The Role And Molecular Mechanism Of Transcription Coactivator YAP In Molecular Targeted Therapy Resistance Lung Cancer

Background and objectivesCurrently,lung cancer is still one of the most common malignant tumors world wide.Molecular targeted therapy targeting driver genes of lung cancer such as epidermal growth factor receptor(EGFR),anaplastic lymphoma kinase(ALK)and c-ros-oncogene 1(ROS1)has become a main treatment method for non-small cell lung cancer(NSCLC)in recent years,especially for advanced stage patients.Although targeted therapy has achieved remarkable clinical efficacy,most patients,including those who are sensitive to the initial treatment,will develop drug resistance to varying degrees,resulting in tumor recurrence and metastasis.Therefore,studying the mechanism of drug resistance to find new therapeutic targets has become a research hotspot in recent years.Hippo-YAP pathway is a newly discovered signal transduction pathway in recent years,which plays an important role in the development of a variety of tumors.YAP,namely yes-associated protein,is a major downstreameffector of Hippo-YAP pathway and a transcriptional co-activator which can interact with different transcription factors to activate the transcription andexpression of target genes.Some studies have demonstrated that Hippo-YAP signaling pathway is involved in the drug resistance of antitumor drugs.Meanwhile,it has been proved that YAP overexpression can lead to epithelial-mesenchymal transition(EMT)of pancreatic cancer cells and is associated with reduced efficacy of anti-tumor chemotherapy drugs.EMT refers to the transformation of epithelial cells into mesenchymal cells under certain physiological and pathological conditions.EMT has been proved to play an important role in the process of anti-tumor drug resistance,including lung cancer.In recent years,studies have found that EMT is associated with targeted therapy resistance to EGFR and ALK inhibitors in lung cancer;however,there has been no relevant report on EMT and resistance of ROS1 inhibitor.Therefore,this study aims to explore the function of YAP in ROS1-rearranged lung cancers and the relationship between YAP and drug resistance of molecular targeted therapy,so as to provide a new theoretical basis for targeted therapy of lung cancer.Materials and methodsIn this study,human histological specimens and in vitro cytological experiments,including immunohistochemical staining,MTT experiment,CCK8 experiment,Western blot,RT-q PCR,and gene silencing technology,were used to explore the expression and clinical significance of YAP in lung adenocarcinoma and ROS1-rearranged lung adenocarcinoma cell line systematically.Meanwhile,the internal molecular mechanisms of YAP-mediated crizotinib resistance in ROS1-rearranged lung adenocarcinoma were further clarified.Results1.YAP was mainly expressed in tumor cells;a small number of normal type ?alveolar epithelial cells were weakly positive;but it was not expressed in type ?alveolar epithelial cells,bronchial epithelial cells,stromal cells and inflammatory cells.In most cases,YAP protein was expressed in nucleus orboth nucleus and cytoplasm;in a few cases,it showed exclusively cytoplasmic staining.Among these patterns,nuclear staining and nuclear and cytoplasmic staining patterns were included in the interpretation of YAP staining results.High expression rate of YAP protein in lung adenocarcinoma was 59%(170/288),and most tumor cells showed diffuse strong positive expression.High expression of YAP was correlated with tumor size,vascular invasion,lymphatic invasion,necrosis and tumor recurrence(P< 0.001).The expression rate of YAP protein was highest in micropapillarypattern lung adenocarcinoma(77.7%),followed by papillary pattern(64.7%),solid pattern(64.3%),acinar pattern(54.8%)and lepidic pattern adenocarcinoma(33.3%).In addition,the positive rate of YAP in lung adenocarcinoma with micropapillary component(> 5%)was significantly higher than that in lung adenocarcinoma without micropapillary component(P< 0.001).2.The expression rate of YAP protein was statistically different in lung adenocarcinomas with different molecular alterations.The expression rate in ROS1-rearranged lung adenocarcinoma was significantly higher than that in cases without ROS1 rearrangement(P = 0.024),and the expression rate in lung adenocarcinoma with EGFR mutation was significantly lower than that in cases without this mutation(P = 0.034).3.Univariate survival analysis showed that progression-free survival(PFS)(P =0.021)and overall survival(OS)(P =0.003)were significantly shorter in patients with high YAP protein expression.Multivariate survival analysis showed that high expression of YAP protein(HR: 2.642,95%CI: 1.550-3.646,P =0.001),tumor size(HR: 1.252,95%CI: 0.803-1.911,P< 0.001),and pleural invasion(HR: 1.831,95%CI:1.090-2.764,P =0.018)were independent prognostic factors for PFS.Pathological stage(HR:3.089,95%CI:1.418-5.431,P =0.003)was an independent prognostic factor for OS.4.High expression rate of YAP protein in ROS1-rearranged lung adenocarcinoma was 78%(7/9),andhigh expression of YAP protein was negatively correlated with E-cadherin expression,and positively correlated with vimentin and snail expression.5.Cell culture and cell climbing experiments showed that ROS1-rearranged lung adenocarcinoma crizotinib-resistant cell line(HCC78CR)demonstrated obvious mesenchymal features morphologically;cells changed from round and epithelioid to spindle in shape,and they were dispersed with decreased intercellular connection.Western blot showed that the expression of YAP protein in HCC78 CR was significantly higher than that in HCC78 cells(P < 0.0001);the expression of EMT-related transcription factors including snail,zeb1 and vimentin significantly increased in HCC78 CR cells(P < 0.01),and the expression of E-cadherin decreased(P<0.01).Electron microscopic observation showed that the number of autophagosomes in crizotinib-resistant HCC78 CR cells increased,and western blot confirmed that the expression of autophagy proteins LC3-?and LC3-?significantly increased in HCC78CR(P < 0.01).6.After silencing YAP in HCC78 CR cells,the sensitivity of HCC78 CR cells to crizotinib treatment was enhanced.The expression of EMT-related proteins was detected usingwestern blot,and the results showed that the expression of snail,zeb1 and vimentin in HCC78 CR decreased significantly(P< 0.01),and the expression level of E-cadherin increased(P< 0.01).RT-q PCR showed that the m RNA levels of snail,zeb1 and vimentin in HCC78 CR decreased significantly,and the m RNA level of E-cadherin increased significantly.Meanwhile,the m RNA expression of LC3-?,LC3-?,and autophagy related genes BECLIN1 and ATG5 also decreased significantly in HCC78 CR cells after silencing YAP expression.Conclusions1.YAP protein is highly expressed in lung adenocarcinoma.High expression of YAP is correlated with clinicopathological features predicting poor prognosis,and is proved to be an independent predictor of poor prognosis in patients with lung adenocarcinoma.The expression rate of YAP in ROS1-rearranged lung adenocarcinoma is higher,and is positively correlated with the expression of vimentin and snail in EMT pathway.2.YAP induces EMT in ROS1-rearranged lung adenocarcinoma cell line HCC78 and its resistance to crizotinib treatment.Resistant cells may prolong survival through autophagy.