TRAF2 Activates The Akt Signaling Pathway Via TRAF4-mediated Ubiquitination Of Akt And Promotes Migration And Invasion Of Breast Cancer Cells

Objective:Breast cancer is a high incidence of malignant tumors in the 21 st century,to explore the cause of breast cancer and the development of molecular mechanisms,then determine the early diagnosis of breast cancer markers and targeted drugs will be the key to solve this problem.Tumor necrosis factor receptor factors(TRAFs)is an important cytoplasmic adapter protein.Its family contains seven members.They involved in the regulation of NF-κB,JNK and other pathways.TRAF2 is one of the classic members of the TRAFs family which is highly expressed in a variety of tumors.We found that TRAF2 promotes the activation of p70s6 k signaling pathway.Upregulation of TRAF2 expression in breast cancer cell line MCF-7 can activate NF-κB nuclear translocation and inhibit the apoptosis of breast cancer cells.Akt,also known as protein kinase B(PKB),as an important signal pathway in tumor cells,regulates cell growth and proliferation,and its abnormal activation is closely related to the occurrence and development of tumor.It has been found that the ubiquitination of Lys63 can promote Akt activation.TRAF4 has E3 ubiquitin ligase activity,can mediate Akt ubiquitination activation.We found that TRAF2 binds to TRAF4 and regulates the cellular localization of TRAF4,so we speculate that whether TRAF2 can affect the activation of Akt signaling by regulating TRAF4.In this study,we confirmed that the high expression of TRAF2 in breast cancer was positively correlated with the activation of Akt signaling pathway,and the prognosis of breast cancer patients with high expression of TRAF2 was unfavourable prognosis.TRAF2 can influence the biological function of breast cancer cells through Akt signaling pathway.We found that TRAF2 activation of this signaling pathway is achieved by TRAF4 to ubiquitination of Akt.Methods:1.Cell culture: The normal mammary epithelial cell line MCF-10 A was cultured in DMEM/F12 medium(Gibco,USA)containing 5% horse serum,10μg/ml insulin and 20 ng/ml EGF.The breast cancer cell lines MDA-MB-231,MDA-MB-468 were cultured in L15 medium(Gibco,USA)containing 10% FBS.The breast cancer cell line MCF-7 was cultured in DMEM medium(Gibco,USA)containing 10% FBS.The breast cancer cell line BT-549 was cultured in 1640 medium(Gibco,USA)containing10% FBS.Except MDA-MB-231 doesn’t need CO2,the culture conditions for all cells were: 37℃,5% CO2.2.Immunohistochemistry: the paraffin-embedded breast tissue specimens were made into 4 μm thick sections and placed on slides.Followed by dewaxing of xylene,gradient elution of benzene,hydration,washing with PBS for 5minutes×3.In the high temperature and pressure citrate buffer for antigen repair,PBS washing 5 minutes×3,dropping peroxidase inhibitor,and incubated in a 37℃ incubator for 15 minutes.PBS washing for 5 minutes×3,add 10% non-immune animal serum,placed in a wet box and placed in a 37℃ incubator for 60 minutes.50-100μl anti-dilution(PBS as solvent,1: 100 dilution ratio of TRAF2 antibody,1: 200 dilution ratio of Akt antibody)was added dropwise to each slide,and PBS was used instead of antibody as the control,placed in a wet box and placed in a 4℃ refrigerator for overnight incubation.Next day,PBS washing for 5 minutes×3,add biotin labeled secondary antibody,placed in a wet box into the 37℃ incubator for 30 minutes.PBS washing for 5minutes×3,adding streptavidin anti-avidin-peroxidase solution,continuing to place in the wet box into the 37℃ incubator for 30 minutes.PBS washing for 5 minutes×3,the use of DAB coloring agent,hematoxylin in the nucleus,hydrochloric acid alcohol rapid differentiation,tap water anti-blue,Gradient ethanol for dehydration,xylene dewaxing transparent,neutral resin seal.3.Transfection,plasmids and siRNAs:HTRAF2pLPCX-HA-Flag/P874(TRAF2 full-length plasmid)was purchased from Addgene Corporation(USA).TRAF4 and Akt interference sequences were purchased from Guangzhou Ruibo Biotechnology Co.,Ltd.Transfection reagents Attractene and interfering reagents HiPerFect were purchased from Qiagen(Germany),according to the manufacturer’s instructions for transient transfection and interference experiments.Use empty plasmid as a negative control.4.Western blot: After transfection of plasmid or interfering for 48 hours,cells were collected and the supernatant was extracted after lysis.Bradford method was used to detect the protein concentration,the same amount of protein was taken up,then the protein was separated by SDS-PAGE,transferred(60V,2hours)to PVDF membrane.Membranes were blocked for 2 hours at room temperature with 5% non-fat milk or 5% BSA and incubated with antibodies at 4℃ for overnight.Secondary antibody was included for 2 hours at room temperature,ECL color rendering,image acquisition,gray value measurement.5.Scratch test: Cells were processed andseeded into 6-well plates,and 200μl of pipettes were drawn vertically.After PBS washed,add serum-free medium and photographed after incubator to continue to culture.Select the different time point to take pictures and analyze migration capabilities with Image J software.6.Transwell: After 24 hours treatment,the cells were hungry for another 24 hours and resuspended averaged on the upper chamber(Matrigel gel).The medium was added in lower chamber.The cells were incubated for 24 hours in the incubator room.Use cotton swabs to wipe the upper chamber.Fixed by methanol then dyeing.Under the microscope,five high magnification images were randomly selected and counted,and the statistical analysis was carried out.7.Immunofluorescence microscopy: Cells were transfected and homogenized on slides in 24-well plates,After 48 hours,the phosphate buffer(PBS)was washed with a dish of 2,4% paraformaldehyde on ice for 20 minutes and washed with PBS for 5 minutes ×3.At room temperature,Triton-X100 was punched for 15 minutes and washed with PBS for 5 minutes ×3.At room temperature,3% BSA or normal goat serum was incubated for 1 hour.Remove the blocking solution,add antibody(TRAF2 dilution 1: 50,p-Akt dilution 1: 100)in a wet box at 4℃ overnight.PBS wash for 5 minutes ×3,At room temperature,adding anti-mouse,anti-rabbit fluorescent secondary anti-earthox 488 and 594(dilution concentration of 1: 100)in the dark room,incubated for 2 hours.PBS was washed for 5 min ×3,DAPI stained nuclei,and fluorescence confocal microscopy were observed.8.Ubiquitination experiment: The cells were transfected by required plasmid and at the same time transfected into HA-Ub.After 24 hours,20 μM proteasome inhibitor MG-132 was added,after another 4 hours the cells were harvested.The supernatant protein was extracted after lysated on ice for 30 minutes then added 1μg of Akt antibody incubated overnight at 4℃.Next day add Protein A+G beads and incubated at 4℃ for 4 hours.The immune complex was eluted and subjected to Western blot to detect ubiquitination level.9.Detection of apoptosis by flow cytometry: After 48 hours of transfection or interference,the cells were stained with Qiagen apoptotic kit and use flow cytometry to detect the apoptosis level.10.Statistical Analysis: Use SPSS 17.0 statistics software to analysis experimental data.Results:1.Immunohistochemistry showed that TRAF2 was negative in normal breast tissues and was highly expressed in breast carcinoma and invasive ductal carcinoma.The expression of TRAF2 was associated with pathologic classification of breast cancer(p<0.05),and positively correlated with the expression of Akt.Kaplan-Meier survival curves showed poor prognosis in patients with positive expression of TRAF2.Western blot showed that Akt and TRAF2 were significantly higher expressed in breast cancer cell lines than in normal breast cell lines(p <0.05).2.The results of flow cytometry showed that TRAF2 in MCF-7 cells’ transfection inhibited the apoptosis of cells.The apoptotic rate of si-Akt was enhanced.Transfection TRAF2 and si-Akt significantly weaken the apoptotic rate caused by TRAF2 overexpression.Scratch experiments and Transwell experiments showed that transfection of TRAF2 promoted the invasion of breast cancer cells.Transfection si-Akt group cells invasive ability was inhibited,and both were co-transfected to weaken the invasion ability of breast cancer cells.3.Western blot showed that elevated levels of Akt phosphorylation were significantly increased after upregulation of TRAF2 expression in MCF-7.The same results were obtained by immunofluorescence.Then introduced Akt activation effect factor EGF and selected the appropriate time points.In the MCF-7 and BT-549 cell lines,the level of Akt phosphorylation was significantly increased after TRAF2 upregulation,and the levels of Akt phosphorylation were significantly decreased after TRAF2 down regulation induced by EGF.4.We overexpressed TRAF2 in breast cancer MCF-7 cell line by ubiquitination assay and found that the ubiquitination level of AKT increased.Down-regulation of TRAF2 expression,AKT ubiquitination level decreased Down-regulation of TRAF4 expression,AKT ubiquitination level decreased.5.Transfecting TRAF2 into MCF-7 and BT-549,p-Akt expression level increased.After interfering with TRAF4,p-Akt expression level decreased.Transfecting TRAF2 and interfering TRAF4 at the same time significantly inhibited p-Akt level which increased by the transfection of TRAF2.MG-132 can synergistically enhance the increase in p-Akt induced by EGF.Ubiquitination experiments show in MCF-7 and BT-549,if upregulate TRAF2,the ubiquitination of Akt level would increase and if downregulate TRAF4,the ubiquitination of Akt level would decrease.Transfecting TRAF2 and interfering TRAF4 at the same time would significantly inhibited the Akt ubiquitination level.Conclusion: TRAF2 and Akt overexpression in breast cancer,TRAF2 expression was positively correlated with histological type and Akt expression.Breast cancer patients whose TRAF2 was high expression were poor prognosis.TRAF2 can inhibit theapoptosis by Akt and promote cell migration invasion.TRAF2 mediates the activation of EGF-induced Akt signaling pathway.TRAF2 affects the ubiquitination and activation of Akt through TRAF4.