Comparative genomics on DKK2 and DKK4 orthologs

WNT family proteins activate the beta-catenin - TCF pathway to induce carcinogenesis through cell fate determination, and also activate the planar cell polarity (PCP) pathway to induce cell motility and metastasis. DKK1, DKK2, DKK3 and DKK4 are secreted-type WNT signaling modulators belonging to the Dickkopf family. Here, we identified and characterized rat Dkk2 and Dkk4 genes by using bioinformatics. Rat Dkk2 and Dkk4 genes, consisting of four exons, were located within AC120263.4 and AC109661.6 genome sequences, respectively. Rat Dkk2 gene encoded a 259-aa protein, showing 95.8% total-amino-acid identity with human DKK2. Rat Dkk4 gene encoded a 221-aa protein, showing 75.4% total-amino-acid identity with human DKK4. Mammalian Dkk family members were secreted proteins with two Cys-rich regions, each containing ten conserved Cys residues. Asn-linked glycosylation site at codon 52 was conserved among mammalian Dkk2 orthologs; however, Asn-linked glycosylation site was not identified among mammalian Dkk4 orthologs. Dkk2 proteins were more conserved than Dkk4 proteins, while Dkk4 promoters were more conserved than Dkk2 promoters. TATA-box was identified within Dkk2 and Dkk4 promoters. MYOD and triple TCF/LEF binding sites were conserved between human DKK4 promoter and rodent Dkk4 promoter. DKK2 mRNA was expressed in Ewing's sarcoma, and fetal heart. DKK4 mRNA was expressed in human embryonic stem (ES) cells differentiated to an early endodermal cell type, breast cancer, and diffuse type gastric cancer. DKK4 orthologs are implicated in the negative feed back mechanism of the WNT/beta-catenin signaling pathway (the canonical WNT signaling pathway).     

Adenomatous polyposis coli (APC) protein expression in primary and metastatic serous ovarian carcinoma

The aim of this study was to investigate protein expression of adenomatous polyposis coli (APC) in primary and metastatic serous ovarian carcinoma. The expression of beta-catenin and E-cadherin was additionally analyzed. One hundred and thirteen primary (n = 56) and metastatic (n = 57) lesions were immunohistochemically stained for APC, E-cadherin, and beta-catenin. Staining extent was scored. Possible differences in immunoreactivity in primary and metastatic sites and the association between the proteins analyzed were evaluated statistically. Cytoplasmic immunoreactivity for APC was found in 67/113 (59%) tumors, most often in the majority (> 50%) of cells. E-cadherin was detected in 102/113 (90%) carcinomas, while beta-catenin was expressed in 109/113 (97%) specimens. Nuclear expression of beta-catenin was seen in 3/113 (3%) specimens, all negative for APC. APC and beta-catenin were often coexpressed, but this finding failed to reach statistical significance (p = 0.11). A significant association was seen between E-cadherin and beta-catenin expression (p = 0.001). APC expression was comparable in primary and metastatic tumors (p > 0.05). In conclusion, APC expression is absent in a considerable number of both primary and metastatic ovarian carcinomas, but this finding is only rarely coupled to nuclear accumulation of beta-catenin. These findings support the role for beta-catenin signaling via the Wingless/Wnt pathway in ovarian carcinoma. The mechanism behind the down-regulated expression of APC in serous ovarian carcinoma and its significance has yet to be elucidated.     

Sporadic childhood hepatoblastomas show activation of beta-catenin, mismatch repair defects and p53 mutations.

Hepatoblastoma, a rare embryonic tumor that may arise sporadically or in the context of hereditary syndromes (familial adenomatous polyposis and Beckwith-Wiedemann's) is the most frequent liver cancer of childhood. Deregulation of the APC/beta-catenin pathway occurs in a consistent fraction of hepatoblastomas, with mutations in the APC and beta-catenin genes implicated in familial adenomatous polyposis-associated and sporadic hepatoblastomas, respectively. Alterations in other cancer-related molecular pathways have not been reported. We investigated a series of 21 sporadic paraffin-embedded hepatoblastoma cases for mutations in the p53 (exons 5-8) and beta-catenin (exon 3) genes, loss of heterozygosity at APC, microsatellite instability and immunohistochemical expression of beta-catenin and of the two main mismatch repair proteins, MLH1 and MSH2. No loss of heterozygosity at APC was detected. We found mutations in beta-catenin and p53 in 4/21 (19%) and 5/21 (24%) cases respectively, beta-catenin protein accumulation in 14/21 cases (67%), microsatellite instability in 17/21 cases (81%), of which eight resulted positive for high-level of microsatellite instability (in four cases associated with loss of MLH1/MSH2 immunostaining). No correlations between involved molecular pathway(s) and hepatoblastoma histotype(s) emerged. This study confirms that beta-catenin deregulation is involved in sporadic hepatoblastoma and also suggests that mismatch repair defects and p53 mutations contribute to this rare liver cancer. Sporadic hepatoblastoma appears to be molecularly and phenotypically heterogeneous and may reflect different pathways of liver carcinogenesis.     

Characterization of the E-cadherin/catenin complex in colorectal carcinoma cell lines

The E-cadherin/catenin complex is a prime mediator of cell-cell adhesion. APC mutations can result in loss of beta-catenin downregulation and an accumulation of beta-catenin in the cell. Beta-CATENIN mutations can have a similar effect. The aim of this study was to investigate the effect of beta-CATENIN and APC mutations on the expression and assembly of the E-cadherin/catenin complex. Five colorectal carcinoma cell lines with different APC and beta-CATENIN gene status were selected and mutations were confirmed. The expression of members of the E-cadherin/catenin complex was studied by immunohistochemistry and Western blotting. Complex assembly was investigated by immunoprecipitation. It is shown that E-cadherin and catenins are expressed in colorectal carcinoma cell lines with the predominant complex assembly being E-cadherin/beta-catenin/alpha-catenin. The subcellular distribution of the proteins is influenced by cell-cell contact, resulting in membranous localization. The expression and assembly of the E-cadherin/catenin complex does not appear to be affected by the presence of APC and or beta-CATENIN mutations.     

Wnt/β-catenin信号通路在实验性大鼠肝癌形成过程中的作用

目的:探讨Wnt/β-catenin信号转导通路与肿瘤发生的关系,为肝癌的防治提供新的思路。方法:选取Wnt/β-catenin信号转导通路中上下游关键因子wnt1、β-catenin、APC、cyclinD1以及c-myc等,应用RT-PCR法观察它们在正常肝脏,不典型增生肝脏和肝癌组织中的转录水平。用免疫组化染色法研究β-catenin、APC和cyclinD1等3个因子有无表达。结果:在正常肝脏中,用RT-PCR未检测到wnt1、cyc-linD1以及c-myc的mRNA转录,免疫组化染色也只观察到β-catenin在细胞膜处有比较弱的表达。诱癌14周后,在发生不典型增生的肝组织中,检测到β-catenin、APC和cyclinD13个基因的转录。通过免疫组化染色也观察到β-catenin蛋白在胞质中的积累,APC和cyclinD1在部分细胞中出现表达。诱癌16周后,在肝癌组织中,除wnt1mRNA外,其他几个因子的mRNA都有转录,免疫组化也印证了上述各个发生转录的因子在蛋白水平都有不同程度的表达。结论:Wnt/β-catenin信号转导通路在大鼠的肝癌形成过程中被激活,其可能是实验性肝癌发生的原因之一。