title: Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice.
abstract: It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing.
  
  
  
  We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be up-regulated after tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines.
  
  
  
  We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation.
  
  
  
  Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Chun-Qing
authors.initials: CQ
authors.lastname: Song
authors.affiliation: Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts; Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, China.
authors.forename: Yingxiang
authors.initials: Y
authors.lastname: Li
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Haiwei
authors.initials: H
authors.lastname: Mou
authors.affiliation: Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Jill
authors.initials: J
authors.lastname: Moore
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Angela
authors.initials: A
authors.lastname: Park
authors.affiliation: Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.
authors.forename: Yotsawat
authors.initials: Y
authors.lastname: Pomyen
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Soren
authors.initials: S
authors.lastname: Hough
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Kennedy
authors.affiliation: Department of Pathology, UMass Memorial Medical Center, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Andrew
authors.initials: A
authors.lastname: Fischer
authors.affiliation: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Hao
authors.initials: H
authors.lastname: Yin
authors.affiliation: David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts; Division of Health Sciences and Technology, Harvard-Massachusetts Institute of Technology, Cambridge, Massachusetts; Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Anderson
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Darryl
authors.initials: D
authors.lastname: Conte
authors.affiliation: Department of Internal Medicine VIII, University Department of Medicine, University Hospital Tübingen, Tübingen, Germany; Department of Physiology I, Institute of Physiology, Eberhard Karls University, Tübingen, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Zender
authors.affiliation: Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.
authors.forename: Xin Wei
authors.initials: XW
authors.lastname: Wang
authors.affiliation: Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.
authors.forename: Snorri
authors.initials: S
authors.lastname: Thorgeirsson
authors.affiliation: Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts; Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, China. Electronic address: Zhiping.Weng@umassmed.edu.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Weng
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts; Program in Molecular Medicine and Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts. Electronic address: Wen.Xue@umassmed.edu.
authors.forename: Wen
authors.initials: W
authors.lastname: Xue
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D064113:CRISPR-Cas Systems; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins; D004273:DNA, Neoplasm / Q000235:genetics; D004791:Enzyme Inhibitors; D015972:Gene Expression Regulation, Neoplastic; D016514:Genes, Neurofibromatosis 1; D055106:Genome-Wide Association Study; D024003:HMGA Proteins / Q000235:genetics; D025743:HMGA2 Protein / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000235:genetics*; D008114:Liver Neoplasms, Experimental / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D008819:Mice, Nude; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology; D011379:Prognosis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D011728:Pyridones / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000235:genetics; D017422:Sequence Analysis, DNA; D000077157:Sorafenib; D016019:Survival Analysis; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics; D018631:ras Proteins / Q000235:genetics
chemical_list: C517975:AZD 6244; C000620227:B9d1 protein, mouse; D001562:Benzimidazoles; D002070:Butadienes; D003598:Cytoskeletal Proteins; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; C514259:FLRT2 protein, mouse; D024003:HMGA Proteins; D025743:HMGA2 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D009570:Nitriles; D010671:Phenylurea Compounds; C492118:Plxnb1 protein, mouse; D047428:Protein Kinase Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D011728:Pyridones; D011744:Pyrimidinones; D011956:Receptors, Cell Surface; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C113580:U 0126; D009536:Niacinamide; C560077:trametinib; D000077157:Sorafenib; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins
keywords: CRISPR Screen; Liver Cancer; Mouse Model; Tumor Suppressor Genes