inhibition in antigen cross- presentation by Igfbp7-/- dendritic cells. Conversely, IGFBP7 overexpression inhibited growth of HCC cells in syngeneic immunocompetent mice. Depletion of CD4+ or CD8+ T lymphocytes abolished this growth inhibition, identifying it as an immune-mediated response. Our findings define an immune component of the pleiotropic mechanisms through which IGFBP7 suppresses HCC. Furthermore, they offer a genetically based preclinical proof of concept for IGFBP7 as a therapeutic target for immune management of HCC. 4 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2885 Introduction Evasion from immune surveillance is an important mechanism facilitating tumor development and progression (1). Three equally important events are necessary for immune evasion, the masking of neo-antigens, downregulation of antigen presentation machinery, and irresponsiveness to Interferon-γ (IFN-γ)-mediated killing by immune cells (2). Tumor antigenic peptides are generally produced in the cytosol via processing by proteasomes that contain interferon-γ-inducible subunits LMP-2, LMP-7 and LMP-10 (3). The 8-9 amino acids antigen peptides thus produced are translocated by transporters associated with antigen processing (TAP-1 and TAP-2) to the endoplasmic reticulum (ER) where the peptides are assembled with MHC class I heavy chain and β2- microglobulin light chain and are transported to the cell surface to be presented to CD8+ cytotoxic T lymphocytes (CTL). The components of the antigen presentation pathway, such as TAP1/2 and LMP2/7, are downregulated in cancers, including HCC, resulting in a loss of immune-surveillance and initiation and progression of the disease (4). Insulin-like growth factor (IGF) signaling plays an important oncogenic role in hepatocellular carcinoma (HCC) (5). Insulin-like growth factor binding protein-7 (IGFBP7) is a secreted protein that binds to IGF-1 receptor (IGF-1R) and blocks activation by IGFs (6). IGFBP7 functions as a tumor suppressor in a variety of cancers, including HCC, where its expression is markedly downregulated (7-9). Genomic deletion and promoter hypermethylation cause downregulation of IGFBP7 in HCC (7,10). Recombinant IGFBP7 (rIGFBP7) protein induces senescence and/or apoptosis and inhibits angiogenesis in diverse cancers either in vitro or in nude mice xenograft models (8,9,11). Stable overexpression of IGFBP7 in aggressive human HCC cells led to inhibition in IGF signaling, induced senescence, inhibited proliferation and resulted in profound inhibition in xenograft growth in nude mice which was accompanied by marked 5 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2885 inhibition in angiogenesis (7). Intratumoral injection of an adenovirus expressing IGFBP7 (Ad.IGFBP7) eradicated both injected tumors as well as non-injected tumors established in the other flank of nude mice indicating that IGFBP7 not only has direct effect on primary cancer but also exerts a ‘by-stander’ anti-tumor effect (12). However, the mechanism by which IGFBP7 exerts this ‘by-stander’ effect remains to be determined. In this manuscript we describe the generation and characterization of an Igfbp7 knockout (Igfbp7-/-) mouse which establishes the tumor suppressor functions of IGFBP7 and unravels a novel role of IGFBP7 in regulating an anti-tumor immune response. These studies indicate that IGFBP7 inhibits cancer by pleiotropic mechanisms and might be an effective therapeutic for HCC and other cancers. Materials and methods Generation of Igfbp7-/- mouse: Igfbp-7-/- mouse was created in a pure C57BL/6 background using a Cre-loxP strategy (Fig. S1). All animal studies were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University, and were performed in accordance with the Animal Welfare Act, the PHS Policy on Humane Care and Use of Laboratory Animals, and the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. Primary cells isolation and culture conditions: All primary cells were used immediately after isolation and were mycoplasma free. Primary mouse hepatocytes were isolated and cultured in Williams E Medium containing NaHCO3, L-glutamine, insulin (1.5 mmol/L), and dexamethasone (0.1 mmol/L) as described (13). Kupffer cells were isolated from liver homogenates by centrifuging at 500 RPM for 10 min (14). The supernatant containing immune cells was sorted for CD11b+F4/80+ cells using FACSAria II (BD Biosciences). Mouse embryonic fibroblasts (MEFs) were isolated as described 6 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2885 from (E13.5 embryos) and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) (15). Bone marrow-derived macrophages and peritoneal macrophages were isolated according to standard protocols (16). Bone marrow cells were isolated from femurs of C57BL/6 mice and were differentiated into macrophages using RMPI-1640 medium supplemented with 10% heat-inactivated FBS and 20% L929 conditioned media for 7days. At day 7, the media was changed to complete RPMI-1640 containing 10% heat-inactivated FBS. Macrophages