IGFBP7 Deletion Promotes Hepatocellular Carcinoma

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 IGF1 receptor (IGF1R), 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 proinflammatory 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 IGF1 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. Cancer Res; 77(15); 4014-25. ©2017 AACR.

Figure 1

Igfbp7−/− mice develop spontaneous tumors. A. Igfbp7 mRNA levels in livers and spleens of Igfbp7+/+ and Igfbp7−/− mice. B. IGFBP7 protein levels in the indicated organs. β-actin: loading control. C. Immunohistochemistry for IGFBP7 in sections of liver and spleen of adult mice. D. Igfbp7 mRNA levels in total liver and in hepatocytes (Hep), stellate cells (Stell) and peritoneal macrophages (Mac). E. H&E staining of sections of the indicated organs in 24 months old mice. Arrows indicate lymphocytic infiltration. F. Afp and Il6 mRNA levels in livers and lungs of 24 months old Igfbp7+/+ and Igfbp7−/− mice. For graphs, data represent mean ± SD. *: p<0.01; #: p<0.05. A.U.: arbitrary unit. For all analyses at least 3 mice or more were used per group.

Figure 2

Loss of Igfbp7 increases proliferation and inhibits senescence by activating IGF signaling pathway. A. Cell proliferation (left) and colony formation assays (right) in mouse embryonic fibroblasts (MEFs). B. Cell proliferation (left) and IGF-1-stimulated BrdU incorporation assays (right) in hepatocytes. C. Senescence-associated β-galactosidase (SA-β-Gal)-positive hepatocytes at day 8 of culture. D. γ-H2AX positive MEFs after 12 h of serum starvation. E. Cell cycle analysis of MEFs at 18 and 36 h after release from serum-free medium. F. Hepatocytes were cultured in insulin-free medium for 12 hours prior to treatment with IGF-1 (20ng/mL) for the indicated time points. Western blot analyses for the indicated proteins were performed. G. Western blot analysis for the indicated proteins in liver lysates of three independent Igfbp7+/+ and Igfbp7−/− mice. H. Western blot analysis for the indicated proteins in the indicated cells isolated from adult mice. For each cell type a representative EF1α blot is shown as loading control. I. Cell proliferation analysis in MEFs upon treatment with OSI-906 (4 µmol/L). For graphs, data represent mean ± SD. *: p<0.01.

Figure 3

Genetic deletion of Igfbp7 accelerates DEN-induced HCC. Mice were treated with DEN (10 µg/gm) at 2 wks of age and were sacrificed at 32 wks. A. Photograph of DEN-treated mouse livers of the indicated genotypes. B. Number and sizes of liver nodules. C. Liver weight of DEN-injected mice. D–E. Total protein (D) and Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) (E) levels in DEN-injected mouse sera. For B–E, n = 15 mice for +/+, 12 mice for +/− and 11 mice for −/−. F. H&E and immunostaining for the indicated proteins in liver sections. Scale bar: 20 µm. For graphs, data represent mean ± SD. *: p<0.01.

Figure 4

Loss of Igfbp7 generates an inflammatory and immunosuppressive environment. A. Immune profile of DEN-treated Igfbp7+/+ and Igfbp7−/− mice at 20 weeks. B. Levels of the indicated mRNAs in the livers of DEN-injected mice at 32 wks (top), in hepatocytes isolated from DEN-injected mice at 24 wks (middle), and in Kupffer cells isolated from DEN-injected mice at 5 wks (bottom). C. Levels of the indicated mRNAs in hepatocytes in vitro treated with DEN for 24 h (top). Levels of the indicated mRNAs in macrophages treated or not with IL-1β (10 ng/ml) for 6 or 12h. D. Immunofluorescence for p65 NF-κB in hepatocytes treated with LPS (500 ng/mL) for 30 min. E. NF-κB luciferase reporter activity was measured in Igfbp7+/+ and Igfbp7−/− hepatocytes. Firefly luciferase activity was normalized by Renilla luciferase activity. The activity of empty pGL3-basic vector was considered as 1. R.L.U.: Relative luciferase units. F. Western blot analysis for the indicated proteins in LPS-treated hepatocytes and bone marrow-derived macrophages. For graphs, data represent mean ± SD. *: p<0.01; **: p<0.05; #: p<0.04. For all analyses at least 3 mice or more were used per group. A.U.: arbitrary unit.

Figure 5

Inhibition of immunosurveillance in Igfbp7−/− mice. A. Top biological pathways downregulated in naive Igfbp7−/− livers. B. Tap1 mRNA levels in total liver. C. Levels of CD86 and CD80 in bone marrow-derived dendritic cells (BMDCs). D. Donor pmel-17 T cells were co-cultured with BMDCs pulsed with gp100 at the indicated molar ratios (DC:TC) for 60 h with 2 h pre-treatment with LPS (500 ng/mL) for priming. ³H-thymidine (³HTdR) incorporation was measured (left). IL-12p40 (middle) and IL-2 (right) levels were measured in the media by ELISA. E. gp100-loaded Igfbp7−/− BMDCs were treated or not with rIGFBP7 protein for 3 h followed by OSI-906 (4 µmol/L) for 2 h and LPS (500 ng/mL) for 2 h. The cells were washed and co-cultured with pmel-17 T cells for 60 h. ³H-thymidine (³HTdR) incorporation was measured (left). IFN-γ (middle) and IL-2 (right) levels were measured in the media by ELISA. F. BMDCs were treated with LPS (500 ng/mL) for the indicated time points and Western blot analysis was performed for the indicated proteins. For graphs, data represent mean ± SD. *: p<0.01.

Figure 6

Overexpression of Igfbp7 decreases tumor growth and stimulates an anti-tumor immune response. A. IGFBP7 protein levels in the conditioned media (top) and Igfbp7 mRNA levels in the cells (bottom) of control and IGFBP7-overexpressing clone (BP7-OE) of Hepa1-6 cells. B. The indicated clones (1X10⁶ cells) were injected s.c. in C57L mice (n=5 per group) and tumor volume was measured. C. Levels of the indicated proteins in tumor lysates at the end of the study. D. IFN-γ-producing CD8⁺ or CD4⁺ T cells and MDSCs in the tumors at the end of the study. E. Il12a (left), Ifng (middle) and Tap1 (right) mRNA levels in the tumors at the end of the study. F. BP7-OE cells were injected s.c. in C57L mice (n=5 per group) and treated with Control IgG and CD8⁺ or CD4⁺ neutralizing antibodies (200 µg i.p.). Tumor volume was measured. For all graphs, the data represent mean ± SD; *: p<0.01. G. Schematic representation of the molecular mechanism by which IGFBP7 suppresses tumors. IGFBP7, secreted from hepatocytes, inhibits IGF and NF-κB signaling in macrophages preventing inflammation. IGFBP7, secreted from the microenvironment cells, inhibits IGF and NF-κB signaling in hepatocytes thus impeding proliferation and inducing senescence. IGFBP7 promotes antigen presentation by dendritic cells resulting in recruitment of CD4+ and CD8+ cells. These effects collectively protect from HCC. In addition to the paracrine effects depicted in this diagram there might be autocrine effects regulating the same end-points.