IGF2 Is Up-regulated by Epigenetic Mechanisms in Hepatocellular Carcinomas and Is an Actionable Oncogene Product in Experimental Models

• Published in: Gastroenterology (New York, N.Y. 1943) Vol. 151; no. 6; pp. 1192 - 1205
• Main Authors: Martinez-Quetglas, Iris, Pinyol, Roser, Dauch, Daniel, Torrecilla, Sara, Tovar, Victoria, Moeini, Agrin, Alsinet, Clara, Portela, Anna, Rodriguez-Carunchio, Leonardo, Solé, Manel, Lujambio, Amaia, Villanueva, Augusto, Thung, Swan, Esteller, Manel, Zender, Lars, Llovet, Josep M
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2016
• Subjects: Animals; Antibodies, Neutralizing - pharmacology; Antibodies, Neutralizing - therapeutic use; Antineoplastic Agents - therapeutic use; Apoptosis; Carcinoma, Hepatocellular - drug therapy; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - metabolism; Carcinoma, Hepatocellular - pathology; Case-Control Studies; Cell Line, Tumor; Cell Movement; Cell Proliferation - drug effects; DNA Methylation; Epi-Driver; Epigenesis, Genetic; Female; Gastroenterology and Hepatology; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hepatocarcinogenesis; Humans; IGF 1 Receptor; Insulin-Like Growth Factor II - genetics; Insulin-Like Growth Factor II - metabolism; Liver Neoplasms - drug therapy; Liver Neoplasms - genetics; Liver Neoplasms - metabolism; Liver Neoplasms - pathology; Male; Mice, Inbred C57BL; MicroRNAs - genetics; Neovascularization, Pathologic - drug therapy; Niacinamide - analogs & derivatives; Niacinamide - therapeutic use; Phenylurea Compounds - therapeutic use; Phosphorylation - drug effects; Proto-Oncogene Proteins c-akt - genetics; Proto-Oncogene Proteins c-akt - metabolism; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - metabolism; Receptors, Somatomedin - metabolism; RNA, Messenger - metabolism; RNA, Small Interfering; Signal Transduction - genetics; Sorafenib; Therapeutic Target; Tumor Stem Cell Assay; Up-Regulation; fold change; insulin-growth factor 1 receptor; Epi-Driver; INSR; HCC; IGF; genetically engineered mouse model; IGF1R; mAb; VEGFA; Therapeutic Target; Hepatocarcinogenesis; sh; IGF Receptor; short hairpin; miRNA; microRNA; insulin-like growth factor; insulin receptor; monoclonal antibody; FC; vascular endothelial growth factor A; hepatocellular carcinoma; GEMM; IGF 1 Receptor
• ISSN: 0016-5085; 1528-0012
• DOI: 10.1053/j.gastro.2016.09.001

Background & Aims Effective treatments are urgently needed for hepatocellular carcinoma (HCC), which is usually diagnosed at advanced stages. Signaling via the insulin-like growth factor (IGF) pathway is aberrantly activated in HCC by IGF2 overexpression. We aimed to elucidate the mechanism of IGF2 overexpression and its oncogenic activities and evaluate the anti-tumor effects of reducing IGF2 signaling. Methods We obtained 228 HCC samples from patients who underwent liver resection, 168 paired non-tumor adjacent cirrhotic liver samples, and 10 non-tumor liver tissues from patients undergoing resection for hepatic hemangioma. We analyzed gene expression, microRNA, and DNA methylation profiles for all samples, focusing on genes in the IGF signaling pathway. IGF2 was expressed in SNU449 and PLC5 HCC cells and knocked down with small hairpin RNAs in Hep3B and Huh7 cell lines. We analyzed these cells for proliferation, apoptosis, migration, and colony formation. We performed studies of mice engineered to express Myc and Akt1 in liver, which develop liver tumors, with or without hepatic expression of Igf2 . Mice with xenograft tumors grown from HCC cells were given a monoclonal antibody against IGF1 and IGF2 (BI 836845), along with sorafenib; tumor growth was measured and tissues were analyzed by immunohistochemistry and immunoblots. Results Levels of IGF2 messenger RNA and protein were increased >20-fold in 15% of human HCC tissues compared with non-tumor liver tissues. Methylation at the fetal promoters of IGF2 was reduced in the HCC samples and cell lines that overexpressed IGF2, compared with those that did not overexpress this gene, and non-tumor tissues. Tumors that overexpressed IGF2 had gene expression patterns significantly associated with hepatic progenitor cell features, stellate cell activation, NOTCH signaling, and an aggressive phenotype ( P < .0001). In mice engineered to express Myc and Akt1 in liver, co-expression of Igf2 accelerated formation of liver tumors, compared to mice with livers expressing only Myc and Akt1 , and shortened survival times ( P  = .02). The antibody BI 836845 blocked phosphorylation of IGF1 receptor in HCC cell lines and reduced their proliferation and colony formation. In mice with xenograft tumors, injection of BI 836845, with or without sorafenib, slowed tumor growth and increased survival times compared to vehicle or sorafenib alone. BI 836845 inhibited phosphorylation of IGF1 receptor and AKT and reduced decreased tumor vascularization compared with vehicle. Conclusions A large proportion of HCC samples were found to overexpress IGF2, via demethylation of its fetal promoter. Overexpression of IGF2 accelerates formation of liver tumors in mice with hepatic expression of MYC and AKT1, via activation of IGF1 receptor signaling. An antibody against IGF1 and IGF2 slows growth of xenograft tumors and increases survival of these mice.