Proteasome Inhibitor Enhances The Anti-tumor Activity Of Bevacizumab By Promoting Ubiquitinated Degradation Of AGR2

BackgroundA gradient gradient protein 2 homolog(AGR2)is one of the members of the endoplasmic reticulum disulfide isomerase(PDI)family and is found expression in normal human tissues such as the lung,breast,stomach,small intestine and prostate.But its physiological function is not yet fully understood,it may be related to the formation of glands,embryonic development,and limb regeneration.The human AGR2 protein contains 175 amino acid residues,and its unique protein structure determines its functional diversity:i)81-84 amino acid residues are PDI activity Motif(CPHS),but compared to the classical PDI structure(CXXC)Contains,it has only one Cys,its PDI activity is not strong,but still has the function of PDI-like chaperones,promotes the correct folding of the protein;ii)most of the PDI protein has a typical H/KDEL endoplasmic reticulum retention signal at the C-terminus,and AGR2 The signal is KTEL,which has weak binding to the KDEL receptor,can be detached from the endoplasmic reticulum,functions as a secretory protein in extracellular and humoral fluids;iii)AGR2 can also be localized in the nucleus,cytoplasm,participate in signal transmission and function through protein-protein interactions.Because AGR2 is highly expressed in various tumor tissues,it has been identified as a proto-oncogene and promotes tumorigenesis,invasion,metastasis,and production of multi-drug resistance and other malignant phenotypes.It is a potential drug therapeutic target and diagnostic marker.Our previous study found that AGR2 is highly expressed in prostate cancer and lung cancer tissues in Chinese Han population.It promotes cancer cell proliferation,angiogenesis,invasion,and metastasis by promoting cell cycle progression and enhancing VEGF/VEGFR signaling.Therefore,AGR2 can be used as a new targeting markers for targeted drug treatment for angiogenesis;and found that prote asome inhibitors downregulate the expression of AGR2 in lung cancer by inducing autophagic degradation mechanisms.Since AGR2 is rich in lysine residues(17/175 amino acid residues),ubiquitination of lysine(Lys)is an essential group for proteasome degradation,so this paper is based on previous studies to further analyze:i)Whether AGR2 can carry out the ubiquitin degradation pathway,whether its Lys is involved in its degradation process;ii)proteasome inhibitors can decrease angiogenesis by down-regulating AGR2,whether its function can increase the efficacy of the bevacizumab(Bev)monoclonal antibody,which a targeted drug for angiogenesis.1.Proteasome inhibitors down-regulate AGR2 expression and promote ubiquitination of its proteinsOur previous results showed that the proteasome inhibitor MG 132 significantly down-regulated the expression of AGR2 and promoted the degradation of AGR2 by inducing autophagy(Supplementary Literature,Dawei Thesis).We first determined this effect using another proteasome inhibitor Bortezomib(Bor).Western blotting results showed that BOR can significantly reduce the expression of AGR2,while the proteasome substrate p53 and p27 expression showed an increasing trend.At the same time,the results of co-immunoprecipitation showed that proteasome inhibitors can increase the polyubiquitylation level of AGR2 protein.Therefore,inhibition of proteasome activity can promote ubiquitylation of AGR2.2.E3 ligase UBR5 mediates the link between AGR2 and ubiquitin K48 sites(1)MG132 can mediate AGR2 polyubiquitylation degradation through K48 ubiquitin chainSince ubiquitin mainly ubiquitylates substrates by K48 and K63,it promotes the degradation of substrates.We transfected 293T cells with only ubiquitin K48,K63 and K48 mutant plasmid K48R and K63 mutant plasmid K63R.The site of polyubiquitylation chain formation of AGR2 was determined by immunoprecipitation experiments.The results showed that under the action of the inhibitor MG 132,ubiquitin K48 mediates the ubiquitination of AGR2,whereas when K48 is mutated(K48R),the ubiquitination of AGR2 is significantly reduced.Western blotting experiments confirmed that overexpression of ubiquitin K48,autophagy inducer rapamycin(RAPA)significantly reduced the level of AGR2.When K48 was mutated,downregulation of AGR2 was significantly attenuated;autophagy inhibitor Chloroquine(CQ)significantly restored the protein level of AGR2 and decreased the ubiquitylation degradation on AGR2.Therefore,MG 132 degrades through multi-ubiquitination of AGR2 mediated by the K48 ubiquitin chain.(2)MG132 mediates the ubiquitination of AGR2 through the E3 ligase UBR5In order to determine the E3 ligase that mediates AGR2 ubiquitination,we used the TCGA database to analyze the interacting proteins of AGR2 through the IPA software and found that the Ubiquitin protein ligase E3 component-recognin 5(UBR5)protein can interact with AGR2.UBR5 functions as an E3 ligase,catalyzing the ubiquitination of its substrate AGR2.Western blotting showed that under the action of MG132,overexpression of UBR5 could significantly down-regulate the protein level of AGR2.When UBR5 ubiquitinated domain was mutated(UBR5-C2768A),the effect of MG 132 on the inhibition of AGR2 was significantly reduced,indicating that UBR5 mediates AGR2 ubiquitination degradation.The result of co-immunoprecipitation showed that AGR2 could bind UBR5 and show obvious bands in its protein precipitation complex,but could not interact with ubiquitination site mutated UBR5-producing protein,further confirming that E3 ligase UBR5 mediated The ubiquitinated degradation of AGR2.3.Proteasome inhibitors promote ubiquitination and degradation by AGR2-Lys89 siteLysine(Lys,K)is the key amino acid for protein ubiquitination,and AGR2 protein contains 17 lysines.In order to determine the key lysine of ubiquitination of AGR2,we previously constructed the expression plasmid which all mutations of Lys in AGR2(Km)and corresponding expression plasmid which K26,K30,K31,K34,K39,K64,K66,K70,K88,K89,K95,K99,K116,K165,K166,K169,K172 site were mutated to arginine(Arg,R).Western blotting results showed that when the four lysine residues K34,K66,K89,and K169 were mutated to R,the effect of MG 132 on downregulation of AGR2 could be attenuated,while the effect of other sites on Lys mutation was not significant,indicating that K34,K66,K89,and K169 may be involved in the connection of AGR2 to ubiquitin.We then constructed an AGR2 mutant plasmid containing only 34,66,89,and 169 lysine,and further analyzed key amino acid residues that mediate AGR2 ubiquitination.The result of co-immunoprecipitation showed that MG 132 could significantly increase the ubiquitination of AGR2 when lysine at K89 was present.After K89 mutation,the ubiquitination level of AGR2 was significantly reduced.The presence of lysine at K34,K66,and K169 did not significantly increase the ubiquitination of AGR2.Therefore,the 89th Lys is the key amino acid that mediates the ubiquitination of AGR2.4.Proteasome inhibitors mediated ubiquitination of AGR2 through autophagy receptors NBR1 and p62The previous results confirmed that MG132 promoted the degradation of AGR2 by inducing autophagy.Because the ubiquitinated target protein needs to be recognized by a specific autophagic receptor,it is further degraded by autophagy lysosome recognition.We first detected changes in the universal autophagy receptor SQSTM1/p62,and found that SQSTM1/p62 did not decrease but slightly increased after treatment with MG 132.Co-immunoprecipitation showed that AGR2 does not bind to SQSTM1/p62.Further analyze other autophagic receptors that mediate AGR2 degradation.The experimental results show that under the action of MG132,the autophagy receptors Calcium binding and coiled-coil domain 2(NDP52)and Optineurin(OPTN)do not form complexes with AGR2,and the Neighbor of BRCA1 gene 1(NBR1)is very significant with AGR2.The binding indicates that NBR1 is an autophagic receptor that induces ubiquitination and degradation of AGR2 by MG132.We then determined whether Bortezomib(Bor)had the same effect and found that Bor,unlike MG132,down-regulated the expression levels of SQSTM1/p62 and NBR1 simultaneously.The result of co-immunoprecipitation confirmed that under the action of Bor,SQSTM1/p62 also formed a precipitation complex with AGR2,showing obvious bands.Therefore,the proteasome inhibitors MG132 and Bor mediate the degradation of ubiquitinated AGR2 through NBR1 and SQSTM1/p62.5.Downregulation of AGR2 by a proteasome inhibitor can enhance the antitumor efficacy of bevacizumabOur previous results showed that high expression of AGR2 increased the level of secreted AGR2,through epithelial-mesenchymal transition,and formed a complex with VEGF to promote tumor invasion and metastasis and block the activity of the angiogenesis targeted drug bevacizumab.Therefore,we speculated that if AGR2 is highly expressed,whether the effect of proteasome inhibitor downregulating AGR2 can increase the sensitivity of tumors to bevacizumab.(1)Bor and bevacizumab have a synergistic effect in combinationThe drug combination curve shows that Bor is used in combination with bevacizumab,and its CI value is less than 0.3,which has a good synergistic effect.We also verified through animal experiments.The dose of Bor is usually 1.2 mg/kg.Considering its toxicity,we used 0.8 mg/kg(labeled high-dose Bor-H)and low-dose Bor(0.2 mg/kg,Bor-L)groups.The results showed that the high-dose group had good efficacy,the tumor weight and tumor volume of the mice were significantly reduced,while the higher-dose group of the low-dose Bor group was reduced.The efficacy of the bevacizumab group was also not significant,but after the low-dose Bor was combined,the tumor weight and tumor volume in the mice were significantly reduced,showing a very good synergistic effect.HE staining showed that the levels of both ki67 and CD31 in the combination group were greatly reduced,further confirming the synergistic efficacy of the combination.(2)Bor and bevacizumab combined to induce autophagy and downregulate AGR2The results of molecular level tests showed that the Bor-H,Bor-L and combination groups all showed obvious apoptosis markers---there was a significant shear band in PARP,and there was no obvious band in bevacizumab group,consistent with the efficacy results..The induction of LC3-II in Bor-H and combination groups was more significant than in Bor-L group.Similarly,bevacizumab had no significant effect on autophagy.In the Bor group,the levels of NBR1 and SQSTM1/p62 were significantly decreased,and the downregulation of SQSTMl/p62 in the combination group was more prominent,while the bevacizumab group had no significant effect on NBRI and SQSTM1/p62.Most importantly,both Bor-H and Bor-L significantly decreased AGR2 levels,while bevacizumab did not affect AGR2 expression,but the combination of Bor-L significantly decreased AGR2 levels.Therefore,low-dose Bor combined with bevacizumab showed good autophagy induction and down-regulated AGR2 protein levels.(3)The combination of Bor and Bevacizumab significantly reduces the toxicity and improves the quality of life of miceWe used conventional indicators and animal behavior test methods to determine drug toxicity.The results showed that the Bor-H group had a significant reduction in body weight of mice,resulting in higher liver function ALT,AST indicators and affect the ability of mice sports ability,which shows that high-dose Bor does produce certain drug toxicity to the body.But the Bor-L,Bev,and Bor-L/Bev did not significantly reduce the weight of the mice and did not affect the mice's feeding ability,and they have similar performance to the control group in ALT and AST indicators of liver function and indicators of sports ability and trajectory of mice.In particular,the combination group was able to maintain a smaller drug impact on the body and ensure the quality of life of the mice while providing significant anti-tumor efficacy.Therefore,low-dose Bor combined with bevacizumab has the effect of improving the efficacy and reducing the toxicity.Among them,the effect of Bor down-regulating AGR2 is an important mechanism for its synergistic effect,and provides experimental basis for clinical drug combination therapy.conclusion:1.Proteasome inhibitors promote the ubiquitination of the endoplasmic reticulum protein AGR2,and down-regulate AGR2 protein levels through autophagic degradation pathways.2.UBR5 is an E3 ligase that ubiquitinate AGR2.3.In the course of ubiquitinated degradation of AGR2,the lysine at position 89 is a key amino acid for ubiquitination,and AGR2 is polyubiquitinated by the function of ubiquitin K48 linkage.Proteasome inhibitors MG 132 and bortezomib recognize ubiquitylated AGR2 through NBR1 and SQSTM1/p62,respectively,and promote autophagy degradation.5.Downregulation of AGR2 by proteasome inhibitors can reduce the pro-angiogenic effect of AGR2 and enhance the anti-tumor activity of the angiogenesis-targeted drug bevacizumab.Combinations of low-dose Bor and bevacizumab enhance the antitumor efficacy and reduces the toxicity.Innovation and deficiencies:Innovation:1.It was first reported that the molecular mechanism of proteasome inhibitors promoting the degradation of AGR2 by ubiquitination.2.It was first reported that UBR5 is an E3 ligase that ubiquitinate AGR2.3.The proteasome inhibitors MG 132 and bortezomib were first reported to mediate the ubiquitylation degradation of AGR2 through NBR1 and SQSTM1/p62.4.The first study on the mechanism of down-regulation of AGR2 expression by proteasome inhibitors provides a theoretical basis for the combined use of bortezomib and bevacizumab drugs,and has a certain guiding role in the clinical combination of drugs.insufficient:1.Proteasome inhibitors downregulate the expression of AGR2 and promote its ubiquitin degradation.Whether it has a similar regulatory effect on other disulfide isomerases has not been discussed.2.Proteasome inhibitors are therapeutic drugs for a variety of tumors.In addition to inhibiting the degradation of substrate proteins,there are other mechanisms involved in its anti-tumor effects,such as c-Myc regulation.Therefore,whether bortezomib is involved in the regulation of AGR2 through other mechanisms still needs further study.