The Mechanism Of Anterior Gradient 2 Expression Suppressed By Proteasome Inhibitor

Lung cancer,the fastest growing incidence and mortality,is becoming the biggest threat to people health and life as one of the malignant tumor.Lung cancer,the 5-year survival rate is only 16.1%in China,can be improved up to 60?80%if the effective surgical excision has been performed in the early phase of after diagnosis.According to the characters of clinical pathology,lung cancer can be divided into small cell lung cancer and non-small cell lung cancer,and 80%of the patients with lung cancer clinically belongs to non-small cell lung cancer,but only about 30%of the patients were diagnosed as early patients which can be completely removed by surgery,and the remaining 70%of patients that are diagnosed as advanced tumor can only be treated by chemotherapy,but the prognosis is poor.So how to find the effective markers and therapeutic targets to diagnose and treat the early phase of non-small cell lung cancer is particularly important.A recent study found that Anterior gradient-2(AGR2)as a new proto-oncogene increased in non-small cell lung cancer tissue,and was more sensitive to diagnose the early stage of lung adenocarcinoma,therefore AGR2 protein can be used as a new prognostic marker and therapeutic target to treat non-small cell lung cancer.AGR2 is located in chromosome7p21.3,and its primary protein structure was very special which contains secretion signal,endoplasmic reticulum retention sequence,protein disulphide isomerase activity and reptin protein binding sites which determine the AGR2 protein wide distribution and diversity function in the cell.In recent years,studies have shown that the main function of AGR2 protein is to promote tumor metastasis,cell survival,etc.However the regulators of AGR2 are not very clear,through early drug screening,we found that the proteasome inhibitor MG132 can significantly lower AGR2 protein expression.Proteasome can degrade damaged denatured protein,short life of regulatory proteins through ubiquitin pathway,which plays an important role in regulation of physiological processes such as cell cycle,apoptosis,differentiation,and signal transduction.So short life of regulatory proteins like p53?p21 can be induced when the proteasome is inhibited,and meantime the compensatory autophagy pathway are able to activate to degrade intracellular accumulation of misfolded proteins.This topic will focus on the possible mechanisms of AGR2 suppression in the presence of MG 132 in aspects of protein transcription and degradation.Result:Part I the relationship between AGR2 expression and prognosis of non-small cell lung cancer1.AGR2 overexpressed in non-small cell lung cancer.We analyzed the existing cancer gene database such as TCGA,oncomine etc to study AGR2 protein expression in non-small cell lung cancer,it is found that AGR2 expression in tumor tissue is 2 times higher than the normal pulmonary bronchial epithelial tissue,at the same time,the NCI-60 database confirmed the copy number of AGR2 gene in non-small cell lung cancer cell line are all above 2 n,therefore AGR2 gene is highly expressed in non-small cell lung cancer.2.AGR2 protein affects cell survival,and its expression level affects the prognosis of patients with non-small cell lung cancer.As a result of AGR2 in non-small cell lung cancer with high expression,we use specific AGR2 siRNA to knock down AGR2 protein levels in non-small cell lung cancer cell line,light microscopy showed that cell growth slowed markedly in the treated cells,MTT experiments also prove the same result.Meanwhile,we used the TCGA database to analyze the survival of non-small cell lung cancer patients after chemotherapy;the results show that high AGR2 protein expression significantly reduced the survival rate.Part II the molecular mechanism of inhibition of proteasome activity in abrogating AGR2 expresson1.MG 132 suppressed AGR2 expression in non-small cell lung cancer.According to the results of gene chip,we found the proteasome inhibitor MG 132 can significantly reduce AGR2 expression in non-small cell lung cancer A549 cells.We first utilized western blotting and quantitative PCR experiment verification that MG132 significantly lower AGR2 mRNA and protein levels in A549 cells,which suggested that MG 132 can restrain AGR2 expression in the transcription level.Transfection experiment results also show that MG 132 can potently inhibit AGR2 promoter(1.4 Kb)or core promoter(including promoter sequences of 0.5 Kb)activity.Concentration gradient experimental results show that low doses of MG132(1?M)can significantly lower AGR2 protein expression,and the substrate protein of proteasome such as p5 3 and p27 continue to rise as the extension of action time,which indicated that the concentration of MG132 can significantly inhibit proteasome activity.More importantly,MG 132 in that concentrations had no significant induction in cell death which eliminated the influence of MG 132 induced cell death in regulation of AGR2 expression.2.MG 132 induction of endoplasmic reticulum stress and elevated ROS levels do not participate in the process of AGR2 expression regulation.Inhibition of proteasome activity can block the degradation of misfolded proteins,thus caused induction of endoplasmic reticulum stress and elevated ROS levels,which can induce some stress protein expression through different mechanisms to resist the external environment impact on cells.So AGR2 as an endoplasmic reticulum disulfide bond isomerase,can its expression be regulated by the endoplasmic reticulum stress?(1)MG132 can significantly induce endoplasmic reticulum stress,lower the expression of AGR2:MG132 make the sign of endoplasmic reticulum stress protein GRP78 continue to increase,and the expression of p-eIF2 alpha(inactivation forms)also presents the same trend without change of total protein eIF2 alpha,the above result evidenced that MG 132 indeed can induce endoplasmic reticulum stress.(2)Alleviation of endoplasmic reticulum stress caused by MG 132 can not protect AGR2 expression:western blotting results substantiate that endoplasmic reticulum stress inhibitors does not affect MG 132 inhibition in AGR2 expression.Similarly,p ?phosphorylation inhibitors Salubrinal can not attenuate AGR2 expression.(3)the classical endoplasmic reticulum stress revulsant increasing expression of AGR2:tunicamycin(TM)and Thapsigargin(TG)are two classical endoplasmic reticulum stress inducers,contrast to the results of MG132,western blotting and quantitative PCR results showed that the two revulsant can induce the expression of AGR2 in transcription and protein levels,and the expression of AGR2 continues to increase with the passenge of time,the results show that MG132 mediated endoplasmic reticulum stress is not the main mechanism in AGR2 depression.(4)ROS induced by MG 132 is not involved in its regulation in AGR2:according to the results of flow cytometry MG132 can improve the level of ROS in the cells in time dependent manner,and the expression level of AGR2 is significantly lower when the cells ROS levels increased significantly at the same time.We further use antioxidants NAC to reduce MG132 induced ROS level,however,the function of MG132 towards AGR2 expression was not affected.Therefore,MG132 caused endoplasmic reticulum stress and ROS is not participated in the AGR2 regualation.3.Transcription factors E2F1 and p65 can raise AGR2 expression,MG 132 mediated E2F1 reduction contributed to AGR2 transcriptional inhibition.Since MG132 can block AGR2 transcription,we further analyze AGR2 transcriptional regulation in the promoter region.On the basis of pilot studies,hormones,transcription factor FOXA1,FOXA2,androgen receptor(AR)can positively regulate the expression of AGR2.We further use Genomatix software to analyze AGR2 promoter regions to predict the potential transcription factor binding sites,and we found that in addition to the above transcription factor,there were a classic transcription factor E2F1 and p65 binding sites,however early data suggest that MG132 does not affect FOXA1,FOXA2 expression.(1)E2F1 and p65 can positively regulate the transcription of AGR2.RNA interference experiment results show that lower E2F1 and p65 expression by siRNA can significantly reduce AGR2 mRNA and protein level,and meanwhile Dual-luciferase reporter gene assay show E2F1 and p65 can significantly enhance the activity of AGR2 promoter which substantiated E2F1 and p65 as two AGR2 regulatory factors.(2)MG132 can stall the expression of E2F1 and AR:quantitative PCR and Western blotting illustrated that MG 132 can significantly lower transcription factor E2F1 and AR expression level.(3)P65 and AR don't participate in MG132 mediated AGR2 inhibition:western blot results show MG132 did not reduce the expression of p65,but can significantly enhance P-p65(ser573)protein levels,thus MG132 in AGR2 regulation rule may not directly depend on p65.RNA interference experiment results show that loss of AR protein can slightly reduce the expression of AGR2,consistent with the early reports regarding the role of AR on the AGR2 expression,however,exogenous AR can not reverse MG132 in the transcription of AGR2 inhibitory effect,therefore,AR is not a key factor of in MG132 mediated AGR2 transcription regulation.(4)E2F1 can be adjusted by MG132 which contributed to AGR2 transcription depression:E2F1 exogenous expression can partially restore MG132 inhibitory facts on AGR2 mRNA.Similarly,overexpression of E2F1 can improve AGR2 promoter activity which contained E2F1 loci,and diplay reversal effects on AGR2 promoter inhibition caused by MG132.Therefore,E2F1 has an important regulating role in MG 132 mediated AGR2 inhibition.4.MG132 induced autophagy can promote AGR2 protein degradation.(1)MG 132 significantly reduced AGR2 protein levels:protein synthesis inhibitors(CHX)can inhibit AGR2 translation,western blotting results illustrated that in the presence of CHX,MG132 still further attenuate AGR2 protein level,which suggested that MG132 not only inhibit the expression of AGR2 in transcription level,but also can potentiate AGR2 protein degradation,thus the AGR2 protein were significantly reduced.(2)MG 132 can induce autophagy in cells:after measuring the half-life of AGR2 protein,we found that AGR2 protein half-life is about 24 h,and the protein was considered as long life protein that may be degraded by autophagy when the half-life is greater than 5 h.Research has confirmed that compensatory autophagy was able to initiate after the proteasome activity is inhibited,so that the accumulation of ubiquitin protein degradation can be degradation to alleviate the toxicity,the function of MG 132 induced autophagy had been evidenced by the results of Western blot and immunofluorescence,which MG132 can induce autophagy marker LC3B protein expression,the transformation of the LC3B-I to the LC3B-II,and induction of LC3 fluorescence spots.(3)MG132 induced autophagy promotes AGR2 protein degradation:according to the Western blotting results,MG132 can significantly improve LC3BII expressed in time dependent manner,and AGR2 protein levels were decreased accompanied with the increase of LC3BII expression.Immunecoprecipitation experiments showed that ubiquitined AGR2 protein was accumulated in the presence of MG132,which indicated that MG 132 induced autophagy can facilitate ubiquitined AGR2 protein degradation.Then laser confocal analysis was utilized to locate LC3B and AGR2 protein,and the results show that after co-transfection the plasmids of pmCherry-AGR2 and pEGFP-LC3 in A549 cells,the red fluorescence AGR2 proteins can be positioned with green LC3 spots.In conclusion,autophagy caused by MG 132 may promote the degradation of AGR2 protein.(4)AGR2 protein was degradated by autophagy pathway:in order to further analysis of autophagy in AGR2 protein degradation,we use the classic autophagy revulsant rapamycin induced autophagy in cells,we found that rapamycin significantly increase LC3BII,and reduce the p62 protein(one of the substrates of autophagy),at the same time make the AGR2 protein decrease.Autophagy inhibitor 3-MA,chloroquine(CQ)which alleviated MG132 induced autophagy level,also inhibit the degradation of AGR2 protein.In addition,silencing the key autophagy regulation protein such as ATG5 and ATG7 reduced autophagy as indicated by slow LC3B swich,can significantly reverse MG132 induced AGR2 protein abrogation.The above results show that AGR2 protein can degrade through the autophagy pathway.(5)Endogenous level of autophagy negatively correlated with AGR2 protein in cells:the AGR2 protein and autophagy level in six kinds of lung cancer cell lines was analyzed,western blotting results indicated that AGR2 protein levels present negative correlation with cell autophagy level.When we treated autophagy inhibitor chloroquine(CQ)in high autophagy cell lines-HBE and H1688,AGR2 protein can significantly increase.In addition,using the Pubmed database,we also compared the expression level of AGR2 and autophagy related protein p62 in non-small cell lung cancer and normal lung tissue,the results showed that the expression of AGR2 and p62 are high in non-small cell lung cancer,so the high AGR2 protein expression in non-small cell lung cancer is possible related to the low level of autophagy,which further confirmed that autophagy can promote AGR2 protein degradation.Conclusion:1.The proteasome inhibitor MG132 can significantly suppress AGR2 expression level independently on MG132 caused cell death.2.Endoplasmic reticulum stress and ROS induced by MG 132 treatment had no effects on regulating AGR2 expression.3.E2F1 and p65 can regulate the expression of AGR2;Restoration of E2F1 expression ameliorates the AGR2 mRNA reduction caused by MG132 treatment.4.MG132 can cause lung cancer cell autophagy,and the level of autophagy in the cell can adjust AGR2 protein abundance.Novelty and defects:1.We first reported MG132 can down-regulate the expression of AGR2,and MG132 can influence AGR2 expression through transcription and protein modification level,and we first reported E2F1 and p65 participated in MG 123 regulation of AGR2 expression;however only E2F1 can partially reversed MG132 caused AGR2 mRNA decrease,so other proteins involved in regulation of AGR2 transcription still needs further explore.2.We first reported human lung cancer cells can sequestrate ubiquitined AGR2 protein by autophagy,and AGR2 protein expression and autophagy level has a negative correlation,but we has not yet found autophagy related receptors to facilitate AGR2 degradation.