5518 MOLECULAR MEDICINE REPORTS 17: 5518-5527, 2018 Cooperation between ETS variant 2 and Jumonji domain‑containing 2 histone demethylases XIAOMENG LI1,2, GENE MOON2, SOOK SHIN2,3, BIN ZHANG1 and RALF JANKNECHT2,3 1China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China; 2Department of Cell Biology, University of Oklahoma Health Sciences Center; 3Stephenson Cancer Center, Oklahoma, OK 73104, USA Received June 2, 2017; Accepted January 3, 2018 DOI: 10.3892/mmr.2018.8507 Abstract. The E26 transformation‑specific (ETS) variant 2 Introduction (ETV2) protein, also designated as ETS‑related 71, is a member of the ETS transcription factor family and is essen- The ETS variant 2 (ETV2) protein, also designated as tial for blood and vascular development in the embryo. The ETS‑related 71 (ER71), is a DNA‑binding transcription role of ETV2 in cancer has not yet been investigated. In the factor (1,2); ETV2 belongs to the E26 transformation‑specific present study, the expression of ETV2 mRNA was identified (ETS) family of proteins, which are characterized by a in a variety of tumor types, including prostate carcinoma. In ~85 amino acid‑long ETS DNA‑binding domain and comprises addition, ETV2 gene amplification was identified in several 28 members in humans (3). The knockout of ETV2 in mice has types of cancer, suggesting that ETV2 plays an oncogenic been reported to cause embryonal lethality around day E9.5, role in tumorigenesis. It was demonstrated that ETV2 indicating that ETV2 is essential during early developmental forms complexes with two histone demethylases: Jumonji stages; in particular, ETV2 is required for the development of domain‑containing (JMJD)2A and JMJD2D; JMJD2A blood and vascular structures (4,5). Mechanistically, ETV2 has been previously reported as a driver of prostate cancer facilitates the upregulation of fetal liver kinase (FLK1), a development. In the present study, it was reported that ETV2 trans‑membrane tyrosine kinase and receptor for vascular exhibited the potential to stimulate the promoters of matrix endothelial growth factor that is crucial for the aforementioned metalloproteinases (MMPs), including MMP1 and MMP7, developmental processes. Notably, a lack of FLK1 expression within LNCaP prostate cancer cells. JMJD2A and JMJD2D phenocopied ETV2 deficiency in mice (6), emphasizing the could synergize with ETV2 to activate the MMP1 promoter, biological relevance of the ETV2‑FLK1 axis in embryonal whereas only JMJD2A stimulated the MMP7 promoter in blood and vessel formation. cooperation with ETV2. Furthermore, ETV2 expression was In adult mice, ETV2 expression was reported to be unde- positively associated with JMJD2A and JMJD2D mRNA tectable in endothelial cells and the conditional knockout levels in neuroendocrine prostate tumors, in which an ETV2 of ETV2 in the endothelial compartment did not affect gene amplification rate of 17.8% was identified. Collectively, adult vasculature or viability. Upon injury, however, ETV2 the results of the present study indicated that ETV2, JMJD2A expression was rapidly upregulated in endothelial cells, and and JMJD2D may jointly promote tumorigenesis, particularly vascular regeneration was dependent on ETV2 in several neuroendocrine prostate tumors. In addition, the interaction models, including the hindlimb ischemic injury paradigm. In with the JMJD2A and JMJD2D epigenetic regulators may be addition, the delivery of lentivirus‑expressing ETV2 into isch- important in the ability of ETV2 to reprogram cells, modulate emic hindlimbs demonstrated positive therapeutic effects by normal and cancer stem cells, and affect spermatogenesis. increasing capillary formation and reducing tissue fibrosis (7). Furthermore, the conditional deletion of ETV2 in adult hema- topoietic cells did not affect the viability of mice, but led to a decrease in the number of hematopoietic stem cells. This may not be relevant under normal conditions; however, the stem cell repopulation potential appeared to be adversely affected Correspondence to: Dr Ralf Janknecht, Department of Cell following damage (8). Thus ETV2 is absolutely critical in Biology, University of Oklahoma Health Sciences Center, 975 NE 10th Street, BRC‑1464, Oklahoma, OK 73104, USA hematopoietic and endothelial cells during embryogenesis, E‑mail: ralf‑[email protected] and is not essential in these respective adult cells, but may facilitate their response to injury. Key words: cancer, ETS‑related 71, ETS variant 2, histone Nor ther n blot analyses have revealed elevated ETV2 expres- demethylase, Jumonji domain‑containing 2, lysine demethylase 4, sion levels in adult mouse testes, whereas other tissues did not transcription exhibit significant levels of ETV2 mRNA expression (1,9). In addition, the ETV2 gene has been identified as a target of the sex‑determining region Y (SRY) protein that is encoded on the Y chromosome. It has been reported that ETV2 expression LI et al: INTERACTION OF ETV2 WITH JMJD2 DEMETHYLASES 5519 becomes upregulated within male mouse gonads following incubation with the indicated primary antibodies followed by the transient expression of SRY during embryogenesis (10), incubation with goat anti‑mouse (cat. no. 1706516; Bio‑Rad indicating that ETV2 may be involved in the differentiation Laboratories, Inc., Hercules, CA, USA) or goat anti‑rabbit (cat. of the initially bipotential gonads into testes. Furthermore, no. 1706515; Bio‑Rad Laboratories, Inc.) polyclonal secondary ETV2 can bind to and activate the promoter of SOX9 (10,11), antibodies coupled to horseradish‑peroxidase and visual- a transcription factor downstream of SRY. SOX9 is important ized utilizing enhanced chemiluminescence, as previously for male differentiation in the embryo, and its expression described (23). persists in adult testes. Therefore, ETV2 may serve a role in the development of male gonads and their function in adults. Reporter gene experiments. Human LNCaP prostate cancer At present, very little is known about how ETV2 is cells (CRL‑1740; American Type Culture Collection) were regulated at the molecular level. For instance, only a few inter- grown in poly‑L‑lysine‑coated 6‑wells to ~30% conflu- action partners of ETV2 have been identified. These include ency (24) and transfected with 1 µg pGL2‑MMP1 (25) or ovo like zinc finger 2, a zinc‑finger transcription factor that pGL2‑MMP7 (26) luciferase reporter plasmid, 1 µg pBluescript cooperates with ETV2 in the regulation of the FLK1 gene KS+, pcDNA‑ETV2 (9) or empty vector pcDNA3, and 100 ng promoter (12), and Jumonji domain‑containing (JMJD)1A, Flag‑tagged JMJD2A or JMJD2D expression vector (27) or a cofactor that may repress the ETV2‑mediated stimulation empty vector pEV3S, with 8 µg polyethylenimine. At 8‑10 h, of matrix metalloproteinase (MMP) 1 transcription (13). cells were washed once with 2 ml PBS and then incubated for Notably, JMJD1A belongs to the JMJD protein family; most another 40 h prior to lysis with 350 µl of 25 mM Tris, 2 mM members of this family have been demonstrated to be capable EDTA (pH 7.8), 1% Triton X‑100, 10% glycerol and 2 mM of demethylating histone lysine residues (14,15). The present dithiothreitol, as previously described (28). Luciferase activi- study explored whether other members of the JMJD protein ties in 100 µl lysate were determined using a luminometer family may modulate ETV2 function, and how these functions ~25 min after lysis, as previously described (29,30). may be associated with cancer. Statistical analysis. For luciferase reporter gene assays, the Materials and methods means of independent replicates with standard deviations are presented. Statistical significance was evaluated with a Bioinformatics. Data regarding the mRNA expression levels of one‑way analysis of variance followed by Tukey's multiple ET V2 i n huma n tissues were obt a i ned f rom t he Genot ype‑Tissue comparison test, and corresponding P‑values were calculated Expression project through the Human Protein Atlas (16), with GraphPad Prism 6.0 h software (GraphPad Software, Inc., accessible from www.proteinatlas .org. ETV2 gene expression La Jolla, CA, USA). For Pearson correlations, statistical signifi- and amplification in various cancers was identified with cbio- cance was estimated using a two‑tailed Student's t‑test. P<0.05 portal (www.cbioportal.org). was considered to indicate a statistically significant difference. Coimmunoprecipitation experiments. 293T cells (CRL‑3216; Results American Type Culture Collection, Manassas, VA, USA) were grown in poly‑L‑lysine‑coated 6‑cm plates to ~25% conflu- Expression levels of ETV2 in normal tissue and cancer. ency in Dulbecco's modified Eagle's medium (10‑013‑CV; Previously, it was reported that ETV2 expression levels in Mediatech; Corning Inc, Corning, NY, USA) supplemented adult mouse tissues, apart from the testes, were undetectable with 10% fetal bovine serum (S11150; Atlanta Biologicals, by northern blotting (1,9); however, analysis was not previously Flowery Branch, GA, USA), as previously described (17). conducted in human tissue. In the present study, downloaded Subsequently, cells were transfected by the calcium phosphate RNA sequencing datasets were employed to analyze ETV2 coprecipitation method (18,19). The following amounts of expression via bioinformatics analyses, as RNA sequencing DNA were used for transfection: 1 µg 6Myc‑ETV2 expres- is more sensitive than northern blotting. As expected, it was sion construct, as previously described (9,13), or pCS3+‑6Myc revealed that the highest levels