ATP1A2 Acts As A Tumor Suppressot Invovled In Proliferation,Invasion And Apoptosis In Breast Cancer By Inhibiting The Src/PI3K/Akt Signaling Pathway

Purpose:As a well-known membrane protein,Na-K-ATPase is widely involved in various physiological and pathological processes.Under normal physiological conditions,it participates in the regulation of ion balance inside and outside the membrane,maintaining resting potential,and material metabolism.Under pathological conditions,such as migraine,familial epilepsy and congestive heart failure,Na-K-ATPase also plays a role.It has also been shown that Na-K-ATPase is important in tumorigenesis.In addition to the classical effect on ion concentration inside and outside the membrane,Na-K-ATPase has been proved to be able to activate Src kinase in binding with its ligand cardiac glycosides(GS),triggering the activation of downstream Raf-MAPK pathway,thereby increasing the synthesis of reactive oxygen species(ROS).Moreover,it can inhibit tumors by inhibiting the activation of Akt and NF-kappa B,etc.Na-K-ATPase has three subunits,α,β and FXYD,and each subunit has different isoforms.The roles of different isoforms are also different.As the main "executor" of Na-K-ATPase,α subunit has four isoforms.Amongthem,α1 subunit(ATP1A1)widely exists in various tissues.It is considered to be highly expressed in some tumors and promote the occurrence and development of tumors.However,other subunits are less concerned,especially the α2 subunit(ATP1A2).This study focused on the effects of ATP1A2 on breast cancer and the related mechanisms.Methods:1.Data from The Cancer Genome Atlas(TCGA)database were downloaded.GraphPad Prism 5.0 and SPSS 19.0 were used to analyze ATP1A2 expression level,survival and the correlation between expression and clinical.2.Fourteen pairs of breast cancer and adjacent tissues were used to extract mRNA,and quantitative PCR(RT-qPCR)was used to estimate the expression level of ATP1A2.Immunohistochemistry was used to detect the expression of ATP1A2 in 17 pairs of breast cancer and adjacent tissues.3.ATP1A2 and empty plasmid were transfected into MDA-MB-231 and SK-BR-3 breast cancer cell lines with low expression of ATP1A2.G418 was added for screening.After 14 days,stable over-expressing ATP1A2 cell lines were confirmed by semi-quantitative PCR and Western blot.CCK-8,Transwell and flow cytometry were used to detect the proliferation,invasion,apoptosis and cell cycle arrest of over-expression groups and control groups.4.Western blot was used to detect the expression of Src,PI3 K,Akt and other related proteins in over-expression groups and control groups.Results:1.Through the analysis of the data from TCGA database,it was found that only ATP1A2 was down-regulated in tumors among the four isoformsof Na-K-ATPase(p < 0.05),and its expression level related with survival analysis,tumor size,and the expression of ER,PR and HER-2(p < 0.05).2.The results of RT-qPCR and immunohistochemistry showed that the expression of ATP1A2 in breast cancer was lower than that in adjacent normal tissues at both transcriptional and translation levels(p < 0.05).3.CCK-8,colony formation,Transwell and flow cytometry assays showed that compared with the control groups,the proliferation and invasion of the overexpression groups were inhibited,while apoptosis increased.Cycle arrest occurred in G1/G0 phase in overexpression groups.The above results have statistical significance(p < 0.05).4.Western blot showed that the phosphorylation of Src,PI3 K,Akt and NF-kappa B,the expression of Bcl-2 and Bcl-xl were inhibited,while Bax and P21 were upregulated in the overexpression groups compared with the control groups.Conclusions:1.Analyzed the data from TCGA,ATP1A2 was downregulated in breast cancer,and the downregulation was correlated with clinical features,indicating that the expression level of ATP1A2 might be related to the prognosis of breast cancer.The high expression group,whether in survival analysis or in correlation analysis of clinical features,suggested a better prognosis than the low expression group.It suggests that ATP1A2 may play an anti-cancer role in breast cancer.2.The results of immunohistochemistry and RT-qPCR of human breast cancer and adjacent tissues confirm the conclusion that ATP1A2 is lowly expressed in breast cancer from TCGA database data analysis.3.The functional assays showed that overexpression of ATP1A2 inhibited the proliferation and invasion of breast cancer cells,increasedapoptosis and cause cell cycle arrest.These results further confirm that ATP1A2 could inhibit breast cancer.4.The results of western blot confirm that overexpression of ATP1A2 inhibits Src/PI3K/Akt signaling pathway,and the activation of this pathway promotes the proliferation,invasion and migration of cancer cells.In conclusion,ATP1A2 is down-regulated in human breast cancer and is associated with the prognosis of breast cancer.It inhibits the proliferation and invasion of breast cancer cells,induces apoptosis and cell cycle arrest,which are achieved by inhibiting Src/PI3K/Akt signaling pathway.This may bring a new way and new perspectives to the treatment of breast cancer.