IFITM3 Regulate Stemness And EMT Process Of Breast Cancer Cell Via PI3K/AKT Signaling Pathway

Research background:Breast cancer(BC)is the most common malignant tumor in women worldwide,and seriously threatens women's health.Generally,the occurrence of breast cancer is related to many factors,including host immunity,genetics,environment and so on.It was found that breast cancer is closely related to epithelial-mesenchymal transition(EMT),which is a biological process in which epithelial cells acquire the motile and invasive characteristics of mesenchymal cells.Activation of EMT can increase tumorigenicity of epithelial cells and became one of the major mechanisms of development of cancer stem cell(CSC).EMT is considered to be the earliest stages of breast cancer.Therefore,this study focuses on elucidating the host factors and molecular mechanisms underlying EMT activation in order to provide experimental evidence and new clues for a deeper understanding of early clinical diagnosis and treatment of breast cancer.Study purposes: Explore the effects of interferon induced transmempbrane protein 3(IFITM3)regulate multiple biological behaviors of breast cancer cells by using breast cancer databases,human breast cancer cell lines,and a variety of experimental techniques.The parameters for examination include the effects of IFITM3 on EMT process,stemness,apoptosis,proliferation,migration and invasion on human breast cancer cells.Finally explore the relationship between the role of IFITM3 in breast cancer and the PI3 K / AKT signaling pathway.Investigate the role of IFITM3 in the tumorigenicity of human breast cancer cells by using nude mice tumor-bearing experiments.Methods: This study first analyzed the expression of IFITM3 in breast cancer by TCGA-BRCA(Breast Invasive Cancer Database).IFITM3 expression was detected in breast cancer cell lines MCF-7 and MDA-MB-231 by using real-time quantitative PCR and western blot.By transient transfection and lentiviral infection in both cell lines,we establish cell lines overexpressing or downregulating IFITM3.Through western blot,cell immunofluorescence analysis,real-time quantitative PCR analysis(q RT-PCR),wound healing assay,colony formation assay,transwell assay,sphere formation assay to examine the effects of IFITM3 on a variety of biological behaviors such as EMT,apoptosis,migration,and stemness in human breast cancer cells.Nude mice tumor-bearing experiments was used to detect the effect of IFITM3 on the tumorigenic ability of breast cancer cells in vivo.Results:1.Overexpressed IFITM3 can promote invasion/migration and proliferation,and inhibit apoptosis in breast cancer cells.Analyze the data using the Breast Invasive Cancer Database(TCGA-BRCA)of The Cancer Genome Atlas(TCGA).Data shows that IFITM3 expression was significantly higher in invasive breast cancer tissue samples(n = 1109)than that in normal breast samples(n = 113),which was 1.402 times(P <0.0001).By using western blot,IFITM3 was found to be highly expressed in MCF-7 and MDA-MB-231 cell lines,and its expression level in MDA-MB-231(with higher malignancy)is higher than MCF-7,suggesting IFITM3 may be involved in breast cancer progression.After the establishment of two cell lines overexpressing or downregulating IFITM3 by lentivirus infection.And named 7-up-IFITM3,7-Sh IFITM3 with their respective controls(7-Ctrl,231-Ctrl),and 231-up-IFITM3,231-Sh IFITM3 with their respective controls(7-Sh Scramble,231-Sh Scramble).Used colony formation assay found that the number and size of cell colonies were larger in the 7-up-IFITM3 and 231-up-IFITM3 group than their control group,the 7-Sh IFITM3 and 231-Sh IFITM3 group was smaller than their control group.This result indicates that IFITM3 can promote cell proliferation ability.Wound healing assays results show the wound healing interval of cells 7-up-IFITM3 and 231-up-IFITM3 group was smaller than that in their control cells,and the cell7-Sh IFITM3 and 231-Sh IFITM3 group was larger than the control in their control cells at 24 h,48 h,and 72 h.Transwell migration assay shows the number of transmembrane cells in the 7-up-IFITM3 and 231-up-IFITM3 group was more than their control group,and the 7-Sh IFITM3 and 231-Sh IFITM3 group was less than their control group.The above results suggested that IFITM3 evidently promotes cell migration capacity in both MCF and MDA-MB-231 cells.Expression of the proteins MMP2 and MMP9,which represent the invading ability of cancer cells was detected by western blot.Compared with control cells,expression levels of MMP2 and MMP9 were significantly increased in 7-up-IFITM3 and231-up-IFITM3 group and decreased in 7-Sh IFITM3 and 231-Sh IFITM3 group.It was further proved that IFITM3 can promote cell invasion.This study also found that the expression of apoptosis-related markers Bcl-2(anti-apoptosis),Bax and cleaved PARP(apoptosis)also changed.In the 7-up-IFITM3 and 231-up-IFITM3 group,BCL2 increased and PARP(Cleaved)and Bax decreased;vice versa,BCL2 decreased and PARP(Cleaved)and Bax increased in the 7-Sh IFITM3 and 231-Sh IFITM3 group,accompanied by increase in the Bcl-2/Bax ratio.Our data indicated that IFITM3 inhibits cell apoptosis,which may be one of an important reason of IFITM3 promoting breast cancer cell proliferation.2.IFITM3 regulates EMT process and stemness in breast cancer cells via PI3K/AKT signaling pathway.To uncover whether IFITM3 affect EMT,we examined typical EMT markers (E-cadherin,N-cadherin,Snail1,Twist1 and Vimentin)by western blot.It was found that overexpressing IFITM3 significantly reduced the expression of E-cadherin,and increased the expression of N-cadherin,Snail1,Vimentin,and Twist1;vice versa,downregulating IFITM3 increased the expression of E-cadherin,while the expression of N-cadherin,Snail1,Vimentin,and Twist decreased.Similar results were also found through immunofluorescence analysis.In 7-up-IFITM3 and 231-up-IFITM3 group,E-cadherin fluorescence intensity decreased,while N-cadherin fluorescence intensity increased.And E-cadherin fluorescence intensity increased in 7-Sh IFITM3 and231-Sh IFITM3 group,while N-cadherin fluorescence intensity decreased.These results indicate that IFITM3 can regulate the EMT process,and overexpressed IFITM3 inhibits the mesenchymal-epithelial transition(MET)process and may increase the ability to migrate and invade in breast cancer cell lines.Tested the expression of main stem cell markers(ALDH1,SOX2 and OCT4)in experimental cells and control cells by western blot analysis.The elevated expression of ALDH1,SOX2 and OCT4 were observed in 7-up-IFITM3 and 231-up-IFITM3 group and the expression of ALDH1 SOX2 and OCT4 were decreased in 7-Sh IFITM3 and231-Sh IFITM3 group compared with their corresponding control cells.Overexpressing IFITM3 significantly increased the number and size of the formation of spheroids of breast cancer cells,compared with their corresponding control cells,and downregulating IFITM3 also inhibited this phenomenon.We were able to demonstrate that the expression level of IFITM3 was positively correlated with the stemness of breast cancer cells.IFITM3 may be essential for the maintenance of cell self-renewal.Downregulating IFITM3 in breast cancer cells were used to examine the effect of IFITM3 on PI3K/AKT signaling pathway activity.The results show that the expression of total AKT did not change significantly,but the expression of P-AKT(ser473)decreased in 7-Sh IFITM3 and 231-Sh IFITM3 group compared with their corresponding control group.It indicated that downregulating IFITM3 can inhibit the expression of phosphorylated AKT,suggesting that the activity of the PI3K/Akt signaling pathway may be weakened by reduced IFITM3,leading to inhibition of migration,invasion, stemness,and EMT process in breast cancer cells.It inferred that the highly expressed IFITM3 may promote the progression of breast cancer by activating the PI3K/Akt signaling pathway.3.IFITM3 knockdown of breast cancer cells inhibits tumorigenicity in vivo Nude mice tumor-bearing experiments results show,during observation period(14 days after transplantation of MDA-MB-231 cell),the average tumor volume and weight of231-Sh IFITM3 group was obviously smaller than that in 231-Sh Scramble group,implying the tumor growth in the 231-Sh IFITM3 group is relatively slow compared with the 231-Sh Scramble group.This result indicates that the tumorigenicity in vivo of triple negative breast cancer cells MDA-MB-231 was inhibited when the expression of IFITM3 was knocked down,suggesting that high expression of IFITM3 may become a marker for early clinical diagnosis.Conclusion:(1)IFITM3 is highly expressed in invasive breast cancer tissues and breast cancer cells,and is an important oncogene for breast cancer diagnosis.(2)IFITM3 has biological characteristics that promote breast cancer cell proliferation,invasion and migration,and inhibit apoptosis.(3)The expression level of IFITM3 affects the negative correlation between E-cadherin and N-cadherin,and can promote the EMT process of breast cancer cells.(4)The expression level of IFITM3 is positively correlated with the stemness of breast cancer cells.(5)Knocking down IFITM3 can reduce the phosphorylated AKT component of the PI3 K / AKT pathway,weaken the tumorigenic capacity of breast cancer cells in nude mice,and delay the progression of breast cancer.IFITM3 can interact with the PI3 K /AKT pathway to regulate its biological behavior characteristics.