Epithelial Membrane Protein 1 Expression Research Articles

EMP1 correlated with cancer progression and immune characteristics in pan-cancer and ovarian cancer.

This study examines the prognostic role and immunological relevance of EMP1 (epithelial membrane protein-1) in a pan-cancer analysis, with a focus on ovarian cancer. Utilizing data from TCGA, CCLE, and GTEx databases, we assessed EMP1 mRNA expression and its correlation with tumor progression, prognosis, and immune microenvironment across various cancers. Our results indicate that EMP1 expression is significantly associated with poor prognosis in multiple cancer types, including ovarian, bladder, testicular, pancreatic, breast, brain, and uveal melanoma. Immune-related analyses reveal a positive correlation between EMP1 and immune cell infiltration, particularly neutrophils, macrophages, and dendritic cells, as well as high expression of immune checkpoint such as CD274, HAVCR2, IL10, PDCD1LG2, and TGFB1 in most tumors. In vivo experiments confirm that EMP1 promotes ovarian cancer cell proliferation, metastasis, and invasion. In conclusion, EMP1 emerges as a potential prognostic biomarker and therapeutic target in various cancers, particularly ovarian cancer, due to its influence on tumor progression and immune cell dynamics. Further research is warranted to elucidate the precise mechanisms of EMP1 in cancer biology and to translate these findings into clinical applications.

SMYD3 drives the proliferation in gastric cancer cells via reducing EMP1 expression in an H4K20me3-dependent manner

Protein lysine methyltransferase SET and MYND domain-containing 3 (SMYD3) is aberrantly expressed in various cancer settings. The mechanisms that SMYD3 activates the expression of critical pro-tumoral genes in an H3K4me3-dependent manner have been well described in previous reports. Besides H3K4me3, H4K20me3 is another catalytic product of SMYD3, however it is a transcriptionally repressive hallmark. Since it is not clear that how SMYD3-elicited transcriptionally repressive program functions in cancer, we used gastric cancer (GC) as a model to investigate the roles of SMYD3-H4K20me3. Herein, online bioinformatics tools, quantitative PCR, western blotting and immunohistochemistry assays demonstrated that SMYD3 expression was markedly increased in GC tissues from our institutional and The Cancer Genome Atlas (TCGA) cohort. Additionally, aberrantly increased SMYD3 expression was closely associated with aggressive clinical characteristics and poor prognosis. Depletion of endogenous SMYD3 expression using shRNAs significantly attenuates the proliferation in GC cells and Akt signaling pathway in vitro and in vivo. Mechanistically, chromatin immunoprecipitation (ChIP) assay showed that SMYD3 epigenetically repressed the expression of epithelial membrane protein 1 (EMP1) in an H4K20me3-dependent manner. Gain-of-function and rescue experiments validated that EMP1 inhibited the propagation of GC cells and reduced p-Akt (S473) level. Based on these data, pharmaceutical inhibition of SMYD3 activity using the small inhibitor BCI-121 deactivated Akt signaling pathway in GC cells and further impaired the cellular viability in vitro and in vivo. Together, these results demonstrate that SMYD3 promotes the proliferation in GC cells and may be a valid target for therapeutic intervention of patients with GC.

Abstract P4-02-28: EMP1 low expressing tumor is associated with cell proliferation and poor overall survival of ER-positive breast cancer patients

Abstract Background: Epithelial membrane protein 1 (EMP1) belongs to PMP22 gene family and is reported to be expressed low in numerous gastrointestinal cancers. However, little is known about its role in breast cancer. EMP1 functions to promote cell proliferation. In this study, we hypothesized that low EMP1 expressing breast cancer is associated with high proliferative characteristics and poor survival. Material and Methods: The clinicopathological data and transcriptome data of breast cancer patients from two independent large publicly available databases, The Cancer Genome Atlas (TCGA, n = 1090) and The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC, n = 1904), were utilized in the current study. Survival analyses; Overall survival (OS), Disease-specific survival (DSS) and Disease-free survival (DFS) were performed by comparing the high and low expression groups. CYT score, xCell, and other immunological factors were used to evaluate intratumoral immune cell composition. Also, gene set enrichment analysis (GSEA) was performed between EMP1 high and low expression groups. Results: We divided each cohort into EMP1 expression high and low groups by utilizing median cutoff. The expression levels of EMP1 were not associated with clinical stage in any subtypes. However, interestingly lower expression of EMP1 was significantly associated with more advanced grades in ER positive/HER2 negative (ER+/HER2) subtype in both TCGA and METABRIC cohorts (p< 0.001 and p< 0.001, respectively). Also, low EMP1 expressing tumors demonstrated higher MKI67 expression levels in ER+/HER2- subtype consistently in both TCGA (p< 0.001) and METABRIC cohorts (p< 0.001). Furthermore, GSEA demonstrated that low EMP1 expressing tumors enriched the gene sets associated with cell proliferation such as MYC Targets, E2F signaling, and G2M Checkpoint signaling, compared with low EMP1 expressing tumors in ER+/HER2- of both TCGA and METABRIC cohorts. On the contrary, high EMP1 expressing tumors enriched the immune related gene sets such as Coagulation, Inflammatory_Response, Complement, and IL-6_JAK_STAT3 Signaling in ER+/HER2- of both TCGA and METABRIC cohorts. To this end, we further hypothesized that high EMP1 tumors were associated with favorable tumor immune microenvironment (TIME) and analyzed TIME utilizing xCell. However, to our surprise high EMP1 expressing tumors were not associated with favorable TIME. Low EMP1 expressing tumors demonstrated worse DFS, DSS, and OS compared with high EMP1 expressing tumors in ER+/HER2- breast cancer patients (p=0.013, p=0.003, and p=0.006) which was not the case in the other subtypes in METABRIC cohort. Conclusion: Low EMP1 expressing tumors were associated with improved OS, DSS, DFS in ER-positive breast cancer patients. Also, low EMP1 expressing tumors were found to associate with advanced grades and enriched gene sets related to cell proliferation and cell cycle, which may explain the poor survival of patients with low EMP1 expressing ER+/HER2- breast cancer. Citation Format: Junko Ukai, Yoshihisa Tokumaru, Masanori Oshi, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. EMP1 low expressing tumor is associated with cell proliferation and poor overall survival of ER-positive breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-28.

Epithelial Membrane Protein 1 is a Potential Prognostic Biomarker for Ovarian Cancer

Background: Epithelial membrane protein 1 (EMP1) is a cell junction protein located in the plasma membrane. The function of EMP1 in cancer cells varies in different tumour types. In the present study we investigated the expression of EMP1 and its clinical significance in ovarian cancer. Methods: Sequencing data from the Cancer Genome Atlas (TCGA) and from several other public databases was used to study the expression of EMP1 and its gene regulation networks in ovarian cancer patients. Oncomine and Gene Expression Profiling Interactive Analysis tools (GEPIA) were used to evaluate EMP1 expression. EMP1 alterations were identified using cBioPortal and the prognostic value of EMP1 was assessed using Kaplan-Meier analysis and OncoLnc online tools. Genes that were co-expressed with EMP1 were identified using LinkedOmics and the Link-Interpreter module was used to analyse pathway enrichment and the possible functions of co-expressed genes. Results: EMP1 was highly expressed in ovarian cancer and was associated with advanced tumour stage and poor overall survival (OS). Amplification of the EMP1 gene was also common in ovarian cancer. EMP1 participates mainly in the biological processes of epidermal development, cell adhesion, peptidyl tyrosine modification and angiogenesis. Functional network analysis suggests that EMP1 regulates tumorigenesis and progression of ovarian cancer through different signalling pathways that include several kinases, microRNAs (miRNAs) and transcription factors related to tumorigenesis. Conclusions: This study confirmed the expression of EMP1 in ovarian cancer and elucidated its regulatory networks, thus providing a theoretical basis for further studies into its functions. EMP1 could potentially serve as a diagnostic and prognostic biomarker in ovarian cancer patients. However, owing to the limited sample size and range of experimental work, further validation studies are needed to confirm the role of EMP1 in ovarian cancer.

High expression of EMP1 predicts a poor prognosis and correlates with immune infiltrates in bladder urothelial carcinoma.

Epithelial membrane protein 1 (EMP1) is a key gene that regulates cell proliferation and metastatic capability in various types of cancer, and serves an important role in tumor-immune interactions. However, the association between EMP1 and clinical prognosis, as well as the presence of tumor-infiltrating lymphocytes in bladder urothelial carcinoma (BLCA) remains unclear. The present study aimed to explore the relationship between EMP1 expression and tumor immune cell infiltration in BLCA. In the present study, EMP1 expression in BLCA was analyzed using the Oncomine database, The Cancer Genome Atlas (TCGA) and the Tumor Immune Estimation Resource (TIMER). The effects of EMP1 on clinical prognosis were evaluated using the Kaplan-Meier plotter and Gene Expression Profiling Interactive Analysis. The correlations between EMP1, cancer immune infiltrates and lymphocyte abundance were determined using the TIMER and Tumor immune system interaction database. In addition, correlations between EMP1 expression and gene markers in immune infiltrates were analyzed using cBioportal. The results demonstrated that, compared with adjacent normal tissues, EMP1 was downregulated in BLCA tissues. High expression of EMP1 was significantly associated with poor overall survival (OS) in BLCA cases obtained from TCGA. Multivariate Cox analysis revealed that EMP1 was an independent predictor of OS in patients with BLCA. Gene set enrichment analysis revealed that EMP1 was associated with cancer-related pathways and was positively correlated with the levels of infiltrating CD8+ T cells, macrophages, neutrophils and dendritic cells in BLCA. Further analysis demonstrated that EMP1 was significantly associated with the enrichment of multiple types of lymphocyte. EMP1 expression exhibited a strong correlation with a range of immune markers in BLCA. In conclusion, the results of the present study demonstrated that EMP1 was associated with a poor prognosis in patients with BLCA, and that the levels of immune infiltration and multiple immunomarker groups were associated with EMP1 expression. These results suggested that EMP1 may be used as a predictive biomarker to determine the prognosis and immune infiltration in BLCA.

CircMACF1 Attenuates Acute Myocardial Infarction Through miR-500b-5p-EMP1 Axis.

It is widely accepted that circular RNA (circRNA) plays an important role in cardiovascular diseases. Therefore, this experiment aimed to investigate the pathogenesis of circMACF1 in acute myocardial infarction (AMI). qRT-PCR and immunoblotting were used to detect the expression levels of circMACF1, miR-500b-5p, and epithelial membrane protein 1 (EMP1). The role of circMACF1, miR-500b-5p, and EMP1 in cardiomyocyte apoptosis was assessed using annexin V-FITC/PI. Echocardiographic assessment, serum creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH), myocardial infarct size, and TUNEL staining were applied in our research. In the MI group, the expression levels of circMACF1 and EMP1 were decreased with the increasing expression level of miR-500b-5p. CircMACF1 upregulated the expression of EMP1 as a sponge of miR-500b-5p, and circMACF1 was a direct target of miR-500b-5p. CircMACF1 impaired the progression of AMI by modulating the miR-500b-5p/EMP1 axis. CircMACF1 may be a potential therapeutic target for treating AMI. Graphical Abstract CircMACF1 upregulated EMP1 expression by sponge miR-500b-5p.

Epithelial membrane protein 1 promotes glioblastoma progression through the PI3K/AKT/mTOR signaling pathway

Glioblastoma multiforme (GBM) is the most malignant intracranial tumor. Although the affected patients are usually treated with surgery combined with radiotherapy and chemotherapy, the median survival time for GBM patients is still approximately 12–14 months. Identifying the key molecular mechanisms and targets of GBM development may therefore lead to the development of improved therapies for GBM patients. In the present study, the clinical significance and potential function of epithelial membrane protein 1 (EMP1) in malignant gliomas were investigated. Increased EMP1 expression was associated with increasing tumor grade (P<0.001) and worse prognosis in patients (P<0.001) based on TCGA, Rembrandt and CGGA databases for human gliomas. In vitro, gene silencing of EMP1 in U87MG and P3 GBM (primary glioma) cells significantly inhibited tumor proliferation and invasion. In addition, it was revealed that activation of the PI3K/AKT/mTOR signaling pathway is the driving force of EMP1-promoted glioma progression. Finally, it was demonstrated, using an intracranial GBM animal model, that EMP1 knockdown significantly inhibits tumor growth in vivo and increases overall survival in tumor-bearing animals. Our research provides new insights into the molecular mechanisms underlying EMP1 knockdown-mediated inhibition of GBM cell invasion and raises the possibility that targeting of EMP1 may represent a promising strategy for the treatment of GBM.

EMP1 regulates cell proliferation, migration, and stemness in gliomas through PI3K-AKT signaling and CD44.

Glioblastoma multiforme (GBM) is an intracranial tumor; the feature is higher malignant and poorer prognosis. The search for therapeutic targets for gliomas has always been a focus of research in the field of neurology. The unusual expression of epithelial membrane protein 1 (EMP1) has been proved in most tumors. In our study, we determined the expression level of EMP1 expression in glioma tissues. There were higher levels of EMP1 in glioma tissues-particularly GBM tissues-than those in normal brain tissues. Then we discovered that silencing EMP1 inhibited glioma cell invasion and proliferation through inhibiting the PI3K-AKT signaling pathway. Subsequently, we investigated the function of EMP1 on glioma stem cells and found that it regulates the expression of CD44 in such cells to promote stemness. Taken together, the new strategies for the treatment of glioma may be provided by these finding, thereby improving the prognosis associated with it.

The Prognostic Value of Epithelial Membrane Protein 1 (EMP-1) in Patients with Laryngeal Carcinoma.

BackgroundIn the present study, we aimed to investigate the prognostic value of epithelial membrane protein 1 (EMP-1) gene in patients diagnosed with laryngeal carcinoma (LC).Material/MethodsPatients who were pathologically diagnosed with LC were enrolled in the present study. The expression levels of EMP-1 in tumor tissues and corresponding normal tissues collected from the LC patients were detected by semi-reverse transcriptase polymerase chain reaction (semi-RT-PCR). Chi-square analysis was used to evaluate the relationship between EMP-1 expression level and clinical characteristics. Survival analysis for the study population was analyzed by Kaplan-Meier method with log rank test. Additionally, Cox regression model was applied to evaluate the prognostic value of EMP-1 in LC patients.Results106 LC patients, including 55 men and 51 women, were enrolled in the present study. Semi-RT-PCR demonstrated that the expression level of EMP-1 was decreased in tumor tissues, compared with adjacent normal tissues (p<0.001). Moreover, the level was significantly associated with lymph node metastasis, histological grade, and clinical stage (p<0.05 for all). In addition, low levels of EMP-1 was significantly correlated with poor survival rate (log rank test, p=0.020). Cox regression analysis indicated that EMP-1 was an independent marker for LC prognosis (HR=2.755, 95% CI=1.123–6.760, p=0.027).ConclusionsThe abnormal expression of EMP-1 may be associated with progression of LC and the gene may act as a prognostic marker for LC.

The expression and function of epithelial membrane protein 1 in laryngeal carcinoma.

In this study, we compared the expression of epithelial membrane protein1 (EMP1) on the steady-state mRNA level (by quantitative real-time PCR) and on the protein level (by western immunoblot and immunohistochemistry) in 51pairs of laryngeal carcinoma tissues and matched cancer-free peritumor tissues, and we analyzed the correlation between EMP1 expression and different clinicopathological factors. Furthermore, we ectopically expressed EMP1 in human laryngeal carcinoma Hep-2 cells and examined the effects on cell viability, apoptosis, colonogenicity, and motility, by MTT assay, flow cytometry, colony formation assay and Transwell migration assay, respectively. EMP1 expression (on both the mRNA and protein levels) was significantly lower in the cancer tissues than in matched peritumor tissues(P<0.05). In laryngeal cancers, the level of EMP1 protein was correlated with histological grade (P<0.05), but not with age, gender, clinical stage, cancer subtype or lymph node metastasis (P>0.05). Functionally, ectopic expression of EMP1 in Hep-2 cells significantly reduced cell viability, colony formation, and migration, but enhanced apoptosis. Therefore, EMP1 is a tumor suppressor in laryngeal carcinoma. Boosting EMP1 expression in laryngeal carcinoma initiates multiple anticancer phenotypes and thus presents a promising therapeutic strategy for laryngeal cancer.

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.

Association of EMP1 with gastric carcinoma invasion, survival and prognosis.

The aim of this study was to determine the expression and function of epithelial membrane protein 1 (EMP1) in gastric carcinoma. Gastric samples were taken from cancer lesions and adjacent normal tissue in gastric cancer patients immediately after endoscopic biopsy. A portion of the sample was either fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry or stored in liquid nitrogen for western blotting. In order to determine protein expression of EMP1 in gastric cancer (n=65) and normal tissue (n=27), semi-quantitative immunohistochemistry and western blotting were utilized. For in vitro studies, the human gastric cancer cell line SGC-7901 was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. Recombinant lentivirus mediated overexpression of EMP1 in SGC-7901 cells was quantified with quantitative polymerase chain reaction (qPCR) and western blotting. Control SGC-7901 cells were transfected with an empty vector. To further study the effect of EMP1 overexpression in SGC-7901 cells, cell proliferation, cell apoptosis and migration and invasion assays were conducted. The expression of EMP1 was significantly lower in gastric cancer tissue compared to normal tissue using both immunohistochemistry (41.5 vs. 70.4% of tissues, P<0.05) and western blotting (0.153 ± 0.012 vs. 0.626 ± 0.058, P<0.05). Decreased expression of EMP1 was significantly correlated with tumor invasion, lymph node metastasis, clinical stage and histological grade of patients with gastric cancer (P<0.05). According to Kaplan-Meier analysis, low EMP1 expression correlated significantly with poor overall 5-year survival (47.4 vs. 70.3% survival, P<0.05). SGC-7901 cells transfected with EMP1 had a lower survival fraction, higher cell apoptosis (13.2 ± 1.5% vs. 2.2 ± 0.5%, P<0.05), significant decrease in migration and invasion (157.0 ± 16.0 and 112.0 ± 12.0, respectively vs. 243.0 ± 21.0 and 203.0 ± 19.0, respectively, P<0.05), higher caspase-9 (0.501 ± 0.050 vs. 0.114 ± 0.010, P<0.05) and lower VEGFC protein expression 0.135 ± 0.011 vs. 0.619 ± 0.074, P<0.05) relative to cells not transfected with EMP1. Low EMP1 expression in gastric cancer is associated with increased disease severity, suggesting that EMP1 may be a negative regulator of gastric cancer.

EMP1, a member of a new family of antiproliferative genes in breast carcinoma

This study aimed to analyze the expression, clinical significance of epithelial membrane protein 1 (EMP1) in breast carcinoma and the biological effect in its cell line by EMP1 overexpression. Immunohistochemistry and western blot were used to analyze EMP1 protein expression in 67 cases of breast cancer and 35 cases of normal tissues to study the relationship between EMP1 expression and clinical factors. Quantitative real-time RT-PCR and western blot were used to detect the mRNA level and protein of EMP1. MTT assay, migration and invasion assays were also conducted as to the influence of the upregulated expression of EMP1 that might be found on MCF-7 cell biological effect. The relative amount of EMP1 protein in breast cancer tissue was found to be significantly lower than in normal tissues (P < 0.05). The level of EMP1 protein expression was correlated with T stages, lymph node metastasis, clinic stage, and histological grade (P < 0.05). Meanwhile, loss of EMP1 expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P < 0.05). The result shown that MCF-7 cell transfected EMP1 had a lower survival fraction, higher cell apoptosis, significant decrease in migration and invasion, higher caspase-9, and lower VEGFC protein expression compared with MCF-7 cell untransfected EMP1 (P < 0.05). EMP1 expression decreased in breast cancer and correlated significantly with lymph node metastasis, clinic stage, histological grade, and poor overall survival, T stages, suggesting that EMP1 may play important roles as a negative regulator to breast cancer MCF-7 cell by regulating the expression of caspase 9 and VEGFC protein.

Epithelial membrane protein 1 negatively regulates cell growth and metastasis in colorectal carcinoma.

To determine the expression and function of epithelial membrane protein 1 (EMP1) in colorectal carcinoma. Colorectal samples were taken from cancer lesions and adjacent normal tissue in colorectal cancer patients immediately after endoscopic biopsy. A portion of the sample was either fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry or stored in liquid nitrogen for Western blot. In order to determine protein expression of EMP1 in colorectal cancer (n = 63) and normal tissue (n = 31), semi-quantitative immunohistochemistry and Western blot were utilized. For in vitro studies, the human colorectal cancer cell line SW-480 was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. Recombinant lentivirus mediated overexpression of EMP1 in SW-480 cells was quantified by real-time reverse transcription-polymerase chain reaction and Western blot. Control SW-480 cells were transfected with an empty vector. To further study the effect of EMP1 overexpression in SW-480 cells, cell proliferation, apoptosis, migration and invasion assays were conducted. Expression of EMP1 was significantly lower in colorectal cancer tissue than in normal tissue using both immunohistochemistry (39.7% vs 90.3% of tissues, P < 0.05) and Western blot (0.126 ± 0.022 vs 0.632 ± 0.053, P < 0.05). The level of EMP1 protein expression was not correlated with gender, age, or tumor location. Decreased expression of EMP1 was significantly correlated with T stage, lymph node metastasis, clinic stage, and histological grade in patients with colorectal cancer (P < 0.05). According to Kaplan-Meier analysis, low EMP1 expression correlated significantly with poor overall five-year survival (34.2% vs 64.0% survival, P < 0.05). SW-480 cells transfected with EMP1 had a lower survival fraction, higher cell apoptosis (12.1% ± 1.3% vs 3.1% ± 0.6%, P < 0.05), a significant decrease in migration and invasion (124.0 ± 17.0 and 87.0 ± 12.0, respectively vs 213.0 ± 29.0 and 178.0 ± 21.0, respectively, P < 0.05), higher caspase-9 (0.635 ± 0.063 vs 0.315 ± 0.032, P < 0.05), and lower VEGFC protein expression (0.229 ± 0.021 vs 0.519 ± 0.055, P < 0.05) relative to cells not transfected with EMP1. Low EMP1 expression in colorectal cancer is associated with increased disease severity, suggesting that EMP1 may be a negative regulator of colorectal cancer.

EMP1 inhibits nasopharyngeal cancer cell growth and metastasis through induction apoptosis and angiogenesis

This study aimed to analyze the expression, clinical significance of epithelial membrane protein-1 (EMP1) in nasopharyngeal carcinoma, and the biological effect in its cell line by EMP1 overexpression. Immunohistochemistry and Western blot were used to analyze the EMP1 protein expression in 75 cases of nasopharyngeal cancer and 31 cases of normal tissues to study the relationship between EMP1 expression and clinical factors. Recombinant lentiviral vector was constructed to overexpress EMP1 and then infect nasopharyngeal cancer CNE2 cell line. Quantitative real-time RT-PCR and Western blot were used to detect the mRNA level and protein of EMP1. MTT assay, cell apoptosis, migration, and invasion assays were also conducted to determine the influence of the upregulated expression of EMP1 that might be found on CNE2 cells' biological effect. Immunohistochemistry and Western blot: The level of EMP1 protein expression was found to be significantly lower in nasopharyngeal cancer tissue than in the normal tissues (P < 0.05). Decreased expression of EMP1 was significantly correlated with T stages, lymph node metastasis, clinic stage, and histological grade of patients with nasopharyngeal cancer (P < 0.05). Meanwhile, the loss of EMP1 expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P < 0.05). The result of biological function has shown that CNE2 cell-transfected EMP1 had a lower survival fraction, higher cell apoptosis, significant decrease in migration and invasion, higher caspase-9, and lower vascular endothelial growth factor C protein expression compared with CNE2 cell-untransfected EMP1 (P < 0.05). EMP1 expression decreased in nasopharyngeal cancer and correlated significantly T stages, lymph node metastasis, clinic stage, histological grade, and poor overall survival, suggesting that EMP1 may play important roles as a negative regulator to nasopharyngeal cancer cell.

EMP1, a Novel Poor Prognostic Factor In Pediatric Leukemia Regulates Prednisolone Resistance, Cellular Proliferation, Migration and Adhesion

Background20% of pediatric precursor B-acute lymphoblastic leukemia (BCP-ALL) and 25% of pediatric T-ALL patients relapse on or after current treatment strategies. Treatment failure has been associated with cellular resistance to prednisolone, the leading drug in multi-drug treatment of ALL. This study aimed to find new druggable targets that may modulate prednisolone resistance. MethodsLeukemic cells from children with newly diagnosed ALL were isolated from bone marrow aspirates, only samples with ≥ 90% leukemic blasts were used. RNA of 178 leukemic patients were hybridized to Affymetrix U133 Plus 2.0 gene expression microarrays and limma analysis was performed. EMP1 knockdown was achieved by lentiviral shEMP1 spin-occulation in four BCP-ALL cell lines and two T-ALL cell lines. Knockdown was confirmed by qRT-PCR and Western blot. Cell proliferation, cell cycle, and cell survival was analyzed by an AnnexinV/PI-staining and by a BrdU/7-AAD-staining. Response to prednisolone was evaluated in an in vitro3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Irradiated human mesenchymal stromal cells (hMSC-TERT) were co-cultured overnight with maroon-labeled leukemic cells, whereafter non-adherent and adherent leukemic cells were counted with the MACSQuant. Maroon-labeled leukemic cells were also inserted in the upper compartment of a 3 µM pore-size transwell system and were allowed to migrate overnight to hMSC-TERT. Migrated leukemic cells were evaluated by trypan blue and the MACSQuant. To assess MSC-mediated prednisolone resistance, leukemic cells alone or in combination with irradiated hMSC-TERT were cultured with or without prednisolone and cell survival was assessed after 72 hours. Cell signaling after shEMP1 knockdown was studied using Milliplex 8-plex Human Src Family kinase kit and the 9-plex Multi-Pathway kit with GAPDH and β-tubuline loading control beads, following the manufacturers' protocol. Cumulative incidence of relapse was calculated as time to relapse with death as a competing event in R using the method of Fine and Gray. Multivariate analysis of event free survival were performed in IBM SPSS Statistics 20 using Cox regression. ResultsGene expression microarray data from 178 primary pediatric ALL patients showed a 3.6-fold increase in epithelial membrane protein 1 (EMP1) expression between in vitro prednisolone-resistant and -sensitive patients (p=0.002). This difference was confirmed with qRT-PCR (p<0.0001). EMP1 is a small hydrophobic four-transmembrane glycoprotein of which the biological function is largely unknown. We show that lentiviral silencing of EMP1 in six different BCP-ALL and T-ALL cell lines increased apoptosis and induced G0/G1 and G2/M arrest leading to 84.1 ± 4.5% reduction in leukemic survival compared to non-silencing control cells (shNSC , p=0.014). Moreover, EMP1 silencing sensitized three out of six cell lines to prednisolone up to 8.8-fold (p<0.001) compared to shNSC. Silencing of EMP1 also abrogated migration and adhesion to mesenchymal stromal cells (MSCs) by 78.3±9.0% and 29.3±4.1% respectively compared to shNSC (p<0.002). Notably, we also discovered that EMP1 contributes to MSC-mediated prednisolone resistance, as prednisolone resistance triggered during leukemic MSC coculture was partially reversed after EMP1 knockdown. Pathway analysis indicated that EMP1 signals through Src kinase family phosphorylation to activate JNK, STAT3, STAT5, CREB and NF-κB. Finally, BCP-ALL patients with high EMP1 mRNA expression showed a higher cumulative incidence of relapse and non-response (p=0.02) and a poorer 5-year event-free survival compared to EMP1-low patients (82%±2 vs. 91%±9 ; p<0.01). Most importantly, Cox regression multivariate analysis with white blood cell count, age, and EMP1 expression as co-variates identified EMP1 as an independent predictor for poor outcome in BCP-ALL (p<0.01, hazard ratio: 2.36 (1.31-4.25) ). ConclusionOur results indicate that EMP1 is a novel poor prognostic factor in pediatric leukemia that regulates prednisolone resistance, cell survival, migration and adhesion. With this study we provide preclinical evidence that EMP1 is an interesting new druggable target to treat pediatric leukemia and sensitize to prednisolone. Disclosures:No relevant conflicts of interest to declare.

Knockdown Epithelial Membrane Protein 1 Suppresses Human Degenerative Intervertebral Disc-Derived Nucleus Pulposus Cell Proliferation.

Objective:Our previous work showed that epithelial membrane protein 1 (EMP1) is highly expressed in nucleus pulposus of the human degenerative intervertebral disc. The present study was designed to investigate the role of EMP1 in nucleus pulposus cells in intervertebral disc degeneration (IDD).Design:Human nucleus pulposus cells derived from degenerative intervertebral discs were cultured. EMP1 expression was knocked down by lentivirus-mediated specific interfering RNA. Cell morphology was observed, and cell proliferation, apoptosis, and cycle were evaluated.Results:Knockdown of EMP1 inhibited cell proliferation, caused cells to shrink, and accelerated the apoptosis induced by serum deprivation or addition of cycloheximide but did not evoke apoptosis in normal culture conditions.Conclusions:These findings suggest that EMP1 promoted chondrocyte proliferation, survival, and morphological change of cells during IDD, implying that EMP1 may be a target for biological therapy for IDD.

Epithelial membrane protein-1 is a biomarker of gefitinib resistance.

We describe a molecular resistance biomarker to gefitinib, epithelial membrane protein-1 (EMP-1). Gefitinib is a small-molecule inhibitor that competes for the ATP-binding site on EGF receptor (EGFR) and has been approved for patients with advanced lung cancers. Treatment with gefitinib has resulted in clinical benefit in patients, and, recently, heterozygous somatic mutations within the EGFR catalytic domain have been identified as a clinical correlate to objective response to gefitinib. However, clinical resistance to gefitinib limits the utility of this therapeutic to a fraction of patients, and objective clinical responses are rare. We aimed to assess the molecular phenotype and mechanism of in vivo gefitinib resistance in xenograft models and in patient samples. We generated in vivo gefitinib-resistance models in an adenocarcinoma xenograft model by serially passaging tumors in nude mice in presence of gefitinib until resistance was acquired. EMP-1 was identified as a surface biomarker whose expression correlated with acquisition of gefitinib resistance. EMP-1 expression was further correlated with lack of complete or partial response to gefitinib in lung cancer patient samples as well as clinical progression to secondary gefitinib resistance. EMP-1 expression and acquisition of gefitinib clinical resistance was independent of gefitinib-sensitizing EGFR somatic mutations. This report suggests the role of the adhesion molecule, EMP-1, as a biomarker of gefitinib clinical resistance, and further suggests a probable cross-talk between this molecule and the EGFR signaling pathway.