Wnt Antagonist Genes Research Articles

Abstract LB301: BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation

Abstract Objective: The BRAF V600E mutation drives the generation of an aggressive subtype of colorectal cancer (CRC). Although activation of WNT signalling is a generic hallmark of CRC, mutations in APC are uncommon in BRAF V600E-mutant CRC. Instead, mutations in RNF43 are the suspected WNT signalling driving event in these tumours. Here, we aimed to uncover how the WNT pathway is activated in BRAF V600E-mutant colon cancer. Design: CRISPR-LbCpf1-corrected BRAF (V600E) and RNF43 (P441fs) organoids were analysed for their growth potential in the presence or absence of growth factors, for their ability to form tumours, and by immunoblotting, RNA sequencing, and DNA methylation assays. Results: BRAF E600V organoids regained dependency on epidermal growth factor (EGF) receptor signalling for ERK pathway activation and proliferation, and completely lost tumorigenic potential. Correction of BRAF V600E, but not RNF43 P441fs, caused suppression of WNT target genes, while genes reflecting epithelial differentiation and negative regulators of WNT signalling were upregulated in BRAF E600V organoids. DNA methylation analysis revealed promoter hypermethylation of WNT antagonist genes in BRAF V600E organoids. Gene body hypermethylation, associated with transcriptional upregulation, was observed in key WNT-driven intestinal stem cell marker genes LGR5 and EPHB2, and in the WNT-effector TCF4. Treatment of BRAF V600E organoids with the DNA demethylating agent 5-aza-2′-deoxycytidine resulted in upregulation of WNT antagonist genes and a marked decrease in WNT target gene expression. Conclusions: BRAFV600E, rather than mutant RNF43, is a prominent force behind WNT pathway activation, and is essential for maintaining tumorigenic capacity. BRAFV600E causes widespread dysregulation of DNA methylation leading to activation of WNT signalling. Citation Format: Layla El Bouazzaoui, Jeroen M. Bugter, Emre Küçükköse, André Verheem, Jasmin B. Post, Nicola Fenderico, Inne H. Borel Rinkes, Hugo J. Snippert, Madelon M. Maurice, Onno Kranenburg. BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB301.

EZH2 activates Wnt/β-catenin signaling in human uterine fibroids, which is inhibited by the natural compound methyl jasmonate

To investigate the link between EZH2 and Wnt/β-catenin signaling and its role in uterine fibroids (UFs) pathogenesis and explore the potential effect of natural compound methyl jasmonate (MJ) against UFs. EZH2 overexpression or inhibition was achieved in human uterine leiomyoma (HuLM) cells using EZH2-expressing adenovirus or chemical EZH2 inhibitor (DZNep), respectively. The HuLM and normal uterine smooth muscle cells were treated with 0.1-3 mM of MJ, and several experiments were employed. Laboratory study. None. Methyl jasmonate. Protein expression of EZH2, β-catenin, and proliferating cell nuclear antigen (PCNA) was measured by Western blot as well as gene expression alterations of Wnt ligands (Wnt5A, Wnt5b, and Wnt9A), WISP1, CTNNB1, and its responsive gene PITX2 using quantitative real-time polymerase chain reaction. The protein and ribonucleic acid (RNA) levels of several markers were measured in MJ-treated or untreated HuLM cells, including EZH2 and β-catenin, extracellular matrix markers collagen type 1 (COL1A1) and fibronectin (FN), proliferation markers cyclin D1 (CCND1) and PCNA, tumor suppressor marker p21, and apoptotic markers (BAX, cytochrome c, and cleaved caspase 3). EZH2 overexpression significantly increased the gene expression of several Wnt ligands (PITX2, WISP1, WNT5A, WNT5B, and WNT9A), which increased nuclear translocation of β-catenin and PCNA and eventually HuLM cell proliferation. EZH2 inhibition blocked Wnt/β-catenin signaling activation where the aforementioned genes significantly decreased as well as PCNA, cyclin D1, and PITX2 protein expression compared with those in untreated HuLM. Methyl jasmonate showed a potent antiproliferative effect on HuLM cells in a dose- and time-dependent manner. Interestingly, the dose range (0.1-0.5 mM) showed a selective growth inhibitory effect on HuLM cells, not on normal uterine smooth muscle cells. Methyl jasmonate treatment at 0.5 mM for 24 hours significantly decreased both protein and RNA levels of EZH2, β-catenin, COL1A1, FN, CCND1, PCNA, WISP1, and PITX2 but increased the protein levels of p21, BAX, cytochrome, c and cleaved caspase 3 compared with untreated HuLM. Methyl jasmonate-treated cells exhibited down-regulation in the RNA expression of 36 genes, including CTNNB1, CCND1, Wnt5A, Wnt5B, and Wnt9A, and up-regulation in the expression of 34 genes, including Wnt antagonist genes WIF1, PRICKlE1, and DKK1 compared with control, confirming the quantitative real-time polymerase chain reaction results. Our studies provide a novel link between EZH2 and the Wnt/β-catenin signaling pathway in UFs. Targeting EZH2 with MJ interferes with the activation of wnt/β-catenin signaling in our model. Methyl jasmonate may offer a promising therapeutic option as a nonhormonal and cost-effective treatment against UFs with favorable clinical utility, pending proven safe and efficient in human clinical trials.

Abstract P6-11-02: Mitochondria-Nuclear Crosstalk Regulates the WNT Pathway in Triple-Negative Breast Cancer

Abstract Compared to hormone receptor (HR) positive breast cancer (BC), basal or triple-negative BC (TNBC) suffers a poor prognosis caused by a limited understanding of its driver signaling pathways. We used the transmitochondrial cybrid approach to understand the metabolic reprogramming and mitochondria-nuclear crosstalk in metastatic TNBC. In cybrids, mitochondria from cancer and benign cells are compared under a commonly defined nuclear background. Using cybrid-based discovery and its validation in cell lines, patient-derived xenografts, and clinical data, we have previously reported the activation of fatty acid oxidation (FAO) in metastatic TNBC. Recently, we developed a unique gene (DEG) signature by integrating gene expression data from cybrids with different omnibus databases such as TCGA and METABRIC. Our DEG could effectively distinguish the PAM50 subtypes of BC. Further analyses nominated negative regulation of the canonical WNT signaling pathway as one of the major pathways altered by mitochondrial character. The WNT pathway is known to regulate cell signaling, metabolism, epithelial-mesenchymal transition (EMT), and cancer stemness. Cybrids with benign mitochondria showed transcriptional activation of negative WNT regulators, including DKK1, SOST, DAB2, and CAV1. Citrate is a key metabolite from the tricarboxylic acid cycle (TCA cycle) and is an intermediate of many other metabolic pathways. Analysis of TNBC cybrids, cell lines, and BC tissues suggest that in TNBC, citrate levels are increased with the activation of CPT1A, the rate-limiting enzyme of FAO. We confirmed the reduction of the beta-catenin protein in TNBC cells after pharmacological and genetic inhibition of CPT1A and mitochondrial citrate transporter (SLC25A1). We further analyzed the gene set enrichment analysis (GSEA) using gene expression data developed from short and long-term CPT1A inhibited TNBC cells and observed that the FAO alters polycomb repressive complex 2 (PRC2) activity. PRC2 is a critical modulator of the H3K27 methylation pattern as well as pathways related to cancer stemness, including WNT/beta-catenin and Hedgehog (SHH) pathways. These results suggest that the mitochondrial FAO transcriptionally inhibits WNT antagonist genes through repressive histone modification and consequently activates the canonical WNT pathway and cancer stemness. Overall, our current observations provide new insights into the regulation WNT pathway by mitochondria-nuclear crosstalk in TNBC. Citation Format: Sukjin Yang, Jun Hyoung Park, Suna Kim, Meron Ghidey, Benny Kaipparettu. Mitochondria-Nuclear Crosstalk Regulates the WNT Pathway in Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-02.

Methotrexate mediates the integrity of intestinal stem cells partly through nitric oxide-dependent Wnt/β-catenin signaling in methotrexate-induced rat ileal mucositis

This study aimed to elucidate the role of nitric oxide (NO) in intestinal stem cells in methotrexate-induced ileal mucositis in rats. Methotrexate induced the mRNA expressions of the Wnt/β-catenin target genes Wnt3a, Sox9, and Lgr5 and the Wnt-antagonist gene sFRP-1 and the protein expressions of Lgr5 and sFRP-1. Methotrexate also induced Lgr5+ cells and lysozyme+ cells. A non-selective NO inhibitor inhibited the methotrexate induction of Wnt/β-catenin target genes and Lgr5+ cells but enhanced that of sFRP-1 expression. Thus, methotrexate mediates the integrity of intestinal stem cells partly through NO-dependent Wnt/β-catenin signaling and may enhance tolerability to methotrexate-induced injury.

CircCNIH4 inhibits gastric cancer progression via regulating DKK2 and FRZB expression and Wnt/\u03b2-catenin pathway

BackgroundCircular RNAs (circRNAs) have been reported to play an important role in tumor progression in various cancer types, including gastric cancer. The aim of this study was to investigate the role of circCNIH4 (hsa_circ_0000190) in gastric cancer and the underlying mechanism.MethodsThe expression levels of circCNIH4 and Wnt antagonist genes were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of β-catenin, Ki67, Dickkopf 2 (DKK2) and Frizzled related protein (FRZB) were measured by western blot. Ectopic overexpression or knockdown of circCNIH4, proliferation, apoptosis, migration and invasion by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry and transwell assay in vitro, and in vivo experiment, were employed to assess the role of circCNIH4 in gastric cancer.ResultsCircCNIH4 was downregulated in gastric cancer tissues and cells. Overexpression of circCNIH4 inhibited gastric cancer cell proliferation, migration and invasion and promoted apoptosis by inactivating Wnt/β-catenin pathway in vitro. CircCNIH4 induced the expression of DKK2 and FRZB in gastric cancer cells. Moreover, silencing of DKK2 or FRZB reversed circCNIH4 overexpression-mediated effects on gastric cancer cells. Additionally, circCNIH4 suppressed tumor growth via regulating DKK2 and FRZB expression in gastric cancer in vivo.ConclusionOur study demonstrated that circCNIH4 played a tumor-inhibiting role through upregulating DKK2 and FRZB expression and suppressing Wnt/β-catenin pathway in gastric cancer, which might provide a potential biomarker for the diagnosis and treatment of gastric cancer.

DNA methylation and gene expression of sFRP2, sFRP4, Dkk 1, and Wif1 during osteoblastic differentiation of bone marrow derived mesenchymal stem cells

ObjectivesBone marrow derived mesenchymal stem cells (BMSCs) are an irresistible choice for use in stem cell therapy and regenerative medicine. BMSCs osteoblastic differentiation is also important in bone development, diseases, malignancies, and cancers studies. Wnt signaling pathway antagonists, Dickkopf-1 (Dkk 1), Secreted Frizzled-Related Proteins (sFRPs), and Wnt Inhibitory Factor 1 (Wif1) play important roles in inducing osteoblastic differentiation. This study is the first to investigate the association between DNA methylation and gene expression of Dkk1, sFRP2, sFRP4, and Wif1 during BMSCs osteoblastic differentiation. MethodsHuman BMSCs were isolated and characterized using flow cytometry. Then, cells were treated with osteo-differentiation medium for three weeks. Alizarin red S staining and polymerase chain reaction (PCR) (alkaline phosphatase/osteocalcin) were performed for confirmation. The expression of Dkk 1, sFRP2, sFRP4, and Wif1 genes was evaluated at days 7, 14, and 21 using real-time PCR. Methylation-specific PCR (MSP) was performed to detect the methylation status of the promoters of the genes. ResultsData showed significant decreases (P < 0.05) during various days of BMSCs differentiation, while the promoters of the genes remained mostly un-methylated. ConclusionsThe down-regulation of Dkk 1, sFRP2, sFRP4, and Wif1 regulates various stages of human BMSCs during osteoblastic differentiation. DNA methylation does not interfere in the down-regulation of these genes, except for Wif1. We propose that the Wnt antagonist gene promoters should remain un-methylated during osteoblastic differentiation of BMSCs and that the down-regulation of these genes may contribute to other epigenetic mechanisms, other than DNA methylation, which implicitly indicates the role of DNA methylation in osteogenic cancers.

Genetic Variants in the Wingless Antagonist Genes (sFRP, DKK, and Axin2) Predict the Overall Survival and Prognosis of North Indian Lung Cancer Patients Treated with Platinum-Based Doublet Chemotherapy.

Background: To investigate the prognostic implication of genetic variants within the wingless (Wnt) antagonist genes (DKK, sFRP, and Axin2) in North Indian lung cancer patients. Materials and Methods: A total of 212 subjects were genotyped using polymerase chain reaction-restriction fragment length polymorphism technique for 18 polymorphic sites in DKK4, DKK3, DKK2, sFRP3, sFRP4, and Axin2. Overall survival (OS) was estimated using the Kaplan-Meier survival analysis, and adjusted hazard ratio (HR) was obtained using the Cox regression method. Results: It was observed that the unfavorable genotypes of the three DKK2 variants collectively (rs447372, rs419558, and rs17037102) exhibited a highly decreased rate of death (adjusted HR = 0.37, p = 0.03). Adenocarcinoma (ADCC) patients carrying the heterozygous (CT) genotype for DKK4 rs2073664 showed a better OS compared with wild genotype (log rank p = 0.01). The two exonic variants (148 and 1386) of Axin2 gene showed contrasting results, where the ADCC subjects having TT genotype for Axin2 148 showed a better prognosis (adjusted HR = 0.48, p = 0.003) and those with TT genotype for Axin2 1386 showed a poor prognosis in small-cell lung carcinoma patients (adjusted HR = 2.33, p = 0.02). The intronic Axin2 1712 + 19 variant on the other hand indicated a highly increased death risk in ADCC patients with GG genotype. Survival tree analysis depicted DKK4 rs2073664 as the major contributor in predicting the survival of the lung cancer patients. Node 3 exhibited the lowest death rate (HR = 0.04, p = 0.008) and better median survival time (9 months vs. 3 months) when compared with reference node. Conclusions: A cumulative effect of three variants of DKK2 gene along with DKK4 rs2073664 can jointly predict the survival as shown by tree analysis.

A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells

Background: Wnt signaling plays an essential role in tumor cell growth, including the development of malignant mesothelioma (MM). Epigenetic silencing of negative Wnt regulators leading to constitutive Wnt signaling has been observed in various cancers and warrants further attention. We have reported that a succinate ether derivative of α-tocotrienol (T3E) has potent cytotoxic effects in MM cells. Thus, in this study, we investigated whether the anti-MM effect of T3E could be mediated via the epigenetic alteration of the Wnt antagonist gene, Dickkopf-1 (DKK1). Methods: WST-1 and cell analyzers were employed to analyze the effects of T3E on cell viability and apoptosis of human MM cell lines (H2452, H28). Real-time PCR and Western blot were performed to evaluate the expression at mRNA and protein levels. Methylation status and epigenetic modifications of DKK1’s promoter regions after T3E treatment in MM cells were studied using methylation-specific PCR and Chromatin immunoprecipitation. Small interfering RNA-mediated knockdown ­(siRNA), and specific inhibitors, were used to validate DKK1 as a target of T3E. Results: T3E markedly impaired MM cell viability, increased the expression of phosphorylated-JNK and DKK1 and suppressed cyclin D, a downstream target gene of Wnt signaling. Knockdown of DKK1 expression by siRNA or a specific JNK inhibitor confirmed the contribution of DKK1 and JNK to T3E-induced cytotoxicity in MM cells. On the other hand, cytoskeleton-associated protein 4 (CKAP4) expression, which promotes cell proliferation as a Wnt-independent DKK1 receptor was inhibited by T3E. Silencing CKAP4 by ­siRNA did not appear to directly affect MM cell viability, thereby indicating that expression of both DKK1 and CKAP4 is required. Furthermore, T3E-mediated inhibition of both DNA methyltransferases (DNMT1, 3A, and 3B) and histone deacetylases (HDAC1, 2, 3, and 8) in MM cells leads to increased DKK1 expression, thereby promoting tumor growth inhibition. MM cells treated with Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC inhibitor) exhibited cytotoxic effects, which may explain the inhibitory action of T3E on MM cells. In addition, an enhanced expression of DKK1 in MM cells following T3E treatment is positively correlated with the methylation status of its promoter; T3E decreased DNA methylation and increased histone acetylation. Moreover, T3E specifically increased histone H3 lysine 4 (H3K4) methylation activity, whereas no effects were observed on histone H3K9 and H3K27. Conclusions: Targeting the epigenetic induction of DKK1 may lead to effective treatment of MM, and T3E has great potential to induce anti-MM activity.

JARID2 Regulates Skeletal Muscle Differentiation through Regulation of Canonical Wnt Signaling Pathway

Myogenesis is a tightly regulated process that is dependent on the precise and dynamic integration of signals from various signaling pathways including the Notch, Sonic hedgehog and Wnt signaling pathways. The Wnt signaling pathway is an essential signaling pathway that has been shown to have diverse roles during both embryonic muscle development and adult muscle regeneration. In this study, we sought to understand the role of JARID2 ‐ an evolutionarily conserved, inactive histone demethylase that often has been shown to function in association with a transcription repressive complex, polycomb repressive complex‐2 (PRC2), during muscle differentiation. Utilizing the widely used C2C12 cell model system, we report that JARID2 is required for muscle differentiation and it regulates muscle differentiation through modulation of Wnt signaling. We found that JARID2 regulates the myogenic regulatory factors (MRFs), MyoD and myogenin. We discovered that JARID2/PRC2 not only regulates expression of the MRFs directly through tri‐methylation of lysine 27 of histone 3 (H3K27me3) but also indirectly, through regulation of canonical Wnt signaling. JARID2 controls MRF expression indirectly through direct repression of the Wnt antagonist gene, secreted frizzled‐related protein‐1 (Sfrp1). We found that activation of canonical Wnt signaling through inhibition of GSK3b rescues the differentiation block of JARID2 depleted cells through the restoration of MyoD and myogenin expression. JARID2 depleted cells showed a reduction in the nuclear levels of the canonical‐Wnt signaling pathway effector, b‐catenin, and we found that inhibition of GSK3b induced translocation of b‐catenin into the nucleus of these cells. Using chromatin immunoprecipitation (ChIP) assays, we also show that b‐catenin positively regulates the transcription of MyoD through direct binding in the distal regulatory region (DRR) of the MyoD promoter. Thus, we establish here that JARID2 and the PRC2 complex are essential regulators of the initiation of skeletal muscle differentiation through modulation of the Wnt signaling pathway. In myoblasts, PRC2 represses the MRFs but also represses Wnt antagonists, which allows the Wnt pathway to activate MyoD and initiate differentiation.Support or Funding InformationNIH/NIAMS AR068622This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Helicobacter pylori-induced modulation of the promoter methylation of Wnt antagonist genes in gastric carcinogenesis

This study aimed to investigate the changes in the promoter methylation and gene expression of multiple Wnt antagonists between the chronic infection and eradication of Helicobacter pylori (H. pylori) in gastric carcinogenesis. The levels of methylation and corresponding mRNA expression of seven Wnt antagonist genes (SFRP1, -2, -5, DKK1, -2, -3, WIF1) were compared among the patients with H. pylori-positive gastric cancers (GCs), and H. pylori-positive and H. pylori-negative controls, by quantitative MethyLight assay and real-time reverse transcription (RT)-polymerase chain reaction (PCR), respectively. The changes of the methylation and expression levels of the genes were also compared between the H. pylori eradication and H. pylori-persistent groups 1year after endoscopic resection of GCs. The methylation levels of SFRP and DKK family genes were significantly increased in the patients with H. pylori-positive GCs and followed by H. pylori-positive controls compared with H. pylori-negative controls (P<0.001). SFRP1, -2, and DKK3 gene expression was stepwise downregulated from H. pylori-negative controls, H. pylori-positive controls, and to H. pylori-positive GCs (P<0.05). Among the Wnt antagonists, only the degrees of methylation and downregulation of DKK3 were significantly reduced after H. pylori eradication (P<0.05). Epigenetic silencing of SFRP and DKK family genes may facilitate the formation of an epigenetic field during H. pylori-associated gastric carcinogenesis. The epigenetic field may not be reversed even after H. pylori eradication except by DKK3 methylation.

Concurrent Hypermethylation of SFRP2 and DKK2 Activates the Wnt/β-Catenin Pathway and Is Associated with Poor Prognosis in Patients with Gastric Cancer.

Aberrant hypermethylation of Wnt antagonists has been observed in gastric cancer. A number of studies have focused on the hypermethylation of a single Wnt antagonist and its role in regulating the activation of signaling. However, how the Wnt antagonists interacted to regulate the signaling pathway has not been reported. In the present study, we systematically investigated the methylation of some Wnt antagonist genes (SFRP2, SFRP4, SFRP5, DKK1, DKK2, and APC) and their regulatory role in carcinogenesis. We found that aberrant promoter methylation of SFRP2, SFRP4, DKK1, and DKK2 was significantly increased in gastric cancer. Moreover, concurrent hypermethylation of SFRP2 and DKK2 was observed in gastric cancer and this was significantly associated with increased expression of β-catenin, indicating that the joint inactivation of these two genes promoted the activation of the Wnt signaling pathway. Further analysis using a multivariate Cox proportional hazards model showed that DKK2 methylation was an independent prognostic factor for poor overall survival, and the predictive value was markedly enhanced when the combined methylation status of SFRP2 and DKK2 was considered. In addition, the methylation level of SFRP4 and DKK2 was correlated with the patient’s age and tumor differentiation, respectively. In conclusion, epigenetic silencing of Wnt antagonists was associated with gastric carcinogenesis, and concurrent hypermethylation of SFRP2 and DKK2 could be a potential marker for a prognosis of poor overall survival.

DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM)

Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples.

Dissecting the Development of Dupuytren Disease Through Human Genetics

Objective: Dupuytren disease (DD) is a polygenic fibroproliferative disorder of the palmar fascia of the hand. Building on our previous work, we aimed to further characterize the genetic predisposition to DD by performing a genome-wide association study (GWAS) on a total of 7934 DD cases and 12°861 controls from the United Kingdom, Holland, and Germany. We then aimed to dissect the functional effects of the statistically associated variant within DD-derived myofibroblasts. Materials and Methods: We extracted DNA from saliva or blood of DD patients who had undergone fasciectomy or dermofasciectomy. The genotypes of each sample were investigated using Illumina HumanExomeCore microarray chips in the discovery phase, and other platforms for replication phase. For our functional analysis, we used single-gene TaqMan assays to measure messenger RNA (mRNA) expression of myofibroblast cells that were disaggregated from DD nodules. Primary myofibroblasts were stimulated with a combination of recombinant WNT3A, SFRP4, and DKK1 proteins for 48 hours, and mRNA expression was measured. Results: Our GWAS meta-analysis revealed a total of 24 DD-associated regions, 15 of which were novel ( P &lt; 5 × 10−8). We observed a 4-fold difference in SFRP4 mRNA expression between the 2 homozygous genotypes of our most associated single-nucleotide polymorphism rs1687975. Interestingly, an inverse correlation was observed in extracellular SFRP4 protein secretion. Our WNT signaling pathway stimulation experiments demonstrated that SFRP4 attenuates WNT3A-induced connective tissue growth factor (CTGF) expression, showing a 20% decrease ( P &lt; .05). SFRP4 alone has no effect on alpha-smooth muscle actin, COL1A1, and COL3A1 mRNA expression. Conclusions: We have performed a large 3-stage GWAS and have discovered 15 novel Dupuytren-associated regions. We have identified that our most significantly associated variant, rs16879765, has an effect on the mRNA expression of a WNT antagonist gene SFRP4. Further functional analysis on SFRP4 has revealed that it affects the expression of CTGF. This implies its anti-fibrotic effect is mediated by the non-canonical WNT signaling pathway, potentially via the YAP/TAZ pathway that is known to interact with transforming growth factor β (TGF BETA) pathway, and this plays a critical role in the development of DD. Our GWAS has also revealed multiple potential new therapeutic targets for DD. Genes within the associated regions include MMP14 and WNT4 and have effects on diverse functions such as inflammation, extracellular matrix remodeling, cellular differentiation, and proliferation, which are likely to be important processes in the development of DD and fibrotic diseases in general.

Tu2065 Effect of Helicobacter pylori Eradication on the Promoter Methylation of Wnt Antagonist Genes in Gastric Cance

Tu2065 Effect of Helicobacter pylori Eradication on the Promoter Methylation of Wnt Antagonist Genes in Gastric Cance

The Epigenetically Regulated Effects of Wnt Antagonists on the Expression of Genes in the Apoptosis Pathway in Human Bladder Cancer Cell Line (T24)

The epigenetic suppression of Wnt antagonists (sFRPs, DKKs, and WIF-1) causes the activation of both β-catenin and target genes, which play an important role in cell proliferation, metastasis, and angiogenesis. This study is aimed to investigate, on transcriptional and protein levels, the synergic effects of unaccompanied and/or combined use of 5-aza-2'-deoxycytidine (DAC, 5-aza-dC), trichostatin A (TSA), and gemcitabine+cisplatin chemotherapeutic agents on the apoptotic pathway of human bladder cancer cell line T24. The anti-tumor effects of gemcitabine (0-500 nM), cisplatin (0-10 μM), DAC (10 μM), and TSA (300 nM) alone and/or together on T24 cells were determined by WST-1. ELISA method was used to analyze the effects of unaccompanied and combined use of gemcitabine+cisplatin, DAC, and TSA on cell proliferation and determine the cytotoxic and apoptotic dosages at the level of H3 histone acetylation. Methylation-specific PCR was used to evaluate methylation profiles of Wnt antagonist gene (WIF-1). In the case of unaccompanied and/or combined use of specified drugs, the variations in the expression levels of CTNNB1, GSK3β, c-MYC, CCND1, CASP-3, CASP-8, CASP-9, BCL2L1, and WIF-1 genes were determined by quantitative real-time PCR. Our results indicate that through inhibition of DNA methylation, expression of β-catenin and Wnt antagonist re-activation and expressions of canonical Wnt/β-catenin pathway target genes, c-myc and cyclin D1 (CCND1), have decreased. In addition, DAC, TSA, and gemcitabine+cisplatin combination caused an increase in GSK3β mRNA levels, which in turn significantly decreased CCND1 mRNA levels. Moreover, BCL2L1, an anti-apoptotic gene, was downregulated significantly. Meanwhile, both CASP-3 mRNA and active caspase-3 protein levels increased with respect to control (p<0.01). The results revealed that use of quadruplicate gemcitabine+cisplatin+DAC+TSA combination led to a reduced inhibition of canonical Wnt/β-catenin pathway and reduced cell proliferation. Our findings may offer a new approach to consider in the treatment of bladder cancer.

Downregulation of sFRP-2 by epigenetic silencing activates the β-catenin/Wnt signaling pathway in esophageal basaloid squamous cell carcinoma

Basaloid squamous cell carcinoma (BSCC) of the esophagus is a rare variant of typical squamous cell carcinoma (SCC) associated with poor survival. A characteristic feature is nuclear accumulation of β-catenin, without a mutation of the gene. We studied the methylation status of Wnt antagonist genes, such as secreted frizzled-related protein (sFRP) gene family members, Wnt inhibitory factor-1 (WIF-1), Dickkopf-1 (Dkk-1), and human Dapper protein-1 (HDPR-1), and alterations of the APC, Axin1, and Axin2 genes in 30 cases of esophageal BSCC. β-catenin and sFRP (sFRP-1, sFRP-2, sFRP-4, sFRP-5) protein expression was examined by immunohistochemistry. APC, Axin1, and Axin2 gene mutations were detected in 3, 2, and 2 cases, respectively, and 6 cases (20%) harbored at least 1 alteration in these genes. Methylation of the sFRP-2 promoter region was observed in all cases, and methylation was frequent in sFRP-1 and sFRP-5, but infrequent in Dkk-1, WIF-1, sFRP-4, and HDPR-1. sFRP-2 expression was almost completely absent in 25 cases (83%), consistent with the methylation status. Nuclear accumulation of β-catenin was observed in all cases. sFRP-5 expression was associated with a low nuclear β-catenin labeling index. These results show that sFRP-2 is a target gene of hypermethylation in esophageal BSCC and suggest that sFRP-2 might contribute to BSCC tumorigenesis through the Wnt/β-catenin signaling pathway.

Detection of promoter hypermethylation of Wnt antagonist genes in fecal samples for diagnosis of early colorectal cancer.

To investigate the feasibility of detecting aberrantly hypermethylated Wnt-antagonist gene promoters (SFRP2 and WIF-1) in fecal DNA as non-invasive biomarkers for early colorectal cancer (CRC). The methylation-specific polymerase chain reaction assay was performed to blindly analyze the methylation status of SFRP2 and WIF-1 gene promoters in fecal samples from 48 subjects with CRC, 35 with adenomas, 32 with hyperplastic polyps and 30 endoscopically normal subjects. Additionally, we compared the diagnostic efficiency of measuring the hypermethylated SFRP2 and WIF-1 genes in the feces to the fecal occult blood test (FOBT) for the early detection of CRC. Hypermethylated SFRP2 was detected in the feces of 56.3% (27/48) of CRC cases, 51.4% (18/35) of adenoma cases and 12.5% (4/32) of patients with hyperplastic polyps. The hypermethylation of WIF-1 was detected in 60.4% (29/48), 45.7% (16/35) and 18.7% (6/32) of fecal samples from CRC, adenoma and hyperplastic polyp patients, respectively. At least one hypermethylated gene was detected in 81.3% (39/48) of CRC and 65.7% (23/35) of adenoma samples. In contrast, only a hypermethylated WIF-1 gene was detected in one case of normal fecal samples. Moreover, no significant associations were observed between SFPR2 and WIF-1 hypermethylation and clinicopathological features. Additionally, 81.8% of CRC cases diagnosed as Dukes A stage or advanced adenomas had at least one hypermethylated gene detected, while the detection rate with the FOBT was only 31.8% (P < 0.001). Hypermethylated SFRP2 and WIF-1 genes in fecal DNA are novel and promising molecular biomarkers that have great diagnostic potential for early CRC.

DNA methyltransferase inhibitor-mediated apoptosis in the Wnt/β-catenin signal pathway in a renal cell carcinoma cell line

The Wnt signaling pathway is activated in most cancer types when Wnt antagonist genes are inactivated. Glycogen synthase kinase 3 (GSK3β) is an important regulator of the Wnt/β-catenin signaling pathway. The mechanisms underlying GSK3β regulation of neoplastic transformation and tumor development are unclear. Studies have raised the possibility that the Wnt signaling pathway may be implicated in renal cell carcinoma (RCC). Therefore, in the present study, we hypothesize that the expression and methylation status of the secreted frizzled-related protein 2 (sFRP2) gene, one of the secreted antagonists that bind Wnt protein, and re-expression of this gene with the demethylation agent (5-aza-2'-deoxycytidine; DAC) may induce apoptosis in RCC cells. To test this hypothesis, we investigated the relationship among epigenetic inactivation of sFRP2 and p-GSK3β (Ser9) and other Wnt antagonists (sFRP1, DKK3, WIF-1) and apoptotic factors (Bax and Caspase3) as well as the anti-apoptotic factor BCL2. Our results indicate that DAC-mediated inhibition of DNA methylation led to a re-activation of sFRP2 expression and increased expression levels of the Wnt antagonists and apoptotic factors. In contrast, the level of β-catenin (CTNNB1) expression decreased. The p-GSK3β (Ser9) protein level in Caki-2 cells was significantly down-regulated, while the DNA fragmentation rate increased after treatment with 5 μM DAC at 96 h. Our data show that sFRP2 functions as a tumor suppressor gene in RCC and that its restoration may offer a new therapeutic approach for the treatment of RCC. Moreover, our study draws attention to the regulatory features of epigenetic molecules and analyses their underlying molecular mechanisms of action and their potential use in clinical practice.

Antagonists of Wnt pathway

Wnts transduce signals through Wnt/β-catenin pathway (canonical pathway) , in which β-catenin stimulates transcription of target genes, as well as several kinds of non-β-catenin Wnt pathways (alternative pathways) . Wnt/β-catenin pathway is essential for cell proliferation and survival, and its activity is strictly regulated by several antagonists. These antagonists inhibit Wnt signaling either in extracellular fluid, in cytoplasm, or in nucleus. Sclerosteosis and van Buchem disease are rare congenital skeletal diseases caused by inactivating mutations in genes encoding Wnt antagonists. Moreover, it has been reported that knockout mice of Wnt antagonist genes show high bone mass with increased bone formation. Therefore, Wnt antagonists are potential drug targets as bone anabolic agents.

Methylation of Wnt antagonist genes: a useful prognostic marker for myelodysplastic syndrome

Activation of the Wnt signaling pathway has been implicated in the pathogenesis of many tumors as well as in leukemia. However, its role in myelodysplastic syndrome (MDS) is unknown. In this study, we employed methylation-specific PCR to examine the methylation status of six Wnt antagonist genes in 144 MDS patients and in the MDS cell line SKM-1. We also used real-time PCR to examine the expression of Wnt antagonist genes and Wnt pathway genes in the SKM-1 cell line after treatment with 5-aza-2'-deoxycytidine. We found that methylation of the gene promoters of each of the six genes were observed in MDS patients at the following methylation frequencies: 41% for sFRP1, 89.6% for sFRP2, 43.1% for sFRP4, 50.7% for sFRP5, 44.4% for DKK-1, and 69.4% for DKK-3. In the SKM-1 cell line, the gene promoters sFRP1, sFRP2, sFRP5, DKK-1, and DKK-3 were methylated, while sFRP4 was not methylated. Treatment of the SKM-1 cell line with 5-aza-2'-deoxycytidine induced re-expression of methylated Wnt antagonists and inactivation of the Wnt pathway. Survival analysis showed that methylation status of sFRP1, sFRP4, and sFRP5 was associated with worse survival in MDS and sFRP5 methylation also predicted a high risk of leukemia evolution (P = 0.018). Our results indicate that epigenetic regulation of the Wnt pathway in MDS cell line, and the methylation status of Wnt antagonists predicts prognoses of MDS patients.