Promoter methylation-mediated silencing of ?-catenin enhances invasiveness of non-small cell lung cancer and predicts adverse prognosis.
ABSTRACT: ?-Catenin plays dual role in adhesion complex formation and the Wnt signaling pathway. Although ?-catenin expression appears to be upregulated and Wnt signaling pathway is activated in the majority of cancers, its expression level seems to be lost in non-small cell lung cancer (NSCLC). We previously reported that the promoter of ?-catenin was hypermethylated in two NSCLC cell lines. In the current study, we expanded our analysis for the methylation status of ?-catenin promoter region and its protein expression in seven NSCLC cell lines and a series of 143 cases of primary human lung cancer with adjacent non-neoplastic tissues. Quantitative methylation specific PCR (qMSP) analysis showed methylation of ?-catenin promoter region in five NSCLC cell lines, with increased ?-catenin protein levels upon 5'-Aza-2'-deoxycytidine (5-aza-dC) treatment. The methylation status in SPC (methylated) and A549 (unmethylated) was confirmed by bisulfite sequencing PCR. 5-Aza-dC treatment inhibited invasiveness of SPC but not A549. Immunofluorescence analysis showed membranous ?-catenin expression was lost in SPC and could be re-established by 5-aza-dC, while Wnt3a treatment led to nuclear translocation of ?-catenin in both SPC and A549. Dual-luciferase assays indicated that 5-aza-dC treatment caused no significant increase in Wnt signaling activity compared with Wnt3a treatment. The effect of demethylation agent in SPC can be reversed by ?-catenin depletion but not E-cadherin depletion which indicated that the methylation mediated ?-catenin silencing might enhance NSCLC invasion and metastasis in an E-cadherin independent manner. Subsequent immunohistochemistry results further confirmed that ?-catenin promoter hypermethylation correlated with loss of immunoreactive protein expression, positive lymph node metastasis, high TNM stage and poor prognosis. The present study implicates ?-catenin promoter hypermethylation in the mechanism of epigenetic changes underlying NSCLC metastasis and progression, thus indicating the potential of ?-catenin as a novel epigenetic target for the treatment of NSCLC patients.
Project description:Prenatal hypoxia (PH) is a common pregnancy complication, harmful to brain development. This study investigated whether and how PH affected Wnt pathway in the brain. Pregnant rats were exposed to hypoxia (10.5% O2 ) or normoxia (21% O2 ; Control). Foetal brain weight and body weight were decreased in the PH group, the ratio of brain weight to body weight was increased significantly. Prenatal hypoxia increased mRNA expression of Wnt3a, Wnt7a, Wnt7b and Fzd4, but not Lrp6. Activated ?-catenin protein and Fosl1 expression were also significantly up-regulated. Increased Hif1a expression was found in the PH group associated with the higher Wnt signalling. Among 5 members of the Sfrp family, Sfrp4 was down-regulated. In the methylation-regulating genes, higher mRNA expressions of Dnmt1 and Dnmt3b were found in the PH group. Sodium bisulphite and sequencing revealed hyper-methylation in the promoter region of Sfrp4 gene in the foetal brain, accounting for its decreased expression and contributing to the activation of the Wnt-Catenin signalling. The study of PC12 cells treated with 5-aza further approved that decreased methylation could result in the higher Sfrp4 expression. In the offspring hippocampus, protein levels of Hif1a and mRNA expression of Sfrp4 were unchanged, whereas Wnt signal pathway was inhibited. The data demonstrated that PH activated the Wnt pathway in the foetal brain, related to the hyper-methylation of Sfrp4 as well as Hif1a signalling. Activated Wnt signalling might play acute protective roles to the foetal brain in response to hypoxia, also would result in disadvantageous influence on the offspring in long-term.
Project description:DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.
Project description:p73 is a member of the p53 tumor suppressor protein family and induces apoptosis in tumor cells that lack functional p53. It has been demonstrated that methylation of CpG islands in the promoter and exon 1 region may result in silencing of the p73 gene. The aim of this study was to investigate the correlation between p73 gene expression and chemosensitivity in non-small cell lung cancer (NSCLC) cell lines. The expression of the p73 transcript in six NSCLC cell lines was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The methylation status in these cell lines was determined by methylation-specific PCR (MSP) analysis. An in vitro demethylation assay was conducted using the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC). Restored expression of p73 in the human lung squamous cell carcinoma cell line C57, both at the mRNA and protein level, was investigated by RT-PCR and immunohistochemistry, respectively. A colony formation assay was used to measure the surviving fraction of the C57 cell line. Transcript silencing of the p73 gene in the six NSCLC cell lines was observed and related to aberrant methylation. The expression of the p73 transcript and protein in the C57 cell line was restored by 5-aza-dC. The surviving fraction for colony formation in C57 cells pre-treated with 5-aza-dC was 0.059±0.006, which was significantly different from that of the control group (0.12±0.008; P<0.05). Our data demonstrated a significant correlation between expression of p73 and cellular chemosensitivity in NSCLC.
Project description:Wnt/?-catenin signaling is essential for normal mammalian development. Wnt3a activates the Wnt/?-catenin pathway through stabilization of ?-catenin; a process in which the phosphoprotein Dishevelled figures prominently. Protein arginine methylation in signaling complexes containing Dishevelled was investigated. Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1. Stimulation of totipotent mouse embryonic F9 cells with Wnt3a provoked increased methylation of G3BP1. We show that G3BP1 is a novel Ctnnb1 mRNA binding protein. Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a. Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA.
Project description:Purpose: The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on microRNA (miRNA) silencing in non-small cell lung cancers (NSCLC). Experimental Design: We performed microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2´-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. MiRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding non-malignant lung tissue samples of 101 stage I-III NSCLC patients. Results: By comparing microarray data of untreated and drug treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analysed. Moreover, miR-9-3 and miR-193a were found to be tumor-specifically methylated in NSCLC patients. We observed a shorter disease-free survival of miR-9-3 methylated lung squamous cell carcinoma (LSCC) patients compared to miR-9-3 unmethylated LSCC patients by multivariate analysis (HR = 3.8, 95% CI = 1.3 to 11.2, p = 0.017) and a shorter overall survival of miR-9-3 methylated LSCC patients compared to miR-9-3 unmethylated LSCC patients by univariate analysis (p = 0.013). Conclusions: Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in LSCC patients. MiRNA expression (LC Sciences, mirBASE12) was analyzed before and after treatment of A549 cells with 5-aza-2´-deoxycytidine (Aza-dC) and a combination of Aza-dC and trichostatin A (TSA). Experiments were performed in duplicates.
Project description:Wnt signaling is initiated upon binding of Wnt proteins to Frizzled proteins and their co-receptors LRP5 and 6. The signal is then propagated to several downstream effectors, mediated by the phosphoprotein scaffold, dishevelled. We report a novel role for arginine methylation in regulating Wnt3a-stimulated LRP6 phosphorylation. G3BP2, a dishevelled-associated protein, is methylated in response to Wnt3a. The Wnt3a-induced LRP6 phosphorylation is attenuated by G3BP2 knockdown, chemical inhibition of methyl transferase activity or expression of methylation-deficient mutants of G3BP2. Arginine methylation of G3BP2 appears to be a Wnt3a-sensitive 'switch' regulating LRP6 phosphorylation and canonical Wnt-?-catenin signaling.
Project description:Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC.Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression.Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and ?-catenin levels.Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/?-catenin signaling.
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:The role of canonical Wnt signaling in myofibroblast biology has not been fully investigated. The C3H10T1/2 mesenchymal cell line recapitulates myofibroblast differentiation in vitro and in vivo, including SM22alpha expression. Using this model, we find that Wnt3a upregulates SM22alpha in concert with TGFbeta(1). Wnt1, Wnt5a and BMP2 could not replace Wnt3a and TGFbeta(1) signals. Chromatin immunoprecipitation identified that Wnt3a enhances both genomic SM22alpha histone H3 acetylation and beta-catenin association, hallmarks of transcriptional activation. By analyzing a series of SM22alpha promoter-luciferase (LUC) reporter constructs, we mapped Wnt3a-regulated DNA transcriptional activation to nucleotides -213 to -192 relative to the transcription initiation site. In gel shift assays, DNA-protein complexes assembled on this element were disrupted with antibodies to beta-catenin, Smad2/3, and TCF7, confirming the participation of known Wnt3a and TGFbeta transcriptional mediators. Mutation of a CAGAG motif within this region abrogated recognition by these DNA binding proteins. Wnt3a treatment increased Smad2/3 binding to this element. Mutation of the cognate within the context of the native 0.44 kb SM22alpha promoter resulted in a 70% decrease in transcription, and reduced Wnt3a+TGFbeta(1) induction. A concatamer of SM22alpha [-213 to -192] conveyed Wnt3a+TGFbeta(1) activation to the unresponsive RSV promoter. Dominant negative TCF inhibited SM22alpha [-213 to -192] x 6 RSVLUC activation. Moreover, ICAT (inhibitor of beta-catenin and TCF) decreased while TCF7L2 and beta-catenin enhanced 0.44 kb SM22alpha promoter induction by Wnt3a+TGFbeta(1). RNAi "knockdown" of beta-catenin inhibited Wnt3a induction of SM22alpha. Thus, Wnt/beta-catenin signaling interacts with TGFbeta/Smad pathways to control SM22alpha gene transcription.
Project description:The Wnt/?-catenin pathway is one of the most conserved signaling pathways across species with essential roles in development, cell proliferation, and disease. Wnt signaling occurs at the protein level and via ?-catenin-mediated transcription of target genes. However, little is known about the underlying mechanisms regulating the expression of the key Wnt ligand Wnt3a or the modulation of its activity. Here, we provide evidence that there is significant cross-talk between the dopamine D2 receptor (D2R) and Wnt/?-catenin signaling pathways. Our data suggest that D2R-dependent cross-talk modulates Wnt3a expression via an evolutionarily-conserved TCF/LEF site within the WNT3A promoter. Moreover, D2R signaling also modulates cell proliferation and modifies the pathology in a renal ischemia/reperfusion-injury disease model, via its effects on Wnt/?-catenin signaling. Together, our results suggest that D2R is a transcriptional modulator of Wnt/?-catenin signal transduction with broad implications for health and development of new therapeutics.