Up-regulation and overproduction of DVL-1, the human counterpart of the Drosophila dishevelled gene,in cervical squamous cell carcinoma

The Dvl-1 gene on chromosome 1p36 belongs to a family of highly conserved secreted proteins which regulates embryonic induction, generation of cell polarity and specification of cell fate through activation of Wnt signaling pathways. Wnt signaling activates the gene encoding DVL-1; the latter suppresses beta-catenin by promoting its degradation through enhanced inactivation of glycogen-synthase-kinase 3 (GSK3). Here we demonstrate increased expression of DVL-1 mRNA in over two thirds of primary cervical squamous cell cancers (11 of 15 cases) when compared to corresponding non-cancerous uterine squamous cell tissues. In addition, we noted up-regulation of cyclin D1, a downstream effector of Wnt signal pathway in cervical cancer. Immunohistochemical staining demonstrated that DVL-1 protein was prominent in the cytoplasm of cancer cells whereas it was unreactive in the surrounding normal cervical squamous cells. These data indicate that amplification and increased expression of the DVL-1 gene may play some role in the development of a portion of human cervical squamous cell cancer through derangement of the Wnt signaling pathway.     

结直肠癌组织中突变型Axin2对Wnt信号转导系统的调控及其机制

背景与目的:Axin2是近期发现的Wnt信号转导系统的新基因,其在结直肠癌中最主要的突变形式为羧基端缺失突变(mutant-type Axin2,mtAxin2).本研究的目的在于探索mtAxin2对Wnt信号转导系统的调控作用及其相关机制.方法:采用免疫组化的方法检测大肠癌组织中β-catenin的表达;构建pCMV-Flag-wtAxin2 和pCMV-Flag-mtAxin2质粒转染293细胞,采用免疫沉淀(co-immunoprecipitation,IP)和Western印迹杂交研究mtAxin2与Wnt信号转导系统中其它关键蛋白的作用;应用TNT T3/T7体外转录翻译系统体外合成mtAxin2 蛋白,用于mtAxin2的体外聚合实验:使用Dual-Luciferase 报告检测系统检测Firefly和Renilla荧光酶活性;构建视网膜受体(retinoid receptor,RR)与蜕皮素受体(ecdysone receptor,ECR)质粒,用于恢复mtAxin2的DIX功能区对mtAxin2功能影响的研究.结果:免疫组化检测显示在有Axin突变的结直肠癌标本中β-catenin蛋白在核内积聚,而无Axin突变者在胞浆内.Westem印迹杂交显示mtAxin2仍具有与GSK-3β、APC、β-catenin、DVL-1和PP2A形成复合体的功能.将Flag-wtAxin2和Flag-mtAxin2共转染293细胞后,mtAxin2与wtAxin2可竞争性地与β-catenin结合.在TNT T3/T7体外转录翻译实验中.wtAxin2与Flag-wtAxin2结合,而与Flag-mtAxin2不结合,mtAxin2不能形成二聚体或多聚体.pCMV-Flag-mtAxin2-RXR质粒转染293细胞后可降低T细胞转录因子报告基因(T-cell factor,TCF)活性,pCMV-Flag-mtAxin2-ECR质粒转染后TCF活性升高;pCMV-Flag-mtAxin2-RXR和pCMV-Flag-mtAxin2-ECR共转染,TCF活性也下降,显示mtAxin2与RXR融合后,融合蛋白可形成二聚体,恢复了Axin2的功能.致TCF报告基因的活性下降.结论:因为缺失DIX区,mtAxin2不能形成二聚体,干扰了β-catenin的降解,引起β-catenin在核内的积聚进而激活Wnt信号转导系统.     

DIXDC1 targets p21 and cyclin D1 via PI3K pathway activation to promote colon cancer cell proliferation

DIXDC1 is the human homolog of Ccd1, a recently identified DIX domain containing protein in zebrafish. It is a positive regulator in the Wnt signaling pathway functioning downstream of Wnt and upstream of Axin. Since Wnt pathway activation is correlated with human colon cancer formation and progression, the biological role of DIXDC1 in human colon cancer was examined. In the current study, up-regulation of DIXDC1 protein was detected in human colorectal adenocarcinoma tissues and was found to be correlated well with high cell proliferation index. Ectopic over-expression of DIXDC1 resulted in increased cell proliferation in vitro and accelerated tumorigenesis on nude mice in vivo . We also showed that DIXDC1 promoted G0/G1 to S phase transition concomitantly with up-regulation of cyclin D1 and down-regulation of p21 protein. DIXDC1 over-expression cells showed activation of the PI3K/AKT pathway. Both siRNA knockdown of DIXDC1 and blocking the PI3K pathway using a specific inhibitor caused G1/S phase arrest, as well as down-regulation of cyclin D1 and up-regulation of p21 in DIXDC1 over-expression colon cancer cells. Collectively, this study demonstrates that over-expression of DIXDC1 might target p21 and cyclin D1 to promote colon cancer cell proliferation and tumorigenesis at least partially through activation of the PI3K/Akt pathway. ( Cancer Sci  2009; 100: 1801–1808)     

Wnt signaling stabilizes the DIXDC1 protein through decreased ubiquitin‐dependent degradation

(Cancer Sci 2010; 101: 700–706) Wnt signaling plays key roles in development, cell growth, differentiation, polarity formation, neural development, and carcinogenesis. DIX Domain Containing 1 (DIXDC1), a novel component of the Wnt pathway, was recently cloned. DIXDC1 is the human homolog of Ccd1, a positive regulator of the Wnt signaling pathway during zebrafish neural patterning. Little has been known about DIXDC1 gene expression regulation. In the present study, we showed that the DIXDC1 protein was induced upon Wnt‐3a stimulation, whereas the DIXDC1 mRNA level was not significantly increased after Wnt‐3a treatment. Positive DIXDC1 staining was detected in colon cancer cells and was colocalized with β‐catenin staining. However, the DIXDC1 mRNA expression decreased in human colon cancer cells compared to the matched normal colon epithelial cells. Our further investigation showed that the DIXDC1 protein was degraded through the proteasome pathway, and the activation of canonical Wnt signaling decreased the ubiquitin‐dependent degradation of both the ectopic and endogenous DIXDC1 protein. In order to explore the possible mechanism of the ubiquitination of DIXDC1, we found that the phosphorylation of DIXDC1 was inhibited by Wnt‐3a. Collectively, these results indicate that canonical Wnt/β‐catenin pathway activation might upregulate DIXDC1 through a post‐translational mechanism by inhibiting the ubiquitin‐mediated degradation of the DIXDC1 protein.     

Expression of Notch and Wnt pathway components and activation of Notch signaling in medulloblastomas from heterozygous patched mice

Summary Hedgehog (Hh), Notch, and Wingless (Wnt) signaling control normal development of the cerebellum, and dysregulation of these signaling pathways are associated with medulloblastoma (MB). As an initial step in the study of the role of interacting signaling pathways in MB pathogenesis, we demonstrate the expression of several components of the Notch and Wnt signaling pathways, and activation of Notch signaling in MB from Ptch ^+/− mice that have elevated Hh signaling. We also show a marked downregulation in the expression of Notch2, Jagged1, Hes1, mSfrp1, and mFrz7 in cerebella of developing mice with reduced Hh signaling, suggesting that Hh signaling regulates the expression of these genes. Together with recent published data, these findings indicate that Hh signaling might synergize simultaneously with Notch and Wnt signaling in MB development by controlling Notch and Wnt pathway ligand, receptor and/or target gene expression.     

Wnt targets genes are not differentially expressed in desmoid tumors bearing different activating β-catenin mutations

IntroductionSporadic desmoid-type fibromatosis (DTF) is a rare soft tissue tumor of mesenchymal origin. It is characterized by local invasive growth and unpredictable growth behavior. Three distinct mutations involving the CTNNB1 (β-catenin) gene have been identified in the vast majority of DTF tumors, which cause activation of the Wnt signaling pathway and impact prognosis. This study examines whether the different CTNNB1 mutants (T41A, S45F) occurring in DTF tumors differentially affect Wnt signaling activity, which might explain the different disease course between DTF patients harboring different CTNNB1 mutations.Materials and methodsReal-time polymerase chain reaction (RT-PCR) on 61 formalin fixed paraffin embedded DTF samples with known CTNNB1 status was used to measure the relative mRNA expression level of Wnt target genes AXIN2, DKK1 and CCND1. Additionally, publicly available mRNA expression data retrieved from the Gene Expression Omnibus of 128 DTF samples were used for an unsupervised cluster analyses based on the expression of a selection of Wnt targets.ResultsNo statistically significant difference in relative expression levels of Wnt target genes AXIN2, DKK1 and CCND1 was identified between either CTNNB1 wild-type, S45F or T41A mutated DTF samples. Moreover, the hierarchical cluster analyses using selected Wnt targets did not discriminate between different CTNNB1 mutation types.ConclusionsNo differences in the expression levels of Wnt target genes were observed between the different CTNNB1 mutation types in DTF tumors. Further studies are needed to decipher the mechanism accounting for the diverse disease courses between DTF patients with different CTNNB1 variants.     

Cathelicidin,an antimicrobial peptide produced by macrophages,promotes colon cancer by activating the Wnt/β-catenin pathway

Here we found that levels of cathelicidin, an antimicrobial peptide, were increased in colon cancer tissues compared to noncancerous tissues. Importantly, cathelicidin was mainly expressed in immune cells. Contact with tumor cells caused macrophages to secrete cathelicidin. Neutralization of cathelicidin, in vivo, significantly reduced the engraftment of macrophages into colon tumors, as well as proliferation of tumor cells, resulting in an inhibition of tumor growth. Furthermore, treatment with cathelicidin neutralizing antibody de-activated the Wnt/β-catenin signaling pathway in tumor cells both in vivo and in vitro. Cathelicidin activated Wnt/β-catenin signaling by inducing phosphorylation of PTEN, leading to activation of PI3K/Akt signaling and subsequent phosphorylation of GSK3β, resulting in stabilization and nuclear translocation of β-catenin. These data indicate that cathelicidin, expressed by immune cells in the tumor microenvironment, promotes colon cancer growth through activation of the PTEN/PI3K/Akt and Wnt/β-catenin signaling pathways.     

CD44+ slow‐cycling tumor cell expansion is triggered by cooperative actions of Wnt and prostaglandin E2 in gastric tumorigenesis

(Cancer Sci 2010; 101: 673–678) Similar to normal tissue stem cells, cancer stem cells (CSCs) are thought to be quiescent or slow‐cycling and, thereby, insensitive to chemo‐ and radiotherapies. CD44, a cell surface component that interacts with the extracellular matrix, has been found to be highly expressed in CSCs of several solid tumors. However, the relevancy between CD44⁺ cells and slow‐cycling cells and the underlying mechanisms for the emergence of CD44⁺ CSCs during tumorigenesis have not been elucidated. Here we show that a gastric gland residing at the squamo‐columnar junction (SCJ) in normal mouse stomach contains CD44⁺ stem cell‐like slow‐cycling cells and that this characteristic CD44⁺ gland was expanded by prostaglandin E2 (PGE₂) and Wnt signaling in K19‐Wnt1/C2mE mouse, a genetic mouse model for gastric tumorigenesis. The analysis of three transgenic mouse lines, K19‐Wnt1, K19‐C2mE and K19‐Wnt1/C2mE, revealed that the expansion of CD44⁺ SCJ cells is triggered by PGE₂‐mediated signaling and is prominently enhanced by the addition of Wnt activation. Furthermore, each expanded CD44⁺ gland in gastric tumor of K19‐Wnt1/C2mE mouse contains a few BrdU label‐retaining quiescent or slow‐cycling cells, suggesting that the CD44⁺ SCJ cells in normal mouse are candidates for the cell‐of‐origin of gastric CSCs. These observations suggest that PGE₂‐mediated inflammatory signaling and Wnt signaling cooperatively trigger the expansion of CD44⁺ slow‐cycling stem‐like cells in SCJ, leading to development of lethal gastric tumors in mice.     

Aberrant expression of Wnt/β-catenin signaling pathway genes in aggressive malignant gastric gastrointestinal stromal tumors

IntroductionRecent reports on gene expression profiling (GEP) show several genes associated with malignant progression of GIST. However, genes associated with malignant transformation have not been clarified. Here, we aimed to reveal distinct genes in aggressive malignant GIST, using comprehensive gene expression analysis.Materials and methodsWe investigated GEP obtained by microarrays for 43 gastric GISTs, which mostly harbored KIT and PDGFRA mutations and integrated clinicopathological risk information. RT-PCR and immunohistochemistry were performed for FZD7, a receptor of Wnt ligands.ResultsGEP divided 43 gastric GISTs into two clusters. A cluster included seven of eight high-risk GISTs (88%) in modified NIH classification and was defined as high-risk cluster; the other cluster was defined as low-risk cluster. The number of probes with over 3-fold changes between the two clusters was 1,177, in which probes corresponding to 16 oncogenes were included. Genes involved in the Wnt signaling pathway were the most abundant among the 16 oncogenes. Focusing on 73 Wnt signaling pathway genes of the 21,578 probes, 12 upregulated and 5 downregulated genes were found in the high-risk cluster. Major cascade genes promoting the Wnt/β-catenin signaling pathway, including WNT11, FZD family, and DVL2, were upregulated in the high-risk cluster. SNAI1, SNAI2, and BIRC5, which are activated by this pathway and increase cell proliferation, were also upregulated. These gene expression alterations were consistent in the positive direction of this pathway. GISTs in high-risk cluster strongly expressed FZD7.ConclusionWnt/β-catenin signaling pathway may play an important role in malignant transformation of indolent GIST.