The Role Of TC-1in The Cell Biological Behavior Of Non-small Cell Lung Cancer

AimsLung cancer is the leading cancer-related cause of morbidity and mortality worldwide.Cell biological behavior plays an important role in the development and progression ofprimary lung carcinoma. Although TC-1is highly expressed in several carcinoms andassociated strongly with cell biological behavior, the role of TC-1in lung cancer is stillunclear. The aim of this study was to investigate the expression and cell biologicalbehavior relevance of TC-1in human non-small cell lung cancer (NSCLC) and to explorethe potential mechanism, which may provide a new target and open up a new way for thestudy of lung cancer.Methods(1) Smaples of122patients with NSCLC including cancer tissues, normal lung tissues,and clinical pathological data were collected. Immunohistochemistry analysis was used toexamine the expressions of TC-1and Ki-67in NSCLC and normal lung tissues. Therelationship between TC-1expression and clinicopathological characteristics including age, gender, histological type, TNM staging, regional lymph node metastasis and Ki-67labelling index were analyzed in NSCLC.(2) The pLenti-TC-1and pLenti-shRNA1were constructed. The TC-1stable overexpressed colonies (NCI-H520-pLenti-TC-1) and the TC-1stable knockdown colonies(A549-pLenti-shRNA1) were established by lentivirus transfection. In order to furtherstudy the effects of TC-1on cell biological behavior of NSCLC cells:①the MTT assayand the Plate colony formation assay were performed to test the effect of TC-1on theproliferation of NSCLC cells in vitro;②the cell cycle assay was performed to test theeffect of TC-1on the cell cycle of NSCLC cells in vitro;③the cell apoptosis assay wasperformed to test the effect of TC-1on the apoptosis of NSCLC cells in vitro;④the cellmigration assay was performed to test the effect of TC-1on the migration of NSCLC cellsin vitro;⑤the cell invasion assay was performed to test the effect of TC-1on the invasionof NSCLC cells in vitro;⑥the tumorigenicity assay and the immunohistochemicalstaining were performed to test the effect of TC-1on the proliferation of NSCLC cells invivo.(3) A549and NCI-H520-pLenti-TC-1cells were treated with FGFR2pathway inhibitorPD173074. RT-PCR and Western Blotting were performed to detect the expression ofTC-1in the PD173074treated cells. In order to further study the role of blockage ofFGFR2signaling pathway in cell biological behavior of NSCLC cells:①the MTT assayand the Plate colony formation assay were performed to test the role of blockage ofFGFR2signaling pathway in the proliferation of NSCLC cells in vitro;②the cell cycleassay was performed to test the role of blockage of FGFR2signaling pathway in the cellcycle of NSCLC cells in vitro;③the cell apoptosis assay was performed to test the roleof blockage of FGFR2signaling pathway in the apoptosis of NSCLC cells in vitro;④the cell migration assay was performed to test the role of blockage of FGFR2signalingpathway in the migration of NSCLC cells in vitro;⑤the cell invasion assay wasperformed to test the role of blockage of FGFR2signaling pathway in the invasion ofNSCLC cells in vitro;⑥the FGFR2signaling pathway assay and the chemotherapeuticsassay were performed to test the role of blockage of FGFR2signaling pathway in the proliferation of NSCLC cells in vivo.(4) Western Blotting were performed to detected the expression of wnt/β-catenin targetproteins (CCND1, c-Myc, c-Met) and Caspase-3.Results(1) TC-1protein exhibited and located in the cytoplasm of positive cells diffusely orlocally by immunostaining with a rate of73.77%(90/122) in NSCLC, which was largelyconfined to the lung cancer cells, but in the normal lung, the non-neoplastic bronchial andalveolar epithelia cells were low-reactive or non-reactive for TC-1. In addition, there wasno difference between gender, age and histological subtypes for the expression of TC-1(P>0.05). However, the expresssion of TC-1was significantly correlated with TNMstaging and regional lymph node metastasis (P<0.05). The rates of TC-1positiveexpression NSCLC cells were47.62%(10/21) in stage I,72.34%(34/47) in stage II and85.19%(46/54) in stage III; and were46.15%(12/26) in N0,73.47%(36/49) in N1and89.36%(42/47) in N2. The cases were divided into the high proliferation group (Ki-67labelling index>10%) and the low proliferation group (Ki-67labelling index≤10%)according to the Ki-67labelling index. There was significant statistical difference forTC-1expression between groups with different proliferation (95.29(81/85)%in highproliferation group and24.32%(9/37) in low proliferation group). These results revealedthat the expression of TC-1was correlated strongly with TNM staging, regional lymphnode metastasis and cell proliferation, but not with gender, age and histological subtypesin NSCLC.(2) To study the effect of TC-1expression on NSCLC cells, the expression of TC-1inhuman NSCLC cell lines A549, SPC-A-1,95D and NCI-H520was examined by usingRT-PCR and Western Blotting, the tunica mucosa bronchiorum epithelium16HBE cellline was used as a control group. Upon Real-Time PCR and Western Blotting, theexpression level of TC-1in A549, SPC-A-1,95D cells was higher than that in16HBEcells; the expression level of TC-1in NCI-H520cells was lower than that in16HBE cells.Based on these results, A549and NCI-H520were representative cells which had the highest or the lower level expression of TC-1and were selected for further study. Andthen the pLenti-TC-1-shRNA1was transfected into A549cells and the pLenti-TC-1wastransfected into NCI-H520cells respectively. Stable colonies were isolated after clonescreening by fluorescence-activated cellsorting or blasticidin, respectively. qRT-PCR andWestern Blotting showed that TC-1expression decreased or increased markedly in treatedcells compared with the control, while the negative control did not changed the level ofTC-1expression. Finally, a series of loss-of-function and gain-of-function studies wereperformed in vitro or in vivo to test the relationship between TC-1expression and cellbiological behavior. The results showed that compared with control group cells,respectively, the proliferation, cell cycle transition, apoptosis resistance, migation andinvasion of A549-pLenti-shRNA1cells were significantly decreased (P<0.05), theproliferation, cell cycle transition, apoptosis resistance, migation and invasion ofNCI-H520-pLenti-TC-1cells were significantly improved (P<0.05).(3) A549and NCI-H520-pLenti-TC-1cells in SITA were treated with FGFR2pathwayinhibitor PD173074to study the FGFR2signaling pathway which if regulates theexpression of TC-1. Real-Time PCR and Western Blotting showed that TC-1expressiondecreased markedly in treated cells compared with control cells. The negative control,which supplemented with no PD173074but equal volume of DMSO, did not change thelevel of TC-1expression. To have an insight of the effect of blockage of FGFR2signalingpathway on TC-1induced cell biological behavior, a series of loss-of-function andgain-of-function studies were performed in vitro and the FGFR2signaling pathway assayand chemotherapeutics assay were performed in vivo. The results of loss-of-function andgain-of-function studies in vitro showed that compared with control group cells, theproliferation, cell cycle transition, apoptosis resistance, migation and invasion ofPD173074treated cells were significantly decreased, resepectively (P<0.05). The resultsof FGFR2signaling pathway assay and chemotherapeutics assay in vivo showed thatcompared with MOCK group, PD(+) group, CIS(+) group and PD+CIS(+) groupsignificantly inhibited cell proliferation and prolonged survival of tumor-bearing animals,especially the PD+CIS group which exerted an additional inhibition effect and survival benefit than others (P<0.05).(4) To investigated the potential downstream proteins which were regulated by TC-1and implicated in cell biological behavior of NSCLC cells, western blotting wasperformed. The results showed that CCND1, c-Myc and c-Met proteins weredown-regulated considerably while the Caspase-3peotein was up-regulated markedly inA549-pLenti-shRNA1cells compared with A549cells. CCND1, c-Myc and c-Metproteins were up-regulated significantly while the Caspase-3peotein was down-regulatedremarkably in NCI-H520-pLenti-TC-1cells compared with NCI-H520cells.Conclusions(1) The expression of TC-1was upregulated in NSCLC tissures and the expression ofTC-1was significantly correlated with TNM staging, regional lymoh nodes metastasis andpeolifertaion, but not with age, gender and histological subtyoes.(2) A549colonies stably inhibition of TC-1expression (A549-pLenti-shRNA1) andNCI-H520-pLenti-TC-1stably overexpression of TC-1were established successfully.TC-1expression had influence on the NSCLC cell biological behavior both in vitro and invivo.(3) TC-1was a downstream target gene of FGFR2pathway in NSCLC cells. Additionof PD173074blocks the FGFR2signaling pathway, which decreases the expression ofTC-1gene, result in inhibition of TC-1mediated cell biological behavior.(4) TC-1regulates cell biological behavior of NSCLC cells by induces the expression ofwnt/β-catenin signaling pathway target proteins and inhibits the expression of Caspase-3.In this study, the expression of TC-1in NSCLC and the role of TC-1in the cellbiological behavior were systematically investigated. It was revelaed that TC-1is adownstream gene of FGFR2signaling pathway and an upstream regulator ofWnt/β-catenin pathway and Caspase-3, which involved in the regulation of cell biologicalbehavior and could be a potential target and a new way for further study.