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Acetylation of CCAR2 Establishes a BET/BRD9 Acetyl Switch In Published OnlineFirst January 14, 2019; DOI: 10.1158/0008-5472.CAN-18-2003 Cancer Genome and Epigenome Research Acetylation of CCAR2 Establishes a BET/BRD9 Acetyl Switch in Response to Combined Deacetylase and Bromodomain Inhibition Praveen Rajendran1, Gavin Johnson1,LiLi1, Ying-Shiuan Chen1, Mohaiza Dashwood1, Nhung Nguyen1, Ahmet Ulusan1, Furkan Ertem1, Mutian Zhang1, Jia Li1, Deqiang Sun1, Yun Huang1, Shan Wang1, Hon-Chiu Leung2, David Lieberman3, Laura Beaver4, Emily Ho4, Mark Bedford5, Kyle Chang5, Eduardo Vilar5, and Roderick Dashwood1,5 Abstract There continues to be interest in targeting epigenetic "read- members of the bromodomain and extraterminal domain ers, writers, and erasers" for the treatment of cancer and other (BET) family. Treatment with the BET inhibitor JQ1 synergized pathologies. However, a mechanistic understanding is fre- with sulforaphane in colon cancer cells and suppressed tumor quently lacking for the synergy observed when combining development effectively in a preclinical model of colorectal deacetylase and bromodomain inhibitors. Here we identify cancer. Studies with sulforaphaneþJQ1 in combination impli- cell cycle and apoptosis regulator 2 (CCAR2) as an early target cated a BET/BRD9 acetyl switch and a shift in the pool of acetyl for acetylation in colon cancer cells treated with sulforaphane. "reader" proteins in favor of BRD9-regulated target genes. N-terminal acetylation of CCAR2 diminished its interactions with histone deacetylase 3 and b-catenin, interfering with Wnt Significance: These results highlight the competition that coactivator functions of CCAR2, including in cells harboring exists among the "readers" of acetylated histone and nonhis- genetically encoded CCAR2 acetylation. Protein domain tone proteins and provide a mechanistic basis for potential arrays and pull-down assays identified acetyl "reader" proteins new therapeutic avenues involving epigenetic combination that recognized CCAR2 acetylation sites, including BRD9 and treatments. Introduction 3 (HDAC3), while also interacting with b-catenin to stabilize b-catenin/Tcf complexes in the nucleus (6, 7). In doing so, CCAR2 Cell cycle and apoptosis regulator 2 (CCAR2), also known as serves as a coactivator of Wnt signaling, a well-studied pathway in DBC1/KIAA1967, has gained attention as a "master regulator" of disease and development (8). metabolism, aging, and cancer (1–4). This designation derives Our attention was drawn to CCAR2 based on two converging from the interactions of CCAR2 with protein partners that observations. First, when CCAR2 is overexpressed in colon exert critical roles in physiology and pathophysiology, including tumors, the corresponding patients exhibit significantly reduced Sirtuin 1 (SIRT1) and CHK2, linking CCAR2 to p53 function and survival (7). Second, as reported here, CCAR2 was identified as an DNA repair (1–7). Less is known about the N-terminal region of early target for acetylation by sulforaphane, an agent that causes CCAR2 that associates with, and inhibits, histone deacetylase inhibition and turnover of HDAC3 in colon cancer cells (9–12). Notably, when sulforaphane was combined with JQ1, an inhib- 1 itor of the bromodomain and extraterminal domain (BET) fam- Center for Epigenetics & Disease Prevention, Texas A&M College of Medicine, – Houston, Texas. 2Mass Spectrometry-Proteomics Core, Baylor College of Med- ily (13 15), CCAR2 no longer served as an effective coactivator of icine, Houston, Texas. 3Division of Gastroenterology and Hepatology, Oregon Wnt/b-catenin signaling in vitro and in vivo. Health & Science University, Portland, Oregon. 4College of Public Health and There is growing interest in targeting epigenetic "readers, Human Sciences, Oregon State University, Corvallis, Oregon. 5The University of writers, and erasers" deregulated in cancer and other patholo- Texas MD Anderson Cancer Center, Houston, Texas. gies (13–16). This investigation combined sulforaphaneþJQ1 Note: Supplementary data for this article are available at Cancer Research to affect CCAR2 acetylation, and in so doing provided new Online (http://cancerres.aacrjournals.org/). mechanistic insights into the competition that exists among the P. Rajendran and G. Johnson contributed equally and are the co-first authors of "readers" of acetylated histone and nonhistone proteins that are this article. regulated during epigenetic combination therapies. Corresponding Authors: Roderick Dashwood, Texas A&M Health Sci Center and MD Anderson Cancer Center, 2121 West Holcombe Blvd., Houston, TX 77030. Phone: 713-677-7806; Fax: 713-677-7784; E-mail: [email protected]; Materials and Methods and Praveen Rajendran, Director, Antibody Biopharmaceutics Core, Center for Cells and treatments Epigenetics & Disease Prevention, Texas A&M Health Sci Center, Houston, TX HCT116, SW480 (human colon cancer cells), and 77030. Phone: 713-677-7803; E-mail: [email protected] CCD841 (nontransformed colonic epithelial cells) were from doi: 10.1158/0008-5472.CAN-18-2003 ATCC, and used within 10–15 passages from receipt. Each cell Ó2019 American Association for Cancer Research. line was confirmed independently to be of human origin, with no 918 Cancer Res; 79(5) March 1, 2019 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst January 14, 2019; DOI: 10.1158/0008-5472.CAN-18-2003 BET/BRD9 Acetyl Switch on CCAR2 mammalian interspecies contamination, and with the correct D1, matrix metalloproteinase 7 (MMP7), PARP, caspase-3, Pin1, genetic profile based on allele-specific markers (IDEXX Bioana- Lamin, and b-actin primary antibodies were from sources lytics Radil; refs. 17, 18). Cells were cultured in McCoy's 5A media reported (9–11, 17, 24). IHC followed the general procedures (Invitrogen) or EMEM (Invitrogen), supplemented with 10% FBS described elsewhere (17, 24). and 1% penicillin/streptomycin, at 37C in a humidified chamber with 5% CO2. All cells were tested routinely for Mycoplasma by Proximity ligation assays DAPI staining, and by using a PCR-based methodology (19). Protein–protein interactions were examined in situ, in cell- JQ1 was purchased from MedChem Express, whereas the other based assays and tissue sections, using the Duolink PLA Fluores- test agents were from the sources noted elsewhere (11). Nominal cence Protocol (Sigma-Aldrich), according to the manufacturer's concentrations were as follows: 15 mmol/L sulforaphane, recommendations. 6-methylsulfinylhexyl isothiocyanate (6-SFN), 9-methylsulfinyl- nonyl isothiocyanate (9-SFN), and allyl isothiocyanate (AITC); Pulldown assays 1 mmol/L trichostatin A (TSA); 10 mmol/L sodium butyrate Immunoprecipitation (IP) methodologies were as reported for (NaB); and 1 mmol/L valproic acid (VPA). For combination index endogenous proteins (10, 11), or Myc-, GST-, and HA-tagged (CI) experiments, sulforaphane, JQ1, and suberoylanilide hydro- proteins (5, 25–27). xamic acid (SAHA) were tested in the range 2–17 mmol/L, 1–60 mmol/L, and 0.1–2 mmol/L, respectively, with DMSO as Mass spectrometry vehicle. In most experiments, cells were treated with test agents 24 Acetylation sites on CCAR2 were identified following the hours after seeding (10, 11), except in HDAC3 siRNA knockdown general approach reported previously (5). In brief, 24 hours after assays, which were conducted according to a published method- seeding, HCT116 cells were treated with sulforaphane or DMSO, ology (11, 12). HDAC3 siRNA (Trilencer-27) and control siRNA and 6 hours later the cell lysates were subjected to IP using CCAR2 were procured from Origene, and cells were transfected with antibody. Following SDS-PAGE separation, the CCAR2 band was RNAiMAX Reagent (Invitrogen) for 24–48 hours, using the man- excised from the gel and digested overnight with trypsin prior to ufacturer's protocol. Two of the target siRNAs, designated as extraction and analysis on an Eksigent cHiPLC with nanoLC siRNA(1) and siRNA(3), produced the most efficient knockdown linked via a nanoflex to an ABSCIEX TripleTOF 5600 of HDAC3, and the data are shown in the corresponding figures. Mass Spectrometer (Mass Spectrometry-Proteomics Core, Baylor Unless indicated otherwise, whole-cell lysates or nuclear and College of Medicine, Houston, TX). Peaks Studio version 7.0 cytoplasmic fractions (10) were harvested 6 hours after treatment (Bioinformatics Solutions Inc.) was used to match spectra to with test agents, followed by RNA or protein expression analyses. peptides using the NCBI nonredundant database, including con- Additional experiments involved CCAR2 deletion from colon sideration of lysine acetylation. Modified peptides were verified cancer cells via CRISPR/Cas9 genome editing (20, 21). The PX459 by manual inspection of MS/MS data. Vector Control (Addgene) included a nontargeting gRNA sequence integrated into the vector. For reintroduction of CCAR2 RNA analyses into CCAR2-null cells, transient transfection was conducted using RNA-sequencing (RNA-seq) and bioinformatics analyses were expression constructs for wild-type (WT) protein or acetylation as reported (28) for adenomatous colon polyps from patients mutants. In the latter case, a Q5 Site-Directed Mutagenesis Kit with familial adenomatous polyposis (FAP; GSE88945 and (New England Biolabs) was used to convert Lys to Arg, starting GSE106500) and the polyposis in rat colon (Pirc) preclinical with CCAR2 plasmid pcDNA Myc DBC1 (Addgene plasmid no. model (29). Library preparation via a NEBNext Ultra Directional 35096; ref. 22), with confirmation
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