Epithelial Membrane Protein-2 Expression Research Articles

Abstract 2508: EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies

Abstract Background: Glioblastoma (GBM) is characterized as the most aggressive and lethal primary brain tumor. One of the hallmarks of GBM tumor growth and progression lies within the proangiogenic tumor microenvironment. Current anti-angiogenic drugs such as the anti-VEGF antibody take effect by inducing tumor cell starvation, leading to a functional normalization of the tumor vasculature. However, the survival benefits of anti-angiogenic drugs have been very limited. To this end, we hypothesized that the tetraspan Epithelial Membrane Protein-2 (EMP2) may help mediate resistance. Objective: We hypothesize that Epithelial Membrane Protein-2 may serve as a targetable biomarker for anti-VEGF resistance. We further explored the effectiveness of combination therapy in SB28 and GL261 GBM mouse models using anti-VEGF drug, Aflibercept, with a novel anti-EMP2 monoclonal antibody (mAb). Results: Tumor tissue was collected prospectively from patients undergoing surgery for suspected glioblastoma and stained for EMP2 and HIF2a using standard immunohistochemistry. Patients had banked tumor tissue both prior to and after bevacizumab treatment with 3 months of clinical and radiologic follow-up available. Bevacizumab treatment increased EMP2 protein expression. This increase in EMP2 correlated with reduced mean survival time post-bevacizumab therapy. Similarly, HIF-2a showed increased expression post-bevacizumab treatment and showed expression in both tumor and stromal cells. Limited animal models exist for anti-VEGF therapy for GBM. To establish syngeneic models, 10 week-old mice were subcutaneously implanted with murine SB28 or GL261 GBM cells. Treatment with either polyclonal sera to VEGF or Aflibercept failed to reduce tumor load or provide a therapeutic benefit. To next investigate if this model could recapitulate the response obtained patients tumors, formalin fixed paraffin embedded tissue was analyzed for both EMP2 and HIF-2a expression. Both markers were significantly upregulated in Aflibercept treated samples compared to the control.To next investigate if combination treatment could produce a significant reduction in tumor load, animals were treated with a combination treatment with Aflibercept and anti-EMP2 mAb. The combination therapy significantly reduced tumor volume and weight in comparison to control-treated tumors. Combination treatment in SB28 tumors resulted in a two fold reduction in tumor load and 50% reduction in tumor weight. Moreover, tumors showed reduced Ki67 expression. Similarly, treated GL261 tumors produced a 2.3 fold reduction in tumor size and ~2.7 fold reduction in tumor weight. Our results suggest that EMP2 can be a promising new target therapeutic target that can be combined with anti-VEGF treatment to potentially increase overall survival in GBM patients. Citation Format: Brian Aguirre, Mahlet Mekonnen, Riddhi Mehta, Meera Suresh, Anubhav Chandla, Karam Han, Isaac Yang, Madhuri Wadehra. EMP2 is a targetable biomarker in GBM tumors resistant to anti-angiogenic therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2508.

89Zr-ImmunoPET for the Specific Detection of EMP2-Positive Tumors.

Epithelial membrane protein-2 (EMP2) is upregulated in a number of tumors and therefore remains a promising target for monoclonal antibody (mAb)-based therapy. In the current study, image guided therapy for an anti-EMP2 mAb was evaluated by positron emission tomography (PET) in both syngeneic and immunodeficient cancer models expressing different levels of EMP2 in order to enable a better understanding of its tumor uptake and off target accumulation and clearance. The therapeutic efficacy of the anti-EMP2 mAb was initially evaluated in high- and low-expressing tumors, and the mAb reduced tumor load for the high EMP2 expressing 4T1 and HEC-1-A tumors. To create an imaging agent, the anti-EMP2 mAb was conjugated to p-SCN-Bn-deferoxamine (DFO) and radiolabeled with 89Zr. Tumor targeting and tissue biodistribution were evaluated in syngeneic tumor models (4T1, CT26, and Panc02) and human tumor xenograft models (Ramos, HEC-1-A, and U87MG/EMP2). PET imaging revealed radioactive accumulation in EMP2 positive tumors within 24h post-injection, and the signal was retained for 5 days. High specific uptake was observed in tumors with high EMP2 expression (4T1, CT26, HEC-1-A, U87MG/EMP2), with less accumulation in tumors with low EMP2 expression (Panc02, Ramos). Biodistribution at 5 days post-injection revealed that the tumor uptake ranged from 2 to ~16 %ID/cc. The results show that anti-EMP2 mAbs exhibit EMP2-dependent tumor uptake with low off-target accumulation in preclinical cancer models. The development of improved anti-EMP2 antibody fragments may be useful to track EMP2 positive tumors for subsequent therapeutic interventions.

ANGI-11. MOLECULAR MECHANISM OF TARGETING EPITHELIAL MEMBRANE PROTEIN-2 IN MALIGNANT MENINGIOMAS

Abstract BACKGROUND Meningiomas comprise approximately one-third of all primary central nervous system neoplasms. While many meningiomas remain benign, as many as 35% of meningiomas are graded as atypical or anaplastic/malignant with a 5-y survival rate as low as 35%. Targeting the tetraspan protein epithelial membrane protein-2 (EMP2)-HIF1α pathway through EMP2 knockdown may inhibit tumor growth, invasion, and neoangiogenesis in malignant meningioma. METHODS In collaboration with the UCLA Brain Tumor SPORE, we gathered a panel of malignant meningiomas (IOMM-LEE, CH157). To determine the function of EMP2, we created EMP2 vector control and knockdown models in both cell lines and analyzed the cells by RNA sequencing, transcriptomic functional analysis, and migration assay. RESULTS Gene functional analysis enriched for biological processes showed that knockdown of EMP2 inhibited multiple malignant pathways in these meningioma cells including the response to hypoxia, cell proliferation, invasion, and stem cell maintenance. Further analyses of EMP2 expression with other differentially expressed genes of interest revealed that VEGFA expression significantly decreased when EMP2 is knocked down (p< 0.001). Moreover, cellular migration and invasion regulated by EMP2 were tested in control and EMP2 knockdown malignant cells using a scratch assay, where knockdown of EMP2 reduced the rate of cell invasion by 50% (p = 0.0003). Additionally, anti-EMP2 mAb treatment in the control malignant cell line significantly reduced EMP2-mediated signal transduction (p = 0.0031). CONCLUSIONS Our study is the first to demonstrate the modulation of EMP2 and its implications in malignant meningioma pathogenesis, VEGFA-HIF1α pathway, and stem cell regulation. Targeting EMP2 may result in therapeutic benefits for malignant meningioma.

ANGI-10. IMPLICATIONS OF EPITHELIAL MEMBRANE PROTEIN-2 (EMP2) IN THE PATHOGENESIS OF MENINGIOMAS

Abstract INTRODUCTION VEGF-A is an important mediator of angiogenesis in meningiomas. However, targeting this protein with bevacizumab has offered only a modest improvement in survival, suggesting that there are other compensatory mechanisms involved, enabling vascularization despite ongoing treatment. Epithelial membrane protein 2 (EMP2), a cell surface protein, promotes angiogenic processes in gynecological cancers and brain tumors including gliomas and glioblastoma. However, little is known about its etiology and mechanism of action within meningiomas. OBJECTIVE We aimed to evaluate EMP2 expression in non-pathologic brain tissue, tumor specimens, and in meningioma cell lines. We also aimed to understand its mechanism within cells with regard to proliferation and invasion. METHODS Non-pathologic brain tissues from routine autopsies and tumor specimens from surgical resection were analyzed. Additionally, four meningioma cell lines were obtained (CH157, BEN-MEN1, SF1335 and IOMM-LEE) and changes in cellular proliferation and invasion were measured. Furthermore, EMP2 expression was modulated using lentiviral infection. A p-value < 0.05 was considered statistically significant. RESULTS EMP2 mRNA and protein expression were significantly higher in the meningioma specimens compared to non-pathologic meninges. Its expression also positively correlated with an angiomatous pattern and VEGF-A mRNA expression. In vitro, high EMP2 protein expression was noted in all cell lines, and quantification demonstrated highest expression in the malignant cell line, the IOMM-LEE. Viral vectors were subsequently utilized to create EMP2 overexpressing and knockdown IOMM-LEE models. Western analysis demonstrated a correlation between EMP2 overexpression and HIF1α, a key mediator of tumor growth and angiogenesis. CONCLUSION Our data from primary tumor samples and in vitro models implicate EMP2 in meningioma pathogenesis. This warrants further investigation by evaluating the effects of anti-EMP2 therapy on tumor growth and invasiveness in both in vitro andin vivo models.

Epithelial membrane protein 2 (EMP2) regulates hypoxia-induced angiogenesis in the adult retinal pigment epithelial cell lines

Pathologic retinal neovascularization is a potentially blinding consequence seen in many common diseases including diabetic retinopathy, retinopathy of prematurity, and retinal vaso-occlusive diseases. This study investigates epithelial membrane protein 2 (EMP2) and its role as a possible modulator of angiogenesis in human retinal pigment epithelium (RPE) under hypoxic conditions. To study its effects, the RPE cell line ARPE-19 was genetically modified to either overexpress EMP2 or knock down its levels, and RNA sequencing and western blot analysis was performed to confirm the changes in expression at the RNA and protein level, respectively. Protein expression was evaluated under both normoxic conditions or hypoxic stress. Capillary tube formation assays with human umbilical vein endothelial cells (HUVEC) were used to evaluate functional responses. EMP2 expression was found to positively correlate with expression of pro-angiogenic factors HIF1α and VEGF at both mRNA and protein levels under hypoxic conditions. Mechanistically, EMP2 stabilized HIF1α expression through downregulation of von Hippel Lindau protein (pVHL). EMP2 mediated changes in ARPE-19 cells were also found to alter the secretion of a paracrine factor(s) in conditioned media that can regulate HUVEC migration and capillary tube formation in in vitro functional angiogenesis assays. This study identifies EMP2 as a potential mediator of angiogenesis in a human RPE cell line. EMP2 levels positively correlate with pro-angiogenic mediators HIF1α and VEGF, and mechanistically, EMP2 regulates HIF1α through downregulation of pVHL. This study supports further investigation of EMP2 as a promising novel target for therapeutic treatment of pathologic neovascularization in the retina.

CircRNA-0002109 promotes glioma malignant progression via modulating the miR-129-5P/EMP2 axis

Glioma is a common intracranial malignant tumor with high mortality and high recurrence rate. In recent years, increasing evidence has demonstrated that circular RNAs (circRNAs) are potential biomarkers and therapeutic targets for many tumors. However, the role of circRNAs in glioma remains unclear. In this study, we found that circRNA-0002109 was highly expressed in glioma tissues and cell lines. Downregulation of circRNA-0002109 expression inhibited the proliferation, migration, and invasion of glioma cells and inhibited the malignant progression of tumors in vivo. Investigations into the relevant mechanisms showed that circRNA-0002109 upregulated the expression of EMP2 through endogenous competitive binding of microRNA-129-5P (miR-129-5P), which partially alleviated the inhibitory effect of miR-129-5P on epithelial membrane protein-2 (EMP2) and ultimately promoted the malignant development of glioma. Our results indicate that circRNA-0002109 plays an important role in the proliferation, invasion, and migration of glioma cells by regulating the miR-129-5P/EMP2 axis, which provides a new potential therapeutic target for glioma.

Characterization of microRNA Profiles in Pasteurella multocida-Infected Rabbits and Identification of miR-29-5p as a Regulator of Antibacterial Immune Response.

Pasteurella multocida is the pathogenic agent for a variety of severe diseases in livestock, including rabbits. MicroRNAs (miRNAs) participate in the immune response to the pathogen. Distinct miRNA expression patterns were explored in rabbit lung by small-RNA deep sequencing to assess dysregulated miRNAs during P. multocida infection. Totally, 571 miRNAs were screened, of which, 62 were novel, and 32 exhibited differential expression (DE). Of the 32 known DE-miRNAs, 13 and 15 occurred at 1 day and 3 days post-infection (dpi); and ocu-miR-107-3p and ocu-miR-29b-5p were shared between the two time points. Moreover, 7,345 non-redundant target genes were predicted for the 32 DE-miRNAs. Putative target genes were enriched in diverse GO and KEGG pathways and might be crucial for disease resistance. Interestingly, upregulation of ocu-miR-29-5p suppresses P. multocida propagation and downregulates expression of epithelial membrane protein-2 (EMP2) and T-box 4 (TBX4) genes by binding to their 3′ untranslated region in RK13 cells. Thus, ocu-miR-29-5p may indirectly inhibit P. multocida invasion by modulating genes related to the host immune response, such as EMP2 and TBX4.

EMP2 induces cytostasis and apoptosis via the TGFβ/SMAD/SP1 axis and recruitment of P2RX7 in urinary bladder urothelial carcinoma.

Urinary bladder urothelial carcinoma (UBUC) is a common malignant disease, and its high recurrence rates impose a heavy clinical burden. The objective of this study was to identify signaling pathways downstream of epithelial membrane protein 2 (EMP2), which induces cytostasis and apoptosis in UBUC. A series of in vitro and in vivo assays using different UBUC-derived cell lines and mouse xenograft models were performed, respectively. In addition, primary UBUC specimens were evaluated by immunohistochemistry. Exogenous expression of EMP2 in J82 UBUC cells significantly decreased DNA replication and altered the expression levels of several TGFβ signaling-related proteins. EMP2 knockdown in BFTC905 UBUC cells resulted in opposite effects. EMP2-dysregulated cell cycle progression was found to be mediated by the TGFβ/TGFBR1/SP1 family member SMAD. EMP2 or purinergic receptorP2X7 (P2RX7) gene expression upregulation induced apoptosis via both intrinsic and extrinsic pathways. In 242 UBUC patient samples, P2RX7 protein levels were found to be significantly and positively correlatedwith EMP2 protein levels. Low P2RX7 levels conferred poor disease-specific and metastasis-free survival rates, and significantly decreased apoptotic cell rates. EMP2 was found to physically interact with P2RX7. In the presence of a P2RX7 agonist, BzATP, overexpression of both EMP2 and P2RX7 significantly increased apoptotic cell rates compared to overexpression of EMP2 or P2RX7 alone. EMP2 induces cytostasis via the TGFβ/SMAD/SP1 axis and recruits P2RX7 to enhance apoptosis in UBUC. Our data provide new insights that may be employed for the design of UBUC targeting therapies.

EMP2 Is a Novel Regulator of Stemness in Breast Cancer Cells.

Little is known about the role of epithelial membrane protein-2 (EMP2) in breast cancer development or progression. In this study, we tested the hypothesis that EMP2 may regulate the formation or self-renewal of breast cancer stem cells (BCSC) in the tumor microenvironment. In silico analysis of gene expression data demonstrated a correlation of EMP2 expression with known metastasis-related genes and markers of cancer stem cells (CSC) including aldehyde dehydrogenase (ALDH). In breast cancer cell lines, EMP2 overexpression increased and EMP2 knockdown decreased the proportion of stem-like cells as assessed by the expression of the CSC markers CD44+/CD24-, ALDH activity, or by tumor sphere formation. In vivo, upregulation of EMP2 promoted tumor growth, whereas knockdown reduced the ALDHhigh CSC population as well as retarded tumor growth. Mechanistically, EMP2 functionally regulated the response to hypoxia through the upregulation of HIF-1α, a transcription factor previously shown to regulate the self-renewal of ALDHhigh CSCs. Furthermore, in syngeneic mouse models and primary human tumor xenografts, mAbs directed against EMP2 effectively targeted CSCs, reducing the ALDH+ population and blocking their tumor-initiating capacity when implanted into secondary untreated mice. Collectively, our results show that EMP2 increases the proportion of tumor-initiating cells, providing a rationale for the continued development of EMP2-targeting agents.

Epithelial Membrane Protein 2 (EMP2) Promotes VEGF-Induced Pathological Neovascularization in Murine Oxygen-Induced Retinopathy.

PurposeRetinopathy of prematurity (ROP) is a leading cause of childhood blindness. ROP occurs as a consequence of postnatal hyperoxia exposure in premature infants, resulting in vasoproliferation in the retina. The tetraspan protein epithelial membrane protein-2 (EMP2) is highly expressed in the retinal pigment epithelium (RPE) in adults, and it controls vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line. We, therefore, hypothesized that Emp2 knockout (Emp2 KO) protects against neovascularization in murine oxygen-induced retinopathy (OIR).MethodsEyes were obtained from wildtype (WT) and Emp2 KO mouse pups at P7, P12, P17, and P21 after normoxia or hyperoxia (P7−P12) exposure. Following hyperoxia exposure, RNA sequencing was performed using the retina/choroid layers obtained from WT and Emp2 KO at P17. Retinal sections from P7, P12, P17, and P21 were evaluated for Emp2, hypoxia-inducible factor 1α (Hif1α), and VEGF expression. Whole mount images were generated to assess vaso-obliteration at P12 and neovascularization at P17.Results Emp2 KO OIR mice demonstrated a decrease in pathologic neovascularization at P17 compared with WT OIR mice through evaluation of retinal vascular whole mount images. This protection was accompanied by a decrease in Hif1α at P12 and VEGFA expression at P17 in Emp2 KO animals compared with the WT animals in OIR conditions. Collectively, our results suggest that EMP2 enhances the effects of neovascularization through modulation of angiogenic signaling.ConclusionsThe protection of Emp2 KO mice against pathologic neovascularization through attenuation of HIF and VEGF upregulation in OIR suggests that hypoxia-induced upregulation of EMP2 expression in the neuroretina modulates HIF-mediated neuroretinal VEGF expression.

Identification of epithelial membrane protein 2 (EMP2) as a molecular marker and correlate for angiogenesis in meningioma.

Although intracranial meningiomas are the most common primary brain tumor in adults, treatment options are few and have traditionally been limited to surgical resection and radiotherapy. Additional targeted therapies and biomarkers are needed, especially as complete surgical resection is frequently not feasible in many patients. Non-pathologic brain tissue from 3 patients undergoing routine autopsies and tumor specimens from 16 patients requiring surgical resection for meningioma were collected. EMP2 protein expression was evaluated by immunohistochemistry and western blot analysis. EMP2 mRNA expression was also investigated using surgical specimens and validated by analysis of several independent NCBI GEO databases. EMP2 mRNA expression levels were found to be higher in meningioma relative to non-pathologic meninges (P = 0.0013) and brain (P = 0.0011). Concordantly, strong EMP2 protein expression was demonstrated in 100% of meningioma specimens from all 16 patients, with no observable protein expression in normal brain tissue samples from 3 subjects (P < 0.001). EMP2 expression was confirmed by western blot analysis in five samples, with EMP2 protein intensity positively correlating with histologic staining score (R2 = 0.780; P = 0.047). No association was found between EMP2 mRNA or protein levels and WHO grade or markers of proliferation. However, EMP2 expression was positively associated with an angiomatous pattern on histologic evaluation (P = 0.0597), VEGF-A mRNA expression (P < 0.001), and clinical markers of tumor vascularity such as operative blood loss (P = 0.037). EMP2 is not found in normal brain tissue, yet has shown consistently high mRNA and protein expression in meningiomas, and may serve as a useful molecular marker for these tumors.

Increased epithelial membrane protein 2 expression in glioblastoma after treatment with bevacizumab.

BackgroundAntiangiogenic therapy with bevacizumab has failed to provide substantial gains in overall survival. Epithelial membrane protein 2 (EMP2) is a cell surface protein that has been previously shown to be expressed in glioblastoma, correlate with poor survival, and regulate neoangiogenesis in cell lines. Thus, the relationship between bevacizumab and EMP2 was investigated.MethodsTumor samples were obtained from 12 patients with newly diagnosed glioblastoma at 2 time points: (1) during the initial surgery and (2) during a subsequent surgery following disease recurrence post-bevacizumab treatment. Clinical characteristics and survival data from these patients were collected, and tumor samples were stained for EMP2 expression. The IVY Glioblastoma Atlas Project database was used to evaluate EMP2 expression levels in 270 samples by differing histological areas of the tumor.ResultsPatients with high EMP2 staining at initial diagnosis had decreased progression-free and overall survival after bevacizumab (median progression-free survival 4.6 months vs 5.9 months; log-rank P = .076 and overall survival 7.7 months vs 14.4 months; log-rank P = .011). There was increased EMP2 staining in samples obtained after bevacizumab treatment in both unpaired (mean H-score 2.31 vs 1.76; P = .006) and paired analyses (mean difference 0.571; P = .019). This expression increase correlated with length of bevacizumab therapy (R2 = 0.449; Pearson P = .024).ConclusionsBevacizumab treatment increased EMP2 protein expression. This increase in EMP2 correlated with reduced mean survival time post-bevacizumab therapy. We hypothesize a role of EMP2 in clinical bevacizumab resistance and as a potential antiangiogenic therapeutic target in glioblastoma.

Abstract 1218: EMP2 serves as a novel target for immunotoxin mediated ovarian cancer therapy

Abstract Ovarian cancer (OC) currently accounts for only ~2% of all new cancer cases per year, but once diagnosed, patients only show a 5 year survival of 30%. Subdivided into four subtypes: serous, endometrioid, clear cell, and mucinous carcinoma, high-grade serous ovarian cancer (HGSOC) accounts for 70-80% of the ovarian cancer deaths. Immunoconjugates represent an innovative therapeutic approach to deliver a deadly payload to a tumor cell with more specificity and potentially less avenues for resistance than either can exert individually. However, it has been a challenge to find candidates as well as implement conjugation strategies to achieve these goals for a number of cancers. Epithelial membrane protein-2 (EMP2) is over-expressed in a number of cancers in women including 70% of ovarian tumors, with up to 88% of advanced serous carcinomas showing high expression of the antigen. With an eye towards therapy, we have recently developed an IgG1 targeting the extracellular domain of EMP2 with single digit nM affinity. As the antibody-antigen complex rapidly internalizes, we sought to improve on the efficacy of the naked antibody. Granzyme B (GrB) is a serine protease that plays a critical role in the body’s defense against viral infection and tumor development by initiating the apoptotic cascade via both caspase-dependent and -independent mechanisms. Accordingly, we developed novel fusion proteins composed of the anti-EMP2 backbone as the targeting moiety and granzyme B as the cytotoxic payload. The designs comprised (a) the anti-EMP2 IgG1 with GrB fused to the Fc domain through a cleavable linker, and (b) active GrB fused to the IgG heavy chain (Fc) domain containing 2 single-chain antibodies against EMP2, a format that results in a longer plasma retention time. The constructs were expressed by transient transfection in HEK293E cells and purified to homogeneity. Flow cytometry and ELISA demonstrated binding properties of the fusion protein with high specificity and sensitivity to EMP2 expression. The enzymatic activity of the fusion construct was similar to commercially-available GrB. Internalization of the immunotoxins was confirmed, and they showed high cytotoxicity to EMP2-positive cells compared to EMP2-negative cells. We predict that these designs may improve therapeutic outcomes for EMP2+ tumors. Citation Format: Khalid Mohamedali, Su-Yin Kok, Lawrence H. Cheung, Michael G. Rosenblum, Madhuri Wadehra. EMP2 serves as a novel target for immunotoxin mediated ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1218.

Epithelial Membrane Protein-2 (EMP2) Antibody Blockade Reduces Corneal Neovascularization in an In Vivo Model.

PurposePathologic corneal neovascularization is a major cause of blindness worldwide, and treatment options are currently limited. VEGF is one of the critical mediators of corneal neovascularization but current anti-VEGF therapies have produced limited results in the cornea. Thus, additional therapeutic agents are needed to enhance the antiangiogenic arsenal. Our group previously demonstrated epithelial membrane protein-2 (EMP2) involvement in pathologic angiogenesis in multiple cancer models including breast cancer and glioblastoma. In this paper, we investigate the efficacy of anti-EMP2 immunotherapy in the prevention of corneal neovascularization.MethodsAn in vivo murine cornea alkali burn model was used to study pathologic neovascularization. A unilateral corneal burn was induced using NaOH, and subconjunctival injection of either anti-EMP2 antibody, control antibody, or sterile saline was performed after corneal burn. Neovascularization was clinically scored at 7 days postalkali burn, and eyes were enucleated for histologic analysis and immunostaining including VEGF, CD31, and CD34 expression.ResultsAnti-EMP2 antibody, compared to control antibody or vehicle, significantly reduced neovascularization as measured by clinical score and central cornea thickness, as well as by histologic reduction of neovascularization, decreased CD34 staining, and decreased CD31 staining. Incubation of corneal limbal cells in vitro with anti-EMP2 blocking antibody significantly decreased EMP2 expression, VEGF expression and secretion, and cell migration.ConclusionsThis work demonstrates the effectiveness of EMP2 as a novel target in pathologic corneal neovascularization in an animal model and supports additional investigation into EMP2 antibody blockade as a potential new therapeutic option.

EMP2 acts as a suppressor of melanoma and is negatively regulated by mTOR-mediated autophagy.

Cutaneous melanoma is one of the most common malignant skin tumors and advanced melanoma is usually associated with a poor prognosis. In the current study, we demonstrated the tumor suppressing role of epithelial membrane protein-2 (EMP2) by inducing apoptosis in a A375 human melanoma cell line. Mechanistically, the low expression of EMP2 in melanoma is partially due to autophagic protein degradation mediated by the mTOR pathway. These results suggest there is regulation of autophagy as well as EMP2 levels might be an interesting novel targeted therapeutic strategy for melanoma. Although the further investigation is needed to deeply understand the regulatory mechanisms of EMP2 in melanoma progression and metastasis, our results clarify the functions and mechanisms of autophagy in melanoma, and shed new light on novel targeted therapeutics for melanoma.

Epithelial membrane protein 2: a novel biomarker for circulating tumor cell recovery in breast cancer.

EpCAM is a common marker used in the detection of circulating tumor cells (CTC). Disseminatedcancer cells display the characteristics of epithelial-to-mesenchymal transition events. The purpose of this study was to assess the potential of epithelial membrane protein 2 (EMP2) as a novel biomarker for CTC retrieval in breast cancer. MCF7 and MDA-MB-231 cells were stained with either anti-EpCAM or anti-EMP2 mAbs, respectively, followed by flow cytometric assay to measure their expression levels. PBMCs isolated from healthy donors were used for breast cancer cell spiking. CD45-depleted PBMCs from breast cancer patients' blood were used for CTC capturing. Immunomagnetic separation was used to enrich breast cancer cells. Cytospincentrifugation was performed to concentrate the captured cells, followed by immunofluorescence staining with anti-CD45 mAb, anti-pan cytokeratin mAb and DAPI. Fluorescent images were taken using a confocal microscope for CTC counts. MDA-MB-231 cells had 2.56 times higher EMP2 expression than MCF7 cells, and EMP2 had a significantly higher capture efficiency than EpCAM for MCF7 cells. Furthermore, anti-EMP2 was capable of capturing MCF7 cells that escaped in the flow-through of anti-EpCAM. Likewise, EMP2 had a significantly higher capture efficiency on MDA-MB-231 cells when compared to MCF7 cells. Most importantly, EMP2 biomarker was successfully used for CTC capture in patients with primary breast cancer. EMP2 is superior to EpCAM for capturing both MCF7 and MDA-MB-231 cells. Additionally, EMP2 is a novel biomarker and capable of capturing breast cancer cells in patient blood samples.

Abstract 4466: Adenomatous polyposis coli transcriptionally regulates epithelial membrane protein 2 to regulate apical-basal polarity

Abstract Adenomatous Polyposis Coli (APC) is a well-known negative regulator of the Wnt pathway; however, it also regulates polarity proteins. Loss of apical-basal polarity disrupts several cellular processes including epithelial structure and intracellular signaling, and is an early marker for tumor development. We previously demonstrated that APC knockdown (APCKD) in Madin-Darby Canine Kidney (MDCK) cells altered cyst size and inverted polarity in 3D culture. Through microarray analysis we made the novel observation that APC loss increased Epithelial Membrane Protein 2 (EMP2) expression. Interestingly, EMP2 knockdown in APCKD cells decreased cyst size and restored apical polarity. These data suggest a previously unknown role for EMP2 in regulating polarity; however, how EMP2 influences APC-mediated polarity remains unknown. Therefore we hypothesize that APC transcriptionally regulates EMP2 to influence apical-basal polarity. In this study, we investigate transcriptional activation of EMP2 using bioinformatics, transcription factor DNA/protein arrays, luciferase reporters, and ChIP assays. EMP2 promoter screens (ConTra v3) identified binding sites for signal transducer and activator of transcription 1 and 3 (STAT1 and 3), and E2F transcription factor 1 (E2F1). Interestingly, APCKD cells exhibited increased expression of STAT1 and E2F1 in DNA/protein arrays compared to controls. Additionally, STAT3 activation was increased in APCKD cells in reporter assays. These studies identified STAT3, STAT1, and E2F1 as possible APC-mediated transcriptional regulators of EMP2. Current studies utilizing luciferase reporter assays aim to determine if E2F1 and STAT1 are transcriptionally active upon APC loss. ChIP assays are being utilized to determine if these candidate transcription factors can bind the canine EMP2 promoter. As CREB is a known transcriptional regulator of EMP2, it was used as a positive control for EMP2 promoter binding. However, no changes in CREB binding are expected between APCKD and MDCK cells, as DNA/protein arrays and luciferase reporter assays did not reveal changes in CREB expression or activity upon APC loss. In preliminary studies, E2F1 was found to weakly bind the canine EMP2 promoter, though this binding was not regulated by APC. Current studies seek to determine if STAT1 and STAT3 bind the EMP2 promoter. Reporter assays using mutant EMP2 promoter constructs are being used to assess if the candidate transcription factors drive EMP2 expression upon APC loss. Future studies will determine if candidate transcription factors influence APC-mediated polarity. Transcription factors will be inhibited in APCKD cells and activated in MDCK cells and EMP2 expression, apical polarity and cyst size will be evaluated in 3D culture. Understanding the interaction of APC and EMP2 and the influence on polarity will identify key players in APC disease progression. Citation Format: Alyssa Lesko, Carolyn Ahlers, Anna T. Lyons, Jocelyn D. Gaona, Jenifer R. Prosperi. Adenomatous polyposis coli transcriptionally regulates epithelial membrane protein 2 to regulate apical-basal polarity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4466.

MiR-133b acts as a tumor suppressor and negatively regulates EMP2 in glioma.

In recent years, the incidence of neuroglioma (glioma) has trended towards a younger age-group. Gene therapy has been widely implemented and a growing number of microRNAs associated with glioma have been identified., Herein, we detected the expression of micro RNA - miR-133b - in glioma by qPCR and also its effect on cell viability, survival and apoptosis of in vitro U87 and A172 cells. The binding effect of miR-133b on epithelial membrane protein-2 (EMP2) was verified and we then investigated the effect of EMP2 on in vitro glioma cells and tested the expression of apoptosis related factors after administration of altered miR-133b and EMP2 expressions. We found that miR-133b was down-regulated in glioma compared to adjacent non-tumorous tissue and also that its over-expression inhibits cell viability and survival and enhances apoptosis in the U87 and A-172 cells. Moreover, miR-133b effectively binds to EMP2, down-regulates its expression and negates its normal function. EMP2 normally promotes cell apoptosis and reduces cell viability and survival while miR-133b over-expression regulates the expression of apoptotic-associated protein and activates the apoptotic pathway, thus counteracting EMP2 regulation of opposite expression effects. Further, miR-133b can be considered a tumor suppressor because of its low expression and effects on cell apoptosis via down-regulating EMP2 expression and activating the apoptotic cell pathway in glioma. EMP2 is a risk factor for glioma, and miR-133b should prove a potential target for glioma clinical prevention and treatment.

Tissue microarray analysis for epithelial membrane protein-2 as a novel biomarker for gliomas.

Epithelial membrane protein-2 (EMP2) expression is noted in many human cancers. We evaluated EMP2 as a biomarker in gliomas. A large tissue microarray of lower grade glioma (WHO grades II-III, n=19 patients) and glioblastoma (GBM) (WHO grade IV, n=50 patients) was stained for EMP2. EMP2 expression was dichotomized to low or high expression scores and correlated with clinical data. The mean EMP2 expression was 1.68 in lower grade gliomas versus 2.20 in GBMs (P=0.01). The percentage of samples with high EMP2 expression was greater in GBMs than lower grade gliomas (90.0 vs. 52.6%, P=0.001). No significant difference was found between median survival among patients with GBM tumors exhibiting high EMP2 expression and survival of those with low EMP2 expression (8.38 vs. 10.98months, P=0.39). However, EMP2 expression ≥2 correlated with decreased survival (r=-0.39, P=0.001). The EMP2 expression level also correlated with Ki-67 positivity (r=0.34, P=0.008). The mortality hazard ratio for GBM patients with EMP2 score of 3 or higher was 1.92 (CI 0.69-5.30). Our findings suggest that elevated EMP2 expression is associated with GBM. With other biomarkers, EMP2 may have use as a molecular target for the diagnosis and treatment of gliomas.

EMP2 is a novel therapeutic target for endometrial cancer stem cells.

Previous studies have suggested that overexpression of the oncogenic protein epithelial membrane protein-2 (EMP2) correlates with endometrial carcinoma progression and ultimately poor survival from disease. To understand the role of EMP2 in the etiology of disease, gene analysis was performed to show transcripts that are reciprocally regulated by EMP2 levels. In particular, EMP2 expression correlates with and helps regulate the expression of several cancer stem cell associated markers including aldehyde dehydrogenase 1 (ALDH1). ALDH expression significantly promotes tumor initiation and correlates with the levels of EMP2 expression in both patient samples and tumor cell lines. As therapy against CSCs in endometrial cancer is lacking, the ability of anti-EMP2 IgG1 therapy to reduce primary and secondary tumor formation using xenograft HEC1A models was determined. Anti-EMP2 IgG1 reduced the expression and activity of ALDH and correspondingly reduced both primary and secondary tumor load. Our results collectively suggest that anti-EMP2 therapy may be a novel method of reducing endometrial cancer stem cells.