The Effects And Mechanisms Of KDM6B In Acquired Resistance To EGFR Tyrosine Kinase Inhibitors In Lung Adenocarcinoma

Objective:Lung cancer is the most deadly carcinoma in the world nowadays.It is paramount important to explore and elucidate the mechanisms of the oncogenesis and progression of lung adenocarcinoma for the development of treatment mode and improvement of patients’life quality and prognosis.Lung adenocarcinoma is the main subtype of NSCLC,and most lung adenocarcinomas have tumor-driven genes.The targeted therapy that aiming at the inhibition of the tumor-driven genes has the advantages of favorable clinical effects and few adverse reactions.This therapy mode is effective in the improvement of patients’life quality and prognosis.However,the development of drug resistance,which leads to the deterioration of disease and poor prognosis,is inevitable in the processes of target therapy.Epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)therapy is the most common form of target therapy.However,the potential molecule mechanisms of the drug resistance formation remain to clarify.It is paramount important to discover the new mechanisms of EGFR-TKIs resistance in lung adenocarcinoma treatment.In the current study,we have mined the tight relationship between lysine demethylase 6B(KDM6B)and the formation of the EGFR-TKIs resistance in the EGFR-TKIs resistance cell lines of lung adenocarcinoma with EGFR mutations.We have elucidated the regulation mechanisms of the high protein expression of KDM6B.EGFR-TKIs combined with the inhibition of KDM6B could be a potential method to overcome the EGFR-TKIs resistance.Our study has provided a new direction for the management of EGFR-TKIs resistance in lung adenocarcinoma.Materials and MethodsChapter I The establishment of EGFR-TKIs resistance cell linesWe established the EGFR-TKIs resistance cell lines including HCC827 gefitinib resistance cell line(HCC827-GR),PC9 osimertinib resistance cell line(PC9-OR),and HCC827 osimertinib resistance cell line(HCC827-OR)via the method of continuously increased drug concentration.The H1975 cell line with characteristics of primary gefitinib resistance was also applied in this study.Then,we conducted the short tandem repeat(STR)experiment to validate the cell types of resistance cells.The half-maximal inhibitory concentration(IC50)of parental and resistant cells was detected by the CCK-8 experiment.The proliferation ability of parental and resistant cells under a certain concentration of EGFR-TKIs was also detected by cloning formation experiment.Chapter II Study on the relationship between KDM6B and EGFR-TKIs resistance in lung adenocarcinomaWe assumed the potential role of KDM6B in the process of lung adenocarcinoma EGFR-TKIs resistance formation based on previous studies.The m RNA and protein expression difference between parental and resistant cells and the effects of nock down experiments were validated by q PCR and western blotting.We used the CCK-8experiment to explore the changes of proliferation ability and IC50 after application of GSK J4(KDM6B inhibitor)or KDM6B nock down combines with EGFR-TKIs in resistant cells.cloning formation experiment was also used to detect the influence of GSK J4 combines with EGFR-TKIs on proliferation rate.Chapter III Study on the roles of KDM6B in EGFR-TKIs resistance cell lines of lung adenocarcinomaWestern blotting was applied to explore the expression changes on related molecules in EGFR downstream and other signaling pathways.And the protein interaction was demonstrated by immunoprecipitation(IP).For the validation of the specific mechanisms,the relative proteins and signal pathways were activated or inhibited by special chemical compounds.And the gene related were overexpressed and silenced by transient transfection and small interfering RNA(si RNA),respectively.Chapter IV The validation of KDM6B roles in acquired EGFR-TKIs resistance of adenocarcinoma in vivoWe used the PC9 and PC9-OR cell lines to plant the subcutaneous tumor modes in nude mice.Osimertinib was administrated by intraperitoneal injection after the tumor size exceed 100mm~3,and the tumor size was recorded on time.Results:Chapter I The establishment of EGFR-TKIs resistance cell lines1.Comparing with PC9 and HCC827,PC9-OR,HCC827-OR,and HCC827-GR had some changes on cell morphology.2.STR experiment pointed out that PC9-OR,HCC827-OR,and HCC827-GR were derived from their parental cell lines.3.CCK-8 and cloning formation experiment demonstrated that PC9-OR,HCC827-OR,and HCC827-GR had higher resistance ability compared with parental cell lines.Chapter II Study on the relationship between KDM6B and EGFR-TKIs resistance in lung adenocarcinoma1.High expression of KDM6B was tightly relative with poor prognosis and drug resistance of lung adenocarcinoma in public database analysis.2.The protein expression of KDM6B in PC9-OR,HCC827-OR,and HCC827-GR was higher than parental cells significantly.3.H1975 had the primary ability of gefitinib resistance.H1975 had a higher resistance ability compared with PC9 and HCC827 under the same concentration of gefitinib.4.GSK J4 combined with gefitinib achieved lower cell resistance and proliferation rate than gefitinib alone in H1975 and HCC827-GR cell lines.5.KDM6B nock down combined with gefitinib achieved lower cell resistance and proliferation rate than gefitinib alone in H1975 cell line.6.GSK J4 combined with osimertinib achieved lower cell resistance and proliferation rate than osimertinib alone in PC9-OR,HCC827-OR,and H1975 cell lines.7.KDM6B nock down combined with osimertinib achieved the lower cell resistance and proliferation rate than osimertinib alone in PC9-OR,HCC827-OR,and H1975 cell lines.Chapter III Study on the roles of KDM6B in EGFR-TKIs resistance cell lines of lung adenocarcinoma1.In the downstream signaling pathways of EGFR,the activation of phosphorylated extracellular regulated kinase1/2(p-ERK1/2)was inhibited in resistant cells.The activation of p-ERK1/2 was negatively correlated with the protein expression of KDM6B.2.The protein expression of KDM6B was influenced by p-ERK1/2.KDM6B protein was upregulated when p-ERK1/2 was inhibited by gefitinib and osimertinib in lung adenocarcinoma EGFR mutation cell lines.And when p-ERK1/2 was activated by EGF and plasmid with MEK activation mutation,the expression of KDM6B protein was reduced.3.In the members of mitogen-activated protein kinase(MAPK)family,KDM6B expression was influenced only by p-ERK1/2 rather than p-P38 and p-JNK.4.KDM6B was ubiquitylated and degraded when p-ERK1/2 was activated.However,the active p-ERK1/2 can’t reduce KDM6B when proteasome was inhibited by MG132.5.The ubiquitylation of KDM6B was regulated by the E3 ubiquitin ligase ubiquitin protein ligase E3 component n-recognin 5(UBR5).Chapter IV The validation of KDM6B roles in acquired EGFR-TKIs resistance of adenocarcinoma in vivo1.The tumor growth rate of PC9 was lower than PC9-OR cell line in nude mice.2.The tumor growth of PC9 was obviously inhibited when osimertinib was administrated in nude mice.3.The tumor growth of PC9-OR sh KDM6B3 and sh KDM6B4 was obviously inhibited when osimertinib was administrated in nude mice.Conclusion:Our study established the lung adenocarcinoma EGFR-TKIs resistant cell lines by continuously increased drug concentration method.Upregulated KDM6B was demonstrated in resistant cells.Inhibition of KDM6B activation and expression could overcome EGFR-TKIs resistance in vitro/vivo studies.Activation of p-ERK1/2 could induce the ubiquitylation and degradation of KDM6B protein.The activation of p-ERK1/2 was inhibited in resistant cells,which led to the inhibition of KDM6B ubiquitylation and degradation and high expression of KDM6B.Besides,UBR5 was the specific E3 ligase of KDM6B.