Maoyong Fu

Diabodies Targeting Epithelial Membrane Protein 2 Reduce Tumorigenicity of Human Endometrial Cancer Cell Lines

Purpose: Endometrial cancer is the most common gynecologic malignancy. One promising biomarker is epithelial membrane protein 2 (EMP2), and its expression is an independent prognostic indicator for tumors with poor clinical outcome expression. The present study assesses the suitability of EMP2 as a therapeutic target. Experimental Design: Human monovalent anti-EMP2 antibody fragments were isolated from a human phage display library and engineered as bivalent antibody fragments (diabodies) with specificity and avidity to both EMP2 peptides and native cell-surface EMP2 protein. Diabodies were assessed using cell death and apoptosis assays. In addition, the efficacy of EMP2 diabodies on endometrial cancer tumors was determined using mouse xenograft models. Results: Treatment of human endometrial adenocarcinoma cell lines with anti-EMP2 diabodies induced significant cell death and caspase-3 cleavage in vitro. These responses correlated with cellular EMP2 expression and were augmented by progesterone, which physiologically induces EMP2 expression. In vivo, treatment of subcutaneous human xenografts of HEC-1A cell lines with anti-EMP2 diabodies suppressed tumor growth and induced cell death in the xenograft. Conclusions: These findings suggest that EMP2 may be a potential pharmacologic target for human endometrial cancer.

Epithelial Membrane Protein-2 Is a Novel Therapeutic Target in Ovarian Cancer

Purpose: The tetraspan protein epithelial membrane protein-2 (EMP2) has been shown to regulate the surface display and signaling from select integrin pairs, and it was recently identified as a prognostic biomarker in human endometrial cancer. In this study, we assessed the role of EMP2 in human ovarian cancer. Experimental Design: We examined the expression of EMP2 within a population of women with ovarian cancer using tissue microarray assay technology. We evaluated the efficacy of EMP2-directed antibody therapy using a fully human recombinant bivalent antibody fragment (diabody) in vitro and ovarian cancer xenograft models in vivo. Results: EMP2 was found to be highly expressed in >70% of serous and endometrioid ovarian tumors compared with nonmalignant ovarian epithelium using a human ovarian cancer tissue microarray. Using anti-EMP2 diabody, we evaluated the in vitro response of nine human ovarian cancer cell lines with detectable EMP2 expression. Treatment of human ovarian cancer cell lines with anti-EMP2 diabodies induced cell death and retarded cell growth, and these response rates correlated with cellular EMP2 expression. We next assessed the effects of anti-EMP2 diabodies in mice bearing xenografts from the ovarian endometrioid carcinoma cell line OVCAR5. Anti-EMP2 diabodies significantly suppressed tumor growth and induced cell death in OVCAR5 xenografts. Conclusions: These findings indicate that EMP2 is expressed in the majority of ovarian tumors and may be a feasible target in vivo. Clin Cancer Res; 16(15); 3954–63. ©2010 AACR.

Epithelial Membrane Protein-2 Promotes Endometrial Tumor Formation through Activation of FAK and Src

Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2), a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK)/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.

EMP2 regulates angiogenesis in endometrial cancer cells through induction of VEGF

Understanding tumor-induced angiogenesis is a challenging problem with important consequences for the diagnosis and treatment of cancer. In this study, we define a novel function for epithelial membrane protein-2 (EMP2) in the control of angiogenesis. EMP2 functions as an oncogene in endometrial cancer, and its expression has been linked to decreased survival. Using endometrial cancer xenografts, modulation of EMP2 expression resulted in profound changes to the tumor microvasculature. Under hypoxic conditions, upregulation of EMP2 promoted vascular endothelial growth factors (VEGF) expression through a HIF-1α-dependent pathway and resulted in successful capillary-like tube formation. In contrast, reduction of EMP2 correlated with reduced HIF-1α and VEGF expression with the net consequence of poorly vascularized tumors in vivo. We have previously shown that targeting of EMP2 using diabodies in endometrial cancer resulted in a reduction of tumor load, and since then we have constructed a fully human EMP2 IgG1. Treatment of endometrial cancer cells with EMP2-IgG1 reduced tumor load with a significant improvement in survival. These results support the role of EMP2 in the control of the tumor microenvironment and confirm the cytotoxic effects observed by EMP2 treatment in vivo.

Rationale and Preclinical Efficacy of a Novel Anti-EMP2 Antibody for the Treatment of Invasive Breast Cancer

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. Mol Cancer Ther; 13(4); 902–15. ©2014 AACR.

Epithelial Membrane Protein-2 (EMP2) Activates Src Protein and Is a Novel Therapeutic Target for Glioblastoma

Background: EMP2 is a tetraspan protein linked with aggressive disease. Results: EMP2 correlates with activated Src in patients with GBM. Using intracranial mouse models, EMP2 promotes tumor cell invasiveness. Antibodies to EMP2 reduce GBM tumor load. Conclusion: EMP2 is a novel therapeutic target in GBM. Significance: The clinical outcome for patients with GBM remains poor, and thus new targeted therapies are needed. 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.

Abstract 4621: EMP2 regulates the tumor microenvironment and is a novel therapeutic target for triple negative breast cancer

Breast cancer remains the most common malignancy in women, with the aggressive triple negative subtype accounting for nearly half of all breast cancer deaths. In this abstract, we describe a novel tetraspan protein, epithelial membrane protein-2 (EMP2) that is highly expressed in the majority of breast cancer tumors examined compared to healthy mammary epithelium. In particular, high levels of EMP2 were observed in over 70% of triple negative breast cancer cases examined. In tumor cells, EMP2 altered the integrin expression profile as well as promoted endothelial cell migration and tube formation through upregulation of VEGF. We produced a fully human intact IgG1 to test their effect on EMP2 expressing breast cancer cell lines. In vitro treatment with anti-EMP2 IgG1 decreased Src and FAK activation and resulted in cell death. Moreover, cultured supernatant from treated cells reduced endothelial cell migration and capillary-like tube formation. To determine the in vivo efficacy of EMP2 treatment, we first characterized potential toxicity from systemic administration. Anti-EMP2 IgG1 produced no observable toxicity (weight, CBC counts, tissue pathology) when administered at 10mg/kg over 7 weeks. Using similar conditions in preclinical xenograft and syngeneic mouse models, anti-EMP2 IgG1 decreased tumor growth with induction of necrosis. This effect was specific as no difference in tumor size was observed in EMP2 negative (Ramos) cell tumors. As in vitro and in vivo cytotoxicity was observed in both triple negative cell lines as well as cells expressing estrogen and progesterone receptors and/or Her-2/neu, these results present compelling evidence that EMP2 is a novel therapeutic target in triple negative breast cancers as well as other variants. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4621. doi:1538-7445.AM2012-4621

Abstract 5310: Positron emission tomography imaging of endometrial cancer using an anti-EMP2 minibody

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Endometrial cancer is increasingly affecting pre-menopausal women, prompting a need to develop fertility-sparing therapy, and monitor response to treatment by quantitative non-invasive strategies. Epithelial membrane protein 2 (EMP2) is a tetraspan protein associated with surface localization and function of cancer-relevant integrin isoforms, and EMP2 expression in endometrial cancer correlates with increasing cancer stage and poor prognosis. As EMP2 is highly expressed in endometrial cancer, this pre-clinical study tested the feasibility of non-invasive detection of human endometrial tumor xenografts using a radiolabeled anti-EMP2 diabody and positron emission tomography (PET). EMP2 biodistribution in murine organ was determined by western blot and immunohistochemistry. Anti-EMP2 minibody (MB83) fragment was designed and transfected into CHO-K1 cells. Purified minibody was DOTA conjugated and the binding activity was tested by flow cytometry using endometrial cancer cell line, Hec-1A/EMP2 cells. Then conjugated minibody was chelated with Cu-64. In vivo biodistribution of Cu-64 labeled MB83 in Balb/c nu/nu mice were determined by microPET imaging and AMIDE sofeware. MB83 location was determined by microPET in mice bearing Hec1a/EMP2 xenograft. EMP2 expression was detectable in the lung but not other organs by either western or immunohistochemistry. The absence of detectable EMP2 protein in the organs reflects that the target tumor with high EMP2 protein could uptake anti-EMP2 antibody which could be observed by microPET. The specific uptake of MB83 minibody within the Hec1a/EMP2 tumor can be seen both visually and quantitatively. Selective tumor uptake became more specific over time. In contrast, 2-deoxy-2-[18F]fluoro-D- glucose (FDG) microPET uptake by HEC-1A/EMP2 was minimal, reflecting the poor detection of gynecologic cancer cell types by conventional FDG-PET imaging. These findings demonstrate the feasibility of imaging endometrial and other gynecological cancers which have upregulated EMP2 expression using recombinant anti-EMP2 antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5310. doi:10.1158/1538-7445.AM2011-5310

Abstract 3624: Therapeutic targeting of glioblastoma multiforme using anti-EMP2 antibodies

Despite recent advances in molecular classification, surgery and radiotherapy, and the integration of novel molecular targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In particular, the prognosis for patients with glioblastoma multiforme (GBM), the most common and aggressive form of brain tumors, yields only a median survival of 12 months; the 5-year survival rate of less than 5%. In this report, we show that epithelial membrane protein-2 (EMP2) is a novel tetraspan protein upregulated at both the mRNA and protein levels in 95% of patients with GBM. We have started to characterize the functional consequence of EMP2 up-regulation in GBM cells. Within these cells, EMP2 appears to orchestrate the membrane expression of proteins required for cell migration and invasion. Specifically, our past results as well as our current preliminary results strongly suggest that EMP2 modulates the repertoire of integrin heterodimers on the cell surface and stabilizes the FAK complex and signaling required for cell migration and GBM progression. We further show that this upregulation is sufficient to induce tumor growth in a number of xenograft models using immunocompromised mice. The high expression of EMP2 on the membrane of GBM cells caused us to predict that we could exploit this increase in expression using anti-EMP2 recombinant antibodies. Using both anti-EMP2 antibody fragments (diabodies) as well as a humanized anti-EMP2 IgG1, we show that anti-EMP2 antibodies specifically induce apoptosis both in vitro and in vivo using GBM xenograft models. We have also started characterizing any potential toxicity that may arise from anti-EMP2 therapy. Importantly, no systemic or histological toxicity was observed at therapeutic doses. Thus, we predict that EMP2 expression can be targeted in GBM to develop a novel therapeutic strategy, and it may have broad applicability given that EMP2 expression is upregulated in a number of other tumors such as ovarian and breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3624. doi:10.1158/1538-7445.AM2011-3624