Published OnlineFirst August 6, 2018; DOI: 10.1158/1541-7786.MCR-18-0313 Genomics Molecular Cancer Research Integrated Genomic Comparison of Mouse Models Reveals Their Clinical Resemblance to Human Liver Cancer Sun Young Yim1,2, Jae-Jun Shim1,3, Ji-Hyun Shin1, Yun Seong Jeong1, Sang-Hee Kang1,4, Sang-Bae Kim1, Young Gyu Eun1,5, Dong Jin Lee1,6, Elizabeth A. Conner7, Valentina M. Factor7, David D. Moore8, Randy L. Johnson9, Snorri S. Thorgeirsson7, and Ju-Seog Lee1 Abstract Hepatocellular carcinoma (HCC) is a heterogeneous dis- able prognoses. Mst1/2 KO and Sav1 KO models best resem- ease. Mouse models are commonly used as preclinical mod- ble human HCC with hepatic stem cell characteristics. Apply- els to study hepatocarcinogenesis, but how well these models ing a genomic predictor for immunotherapy, the six-gene recapitulate molecular subtypes of human HCC is unclear. IFNg score, the Mst1/2 KO, Sav1 KO, SV40, and DEN models Here, integration of genomic signatures from molecularly were predicted to be the least responsive to immunotherapy. and clinically defined human HCC (n ¼ 11) and mouse Further analysis showed that elevated expression of immune- models of HCC (n ¼ 9) identified the mouse models that best inhibitory genes (Cd276 and Nectin2/Pvrl2)inMst1/2 KO, resembled subtypes of human HCC and determined the Sav1 KO, and SV40 models and decreased expression of clinical relevance of each model. Mst1/2 knockout (KO), Sav1 immune stimulatory gene (Cd86) in the DEN model might KO, and SV40 T antigen mouse models effectively recapitu- be accountable for the lack of predictive response to lated subtypes of human HCC with a poor prognosis, where- immunotherapy. as the Myc transgenic model best resembled human HCCs with a more favorable prognosis. The Myc model was also Implication: The current genomic approach identified the associated with activation of b-catenin. E2f1, E2f1/Myc, E2f1/ most relevant mouse models to human liver cancer and Tgfa, and diethylnitrosamine (DEN)-induced models were suggests immunotherapeutic potential for the treatment of heterogeneous and were unequally split into poor and favor- specific subtypes. Mol Cancer Res; 16(11); 1713–23. Ó2018 AACR. Introduction (5-year survival rates of 0% to 10%; ref. 1). Currently, sorafenib and regorafenib are the only approved molecular-targeted ther- Hepatocellular carcinoma (HCC) represents 75% of cases of apies for HCC (4, 5). Therefore, improved treatments are still primary liver cancer (1), which is the seventh most common needed, and much remains to be discovered in clinical and cancer globally (2). Despite the implementation of surveillance experimental studies. programs for at-risk populations, 30% to 60% of HCCs are Hepatocarcinogenesis is a multistep process involving the detected at an advanced stage (3), resulting in a dismal prognosis accumulation of genetic changes that result in altered expression of cancer-related genes, such as oncogenes and tumor-suppressor genes, and their related molecular signaling pathways (6). Genet- 1Department of Systems Biology, The University of Texas MD Anderson Cancer ically engineered mouse models recapitulate the complex multi- 2 Center, Houston, Texas. Department of Internal Medicine, Korea University step process of hepatocarcinogenesis so that researchers can College of Medicine, Seoul, Korea. 3Department of Internal Medicine, School of understand it and design therapeutic experiments. Although Medicine, Kyung Hee University, Seoul, Korea. 4Department of Surgery, Korea University College of Medicine, Seoul, Korea. 5Department of Otolaryngology- mouse models are extensively used in cancer research, no one Head and Neck Surgery, School of Medicine, Kyung Hee University, Seoul, Korea. mouse model fits all purposes, and each model can only recapit- 6Department of Otolaryngology-Head and Neck Surgery, Hallym University ulate part of the process of hepatocarcinogenesis in humans. Medical Center, Seoul, Korea. 7Laboratory of Experimental Carcinogenesis, Furthermore, genomic studies have identified molecularly dis- National Cancer Institute, National Institutes of Health, Bethesda, Maryland. tinct subtypes of HCC with different clinical outcomes (7–12). 8Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Therefore, establishing the molecular and clinical resemblance of Texas. 9Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas. mouse cancer models to these subtypes of human HCC (or vice versa) will enable the appropriate selection of mouse models for Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). use in investigations of the functional roles of newly discovered cancer genes and validation of potential therapeutic targets. S.Y. Yim, J.-J. Shim, and J.-H. Shin contributed equally to this article. However, there have been no systematic comparisons of human Corresponding Author: Ju-Seog Lee, The University of Texas MD Anderson HCCs and mouse models at the molecular, genomic, and clinical Cancer Center, 6565 MD Anderson Blvd, Houston, TX 77054. Phone: 713-834- levels. 6154; Fax: 713-563-4235; E-mail: [email protected] Currently, genomic studies of human HCC are progressing, and doi: 10.1158/1541-7786.MCR-18-0313 systematic analyses of gene expression patterns are providing Ó2018 American Association for Cancer Research. insight into the biology and pathogenesis of HCC. Many gene www.aacrjournals.org 1713 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 6, 2018; DOI: 10.1158/1541-7786.MCR-18-0313 Yim et al. signatures associated with HCC progression and recurrence have and wild-type normal livers was used for labeling and hybridiza- been and are being explored (8, 11, 13). In previous studies, we tion according to the manufacturer's protocol. Collected gene and others demonstrated that gene expression signatures reflect- expression data were transformed and normalized as described ing the clinical and molecular characteristics of tumors are highly previously (7–9). Because gene expression data from tumors from conserved in human and mouse tumors (7, 9). The purpose of the the DEN, Myc, E2f1, Myc/E2f1, Myc/Tgfa, and SV40 models were present study, therefore, was to determine how well mouse defined as expression ratio to normal liver, gene expression from models recapitulated human HCCs. We determined whether tumors from the Mst1/2 KO, Sav1 KO, and Ctnnb1 models was 11 human gene signatures associated with prognosis, activation renormalized as expression ratio to normal liver by dividing by of oncogenes, and activation of the immune system in HCCs were the expression value from normal liver. found in gene expression data from nine mouse HCC models. fi Materials and Methods Clinically de ned liver cancer subtypes and associated gene expression signatures Mouse HCC models and tumors The National Cancer Institute proliferation (NCIP; refs. 7, 8, Nine genetically engineered or chemically induced mouse HCC 23), hepatic stem cells (HS; ref. 9), Seoul National University models were used in the current study. A total of 108 HCC tumors recurrence (SNUR; ref.12), Cholangio-like HCC (CLHCC; ref. 10), were collected for the study. Tumors from transgenic mice expres- silence of Hippo (SOH; ref. 11), recurrence-risk score (RS; ref. 13), sing Myc (n ¼ 15), Myc/Tgfa (n ¼ 15), E2F transcription factor b-catenin (BC; ref. 20), EPCAM (24), and Hoshida (25). HCC 1(E2f1; n ¼ 18), or Myc/E2f1 (n ¼ 15) were collected as described subtypes and associated gene signatures were described in earlier in previous studies (7, 14–16). The diethylnitrosamine (DEN) studies. IDH-like signatures were identified in a recent analysis of model (n ¼ 16) was described in an earlier study (17). The SV40 liver cancer genomic data from The Cancer Genome Atlas (26). T antigen transgenic model (n ¼ 10) was also described previously The six-gene IFNg (IFNG6) composite score was calculated on the (18). Generation of liver-specific Mst1/2 knockout (KO) and basis of genes reported in a recent study (27). The numbers of Salvador family WW domain containing protein 1 (Sav1)KO genes in each signature are listed in Table 1. mice was described in a previous study (19). HCC tumors were collected from mice ages 6 to 7 months in the Mst1/2 double-KO Analysis of data and stratification of mouse tumors model (n ¼ 4) and ages 13 to 14 months in the Sav1 KO model The BRB Array Tools software program (http://linus.nci.nih. (n ¼ 11). The mouse model with activated catenin beta 1 (Ctnnb1) gov/BRB-ArrayTools.html) was used for analysis of the gene was generated as described previously (20). Briefly, mice carrying expression data and construction of a prediction model (28). A loxP sites flanking Ctnnb1 exon 3 (Ctnnb1loxP(ex3)/loxP(ex3)), which heatmap was generated using the Cluster and TreeView software contains the negative regulatory phosphorylation sites for degra- programs (29), and further statistical analysis was performed dation of b-catenin (21), were treated with Ad-Cre (Cre-expressing using the R language (http://www.r-project.org). Data on genes adenovirus) to make constitutively activated b-catenin in the liver. with more than 30% of their expression data missing across tissue Mice were further treated with a single dose of TCPOBOP (3 mg/ samples were excluded and then transformed and normalized (8). kg body weight), which is a constitutive androstane receptor (Car) When a gene was represented more than once on the microarray agonist, and liver tumor promoters and tumors were collected at 8 platform, the genes with the greatest variance in expression were months of age. All animals received humane care according to the selected. To identify mouse genes whose expression changed criteria outlined in the "Guide for the Care and Use of Laboratory nontrivially, we selected expression ratios with at least a 2-fold Animals" prepared by the National Academy of Sciences and difference relative to a reference in at least nine tissues (880 published by the National Institutes of Health (NIH publication genes).