Epithelial Membrane Protein 1 Promotes Glioblastoma Progression Through The PI3K/AKT/mTOR Signaling Pathway

BackgroudGlioblastoma multiforme(GBM)is the most malignant primary human brain tumor.Tumors are characterized by a high proliferation rate and chemoresistance.Although the combination of radiotherapy and chemo-therapy has progressed after surgical resection,the 5-year survival rate of WHO grade ? glioblastoma is still lower than 5%.Although significant progress has been made in understanding the molecular status of this type of tumor,there is still a need for new and effective therapeutic approaches.Epithelial membrane protein 1(EMP1)is a member of the EMP family that has been implicated as a cell junc-tion protein on the plasma membrane.However,little is known about its specific functions and mechanisms.Recently,several members of the EMP family have been indicated to participate in cancer progression.EMP1 has been revealed to be a novel poor prognostic factor in pediatric leukemia and gastric carcinoma by regulating cell proliferation,migration and invasion,and was demonstrated to be involved in gefitinib resistance in lung cancer.EMP1 was also identified as a novel MYC-interacting gene with cancer-related functions in GBM.However,the biological role of EMP 1 in GBM remains unclear.To the best of our knowledge,we are the first to determine the important role of EMP1 in GBM.In the present study,it was revealed that knockdown of EMP1 inhibited GBM cell prolif-eration,migration and invasion.In addition,it was determined that EMP 1 is an independent predictor of poor prognosis in GBM patients.To sum up,our data indicated that EMP1 is a potential therapeutic target for the treatment of GBM.Part I Expression and prognosis of EMP1 in glioma patients ObjectiveTo explore the expression of EMP1 gene in human normal brain tissue and human gliomas and its relationship with the prognosis of glioma patients.Methods1.Explore the relationship between EMP1 gene expression and glioma grade by Oncomine,TCGA,REMBRANDT and CGGA databases.2.We collected human normal brain tissue,human glioma specimens of different grades and made paraffin sections,and explored the relationship between EMP1 gene expression and glioma grade by immunohistochemistry.3.We collected human normal brain tissue,human glioma specimens of various grades and extracted tissue specimen proteins.Western blot was used to explore the relationship between EMP1 gene expression and glioma grade.4.To explore the relationship between EMP1 gene expression and prognosis in patients with glioma through TCGA,REMBRANDT and CGGA databases.Results1.The expression of EMP1 gene in glioma is significantly up-regulated compared with normal brain tissue.To begin,to determine whether EMP1 was differen-tially expressed between glioblastoma and normal tissues,microarray data from patient samples were extensively examined in the Oncomine database.A meta-analysis of five independent glioblastoma data sets including 829 human glioblastoma samples and 47 normal brain tissues revealed that EMP1 was significantly and consistently present in glio-blastoma in all data sets.To further verify the level of EMP1 in normal brain tissues and different grade glioma tissues,the publicly available databases,The Cancer Genome Atlas(TCGA),Rembrandt,and the Chinese Glioma Genome Atlas(CGGA)were systematically retrieved and a relatively higher mRNA level of EMP1 was revealed in high grade gliomas compared to low grade gliomas.Immunohistochemistry results revealed that increased EM PI levels were associatedwith increased glioma grade.EMP1 was highly expressed in grade ?/? astrocytoma and glioblastoma(P<0.01),whereas staining was weak or absent in peri-tumor tissues.Western blotting results corroborated these results.EMP1 protein levels were increased in glioma cases relative to peri-tumor tissues.Collectively,these results demonstrated that EMP1 levels were increased in glioma compared to normal brain tissues.2.EMP1 gene expression in gliomas is associated with poor prognosis in patients with glioma.The differences in expression levels of EMP1 in glioma and normal brain tissues prompted us to further investigate whether EMP1 can be used as a prognostic marker in glioma patients.Data from the TCGA,Rembrandt,and CGGA databases was used to determine the relationship between EMP1 levels and overall survival(OS)in glioma patients.Each sample was classified as EMP1-high expres-sion if the signal was above the median expression for the population.These data revealed significant differences in OS and progression-free survival(PFS)between glioma patients with low EMP1 expression and those with high expression.ConclusionThe expression level of EMP1 is positively correlated with the degree of malignancy of glioma,and the high expression of EMP1 can be used as an independent factor for poor prognosis of glioma patients.It can be used for clinical diagnosis and prognosis evaluation of glioma patients.Part ? Epithelial membrane protein 1 promotes glioblastoma progression through the PI3K/AKT/mTOR signaling pathwayObjectiveTo investigate the effect and molecular mechanism of EMP1 on the malignant behavior of glioblastoma cells in vitro and in vivo.Methods1.Analyze the highly positive and negative genes associated with EMP1 expression in the TCGA human glioma database by bioinformatic analysis.Furthermore,functional enrichment analysis and KEGG analysis were performed on genes positively and negatively correlated with EMP1 expression.2.Western blot was used to investigate the expression of EMP1 in human normal astrocytes Astrocyte and glioblastoma cell lines P3 GBM,T98,A172,U87MG and U251.3.Lentiviral transfection(sh-NC and sh-EMP1)and knockdown of EMP1 gene expression in U87MG and P3 GBM,and Western blot to detect EMP1 knockdown efficiency.4.CCK-8 method was used to investigate the changes of cell viability in sh-NC and sh-EMP1 groups.5.Cell scratch assay was used to investigate the changes of cell migration ability in sh-NC and sh-EMP1 groups.6.Transwell assay was used to investigate the changes of cell invasion ability in sh-NC and sh-EMP1 groups.7.Western blot was used to investigate the expression of AKT,p-AKT,mTOR and p-mTOR proteins in the sh3 and AKT signaling pathways of sh-NC and sh-EMP 1.8.After using AKT enhancer SC79,Western blot was used to investigate the expression of AKT,p-AKT,mTOR and p-mTOR proteins in sh-NC and sh-EMP lgroups.9.After using AKT enhancer SC79,the CCK-8 method was used to investigate the changes in cell viability of the sh-NC and sh-EMP1 groups.10.After using the AKT enhancer SC79,the Transwell assay was used to investigate the changes in cell invasion ability of sh-NC and sh-EMP1 groups.11.The luciferase-stable sh-NC or sh-EMP1 P3 GBM cells were implanted into the brain of 4-week old nude mice by stereotaxic apparatus.The intracranial tumor growth of nude mice was monitored weekly using a small animal imager.12.We monitored the weight of nude mice daily,anesthetized the nude mice when the weight loss is?20%,and took the brain out after infusion of 4%paraformaldehyde.The brain was made into frozen sections and paraffin sections.13.Immunohistochemical Ki67,MMP2 and MMP9 staining were used to investigate the effects of EMP1 on proliferation and invasion in GBM cells.Results1.Pathway analysis of EMP1 and co-regulated genes.To further understand the biological implications of EMP1 in gliomas,correlation analysis of EMP 1 expression in whole genome gene profiling was performed in TCGA.The results revealed that 5,604 genes were correlated with EMP1 expres-sion in TCGA database.As illustrated in the volcano plot and heatmaps,these significant correlated genes were separated into positively correlated and negatively correlated genes.Subsequently,GO analysis revealed that EMP1 positively-correlated genes were strongly associated with biological processes including positive regulation of cell proliferation,negative regulation of apoptotic process,cell adhesion and extracellular matrix organization.In KEGG analysis,EMP 1-correlated genes were enriched in pathways in cancer,especially in the PI3K/AKT signaling pathway.2.Knockdown of EMP1 inhibits proliferation,migration and invasion in glioma cells in vitro.To determine the biological roles in glioma,the expression level of EMP1 was first verified in several glioma cell lines.Western blots results confirmed that the expression levels of EMP1 protein were increased in several glioma cell lines,especially in P3 GBM,which was derived from a primary GBM through orthotopic passage in mice,and U87MG cells.shRNA targeting EMP1 lentiviral constructs were designed for stable knock-down of expression.EMP1 protein levels in U87MG and P3 GBM cells were significantly downregulated after infection with three different shRNAs against EMP1 compared to NC constructs,especially sh-EMPl-2.Therefore,this shRNA was selected for the subsequent functional assays.It was then determined whether EMP1 knockdown may be effective against GBM,using the cell viability assay,CCK-8.Knockdown of EMP1 led to significant decreases in cell viability in both U87MG and P3 GBM cells.Having observed a marked change in cell morphology and retraction of pseudopodia after knockdown of EMP1,a wound healing assay was used to examine whether knockdown of EMP1 affected migration in GBM cells.Knockdown of EMP1 led to a significant lower migratory rate both in U87MG and P3 GBM cells.Transwell analysis was further applied to assess the inhibitory effect of EMP1 knockdown on cell invasion.In order to mimic the extracel-lular matrix around glioma,U87GBM and P3 GBM cells were plated in the upper chambers of a Transwell system coated with Matrigel.The results revealed that the invasive ability of GBM cell was significantly decreased after knockdown of EMP1.3.EMP1 promotes human glioma progression through activation of the PI3K/AKT/mTOR signaling pathway.It is well known that abnormal activation of the PI3K/AKT/mTOR signaling pathway promotes tumorigenesis,and in KEGG analysis,EMP1-correlated genes were enriched in the PI3K/AKT signaling pathway(Fig.3D).Therefore,phosphorylation status of AKT and mTOR proteins after knockdown of EMP1 was examined by western blotting to determine whether the observed responses could be due to a decrease in PI3K/AKT/mTOR signaling.Phosphorylated AKT and mTOR decreased compared to sh-NC following knockdown of EMP1 in modified cells,demonstrating that the PI3K/AKT/mTOR signaling pathway was inhibited after EMP1 knockdown.After being treated simultaneously with the novel AKT activator SC79,the activation of the PI3K/AKT/mTOR signaling pathway in GBM cells was partially restored.SC79 increased phosphorylation of AKT and mTOR in EMP1-knockdown GBM cells,and therefore further confirmed PI3K/AKT/mTOR signaling as a molecular target for EMP1 knockdown of GBM growth.CCK-8 and Transwell invasion assays were repeated to assess whether SC79 could restore proliferation and invasion in EMP1-knockdown GBM cells.The results demonstrated that SC79 increased proliferation and invasion in EMPI-knockdown GBM cells.4.EMP1 enhances growth of GBM cells in vivo.Considering the heterogeneity of GBM,P3 GBM,which is an in vivo-propagated primary GBM tumor cell line,was applied to investigate EMP1 function.Athymic nude mice(n=10)were implanted with lucif-erase-stable P3 cells in an intracranial tumor model and tumor growth was monitored over time using bioluminescence values.The results demonstrated that knockdown of EMP1 significantly reduced tumor growth.Kaplan-Meier analysis of the survival data demonstrated a statistically significant difference between sh-NC and sh-EMP1 mice.Immunohistochemistry(IHC)was performed on tissue sections from animals to examine proliferation and invasion.Ki-67,a marker for proliferation,MMP2 and MMP9,markers for invasion were decreased after EMP1 knockdown.Conclusion1.Epithelial membrane protein 1(EMP1)promotes glioblastoma progression through the PI3K/AKT/mTOR signaling pathway2.EMP1 enhances growth of GBM cells in vivo.