The Mechanism Of TAZ On The Regulation Of Vasculogenic Mimicry In Gastric Cancer

Objective:According to the latest global cancer statistics,gastric cancer(GC)is the fifth most common cancer type in the world and the third leading cause of cancer-related death.Currently,although the incidence of GC has stabilized in recent years,the prognosis and survival rate of patients with advanced GC remain low due to the high rates of local and distant metastases.Because angiogenesis is regarded as an an important contributor to tumor growth and metastasis,it was once considered a potential therapeutic target for blocking tumor growth.However,some anti-angiogenic drugs have not yet achieved significant clinical effects in GC patients,and this finding might be related to the ability of cancer to exhibit different blood vessel formation patterns.Vasculogenic mimicry(VM),a new tumor blood supply model,is a functional vascular-like channel produced by invasive cancer cells in the absence of endothelial cells.VM,which has been observed in most malignant tumors,has been correlated with tumor invasion and metastasis and is thereby considered significant for the low survival rate and poor prognosis associated with these tumors.In addition,traditional anti-angiogenic agents have little effect on inhibiting VM and are sometimes even counterproductive.Therefore,many studies have attempted to explore the potential mechanism of VM formation and have identified several key regulatory factors,including VE-cadherin,MMP2,and MMP9.Furthermore,epithelial-mesenchymal transition(EMT)and cancer stem cells(CSCs)have been shown to be involved in VM formation.However,the molecular mechanisms of VM formation in GC are unclear,and elucidation of these mechanisms may provide new avenues for the development of angiogenic therapies.TAZ is a downstream effector molecule in the Hippo signaling pathway.It is found to be highly expressed in many tumors,and binding to the TEAD family can promote tumor proliferation and metastasis.However,the molecular mechanism of the relationship between TAZ and VM formation in gastric cancer is not clear.In this study,we explore the role of TAZ in VM formation,EMT and CSC by immunohistochemistry,cell experiments in vitro and various molecular biology experiments,which provide a new insight for gastric cancer anti-angiogenic therapy.Methods:1.The correlation between TAZ and TEAD4 in human gastric cancer tissues and its relationship with VM.1)A total of 228 specimens of gastric cancer tissue from Tianjin Medical UniversityGeneral Hospital and Cancer Hospital were collected.The pathologic diagnosiswas confirmed by at least two trained pathologists.Detailed pathologic and clinicaldata were collected for all samples.The CD34/PAS double staining andimmunohistochemical staining were performed on the sections to observe theexpression level of TAZ and TEAD4 in gastric cancer and the presence of VM,andto analyze the correlation between TAZ and TEAD4,VM and clinicopathologicaldata.In addition,Kaplan-Meier survival analysis showed the relationship betweenrelevant indicators and patients prognosis.2)Immunohistochemistry was used to detect the expression of VE-cadherin,E-cadherin and CD44 in 228 gastric cancer tissues.The relationship between TAZ and VE-cadherin,E-cadherin and CD44 were further analyzed by Pearson method andc~2 test.2.We explore the role of TAZ in gastric cancer VM,EMT,CSC in vitro. 1)The expression of TAZ in four gastric cancer cell lines(MGC803,MKN45,MKN28,MKN74)was detected by Western blot,and two cell lines were selected.The plasmids were transfected with TAZ in the corresponding gastric cancer cell lines,and we established the corresponding stable cell lines with puromycin. 2)The effects of TAZ on the tube formation,migration,proliferation,and sphere formation ability of gastric cancer cells were examined by three-dimensional(3D)culture,wound-healing assay,invasion and migration assay,3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)proliferation,and tumor 3D spheroid culture.3)The expression of TEAD4,VM-related protein markers(VE-cadherin,MMP2, MMP9),EMT-related protein markers(E-cadherin,Vimentin,snail),CSC-related protein markers(ALDH1,CD44,c-Myc,SOX2)were tested by Western blot.The localization and expression of TAZ,TEAD4,VE-cadherin,E-cadherin,Vimentin, ALDH1 and CD44 were further confirmed by immunofluorescence experiments.4)We transfected the sh-TEAD4 expression plasmid and the TAZ overexpression plasmid in MKN28 cell lines,and transfected the TEAD4 overexpression plasmid and sh-TAZ expression plasmid in MGC803 cell lines.The interaction between TAZ and TEAD4 was verified by western blot,invasion and migration assay and 3D culture.In addition,the protein interaction between TAZ and TEAD4 was examined by Co-IP assay.Results:1.TAZ and TEAD4 were highly expressed in human gastric cancer tissues,and were associated with poor prognosis of gastric cancer patients.TAZ and TEAD4 were positively correlated with VM. 1)TAZ was highly expressed in 101 of 228(101/228)human gastric cancer tissues. TEAD4 was highly expressed in 95 of 228(95/228)human gastric cancer tissues.TAZ and TEAD4 expression were positively correlated with VM(P<0.05).Immunohistochemistry and clinicopathological data showed that TAZ and TEAD4 were mainly expressed in the nucleus and correlated with tumor grade,TNM stage and metastasis(P<0.05).Kaplan-Meier analysis showed that patients with gastric cancer with TAZ and TEAD4 positive had a poor prognosis(P<0.05).Pearson analysis showed that the expression of TAZ was positively correlated with TEAD4 in 228 GC samples(r=0.3331,P=0.000). 2)c~2 test results showed that TAZ expression was positively associated with VM related indicator VE-cadherin(c~2=10.525,P=0.001)and CSC related indicator CD44(c~2=4.635,P=0.031),and was negatively associated with EMT related indicator E-cadherin(c~2=1.282,P=0.039),and the difference was statistically significant. 2.TAZ promotes VM,EMT and CSC formation in gastric cancer cells through interaction with TEAD4. 1)The gastric cancer cell line MGC803 with high expression of TAZ and MKN28 with low expression of TAZ were selected by Western blot.The results of cell function test showed that overexpressing TAZ MKN28 had enhanced the ability of tube formation migration wound healing proliferation and sphere formation compared with the control group(P<0.05).On the contrary,MGC803 cell lines that down-regulated TAZ had weaker the ability ability of tube formation migration wound healing proliferation and sphere formation(P<0.05). 2)Western blot and immunofluorescence showed that the expression of VE-cadherin, MMP2,MMP9,Vimentin,snail,ALDH1,CD44,c-Myc and SOX2 increased with the overexpression of TAZ.The expression of E-cadherin was decrease than the control group(P<0.05).MGC803 cell lines that were transfected with sh-TAZ plasmid had the opposite result. 3)The results of immunoprecipitation experiments showed that there was protein interaction between TAZ and TEAD4.To further verify the interaction between TAZ and TEAD4,TEAD4 was down-regulated while MKN28 was up-regulated TAZ,and the TAZ down-regulated plasmid was co-transfected with TEAD4 up- regulated plasmid in MGC803.Western blot analysis showed that the expression of VE-cadherin,vimentin and snail was decreased and the expression of E-cadherin was increased after up-regulation of TAZ in MKN28 cells and down-regulation of TEAD4.In contrast,overexpression of TEAD4 enhanced the expression of VE- cadherin,vimentin,and snail proteins in MGC803 cells after TAZ down-regulation, and decreased the expression of E-cadherin,but no change was observed in the control group. 4)After co-transfection of cells,overexpression of TEAD4 in MGC803-shTAZ cells significantly rescued the inhibition of TAZ downregulation on tube formation and migration.In addition,TEAD4 down-regulation inhibited the enhancement of the ability to tube formation and migration in MKN28 cells after TAZ overexpression.Conclusions:1.The expression of TAZ and TEAD4 are associated with tumor grade,TNM stage, metastasis and VM,and are correlated with poor prognosis.In addition,TAZ was positively correlated with TEAD4 in 228 gastric cancer tissue samples. 2.TAZ not only promotes the ability of gastric cancer cells tube formation,migration, proliferation,and sphere formation,but also promotes VM,EMT and CSC and the expression of related proteins in vitro. 3.TAZ promotes VM formation by interacting with TEAD4 in gastric cancer cells.