The Effects And Molecular Mechanisms Of Autophage Regulated By PSMC2 Through UPS In Acquired EGFR-TKI Resistance Of Non Small-cell Lung Cancer (NSCLC)

OBJECTIVE: Lung cancer is the commonest malignant disease and has the highest mortality.The percent of non small-cell lung cancer(NSCLC)is about 85%,it does great harm to national health.Most NSCLC patients have been terminal stage when the diagnosis is definite and have lose the chance of curation by operation.The objective reaction rate is about 25% of kinds of chemotherapy schemes in medcine but the side effects are severe.In recent years,the epidermal growth factor(EGFR)-tyrosine kinaseinhibitors(TKI)has got great success in the first-line therapy of advanced NSCLC,the effective ratio of EGFR mutation patients is about 62-83%.Their survival times have been prolonged dramatically.But almost all patients who have taken TKI will have a result of drug resistance in succession.The EGFR-TKI acquired resistance has became a bottleneck and a key problem in clinical practices.So,expliciting the mechanism of EGFR-TKI acquired resistance is urgent in lung cancer treatment.Now many valuable researches have been done on it including the 20 th exon of EGFR secondary mutation,the structural activation of downstream effector mocules,genes amplification of EMT and c MET,angiogenesis and tumor microenvironment.In addition,PLK3 CA mutation and AXL kinase activation are inclued.But ingrettably,they only give partal reaons of EGFR-TKI acquired resistance.Late researches found that the autophage of colon cell line dealed with chloroquine was weaken accompanied by the activity of UPS enhanced.The expression of Proteasome Subunit m RNA was high and cells became resistant to chemotherapy.It confirmed that autophage modulated by Ubiquitin Proteasome System(UPS)play a vital role in EGFR-TKI acquired resistance.But the definite mechanism is still unknown.Preliminary studies have shown that the expression of PSMC2 in EGFR-TKI acquired resistant cell lines is distinctly higher than in the sensitive ones.Chymotrypsin(Ct-L,one landmark of UPS)activity detected by continuous fluorescence was intensive too.The levels of landmark proteins of LC3 and Beclin were declined..Meanwhile the expression of Akt,downstream effective protein of EGFR was declined too.After using RNAi to restrain the expression of PMSC2 in EGFR-TKI acquired resistant cells,UPS activity in intraelluar region was down.The level of autophage elevated.Resistant cells were sensitiver to EGFR-TKI.It suggested that PSMC2 may play a key role in EGFR-TKI acquired resistance by modulating autophage through activating UPS.Clarify the mechanisms that PSMC2 involved in EGFR-TKI acquired resistance in NSCLC needs intensive studies.METHOD: EGFR is a kind of transmembrane protein structurally.It is a family member of epidermal growth factor(Erb).It contains three regions:extracelluar ligand binding region,intracelluar tyrosine kinase activity region and transmembrane region between the two.Among them,the amino acids located 294 to 543 in extracelluar ligand binding region are the first.Intracelluar tyrosine kinase activity region is divided into three subregions because of their different structures and functions: near membrane,tyrosine kinase and carbon terminal.Tyrosine kinase subregion is the key subregion because it has ATP binding site which can be binded by EGFR-TKI.The mass effect can block the follow-up signal transduction tiggered by the binding of ligand and receptor in usual.Clinical researches found that tyrosine kinase mutations located mainly at 18-21 exons regions.The mutations include 21 exon point mutation(L858R)probably 40-45%,19 exon deletion mutation(del E46-A750),the probability is equal to the former,18 exon point mutation is found too,but the probability is only about 5%.20 exon mutation is less too,about 5% and the main mutation style is insertion mutation.Among all mutations having been found,21 exon point mutation(L858R)is the most common.It can activate tyrosine kinase(TKI)and can make TKI being in state of activation in a long time.19 exon deletion mutation region mainly located at the binding region of ATP of EGFR.The mutation can make the structure of selfl ATP binding point change;When 18 exon G791X mutation can make the conformation of EGFR lose stabilization,EGFR is activated and continously activated.The mechanism of EGFR-TKI is that it bind with ATP binding point of EGFR instead of ATP and the binding is earlier than ATP.This binding not only not activate the downstream signal pathway,but also block the signal transmission.Thus the signals of cell growth can be suspended so can inhabit tumor growth.The several mutations of EGFR mentioned above are all indications of EGFR-TKI in NSCLC treatment.Previous investigations have acknowledged that the effective ratio is about 75% when using TKI on above-mentioned EGFR mutations patients.So,to improve the efficiency clinically,it is necessary to detect if the EGFR mutation exsist first before using TKI.If the mutations exist in NSCLC,TKI is the first option to the patients who are in late stage clinically and not optimal to operation.TKIs which act on EGFR intracelluar region are usually used such as gefitinib and erlotinib.They are hypotoxicity,efficient and have good patient compliances.But after a spell,patients will get drug resistance inevitably including primary resistance and secondary resistance,the latter is also called acquired resistance.Now the specific mechanisms of the acquired resistance to EGFR-TKI remain unkown,need to be intensive researches.No studies have seen that how the specific mechanisms of PSMC2 activate UPS to inhabit autophage which invovled in EGFR-TKI acquired resistance.But some earlier studies have seen that PSMC2 highly express in NSCLC cells which have EGFR-TKI acquired resistance and activate UPS,inhabit intracelluar autophage.On this basis,we will figure out the particular mechanisms of how PMSC2 modulate autophage in EGFR-TKI acquired resistance.1.The expressions of PMSC2 and activities of UPS and the changes of autophage in resistant NSCLC cell line model.The acquired Gefitinib-resistant cell line PC9/R used in previous experiments have been established successfully by us firstly.We tested the expression of PSMC2 in them,proteasome activities of relevant indicators of UPS(includingchymotrypsin-like,CT-L;peptidly-glutamylpeptide-hydrolyzing-like,PGPH-L;trypsin-like,T-L),EGFR,the vital factors of autophagy regulatory pathway PI3K-Akt-m TOR(PI3K,p-PI3 K,Akt,m TOR,p70s6,e IF4 E et al),the changes of autophage biomarkers(Atg5,Bcelin1,LC3-II).UPS have three main protease activities:CT-L,PGPH-L and T-L respectively.Their activities represent the activity of UPS and are necessary to maintain celluar homeostasis.PI3 K,p-PI3 K,Akt,m TOR,p70s6 and e IF4 E are very important regulatory signals and dowmstream effective factors of autophage and EGFR.Atg5,Bcelin1,LC3-II are biomarks of autophagosome.The ralationship between PSMC2 and the biomarks decide its important effects and significance.Analyse the regulating effects and directions of PMSC2 acting on each above-mentioned factors invoved in UPS,autophage and TKI resistance during the course of EGFR-TKI acquired resistance.2.Signal transduction pathway mediated by PSMC2 in PC9/R cells and the interaction of all the segments.UPS is a vital selective degradation passage of intracelluar proteins.The passage of PI3K-Akt-m TOR is a important signal passage of autophage.But it is still unknown that how PSMC2 conduct signals through above-mentioned passage in PC9/R cells.Previous researches detected the changes of expressions of signal factors after adopting methods of loss-of-function and gain-of-function to targeting genes,specific inhabitors and activators to intefere or overexpress the above-mentioned passages signal factors from three levels including related protease activity,PSMC2 and autophage.To make clear the ralationship and interaction of PSMC2,UPS and autophage in PC9/R cells which are resistant to EGFR-TKI.Meanwhile detected the changes of related indexes of UPS(the activities of CT-L,PGPH-L and T-L);EGFR;vital factors of autophage passage PI3K-Akt-m TOR(PI3K,p-PI3 K,Akt,m TOR,p70s6,e IF4E);biomarkers of autophage(Atg5,Bcelin1,LC3-II).Analyse the main effects of every passage.3.The expressions of PSMC2 in nude mices and the ralationship between UPS,autophage and TKI resistance.Analyze the expressions of PSMC2 at gene level and protein level firstly.Meanwhile detected the changes of expressions including UPS,EGFR and related factors of autophage.Analyze the effects of PMSC2 in EGFR-TKI resistant tumor models.Then deal with animal models using PSMC2 genes loss-of-function and gain-of-function techniques.Observing the influences on changes of PSMC2 expressions in UPS,EGFR,autophage and EGFR-TKI resistance.Discuss the effects of PSMC2 on reversal of drug resistance target.Previous researches of this study adopted techniques such as RNA inteference,q PCR,Western blot,flow cytometry,confocal laser scanning microscope,on molecule,cell,tissue and animal levels,make it clear that PSMC2 play roles through UPS regulating autophage.Discussed the regulatory signal passages and molecule mechanisms of PSMC2 in the course of EGFR-TKI resistance.Explicited the ralationship between PSMC2,UPS and autophage.The purpose of this study is to verify the former research.98 specimens were selected with pathological confirmation of adenocarcinoma of lung.Among them,49 specimens are sensitive to EGFR-TKI,the others are resistant.PCR was used to quantitatively detect PMSC2,m TOR(one critial factor of autophage),Beclin 1(a biomarker of autophage)and immunohistochemistry and western blot techniques were used to detect protein expressions of the same factors.To explicit the relationships between PMSC2,UPS and autophage in NSCLC specimens which have acquired resisitance to EGFR-TKI.RESULT:73.50%,77.60%and24.49% patients resistant to EGFR-TKI have a positive PMSC2、m TOR and Beclin1 expression respectively,as well as the data are 18.37%、14.49%and 83.67% in the sensitive ones by immunohistochemistry.The expressions of PMSC2、m TOR m RNA are lower in resistant group than in sensitive group by RT-PCR.As to Beclin1,the situation is contrary.By using Western blot,the expressions of Beclin1 in the resistant group are lower than in the sensitive group too[0.315±0.037 to 1.226±0.017 and0.420±0.016 to 1.023±0.014],but the expressions of PMSC2,m TOR in the resistant group are higher than in the sensitive group[0.986±0.032 to 0.282±0.021].The differences are all statistically significant too(p<0.05).m TOR and Beclin1,m TOR and PMSC2 are in positive correlation whether in the resistant ones or in the sensitive ones,but Beclin1 are in negative correlation with m TOR.CONCLUSION:The EGFR-TKI acquired resistance has became a bottleneck and a key problem in clinical practices.Until now many researches have been done on mechnisms of target medcine resistance,among them autophage is the vital factor of regulating tumor cell resistance.But little done about it.PMSC2 is one subunit of regulatory granule of UPS.Its expressions markedly elevates in EGFR-TKI acquired resistant cells.On contrary,autophagy marker gene and protein expression decline.PMSC2 probably bring out EGFR-TKI acquired resistance by regulating autophage through UPS.This is may only a part of mechnisms of EGFR-TKI acquired resistance in NSCLC.It can help us to seek potential resistant biomarks and give an important target for reversal of lung cancer resistance and individualized treatment.Further studies are needed about how PMSC2 induce EGFR-TKI acquired resistance by regulating autophage through UPS,which regulating factors and passages take part in it and their relationships of mutual restriction and cooperation.