Mechanism Of Targeting YAP1 Increased DUSP1 To Reverse Osimertinib Resistance In Non-Small Cell Lung Cancer

Background:Non-small cell lung cancer(NSCLC)is one of the most common malignancies in the world.About 70% of NSCLC patients are diagnosed in the advanced stage,and have a poor prognosis.Recently,targeted therapy has become one of the most important treatment options for cancer,and the third generation EGFR-TKI osimertinib(AZD9291)has replaced the first generation EGFR-TKIs,gefitinib and erlotinib,due to a favorable and durable treatment response showing superior survival benefits and improved quality of life.As first-line therapy for patients with EGFR mutated NSCLC.However,the emergence of acquired resistance in the majority of patients poses great clinical challenges.The mechanism of acquired osimertinib resistance is heterogeneous,involving EGFR-dependent and EGFR-independent mechanisms.EGFR independent resistance mechanisms include changes in EGFR downstream signaling pathways(e.g.PI3K/AKT,RAS/MAPK/ERK and JAK/STAT).The Hippo pathway regulates cell proliferation,differentiation,and organ size during development,and is also involved in carcinogenesis,drug resistance,and tumor metastasis.YAP1 and its accessory TAZ are the final effectors of the Hippo signaling pathway.When combined with TEADs,they form transcription complexes to regulate transcription.YAP1 is overexpressed in 60% to 70% of NSCLC,and copy number amplification of YAP1 occurs in 15% of squamous lung cancers.YAP1 overexpression was positively correlated with poor clinical prognosis in lung cancer patients.Recent studies have demonstrated that YAP promotes resistance in both conventional chemotherapy and targeted therapies.In EGFR-TKI resistant cells,YAP not only activates ERK and AXL signaling but also induces EMT to make NSCLC resistant to EGFR-TKI.EGFR-TKI combined with YAP inhibitors can inhibit EGFR-TKI resistance.In addition,AXL,a downstream target of YAP1,was found to be a pathogenic factor in the third generation of EGFR-TKI(Osimertinib persistent cells).But so far,the role of YAP1 in acquired osimertinib resistance has not been studied.This study aims to explore the role of YAP1 in osimertinib resistance in NSCLC,explore whether CA3,a new inhibitor of YAP1 combined with osimertinib,can overcome osimertinib resistance in NSCLC and its specific molecular mechanism,and provide a new treatment strategy for the follow-up treatment of osimertinib resistant NSCLC patients.Research aims:1.To study the expression of YAP1 in patients with EGFR mutation in non-small cell lung cancer and its correlation with osimertinib resistance.2.To explore whether CA3,a new inhibitor of YAP1,can play an antitumor role in NSCLC cells by inhibiting YAP1.3.Confirm whether CA3 combined with osimertinib overcomes and delay the acquired resistance of osimertinib in NSCLC.4.Determine whether YAP1/YY1 synergistically regulates DUSP1 transcription and activates EGFR/MAPK pathway.Research methods:1.Expression of YAP1 in EGFR mutated non-small cell lung cancer patients and its correlation with osimertinib resistance(1)Bioinformatics analysis of the relationship between YAP1 and osimertinib resistance using GEO database.(2)q PCR and Western Blot to detect the expression of YAP1 in osimertinib-resistant cells in NSCLC.(3)Cell viability assay to examine the sensitivity of NSCLC osimertinib resistant cells with lentiviral knockdown of YAP1 to osimertinib.(4)Immunohistochemical detection of YAP1 protein expression in NSCLC tissues before and after treatment with osimertinib and YAP1 expression in NSCLC patients with EGFR mutations and its relationship with clinicopathological features and prognosis.2.CA3,a novel inhibitor of YAP1,can act in NSCLC cells by inhibiting YAP1.(1)CCK8,Transwell,and apoptosis assays were performed to detect the effects of CA3 inhibition of YAP1 on the proliferation,migration ability,and apoptosis of NSCLC cells in vitro.(2)q PCR and Western Blot to examine the m RNA and protein expression of YAP1 in CA3-treated NSCLC cells.3.CA3 combination with osimertinib overcome and delay osimertinib acquired resistance of NSCLC.(1)Cell viability assay to detect whether there is a synergistic effect of CA3 in combination with osimertinib in osimertinib-resistant cells of NSCLC.(2)Cell nucleoplasm isolation assay and immunofluorescence assay were performed to recognize the localization of CA3 combined with osimertinib on YAP1 in osimertinib-resistant NSCLC.(3)The effects of CA3 combined with osimertinib on the proliferation,migration ability,and apoptosis of osimertinib-resistant NSCLC cells were investigated by CCK8,Colony Formation,Transwell and apoptosis assays.(4)The effects of CA3 combined with osimertinib on the autophagy of osimertinib-resistant NSCLC cells were examined by electron microscopy,immunofluorescence,and Lyso-Tracker assay.(5)The effects of autophagy on the migration ability and apoptosis of osimertinib-resistant NSCLC cells were investigated by Transwell and apoptosis assays.(6)Regularly cell morphology observed under microscope to understand the production process and specific changes of DTP cells,and the expression of YAP1 in DTP cells was detected by Western blot.Cell morphology and Colony Formation were observed regularly under microscope to detect the killing of CA3 on DTP cells.The effect of early CA3 use on acquired Osimertinib resistance in NSCLC cells was observed by regular cell morphology under microscope.(7)To construct a subcutaneous tumor model in nude mice to verify in vivo the effect of YAP1 on the proliferation ability of osimertinib-resistant NSCLC.4.YAP1/YY1 synergistically regulates DUSP1 transcription and activates the EGFR/MAPK pathway.(1)RNA-seq sequencing assay to conceal changes in gene m RNA expression in osimertinib-resistant NSCLC cells after CA3 knockdown of YAP1.(2)The effects of DUSP1 on proliferation,migration ability,and apoptosis of osimertinib-resistant NSCLC cells in vitro were discoveried by CCK8,Colony Formation,Transwell,and apoptosis assays.(3)To investigate the interaction between YAP and DUSP1 in osimertinib-resistant NSCLC cells by Chip,IP,Western blot,immunofluorescence,dual-luciferase assay,and bioinformatics methods.(4)To examine the interaction between YAP and EGFR and the regulation of EGFR/MAPK pathway by DUSP1 in osimertinib-resistant NSCLC cells by IP,Western blot,immunofluorescence,and bioinformatics methods.(5)The effects of CA3 combined with osimertinib and transiently transfected with disrupted DUSP1 on the proliferation,migration ability,and apoptosis of osimertinib-resistant NSCLC cells in vitro were investigated by CCK8,Colony Formation,Transwell,and apoptosis assays.Research results:1.YAP1 was involved in the regulation of osimertinib resistance in NSCLC,and knocking down YAP1 can restore the sensitivity of NSCLC to osimertinib.(1)YAP1 was highly expressed in osimertinib-resistant cells of NSCLC and patients with EGFR mutations treated with osimertinib.(2)YAP1 was highly expressed in NSCLC patients with EGFR mutation.YAP1 was elevated in EGFR-mutated NSCLC patients treated with EGFR-TKI.(3)High expression of YAP1 was associated with poor prognosis in NSCLC patients with EGFR mutations.2.CA3 can act as an inhibitor of YAP1 in NSCLC.(1)CA3 inhibited the proliferation and migration of NSCLC cell lines and promote apoptosis.(2)CA3 inhibited YAP1 expression in NSCLC cell lines.3.CA3 collaborated with osimertinib to inhibit proliferation and migration of osimertinib acquired resistant NSCLC cells,and promote apoptosis and autophagy.(1)CA3 and osimertinib had synergistic effects in Osimertinib-resistant NSCLC cells and could inhibit YAP1 nuclear translocation.(2)CA3 combined with osimertinib could inhibit the proliferation and migration of osimertinib-resistant NSCLC cells and promote apoptosis and autophagy.(3)CA3 combined with osimertinib regulated the proliferation and migration of osimertinib-resistant cells in NSCLC through autophagy.(4)CA3 combined with osimertinib killed DTP cells generated in the process of acquired osimertinib resistance in NSCLC,and delayed the acquired of osimertinib resistance in NSCLC.(5)The use of lentiviral knockdown of YAP1 or the use of YAP1 inhibitor CA3 in combination with osimertinib inhibited subcutaneous tumor growth in nude mice with osimertinib-resistant cells and potentiated the antitumor effect.4.CA3 combined with osimertinib reversed acquired osimertinib resistance by inducing upregulation of DUSP1 and inhibiting activation of the EGFR/MAPK pathway.(1)CA3 combined with osimertinib induced upregulation of DUSP1 and inhibited the activation of the EGFR/MAPK pathway.(2)Overexpression of DUSP1 inhibited the proliferation and migration of osimertinib-resistant NSCLC cells and promoted apoptosis.(3)YAP1 collaborated with YY1 to bind DUSP1 promoter and3’-UTR to inhibit DUSP1 transcription.(4)DUSP1 feedback inhibited YAP1 phosphorylation.(5)DUSP1 bound to EGFR and inhibited the activation of downstream MAPK pathways.(6)CA3 combined with osimertinib partially inhibited the proliferation and migration of osimertinib-resistant NSCLC cells by inducing up-regulation of DUSP1 and promoting apoptosis.Research conclusion:1.YAP1 is involved in the regulation of osimertinib resistance in NSCLC,and targeting YAP1 regulates the proliferation and migration of osimertinib-resistant NSCLC cells through the autophagy pathway.2.CA3 acted as a targeted inhibitor of YAP1 to inhibit the proliferation and migration of NSCLC cells and promote cell apoptosis.3.YAP1 cooperates with YY1 to bind to the promoter and 3’-UTR of DUSP1 and repress DUSP1 transcription.DUSP1 regulates YAP1 phosphorylation in a feedback manner.4.CA3 combined with osimertinib can inhibit the proliferation and migration of osimertinib-resistant NSCLC cells,promote cell apoptosis,and overcome the acquired resistance of NSCLC to osimertinib by up-regulating DUSP1 and binding EGFR,resulting in the inactivation of its downstream MAPK signaling pathway.