The Function And Molecular Mechanism Of Src And GSTA1 In Lung Adenocarcinoma Metastasis

Background and significanceLung cancer is one of the major malignancies in the world.According to 2011 global cancer statistics,the incidence and mortality of lung cancer ranks first in all male cancer types,accounting for 17%of total new cancer cases and 23%of all cancer-related deaths.In addition,the incidence of female lung cancer continues to rise in recent years,and lung cancer has become the second most common type of cancer.The occurrence of lung cancer may be caused by indoor and outdoor air pollution and the exposure of occupational or environmental carcinogens.Lung cancer develops from normal lung and airway epithelial cells,undergoing multiple genetic damages and eventually transforming into uncontrolled,proliferating cells with invasion behavior.There are two main types of lung cancer:small cell lung cancer(SCLC),which accounts for about 15%of lung cancer cases,and about 85%of non-small cell lung cancer(NSCLC).There are three major histological types of NSCLC:adenocarcinoma,squamous cell carcinoma,and large cell carcinoma.Adenocarcinoma is the most common histologic subtype of lung cancer,accounting for about half of NSCLC cases,originating in bronchioalveolar stem cells,club cells,or type II alveolar cells.Lung adenocarcinoma is also a major subtype of female and non-smoking patients.Lung squamous cell carcinoma accounts for about 30%of NSCLC,mainly in big airway and segmental bronchus,and is closely related to smoking history.Small cell lung cancer,whose origin has not been determined,has been speculated to originate from neuroendocrine cells or non-neuroendocrine cells that acquire neuroendocrine differentiation.Lung cancer is a disease whose clinical burden is gradually increasing worldwide.Despite advances in diagnosis and treatment,the 5-year survival rate of lung cancer patients is still lower than 17%,and the therapeutic effect is far from satisfactory.One of the main reasons is the metastasis of lung cancer.The absolute damage caused by distant metastasis of lung cancer is more than local lesions.Metastasis is the main cause of cancer-related mortality in lung cancer.Statistically,metastasis-related deaths are as high as 90%of all lung cancer deaths.Unfortunately,approximately 70%of lung cancer patients were diagnosed with regional lymph node metastasis or distant metastases at the time of their first diagnosis,and approximately 65%of patients with NSCLC applicable for surgery will relapse within two years and subsequently will die of cancer metastasis.Metastasis is the dynamic interaction between cancer cells and the microenvironment.It was previously thought that this is an advanced event and only occurs when the primary lesion locally progresses.However,recent evidence suggests that the spread of tumor cells in circulation system also occurs in the early stages of lung cancer.In the distant metastasis of NSCLC,brain,bone,liver and adrenal gland metastasis ranked in the top four incidence rates.Among them,the most important metastatic organ is the brain.Up to 68%of mediastinal lymph node metastases eventually develop to brain metastases.Under such circumstances,metastasis has become a main obstacle to the successful treatment of lung cancer.However,the mechanism of lung cancer metastasis is still poorly understood and there is still a lack of specific effective therapies.Therefore,understanding the underlying molecular mechanism of regulating lung cancer metastasis can help discover new therapeutic targets.Src has been reported to be involved in the development of non-small cell lung cancer and other cancers.Several Src inhibitors with potential therapeutic value are in preclinical or clinical trials.The Src kinase is a member of the Src family and is a tyrosine kinase involved in the regulation of a variety of cellular signal transduction.It plays a key role in tumor growth,metastasis,and angiogenesis,and is therefore considered to be a promising therapeutic target for many solid tumors.For non-small cell lung cancer,Src kinase upregulation is frequently detected,especially in squamous cell lung cancer in frequent smokers.Src expression elevated was detected in 60-80%of lung adenocarcinomas and bronchioloalveolar carcinomas,as well as in 50%of lung squamous cell carcinomas.Studies have found that the role of Src in the invasion of non-small cell lung cancer cells is mediated by Fn14,a member of the cell surface receptor tumor necrosis factor superfamily.Fn 14 is the smallest member of the tumor necrosis factor receptor superfamily and is a receptor for TNF-like weak inducer of apoptosis(TWEAK).It has the function of inducing apoptosis and proliferation,and is involved in inflammatory reactions and can promote the secretion of cytokines,and participates in immune regulation and other effects.In addition,Fn14 is also involved in the regulation of cell migration and differentiation.By connecting different receptor proteins or cytoplasmic proteins containing the TNF receptor-associated factor(TRAF)domain,multiple signal transduction pathways in the cell are initiated and the corresponding gene activation is induced.NF-?B is a protein complex containing five subunits:p105,p100,Rel-A,Rel-B,and c-Rel.NF-?B controls more than 400 genes involved in normal physiological and pathological conditions.NF-?B controls DNA transcription,cytokine production and cell survival,and regulates the immune response to infection.Improper regulation of NF-?B can lead to cancer and metastasis,inflammation,and autoimmune diseases.Src can regulate NF-?B signaling has been demonstrated in a variety of tumors.In view of the fact that Fnl4 regulates the growth of NSCLC and SCLC through NF-?B signaling,we speculated that Src activates the NF-?B signaling pathway in NSCLC cells via Fn14 to promote the metastasis of lung cancer.And this is validated by in vitro and in vivo experiments in the first part of this study.Glutathione S-transferase A1(GSTA1)is a major isoform of GSTs and is primarily involved in the detoxification of electrophilic compounds by combining glutathione.Newly discovered evidence suggests that changing the expression of the GST gene increases the risk of cancer.Recently,Pan et al.pointed out the potential of GSTA1 in the early diagnosis and treatment of lung cancer.Studies have shown that the expression of GSTA1 is much higher in lung cancer tissues and cells than in normal tissues and cells,and GSTA1 suppression can inhibit the proliferation of lung cancer cells.However,the potential role of GSTA1 in lung cancer metastasis has not been studied until now.Common steps in the metastasis of lung cancer and most solid tumors are:(1)detachment from the extracellular matrix(ECM),(2)local migration and invasion,(3)infiltration into blood or lymphatic system,(4)survival in the circulatory system,(5)extravasation of metastatic sites and(6)proliferation and formation of new tumors.This is a complex process.Metastatic cells require special properties to overcome obstacles.Epithelial mesenchymal transition(EMT)is an important feature of most cancer cell metastases.EMT is the process by which epithelial cells lose epithelial cell characteristics and develop into mesenchymal cell phenotype.In lung cancer,abnormal activation of EMT leads to tumor invasion,metastatic dissemination,and treatment resistance,resulting in a poor prognosis.EMT is characterized by loss of epithelial markers(such as E-cadherin and keratin),upregulation of mesenchymal markers(such as vimentin and N-cadherin),and increase of mobility,invasiveness and metastatic potential.In the second part of this study,we transfected A549 cells with GSTA1-siRNA to knockdown the expression of GSTA1,studied the effect of GSTA1 on proliferation,invasion and adhesion of lung cancer cells,and illustrated GSTA1 promotes lung cancer metastasis through EMT,a process closely related to cancer metastasis.We studied the roles of Src/Fn14/NF-?B and GSTA1/EMT signaling pathways in the metastasis of lung adenocarcinoma cells and related molecular mechanisms to further understand the underlying molecular basis for the regulation of lung cancer metastasis.Exploring potential therapeutic value in these signal pathways in order to find new therapeutic targets for the future treatment of lung cancer.The following is the abstracts of the two parts of this study.Part ?:Src promotes the metastasis of lung adenocarcinoma through Fn14-mediated NF-?B signaling pathwayObjective:Src is a tyrosine kinase involved in the regulation of a variety of cellular signal transduction and plays a key role in tumor growth,metastasis,and angiogenesis.Studies have found that the role of Src in the invasion of non-small cell lung cancer cells is mediated by Fn14,a member of the cell surface receptor tumor necrosis factor superfamily.And Src regulates NF-?B signaling has been confirmed in a variety of tumors.Fnl4 can modulate the growth of non-small cell lung cancer and small cell lung cancer through NF-?B signaling.Therefore,we speculated that Src may activate NF-?B signaling pathway in non-small cell lung cancer cells through Fn14 to promote lung cancer metastasis.The main purpose of this study was to verify the role of Src/Fn14/NF-?B signaling pathway in lung adenocarcinoma metastasis.Methods:The effect of Src knockdown on proliferation,migration,and invasion of HCC827 cells was examined in vitro.Western blot was used to detect the expression of Fn14 and related markers of NF-?B signaling pathway in Src-silenced HCC827 cells.The role of Fn14 in regulating cell migration,invasion,and activation of NF-?B signaling was investigated by overexpressing Fn14 in Src knockdown lung adenocarcinoma cells.In addition,mouse lung cancer metastasis models were established,and the effects of Src promoting cancer metastasis in mouse models were detected.Results:Src suppression inhibited the proliferation,migration and invasion of HCC827 cells,and the expression levels of Fnl4,p-I?B?,p-IKK? and nuclear NF-?B p65 decreased.Overexpression of Fn14 restored the migration and invasive ability of Src-silenced NSCLC cells,as well as the activation of NF-?B signaling.In addition,experiments in vivo showed that Src silence inhibited the metastasis of lung cancer HCC827 cells in mouse models and inhibited the expression of Fnl4 and nuclear translocation of NF-?B p65.Conclusion:Src plays a key role in the occurrence and metastasis of lung adenocarcinoma.Knockdown of Src suppresses proliferation,migration and invasion of human lung adenocarcinoma cells and reduces NF-?B signaling.Overexpression of Fn14 in Src-silenced NSCLC cells restores cell phenotype and activation of the NF-?B pathway.Our study firstly demonstrates that the key role of Src/Fn14/NF-?B axis in the growth and metastasis of non-small cell lung cancer,and the potential therapeutic value of this axis deserves further study in the future.Part ?:GSTA1 promotes the metastasis of lung adenocarcinoma by mediating epithelial-mesenchymal transition(EMT)Objective:Glutathione S-transferase A1(GSTA1)is a major isoform of GSTs.And altering the expression of the GST gene increases the risk of cancer.The expression of GSTA1 is much higher in lung cancer tissues and cells than in normal tissues and cells.Suppression of GSTA1 can inhibit the proliferation of lung cancer cells.Epithelial mesenchymal transition(EMT)is a prominent feature of most cancer metastases.Abnormal activation of EMT in lung cancer results in tumor invasion,metastatic dissemination,and treatment resistance,leading to a poor prognosis.In this study,we transfected A549 cells with GSTA1-siRNA to knockdown the expression of GSTA1 in order to investigate the effect of GSTA1 on proliferation,invasion and adhesion of lung cancer cells.In addition,we investigated the effect of GSTAI on the process of EMT,which is closely related to the metastasis of lung cancer.Methods:A549 cells were treated with different concentrations of nicotine(0.01,0.1,1 and 10 ?M),and mRNA and protein expression of GSTA1 was detected by RT-qPCR and Western Blot.To reduce GSTA1 expression,GSTA1-siRNA was transfected into A549 cells.MTT,Transwell-Matrigel invasion and cell adhesion analysis were used to determine cell viability,invasiveness,and adhesion ability,respectively.The expression of epithelial cell markers(E-cadherin and Keratin-pan)and mesenchymal cell markers(Vimentin and N-cadherin)in A549 cells was detected by Western Blot analysis.Results:Our study found that GSTA1 expression of A549 cells was induced by nicotine treatment.Suppression of GSTA1 expression can inhibit lung cancer cell proliferation,invasion,and adhesion.After GSTA1 knockdown,the E-cadherin and keratin levels of epithelial cells in lung cancer cells were significantly increased,while the vimentin and N-cadherin levels of mesenchymal cell markers were significantly reduced.Our studies show that GSTA1 promotes the metastasis of lung cancer cells through EMT.Conclusion:This study firstly demonstrats that GSTA1 can promote the invasion and adhesion of lung cancer cells,and also mediate the effect of nicotine on lung cancer cell metastasis.We also demonstrate that GSTA1 influences the metastasis of lung adenocarcinoma by promoting EMT.