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Published OnlineFirst September 13, 2019; DOI: 10.1158/0008-5472.CAN-19-1034 Cancer Genome and Epigenome Research MUC1-C Activates the NuRD Complex to Drive Dedifferentiation of Triple-Negative Breast Cancer Cells Tsuyoshi Hata1, Hasan Rajabi1, Hidekazu Takahashi1, Yota Yasumizu1, Wei Li1, Caining Jin1, Mark D. Long2, Qiang Hu2, Song Liu2, Atsushi Fushimi1, Nami Yamashita1, Ling Kui1, Deli Hong1, Masaaki Yamamoto1, Masaaki Miyo1, Masayuki Hiraki1, Takahiro Maeda1, Yozo Suzuki1, Mehmet K. Samur1, and Donald Kufe1 Abstract The NuRD chromatin remodeling and deacetylation com- formed complexes with these NuRD components on the ESR1 plex, which includes MTA1, MBD3, CHD4, and HDAC1 promoter. Downregulating MUC1-C decreased MTA1/MBD3/ among other components, is of importance for development CHD4/HDAC1 occupancy and increased H3K27 acetylation and cancer progression. The oncogenic mucin 1 (MUC1) C- on the ESR1 promoter, with induction of ESR1 expression and terminal subunit (MUC1-C) protein activates EZH2 and BMI1 downstream estrogen response pathways. Targeting MUC1-C in the epigenetic reprogramming of triple-negative breast and these NuRD components also induced expression of cancer (TNBC). However, there is no known link between FOXA1, GATA3, and other markers associated with the lumi- MUC1-C and chromatin remodeling complexes. Here, we nal phenotype. These findings support a model in which showed that MUC1-C binds directly to the MYC HLH-LZ MUC1-C activates the NuRD complex to drive dedifferentia- domain and identified a previously unrecognized MUC1- tion and reprogramming of TNBC cells. C!MYC pathway that regulates the NuRD complex. MUC1-C/MYC complexes selectively activated the MTA1 and Significance: MUC1-C directly interacts with MYC to acti- MBD3 genes and posttranscriptionally induced CHD4 expres- vate the NuRD complex, mediating regulation of the estrogen sion in basal- but not luminal-type BC cells. In turn, MUC1-C receptor in triple-negative breast cancer cells. Introduction also activates the IKK!NF-kB p65 pathway, binds to NF-kB p65, and promotes expression of the downstream BCL-XL and ZEB1 The mucin 1 (MUC1) C-terminal subunit (MUC1-C) is an genes (8, 9). MUC1-C activates gene expression in part by increas- oncogenic transmembrane protein that is overexpressed in triple- ing TF occupancy on target genes and by recruiting the p300 negative breast carcinomas (TNBC; refs. 1, 2). MUC1-C interacts acetyltransferase (5, 6). Other studies have shown that MUC1-C with receptor tyrosine kinases, such as EGFR, at the cell membrane induces DNA methyltransferase 1 (DNMT1) and DNMT3b, and and transduces prosurvival signals to the nucleus (2). In this thereby DNA methylation and repression of the CDH1 tumor- capacity, MUC1-C directly interacts with transcription factors (TF) suppressor gene (TSG; refs. 3, 4). MUC1-C also induces expression and contributes to the regulation of their target genes (2–4). For of the BMI1, RING1, and RING2 components of the polycomb instance, MUC1-C associates with b-catenin/TCF4 and activates repressive complex 1 (PRC1) and contributes to BMI1-mediated the WNT pathway CCND1 and MYC target genes (5–7). MUC1-C repression of the CDKN2 TSG (3, 4). These observations were extended by the demonstration that MUC1-C induces the EZH2, 1Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts. SUZ12, and EED subunits of PRC2 and binds directly to 2 Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive EZH2 (3, 4). In turn, MUC1-C increases H3K27 trimethylation Cancer Center, Buffalo, New York. on the BRCA1 TSG and downregulates BRCA1 expression (3, 4). In Note: Supplementary data for this article are available at Cancer Research concert with involvement in epigenetic repression of TSGs, MUC1- Online (http://cancerres.aacrjournals.org/). C has been linked to plasticity as characterized by induction of Current address for T. Hata, H. Takahashi, M. Yamamoto, M. Miyo, M. Hiraki, and the epithelial–mesenchymal transition (EMT), the cancer stem cell Y. Suzuki: Department of Gastroenterological Surgery, Graduate School of (CSC) state, and drug resistance (3, 4). These findings suggested Medicine, Osaka University, Suita, Osaka 565-0871, Japan; and current address that MUC1-C may play a role in other epigenetic pathways of gene for Y. Yasumizu and T. Maeda: Department of Urology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan. repression and activation associated with hallmarks of the cancer cell. Of importance for the present work, there is no available Corresponding Author: Donald Kufe, Dana-Farber Cancer Institute, Harvard evidence for involvement of MUC1-C in the regulation of ATP- Medical School, 450 Brookline Avenue, D830, Boston, MA 02215. Phone: 617- 632-3141; Fax: 617-632-2934; E-mail: [email protected] dependent chromatin-remodeling complexes (10). The nucleosome remodeling and histone deacetylase (NuRD) Cancer Res 2019;79:5711–22 complex regulates chromatin assembly and reorganization, doi: 10.1158/0008-5472.CAN-19-1034 and is of importance for metazoan development and cancer Ó2019 American Association for Cancer Research. progression (10). The highly conserved NuRD complex consists www.aacrjournals.org 5711 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst September 13, 2019; DOI: 10.1158/0008-5472.CAN-19-1034 Hata et al. of different subunits with enzymatic and nonenzymatic functions Hospital for Sick Children) of genomic DNA purified with the that broadly occupy active promoters and enhancers in different GenElute Mammalian Genomic DNA Miniprep Kit (Millipore cell types (11, 12). The core chromodomain-helicase-DNA- Sigma). Cells were monitored for Mycoplasma contamination binding protein 3 (CHD3) and CHD4 catalyze ATP-dependent using the MycoAlert Mycoplasma Detection Kit (Lonza). chromatin remodeling in association with activity of histone deacetylase 1 (HDAC1) and HDAC2 subunits. Nonenzymatic Tetracycline-inducible and stable gene silencing and NuRD components include, among others, the (i) methyl-CpG– expression binding domain 2 (MBD2) and MBD3, (ii) metastasis-associated MUC1shRNA (MISSION shRNA TRCN0000122938 and gene 1 (MTA1), MTA2, and MTA3, and (iii) retinoblastoma- shRNA#2 TRCN0000430218), MYCshRNA (MISSION shRNA binding protein 4 (RBBP4) and RBBP7 proteins. MBD3 links the TRCN0000039642 and shRNA#2 TRCN0000039640), MTA1shRNA histone deacetylase subcomplex containing MTA1, HDAC, and (MISSION shRNA TRCN0000230496), MBD3shRNA (MISSION RBBP proteins with the chromatin-remodeling subcomplex con- shRNA TRCN0000274441), CHD4shRNA (MISSION shRNA taining CHD4 and GATAD2A/B (11). Of these NuRD compo- TRCN0000021361), or a control scrambled shRNA (CshRNA, nents, MTA1 is widely overexpressed in human cancers by Millipore Sigma) was used in pLKO-puro vector or inserted into mechanisms associated, at least in part, with activation of the the pLKO-tet-puro vector (Plasmid #21915; Addgene). The TGFb1 and WNT/b-catenin pathways (13, 14). MTA1 acts as a MUC1-C cDNA was inserted into the pinducer20 vector (Plasmid corepressor by interacting with ERa and repressing ERa-activated #44012; Addgene). The viral vectors were produced in 293T cells gene transcription (15, 16). MTA1 associates with MBD3 and as described (9). Cells transduced with the vectors were selected recruits HDAC1 to deacetylate TFs, such as HIF1a, and regulates for growth in 1 to 3 mg/mL puromycin or 100 mg/mL geneticin. their transactivation functions (17). In addition, MTA1 interacts Cells were treated with 500 ng/mL doxycycline (DOX; Millipore with the CHD subunits in integrating their chromatin remodeling Sigma). functions (18). The pleotropic roles of MTA1 in regulating gene transcription extend to induction of EMT in cancer cells (13, 14) Quantitative reverse-transcription PCR and promotion of invasion and metastases (19). MTA1 has also Total cellular RNA was isolated using Trizol reagent (Invitro- been linked to chromatin remodeling during DNA repair by gen). cDNAs were synthesized using the High Capacity cDNA recruitment to sites of DNA lesions and interaction with Reverse Transcription Kit (Applied Biosystems). The cDNA sam- fi PARP1 (20). ples were ampli ed using the Power SYBR Green PCR Master Mix MUC1-C activates EZH2 and BMI1 in the epigenetic repro- (Applied Biosystems) and the CFX96 Real-Time PCR System gramming of TNBC cells; however, there is no known link (BIO-RAD) as described (9). Primers used for qRT-PCR are listed between MUC1-C and chromatin remodeling complexes, such in Supplementary Table S1. as NuRD. The present studies demonstrate that MUC1-C is Immunoblotting and immunoprecipitation necessary for induction of the MTA1, MBD3, and CHD4 subunits Total and nuclear lysates prepared from subconfluent cells were of the NuRD complex in TNBC cells. The results show that MUC1- subjected to immunoblot analysis using anti–MUC1-C (#HM- C interacts directly with MYC and thereby (i) activates the MTA1 1630-P1ABX; Thermo Fisher Scientific), anti-MYC (#ab32072; and MBD3 genes, and (ii) induces posttranscriptional expression Abcam), anti-ERa (#ab108398; Abcam), anti–b-actin (#A5441; of the CHD4 protein. Of functional significance, we demonstrate Sigma), anti-MTA1 (#5647), anti-MBD3 (#14540), anti-CHD4 that the MUC1-C!MYC!NuRD pathway drives luminal!basal (#11912), anti-HDAC1 (#5356), anti-SOX2 (#D6D9), anti-KLF4 dedifferentiation of TNBC cells. (#D1F2), anti-BMI1 (#D20B7), anti-CD44
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  • Human Urinary Exosomes As Innate Immune Effectors

    Human Urinary Exosomes As Innate Immune Effectors

    BASIC RESEARCH www.jasn.org Human Urinary Exosomes as Innate Immune Effectors † † ‡ Thomas F. Hiemstra,* Philip D. Charles, Tannia Gracia, Svenja S. Hester,§ † ‡ | Laurent Gatto, Rafia Al-Lamki,* R. Andres Floto,* Ya Su, Jeremy N. Skepper, † ‡ Kathryn S. Lilley, and Fiona E. Karet Frankl *Department of Medicine, †Cambridge Centre for Proteome Research and Cambridge Systems Biology Centre, Department of Biochemistry, ‡Department of Medical Genetics, and |Multi-Imaging Centre, Department of Anatomy, University of Cambridge, Cambridge, United Kingdom; and §Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom ABSTRACT Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm–Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans.