Effect Of FOXC1 On Gefitinib Sensitivity In Non-small Cell Lung Cancer

Cancer has become a serious disease that threatens human health with increasing incidence and mortality worldwide.Lung cancer has the highest morbidity and mortality among all cancers.As the most common type of lung cancer,non-small cell lung cancer(NSCLC)has a rapid progression and is prone to recurrence and metastasis.Most patients are in advanced stage when diagnosed,resulted in poor prognosis and low five-year survival rate.Clinical treatment of lung cancer patients usually includes surgery,chemotherapy,radiotherapy and molecular targeted therapy.The scope of surgery is limited,and the toxic effect of chemotherapy and radiotherapy are large.Therefore,molecular targeted therapy is increasingly prominent.Gefitinib(Gef),as a classic small molecule targeted drug of EGFR-TKIs,has been widely used in clinical practice,but most patients eventually will occur drug resistance,leading to treatment failure and worsening of the disease.Therefore,it is urgent to explore mechanisms to overcome the resistance of EGFR-TKIs and develop effective treatment strategies.In recent years,with the in-depth study of tumor molecular biology and genomics,looking for new key genes in the development of cancer and targeted therapy for these key genes is expected to provide new treatment for overcoming EGFR-TKIs resistance.The human forkhead box(FOX)protein family is a family of evolutionarily highly conserved transcriptional regulators characterized by a DNA binding domain with a forkhead or a winged helix.FOX proteins play a key role in a variety of biological processes,including development,metabolism,differentiation,proliferation,apoptosis,invasion and migration.Multiple FOX proteins such as FOXC2,FOXD1 and FOXO3 have been shown to promote chemotherapy resistance of malignant tumor.Forkhead box C1(FOXC1)is an important member of the FOXC subfamily.Recent studies have shown that FOXC1 may promote the growth,proliferation,migration and invasion of tumor cells to reduce he sensitivity of breast cancer,colorectal cancer and ovarian cancer cells.However,whether FOXC1 affects the chemosensitivity of NSCLC has not been reported.Previous laboratory studies have shown that FOXC1 could significantly enhance the stem cell-like properties of NSCLC and induces chemotherapy resistance to docetaxel and cisplatin in A549 and H1299 cells.This indicates that FOXC1 acts as a cancer-promoting factor in NSCLC.Therefore,based on the previous research,NSCLC gefitinib-resistant strain PC9/G and sensitive strain PC9 were used as the research object in this study.The lentiviral packaging was used to construct FOXC1 low expression and high expression stable cell lines,and to analyze the effect of FOXC1 on gefitinib sensitivity of NSCLC cells and its mechanism from cell biological processes such as cell proliferation ability,apoptosis,migration and invasion ability,which in order to expand our understanding of the EGFR-TKIs resistance process and mechanism,and provide a new theoretical basis for overcoming gefitinib-resistant molecular targeted therapy.The study was divided into the following two parts:Part Ⅰ Effect of FOXC1 on the sensitivity of PC9/G and PC9 cells to gefitinibObjective: To investigate the effects of FOXC1 on gefitinib sensitivity in PC9/G and PC9 cells.Methods: 1)The FOXC1 knockdown and overexpression stable cell lines were constructed by lentiviral packaging technology,and the expression of FOXC1 protein in stable cell lines was detected by Western Blot;2)CCK-8 assay was used to detect the changes in sensitivity to gefitinib of NSCLC cells after knockdown and overexpression of FOXC1;3)Clony formation assay was used to detect the effect of gefitinib on proliferation of NSCLC cells;4)Flow cytometry(FCM)was used to detect the effect of gefitinib on the proportion of apoptotic cells of NSCLC cells;5)Transwell assay was used to detect the effect of gefitinib on the migration and invasion of NSCLC cells.RESULTS:(1)FOXC1 knockdown enhanced the sensitivity of PC9/G cells to gefitinib.1)Compared with the control cells PC9/G-LV-sh Control,FOXC1 protein expression was significantly decreased in FOXC1 knockdown stable cells PC9/G-LV-sh FOXC1(P<0.001);2)FOXC1 knockdown enhanced the gefitinib inhibition of PC9/G cells,decreased the IC50 value of gefitinib(P<0.05);3)inhibited the proliferation of PC9/G cells and enhanced the inhibitory effect of gefitinib on the proliferation of PC9/G cells;4)increased the proportion of apoptotic cells in PC9/G cells(P<0.01),significantly increased the proportion of apoptotic cells induced by gefitinib(P<0.05);5)decreased the migrated and invasive ability of PC9/G cells(P<0.01),and enhanced the inhibitory effect of gefitinib on migration and invasion of PC9/G cells(P<0.01).(2)FOXC1 overexpression reduced the sensitivity of PC9 cells to gefitinib.1)Compared with the control cells PC9-LV-Control,FOXC1 protein expression was significantly increased in FOXC1 overexpression stable cells PC9-LV-FOXC1(P<0.001);2)FOXC1 overexpression decreased the gefitinib inhibition of PC9 cells,increased the IC50 value of gefitinib(P<0.05);3)enhanced the proliferation of PC9 cells and decreased the inhibitory effect of gefitinib on the proliferation of PC9 cells(P<0.05);4)decreased the proportion of apoptotic cells in PC9 cells(P<0.05),significantly decreased the proportion of apoptotic cells induced by gefitinib(P<0.05);5)enhanced the migrated and invasive ability of PC9 cells(P<0.01),and attenuated the inhibitory effect of gefitinib on migration and invasion of PC9 cells(P<0.05).Conclusion: FOXC1 knockdown can attenuate the proliferation of PC9/G cells,promote apoptosis,reduce cell migration and invasion,and enhance the sensitivity of PC9/G cells to gefitinib.FOXC1 overexpression can promote the proliferation of PC9 cells,inhibit cell apoptosis,enhance cell migration and invasion,and reduce the sensitivity of PC9 cells to gefitinib.It is suggested that FOXC1 is involved in the regulation of the resistance of NSCLC cells to gefitinib.Part Ⅱ Mechanism of FOXC1 regulating the sensitivity of PC9/G and PC9 cells to gefitinibObjective: To explore the molecular mechanism of FOXC1 regulating the proliferation,apoptosis,invasion and metastasis of PC9/G and PC9 cells.METHODS: After FOXC1 knockdown and overexpression,expression of related molecular protein was detected by Western Blot: cleaved caspase-3 and proapoptotic protein BIM,anti-apoptotic protein Bcl-2 and survival protein Survivin;invasion and metastasis-associated proteins N-cadherin,vimentin,MMP9 and E-cadherin;ABC transporter family proteins ABCG2 and ABCB5.RESULTS:(1)Compared with the control cells PC9/G-LV-sh Control,the expression of cleaved caspase-3 and BIM was increased(P<0.01,P<0.001),but Bcl-2 and Survivin was decreased(P<0.01,P<0.001);the expression of interstitial markers N-cadherin and vimentin was decreased(P<0.01,P<0.01),MMP9 was decreased(P<0.01),the expression of epithelial marker E-cadherin was increased(P<0.001);and the expression of ABC transporter family proteins ABCG2 and ABCB5 expression were significantly decreased in FOXC1 konckdown stable cells PC9/G-LV-sh FOXC1(P<0.01,P<0.01).(2)Compared with the control cells PC9-LV-Control,the expression of cleaved caspase-3 and BIM was decreased(P<0.01,P<0.01),but Bcl-2 and Survivin was increased.(P<0.01,P<0.01);N-cadherin and vimentin was increased(P<0.01,P<0.001),MMP9 was increased(P<0.001),but E-cadherin was decreased(P<0.01);ABCG2 and ABCB5 was significantly increased in FOXC1 overexpression stable cells PC9-LV-FOXC1(P < 0.001,P < 0.001).Conclusion: FOXC1 may inhibit apoptosis of NSCLC cells by regulating apoptosis and survival-related protein expression levels(cleaved caspase-3,BIM,Bcl-2,and Survivin);and regulate epithelial and mesenchymal marker-related protein expression levels(E-cadherin,N-cadherin,vimentin,and MMP9)to enhance cell invasion and migration;and promote ABC transporter protein expression levels(ABCG2 and ABCB5)to increase drug efflux and reduce intracellular druga ccumulation,thereby reduce the sensitivity of NSCLC cells to gefitinib.