Beta-catenin is known as to be always a main contributor to great metastatic potential, a feature of CSCs [41, 57], and FOXC1 provides been proven to market metastasis in NSCLC [18, 19]. Proteins Atlas. CSC-like properties had been analyzed predicated on CSC marker-positive Rabbit polyclonal to ALOXE3 cell people, self-renewal capability, stemness-related gene appearance, paederoside drug and tumorigenicity resistance. The percentage of Compact disc133+ cells was examined by circulation cytometric analysis. Self-renewal ability was detected by sphere-formation analysis. Real-time PCR, western blotting and immunohistochemical staining were employed to detect mRNA and protein levels. Tumorigenicity was decided based on a xenograft formation assay, and effects of FOXC1 on drug resistance were assessed by cell viability and apoptosis assays. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to investigate the binding of FOXC1 to beta-catenin promoter. Results FOXC1 expression was found to be elevated in NSCLC tissues and negatively correlated with patient survival. FOXC1 knockdown reduced CD133+ cell percentage, suppressed self-renewal ability, decreased expression of stemness-related genes (Oct4, NANOG, SOX2 and ABCG2) and inhibited NSCLC cell tumorigenicity in vivo. Moreover, FOXC1 knockdown increased cisplatin and docetaxel sensitivity and reduced gefitinib resistance, whereas FOXC1 overexpression enhanced CSC-like properties. Luciferase reporter and ChIP assays showed beta-catenin to be a direct transcriptional target of FOXC1. Furthermore, overexpression of beta-catenin reversed the CSC-like house inhibition induced by FOXC1 knockdown, and knockdown of beta-catenin attenuated the CSC-like properties induced by FOXC1 overexpression. Conclusions This study demonstrates that FOXC1 induces CSC-like properties in NSCLC by promoting beta-catenin expression. The findings indicate that FOXC1 is usually a potential molecular target for anti-CSC-based therapies in NSCLC. values. ** em P /em ? ?0.01 FOXC1 enhances stemness of NSCLC cells in vitro We found FOXC1 to be widely paederoside expressed in NSCLC cells, and FOXC1 expression was significantly higher in gefitinib-resistant PC9/G cells than in gefitinib-sensitive PC9 cells (Fig.?2a). High (A549 and PC9/G) and low (NCI-H1299 and PC9) FOXC1-expressing cell lines were used paederoside for further studies. We established an A549-LV-shFOXC1 stable cell collection with stable knockdown of FOXC1 expression (Fig. ?(Fig.2b),2b), and a NCI-H1299-LV-FOXC1 stable cell line with constant FOXC1 expression (Fig. ?(Fig.2c).2c). FOXC1 knockdown reduced the percentage of CD133+ cells (Fig. ?(Fig.2d),2d), inhibited sphere formation (Fig. ?(Fig.2f)2f) and downregulated mRNA and protein levels of stemness-related genes (SOX2, Oct4, NANOG paederoside and ABCG2) (Fig. ?(Fig.2h).2h). Conversely, FOXC1 overexpression increased the CD133+ cell percentage (Fig. ?(Fig.2e),2e), promoted sphere formation (Fig. ?(Fig.2g)2g) and upregulated mRNA and protein levels of SOX2, Oct4, NANOG and ABCG2 (Fig. ?(Fig.2i2i). Open in a separate windows Fig. 2 FOXC1 induces stemness of NSCLC cells in vitro. a FOXC1 protein levels in NSCLC cells were detected by western blotting. b and c FOXC1 mRNA and protein levels were stably downregulated in A549 cells and upregulated in NCI-H1299 cells. d and e The percentage of CD133+ cells was analyzed by circulation cytometry. f and g Representative images (left) and figures (right) of spheres (diameter? ?100?m). h and i Protein and mRNA levels of SOX2, Oct4, NANOG and ABCG2. All experiments were independently repeated three times. The bar graph presents the mean??SD. *P? ?0.05, **P? ?0.01 FOXC1 enhances tumorigenicity of NSCLC cells in vivo To investigate whether FOXC1 influences NSCLC cell tumorigenicity in vivo, we subcutaneously inoculated a series of NSCLC cells (5??105, 5??104 and 5??103) into BALB/c nude mice. FOXC1 knockdown decreased tumor incidence rate (Fig.?3a), tumor volume (Fig. ?(Fig.3c3c and ?ande)e) and tumor weight (Fig. ?(Fig.3g),3g), whereas, FOXC1 overexpression had the opposite effects (Fig. ?(Fig.3b,3b, ?,d,d, ?,ff and ?andhh). Open in a separate windows Fig. 3 FOXC1 enhances the tumorigenicity of NSCLC cells in vivo. A series of cells (5??105, 5??104 and 5??103) were subcutaneously inoculated into BALB/c nude mice ( em n /em ?=?8/group). a and b The paederoside tumor incidence of each group. c-f Images and growth curves of tumor xenografts. g and h Histograms show the tumor weights of each group. The bar graph presents the mean??SD. ** em P /em ? ?0.01 FOXC1 confers drug resistance in NSCLC cells As the presence of CSCs is one of the major causes of resistance to therapy , we investigated whether FOXC1 is involved in drug resistance in NSCLC. Cisplatin and docetaxel are widely used cytotoxic anti-cancer brokers in NSCLC treatment [38, 39]. FOXC1 knockdown enhanced the cell killing effects of cisplatin and docetaxel on A549 cells (Fig.?4a and ?andb)b) and increased the percentage of apoptotic cells (Fig. ?(Fig.4e).4e). In contrast, FOXC1 overexpression attenuated cisplatin and docetaxel-mediated killing of NCI-H1299 cells (Fig. ?(Fig.4c4c and ?andd)d) and reduced apoptotic cell percentage (Fig. ?(Fig.4f).4f). Gefitinib is usually a classic molecularly targeted anti-NSCLC.