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.     

Aberrant localization of beta-catenin correlates with overexpression of its target gene in human papillary thyroid cancer

Alterations of the Wnt/beta-catenin signaling pathway are known to occur in mutations of the component genes such as APC, Axin, and beta-catenin, and play a pathogenetic role in tumorigenesis. Activated Wnt signaling stabilizes beta-catenin, which associates with T cell factor, resulting in transactivation of the downstream target genes including c-myc and cyclin D1. To investigate the involvement of Wnt/beta-catenin signaling pathway in thyroid tumorigenesis, we analyzed its activation and localization in 5 human thyroid cancer cell lines and 132 thyroid tumor tissue samples. Dislocalization of beta-catenin was observed in all cell lines. Constitutive activation of T cell factor in two of four thyroid cancer cell lines was observed using reporter gene assay. Furthermore, high expression levels of c-Myc and cyclin D1 were observed in cell lines that showed cytoplasmic or nuclear accumulation of beta-catenin. In 132 paraffin-embedded thyroid carcinoma tissue samples, cytoplasmic beta-catenin was immunohistochemically observed in 52 out of 78 (67%) papillary thyroid cancers, but only in 3 of 34 (9%) follicular adenomas and 5 of 20 (25%) follicular cancers. Cytoplasmic localization of beta-catenin significantly correlated with overexpression of cyclin D1 in papillary carcinomas. Our results suggest that aberrant activation of Wnt/beta-catenin signaling is strongly involved in thyroid tumorigenesis.     

Wnt pathway inhibitors are strongly down-regulated in pituitary tumors

The etiology of sporadic pituitary tumors is currently unknown. The Wnt pathways have been implicated in the pathogenesis of a variety of human tumors, but the role of these pathways in pituitary tumors is unclear. Microarray analysis using the Affymetrix HG U133 plus 2.0 GeneChips identified four secreted frizzled-related protein (sFRP) family members of Wnt pathway inhibitors that were differentially expressed in both nonfunctioning and clinically functioning pituitary tumors (n = 20) compared with normal pituitary controls (n = 3). Reduced tumor expression of Wnt inhibitory factor-1 (WIF1), sFRP2, and sFRP4 mRNA was confirmed by real-time quantitative RT-PCR (P <0.001 and P = 0.002 and 0.013, respectively) in all pituitary subtypes. Hypermethylation of the WIF1 promoter was present in 88% of the pituitary tumors (n = 41). Seventy-six percent of pituitary tumors demonstrated absent or weak cytoplasmic WIF1 staining by immunohistochemistry (n = 41), although preserved staining was seen in some functioning tumors, with strong staining in 92% of normal pituitary controls (n = 13). The Wnt pathway target gene cyclin D1 was found to be up-regulated specifically in the nonfunctioning pituitary tumors compared with controls at both mRNA and protein level, supportive of activation of the Wnt-beta-catenin pathway. Nuclear accumulation of beta-catenin, however, was not observed in any pituitary tumors (n = 70). By transfecting GH3 cells with WIF1, decreased cell proliferation and colony formation was observed compared with empty vector controls. In conclusion, our data suggest that WIF1 may be a tumor suppressor, specifically in nonfunctioning pituitary tumors, and that the Wnt pathways are important in pituitary tumorigenesis.     

Prickle-1 negatively regulates Wnt/beta-catenin pathway by promoting Dishevelled ubiquitination/degradation in liver cancer

BACKGROUND & AIMS: Aberrant activation of Wnt signaling due to accumulation of beta-catenin has been linked to tumorigenesis. Mutations of beta-catenin, APC, and axins are important but not frequent enough to be accountable for the accumulation of beta-catenin in human hepatocellular carcinoma (HCC). In this study, we characterized the roles of Prickle-1, a Dishevelled (Dvl)-associated protein, in regulation of Wnt/beta-catenin activity in HCC. METHODS: The expression levels of human Prickle-1 and Dvl3 were examined in HCC cell lines and human HCC samples. The interaction and effects of Prickle-1 on Dvl3, the Wnt/beta-catenin pathway, and cell growth were assessed in HCC cell lines. RESULTS: We showed that Prickle-1 bound with Dvl3 and facilitated Dvl3 ubiquitination/degradation, and this was through its destruction box (D-box) motifs. Enforced expression of Prickle-1 significantly reduced the Wnt/beta-catenin activity and tumorigenic properties of HCC cells. Clinicopathologic analysis showed that underexpression of Prickle-1 was significantly associated with overexpression of Dvl3, beta-catenin accumulation (P = .023), and larger tumor size (P = .030). CONCLUSIONS: Our results have elucidated a novel mechanistic relationship between Prickle-1 and Dvl3 in the Wnt/beta-catenin pathway. The facilitation of Prickle-1 on Dvl3 degradation and the suppression of beta-catenin activity and cell growth suggest that Prickle-1 is a negative regulator of the Wnt/beta-catenin signaling pathway and is a putative tumor suppressor in human HCCs.     

Wnt-beta-catenin signaling in the pathogenesis of osteoarthritis

Osteoarthritis (OA) is a progressively degenerative joint condition that is influenced by various metabolic and structural factors. The canonical Wnt-frizzled-beta-catenin pathway has been implicated in the pathogenesis of OA. Products of the Wnt, frizzled, secreted frizzled-related protein (sFRP), Dickkopf and LDL-receptor-related protein gene families have crucial roles in the development and maintenance of bone, cartilage and joints. Increased levels of beta-catenin have been observed in degenerative cartilage, suggesting that a diminished capacity to limit Wnt signaling might contribute to cartilage loss. Polymorphisms in genes involved in Wnt signaling-particularly in the gene encoding sFRP-3-are associated with an increased susceptibility to the development of OA. At least one of these polymorphisms in the gene encoding sFPR-3 is associated with a reduced ability to limit beta-catenin signaling. In addition, the canonical Wnt signaling pathway is influenced by local factors, including alterations in glycosaminoglycan sulfation, cartilage matrix content, transforming growth factor beta and vitamin D. A higher circulating level of the Wnt inhibitor Dickkopf-1, for instance, is associated with slowed progression of hip OA. Hence, the sum of local and systemic factors contributes to the outcome of the Wnt-frizzled pathways. Further investigation is needed to fully define the role of Wnt signaling in OA.     

Wnt signaling interacts with Shh to regulate taste papilla development

Wnt and Shh signaling pathways are critical for the development and maturation of many epithelial tissues. Both pathways have roles in stem cell maintenance, tissue development, and tumorigenesis. However, linkage between these pathways in mammalian systems had not been well established. Here, we report that Shh expression in fungiform papillae and formation of normal mature fungiform papillae depend on signaling through Wnt and beta-catenin. We observed that during fungiform papilla formation in mice, Shh and components of the Wnt/beta-catenin signaling pathway are expressed together in the developing placode. The elimination of Wnt/beta-catenin signaling in either Lef1 or Wnt10b knockout mice resulted in down-regulation of Shh expression. In addition, the size and number of fungiform papillae were greatly reduced in Lef1 knockout mice. By examining embryonic mouse tongues in culture we determined that activation of Wnt/beta-catenin signaling up-regulates Shh expression. We observed that blocking Shh signaling in cultured tongue explants enhanced papillae formation and was accompanied by an up-regulation of Wnt/beta-catenin signaling, indicating that Shh inhibits the Wnt/beta-catenin pathway. Exogenously added Shh suppressed expression of endogenous Shh and inhibited Wnt/beta-catenin signaling (assessed in TOPGAL mice), further implicating Shh as an inhibitor of the Wnt/beta-catenin pathway. Our observations indicate that Wnt/beta-catenin signaling and interactions between the Wnt and Shh pathways play essential roles in the development of fungiform papillae.     

Mutations of the APC,beta-catenin,and axin 1 genes and cytoplasmic accumulation of beta-catenin in oral squamous cell carcinoma

Purpose: The Wnt pathway is involved in carcinogenesis and three regulatory genes of the Wnt pathway, APC, beta-catenin and Axin are mutated in some primary human cancers. Mutations in these genes can impair the down regulation of beta-catenin, which results in the stabilization of beta-catenin, accumulation of free beta-catenin and subsequent activation of the Wnt pathway. To clarify the genetic alterations of components of the Wnt pathway in oral squamous cell carcinoma (SCC), we examined mutations in the APC, beta-catenin and Axin genes and subcellular localization of beta-catenin. Methods: 20 oral SCC tissues and four cell lines derived from oral SCC were used. Mutational analysis was performed by a single-strand conformation polymorphism (SSCP) method and direct sequecing analysis. The samples were also examined by immunohistochemical staining and immunoblot analysis. Results: In 3 of 4 cell lines, mutations were observed in the APC and Axin1 genes without amino acid substitutions. In a clinical sample, a mutation in the Axin1 gene was detected; a T insertion at codon 250 resulted in the formation of a stop codon at codon 259. In addition, cytoplasmic accumulation of beta-catenin was observed in 3 (75%) of 4 cell lines and 18 (90%) of 20 cancer tissue samples. Conclusion: The Axin1 gene may be one of the mutational target in oral SCC. In addition, the cytoplasmic accumulation of beta-catenin is a common characteristic of oral SCC, but is not closely associated with mutational alterations in the APC, beta-catenin and Axin1 genes.     

Small-molecule synergist of the Wnt/beta-catenin signaling pathway

The Wnt/beta-catenin signaling pathway regulates cell fate and behavior during embryogenesis, adult tissue homeostasis, and regeneration. When inappropriately activated, the pathway has been linked to colorectal cancer and melanoma, and when attenuated it may contribute to Alzheimer's disease and osteoporosis. Small molecules that modulate Wnt signaling will likely provide new insights into the regulation of this key developmental pathway and ultimately provide pharmacological agents to control Wnt signaling in vivo. To this end, we screened a library of 100,000 small molecules for activity in a cell-based assay of Wnt/beta-catenin signaling and discovered a purine derivative, QS11, that synergizes with Wnt-3a ligand in the activation of Wnt/beta-catenin signal transduction. Through affinity chromatography and subsequent functional assays, we showed that QS11 binds and inhibits the GTPase activating protein of ADP-ribosylation factor 1 (ARFGAP1), suggesting that QS11 modulates Wnt/beta-catenin signaling through an effect on protein trafficking. Consistent with its function as an ARFGAP inhibitor, QS11 inhibits migration of ARFGAP overexpressing breast cancer cells.     

Adenomatous polyposis coli alteration in digestive endocrine tumours: correlation with nuclear translocation of beta-catenin and chromosomal instability

The role of Wnt pathway in digestive endocrine tumours is debated. The aim of this work is to investigate key players in Wnt pathway by a multimodal approach. Sixty cases (49 well-differentiated and 11 poorly differentiated) were investigated for methylation of adenomatous polyposis coli (APC) and E-cadherin promoters, the loss of heterozygosity (LOH) at APC locus and beta-catenin and E-cadherin expression by immunohistochemistry. Tumours showing altered beta-catenin localization were tested for beta-catenin and APC mutations. APC promoter methylation was restricted to gastroduodenal tumours (21 out of 59, 36%), prevalent in poorly differentiated carcinomas (P=0.042) and correlating with aggressive features (high histology grade, P<0.02; tumour death, P=0.026; high fractional allelic loss, P=0.002, in turn correlating with short survival, P=0.017). LOH at APC locus was found in 14 out of 53 cases (26%, 10 gastroduodenal and 4 colorectal), prevalent in poorly differentiated carcinomas (P=0.002) and correlating with histology grade (P=0.012). beta-catenin abnormal expression was found in 41 out of 54 cases (76%), with nuclear staining correlating with APC alteration (P=0.047) and short survival (P=0.006). APC, but not beta-catenin, gene mutations were found (7 out of 35 tumours), 4 of which in the midgut. E-cadherin promoter methylation was rarely detected (2 out of 52 cases), with cytoplasmic expression in 18 out of 43 cases (42%), not correlating with any clinico-pathological feature. In conclusion, Wnt pathway alterations, as represented by abnormal beta-catenin localization, are common events in digestive endocrine tumours, but only nuclear expression correlates with tumour aggressiveness. Though with different alteration mechanisms according to anatomical site, APC plays a major role in Wnt pathway activation and in determining the high chromosomal instability observed in aggressive endocrine carcinomas.     

Immunohistochemical and sequencing analyses of the Wnt signaling components in Japanese anaplastic thyroid cancers.

We investigated the status of the components and target genes of the Wnt signaling pathway in Japanese anaplastic thyroid cancers (ATCs) in the present study. Nuclear and cytoplasmic positive staining of beta-catenin, which might indicate the existence of alterations in the Wnt signaling pathway, were found in 40.9% and 63.6% of the 22 ATC samples, respectively. The beta-catenin, adenomatous polyposis coli (APC) and Axin 1 gene mutations were observed in 4.5%, 9.0%, and 81.8% of the 22 ATC samples, respectively. Overexpression of cyclin D1 and c-myc, which are the target genes of the Wnt signaling pathway, was observed in 27.3% and 59.1% of the ATC samples, respectively. There was no significant correlation between nuclear or cytoplasmic positive staining of beta-catenin and nuclear positive staining of cyclin D1 or c-myc. Taken together, the results of beta-catenin immunohistochemistry suggest that alterations in the Wnt signaling pathway are associated with carcinogenesis of ATC, but the frequency of beta-catenin gene mutation in our series is lower than that previously reported. Furthermore, cyclin D1 and c-myc frequently accumulated in ATC, independently of dysfunction in the Wnt signaling pathway.