Wnt signaling activation and WIF-1 silencing in nasopharyngeal cancer cell lines

Aberrant activation of Wingless-type (Wnt) signaling pathway plays a critical role in oncogenesis of various human cancers. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist of Wnt signaling and acts through direct binding to Wnt in the extracellular space. Recently, we reported Wnt signaling in various human malignancies. In addition, we identified in lung cancer that WIF-1 is silenced due to promoter hypermethylation. In this study, we found constitutive activation of Wnt signaling and WIF-1 silencing in nasopharyngeal carcinoma (NPC) cell lines. Furthermore, by utilizing methylation-specific PCR and sequence analysis, we demonstrated that frequent hypermethylation of the WIF-1 promoter correlates with WIF-1 silencing in NPC cell lines. Our results indicate that aberrant Wnt signaling is a common event in NPC carcinogenesis linked with WIF-1 silencing in at least cell lines. Strategies targeting these molecules should be potentially promising in treating NPC.     

Epigenetic regulation of DACH1, a novel Wnt signaling component in colorectal cancer

Colorectal cancer (CRC) is one of the common malignant tumors worldwide. Both genetic and epigenetic changes are regarded as important factors of colorectal carcinogenesis. Loss of DACH1 expression was found in breast, prostate, and endometrial cancer. To analyze the regulation and function of DACH1 in CRC, 5 colorectal cancer cell lines, 8 cases of normal mucosa, 15 cases of polyps and 100 cases of primary CRC were employed in this study. In CRC cell lines, loss of DACH1 expression was correlated with promoter region hypermethylation, and re-expression of DACH1 was induced by 5-Aza-2'-deoxyazacytidine treatment. We found that DACH1 was frequently methylated in primary CRC and this methylation was associated with reduction in DACH1 expression. These results suggest that DACH1 expression is regulated by promoter region hypermethylation in CRC. DACH1 methylation was associated with late tumor stage, poor differentiation, and lymph node metastasis. Re-expression of DACH1 reduced TCF/LEF luciferase reporter activity and inhibited the expression of Wnt signaling downstream targets (c-Myc and cyclinD1). In xenografts of HCT116 cells in which DACH1 was re-expressed, tumor size was smaller than in controls. In addition, restoration of DACH1 expression induced G2/M phase arrest and sensitized HCT116 cells to docetaxel. DACH1 suppresses CRC growth by inhibiting Wnt signaling both in vitro and in vivo. Silencing of DACH1 expression caused resistance of CRC cells to docetaxel. In conclusion, DACH1 is frequently methylated in human CRC and methylation of DACH1 may serve as detective and prognostic marker in CRC.     

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.     

IDKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.     

Dickkopf-1 antagonizes Wnt signaling independent of beta-catenin in human mesothelioma

Dickkopf-1 (Dkk-1) is a secreted protein that acts as a potent inhibitor of the Wnt signal transduction pathway. It is thought that the antagonistic effect of Dkk-1 is specific to the canonical (Wnt/beta-catenin) pathway. In this study, we demonstrate that restoration of Dkk-1 expression suppresses cell growth and induces apoptotic cell death in beta-catenin-deficient mesothelioma cell lines H28 and MS-1. Furthermore, we found that a small-molecule inhibitor of JNK inhibited the apoptosis induced by Dkk-1 overexpression in these cells. Together, our data suggest that Dkk-1 may be able to antagonize Wnt signaling and exert its tumor suppressive effects through beta-catenin-independent non-canonical pathways (i.e., the Wnt/JNK pathway).     

食管鳞状细胞癌中DICKKOPF-3基因的甲基化状态及表达

目的检测食管鳞状细胞癌(esophageal squamous cell cancer,ESCC)中Wnt通路拮抗基因DICKKOPF-3(DKK3)的甲基化状态,探讨其与ESCC发生的关系。方法应用甲基化特异性PCR(methylation specificPCR,MSP)及RT-PCR的方法检测78例ESCC及相应癌旁非肿瘤组织中DKK3基因的甲基化状态及mRNA表达情况,应用免疫组化的方法检测通路中心因子-βcatenin蛋白及下游靶基因cyclinD1的表达,并分析其与食管癌发生的关系。结果在ESCC组织中,DKK3基因的甲基化频率为37.2%(29/78),明显高于癌旁非肿瘤组织(χ2=35.622,P=0.000);癌组织中该基因的甲基化率与肿瘤患者临床分期相关(χ2=4.705,P=0.030),而与组织学分级无关;食管癌中DKK3基因mRNA的阳性表达率为65.4%(51/78),明显低于癌旁非肿瘤组织(χ2=13.298,P=0.000)。在发生甲基化的食管癌组织中该基因的mRNA表达缺失及-βcatenin蛋白的异质表达率均明显高于未发生甲基化的癌组织,且差异有统计学意义(χ2mRNA=29.141,P=0.000;χ2-βcatenin=6.245,P=0.012)。食管癌中cyclinD1的表达明显高于癌旁组织,且癌组织中该蛋白的表达与DKK3基因的甲基化状态相关(χ2=4.921,P=0.027)。结论 ESCC组织中DKK3基因高甲基化导致的转录沉默可能与食管癌的发生有关,并可能通过活化Wnt/-βcatenin信号转导通路促进下游靶基因cyclinD1的过表达发挥作用。     

WIF-1研究进展

WIF-1是Wnt信号通路上的拮抗物之一,可以阻断Wnt的经典通路和非经典通路。目前在人类多种肿瘤的研究发现WIF-1表达异常。WIF-1（Wnt inhibitory factor-1）,sFRP（Frizzled related protein）和CER（Cerberus）属于Wnt拮抗物家族,通过直接与Wnt蛋白相连从而阻止Wnt与受体蛋白复合物相连,使细胞质中的β-catenin由于磷酸化而不能积累,进而阻断了经典通路和非经典通路。WIF-1可能与中胚层的发生以及肿瘤细胞的生长分化有关。Wnt家族其他成员已经被证实与早期冠心病、Ⅱ型糖尿病、肥胖症、骨质疏松症等相关。因此了解WIF-1在通路上的更多信息,解释WIF-1调节Wnt信号通路的机理和过程,为疾病治疗和预防以及药物开发提供新的方法。     

The Wnt signaling pathway has tumor suppressor properties in retinoblastoma

Retinoblastoma is a pediatric retinal tumor caused by mutational inactivation of the tumor suppressor pRb. Additional genetic changes, as yet unidentified, are believed to be required for tumor initiation. Mutations in the Wnt signaling pathway have been implicated in the pathogenesis of many cancers. Multiple Wnt pathway genes are expressed in the retina and the pRb and Wnt pathways interact biochemically, raising the possibility that alterations in the Wnt pathway contribute to retinoblastoma. Our studies showed that Wnt signaling activation significantly decreased the viability of retinoblastoma cell lines by inducing cell cycle arrest, which was associated with upregulated p53. Furthermore, immunolocalization of the Wnt signaling mediator beta-catenin in human and mouse retinoblastoma tissue indicated that canonical Wnt signaling is suppressed in tumors in vivo. These studies are consistent with the Wnt pathway acting as a tumor suppressor in retinoblastoma and suggest that loss of Wnt signaling is tumorigenic in the retina.     

Epigenetic regulation of Wnt signaling pathway in urological cancer

Constitutive activation of the Wnt signaling pathway is a common feature of solid tumors and contributes to uncontrolled cell-growth and impaired differentiation. We hypothesized that gene silencing mediated through aberrant promoter methylation of upstream Wnt antagonist genes might result in β-catenin accumulation, resulting in constitutive Wnt activation. Wnt antagonist genes (SFRP1, WIF1, APC and CDH1) and CTNNB1 promoter methylation was examined in genomic DNA extracted from 12 urological cancer cell lines and correlated with CTNNB1 mRNA expression. Promoter methylation status was then assessed in 36 BCa, 30 PCa, 31 RCT, and normal bladder mucosa (15), prostate (10) and renal (5) tissue samples. Finally, CTNNB1 mRNA relative expression levels were correlated with Wnt antagonist gene methylation status in RCT. Methylation was found in at least one Wnt antagonist gene and the CTNNB1 promoter was unmethylated in all cancer cell lines tested. When gene methylation levels were compared between cancer cell lines with high and low CTNNB1 mRNA expression, a trend was found for increased CDH1 promoter methylation levels in the former. BCa and PCa tumors demonstrated high frequency of promoter methylation at all tested genes. In RCT, CTNNB1 was unmethylated in all cases and the overall frequency of promoter methylation at the remainder genes was lower. Interestingly, median CTNNB1 mRNA expression levels were significantly higher in RCTs methylated in at least one Wnt antagonist gene promoter. We concluded that epigenetic deregulation of Wnt pathway inhibitors may contribute to aberrant activation of Wnt signaling pathway in bladder, prostate and renal tumors.     

Secreted Wnt antagonists in scrub typhus

BackgroundThe mechanisms that control local and systemic inflammation in scrub typhus have only been partially elucidated. The wingless (Wnt) signaling pathways are emerging as important regulators of inflammation and infection, but have not been investigated in scrub typhus.Methodology/Principal findingsPlasma levels of secreted Wnt antagonists (i.e. DKK-1, sFRP-3, WIF-1 and SOST) were analyzed in patients with scrub typhus (n = 129), patients with similar febrile illness without O. tsutsugamushi infection (n = 31), febrile infectious disease controls, and in healthy controls (n = 31) from the same area of South India, and were correlated to markers of inflammation, immune and endothelial cell activation as well as for their association with organ specific dysfunction and mortality in these patients. We found i) Levels of SOST and in particular sFRP-3 and WIF-1 were markedly increased and DKK-1 decreased in scrub typhus patients at admission to the hospital compared to healthy controls. ii) In recovering scrub typhus patients, SOST, sFRP-3 and WIF-1 decreased and DKK-1 increased. iii) SOST was positively correlated with markers of monocyte/macrophage and endothelial/vascular activation as well as with renal dysfunction and poor outcome iv) Finally, regulation of Wnt pathways by O. tsutsugamushi in vitro in monocytes and ex vivo in mononuclear cells isolated from patients with scrub typhus, as evaluated by gene expression studies available in public repositories, revealed markedly attenuated canonical Wnt signaling.Conclusions/SignificanceOur findings suggest that scrub typhus is characterized by attenuated Wnt signaling possibly involving dysregulated levels of several secreted pathway antagonists. The secreted Wnt antagonist SOST was strongly associated with renal dysfunction and poor prognosis in these patients.     

Wif1 hypermethylation as unfavorable prognosis of non-small cell lung cancers with EGFR mutation

Lung cancer is a leading cause of cancer-related mortality across the world and tobacco smoking is the major risk factor. The Wnt signaling pathway is known to be involved in smoke-induced tumorigenesis in the lung. Promoter hypermethylation of Wnt inhibitory factor 1 (Wif1) has become a common event in a number of human tumors. Using a methylation-specific PCR, hypermethylation of the Wif1 gene promoter was evaluated in 139 primary nonsmall cell lung cancers (NSCLCs) and its correlation with clinicopathological and prognostic parameters was evaluated. Methylation of Wif1 was observed in 47.5% and 20.9% of neoplastic and adjacent normal lung tissues, respectively. Its methylation rate tended to be higher in stage I than stages II-IIIA. Results of Kaplan-Meier analysis showed no significant difference in overall survival according to Wif1 methylation status. However, Wif1 methylation showed an association with unfavorable prognosis of adenocarcinoma (AC) patients with EGFR mutation. According to our current findings, Wif1 promoter methylation is an early, frequent event as an epigenetic field manner and could be considered as a useful prognostic marker for AC patients with EGFR mutation. Further investigation into the therapeutic potential of this finding is warranted.     

Wnt/beta-catenin 信号通路相关蛋白在胃癌组织中的表达和 肿瘤转移的关系

目的：探讨了Wnt信号通路相关蛋白在胃癌组织中的表达及与肿瘤转移的关系。方法：选取2011 年6 月到2012 年6 月我 院胃癌术后47 例肿瘤标本作为研究对象,并选取同一患者的正常胃组织作为对照研究。采用实时荧光定量PCR 和Western blot 对胃癌组织和正常胃组织Wnt 信号通路相关蛋白进行分析,并分析了肿瘤转移和非转移患者Wnt信号通路相关蛋白的变化。结 果：与正常胃组织比较,胃癌组织中Wnt1、Wnt3、Wnt3a、beta-catenin、CyclinD1 和c-Myc 等分子的mRNA 水平明显上调,差异有显 著统计学意义（P<0.05）。胃癌组织中总beta-catenin 和核内beta-catenin蛋白较正常胃组织明显增加,而磷酸化beta-catenin 较正常组明显 下降、差异有显著统计学意义（P<0.05）。与非转移组比较,转移组患者胃癌组织中Wnt1、Wnt3、Wnt3a 等分子mRNA水平显著上 调,差异有统计学意义（P<0.05）。结论：Wnt信号通路异常激活在胃癌发生和癌细胞转移中发挥着重要的作用,为临床治疗提供了 一定靶点。     

Methylation of Wnt7a is modulated by DNMT1 and cigarette smoke condensate in non-small cell lung cancer

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2'-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC.     

Non-canonical Wnt Signaling Induces Ubiquitination and Degradation of Syndecan4

Dynamic regulation of cell adhesion receptors is required for proper cell migration in embryogenesis, tissue repair, and cancer. Integrins and Syndecan4 (SDC4) are the main cell adhesion receptors involved in focal adhesion formation and are required for cell migration. SDC4 interacts biochemically and functionally with components of the Wnt pathway such as Frizzled7 and Dishevelled. Non-canonical Wnt signaling, particularly components of the planar cell polarity branch, controls cell adhesion and migration in embryogenesis and metastasic events. Here, we evaluate the effect of this pathway on SDC4. We have found that Wnt5a reduces cell surface levels and promotes ubiquitination and degradation of SDC4 in cell lines and dorsal mesodermal cells from Xenopus gastrulae. Gain- and loss-of-function experiments demonstrate that Dsh plays a key role in regulating SDC4 steady-state levels. Moreover, a SDC4 deletion construct that interacts inefficiently with Dsh is resistant to Wnt5a-induced degradation. Non-canonical Wnt signaling promotes monoubiquitination of the variable region of SDC4 cytoplasmic domain. Mutation of these specific residues abrogates ubiquitination and results in increased SDC4 steady-state levels. This is the first example of a cell surface protein ubiquitinated and degraded in a Wnt/Dsh-dependent manner.     

Decreased Serum Wnt Antagonist Levels in Patients With Active Acromegaly

ObjectiveThe Wnt signaling pathway is an important modulator of bone metabolism. This study aims to clarify the changes in Wnt antagonists in active and biochemically controlled acromegalic patients.MethodsWe recruited 77 patients recently diagnosed with acromegaly. Of those, 41 patients with complete follow-up data were included. Thirty healthy patients matched for age, sex, and body mass index served as controls. At baseline and posttreatment, Wnt antagonists (sclerostin [SOST], dickkopf-related protein 1 [DKK-1], and Wnt inhibitory factor 1 [WIF-1]), bone turnover markers (osteocalcin, procollagen type 1 N-terminal propeptide [P1NP], and C-terminal telopeptide of type 1 collagen [CTX]) and the bone remodeling index were investigated.ResultsAcromegalic patients had higher serum osteocalcin, P1NP, and CTX and a higher bone remodeling index than controls (P < .01). Serum SOST, DKK-1, and WIF-1 levels were significantly decreased in patients compared to controls (all P < .01). Serum SOST and WIF-1 levels were negatively correlated with growth hormone levels; SOST levels were positively correlated with WIF-1. After treatment, serum bone turnover markers and the bone remodeling index decreased, while SOST and WIF-1 significantly increased (P < .05). DKK-1 levels did not change compared to baseline (P > .05). In biochemically controlled patients, SOST and WIF-1 levels and bone turnover markers were restored and did not differ from those of the control participants (all P > .05).ConclusionPatients with active acromegaly exhibited significantly decreased Wnt antagonist levels. The reduction in Wnt antagonists is a compensatory mechanism to counteract increased bone fragility in active acromegaly.     

巢式甲基化特异性PCR检测肺癌病人WIF-1基因启动子区异常甲基化

为了检测肺癌患者血浆中WIF-I基因启动子区的甲基化状态,收集肺癌患者及健康对照者的血浆标本,采用巢式甲基化特异性PCR（nMSP）法检测WIF-I基因启动子区甲基化状态,并与普通甲基化特异性PCR（MSP）法进行了比较,结果在58例肺癌患者血浆样品中经nMSP法发现20例WIF-I基因启动子的过甲基化,用MSP法只检出10例,有吸烟史组WIF-1基因的甲基化率高于无吸烟史组（P〈0．05）．而20例正常对照血浆中都未检测到形胆J基因启动子的过甲基化;表明利用巢式MSP（nMSP）法检测外周血血浆中WIF-1基因启动子的甲基化,可为非损伤性筛选和早期诊断肺癌提供有价值的信息．