The Wnt-dependent signaling pathways as target in oncology drug discovery

Summary Our current understanding of the Wnt-dependent signaling pathways is mainly based on studies performed in a number of model organisms including, Xenopus, Drosophila melanogaster, Caenorhabditis elegans and mammals. These studies clearly indicate that the Wnt-dependent signaling pathways are conserved through evolution and control many events during embryonic development. Wnt pathways have been shown to regulate cell proliferation, morphology, motility as well as cell fate. The increasing interest of the scientific community, over the last decade, in the Wnt-dependent signaling pathways is supported by the documented importance of these pathways in a broad range of physiological conditions and disease states. For instance, it has been shown that inappropriate regulation and activation of these pathways is associated with several pathological disorders including cancer, retinopathy, tetra-amelia and bone and cartilage disease such as arthritis. In addition, several components of the Wnt-dependent signaling pathways appear to play important roles in diseases such as Alzheimer’s disease, schizophrenia, bipolar disorder and in the emerging field of stem cell research. In this review, we wish to present a focused overview of the function of the Wnt-dependent signaling pathways and their role in oncogenesis and cancer development. We also want to provide information on a selection of potential drug targets within these pathways for oncology drug discovery, and summarize current data on approaches, including the development of small-molecule inhibitors, that have shown relevant effects on the Wnt-dependent signaling pathways.     

Enfermedad de Pyle: un modelo humano de homeostasis corticotrabecular diferenciada

Pyle's disease (OMIN number 265900) is a metaphyseal dysplasia of benign course, inherited with an autosomal recessive pattern. Some 30 genuine cases have been described so far. The cause of this process has been known since 2016, when its relationship to mutations in the gene encoding the sFRP protein, a known inhibitor of the Wnt pathway, was discovered. We report the case of a 58-year-old man, diagnosed with Pyle's disease based on his clinical and radiographic characteristics, whose phenotype suggested a differential control of cortical and trabecular bone homeostasis.     

散发性结直肠癌患者血RASSF2和sFRP1启动子区甲基化检测

目的检测散发性结直肠癌患者血清RASSF2和sFRP1启动子区甲基化情况．从而为散发性结直肠癌的早期筛查提供参考。方法使用甲基化特异性PCR检测59例散发性直结肠癌患者和59例健康对照血清sFRP1和RASSF2基因启动子区甲基化的情况,并分析其与结直肠癌临床病理特征的关系。结果59例结直肠癌患者中RASSF2和sFRP1甲基化者分别为16例（27．1％）和18例（30．5％）;而59例健康对照无一例发现RASSF2或sFRP1基因甲基化,差异有统计学意义（均P〈0．01）。29例（49．2％）结直肠癌患者有至少1个基因甲基化,其甲基化率明显高于RASSF2和sFRP1单-基因甲基化率（均P〈0．05）。结直肠癌患者血清RASSF2和sFRP1基因甲基化率与结直肠癌临床病理特征均无明显关系（均P〉0．05）。结论血清RASSF2和sFRP1基因启动子区甲基化水平在结直肠癌组织异常升高,联合两基因的血清甲基化检测可为散发性结直肠癌的早期筛查提供参考。     

Secreted Frizzled Related Protein 2 (sFRP2) Decreases Susceptibility to UV-Induced Apoptosis in Primary Culture of Canine Mammary Gland Tumors by NF-κB Activation or JNK Suppression

Tumor formation can result from a decrease in cell death, as well as an increase in cell proliferation. In spite of the high incidence of mammary gland tumors (MGTs) in female dogs, the understanding of its etiology is still poor. Consistent with several proto-oncogenes (such as Wnt) for the mammary gland, sFRP2 is expressed in canine MGTs which is normally silent in the mammary gland. To elucidate the roles of SFRP2 in the tumorigenesis of MGTs, apoptosis regulation mediated by sFRP2 was investigated by overexpression of sFRP2 in MGT cells. DNA fragmentation and TUNEL assays showed a decreased susceptibility of the cells to UV-induced apoptosis in the context of sFRP2 overexpression. To analyze the pathways through which sFRP2 transduces anti-apoptosis signals, multiple-color immunofluorescence staining, immunoprecipitation, and immunoblotting were carried out. sFRP2 was found co-localized in the extracellular matrix of MGTs and the tyrosine phosphorylation of FAK was enhanced. Moreover, JNK was suppressed and NF-kB was activated in the cells expressing sFRP2 after UV-induced apoptosis analyzed by immunoblotting and electrophoretic mobility shift assay (EMSA). Taken together, these results suggest that sFRP2 exerts its anti-apoptotic function in mammary cancer cells through NF-κB activation or JNK suppression.The first two authors contributed equally.     

外源性FrzB拮抗Wnt信号转导并抑制人骨肉瘤细胞143B增殖

目的 Wnt信号异常激活与肿瘤发生有关,观察Wnt信号的天然拮抗剂FrzB对Wnt信号转导的影响和对肿瘤细胞增殖抑制。方法采用AdEasy系统构建重组腺病毒AdFrzB;卡那霉素抗性筛选、酶切鉴定及荧光显微镜和Western blot检测标记基因GFP和His表达等方法鉴定AdFrzB。AdFrzB与AdWnt3A共感染人骨肉瘤细胞株143B,AdGFP与AdWnt3A共感染为对照,β-catenin/Tcf荧光素酶反应系统检测FrzB对Wnt信号转导的影响;MTT实验检测FrzB对细胞增殖的抑制。结果卡那霉素抗性筛选及酶切鉴定获得pAdFrzB-His;检测到报告基因GFP和His表达,确证AdFrzB构建成功。FrzB抑制了Wnt信号转导并抑制了143B细胞增殖。结论 AdEasy系统有效构建了AdFrzB;外源性FrzB拮抗Wnt信号转导并抑制143B细胞增殖,FrzB可能是一种天然抗肿瘤因子。     

Epidermal growth factor receptor: a novel target of the Wnt/beta-catenin pathway in liver

BACKGROUND & AIMS: Wnt/beta-catenin activation is observed in normal liver development, regeneration, and liver cancer. Our aim was to elucidate the regulation and mechanism of this pathway in liver. METHODS: We report the generation and characterization of liver-specific nonmutated beta-catenin-overexpressing transgenic mice. Transgenic livers were examined for their morphology and phenotype by histology, proliferation, apoptosis, and microarray analysis. RESULTS: Transgenic livers displayed a significant increase in cytoplasmic, membranous, and nuclear beta-catenin in hepatocytes as compared with their wild-type littermates, which display a predominant membranous localization only. A 15%-20% increase in the liver weight-body weight ratio was evident in transgenic mice secondary to increased hepatocyte proliferation. Microarray analysis showed differential expression of approximately 400 genes in the transgenic livers. Epidermal growth factor receptor RNA and protein and increased levels of activated epidermal growth factor receptor and Stat3 were observed in the transgenic livers. Epidermal growth factor receptor promoter analysis showed a T-cell factor-binding site, and subsequent reporter assay confirmed epidermal growth factor receptor activation in response to Wnt-3A treatment that was abrogated by frizzled related protein 1, a known Wnt antagonist. Epidermal growth factor receptor inhibition successfully decreased liver size in transgenic mice. Next, 7 of 10 hepatoblastomas displayed simultaneous beta-catenin and epidermal growth factor receptor up-regulation, thus suggesting a strong relationship between these 2 proteins in tumors. CONCLUSIONS: beta-Catenin transgenic mice show an in vivo hepatotrophic effect secondary to increased basal hepatocyte proliferation. Epidermal growth factor receptor seems to be a direct target of the pathway, and epidermal growth factor receptor activation might contribute toward some mitogenic effects of increased beta-catenin in liver: epidermal growth factor receptor inhibition might be useful in such states.     

DNA methylation and histone modifications cause silencing of Wnt antagonist gene in human renal cell carcinoma cell lines

Secreted frizzled-related protein 2 (sFRP2) is a negative modulator of the Wingless-type (Wnt) signaling pathway, and shown to be inactivated in renal cell carcinoma (RCC). However, the molecular mechanism of silencing of sFRP2 is not fully understood. Our study was designed to elucidate the silencing mechanism of sFRP2 in RCC. Expression of sFRP2 was examined in 20 pairs of primary cancers by immunohistochemistry. Kidney cell lines (HK-2, Caki-1, Caki-2, A-498 and ACHN) were analyzed for sFRP2 expression using real-time RT-PCR and Western blotting. The methylation status at 46 CpG sites of the 2 CpG islands in the sFRP2 promoter was characterized by bisulfite DNA sequencing. Histone modifications were assessed by chromatin immunoprecipitation (ChIP) assay using antibodies against AcH3, AcH4, H3K4 and H3K9. sFRP2 was frequently repressed in primary cancers and in RCC cells. The majority of sFRP2 negative cells had a methylated promoter. Meanwhile, sFRP2 expression was repressed by a hypomethylated promoter in Caki-1 cells, and these cells had a repressive histone modification at the promoter. In Caki-1 cells, sFRP2 was reactivated by trichostatin A (TSA). Repressive histone modifications were also observed in RCC cells with hypermethylated promoters, but sFRP2 was reactivated only by 5-aza-2'-deoxycytidine (DAC) and not by TSA. However, the activation of the silenced sFRP2 gene could be achieved in all cells using a combination of DAC and TSA. This is the first report indicating that aberrant DNA methylation and histone modifications work together to silence the sFRP2 gene in RCC cells.     

多基因联合提高结直肠癌甲基化检测阳性率的研究

目的探讨联合检测Vimentin、sFRP1和HPP1基因甲基化对提高结直肠癌甲基化阳性率的意义。方法收集90例结直肠癌（结直肠癌组）和60例腺瘤性息肉（腺瘤组）患者及20例结直肠正常组织（正常对照组）标本,提取组织标本的DNA,采用甲基化特异性PCR（MSP）检测Vimentin、sFRP1和HPP1基因甲基化状态。分析其与结直肠癌的关系及甲基化阳性率。结果结直肠癌组Vimentin、sFRP1和HPP1基因甲基化率分别为66．7％（60／90）、68．9％（62／90）和72．2％（65／90）;腺瘤组则分别为53.3％（32／60）、55．0％（33／60）和50．0％（30／60）;正常对照组分别为0、0和5．0％（1／20）。结直肠癌组3个基因甲基化阳性率均高于腺瘤组和正常对照组（P〈O．05）。3个基因甲基化联合检测诊断结直肠癌和腺瘤的阳性率分别为93．3％（84／90）和76．7％（46／60）,高于单个基因检测的甲基化阳性率（P〈0．05）。3个基因甲基化状态与本组结直肠癌患者的性别、年龄、肿瘤部位、淋巴结转移、远处转移及TNM分期无关（P〉0．05）。结论Vimentin、sFRP1和HPP1基因启动子甲基化水平在结直肠癌组织中升高．其联合检测明显提高了结直肠癌甲基化检测阳性率．有可能成为结直肠癌早期诊断的甲基化检测方案。