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



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 in 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 (xentuzumab), along with sorafenib; tumor growth was measured and tissues were analyzed by immunohistochemistry and immunoblots.



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 xentuzumab blocked phosphorylation of IGF1 receptor in HCC cell lines and reduced their proliferation and colony formation. In mice with xenograft tumors, injection of xentuzumab, with or without sorafenib, slowed tumor growth and increased survival times compared to vehicle or sorafenib alone. Xentuzumab inhibited phosphorylation of IGF1 receptor and AKT and reduced decreased tumor vascularization compared with vehicle.



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.
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain.
Iris
I
Martinez-Quetglas
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain.
Roser
R
Pinyol
Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tübingen, Tübingen, Germany.
Daniel
D
Dauch
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain.
Sara
S
Torrecilla
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain.
Victoria
V
Tovar
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain.
Agrin
A
Moeini
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain.
Clara
C
Alsinet
Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, Barcelona, Catalonia, Spain.
Anna
A
Portela
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain.
Leonardo
L
Rodriguez-Carunchio
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain.
Manel
M
Solé
Liver Cancer Program, Division of Liver Diseases and Pathology Department, Tisch Cancer Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Oncological Sciences Department, Icahn School of Medicine at Mount Sinai, New York, New York.
Amaia
A
Lujambio
Liver Cancer Program, Division of Liver Diseases and Pathology Department, Tisch Cancer Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Division of Hematology and Medical Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York.
Augusto
A
Villanueva
Liver Cancer Program, Division of Liver Diseases and Pathology Department, Tisch Cancer Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York.
Swan
S
Thung
Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, Barcelona, Catalonia, Spain; Institució Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain.
Manel
M
Esteller
Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tübingen, Tübingen, Germany; Translational Gastrointestinal Oncology Group within the German Center for Translational Cancer Research, German Cancer Research Center, Heidelberg, Germany.
Lars
L
Zender
Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer Group, Liver Unit, IDIBAPS-Hospital Clínic de Barcelona, CIBERehd Universitat de Barcelona, Catalonia, Spain; Liver Cancer Program, Division of Liver Diseases and Pathology Department, Tisch Cancer Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York; Institució Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain. Electronic address: jmllovet@clinic.cat.
Josep M
JM
Llovet
D000818:Animals; D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing / Q000494:pharmacology* / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000627:therapeutic use; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism*; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D009536:Niacinamide / Q000031:analogs & derivatives / Q000627:therapeutic use; D010671:Phenylurea Compounds / Q000627:therapeutic use; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering; D017526:Receptor, IGF Type 1; D017451:Receptors, Somatomedin / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D000077157:Sorafenib; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation
D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing; D000970:Antineoplastic Agents; C000597236:IGF1R protein, human; C035170:IGF2 protein, human; D035683:MicroRNAs; C489428:Myc protein, mouse; D010671:Phenylurea Compounds; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017451:Receptors, Somatomedin; D009536:Niacinamide; D007335:Insulin-Like Growth Factor II; D000077157:Sorafenib; D017526:Receptor, IGF Type 1; C514862:Akt1 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; C588089:xentuzumab
Epi-Driver; Hepatocarcinogenesis; IGF 1 Receptor; Therapeutic Target