Published OnlineFirst May 29, 2018; DOI: 10.1158/0008-5472.CAN-18-0342 Cancer Genome and Epigenome Research The Tumor Suppressor CIC Directly Regulates MAPK Pathway Genes via Histone Deacetylation Simon Weissmann1,2, Paul A. Cloos1,2, Simone Sidoli2,3, Ole N. Jensen2,3, Steven Pollard4, and Kristian Helin1,2,5 Abstract Oligodendrogliomas are brain tumors accounting for approximately 10% of all central nervous system can- Low MAPK signaling High MAPK signaling cers. CIC is a transcription factor that is mutated in most RTKs patients with oligodendrogliomas; these mutations are believed to be a key oncogenic event in such cancers. MEK Analysis of the Drosophila melanogaster ortholog of ERK SIN3 CIC, Capicua, indicates that CIC loss phenocopies CIC HDAC activation of the EGFR/RAS/MAPK pathway, and HMG C-term studies in mammalian cells have demonstrated a role SIN3 CIC HDAC for CIC in repressing the transcription of the PEA3 HMG C-term Ac Ac subfamily of ETS transcription factors. Here, we address Ac Ac the mechanism by which CIC represses transcription and assess the functional consequences of CIC inacti- vation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched ETV, DUSP, CCND1/2, SHC3,... for MAPK effector genes involved in cell-cycle regulation Normal Astrocytoma and proliferation. CIC binding to target genes was Glioblastoma multiforme abolished by high MAPK activity, which led to their 1p/19q deletion CIC-Mutants transcriptional activation. CIC interacted with the SIN3 SIN3 deacetylation complex and, based on our results, we SIN3 CIC suggest that CIC functions as a transcriptional repressor HDAC HDAC HMG C-term through the recruitment of histone deacetylases. Inde- CIC R215W SIN3 pendent single amino acid substitutions found in oli- HMG C-term CIC HDAC godendrogliomas prevented CIC from binding its target HMG C-term genes. Taken together, our results show that CIC is a R1515C transcriptional repressor of genes regulated by MAPK signaling, and that ablation of CIC function leads Ac Ac Ac Ac Ac Ac Ac Ac to increased histone acetylation levels and trans- cription at these genes, ultimately fueling mitogen- independent tumor growth. Significance: Inactivation of CIC inhibits its direct ETV, DUSP, CCND1/2, SHC3,... ETV, DUSP, CCND1/2, SHC3,... repression of MAPK pathway genes, leading to their increased expression and mitogen-independent growth. Oligodendroglioma Graphical Abstract: http://cancerres.aacrjournals. The tumor suppressor CIC represses its target genes via histone deacetylation. High MAPK signaling or loss of CIC function leads to hyperacetylation and gene activation. org/content/canres/78/15/4114/F1.large.jpg. Cancer Res; © 2018 American Association for Cancer Research 78(15); 4114–25. Ó2018 AACR. 1Biotech Research and Innovation Centre (BRIC), University of Note: Supplementary data for this article are available at Cancer Research Copenhagen, Copenhagen, Denmark. 2Centre for Epigenetics, University Online (http://cancerres.aacrjournals.org/). of Copenhagen, Copenhagen, Denmark. 3Department of Biochemistry S. Weissmann and P.A. Cloos contributed equally to this article. and Molecular Biology, VILLUM Centre for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark. 4MRC Centre for Corresponding Authors: Kristian Helin and Paul A. Cloos, University of Copen- Regenerative Medicine and Edinburgh Cancer Research UK Centre, The hagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark. Phone: 453-532-5666; University of Edinburgh, Edinburgh, United Kingdom. 5The Novo Nordisk Fax: 453-532-5669; E-mail: [email protected]; [email protected] Foundation Center for Stem Cell Biology (Danstem), University of doi: 10.1158/0008-5472.CAN-18-0342 Copenhagen, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Ó2018 American Association for Cancer Research. 4114 Cancer Res; 78(15) August 1, 2018 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst May 29, 2018; DOI: 10.1158/0008-5472.CAN-18-0342 Direct Regulation of MAPK Pathway Genes by CIC Introduction 1 mmol/L sodium pyruvate, penicillin/streptomycin, leukemia inhibitory factor, 1 mmol/L MEK inhibitor (PD0325901), and Oligodendrogliomas represent a subgroup of low-grade glioma 3 mmol/L GSK inhibitor (CT-99021). with a distinct mutational spectrum. The vast majority of oligo- Mouse neural progenitor cells derived from mESCs were dendroglioma cases contain chromosomal deletions of 1p and cultured on PDL/laminin in complete NSC medium: 50:50 19q and 70% harbor mutations in CIC, located on chromosome DMEM/F12:Neurobasal (Invitrogen) supplemented with 19q13.2 (1). CIC encodes a transcription factor containing a N2/B27 (Invitrogen), 0.05 mmol/L b-mercaptoethanol, SOX-like high mobility group (HMG) domain. At least two iso- 2 mmol/L GlutaMAX, 0.1 mmol/L nonessential amino acids, forms of CIC have been identified in humans: a short form CIC-S penicillin/streptomycin, 1 mmol/L sodium pyruvate, 5 mmol/L and a longer isoform CIC-L that shares the HMG domain but HEPES, 2 mg/mL heparin, 50 mg/mL BSA, 10 ng/mL bFGF/EGF. contains an extended N-terminal part (2). Mutations in CIC Human GNS line G144 (19) and human fetal neural stem cell frequently lead to truncations or amino acid substitutions in lines 18.5 and 21.5 (a kind gift of Dr. Steven Goldman, University residues that are thought to be essential for DNA binding of Rochester Medical Center) were cultured on PDL/Laminin in (Supplementary Fig. S1A and S1B). Together with 1p/19q chro- complete NSC medium. mosomal deletions, these recurrent CIC mutations are thought to Flp-In-T-REX-293 cells were cultured in DMEM (Invitrogen) represent an ablation of CIC function (3), being causally involved with 10% FBS (HyClone) and penicillin/streptomycin. Cells in oligodendroglioma formation. Of further interest, CIC muta- were authenticated by measuring the expression of specific tions are mutually exclusive with alterations of EGFR, TP53, ATRX, cellular markers and by observing their morphology and func- and CDKN2A (4, 5). tionality in specific assays. All the cell lines used were routinely Most of our knowledge about CIC stems from studies in (every 4 weeks) tested negative for mycoplasma by PCR. Cell Drosophila melanogaster, where it was genetically defined as a culture time between thawing and experiment did not exceed transcriptional repressor acting downstream of receptor tyrosine five passages. kinase (RTK) signaling regulating developmental patterning (6, 7). MAPK-mediated phosphorylation of Cic leads to its nuclear CRISPR KO export and/or degradation of Cic (8) and expression of Cic target sgRNAs were cloned into pSpCas9(BB)-2A-GFP (Addgene genes. In mammals, RTK signaling has similar effects on 48138), using the protocol described in ref. 20 and transfected CIC-mediated gene repression, possibly involving MAPK and into mESCs (Lipofectamine 2000, Invitrogen), or G144 cells p90RSK (9). How CIC is acting as a transcriptional repressor is (Amaxa Nucleofector II, Program A033). GFP-positive cells were currently not well understood. In Drosophila, Cic-mediated repres- single-cell sorted 48 hours posttransfection, expanded, and sion is dependent on the corepressor protein Groucho (6, 10). screened by immunoblotting for Cic deletion. Gene targeting was Mouse studies have suggested that CIC-mediated repression is in confirmed by PCR amplification and Sanger sequencing. Mouse part dependent on the binding to Ataxin1 (ATXN1) or a related Cic KO clones (KO1, KO2, and KO3) all harbored out-of-frame factor ATXN1L (11–13). mutations causing complete protein depletion. Human CIC KO Several mouse knockout studies have shown that germline clones (KO8 and KO9) harbored a mixture of out-of-frame and deletion of murine Cic leads to perinatal lethality (13–15) and large in-frame mutations, resulting in expression of an HMG deletion of Cic after birth has been linked to T-cell lymphoblastic domain-deleted truncated protein. Primers and sgRNAs are listed lymphoma formation (14, 16). In Ewing sarcomas, a genetic in Supplementary Table S1. fusion of CIC to the exons coding for the transcriptional acti- vator domain of DUX4 causes derepression of PEA3 (polyoma Plasmids and cDNA enhancer activator 3)-family genes (17). Furthermore, CIC dele- The open-reading frame of human CIC-L was amplified from a tion and mutation was shown to influence metastasis in an EGFR cDNA library derived from human fetal brain and cloned into inhibitor-resistant model of lung adenocarcinoma, through TOPO/pCR8, yielding a flawless 7554 bp clone encoding a 2513 the deregulation of the PEA3-family gene Etv4 (18). CIC may amino acid protein (splice variant ENST00000572681, ProteinID therefore act as an important tumor suppressor through the ENSP00000459719). transcriptional repression of its target genes, including the PEA3 The CIC-R215W and R1515C mutants were generated using subfamily, whose deregulation was linked to cancer develop- the Quickchange II Site Directed Mutagenesis Kit (Agilent ment (17, 18). However, the mechanistic consequences of Technologies) and expressed from pPB-CAG-Dest-pA-pgk-bsd inactivation or mutation of CIC and their contribution to (Addgene 74918) by cotransfection with transposase pCyL43 tumorigenesis of oligodendrogliomas are not well understood. (PBase; Sanger Institute's plasmid repository) into mESCs and Furthermore, the mechanism through which CIC achieves gene selected with