A Study On The Role Of AAMP In The Proliferation And Metastasis Of Non-small Cell Lung Cancer Cells

BackgroundAAMP is initially found in hematopoietic system cells and contains two immunoglobulin-like domains,one heparin-binding sequence and one WD40 repeat domain.WD40 domain-rich proteins play an important role in signal transduction,epigenetic modification,protein degradation,gene expression regulation,DNA damage repair and cell cycle regulation,which indicates that AAMP may also play a regulatory role in a variety of cell activities.Meanwhile subcells of AAMP are widely distributed and expressed in extracellular matrix,cell membrane and cytoplasmic matrix,which also indicates that AAMP is probably a multifunctional protein.AAMP is expressed in various cells,such as endothelial cells,trophoblast cells and activated T lymphocytes,and plays an important role in promoting the adhesion and migration of vascular endothelial cells and immune regulation.Compared with normal tissues,AAMP has a higher expression level in tumor cells,which has aroused people's interest in research.However,the role of AAMP in tumor cells is still unclear,and more functions need to be further explored.Proliferation is one of the basic life activities of cells,and it is vital in the growth,development and reproduction of individuals.The process of proliferation is a periodic activity and abnormal proliferation of tumor cells leads to unlimited proliferation and formation of tumor tissue,which eventually affects the normal physiological function of the body.Therefore,more exploration of the mechanism of tumor cell proliferation out of control has important theoretical significance for the discovery of potential targets and drug development.EGFR is an important tyrosine kinase receptor that activates multiple signaling pathways including RAS-ERK1/2,PI3K-PKB,PLCy,and SRC,thereby promoting cell survival,proliferation and anti-apoptosis.In the process of EGFR activation and inactivation,the formation and depolymerization of dimerization plays an important"switch" role.Tumor occurs when EGFR mutation developed and leading to continuous dimerization and depolymerization out of controll.However,the regulatory mechanism of EGFR dimerization is not very clear at present and further studies are needed.EGFR mutations are common in non-small cell lung cancer cells.Classic mutations are L858R point mutation and exon 19 partial deletion,which leads to continuous activation of EGFR and promotes tumor cell proliferation.EGFR Tyrosine Kinase Inhibitor(EGFR-TKI),a targeted drug targeting mutant EGFR,has been widely used in clinical practice.Although the early efficacy of EGFR TKI was significant,all patients with long-term treatment developed resistance to the drug for a variety of reasons,among which 60%of patients with drug resistance were caused by T790M point mutation.Therefore,it is of great significance to find more effective therapeutic targets.In addition to the ability to proliferate indefinitely,metastasis is another characteristic of tumors and is the leading cause of death in cancer patients.Metastasis mainly includes migration and invasion which depend on the formation of pseudopodia.The formation and disappearance of pseudopodia depend on the assembly and deassembly of cytoskeleton,in which microfilaments are the main components.The assembly and deassembly of microfilaments is a complex dynamic process,including nucleation,extension,stabilization and depolymerization,etc.,during which numerous proteins are involved like GTPase.The most studied GTPase families are RAC,CDC42 and RHO,among which CDC42 is involved in nucleation and extension of microfilaments,In addition,the CDC42-PAK-LIMK-CFL signaling pathway is also involved in the stabilization of microfilaments.GTPase has two forms:activation and inactivation.When GTPase combined with GTP,it is active,while GTPase is inactive when combined with GDP.The switching between the two states of GTPase is also regulated by various proteins,including GEF,GAP and GDI,etc.The GAP protein is the GTPase activation protein,which promotes the hydrolysis of GTP to GDP,and plays a negative regulatory role.Due to the complexity of metastasis,the regulatory mechanism of it is not very clear at present and it is urgent to deepen the understanding of metastasis and find the new targets.Methods and ResultsKaplan-meier Plotter database analysis showed that the expression level of AAMP was negatively correlated with the survival rate of patients with lung cancer.Subsequently,we investigated NSCLC cells as experimental materials.AAMP knockdown and overexpression were performed in several cell lines respectively.Then experiments such as photography by inverted phase contrast microscope and living cell workstation,EdU admixed experiment,SRB survival test and clone formation test were performed.Meanwhile,H1792 cell lines stably knocking down AAMP were successfully constructed by lentiviral infection and injected subcutaneous into immune-deficient B-NSG mice.The formation and growth of tumors were observed in long-term feeding.The above experimental results showed that AAMP can promote the proliferation,survival and tumorigenesis of NSCLC cellsNext,we explored the regulation mechanism of AAMP.The expression of AAMP was impaired in four NSCLC cell lines Calu-1,H1792,H1299 and H157.Western Blot results showed that after AAMP was knocked down,the expression level of CCND1 decreased and the proliferation ability of the cells was weakened.S ubsequent plate clone assay and EdU experiments showed that when CCND1 expression was restored,AAMP knockdown did not inhibit cell proliferation.This indicates that the function of AAMP knockdown in proliferation inhibition was performed by down-regulating CCND1.Next,Western Blot results showed that phosphorylation of ERX1/2 was inhibited when AAMP was knocked down in Calu-1 and H1792 cells,while p-ERK1/2 was enhanced when AAMP was overexpressed in A549 and H460 cells.At the same time,the ability of AAMP to up-regulate CCND1 was impaired after MEK inhibitor U0126 was used to inhibit the activation of ERK1/2.The above experiments showed that AAMP promoted cell proliferation in NSCLC cells by activating the ERK1/2-CCND1 signaling pathway.So how does AAMP promote the activation of ERK1/2?Through data base,it is found that there is likely to be an interaction between AAMP and EGFR.Subsequently,immunocoprecipitation was performed to verify the results and showed that AAMP can interacted with the intracellular domain of EGFR.WB experiments showed that AAMP knockdown and overexpression had no effect on EGFR protein level.However,results showed that AAMP knock down can inhibit EGFR phosphorylation at active site Y1173 and ERK1/2 signaling pathway after EGF treatment.While AAMP overexpression can promote EGFR phosphorylation and activate ERK1/2 signaling pathway.Besides,EGFR signaling is also inhibited in vivo tumors formed by H1792 cell lines with stable AAMP knockdown compared with the control group.Subsequently,revert experiment showed that high expression of EGFR can restore down-regulation of p-ERK1/2 caused by AAMP knockdown in Calu-1.The vitro and in vivo results indicated that AAMP can bind to EGFR and activate ERK1/2 by activating EGFR,thereby promoting cell proliferation.Next,co-IP experiments showed that knockdown of AAMP inhibited dimerization of EGFR and then impairing EGFR activity.AAMP can activate EGFR.Is there a feedback regulation of EGFR on AAMP?Knockdown of EGFR expression in wild-type EGFR cell lines H1 299 and H1 792,as well as EGFR mutant cell lines H1 650 and HCC827,and overexpression of EGFR sustainably activated plasmids in A549 and H1792.WB results showed that EGFR knockdown or overexpression had no effect on AAMP protein levels.EGFR mutations often occur in NSCLC patients,and the mutant EGFR can be activated ligand-independent and then continuously activated.Although targeted drugs have been designed for EGFR mutations,drug resistance has significantly weakened drug efficacy.Since AAMP is involved in the activation of EGFR,can the down-regulation of its expression enhance the efficacy of targeted drug TKI?AAMP knockdown was performed in EGFR mutant drug-resistant cell line H1975 and treated with icotinib.The results of flow cytometry,Western Blot and SRB showed that the AAMP knockdown reduced the resistance of H1975 to icotinib.Subsequent co-IP experiments showed that AAMP interacts with the ATP binding region of the intracellular segment of EGFR and binds more closely to the mutant EGFR.Because the binding site of TKI to EGFR was ATP binding site,we propose a hypothesis that after EGFR mutation,AAMP will enhance its binding ability to further promote dimerization activation of EGFR,on the one hand,and on the other hand,AAMP may bind to EGFR in a competitive manner to further inhibit TKI and EGFR chimerism,and thus enhance TKI resistance.In addition,the activation of EGFR also shows resistance to chemotherapy drugs,so does AAMP also affect the killing of NSCLC cells by chemotherapy drugs?AAMP knockdown and overexpression were performed respectively in EGFR wide-type cell lines Calu-1,H1792 and H460,A549 cells and doxorubicin was treated at the same time.The apoptosis rate and survival rate of cells were detected by flow cytometry,Western Blot and SRB experiments.Results demonstrated that AAMP can enhance the resistance of agents.In the process of exploring the impact of AAMP knockdown on NSCLC phenotype,it was found through scratch and invasion experiments that AAMP knockdown could also inhibit the migration and invasion of NSCLC cells.Next,we explore the mechanisms involved.Cell movement cannot be separated from the formation of pseudopodia.Does AAMP affect this process?Next,AAMP was silenced in Calu-1 and overexpressed in H460.TRITC-phalloidin was used to label the microfilament skeleton.Fluorescence confocal microscopy showed that AAMP could promote the formation of filamentous pseudopods in tumor cells.co-IP experiments showed that AAMP and CDC42 interact with each other.CDC42 activity was detected by CDC42 activity detection kit when AAMP was knocked down or overexpressed.The results showed that AAMP knockdown could inhibit CDC42 activation,while overexpression promoted CDC42 activation.The results indicated that AAMP could regulate the activity of CDC42.CDC42 has many active regulatory proteins,among which GAP and GEF play important roles.Through the co-IP experiment,it is found that AAMP and ARHGAP1 are interlinked and in H460 and A549 cells,AAMP inhibited the binding of ARHGAP1 and CDC42.Those indicated that AAMP can promote the activity of CDC42 by inhibiting the binding of ARHGAP1 and CDC42.ConclusionsIn summary:1.AAMP promote the proliferation,clonal formation and tumorigenesis of NSCLC cells.AAMP promotes dimerization by binding to EGFR to activate the downstream ERK1/2 signaling pathway.Activation of ERK1/2 can up-regulate the expression of CCND1 and ultimately promote tumor cells proliferation.2.In addition,AAMP can also enhances the resistance of tumor cells to EGFR-TKI and chemotherapy agents.3.Besides,AAMP activates CDC42 by inhibiting the binding of ARHGAP1 and CDC42,thereby promoting the formation of filopodia and ultimately promoting the migration and invasion of tumor cells.All in all,these results provide theoretical basis for further understanding the specific mechanism of proliferation and metastasis of non-small cell lung cancer cells and developing new tumor therapeutic targets.