The Study On The Effect Of Tumor Necrosis Factor-alpha-induced Protein 8-like 3(TIPE3)on Growth And Metastasis Of Breast Cancer And Its Underlying Mechanism

Objective: To investigate the role of tumor necrosis factor-alpha-induced protein 8-like 3(TNFAIP8L3)(TIPE3)and its molecular mechanism in breast cancer.Methods:(1)A total of 100 pairs of breast cancer tumor and adjacent non-tumor tissues were obtained from randomly selected 100 patients who have undergone surgery for breast cancer.The clinicopathological characteristics of these patients were collected based upon medical history.The expression of TIPE3 in human breast cancer tumor and adjacent non-tumor clinical tissues was analyzed by immunohistochemistry.The expression level of TIPE3 in breast cancer tumor tissues and its correlation with clinicopathological parameters features of breast cancer patients was analyzed.(2)The total cellular protein derived from a panel of human breast cancer cell lines such as MCF7,SKBR3,MDA-MB-231 and MDA-MB-468 as well as a human normal mammary epithelial cell line MCF10A(used as a control)was extracted.The expression of TIPE3 in breast cancer cells was detected by q RT-PCR and Western blot analysis.(3)The p Lenti6.3/TIPE3/IRES/GFP expressing human TIPE3 and the p Lenti6.3/sh RNA-TIPE3/IRES/GFP harboring sh RNA-TIPE3 lentiviral plasmids were constructed,respectively.Then the p Lenti6.3/TIPE3/IRES/GFP,the p Lenti6.3/IRES/GFP,the p Lenti6.3/sh RNA-TIPE3/IRES/GFP or the p Lenti6.3/sh RNA-control/IRES/GFP lentiviral plasmid was cotransfected into 293 T human embryonic kidney cells with helper packaging plasmids including p LP1,p LP2 and VSVG by Lipofectamine 2000.The lentivirus expressing TIPE3(LV-TIPE3)and the blank lentivirus(LV);and the lentivirus harboring sh RNA-TIPE3(LV-sh TIPE3)and the lentivirus harboring sh RNA-control(LV-shcontrol)were consequently generated and the titre was then determined,respectively.(4)A lowly metastatic breast cancer cell line MCF7 that expressed relative low level of TIPE3 was infected with LV-TIPE3 or LV at a multiplicity of infection(MOI)of 50.A highly metastatic breast cancer cell line MDA-MB-231 that expressed relative high level of TIPE3 was infected with LV-sh TIPE3 or LV-shcontrol at a multiplicity of infection(MOI)of 50.The above cells were then selected with drug Blasticidin S(BSD),leading to the generation of MCF7-TIPE3 and MCF7-mock(control);and MDA-MB-231-sh TIPE3 and MDA-MB-231-shcontrol(control)breast cancer cells.The transgene efficiency was analyzed according to GFP reporter by fluorescence microscopy and flow cytometry.The overexpression or knockdown efficiency of TIPE3 in MCF7 or MDA-MB-231 breast cancer cells was determined by q RT-PCR and Western blot analysis.The effect of lentivirus-mediated TIPE3 overexpression or silence on in vitro proliferation,cell-cycle,migration and invasion of breast cancer cells was evaluated by Cell Counting Kit-8(CCK-8)assay,colony formation assay,propidium iodide(PI)cell cycle assay,wound healing assay and Transwell migration and invasion assays.The effect of TIPE3 on breast cancer cell growth in vivo was monitored by measurement of tumor volume and tumor weight using a breast cancer subcutaneously(s.c.)transplanted tumor model in athymic BALB/c nude mouse.The effect of TIPE3 on breast cancer cell metastasis to the lung of athymic BALB/c nude mouse in vivo was detected by intravenous(i.v.)injection of breast cancer cells through murine tail vein.(6)The effect of TIPE3 overexpression or silence on expression of p-AKT(T308),p-AKT(S473),AKT,p-GSK3β(S9),GSK3β,β-catenin,Snail1,Snail2(Slug),Cyclin D1,E-cadherin,N-cadherin and Vimentin in breast cancer cells was analyzed by Western blot.The effect of TIPE3 on β-catenin,Snail1 and Slug was further assessed by immunofluorescence using confocal microscopy.The effect of TIPE3 on Cyclin D1,E-cadherin,N-cadherin and Vimentin m RNA was also analyzed by q RT-PCR.Moreover,the effect of TIPE3 on in vivo expression level of p-AKT(T308),p-AKT(S473),AKT,p-GSK3β(S9),GSK3β,β-catenin,Snail1 and Slug in breast cancer s.c.transplanted tumor tissues in athymic BALB/c nude mouse was detected by immunohistochemistry analysis.Results:(1)We demonstrated that the expression of TIPE3 in human breast cancer tumor tissues was significantly higher than that in adjacent non-tumor tissues,which was positively associated with the tumor size,the presence of lymph node metastasis and the TNM stage.(2)Compared with a normal mammary epithelial cell control group,the expression of TIPE3 in human breast cancer cells was elevated.(3)The stable TIPE3-overexpressed MCF7-TIPE3 vs MCF7-mock,and the TIPE3-knocked down MDA-MB-231-sh TIPE3 vs MDA-MB-231-shcontrol breast cancer cell lines were successfully established.(4)The lentivirus-mediated TIPE3 overexpression remarkably promoted in vitro proliferation,G1/S phase cell cycle transition,migration and invasion in MCF7 breast cancer cells,whereas knockdown of TIPE3 suppressed the proliferation,migration and invasion as well as induced G1 phase cell cycle arrest in MDA-MB-231 breast cancer cells.Knockdown of TIPE3 also repressed in vivo growth and metastasis to lung in MDA-MB-231 breast cancer cells transplanted in athymic BALB/c nude mouse.(5)Overexpression of TIPE3 obviously upregulated level of p-AKT,p-GSK3β,β-catenin,Snail1 and Slug as well as nuclear β-catenin,Snail1 and Slug in MCF7 breast cancer cells.TIPE3 upregulated Cyclin D1,N-cadherin and Vimentin while downregulated E-cadherin in MCF7 breast cancer cells.However,knockdown of TIPE3 exhibited an opposite effect in MDA-MB-231 breast cancer cells in vitro and in vivo in athymic BALB/c nude mouse.Conclusions: TIPE3 is upregulated in breast cancer.TIPE3 promotes growth and metastasis in breast cancer very probably via a AKT-GSK3β-β-catenin/Snail pathway.TIPE3 may be a novel therapeutic target for breast cancer.