| BackgroundLung cancer is the leading cause of cancer-related death,with a high incidence,a strong tendency to metastasis,and resistance to treatment.The ability of local invasion and distant metastasis is the most important feature of malignant tumors and is associated with poor prognosis of the tumor.Tumor metastasis refers to the process in which malignant tumor cells are detached from the primary site and are taken to a distant place through lymphatic vessels,blood vessels or other pathways to form a tumor of the same type as the primary tumor.In the early stage of tumor growth,some of the tumor cells have decreased adhesion and gradually formed vasculogenic mimicry(VM)with abnormal expression of vascular endothelial(VE)-cadherin.VM is used as a means of supporting tumor growth in the early stages of tumorigenesis,describing the process by which highly aggressive and metastatic melanoma cells could form vascular channel structures by tumor cells,without the existence of endothelial cells.VM has been vividly described in various tumors and is involved in the spread and metastasis of cancer progression,and VM-rich regions have been shown to be resistant to conventional chemotherapeutic agents.VM formation involves a variety of molecular mechanisms and signaling pathways.Epithelial-mesenchymal transition(EMT)and cancer stem cells(CSCs)are thought to be involved in tumor cell metastasis and VM formation processes.Tumor cells can be transdifferentiated due to a multipotent,stem cell-like phenotype and forming extracellular matrix(ECM)-rich,CD34 negative and PAS positive vascular networks.The EMT-related transcription factor ZEB1 is up-regulated in VM-formed bladder cancer cells.Studies have shown that ZEB1 knockdown in tumor cells eliminates VM formation,leads to recovery of the epithelial phenotype,and significantly inhibits tumor migration and invasion.Overexpression of many signaling molecules promotes the formation of tumor VM,such as PI3K,VE-Cadherin,HIF-1?,VEGF.The PI3K signaling pathway has been shown to be essential in normal cellular processes such as proliferation,differentiation,survival,metabolism and movement.The PI3K/AKT signaling pathway is also involved in the formation of VM by modulating the cleavage activity of MMPs,MMP-2 and Ln-5c2 chains.Furthermore,the aberrantly activated PI3K pathway targets ZEB1 transcription via the GSK-3?/?-catenin signaling cascade.Protein phosphatase 2A(PP2A)is one of the major Ser/Thr phosphatases involved in the regulation of various cellular processes.Inactivation of PP2A contributes to the initiation or progression of tumors in many different tissues and cells,and controls many cellular signaling pathways such as AKT,p53,c-Myc and p-catenin by triggering dephosphorylation of various signaling proteins to regulate the process of tumor invasion and metastasis.However,the effects and mechanism of PP2A in lung cancer metastasis and VM formation is still unclear.BENC511 is a PI3K inhibitor that acts mainly on the PI3K/AKT signaling pathway and significantly inhibits the phosphorylation of AKT and the expression of downstream signaling proteins in tumor cells.BENC-511 has been shown to regulate the PI3K/AKT/mTOR signaling pathway and inhibit the transcription of Wnt signaling pathway and EMT-inducing factor ZEB1 by inhibiting nuclear translocation of ?-catenin protein,thereby inhibiting tumor invasion and metastasis.However,the effects of BENC-511 on VM formation in lung cancer have not been reported.This study focused on the role of PP2A/ZEB1 in the formation of VM in non-small cell lung cancer and its related mechanisms and the effects of compound BECN-511 intervention in vitro and in vivo.PP2A inhibitor(OA)and agonist(FTY720)and high/low expression of PP2A adenovirus were used to further detect the expression of PP2A/ZEB1/MMP-2 signaling pathway protein and protein interactions and elucidate its mechanism of action.By investigating the effects of the compound BENC-511 on the VM formation in lung cancer,and further improving its anti-tumor activity,it provides a theoretical basis for finding new targets and developing new anti-lung cancer metastasis drugs.Methods and Results1.In vitro studies,according to different invasion and metastasis ability,lung cancer cells H460,A549,H1299 have different VM formation ability.We selected high-invasive and VM-formed lung adenocarcinoma H1299 cells as the study object.The appropriate concentration of PP2A inducer FTY720 and PP2A inhibitor OA was screened by MTT assay,which did not affect proliferation.The concentration of FTY720 and OA is chosen as 5 ?mol/L and 10 nmol/L respectively.The expression of PP2A was by detected by Western blotting after cells were transfected with Ad-PP2A or Ad-dn-PP2A.The results showed that Ad-PP2A significantly promoted the expression of PP2A,and Ad-dn-PP2A significantly inhibited the expression of PP2A.The effects of FTY720(5 ?mol/L),OA(10 nmol/L),Ad-PP2A and Ad-dn-PP2A on cell migration were observed by scratch test.The effects of FTY720(5 ?mol/L),OA(10 nmol/L),Ad-PP2A and Ad-dn-PP2A on cell invasion were detected by Transwell chamber test.Tube formation assays examined the effects of FTY720(5?mol/L),OA(10 nmol/L),Ad-PP2A,and Ad-dn-PP2A on VM formation.The results showed that PP2A inducer FTY720 and Ad-PP2A could significantly inhibit the migration,invasion and VM formation in H1299 cells.PP2A inhibitors OA and Ad-dn-PP2A can significantly promote the migration and invasion and VM formation in H1299 cellsTo investigate the mechanisms of PP2A on invasion and VM formation in lung cancer cells,immunofluorescence assay was used to detect the expression of ZEB1 and VE-cadherin after cells were treated with FTY720(5 ?mol/L),OA(10 nmol/L),Ad-PP2A,and Ad-dn-PP2A.Compared with the control group,FTY720 and Ad-PP2A inhibited the expression of ZEB1 and VE-cadherin,OA(10 nmol/L)and Ad-dn-PP2A promoted the expression of ZEB1 and VE-cadherin;Western Blotting was used to detect the expression of ZEB1 and related signaling pathway proteins.The results showed that the expressions of p-AKT,ZEB1,MMP-2 and VE-cadherin were down-regulated,and the expressions of E-cadherin and BAX were up-regulated after cells were treated with different concentration of FTY720.Compared with FTY720,OA up-regulated the expressions of p-AKT,ZEB1,MMP-2,VE-cadherin and inhibited the expressions of E-cadherin and BAX.To further investigate whether the mechanism of compound BENC-511 on invasion and metastasis and VM formation in lung cancer cells is related to PP2A.Tube formation assay was used to detect the effect of BENC-511 on VM formation.The results showed that BENC-511 could effectively inhibit VM formation,BECN-511+Ad-dn-PP2A could restore VM formation.The expressions of P-AKT,ZEB1,PP2A and VE-cadherin were detected by Western blotting after cells were treated with BENC-511(5 ?mol/L),BENC-511(10 ?mol/L),BENC-511(5?mol/L)+Ad-dn-PP2A,BENC-511(10 ?mol/L)+Ad-dn-PP2A.The results showed that the BENC-511 group significantly down-regulated the expression of P-AKT,ZEB1,MMP-2,VE-cadherin,and up-regulated the expression of PP2A,while the BENC-511+Ad-dn-PP2A group down-regulated the expression of PP2A and up-regulated the expression of P-AKT,ZEB1,MMP-2 and VE-cadherin.2.In vivo studies,nude mice were transplanted with tumors,and the mice were inoculated with H1299-GFP cells under the armpits to construct a tumor-bearing mouse model.The nude mice were continuously administered drugs for 16 days to observe the effects of BENC-511 and PP2A on tumor growth.Immunohistochemical staining was used to detect the expressions of ZEB1,VE-cadherin,VEGFR-2,and E-cadherin.CD34/PAS double staining was used to observe the ability of VM formation of each group.The results showed that BENC-511 and BENC-511+FTY720 significantly inhibited tumor growth compared with the control group,and BENC-511+FTY720 had a better inhibition than BENC-511.The tumor inhibition rate of BENC-511+OA was significantly lower than BENC-511,and OA reversed the inhibitory effect of BENC-511 on tumor growth.Compared with the LacZ group,the Ad-PP2A treatment group significantly inhibited tumor growth,and the Ad-dn-PP2A group promoted tumor growth.Western blotting results showed that BENC-511 and BENC-511+FTY720 up-regulated PP2A expression and down-regulated the expressions of ZEB1,MMP-2 and VE-Cadherin compared with the control group.BENC-511+OA reversed the inhibitory effect of BENC-511 and restored the expressions of ZEB1,MMP-2 and VE-Cadherin.In the gene therapy,Ad-dn-PP2A significantly down-regulated PP2A expression and up-regulated the expressions of ZEB 1,MMP2 and VE-cadherin.Ad-PP2A significantly up-regulated the expression of PP2A and down-regulated the expressions of ZEB1,MMP-2 and VE-cadherin.Compared with the BENC-511 group,the BENC-511+OA group restored the expressions of ZEB1,VE-cadherin,VEGFR-2,and decreased the expression of E-cadherin,indicating that inhibition of PP2A activity reversed BENC-511-induced endothelial phenotype acquisition and VM inhibition.Ad-PP2A down-regulated the expressions of ZEB1,VE-cadherin,VEGFR-2 and up-regulated the expression of E-cadherin compared with the LacZ group.Ad-dn-PP2A up-regulated the expression of ZEB1,VE-cadherin,VEGFR and down-regulated the expression of E-cadherin.The CD34/PAS double staining results indicated that BENC-511 and BENC-511+FTY720 formed fewer VM channels than the control group.BENC-511+OA promoted VM formation compared with the control group.Compared with the LacZ group,it was difficult to find the presence of VM in the Ad-PP2A group,while the VM was significantly increased in the Ad-dn-PP2A group,indicating that inhibiting the activity of PP2A could promote the formation of VM.Conclusions(1)In vitro and in vivo experiments confirmed that PP2A played an important role in lung cancer metastasis and VM formation.PP2A could reduce H1299 cell metastasis,promote apoptosis and inhibit the formation of VM.Meanwhile,PP2A could reduce the tumor growth and VM formation in H1299 xenograft models.(2)PP2A could inactivate the PI3K/AKT signaling pathway by p-AKT dephosphorylation,inhibit the transcription of ZEB1,and then affect the EMT process,and negatively regulate the expressions of VE-cadherin and MMP-2 to inhibit VM formation.(3)BENC-511 inhibited invasion,metastasis and VM formation by inducing PP2A expression and significantly reversing the phosphorylation of PI3K and AKT,down-regulating the expression of ZEB1 and MMP-2 in lung cancer.SignificanceThe role of PP2A/AKT/ZEB1/MMP-2 in invasion,metastasis and VM formation of NSCLC was first studied and was expected to become a new target for lung cancer treatment. |
PDF Full Text Request