FOXP3 promotes tumor growth and metastasis by activating Wnt/?-catenin signaling pathway and EMT in non-small cell lung cancer.
ABSTRACT: The role of cancer cell FOXP3 in tumorigenesis is conflicting. We aimed to study FOXP3 expression and regulation, function and clinical implication in human non-small cell lung cancer (NSCLC).One hundred and six patients with histologically-confirmed NSCLC who underwent surgery were recruited for the study. Tumor samples and NSCLC cell lines were used to examine FOXP3 and its related molecules. Various cell functions related to tumorigenesis were performed. In vivo mouse tumor xenograft was used to confirm the in vitro results.NSCLC patients with the high level of FOXP3 had a significant decrease in overall survival and recurrence-free survival. FOXP3 overexpression significantly induced cell proliferation, migration, and invasion, whereas its inhibition impaired its oncogenic function. In vivo studies confirmed that FOXP3 promoted tumor growth and metastasis. The ectopic expression of FOXP3 induced epithelial-mesenchymal transition (EMT) with downregulation of E-cadherin and upregulation of N-cadherin, vimentin, snail, slug, and MMP9. The oncogenic effects by FOXP3 could be attributed to FOX3-mediated activation of Wnt/?-catenin signaling, as FOXP3 increased luciferase activity of Topflash reporter and upregulated Wnt signaling target genes including c-Myc and Cyclin D1 in NSCLC cells. Co-immunoprecipitation results further indicated that FOXP3 could physically interacted with ?-catenin and TCF4 to enhance the functions of ?-catenin and TCF4, inducing transcription of Wnt target genes to promote cell proliferation, invasion and EMT induction.FOXP3 can act as a co-activator to facilitate the Wnt-b-catenin signaling pathway, inducing EMT and tumor growth and metastasis in NSCLC.
Project description:BACKGROUND:Epithelial-mesenchymal transition (EMT)-associated proteins play key roles in cancer progression and metastasis with the involvement of microRNAs (miRNAs). This study aims to assess the role of miR-506 working in tandem with LIM Homeobox 2 (LHX2) in EMT and metastasis through the Wnt/?-catenin signaling pathway in nasopharyngeal carcinoma (NPC). METHODS:Differentially expressed genes associated with NPC were screened using microarray analyses, from which LHX2 was identified. Next, the potential relationship between miR-506 and LHX2 was analyzed. In order to explore the effect of miR-506 or LHX2 on NPC cell proliferation, migration, invasion and apoptosis, serials of mimics, inhibitors or siRNA against LHX2 were transfected into NPC cells. Then, the expression patterns of LHX2, Wnt1, ?-catenin, E-cadherin, Vimentin, TCF4 and Twist were determined to assess the influence of miR-506 or LHX2 on EMT as well as the relationship between the Wnt/?-catenin signaling pathway and TCF4. The tumorigenicity and lymph node metastasis (LNM) in xenograft tumors of nude mice were observed. RESULTS:The has-miR-506-3p was identified as the down-regulated gene in NPC based on the microarray data while LHX2 was negatively regulated by miR-506. Over-expression of miR-506 or silencing of LHK2 inhibited NPC cell proliferation, migration, invasion, tumorigenicity and LNM but promoted apoptosis indicated by decreased Wnt1, ?-catenin, Vimentin, TCF4 and Twist expressions along with increased E-cadherin expressions. CONCLUSIONS:miR-506 inhibits tumor growth and metastasis in NPC via inhibition of Wnt/?-catenin signaling by down-regulating LHX2, accompanied by decreased TCF4. Taken together, miR-506 targeted-inhibition LHX2 presents a promising therapeutic strategy for the treatment of NPC. TRIAL REGISTRATION:ChiCTR1800018889 . Registered 15 October 2018.
Project description:Wnt signaling contributes to the reprogramming and maintenance of cancer stem cell (CSC) states that is activated by the epithelial-mesenchymal transition (EMT) program. However, the mechanistic relationship between the EMT and Wnt pathway in CSCs remains unclear. Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) indicated that EMT induces a switch from the β-catenin/E-cadherin/Sox15 complex to the β-catenin/Twist1/TCF4 complex, which then binds to CSC-related gene promoters. In tandem co-IP and re-ChIP experiments using epithelial-type cells, Sox15 associated with the β-catenin/E-cadherin complex and then bound to the proximal promoter region of CASP3, consequently resulting in Twist1 cleavage and negatively regulating the β-catenin–elicited promotion of the CSC phenotype. During the EMT, Twist1 in complex with β-catenin enhanced β-catenin/TCF4 transcriptional activity, which includes binding to the proximal promoter region of ABCG2, a marker of CSCs. For clinical application, the five-gene signature nuclear β-cateninHigh/nuclear Twist1High/E-cadherinLow/Sox15Low/CD133High may be a valuable prognostic marker in patients with human lung cancer. Total cellular RNA was extracted from LM and HM20 cells after treatment with or without Wnt3a for 24hours. Human lung cancer A549 cell–derived spheres were transferred back to adhesive tissue culture plates (LM cells). To establish an EMT/metastasis cell model, LM cells were seeded onto Matrigel-coated Boyden chamber membranes. HM20 cells were serially selected for Matrigel invasion 20 times.
Project description:Wnt signaling contributes to the reprogramming and maintenance of cancer stem cell (CSC) states that is activated by the epithelial-mesenchymal transition (EMT) program. However, the mechanistic relationship between the EMT and Wnt pathway in CSCs remains unclear. Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) indicated that EMT induces a switch from the ?-catenin/E-cadherin/Sox15 complex to the ?-catenin/Twist1/TCF4 complex, which then binds to CSC-related gene promoters. In tandem co-IP and re-ChIP experiments using epithelial-type cells, Sox15 associated with the ?-catenin/E-cadherin complex and then bound to the proximal promoter region of CASP3, consequently resulting in Twist1 cleavage and negatively regulating the ?-catenin–elicited promotion of the CSC phenotype. During the EMT, Twist1 in complex with ?-catenin enhanced ?-catenin/TCF4 transcriptional activity, which includes binding to the proximal promoter region of ABCG2, a marker of CSCs. For clinical application, the five-gene signature nuclear ?-cateninHigh/nuclear Twist1High/E-cadherinLow/Sox15Low/CD133High may be a valuable prognostic marker in patients with human lung cancer. Total cellular RNA was extracted from LM and HM20 cells after treatment with or without Wnt3a for 24hours. Human lung cancer A549 cell–derived spheres were transferred back to adhesive tissue culture plates (LM cells). To establish an EMT/metastasis cell model, LM cells were seeded onto Matrigel-coated Boyden chamber membranes. HM20 cells were serially selected for Matrigel invasion 20 times.
Project description:Dehydroeffusol (DHE) is a phenanthrene compound that possesses anti-tumor activity. However, the effect of DHE on non-small cell lung cancer (NSCLC) has not been investigated previously. Therefore, the objective of our study was to explore the role of DHE in NSCLC and the underlying mechanism. Our results showed that DHE significantly inhibited the cell viability of A549 cells in a dose- and time-dependent manner under normoxic condition. Moreover, A549 cells were more sensitive to DHE under hypoxic condition compared with the A549 cells cultured in normoxic condition. Hypoxia-induced increased migration and invasion abilities were mitigated by DHE in A549 cells. Treatment of DHE caused increased E-cadherin expression and decreased N-cadherin expression in hypoxia-induced A549 cells. DHE also suppressed hypoxia-induced increase in both protein and mRNA levels of hypoxia inducible factor-1? (HIF-1?) expression in A549 cells. Furthermore, DHE inhibited hypoxia-induced activation of Wnt/?-catenin pathway in A549 cells. The inhibitory effect of DHE on hypoxia-induced EMT was reversed by LiCl, which is an activator of Wnt/?-catenin signaling pathway. In conclusion, these findings demonstrated that DHE prevented hypoxia-induced EMT in NSCLC cells by inhibiting the activation of Wnt/?-catenin pathway, suggesting that DHE might serve as a therapeutic target for the NSCLC metastasis.
Project description:BACKGROUND:The distant metastasis of cancer cells is a risk factor for tumor lethality and poor prognosis in non-small-cell lung carcinoma (NSCLC). Increased SOX9 expression has been associated with clinical stage and poor prognosis in NSCLC, but the molecular mechanisms by which SOX9 promotes metastasis in NSCLC are still unknown. METHODS:The relationship between SOX9 expression and T, N, M classification was assessed using the χ2 test and Spearman's analysis in 142 immunohistochemically diagnosed specimens of NSCLC. We also generated SOX9-overexpression and SOX9-knockdown cells lines and their corresponding control cell lines by transfection with lentiviral constructs. In vivo assay, SOX9-overexpressing and SOX9-knockdown NSCLC cells were injected in zebrafish to examine distance metastasis. Gene set enrichment analysis (GSEA) was applied to analysis the correlation between SOX9 overexpression and Wnt/β-catenin pathway. Luciferase assay was used to check transcriptional activity of TCF/LEF and western blot and immunofluorescence was employed to detect β-catenin translocation in SOX9-overexpression, SOX9-knockdown and their corresponding control cell lines. RESULTS:We found that SOX9 overexpression correlates with the T, N and M stage significantly (p = 0.03, 0.000, and 0.032 respectively) in 142 immunohistochemically diagnosed specimens of NSCLC. SOX9 overexpression was found to decrease the expression of the epithelial cell markers E-cadherin and γ-catenin and increase the expression of the mesenchymal cell markers N-cadherin and vimentin. An in vivo assay showed distant metastasis of the SOX9-overexpressing cells, which was not observed in the SOX9-knockdown cells. These findings indicate that SOX9 promotes distant metastasis by promoting EMT in NSCLC cells. GSEA showed that SOX9 overexpression was significantly correlated with the Wnt/β-catenin pathway which was corroborated by the expression of EMT-associated proteins in this pathway and its downstream target genes. SOX9 overexpression was also found to enhance the transcriptional activity of TCF/LEF, promote the nuclear translocation of β-catenin and increase the phosphorylation of GSK3β at Ser9. Further, inhibition of β-catenin suppressed the metastasis-promoting effects of SOX9 overexpression. CONCLUSIONS:This study is the first to report that SOX9 is associated with clinical TNM stage and indicates that SOX9 promotes migration, invasion and the EMT process through the Wnt/β-catenin pathway.
Project description:Several studies suggest that Wnt signaling contributes to reprogramming and maintenance of cancer stem cell (CSC) states activated by loss of membranous E-cadherin expression. However, E-cadherin's exact role in Wnt/?-catenin-mediated promotion of the CSC phenotype remains unclear. Recently, a significant positive correlation has been observed between the expression of nuclear (an aberrant nuclear localization) E-cadherin and ?-catenin in gastric and colorectal carcinomas. Here we conducted a series of in-vitro and in-vivo studies to show that the ?-catenin/TCF4 interaction was abolished by E-cadherin and was correlated with its nuclear localization, and consequently decreased ?-catenin/TCF4 transcriptional activity. Nuclear E-cadherin was a negative regulator of Wnt/?-Catenin-elicited promotion of the CSC phenotype. Using immunohistochemistry on lung cancer tissue microarrays, we found that changes in subcellular location of E-cadherin may be described by tumor grade and stage, suggesting cellular redistribution during lung tumorigenesis. Furthermore, nuclear E-cadherin expression was more significantly inversely correlated with CD133 (a lung CSC marker) expression (P<0.005) than total E-cadherin expression (P<0.05), suggesting that lung cancer as defined by nuclear E-cadherin(Low)/nuclear ?-catenin(High)/CD133(High) biomarkers has superior prognostic value over total E-cadherin(Low)/nuclear ?-catenin(High)/CD133(High).
Project description:Cadherin subtype switching from E-cadherin to N-cadherin is associated with the epithelial-to-mesenchymal transition (EMT), a process required for invasion and dissemination of carcinoma cells. We found that N-cadherin is expressed in human and mouse pancreatic intraepithelial neoplasia (PanIN), suggesting that N-cadherin may also have a role in early-stage pancreatic cancer. To investigate the role of N-cadherin in mouse PanIN (mPanIN), we simultaneously activated oncogenic K-ras(G12D) and deleted the N-cadherin (Cdh2) gene in the murine pancreas. Genetic ablation of N-cadherin (N-cad KO) caused hyperproliferation, accelerated mPanIN progression, and early tumor development in K-ras(G12D) mice. Decreased E-cadherin and redistribution of ?-catenin accompanied the loss of N-cadherin in pancreatic ductal epithelial cells (PDEC). Nuclear accumulation of ?-catenin and its transcription co-activator Tcf4 led to activation of Wnt/?-catenin target genes. Unexpectedly, loss of N-cadherin in the K-ras(G12D) model resulted in increased mPanIN progression and tumor incidence. These in vivo results demonstrate for the first time that N-cadherin functions as a growth suppressor in the context of oncogenic K-ras.
Project description:BACKGROUND: The class 1 carcinogen cadmium (Cd2+) disrupts the E-cadherin/beta-catenin complex of epithelial adherens junctions (AJs) and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/beta-catenin signaling are known to contribute to carcinogenesis. RESULTS: We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC). Cd2+ (25 microM, 3-9 h) caused nuclear translocation of beta-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of beta-catenin with AJ components (E-cadherin, alpha-catenin) and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1) were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. CONCLUSIONS: Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.
Project description:Wnt/?-catenin signaling plays a critical role during development of both normal and malignant colorectal cancer tissues. Phosphorylation of ?-catenin protein alters its trafficking and function. Such conventional allosteric regulation usually involves a highly specialized set of molecular interactions, which may specifically turn on a particular cell phenotype. This study identifies a novel transcription modulator with an FLYWCH/Zn-finger DNA-binding domain, called "FLYWCH1." Using a modified yeast-2-hybrid based Ras-Recruitment system, it is demonstrated that FLYWCH1 directly binds to unphosphorylated (nuclear) ?-catenin efficiently suppressing the transcriptional activity of Wnt/?-catenin signaling that cannot be rescued by TCF4. FLYWCH1 rearranges the transcriptional activity of ?-catenin/TCF4 to selectively block the expression of specific downstream genes associated with colorectal cancer cell migration and morphology, including ZEB1, EPHA4, and E-cadherin. Accordingly, overexpression of FLYWCH1 reduces cell motility and increases cell attachment. The expression of FLYWCH1 negatively correlates with the expression level of ZEB1 and EPHA4 in normal versus primary and metastatic colorectal cancer tissues in patients. Thus, FLYWCH1 antagonizes ?-catenin/TCF4 signaling during cell polarity/migration in colorectal cancer. IMPLICATIONS: This study uncovers a new molecular mechanism by which FLYWCH1 with a possible tumor suppressive role represses ?-catenin-induced ZEB1 and increases cadherin-mediated cell attachment preventing colorectal cancer metastasis.
Project description:Oral cancer belongs to the group of head and neck cancers. If not diagnosed or treated early, it can be life threatening. Epithelial-mesenchymal transition (EMT) plays an important role in tumor formation and progression. An increase in the presence of the EMT phenotype causes tumor cell proliferation, migration, invasion, and poor prognosis. Therefore, attenuating carcinogenesis via EMT inhibition is a good strategy. Herein, we will determine the pharmacological effects of chrysophanol on the EMT in FaDu cells. To analyze EMT, we detected the expression EMT markers, including ?-SMA, ?-catenin, vimentin, N-cadherin, E-cadherin, phospho-GSK-3?, and nuclear translocations of p65 and ?-catenin by western blotting. Additionally, accumulating evidence indicates that reactive oxygen species (ROS) mediate EMT. Our results showed that the level of ROS was significantly increased after chrysophanol treatment. We further speculated that chrysophanol-mediated EMT and metastasis are involved in the Wnt-3-dependent signaling pathway. The inhibition of the EMT phenotype and metastasis and accumulation of ROS caused by chrysophanol was reversed by treatment with the Wnt-3 agonist Bml 284. Therefore, our findings indicated that chrysophanol altered EMT formation, ROS accumulation, and metastasis via the Wnt-3-dependent signaling pathway.