The Role And Molecular Mechanisms Of NETO2-modulating Invasion And Metastasis In Gastric Cancer Cells

Background and objective:Gastric cancer（GC）is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide.Currently,the progress of comprehensive therapeutic strategies has greatly improved the treatment effect of GC patients.However,the progn osis of most GC patients is still poor,mainly due to advanced stage of disease at diagnosis and limited understanding of the molecular mechanisms underlying the invasion and metastasis of GC.Therefore,a better insight into the molecular basis for invasion and metastasis of GC would facilitate the development of more effective therapeutic strategies for the patients.Neuropilin and tolloid-like 2（NETO2）was identified as an auxiliary protein of neuronal kainate receptors（KARs）,and played critical roles in regulating the functions of KARs.It was also able to bind to the active oligomeric form of K⁺-Cl^-cotransporter（KCC2）to enhance its recycling in hippocampal neurons.Recently,elevated mRNA levels of NETO2 were detected in several types of tumors.In patients with colorectal cancer（CRC）,NETO2 upregulation was significantly correlated with advanced TNM stages and poor survival.In hepatocellular carcinoma,NETO2 has been identified as a member of the five-gene transcriptomic signature which predicted poor outcome of the patients.However,little is known about the expression pattern and role of NETO2 in GC.This study aims to illustrate the expression pattern,molecular function and the mechanism of NETO2-modulating invasion and metastasis in GC and to explore the feasibility and possibility of NETO2 to be served as a new prognostic indicator and a potential therapeutic target for human GC.Materials and Methods:1.IHC staining and scoring,NCBI GEO and TCGA database analyses,western blotting and qRT-PCR assays were performed to explore the expression pattern of NETO2in gastric cancer tissues and normal tissues.2.Chi-square test was performed to test the relationship between NETO2 expression and clinicopathological parameters in patients with gastric cancer.3.Kaplan–Meier survival,univariate and multivariate analyses and investigation of KMPLOT and TCGA database were performed to test the relationship between NETO2expression and survival rate in patients with gastric cancer.4.We examined the expression of NETO2 in gastric epithelial cell line（GES-1）and 5different GC cell lines（SGC7901,BGC823,MKN-45,AGS and MGC803）as well as a primary GC cell line（XN0422）by qRT-PCR and western blotting analyses.Two gastric cancer cell lines with the highest expression of NETO2 were selected to establish NETO2-knockdown and NETO2-overexpression models and the knockdown and overexpression efficiency were analyzed by western blotting and qRT-PCR analyses.5.Wound healing assay was performed to explore the effects of knockdown or overexpression of NETO2 on the migration ability in gastric cancer cell line MGC803 and a primary gastric cancer cell line XN0422.6.Transwell invasion assay was performed to explore the effects of knockdown or overexpression of NETO2 on the imvasive ability in gastric cancer cell line MGC803 and a primary gastric cancer cell line XN0422.7.The intraperitoneal metastasis model was performed to explore the effects of knockdown or overexpression of NETO2 on the in vivo metastatic ability in gastric cancer cell line MGC803 and a primary gastric cancer cell line XN0422.8.Cell microphotography was performed to explore the effects of knockdown or overexpression of NETO2 on cellular morphology in gastric cancer cell line MGC803 and a primary gastric cancer cell line XN0422.9.qRT-PCR and western blotting analyses were performed to explore the effects of knockdown of NETO2 on epithelial-mesenchymal transition markers in gastric cancer cell line MGC803 and a primary gastric cancer cell line XN0422.10.Gene expression profiling and Ingenuity Pathway Analysis（IPA）analyses were performed to explore the effects of knockdown of NETO2 on the signaling pathways responsible to regulate NETO2-promoted invasion and metastasis of GC cells.11.Western blotting analysis was performed to to explore the effects of knockdown or overexpression of NETO2 on PI3K/AKT signaling pathway.12.Western blotting and transwell invasion assays were performed to test the effects of treatments with PI3K inhibitor LY294002,AKT inhibitor MK2206 or transfection with myristoylated AKT on PI3K/AKT pathway,invasive ability or EMT phenotype with knockdown or overexpression of NETO2.13.Western blotting and luciferase assays were performed to explore the effects of knockdown or overexpression of NETO2 on NF-κB pathway.14.Western blotting,qRT-PCR,transwell invasion and luciferase assays were performed to test the effects of treatment with NF-κB agonist TNF-α,NF-κB inhibitor JSH-23 and BAY 11-7082 on NF-κB pathway,Snail expression,invasive ability or EMT phenotype with knockdown or overexpression of NETO2.15.Western blotting and qRT-PCR assays were performed to explore the effects of knockdown or overexpression of NETO2 on the expression of TNFRSF12A.16.Western blotting and luciferase assays were performed to explore the effects of knockdown of TNFRSF12A on PI3K/AKT and NF-κB pathways.17.Western blotting and luciferase assays were performed to explore the influences of knockdown of TNFRSF12A in NETO2-overexpression cells on PI3K/AKT and NF-κB pathways.Results:1.NETO2 expression was examined in 220 GC samples and paired adjacent non-tumor tissues by IHC.The staining of NETO2 was significantly higher in cancer tissues and metastatic lymph nodes than that in normal gastric mucosa（p<0.0001）.High expression of NETO2 was more frequent in GC tissues（128/220,58.18%）compared with adjacent non-tumor tissues（82/220,37.27%）（p<0.001）（Fig.1C）.These results were supported by analysis on six individual datasets in NCBI GEO database,including GSE29272（p<0.0001）,GSE65801（p=0.0002）,GSE63089（p<0.0001）,GSE54129（p=0.0002）,GSE27342（p<0.0001）and GSE13911（p<0.0001）as well as on TCGA database（p<0.05）.We also detected NETO2 mRNA in 20 pairs of fresh GC tissues by qRT-PCR as well as NETO2 protein in 8 pairs of fresh GC tissues through western blotting.Bot h mRNA and protein levels of NETO2 were significantly elevated tumor tissues compared to adjacent non-tumor counterparts.These results indicate that NETO2 is highly expressed in GC tissues.2.We analyzed the correlation between NETO2 expression and clinicopathological features of patients with GC.The protein level of NETO2 was markedly correlated with TNM stage（p=0.004）,tumor invasion depth（p=0.016）,lymph node metastasis（p=0.022）and tumor size（p=0.045）,but not with age（p=0.387）,sex（p=0.585）,histological grade（p=0.424）,M stage（p=0.112）or tumor location（p=0.467）.3.Kaplan–Meier survival analysis showed that NETO2^high patients had a significantly lower overall survival（OS,p=0.001）and disease-free survival（DFS,p=0.009）rates than that of NETO2^low patients.Univariate and multivariate Cox regression analyses revealed that NETO2 expression was an independent prognostic factor for OS（p=0.001 and 0.011,respectively）and DFS（p=0.006 and 0.041,respectively）in GC patients.Analyses on GC data from KMPLOT（p=0.0379 for OS and p=0.0106 for DFS）and TCGA（p=0.0061）databases also supported that high NETO2 expression acted as an indicator for poor survival of GC patients.4.Results from western blotting and qRT-PCR analyses indicated that the expression of NETO2 in gastric cancer cells was significantly higher than that in gastric epithelial cells.MGC803 and XN0422 showed relatively higher levels of NETO2 expression and were used to establish the cell models of NETO2-knockdown and-overexpression successfully.5.Results from wound healing assay showed that compared with mock cells,silencing NETO2 significantly decreased the migratory capabilities（P<0.05）,while overexpression of NETO2 markedly enhanced the migratory abilities in gastric cancer cell line MGC803and primary gastric cancer cell line XN0422（P<0.05）.6.Results from transwell invasion assay showed that compared with mock cells,silencing NETO2 significantly decreased the invasive capabilities（P<0.05）,while overexpression of NETO2 markedly enhanced the invasive abilities in gastric cancer cell line MGC803 and primary gastric cancer cell line XN0422（P<0.05）.7.Results from intraperitoneal metastasis assay showed that compared with mock cells,silencing NETO2 significantly decreased the metastatic capabilities（P<0.05）,while overexpression of NETO2 markedly enhanced the metastatic abilities in gastric cancer cell line MGC803 and primary gastric cancer cell line XN0422（P<0.05）.HE staining further confirmed that the metastatic nodules were derived from GC cells.8.We observed that NETO2 knockdown altered the GC cells from mesenchymal-like morphology to epithelial morphology,whereas overexpression of NETO2 turned GC cells into more spindle-shaped morphology with more protrusions.9.Results from western blotting and qRT-PCR analyses indicated that knockdown of NETO2 led to inhibition of epithelial-mesenchymal transition,featured with upregulation of E-cadherin,downregulation N-cadherin,Snail and ZEB1 in gastric cancer cell line MGC803 and primary gastric cancer cell line XN0422.10.Results from gene expression profiling showed that a total of 1243 genes（|log FC|≥1.5 and FDR<0.05）were identified as differentially expressed genes,including 580upregulated genes and 663 downregulated genes.Ingenuity Pathway Analysis（IPA）revealed that knockdown of NETO2 affected a wide range of cellular functions and human diseases.The most significantly enriched disease was cancer and the most affected cellul ar functions were invasion,migration and EMT of tumor cells.IPA also indicated that PI3K/AKT pathway was one of the most enriched pathways after knockdown of NETO2（z score=-2.111,p=0.0269）.11.Knockdown of NETO2 significantly inhibited the phosphorylation of p85 and AKT compared to mock cells,while overexpression of NETO2 markedly increased the phosphorylation of p85 and AKT,indicating that PI3K/AKT signaling pathway participates in NETO2-regulated invasion and metastasis in GC cells.12.Treatment with LY294002,a specific inhibitor for PI3K,significantly abrogated the enhanced invasive ability and phosphorylation of AKT,as well as upregulated N-cadherin and downregulated E-cadherin induced by overexpression of NETO2.Similar results were observed when treated with MK2206,an AKT inhibitor.Exogenous expression of myristoylated AKT（myr-AKT）,a constitutive activation form of AKT,reversed the inhibited invasive ability,downregulated N-cadherin and phosphorylation of AKT,as well as,upregulated E-cadherin induced by knockdown of NETO2.13.Knockdown of NETO2 suppressed the phosphorylation of NF-κB p65,IKKβand IκBα.Furthermore,overexpression of NETO2 significantly increased the nuclear accumulation of p65 in MGC803 and XN0422 cells.Luciferase assay showed that overexpression of NETO2 significantly enhanced NF-κB transcription activity,while knockdown of NETO2 led to an opposite result.14.Treatment with tumor necrosis factor-α（TNF-α）,a typical cytokine to activate NF-κB,significantly rescued the inhibitory effects of NETO2 knockdown on Snail expression and p65 phosphorylation.TNF-αtreatment also restored the transcription activity of NF-κB and invasive capability in NETO2-knockdown cells.Treatment with JSH-23,an inhibitor of NF-κB,suppressed NETO2-induced EMT,Snail expression,transcription activity of NF-κB,and invasive capability in NETO2 overexpression cells.Treatment with Bay 11-7082,an IKK inhibitor,showed similar results with JSH-23treatment.15.Our results of IPA showed that TNFRSF12A was significantly downregulated after knockdown of NETO2（fold change=-3.03,p<0.0001）.Results from western blotting and qRT-PCR showed that TNFRSF12A was markedly downregulated by NETO2 knockdown and significantly upregulated by NETO2 overexpression in gastric cancer cell line MGC803and primary gastric cancer cell line XN0422.16.Depletion of TNFRSF12A in gastric cancer cell line MGC803 and primary gastric cancer cell line XN0422 cells impaired the phosphorylation of AKT,p85 and p65 as well as nuclear accumulation of p65.Furthermore,the NF-κB transcription activity in siTNFRSF12A cells was significantly inhibited.17.Depletion of TNFRSF12A in gastric cancer cell line MGC803 and primary gastric cancer cell line XN0422 blocked NETO2-induced phosphorylation of AKT,p85 and p65 as well as the nuclear accumulation of p65 and luciferase assay showed the requirement of TNFRSF12A for NETO2-activating NF-κB signaling.Conclusion:1.NETO2 is significantly upregulated in GC tissues and its expression level is closely associated with TNM stage,tumor invasion depth,lymph node metastasis,tumor size and poor overall and disease-free survival in patients with gastric cancer as an independent prognostic factor.2.High expression of NETO2 promotes the migration,invasion and metastasis of gastric cancer cells.3.NETO2 promotes invasion and metastasis of GC cells in association with induction of EMT.4.NETO2 activates PI3K/AKT pathway to induce EMT in GC cells.5.PI3K/AKT-activated NF-κB/Snail axis contributes to the NETO2 function in GC.6.TNFRSF12A mediates NETO2-induced activation of PI3K/AKT/NF-κB/Snail axis in GC cells.