The Mechanism Of Extracellular Heat Shock Protein 90α Involved In Epithelial Mesenchymal Transition In Gefitinib-sensitive Non-small-cell Lung Cancer

IntroductionLung cancer is the malignant tumor with the highest morbidity and mortality in the world,and non-small cell lung cancer is the most common phenotype.In Asia,about 50%of non-small cell lung cancer patients have mutations in the epidermal growth factor receptor(EGFR)gene,which can be sensitive to EGFR-TKI(epidermal growth factor receptor tyrosine kinase inhibitors,such as gefitinib).Even if the initial effect is satisfactory,acquired resistance to EGFR-TKIs is unavoidable in the later stages.Once resistance is developed,the therapeutic effect of drugs will obviously affected.Usually the mechanism of drug resistance is mainly about the EGFR T790M mutation,and patients often switch to another advanced EGFR-TKIs.However,there are some EGFR-TKI-resistance patients who do not have a mutation and devolop EMT(Epithelial mesenchymal transition)have no good choice.Therefore,overcoming acquired resistance to EGFR-TKIs has become one of the most important issues in the EGFR-targeted treatment of non-small cell lung cancer in recent years.EMT is an important biological process in which epithelial phenotype cells are transformed into cells with interstitial phenotype.It is manifested by the reduction of epithelial phenotypic markers E-cadherin and by the upregulation of mesenchymal phenotype markers like N-cadherin and Vimentin.Meanwhile,it manifests in cell behavioral loss of cell adhesion ability and cell polarity,as well as acquisition of migration and invasion ability.Epithelial mesenchymal transition is also one of the drug resistance mechanisms.Some studies have shown that the more EMT of tumor cells obtain,the more drug resistance the cancer cells develop.Studies have shown that cells with interstitial phenotype can provide more compensation axis signals than cells with epithelial phenotype to promote growth inhibition specific protein 6(Gas6)and cells.Surface receptors bind to escape the proapoptotic signals of erlotinib in order to enable tumor cells to survive and promote drug resistance.This shows that EMT may play a certain role role in the occurrence of acquired resistance.Therefore,studying the molecular mechanism between EMT and acquired resistance is of great significance in delaying the occurrence of acquired resistance.Heat shock protein 90α(HSP90α)is an important molecular chaperone that regulates protein function and stabilizes protein structure stably expressed in cells with a molecular weight of 90kDa.Heat shock protein 90 was found to be elevated in the serum of lung cancer and related to the occurrence and development of the disease.At the same time,heat shock protein 90 was also found to be elevated in other tumors,and also related to the disease.Therefore,HSP90 inhibitors including 17-AGG and ganestepid have emerged endlessly,and most of them have started clinical trials.Unluckily,they always terminate with side effects and toxicity.At the same time,heat shock proteins not only participate in the cell’s biological functions,but it can also be released outside the cell to perform certain functions.In tumor cells,HSP90 can be continuously released outside the cell,and even exosome can be released outside the cell to perform the function of promoting invasion and migration.However,there is little research on extracellular HSP90α in resistance to gefitinib in non-small cell lung cancer.Ferroptosis is a form of atypical cell death which is characterized as accumulation of lipid peroxides and depending on iron ions.At the same time,ferroptosis is also involved in the occurrence and development of drug resistance in tumors.It has been elaborated that metallothionein 1G promotes liver cancer cells by inhibiting iron death Sorafenib resistance also further considers whether iron death is related to drug resistance.It is meaningful to study the role of ferroptosis in EGFR mutation NSCLC to delay and overcome the acquired resistance.In our study,it was found that HSP90 protein is highly expressed in the serum of patients with EGFR-mutated NSCLC.At the same time,it was found that extracellular HSP90α promotes the occurrence of EMT,and also promotes the invasion and migration of cells;HSP90α causes the activation of downstream AKT and other signaling pathways through LRP1,while the cells HSP90α inhibits the occurrence of ferroptosis.Furthermore,inhibiting ferroptosis can promote the occurrence of EMT.At the same time,an xenograft tumor of animal models in vivo is constructed.The exosome inhibitor GW4869 enhance the antitumor activity of gefitinib.The purpose of our research is to explore the role of extracellular heat shock protein 90α in EGFR-mutated non-small cell lung cancer,which may provide new ideas for delaying the emergence of acquired resistance in non-small cell lung cancer and provides a brand new sight for individualized treatment.Methods1.Clinical specimen:The expression of HSP90α in plasma samples from 22 patients with EGFR mutations in non-small cell lung cancer and 10 healthy people was detected by ELSIA.Plasma HSP90a levels were higher in EGFR-mutated NSCLC patients than in controls.2.Western blot:Extracellular HSP90α,EMT biomarkers(E-cadherin,N-cadherin and Vimentin)and ferroptosis biomarkers(Cox-2 and GPX4)was detected by western blot.3.Wound healing assay:Scrapping the cells at the bottom of the dish with a 200μl pipette tip,gently wash the remaining cells at the bottom of the dish with PBS,and incubate the cells for a certain period of time with a light microscope to take a picture of the cell migration.The relative mobility is analyzed with Image J software.4.Transwell:Spreading cells in a density of 7×104 in the upper chamber of a transwell filled with matrigel.After incubation for a certain time,fixing in 4%paraformaldehyde,washing with PBS,and stainning with crystal violet.Image was obtained under a microscope,and the number of cells passing through the upper chamber to the lower chamber membrane was counted.5.Liperfluo:The cell suspension with a density of 5×104 is seeded in a dish,and TGF-β1 and hrHSP90α are pretreated and incubated.After mixing Lipferfuo with DMSO,add it to the culture medium as needed,and place in a cell incubator in the dark for 20 minutes,and then shoot with a confocal microscope.6.Nude xenograft tumors:Balb/c nude mice(15-20g,4-6 weeks of age)can form tumors 4 days after subcutaneous injection of HCC827 cells under their scapulae.intragastric administration once a day.On the day 28,After the injection of anesthesia in abdominal cavity,blood was collected from the eyeballs.After the blood collection was completed,the tumor was removed and separated,weighed,and photographed.The tumor tissue was fixed with 4%paraformaldehyde for 24h to 48h and immunohistochemistry was performed.7.Immunohistochemistry:Dewaxing tumor sections:antigen retrieval,blocking,endogenous blocker,primary antibody(E-cadherin,Vimentin)at 4℃ overnight,secondary antibody incubation at room temperature,DAB staining,discolors,dehydrates,covering slids,observes under the microscope and takes pictures.8.Statistics:Data are calculated as mean ± standard deviation(S.D.)and then processed by Graphpad Pris 5.0 software.The data were analyzed using Student’s t-test.Paired t test and one-way analysis of variance(ANOVA)were used to determine the statistical significance between different groups.Values of*p<0.05 are considered statistically significant.Result1.Plasma HSP90α levels were higher in EGFR-mutated NSCLC patients than in controls.Extracellular HSP90α has a concentration-dependent effect on TGF-β1 and promotes EMT.Hrhsp90α enhances the ability of TGF-β1 to promote EMT,and silencing intracellular Hsp90α by siRNA can significantly reduce the protein levels of N-cadherin and Vimentin.The results of scratch test and Transwell showed that extracellular HSP90α promoted cell migration and invasion.2.It is interesting that the tumor volume of GW4869 and gefitinib group is smaller than that of gefitinib group.Among serum samples,the expression of HSP90α of GW4869 and gefitinib combinal group is the lowest.Immunohistochemical analysis also showed that the expressions of N-cadherin in GW4869 and gefitinib group were lower than those in gefitinib group.E-cadherin expression was elevated.3.TGF-β1 promotes the upregulation of LRP1 in NSLC,and silencing of LRP1 by si RNA promotes the expression of E-cadherin,reduces the expression of N-cadherin and Vimentin.Meanwhile they reduces the level of GPX4 protein.EMT and ferroptosis were reversed when pretreated with hrHSP90α in si-LRP1 cells.The IC50 pre-pretreated with hrHSP90α and Ferrostatin-1 was higher than the hrHSP90αpretreated group,and the IC50 pretreated with hrHSP90α was higher than that of the control group alone.In lentivirus-transfected shHSP90α and shNC PC9,p-ERK,p-AKR,p-EGFR and p-MET protein levels were down-regulated.4.HrHSP90α can enhance the ability of inhibiting ferroptosis induced by TGF-β1.Treating with Ferrostain-1 and TGF-β1,it can inhibit ferroptosis and promote EMT.Silencing GPX4 by siRNA suppressed EMT.The si-GPX4 group treated with hrHSP90α could promote the reduction of E-cadherin and the increase of GPX4,Vimentin and N-cadherin.Taken together,these results indicate that extracellular HSP90α enhances EMT by up-regulating GPX4.The combination of Ferrostatin-1 and hrHSP90α promote the EGFR-TKIs-resistance in NSCLCConclusion1.The high level of HSP90α in the plasma of patients with non-small cell lung cancer developed EGFR mutation may be related to the occurrence of NSCLC.2.Exosome inhibitor GW4869 enhances the sensitivity to gefitinib in an xenograft model.3.Extracellular HSP90α can upregulate of the expression of LRP1 and inhibit ferroptosis.And then they can promote EMT as well as cell invasion and migration and acquire resistance to gefitinib in non-small cell lung cancer.