Epithelial Membrane Protein Research Articles

Construction of epithelial membrane protein 1 eukaryotic expression vector and its influence on migration and invasion of human oral tongue squamous carcinoma cells

This study aimed to construct a eukaryotic expression vector pEGFP-N1-EMP1 of epithelial mem-brane protein 1 (EMP1) and investigate its influence on migration and invasion of human oral tongue squamous carcinoma cells. The human EMP1 gene was amplified by reverse transcription polymerase chain reaction and then ligated into the pEGFP-N1 vector by double restriction endonuclease digestion to construct pEGFP-N1-EMP1 recombinant plasmid. After sequencing identification, pEGFP-N1-EMP1 recombinant plasmid and pEGFP-N1 plasmid were transfected into human oral tongue squamous carcinoma Tb3.1 cell line. The expression of green fluorescent protein in cells was observed after transfection using an inverted fluorescence microscope. The overexpression of EMP1 mRNA was identified at 24, 48, and 72h after transfection by real-time fluorescence quantitative polymerase chain reaction. The effect of EMP1 overexpression on migration and invasion of Tb3.1 cells was detected by Transwell assay. The full-length EMP1 gene sequence was successfully obtained. Sequence analysis showed that the EMP1 gene was inserted into the pEGFP-N1 vector correctly. Green fluorescence was observed in the transfected cells under fluorescence microscopy. The results of real-time fluorescence quantitative polymerase chain reaction indicated that the expression of EMP1 at 24h after pEGFP-N1-EMP1 transfection was significantly higher than the other groups. Transwell assays indicated that overexpression of the EMP1 gene could significantly inhibit the migration and invasion ability of Tb3.1 cells. The eukaryotic expression vector of EMP1 was successfully constructed, and EMP1 overexpression was confirmed to inhibit the migration and inva-sion of oral tongue squamous carcinoma cells in vitro. This study laid a foundation for further investigation on the influence of the EMP1 gene on the metastasis of oral tongue squamous carcinoma and its molecular mechanism. .

Abstract 1478: Anti-EMP2 IgG1 combined with anti-PD1/PDL1 antibodies synergistically reduce tumor load in animal models of breast cancer

Abstract Purpose: Despite significant advances in detection and medicine, the incidence and mortality due to breast cancer world-wide is still unacceptably high. Our recent work reveals that tetraspan protein epithelial membrane protein-2 (EMP2) is up-regulated in up to 70% of patients with invasive breast cancer and its expression is further augmented in metastatic lesions. New data suggests that EMP2 expression correlate with an increase in tumor associated PDL1 expression. We thus hypothesized that EMP2 expression regulated tumor immunogenicity through either direct control of PDL1 expression or MHC Class I processing. Recently, to determine the therapeutic potential of EMP2, our group created a panel of anti-EMP2 antibodies and new data shows that tumors treated with anti-EMP2 IgG1 show an increase in immune infiltrates. We thus proposed that EMP2 positive tumors will be amendable to anti-PD1/PDL1 therapy. Methods:A panel of breast cancer cell lines, both TNBC as well as hormone positive cell lines, were transfected with a vector to overexpress EMP2 or to reduce its levels. To determine if EMP2 expression influenced the expression of immune associated genes, RNAseq was performed. In addition, PDL1 protein expression was measured using standard immunohistochemistry or western blot analysis. Immune cell populations in various xenografts models were quantitated using standard immunohistochemistry. To determine the efficacy of combination anti-EMP2 and anti-PD1 therapy, syngeneic 4T1/firefly luciferase mouse models were created in BALB/c mice. Briefly, cells were injected into the mammary fat pad of female BALB/c mice, and once they approached 100 mm3, were injected IP with a)10 mg/kg dose twice a week of anti-EMP2 antibody, b) 5mg/kg twice a week with anti-PD-1 antibody (BioXCell), c) 10mg/kg ant-EMP2 and 5mg/kg of anti-PD-1 antibody twice a week, or d) saline control. Results: In multiple breast cell lines, EMP2 levels correlated with an increase in PDL1 expression, and treatment with anti-EMP2 IgG1 increased the number of infiltrating leukocytes. Using syngeneic mouse models, tumors treated with a combination of anti-EMP2 and anti-PD-1 antibodies grew slowly and had the smallest total volume compared to all other treatment groups. Anti-EMP2 IgG1 treatment was superior in reducing overall tumor volume compared to anti-PD-1 antibody treatment alone. Conclusions: Our preliminary data strongly supports the synergistic efficacy of anti-EMP2 IgG1 and anti-PD-1 antibody in reducing tumor load, suggesting that using anti-EMP2 treatment with an anti-PD1/PDL1 antibody may improve survival without inducing systemic toxicity as seen with chemotherapy. We predict that this is the direct result of EMP2 expression altering the immunogenicity of the tumor and in support of this, RNAseq data and western blot data suggest that EMP2 levels regulate PDL1 and MHC associated gene expression. Citation Format: Negin Ashki, Jessica Tsui, Madhuri Wadehra. Anti-EMP2 IgG1 combined with anti-PD1/PDL1 antibodies synergistically reduce tumor load in animal models of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1478.

Regulation of Laminin γ2 Expression by CDX2 in Colonic Epithelial Cells Is Impaired During Active Inflammation.

The expression of Caudal-related homeobox transcription factor 2 (CDX2) is impaired by tumor necrosis factor-α (TNF-α)-mediated activation of nuclear factor-κB (NF-κB) in ulcerative colitis (UC). Laminin subunit γ2 (LAMC2) is an epithelial basement membrane protein implicated in cell migration, proliferation, differentiation, as well as tumor invasion and intestinal inflammation, and its expression is enhanced by TNF-α in a NF-κB-dependent regulation of the recently identified LAMC2 enhancer. The aim was to determine whether CDX2 is involved in the basal regulation of LAMC2 in epithelial cells and to assess the influence of inflammation. Transcriptional regulation of LAMC2 was examined by reporter gene assays, overexpression, and shRNA-mediated knock-down of CDX2. CDX2-DNA interactions were assessed by chromatin immunoprecipitation on Caco-2 cells without or with TNF-α, as well as in purified colonic human epithelial cells. Immunohistochemical staining and quantitative reverse-transcription polymerase chain reaction analyses were used to measure the expression of CDX2 and LAMC2 in colonic biopsies from healthy controls and patients with UC. These data indicate that CDX2 directly regulates LAMC2 gene expression through interaction with elements in the LAMC2 promoter region. We further revealed an inverse effect of inflammation on CDX2 and LAMC2. The data presented provide a novel insight into how CDX2 is implicated in the transcriptional regulation of LAMC2 in intestinal epithelial cells, a function that is impaired during mucosal inflammation where a high level of TNF-α is present. J. Cell. Biochem. 118: 298-307, 2017. © 2016 Wiley Periodicals, Inc.

Epithelial Membrane Protein-2 in Human Proliferative Vitreoretinopathy and Epiretinal Membranes.

PurposeTo determine the level of epithelial membrane protein-2 (EMP2) expression in preretinal membranes from surgical patients with proliferative vitreoretinopathy (PVR) or epiretinal membranes (ERMs). EMP2, an integrin regulator, is expressed in the retinal pigment epithelium and understanding EMP2 expression in human retinal disease may help determine whether EMP2 is a potential therapeutic target.MethodsPreretinal membranes were collected during surgical vitrectomies after obtaining consents. The membranes were fixed, processed, sectioned, and protein expression of EMP2 was evaluated by immunohistochemistry. The staining intensity (SI) and percentage of positive cells (PP) in membranes were compared by masked observers. Membranes were categorized by their cause and type including inflammatory and traumatic.ResultsAll of the membranes stained positive for EMP2. Proliferative vitreoretinopathy–induced membranes (all causes) showed greater expression of EMP2 than ERMs with higher SI (1.81 vs. 1.38; P = 0.07) and PP (2.08 vs. 1.54; P = 0.09). However all the PVR subgroups had similar levels of EMP2 expression without statistically significant differences by Kruskal-Wallis test. Inflammatory PVR had higher expression of EMP2 than ERMs (SI of 2.58 vs. 1.38); however, this was not statistically significant. No correlation was found between duration of PVR membrane and EMP2 expression. EMP2 was detected by RT-PCR in all samples (n = 6) tested.ConclusionsAll studied ERMs and PVR membranes express EMP2. Levels of EMP2 trended higher in all PVR subgroups than in ERMs, especially in inflammatory and traumatic PVR. Future studies are needed to determine the role of EMP2 in the pathogenesis and treatment of various retinal conditions including PVR.

E-Cadherin Facilitates Protein Kinase D1 Activation and Subcellular Localization.

Protein kinase D 1 (PKD1) is a serine/threonine kinase implicated in the regulation of diverse cellular functions including cell growth, differentiation, adhesion and motility. The current model for PKD1 activation involves diacylglycerol (DAG) binding to the C1 domain of PKD1 which results in the translocation of PKD1 to subcellular membranes where PKD1 is phosphorylated and activated by protein kinase C (PKC). In this study, we have identified a novel regulation of PKD1 activation. The epithelial cell membrane protein E-cadherin physically binds to PKD1 which leads to a subcellular redistribution of PKD1. Furthermore, artificial targeting of PKD1 to the membrane leads to PKD1 activation in a PKC-independent manner, indicating that membrane attachment is sufficient enough to activate PKD1. The presence of E-cadherin dynamically regulates PKD1 activation by Bryostatin 1, a potent activator of PKD1, and its substrate phosphorylation specificity, implying a loss of E-cadherin during cancer metastasis could cause the re-distribution PKD1 and re-wiring of PKD1 signaling for distinct functions. The knocking down of PKD1 in lung epithelial cell line A549 results in an epithelial to mesenchymal transition with changes in biomarker expression, cell migration and drug resistance. These results extend our previous understanding of PKD1 regulation and E-cadherin signaling functions and may help to explain the diversified functions of PKD1 in various cells. J. Cell. Physiol. 231: 2741-2748, 2016. © 2016 Wiley Periodicals, Inc.

Epithelial membrane protein 2 regulates sphingosylphosphorylcholine-induced keratin 8 phosphorylation and reorganization: Changes of PP2A expression by interaction with alpha4 and caveolin-1 in lung cancer cells

Sphingosylphosphorylcholine (SPC) is found at increased in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. However, the detailed mechanism of SPC-induced K8 phosphorylation and reorganization is not clear.We observed that SPC dose-dependently reduced the expression of epithelial membrane protein 2 (EMP2) in lung cancer cells. Then, we examined the role of EMP2 in SPC-induced phosphorylation and reorganization of K8 in lung cancer cells. We found that SPC concentration-dependently reduced EMP2 in A549, H1299, and other lung cancer cells. This was verified at the mRNA level by RT-PCR and real-time PCR (qPCR), and intracellular variation through confocal microscopy. EMP2 gene silencing and stable lung cancer cell lines established using EMP2 lentiviral shRNA induced K8 phosphorylation and reorganization. EMP2 overexpression reduced K8 phosphorylation and reorganization. We also observed that SPC-induced loss of EMP2 induces phosphorylation of JNK and ERK via reduced expression of protein phosphatase 2A (PP2A). Loss of EMP2 induces ubiquitination of protein phosphatase 2A (PP2A). SPC induced caveolin-1 (cav-1) expression and EEA1 endosome marker protein but not cav-2. SPC treatment enhanced the binding of cav-1 and PP2A and lowered binding of PP2A and alpha4. Gene silencing of EMP2 increased and gene silencing of cav-1 reduced migration of A549 lung cancer cells.Overall, these results suggest that SPC induces EMP2 down-regulation which reduces the PP2A via ubiquitination induced by cav-1, which sequestered alpha4, leading to the activation of ERK and JNK.

Epithelial membrane protein 1 expression in ovarian serous tumors.

The present study aimed to analyze the clinical significance of epithelial membrane protein 1 (EMP1) expression in ovarian serous tumors. A total of 84 cases of ovarian serous tumor (50 patients with malignant ovarian serous tumors and 34 patients with borderline and benign serous tumors) were retrospectively analyzed. Differences in the expression levels of EMP1 between the malignant and non-malignant tumor groups were evaluated by immunohistochemical staining. In addition, the association between EMP1 expression and prognostic factors in malignant ovarian serous tumors was investigated. The expression levels of EMP1 were significantly reduced in all the 50 malignant ovarian serous tumors, compared with the 34 non-malignant ovarian serous tumors (P<0.000). Reduced expression of EMP1 was correlated with high grade (P=0.009) and stage (P<0.000) of malignant tumors. EMP1 expression was not observed to be correlated with any other investigated parameters, including surgery, type of operation and chemotherapy response (P>0.005). These results indicated that EMP1 may have a significant role as a negative regulator in ovarian serous tumors, and reduced EMP1 expression in serous tumors may be associated with increased disease severity.

MPTH-27A LARGE-SCALE UCLA GLIOMA TISSUE MICROARRAY IDENTIFIES EMP2 AS A NOVEL BIOMARKER FOR GBM THAT CORRELATES WITH OVERALL PROGNOSIS

BACKGROUND: Glioblastoma multiforme (GBM) is a deadly disease with a survival of approximately 15 months. Epithelial membrane protein 2 (EMP2) is correlated with advanced disease and adverse outcomes in several cancers and has recently been found to be expressed in GBM. Mechanistically, EMP2 promotes αvβ3 integrin surface expression, resulting in an activation of focal adhesion kinase and Src. The consequences of this activation are an increase in tumor growth, cell migration, and invasion. Our preliminary studies suggest that targeting EMP2 with anti-EMP2 antibody results in GBM cell death. METHODS: A custom large tissue microarray (TMA) containing glioma tumor samples from 184 consecutive patients at UCLA was stained for EMP2 expression using human EMP2 antisera. Staining intensity was quantified by an independent pathologist on a 0-3 histological score scale. EMP2 expression level for each sample was correlated with patients' clinical data and outcomes. RESULTS: In the large-scale glioma TMA, EMP2 was expressed strongly in over 90% of GBM samples. EMP2 expression level correlated with increasing grade of glioma, with GBM expressing the highest levels. Furthermore, EMP2 overexpression was significantly associated with increased tumor malignancy and statistically worse patient outcomes. CONCLUSION: Our data presents an extremely novel biomarker and therapeutic target for GBM. These finding suggest that EMP2 expression can be utilized with high validity to determine prognosis in patients with GBM and represents a highly attractive target for anti-glioblastoma therapies. Future prospective, multicenter studies are needed to further validate this large-scale UCLA tissue microarray analysis.

ATPS-62TARGETING OF NOVEL GBM BIOMARKER EMP2 RESULTS IN DECREASED TUMOR GROWTH AND INCREASED TUMOR NECROSIS

INTRODUCTION: Glioblastoma multiforme (GBM) is a deadly disease with a survival of approximately 15 months. Epithelial membrane protein 2 (EMP2) is correlated with advanced disease and adverse outcomes in several cancers and has recently been found to be expressed on the surface of GBM cells. Its expression in GBM increases integrin surface expression resulting in activation of focal adhesion kinase and Src and promoting increased tumor growth, cell migration, and invasion. METHODS: Subcutaneous and orthotopic mouse models were utilized for this study. In the subcutaneous model, n = 4 BALB/c mice were inoculated with U373 cells and treated with weekly systemic treatments of either anti-EMP2 or control IgG1. Tumor size was measured weekly. In the intracranial model, n = 6 BALB/c nude mice were inoculated with U87 cells overexpressing EMP2 and treated with weekly systemic injections of either anti-EMP2 or control IgG1. Tumor size was followed with bioluminescence imaging. RESULTS: In the subcutaneous model, tumor growth was significantly reduced at 50 days when treated with anti-EMP2 antibody (19 mm3 vs. 45 mm3, p < 0.05), and the residual tumors displayed significant necrosis compared to the control group. In the intracranial model, the treatment group showed a trend toward reduced tumor growth compared to the control group. The mean fold change in average tumor radiance in the treatment group was 402 compared to 543 in the control group. CONCLUSION: Our data present an extremely novel biomarker and therapeutic target for GBM. These findings suggest that targeting EMP2 may be an effective treatment modality for GBM. Further studies are needed to fully elucidate the role of targeting EMP2 in the treatment of GBM.

Targeting EMP3 suppresses proliferation and invasion of hepatocellular carcinoma cells through inactivation of PI3K/Akt pathway.

Epithelial membrane protein-3 (EMP3), a typical member of the epithelial membrane protein (EMP) family, is epigenetically silenced in some cancer types, and has been proposed to be a tumor suppressor gene. However, its effects on tumor suppression are controversial and its roles in development and malignancy of hepatocellular carcinoma (HCC) remain unclear. In the present study, we found that EMP3 was highly expressed in the tumorous tissues comparing to the matched normal tissues, and negatively correlated with differentiated degree of HCC patients. Knockdown of EMP3 significantly reduced cell proliferation, arrested cell cycle at G1 phase, and inhibited the motility and invasiveness in accordance with the decreased expression and activity of urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) in HCC cells. The in vivo tumor growth of HCC was effectively suppressed by knockdown of EMP3 in a xenograft mouse model. The EMP3 knockdown-reduced cell proliferation and invasion were attenuated by inhibition of phosphatidylinositol 3-kinase (PI3K) or knockdown of Akt, and rescued by overexpression of Akt in HCC cells. Clinical positive correlations of EMP3 with p85 regulatory subunit of PI3K, p-Akt, uPA, as well as MMP-9 were observed in the tissue sections from HCC patients. Here, we elucidated the tumor progressive effects of EMP3 through PI3K/Akt pathway and uPA/MMP-9 cascade in HCC cells. The findings provided a new insight into EMP3, which might be a potential molecular target for diagnosis and treatment of HCC.

Epithelial membrane protein 3 functions as an oncogene and is regulated by microRNA-765 in primary breast carcinoma.

Epithelial membrane protein 3 (EMP3) is a transmembrane signaling molecule, which is important in the regulation of apoptosis, differentiation and invasion of cancer cells. However, the specific function and regulatory mechanism of EMP3 in primary breast carcinoma remain to be elucidated. In the present study, the mRNA and protein levels of EMP3 were observed to be upregulated in primary breast carcinoma tissues, compared with normal tissues. It was hypothesized that the overexpression of EMP3 was correlated with the downregulation of microRNA-765 (miR-765), an underexpressed miRNA in primary breast carcinoma tissues. Functional analysis demonstrated that EMP3 was regulated by miR-765 through binding to its 3′untranslated region. In addition, the knockdown of EMP3 and miR-765 had similar effects on the inhibition of proliferation and invasion in SK-BR-3 cells. These results provided novel insight into the regulatory mechanism of EMP3 in primary breast carcinoma.

Loss of Epithelial Membrane Protein 2 Aggravates Podocyte Injury via Upregulation of Caveolin-1.

Nephrotic syndrome is a CKD defined by proteinuria with subsequent hypoalbuminemia, hyperlipidemia, and edema caused by impaired renal glomerular filtration barrier function. We previously identified mutations in epithelial membrane protein 2 (EMP2) as a monogenic cause of this disease. Here, we generated an emp2-knockout zebrafish model using transcription activator-like effector nuclease-based genome editing. We found that loss of emp2 in zebrafish upregulated caveolin-1 (cav1), a major component of caveolae, in embryos and adult mesonephric glomeruli and exacerbated podocyte injury. This phenotype was partially rescued by glucocorticoids. Furthermore, overexpression of cav1 in zebrafish podocytes was sufficient to induce the same phenotype observed in emp2 homozygous mutants, which was also treatable with glucocorticoids. Similarly, knockdown of EMP2 in cultured human podocytes resulted in increased CAV1 expression and decreased podocyte survival in the presence of puromycin aminonucleoside, whereas glucocorticoid treatment ameliorated this phenotype. Taken together, we have established excessive CAV1 as a mediator of the predisposition to podocyte injury because of loss of EMP2, suggesting CAV1 could be a novel therapeutic target in nephrotic syndrome and podocyte injury.

Abstract LB-066: Adenomatous polyposis coli regulates epithelial morphogenesis and migration through FAK/Src signaling

Abstract Adenomatous Polyposis Coli (APC) is a multi-functional protein that is lost or mutated in many epithelial cancers including breast, colorectal, and pancreatic cancer. Although APC is well known as a negative regulator of the Wnt/β-catenin signaling pathway, it also binds to the cytoskeleton, microtubules and polarity proteins, such as Dlg and Scribble, suggesting functions in regulation of epithelial polarity and cell migration. Our lab has previously determined that the mammary glands of ApcMin/+ mice demonstrate mis-regulation of epithelial polarity, exhibit early neoplastic changes, and develop more aggressive mammary tumors when crossed to the MMTV-PyMT model of breast cancer. Cells isolated from these tumors displayed activated FAK/Src signaling. Our lab has also shown that APC knockdown in the Madin-Darby Canine Kidney (MDCK) model altered epithelial morphogenesis, resulted in inverted polarity in 3D culture, and up-regulated gene expression of epithelial membrane protein 2 (EMP2). While restoration of the middle β-catenin binding domain was unable to rescue the phenotype, introduction of either full-length or a c-terminal fragment of APC partially restored these phenotypes. The current studies investigate the Wnt-independent mechanisms by which APC regulates these processes using the MDCK model and primary mammary epithelial cells (MECs) isolated from Apc mutant mice. We hypothesize that the c-terminal fragment of APC mediates FAK/Src signaling to regulate 3D morphogenesis, polarity, and migration. Treatment of APC knockdown MDCK cells with PP2, a Src kinase inhibitor, or AIIB2, a β1-integrin inhibitor, eliminated the drastic cyst size changes produced by APC knockdown. Furthermore, inhibition of Src partially restored the polarity phenotype in cysts with APC loss. In addition, shAPC-MDCK cells and MECs isolated from ApcMin/+ mice exhibited increased cell migration compared to control cells indicating a role for APC in cell motility. Preliminary data demonstrates that treatment of shAPC-MDCK cells with PP2, AIIB2, and a FAK inhibitor decreases migration suggesting FAK/Src signaling as a possible mechanism by which APC mediates cell migration. Interestingly, EMP2 has been shown to bind integrin to activate FAK signaling suggesting an interaction in these pathways, and preliminary studies show EMP2 expression is increased in shAPC-MDCK cells during migration. Future studies will aim to dissect the role of the c-terminal fragment and further devise the mechanism by which FAK/Src signaling and EMP2 play a role in APC regulating gene expression, cell migration, and polarity and 3D morphogenesis in MDCK cells and MECs isolated from Apc mutant mice. Investigating the interactions of APC with several targets such as those in the FAK/Src signaling pathway and EMP2 will help identify key players in the role of APC in Wnt-independent tumor development. Citation Format: Alyssa Lesko, Carolyn Ahlers, Jenifer R. Prosperi. Adenomatous polyposis coli regulates epithelial morphogenesis and migration through FAK/Src signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-066. doi:10.1158/1538-7445.AM2015-LB-066

Abstract 654: PG-101 in combination with docetaxel or Herceptin improves breast cancer survival

Abstract Background: Breast cancer is a heterogeneous disease with several subtypes which dictate which course of treatment each patient will receive. However, resistance remains a significant challenge. In triple negative breast cancer (TNBC), an aggressive histological subtype with limited treatment options and very poor prognosis, resistance to current standard therapies such as taxanes severely limits the available options for previously treated patients. This problem also occurs with targeted therapies. Trastuzumab is the standard of care for HER2+ tumors. However, de novo or acquired Trastuzumab resistance is a major limitation for its clinical efficacy. Thus, there remains an urgent unmet need for improved targeted agents for this patient population. Methods: To this end, we define another marker epithelial membrane protein-2 (EMP2) as a novel target for Trastuzumab and taxane resistant tumors. EMP2 expression was determined using standard immunohistochemistry. We have recently developed a novel IgG1 monoclonal antibody to EMP2 termed PG-101. Acquired Trastuzumab and docetaxel resistant tumors were treated with PG-101 using human xenograft models. Results: EMP2 is an oncogenic protein whose expression has been shown to correlate with tumor progression as well as the formation of breast cancer stem cells. In this study, we present evidence that EMP2 is highly expressed in acquired resistant tumor cells both in culture as well as in patient samples. We also tested the efficacy of PG-101 in combination with docetaxel, and we have evidence that the combination significantly improves tumor-free survival. Moreover, preliminary data also strongly suggests that PG-101 can be used as an adjuvant therapy for docetaxel and Trastuzumab resistant tumors whereby PG-101 reduced both tumor load in both models in vivo. Conclusions: Given that EMP2 is also overexpressed in endometrial and ovarian cancers, our data collectively suggest that PG-101 therapy has the potential to revolutionize the outcomes for women with cancer. Citation Format: Madhuri Wadehra, Meagan Kiyohara, Christen Dillard, Negin Ashki, Christie Qin. PG-101 in combination with docetaxel or Herceptin improves breast cancer survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 654. doi:10.1158/1538-7445.AM2015-654

MicroRNA-31 Promotes Skin Wound Healing by Enhancing Keratinocyte Proliferation and Migration

Wound healing is a basic biological process restoring the integrity of the skin. The role of microRNAs during this process remains largely unexplored. By using an in vivo human skin wound healing model, we show here that the expression of miR-31 is gradually upregulated in wound edge keratinocytes in the inflammatory (1 day after injury) through the proliferative phase (7 days after injury) in comparison with intact skin. In human primary keratinocytes, overexpression of miR-31 promoted cell proliferation and migration, whereas inhibition of miR-31 had the opposite effects. Moreover, we identified epithelial membrane protein 1 (EMP-1) as a direct target of miR-31 in keratinocytes. The expression of EMP-1 in the skin was negatively correlated with the level of miR-31 during wound healing. Silencing of EMP-1 mimicked the effects of overexpression of miR-31 on keratinocyte proliferation and migration, indicating that EMP-1 is a critical target mediating the functions of miR-31 in keratinocytes. Finally, we demonstrated that transforming growth factor-β2, which is highly expressed in skin wounds, upregulated miR-31 expression in keratinocytes. Collectively, we identify miR-31 as a key regulator for promoting keratinocyte proliferation and migration during wound healing.

Autologous transplantation of oral mucosal epithelial cell sheets cultured on an amniotic membrane substrate for intraoral mucosal defects.

The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects.

Mast cells play an important role in chlamydia pneumoniae lung infection by facilitating immune cell recruitment into the airway.

Mast cells are known as central players in allergy and anaphylaxis, and they play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae is an important human pathogen, but it is unclear what role mast cells play during C. pneumoniae infection. We infected C57BL/6 (wild-type [WT]) and mast cell-deficient mice (Kit(W-sh/W-sh) [Wsh]) with C. pneumoniae. Wsh mice showed improved survival compared with WT mice, with fewer cells in Wsh bronchoalveolar lavage fluid (BALF), despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT mice. Cromolyn, a mast cell stabilizer, reduced BALF cells and bacterial burden similar to the levels seen in Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BALF cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation, whereas Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of matrix metalloprotease-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during C. pneumoniae infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for C. pneumoniae.

The cAMP responsive element binding protein 1 transactivates epithelial membrane protein 2, a potential tumor suppressor in the urinary bladder urothelial carcinoma.

In this study, we report that EMP2 plays a tumor suppressor role by inducing G2/M cell cycle arrest, suppressing cell viability, proliferation, colony formation/anchorage-independent cell growth via regulation of G2/M checkpoints in distinct urinary bladder urothelial carcinoma (UBUC)-derived cell lines. Genistein treatment or exogenous expression of the cAMP responsive element binding 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.

Sperm fucosyltransferase-5 mediates spermatozoa-oviductal epithelial cell interaction to protect human spermatozoa from oxidative damage.

Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. Interaction between spermatozoa and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. Our previous data showed that oviductal epithelial cell membrane proteins interact with the human spermatozoa and protect them from ROS-induced reduction in sperm motility, membrane integrity and DNA integrity. Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human spermatozoa. In this study, we demonstrate for the first time that sFUT5 is involved in human spermatozoa-oviduct interaction and the beneficial effects of such interaction on the fertilizing ability of human spermatozoa. Anti-sFUT5 antibody-treated spermatozoa had reduced binding to oviductal membrane proteins. It is consistent with the result that affinity-purified sFUT5 is bound to the epithelial lining of human oviduct and to the immortalized human oviductal epithelial cell line, OE-E6/E7. Pretreatment of spermatozoa with anti-sFUT5 antibody and oviductal membrane proteins with sFUT5 suppressed the protective action of oviductal membrane proteins against ROS/cryopreservation-induced oxidative damage in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of oviductal epithelial cell membrane proteins on sperm motility, membrane and DNA integrity. The results enhance our understanding on the protective mechanism of oviduct on sperm functions.

MP49-19 EMP2 AS A NOVEL TARGET OF BLADDER CANCER IMMUNOTHERAPY

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-19 EMP2 AS A NOVEL TARGET OF BLADDER CANCER IMMUNOTHERAPY Wujiang Liu, Jie Jin, liqun Zhou, and Yinglu Guo Wujiang LiuWujiang Liu More articles by this author , Jie JinJie Jin More articles by this author , liqun Zhouliqun Zhou More articles by this author , and Yinglu GuoYinglu Guo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2814AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES EMP2, epithelial membrane protein 2, is supposed to be upregulated in cancer (Clin Cancer Res. 2008: 7367). Antibody targeting EMP2 shows benefit on cancer growth arrest (Mol Cancer Ther. 2014: 902). Here we investigated EMP2 expression in bladder cancer tissue and the potential for using as a target for cancer immunotherapy. METHODS 31 muscle invasive bladder cancer tissue and adjacent normal bladder tissue were collected from patient underwent cystectomy with informed consent. Protein expression of EMP2 was assayed by immmunohistochemical stain. T24, 5637 and SV-HUC-1 were used as cell lines for bladder cancer cells and normal bladder epithelium. Real-time PCR and Western-blot were performed to measure the mRNA and protein expression of EMP2. Chimeric antigen receptors of EMP2 (CARs-EMP2) were successfully constructed from multiple murine antibodies using second generation (CD28.CD3£a) signaling domains. Gamma-retroviral vector were used for carrying CAR and CD28.CD3£a sequences and subsequently for peripheral blood lymphocytes (PBL) transduction. Anti-tumor effect of T cells expressing CARs-EMP2 was assayed by cytolysis and cytokine release (IL-6, IFN-ƒ× and TNF-ƒÑ)ƒw following co-culture with target cell lines T24 and 5637. In vivo anti-tumor effect of T cells expressing anti-EMP2 CARs was tested on orthotopic mouse bladder cancer model. Tumor growth curve and tumor cell apoptosis were used to test the anti-tumor effect. RESULTS EMP2 was significantly increased in bladder tumor tissue compared to adjacent normal bladder tissue (P<0.01). The location of EMP2 is bladder cancer cell membrane. RNA and protein expression of EMP2 were more intensive in T24 and 5637 cells than that in SV-HUC-1 (P<0.05). T cells expressing CARs-EMP2 were able to lysis T24 and 5637 cells in a dose dependent manner and increase the cytokine release especially IL-6 and IFN-ƒ×ƒ|ƒnƒnCompared with T cells transfected with empty vector, adoptive transfer therapy with T cells transfected with CARs-EMP2 dramatically delay the tumor growth and increase the cancer cell apoptosis (P<0.01). CONCLUSIONS EMP2 is a potential target of bladder cancer immunological therapy. Adoptive transferring T cells expressing anti-EMP2 chimeric antigen receptors could be an alternative option to treat bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e610 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wujiang Liu More articles by this author Jie Jin More articles by this author liqun Zhou More articles by this author Yinglu Guo More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...