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Isolated mouse embryonic stem cells (mESCs) retain the capacities to self-renew limitlessly and to give rise to all tissues of an adult mouse. A precise understanding of the relationships, mechanisms of action and functions of novel genes involved in mESCs differentiation is crucial to expand our knowledge of vertebrate development. The epithelial membrane protein 2 (EMP2) is a membrane-spanning protein found in epithelial and endothelial cell-cell junctions that has been implicated in the regulation of cell proliferation and migration in normal and tumor tissues. In this study, Emp2 was disrupted in mESCs using the CRISPR/Cas9 technology. We subsequently assessed Emp2 functions by using mouse embryoid bodies (EBs) capable of forming the three germ layers of an embryo in vitro and by further analyzing the emergence of the future cardiac tissue in these EB models. We found that when Emp2 is disrupted, expression of pluripotency markers was up-regulated and/or longer retained in EBs. Additionally, the formation of each germ layer was variously affected during gastrulation and in particular, the formation of mesoderm was delayed. Besides, we discovered that Emp2 was involved in the regulation of the epithelial-mesenchymal transition (EMT) process and in the differentiation of cells into functional cardiomyocytes.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor and is associated with an extremely poor clinical prognosis. One pathologic hallmark of GBM is excessive vascularization with abnormal blood vessels. Extensive investigation of anti-angiogenic therapy as a treatment for recurrent GBM has been performed. Bevacizumab, a monoclonal anti-vascular endothelial growth factor A (VEGF-A), suggests a progression-free survival benefit but no overall survival benefit. Developing novel anti-angiogenic therapies are urgently needed in controlling GBM growth. In this study, we demonstrate tumor expression of epithelial membrane protein-2 (EMP2) promotes angiogenesis both in vitro and in vivo using cell lines from human GBM. Mechanistically, this pro-angiogenic effect of EMP2 was partially through upregulating tumor VEGF-A levels. A potential therapeutic effect of a systemic administration of anti-EMP2 IgG1 on intracranial xenografts was observed resulting in both significant reduction of tumor load and decreased tumor vasculature. These results suggest the potential for anti-EMP2 IgG1 as a promising novel anti-angiogenic therapy for GBM. Further investigation is needed to fully understand the molecular mechanisms how EMP2 modulates GBM pathogenesis and progression and to further characterize anti-EMP2 therapy in GBM.

// Chien-Feng Li 1, 2, 3, * , Wen-Jeng Wu 4, 5, 6, 7, * , Wen-Ren Wu 8 , Yu-Jing Liao 9 , Lih-Ren Chen 3, 9 , Chun-Nung Huang 6, 7, 8 , Ching-Chia Li 6, 7, 10 , Wei-Ming Li 4, 5, 7, 11 , Hsuan-Ying Huang 12 , Yi-Ling Chen 8 , Shih-Shin Liang 8, 13 , Nan-Haw Chow 14, 15 , Yow-Ling Shiue 8, 16, 17 1 Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan 2 National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan 3 Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan 4 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 7 Department of Urology, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung, Taiwan 8 Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan 9 Division of Physiology, Livestock Research Institute, Tainan, Taiwan 10 Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan 11 Department of Urology, Ministry of Health and Welfare Pingtung Hospital, Pingtung, Taiwan 12 Department of Pathology, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan 13 Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan 14 Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan 15 Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan, Taiwan 16 Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan 17 Doctoral degree program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan * These authors have contributed equally to this work Correspondence to: Yow-Ling Shiue, e-mail: ylshiue@mail.nsysu.edu.tw Keywords: EMP2, CREB1, urinary bladder urothelial carcinoma, tumor suppressor Received: October 20, 2014 Accepted: February 08, 2015 Published: April 13, 2015 ABSTRACT In this study, we report that EMP2 plays a tumor suppressor role by inducing G 2 /M cell cycle arrest, suppressing cell viability, proliferation, colony formation/anchorage-independent cell growth via regulation of G 2 /M checkpoints in distinct urinary bladder urothelial carcinoma (UBUC)-derived cell lines. Genistein treatment or exogenous expression of the c AMP r esponsive e lement b inding protein 1 ( CREB1 ) gene in different UBUC-derived cell lines induced EMP2 transcription and subsequent translation. Mutagenesis on either or both cAMP-responsive element(s) dramatically decreased the EMP2 promoter activity with, without genistein treatment or exogenous CREB1 expression, respectively. Significantly correlation between the EMP2 immunointensity and primary tumor, nodal status, histological grade, vascular invasion and mitotic activity was identified. Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival. Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models. Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level. Afterward, pCREB1(S133) transactivated the tumor suppressor gene, EMP2 , in vitro and in vivo . Our study identified a novel transcriptional target, which plays a tumor suppressor role, of CREB1.

Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by up-regulating αvβ3 integrin surface expression, activating focal adhesion kinase and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine whether EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.

Despite significant advances in biology and medicine, the incidence and mortality due to breast cancer worldwide is still unacceptably high. Thus, there is an urgent need to discover new molecular targets. In this article, we show evidence for a novel target in human breast cancer, the tetraspan protein epithelial membrane protein-2 (EMP2). Using tissue tumor arrays, protein expression of EMP2 was measured and found to be minimal in normal mammary tissue, but it was upregulated in 63% of invasive breast cancer tumors and in 73% of triple-negative tumors tested. To test the hypothesis that EMP2 may be a suitable target for therapy, we constructed a fully human immunoglobulin G1 (IgG1) antibody specific for a conserved domain of human and murine EMP2. Treatment of breast cancer cells with the anti-EMP2 IgG1 significantly inhibited EMP2-mediated signaling, blocked FAK/Src signaling, inhibited invasion, and promoted apoptosis in vitro. In both human xenograft and syngeneic metastatic tumor monotherapy models, anti-EMP2 IgG1 retarded tumor growth without detectable systemic toxicity. This antitumor effect was, in part, attributable to a potent antibody-dependent cell-mediated cytotoxicity response as well as direct cytotoxicity induced by the monoclonal antibody. Together, these results identify EMP2 as a novel therapeutic target for invasive breast cancer. Fil: Fu, Maoyong. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Maresh , Erin L.. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Helguera, Gustavo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires; Argentina Fil: Kiyohara, Meagan . University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Qin, yu. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Ashki, Negin. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Daniels Wells, Tracy R.. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Aziz, Najib. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Gordon, Lynn K.. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Braun, Jonathan. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Elshimali, Yahya. Charles Drew University. Department of Pathology; Estados Unidos Fil: Soslow, Robert A.. Memorial Sloan-Kettering Cancer Center. Department of Pathology; Estados Unidos Fil: Penichet, Manuel L.. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Goodglick, Lee. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos Fil: Wadehra, Madhuri. University of California Los Angeles. David Geffen School of Medicine at UCLA; Estados Unidos