Published OnlineFirst February 26, 2016; DOI: 10.1158/0008-5472.CAN-14-3816 Cancer Molecular and Cellular Pathobiology Research Mutational Landscape and Antiproliferative Functions of ELF Transcription Factors in Human Cancer Mizuo Ando1,2, Masahito Kawazu3, Toshihide Ueno1, Daizo Koinuma4, Koji Ando5, Junji Koya6, Keisuke Kataoka6, Takahiko Yasuda1, Hiroyuki Yamaguchi1, Kazutaka Fukumura1, Azusa Yamato1, Manabu Soda1, Eirin Sai3, Yoshihiro Yamashita1, Takahiro Asakage2, Yasushi Miyazaki5, Mineo Kurokawa6, Kohei Miyazono4, Stephen D. Nimer7, Tatsuya Yamasoba2, and Hiroyuki Mano1,8 Abstract ELF4 (also known as MEF) is a member of the ETS family of ELF1 and ELF2, as in ELF4, were widespread across human transcription factors. An oncogenic role for ELF4 has been cancers, but were almost all mutually exclusive. Moreover, demonstrated in hematopoietic malignancies, but its function chromatin immunoprecipitation coupled with high-throughput in epithelial tumors remains unclear. Here, we show that ELF4 sequencing revealed ELF4-binding sites in genomic regions can function as a tumor suppressor and is somatically inacti- adjacent to genes related to cell-cycle regulation and apoptosis. vated in a wide range of human tumors. We identified a Finally, we provide mechanistic evidence that the antiprolifera- missense mutation affecting the transactivation potential of tive effects of ELF4 were mediated through the induction of ELF4 in oral squamous cell carcinoma cells. Restoration of the HRK, an activator of apoptosis, and DLX3, an inhibitor of cell transactivation activity through introduction of wild-type ELF4 growth. Collectively, our findings reveal a novel subtype of significantly inhibited cell proliferation in vitro and tumor human cancer characterized by inactivating mutations in the xenograft growth. Furthermore, we found that ELF1 and ELF2, ELF subfamily of proteins, and warrant further investigation of closely related transcription factors to ELF4, also exerted anti- the specific settings where ELF restoration may be therapeuti- proliferative effects in multiple cancer cell lines. Mutations in cally beneficial. Cancer Res; 76(7); 1–11. Ó2016 AACR. Introduction prostate tumors (3). Forced expression of TMPRSS2-ERG in the context of PI3K pathway activation in prostate epithelial cells ELF4, also known as myeloid Elf-1–like factor (MEF), belongs results in the formation of prostatic intraepithelial neoplasia in to the ETS family of transcription factors that are encoded by 28 mice, demonstrating an oncogenic role for the fusion gene (4). In independent genes in the human genome (1, 2). ETS proteins are addition, fusion of EWSR1 with the ETS family gene FLI1 or implicated in the proliferation, differentiation, and malignant related ones occurs frequently in Ewing's sarcoma (5). Such transformation of various cell lineages. The genes for ERG or fusions are promising therapeutic targets, given that knockdown members of the PEA3 subfamily (ETV1, ETV4, and ETV5) of ETS of EWSR1–FLI1 expression suppresses sarcoma growth. proteins, for instance, are fused to TMPRSS2 in >50% of human ELF4 as well as ELF1 and ELF2 constitute the ELF subfamily of ETS proteins. Elf4-null mice manifest severe defects in the devel- 1Department of Cellular Signaling, Graduate School of Medicine, The opment and function of natural killer cells and NK-T cells (6). University of Tokyo, Tokyo, Japan. 2Department of Otolaryngology Furthermore, the hematopoietic system of such mice harbors an and Head and Neck Surgery, Graduate School of Medicine, The Uni- increased population of quiescent hematopoietic stem cells com- versity of Tokyo, Tokyo, Japan. 3Department of Medical Genomics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. pared with wild-type animals, suggesting that ELF4 plays a key 4 Department of Molecular Pathology, Graduate School of Medicine, role in the transition from G0 to G1 phases of the cell cycle (7). The 5 The University of Tokyo, Tokyo, Japan. Department of Hematology, activity of ELF4 is selectively increased in G1 phase and is sup- Nagasaki University Graduate School of Biomedical Sciences, Naga- pressed by the complex of cyclin A and cyclin-dependent kinase 2 saki, Japan. 6Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 7Sylvester (CDK2), further linking ELF4 to cell-cycle regulation (8). Comprehensive Cancer Center, University of Miami, Miami, Florida. An oncogenic role for ELF4 has been suggested by several 8 Strategic Basic Research Program, Japan Science and Technology observations. ELF4 is highly expressed in clinical specimens of Agency, Saitama, Japan. leukemia and ovarian cancer (9,10). Elf4 induces Mdm2, and also Note: Supplementary data for this article are available at Cancer Research inhibits Cdkn2a activation, and thereby cooperates with onco- Online (http://cancerres.aacrjournals.org/). genic HRAS(G12V) in malignant transformation (11). Further- Corresponding Author: Hiroyuki Mano, The University of Tokyo, 7-3-1 Hongo, more, cells from Elf4-null mice are impaired in the ability to form Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81-3-5841-0633; Fax: 81-3-5841- neurospheres, and knockdown of ELF4 was found to inhibit 0634; E-mail: [email protected] neurosphere formation by human primary glioma stem-like cells, doi: 10.1158/0008-5472.CAN-14-3816 suggesting that ELF4 promotes stemness in glioma cells (12). On Ó2016 American Association for Cancer Research. the other hand, however, one article shows a possibility for the www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 26, 2016; DOI: 10.1158/0008-5472.CAN-14-3816 Ando et al. growth-suppressive role of ELF4 in a human lung cancer cell line resulted in the identification of 12 missense mutations including A549 (13). ELF4(L211M) (20). Given that L211 maps within the conserved We now describe an L211M amino acid substitution in the ETS ETS domain of ELF4 (Fig. 1A), we chose this nonsynonymous domain of ELF4 that is present in the human squamous cell mutation for further analysis. A homozygous V382I mutation of carcinoma cell line T3M-1 Cl-10 (14). Forced expression of wild- ELF4 was also identified in the genome of the human breast type ELF4 resulted in marked inhibition of cell proliferation not cancer cell line MDA-MB-231 (data not shown). only in this cell line but also in a human fibrosarcoma cell line Further searching for nonsynonymous mutations of ELF4 in HT1080 (15) and a human endometrial carcinoma cell line the Catalogue of Somatic Mutations in Cancer (COSMIC; http:// HEC59 (16) as well as in the nontransformed human mammary cancer.sanger.ac.uk/cancergenome/projects/cosmic), Cancer Cell epithelial cell line MCF10A (17). We further demonstrate that Line Encyclopedia (CCLE; http://www.broadinstitute.org/ccle/ somatic mutations of ELF1, ELF2, and ELF4 are present and home), International Cancer Genome Consortium (ICGC; mutually exclusive in a wide range of human tumors, and that http://icgc.org), and The Cancer Genome Atlas (TCGA; https:// the antiproliferative effect of ELF4 is mediated through the induc- tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) databases revealed tion of HRK and DLX3 expression. >40 mutations widely distributed throughout the protein struc- ture of ELF4 (Fig. 1A). In addition, Totoki and colleagues (21) fi BCORL1–ELF4 Materials and Methods identi ed a fusion gene in hepatocellular carcinoma. Cell lines We examined the transactivation potential of ELF4(L211M), T3M-1 Cl-10 or HEC59 was obtained from RIKEN Cell Bank or ELF4(V382I), and BCORL1-ELF4 in comparison with that of the Japanese Collection of Research Bioresources Cell Bank, respec- wild-type protein (Fig. 1B). The activity of a reporter construct tively, and human embryonic kidney 293T (HEK293T), HT1080, containing the ETS-binding regions within the IL3 promoter MDA-MB-231, KATO-III, A549 and MCF10A were obtained (APET; ref. 19) was markedly increased by expression of wild- from ATCC. MCF10A was maintained in DMEM-F12 supplemen- type ELF4. Whereas the V382I substitution did not substantially ted with 5% horse serum (Biowest), recombinant human EGF affect the transactivation activity of ELF4, the activities of both (20 ng/mL; Peprotech), bovine insulin (10 mg/mL; Sigma- ELF4(L211M) and BCORL1-ELF4 were significantly attenuated Aldrich), hydrocortisone (0.5 mg/mL; Sigma-Aldrich), and cholera compared with that of the wild-type protein. The observed tran- toxin (100 ng/mL; Sigma-Aldrich). All other cells were main- scriptional activity was dependent on the ETS-binding regions of tained in DMEM-F12 supplemented with 10% FBS and 2 mmol/L the IL3 promoter, given that substitution of nucleotides within the L-glutamine (Invitrogen). ETS-binding sites (the mAPET reporter) abolished all activity. Furthermore, expression level of endogenous as well as artificially Functional analysis of ELF4 and other proteins introduced ELF4 mRNA was determined by reverse transcription We inserted an internal ribosomal entry site (ires) and (RT) and real-time PCR (PCR) analysis. As demonstrated in enhanced green fluorescent protein (EGFP) cDNA (Clontech) Supplementary Fig. S1, almost 20 times larger amounts of ELF4 into pMXS (18) to yield the pMXS-ires-EGFP plasmid, which mRNA were expressed in the transfected cells compared with the allows simultaneous expression of inserted cDNAs together with control, suggesting that competitive effects of endogenous ELF4 EGFP. For reporter assays of ELF4 activity, HEK293T cells were are negligible. transfected with pMXS- or pMXS-ires-EGFP-based expression We next examined whether such loss of function of ELF4 plasmids for various cDNAs, the firefly luciferase–based APET might contribute to carcinogenesis. Whereas introduction of reporter plasmid (19), and the pGL-TK plasmid for Renilla lucif- ELF4(L211M) or BCORL1-ELF4 had no marked effect on the erase (Promega). As a negative control for ELF4 activity, cells were proliferation of T3M-1 Cl-10 cells, expression of wild-type ELF4 transfected with the mAPET plasmid (19) instead of APET. Quan- or ELF4(V382I) greatly inhibited cell growth (Fig.