Effect And Mechanism Of ARHGEF5on Proliferation, Invasion And Migration In NSCLC

Background:Lung cancer is currently the most common cause of death from cancer in the world.Non-small-cell lung cancer （NSCLC） accounts for approximately80%of all cases of cancers.Although many methods have been developed for the treatment of NSCLC, such as surgicalresection, chemotherapy and radiotherapy, the prognosis of NSCLC remains poor and the5-year survival rate is only about10-15%. In recent years, many single tumor biomarkerswhich may be predictive of response and prognosis, including epidermal growth factorreceptor （EGFR）, the canonical Wnt pathway and Notch3have been developed, but there isstill lack of specific and sensitive enough molecular biomarkers to accurately predict thesurvival of NSCLC patients. Therefore, screening and identifying the lung cancer-relatedgenes and proteins, and further studying their molecular mechanisms are important directionsfor the lung cancer research.Src is a particularly promising target for anti-cancer molecules, which regulates thecytoskeleton recombination by the phosphorylated190RhoGAP and focal adhesion kinase,regulates the cell adhesion and movement, and plays an important role in cancer invasionand metastasis. Although Dasatinib, as a src inhibitor, has entered onto Phase Ⅲ clinicaltrials, it wouldn’t achieve the desired results without EGFR molecular inhibitors, whichsuggests that src downstream key signaling proteins and signal transduction pathwaysrequire further research and exploration.ARHGEF5is is a member of the Dbl family of guanine nucleotide exchange factors.Its gene is located on human chromosome7q33-q35and its encoded protein has heavy andlight chain isoforms, which was encoded by a single mRNA, and consisted of DH region,PH region, SH3domain, and the C-terminal proline. The light chain contains519aminoacids with a molecular weight of60KDa; the heavy chain contains1597amino acids with amolecular weight of about170²00KDa. TIM is a short-chain isoform which is identified and isolated from human breast cancer cell lines. Spliceosome mutation of TIM wasreported to play an important role in the metastasis of breast cancer cells. Recently, it hasbeen reported that ARHGEF5heavy chain isoform can be activated by src and it plays animportant role in pseudopod formation, and the over-expressed ARHGEF5heavy chainisoforms promote the remodeling of actin stress fibers through the activated RhoA.Moreover, src induces the activation of pseudopod cytorrhyctes RhoA or Cdc42. Previousstudies only focus on apparent phenomenons such as changes in cell skeleton andpseudopod formation, and there is no relevant report about the possible downstream signaltransduction of this signaling pathway and the resulted tumor invasion and metastasismechanisms.The tumorigenic characteristics of proto-oncogenes are associated with proliferation,adhesion, invasion and metastasis. Although ARHGEF5gene has been identified as anoncogene, few studies are reported in the tumor field, as there are more than70members inARHGEF5Dbl family and the majority functions are unknown. The GEFs of the Dblfamily proteins has been attached more and more importance and become a hot target forcancer therapy. ARHGEF5is isolated from breast cancer cells, and its mutant spliceosomeplays an important role in breast cancer cells. The tumorigenesis of ARHGEF5has beenverified, but the expression and mechanism of NSCLC in human have not been reportedworldwide.Objective:To elucidate the role of an oncogene gene ARHGEF5in tumorigenesis includingproliferation, invasion and migration of NSCLC. Further more, to explore the function ofARHGEF5gene mechanism in NSCLC development, so as to provide new theoretical basisfor the NSCLC diagnosis and treatment.Methods:1．Expression of ARHGEF5and its role in NSCLCImmunohistochemistry SP method was used to detect ARHGEF5and src proteinexpression in193cases of NSCLC tissues, and Western Blot was used to detect theARHGEF5protein expression in two cases of lung adenocarcinoma and squamous cellcarcinoma and five NSCLC cell lines and HBE. Meanwhile, immunofluorescence wasapplied to detect the co-expression of ARHGEF5and src in human NSCLC tissues. 2．Effect of ARHGEF5gene silencing in NSCLC cell proliferation, invasion andmetastasis and its related signaling pathwayAccording to the ARHGEF5gene expression in human NSCLC cell lines, A549andNCI-H1650cell lines were selected, which were moderately expressed in ARHGEF5geneand had abilities of tumorigenesis and metastasis. Import ARHGEF5siRNA into the A549and NCI-H1650cell lines, RT-PCR and Western blot method were conducted to identifythe transfected ARHGEF5genes in cells. Moreover, in vitro functional verificationexperiments were performed, including cell proliferation, apoptosis, changes in the cellcycle, as well as adhesion, invasion and migration. Double-labeling immunofluorescenceand co-immunoprecipitation techniques were performed to verify the co-localizedexpression of ARHGEF5and src in human NSCLC tissues and cells and their physicalbinding. Western Blot and PCR methods were applied to detect the downstream effectorprotein expression. With the above results and literatures review, many target proteinswhich may down-regulate the ARHGEF5expression were inferred, and then verified byselecting the CyclinD1and MMPs proteins which were closely related with proliferation,invasion and metastasis.3．The role of ARHGEF5in nude miceRNA-interfered lentiviral vector was constructed according to the target gene sequenceARHGEF5（NM₀05435.3）.Lentivirus with ARHGEF5interference sequence was used toinfect human NSCLC cells, meanwhile, lentiviral negative control group and blank controlgroup were designed. Nude mice models with A549cells transfected by lentiviral vectorswere established and the effect of ARHGEF5on NSCLC cells was investigated.Results:1．Expression of ARHGEF5in NSCLC tissues and cell linesThe immunohistochemical results showed that ARHGEF5was positively expressed inthe majority of lung cancer tissues, with the positive rate of68.91%（69/193）. Its positiverate was68.09%（64/94） in adenocarcinoma and69.70%（69/99） in squamous cellcarcinoma, while it was negatively expressed in the corresponding non-cancerous tissues.Western Blot results also showed a low expression of ARHGEF5in normal humanbronchial epithelial cells HBE and a high expression in four NSCLC cell lines. Asignificant difference was observed between ARHGEF5positive staining and age, differentiation and tumor stage （p<0.05）; but no significant difference with the pathologicaltype, gender, and lymph node metastasis. The co-expression of ARHGEF5and src inadenocarcinoma tissues was significantly higher than in squamous cell carcinoma,indicating a significant difference in the correlation （p <0.05）2．Effect of ARHGEF5gene silencing in NSCLC cell proliferation, adhesion, invasionand migration and its possible signal pathwayThe ARHGEF5siRNA was successfully transiently transfected into human NSCLCcell lines （A549and NCI-H1650）, and the expression of ARHGEF5protein wassignificantly reduced; infecting A549and NCI-H1650cells with ARHGEF5siRNA canreduce the proliferation, invasion and migration of lung cancer cells, but has no effect onthe apoptosis. The down-regulated ARHGEF5genes resulted in decreases of p-src proteinlevel, p-Akt, NF-κB, CyclinD1and MMP2expressions; PCR detection also indicated asignificant decrease of CyclinD1and MMP2mRNA.3．ARHGEF5gene silencing inhibited the A549subcutaneous tumor growthThe lentiviral vectors, A549cell lines and human NSCLC cell lines were successfullyconstructed, in which ARHGEF5genes were down regulated. Then human NSCLC cellline A549, which was transfected with lentiviral vector, was subcutaneously injected innude mice. In the established mice models, lentiviral vector with ARHGEF5interferenceeffectively reduced the expression of ARHGEF5protein in the screened cells andeffectively inhibited the growth of transplantation tumor in nude mice model of human lungcancer.Conclusions:1．In NSCLC patients with tumor resection surgery, ARHGEF5positive staining isclosely related to age, differentiation and tumor stage （p <0.05）. The co-expression ofARHGEF5and src in adenocarcinoma tissues was significantly higher than in squamouscell carcinoma （p <0.05）.2．ARHGEF5is an important proto-oncogene to promote A549and NCI-H1650cellproliferation, adhesion, invasion and metastasis.3．In NSCLC tissues, ARHGEF5may up-regulate the p-Akt, NF-κB signaling pathway,increase the transcription activity of tumor cells CyclinD1and MMP2and the expression oftheir corresponding proteins, thus speeding up the cell proliferation and extracellular matrix degradation4．Transfection of RNAi targeting ARHGEF5in A549cells reduced tumorproliferation in-vivo nude mice.