Endogenous Thrombopoietin Promotes Non-small Cell Lung Carcinoma Cell Proliferation And Migration By Regulating EGFR Signaling

Lung cancer is the leading cause of cancer-related deaths worldwide.There are two main subtypes of lung cancer,small cell lung carcinoma and non-small-cell lung carcinoma(NSCLC),with NSCLC accounting for approximately 85 % of all cases.Surgery is the most effective treatment strategy for NSCLC;however,many cases are diagnosed at an advanced stage when surgery is no longer possible.Although other treatment options are available,including chemotherapy,radiotherapy,immunotherapy,and molecularly targeted therapy,lung cancer exhibits a very poor prognosis and nearly half of all patients die within one year of diagnosis,with a five-year survival rate of 11 %.A better understanding of NSCLC initiation and development mechanisms is urgently needed and studies are required to identify more oncogenes and suppressor genes as well as targetable gene alterations.Thrombopoietin(TPO)is a hematopoietic cytokine that is mainly produced by the liver and kidneys and whose main function is to regulate megakaryocyte progenitor expansion and differentiation.In the past decade,numerous studies have investigated the effects of TPO outside the hematopoietic system;however,the role of TPO in the progression of solid cancer,particularly lung cancer,has not been well studied.Exogenous TPO acts by binding to the MPL proto-oncogene(C-MPL)and initiating various signal transduction pathways.Most solid tumor tissues and cell lines,including NSCLC,do not express C-MPL or exhibit extremely low expression,so exogenous TPO does not affect non-small cell lung cancer(NSCLC)cells.During our study,we found that TPO is highly expressed in NSCLC tissues and cell lines but is not secreted,which is different from the TPO expression in hepatic or renal cells.Therefore,in this study,we focused on endogenous TPO produced by NSCLC cells.Objective: We aimed to explore the expression of TPO in NSCLC tissues and its correlation with the clinicopathological parameters of NSCLC patients.And also to explore the effect and molecular mechanism of TPO act on NSCLC cells.Methods: A total of 150 paired NSCLC/normal specimens were obtained from patients with NSCLC who underwent surgical resection at the Department of Thoracic Surgery of The First Hospital of China Medical University between 2014 and 2016.No patients underwent chemotherapy or radiotherapy prior to surgery.TPO expression in lung cancer tissue was detected by immunohistochemistry assays.The relationship between TPO expression and lymph node metastasis,tumor size,TNM stage and degree of differentiation was analyzed by SPSS software.The expression of TPO protein and m RNA in NSCLC cells and normal bronchial epithelial HBE cell was detected by Western Blot and RT-PCR assays.ELISA was used to detect the secreted TPO in the medium of NSCLC cell lines.We used CCK8?Transwell and colony formation assay to detect the influence of TPO expression on NSCLC cell proliferation and migration.We also used western blot to detect the proliferation-,and migration related proteins and the expression of key molecules in some signaling pathways.Mass spectrometric analysis was used to predict the possible protein which can interact with TPO,and co-immunoprecipitation assay was used to verify the interaction between proteins.The localization of TPO in NSCLC cells and its correlation with the localization of EGFR protein was detected by immunofluorescence assays.RT-PCR and western blot assays were used to detect the effect of TPO on EGFR transcription and degradation.Results:1.Immunohistochemical analyses on 150 paired NSCLC/normal tissues indicate that TPO is highly expressed in NSCLC tissues,and its expression is positively correlated with clinicopathological parameters of NSCLC patients,including differentiation,P-TNM stage,lymph node metastasis,and tumor size.2.TPO expression was increased in A549,H1299,SK?MES?1,and H292 cells compared to that in HBE cells but was weakly expressed in H460 cells.And there was no detectable TPO secreted from NSCLC or HBE cells.3.CCK8 ? Transwell and colony assay revealed that suppressing TPO inhibits NSCLC cell proliferation and migration.Cyclin E1,Cyclin E2,CDK2,Rho A,Rho C,and c-Myc were downregulated and P27 was upregulated when TPO was suppressed.4.CCK8?Transwell and colony assay revealed that overexpressing TPO does not affect NSCLC cell proliferation and migration.5.We evaluated the changes in some critical signaling pathways related to cell proliferation and migration and found that P-AKT(Ser473)and P-mTOR(Ser2448) protein levels were significantly downregulated when TPO was suppressed.Meanwhile,no visible changes were observed in P-ERK,P-MEK,P-P65,P-JNK,or active ?-catenin levels.6.Basing on the result of mass spectrometry and the change of PI3K/AKT/mTOR pathway,co-immunoprecipitation assays verified the interaction between TPO and EGFR in A549 and H1299 cells.We then detected the change in P-EGFR(Tyr1068)and EGFR levels when TPO was suppressed and found P-EGFR level downregulated significantly with TPO expression but the EGFR level showed no notable change.7.RT-PCR results showed that EGFR m RNA level was upregulated when TPO was knocked down,which indicated that TPO influences EGFR signaling at the post-translational level.We then explored the effect of TPO on ligand-induced EGFR activation and degradation.NSCLC cells were serum-starved overnight and then stimulated with EGF for different lengths of time,with TPO overexpressed or knocked down at each time point.Cycloheximide was used to block protein synthesis before EGF stimulation.EGFR and P-EGFR(Tyr1068)levels were detected by western blot at the indicated time points(0,10,30,60,90,and 120 min).We found that EGF-induced EGFR and P-EGFR degradation was delayed and impaired in TPO overexpressed cells,whereas the opposite effects were observed when TPO was suppressed.8.Since A549 and H1299 cells contain wild-type EGFR,TPO overexpression may not promote their proliferation and migration as EGFR signaling and endocytic trafficking activity is relatively low.Thus,we stimulated A549 and H1299 cells with 100ng/m L EGF,with TPO overexpression enhancing the proliferation and migration of these NSCLC cells.Western blot results showed that alterations in Cyclin E1,Cyclin E2,CDK2,c-Myc,Rho A,Rho C,P-EGFR(Tyr1068),P-AKT(Ser473),and P-mTOR(Ser2448)protein levels were consistent with TPO expression in EGF-stimulated NSCLC cells,whereas the opposite effect was observed on P27 levels,as expected.Conclusion:1.TPO is highly expressed in NSCLC tissues and is positively correlated with differentiation,P-TNM stage,lymph node metastasis,and tumor size.2.TPO promotes NSCLC cell proliferation and migration through upregulating EGFR/ PI3K/AKT/mTOR signaling.3.TPO delays ligand-induced EGFR degradation,thus enhances EGFR stability and increases the duration and intensity of EGFR signaling.