The Mechanism Of TP53INP1 On The Inhibition Of Hypoxia Induced Epithelial Mesenchymal Transition And Vasculogenic Mimicry In Breast Cancer

ObjectiveBreast cancer is the most common malignant tumors in women,most breast cancer patients die primarily as a result of distant metastasis.Cancer cells could accelerate breast cancer development and metastasis in hypoxia environment.vasculogenic mimicry,a new vascular pattern,consists of cancer cells without endothelial cells,and it positively correlated with the poor clinical prognosis and tumor metastasis.Tumor p53 inducible nuclear protein 1(TP53INP1),as a tumor suppress protein,is a stress induced protein and play critical role in the progress of cancer.The study found that down-regulation TP53INP1 promoted metastasis of hepatocellular carcinoma and pancreatic cancer.However,the molecular mechanism of the relationship between TP53INP1 and breast cancer VM formation is unknown.Here,we explored the underlying mechanism by which TP53INP1 regulates not only VM formation but also epithelial-mesenchymal transition(EMT)in vitro and in vivo.Furthermore,we established a hypoxia model induced by Co Cl2 and assessed the effect of TP53INP1 in VM formation and the EMT process by the related signaling pathway,which offers a new idea for breast cancer antiangiogenic drugs and inhibiting tumor metastasis so as to improve patients survival rate.Methods1.100 breast cancer specimens were obtained from General Hospital of Tianjin Medical University by excision.These diagnosis of breast cancer samples were verified by pathologists.we analyzed the clinicopathological data by observing the expression of TP53INP1 and the presence of VM in breast cancer tissue specimens by CD31/PAS double staining.We analyzed the relationship of TP53INP1 and VM in breast cancer.Kaplan-Meier survival analysis showed the influence of TP53INP1 and VM on patients survival.2.We detected the expression of HIF-1?,VE-cadherin,Snail by immunohistochemistry in 100 breast cancer specimens.The relationship between TP53INP1 and HIF-1?,VE-cadherin were analyzed by ?2 test and Pearson test,and we only analyzed the relationship between TP53INP1 and Snail by ?2 test.3.We detected the constitutive expression of TP53INP1 by Western blot in four breast cancer cell lines and selected the two objective cell lines.These objective cell lines were transfected by putting the lentiviral vectors into 293 T cells and adding the upregulation and downregulation TP53INP1 expression plasmids,and we established stable breast cancer cell lines via puromycin selection.4.Wound-healing assay,invasion and migration assay,Three-dimensional(3D)cultures confirmed the migration ability and vascular forming ability of TP53INP1 in breast cancer cells.The expression of EMT(E-cadherin,Vimentin,Snail,GSK-3?,p-GSK-3?)and VM(VE-cadherin,MMP2,HIF-1?)related markers were tested by Western blot and immunofluorescence assay in overexpressing and low expressing TP53INP1 breast cancer cells.5.These transfected breast cancer cells were treated with Co Cl2 by building hypoxia environment and repeated the above experiments to confirm the influence of TP53INP1 about the ability of migration and invasion,EMT and VM forming in hypoxia inducible breast cancer cells.6.The ROS level was measured by flow cytometry analysis in nomaxia or hypoxia transfected breast cancer cells.We analyzed VM channel formation and ROS generation with the different NAC concentrations in MDA-MB-231 cells and the expression of VE-cadherin,HIF-1? by Western blot.We analyzed the EMT and VM-related protein expression by western blot in these transfected cells treated with oxidant scavenger ROS and verified that a ROS mediated signaling pathway participates in the process of inhibiting EMT and VM formation by TP53INP1.7.We performed a experiment in which HIF-1? was downregulated in TP53INP1-overexpressing MDA-MB-231 cells and TP53INP1-silencing MCF-7 cells.these cells were observed the expression of VM related markers by Western blot so as to verify whether there is an intimate connection between TP53INP1 and HIF-1?.8.Tientsin Albinao 2(TA2)mice from the Animals Center of Tianjin Medical University were used to establish a spontaneous breast cancer model.We selected two of five different tumors which approximately equivalent to the MDA-MB-231 and MCF-7 cell lines.After injected overexpressing and downexpressing TP53INP1 plasmids,we measured the volume of these tumors.Immunohistochemistry analysisand endomucin/PAS double staining analyzed the expression of TP53INP1,existence of VM and the expression of EMT/VM related markers(E-cadherin,Snail,HIF-1?,MMP2,VE-cadherin)in these mice model.Results1.The expression of TP53INP1 was low in 64 of 100 breast cancer specimens.The low expression of TP53INP1 was corrrelated with TNM stage(P=0.005),lymphatic metastasis(P=0.015)and triple-negative status(P=0.012).The TP53INP1 expression in 73 breast cancer tissues was lower than those in matched adjacent tissues and the difference had statistical significance(P=0.045).TP53INP1 expression was negatively associated with VM(P=0.009),Kaplan-Meier analysis that the VM and low expression of TP53INP1 were associated with poor overall survival in 100 breast cancer patients.2.?2 test analysis showed TP53INP1 expression was negatively correlated with HIF-1?(P=0.012),VE-cadherin(P=0.044),Snail(P=0.001)and the difference had statistical significance.Pearson test showed that there was correlation between TP53INP1 and HIF-1?(r=-0.0293,P=0.003),VE-cadherin(r=-0.0227,P=0.023).3.The expression of TP53INP1 is the highest in the MCF-7 breast cancer cell line,whereas the expression of TP53INP1 is the lowest in the MDA-MB-231 cell line among the two triple-negative breast cancer cell lines(MDA-MB-231 and HS-578T)and two non-triple negative breast cancer cell lines(MCF-7 and T-47D).Breast cancer cells lines MDA-MB-231 was transfected with TP53INP1 plasmid and MCF-7was transfected with sh-TP53INP1 plasmid.Both cell line were transfected with non-target plasmids to serve as the controls.Whether plasmids transfected successfully were tested by Western blot.4.The results showed overexpressing TP53INP1 MDA-MB-231 had weaker the ability of migration,wounding healing and tube formation than the control(P<0.005).Similarly,compared with those of the controls,MCF-7 cell line that was transfected with sh TP53INP1 plasmid had a higher invasion,migration,wounding healing and channel formation ability(P<0.005).The expression of E-cadherin,GSK-3? were higher in the overexpressing TP53INP1 MDA-MB-231 cell line,the protein expression of Vimentin,Snail,p-GSK-3?,VE-cadherin,MMP2,HIF-1? were lowerthan those in the control.MCF-7 cell lines knocking down TP53INP1 had the opposite result(P<0.005).5.We found that TP53INP1 overexpression reduced the ability of cell migration and invasion and tube formation in a Co Cl2 treated model.Western blot showed TP53INP1-overexpressing cells with Co Cl2-treated exhibited reduced Vimentin,Snail,p-GSK-3?,VE-cadherin,MMP2,HIF-1? expression,increased E-cadherin and GSK-3? expression in Co Cl2-treated TP53INP1-overexpressing cells compared to that in control cells(P<0.005).In contrast,TP53INP1 depletion in MCF-7 cells resulted in a higher invasion ability and tube formation ability.Western blot analysis demonstrated cells with TP53INP1 knocked down with Co Cl2-treated increased EMT and VM related protein expression(P<0.005).6.Flow cytometry analysis showed that the cell under hypoxic conditions with treated-Co Cl2 increased the ROS level than in control and were decreased by NAC(an oxidant scavenger).NAC inhibited the VM channel formation and decreased ROS levels.Additionally,the protein levels of VE-cadherin and HIF-1? were significantly decreased after 20 m M NAC inhibition.The ROS level was not affected by treatment with Co Cl2 or NAC after TP53INP1 upregulation.However,the levels of ROS was increased in the TP53INP1-silenced MCF-7 cell groups under hypoxia than in the control group but decreased with NAC.7.The expression of VE-cadherin and MMP2 decreased when HIF-1? were downregulated in TP53INP1-overexpressing cells regardless of adding Co Cl2,The VE-cadherin and MMP2 protein levels were increased with sh HIF-1? treatment in TP53INP1-silencing MCF-7 cells compared to the level in the control group.8.After establishing TA2 mice model,the speed of tumor size in high-metastasis group with overexpressing TP53INP1 was decreased than the control(P<0.005),TA2 mouse tumor growth with the sh-TP53INP1 plasmid increased than the control;VM formation decreased in the TP53INP1-overexpressing mice.However,there were more mice that showed VM formation in the TP53INP1-silenced group than in the control group by endomucin/PAS double staining.Immunohistochemistry showed that expression of E-cadherin increased,expression of HIF-1?,Snail,VE-cadherin and MMP2 were decreased in tumors with overexpressing TP53INP1,which showeda consistent result.In contrast,compared with the expression in the control group,these EMT and VM related protein expression were increased in the TP53INP1-silenced tumor group.Conclusions1.The expression of TP53INP1 was negatively related to the presence of VM formation,the TNM stage and lymphatic metastasis.There are negative correlation between TP53INP1 and poor prognosis.2.TP53INP1 inhibited not only the ability of breast cancer cells invasion and tube formation,but also VM and EMT formation by vitro and vivo.3.TP53INP1 inhibited hypoxia induced the ability of breast cancer cells invasion and tube formation,decreased EMT related protein expression by establishing a hypoxia model induced by Co Cl2 so as to assess the effect of TP53INP1 in VM formation.We preliminarily assessed that TP53INP1 inhibited HIF-1? mediated VM formation.4.ROS could facilitate the VM formation in breast cancer cells.5.We preliminarily confirmed that ROS mediated GSK-3?/Snail signaling pathway participated in the process of TP53INP1 inhibiting hypoxia-induced EMT and VM formation in breast cancer cells.