Dissecting the mechanism of Wnt signal transduction using chemical probes

使用化学探针剖析 Wnt 信号转导机制

• 批准号: BB/G016887/1
• 金额: $ 9.48万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG016887%2F1
• 关键词: Dissecting; mechanism; Wnt; signal; transduction

The Wnt ligand signaling pathway is considered a key therapeutic target in conditions including cancer, heart disease, arthritis, schizophrenia and Alzheimer's disease. Mutations to 'core' Wnt signaling components including b-catenin, APC and Axin inappropriately activate Wnt signaling in cancers of the colon and liver. Many further cancers show signs of inappropriate Wnt pathway activation (Eg. stabilized nuclear b-catenin in 50% of breast cancers), although the mechanisms leading to pathway activation remain unclear. Similar uncertainty surrounds the mechanism(s) that lead to pathogenic Wnt pathway deregulation in other diseases. To develop drug-like small molecules against the Wnt pathway, we have established a consortium of groups that have expertise ranging from medicinal chemistry, Wnt signal transduction and cell biology through to mouse models of deregulated Wnt signalling in colon cancer and clinical trials of metastatic disease. In previous work, we screened a library of 70,000 small molecule drug like compounds in a cell based assay for inhibitors of Wnt/TCF-dependent transcription. Following a series of deconvolution assays, we identified 4 small molecule inhibitors that blocked tumour cell growth and appeared to operate at distinct points in the Wnt signal transduction cascade. Using biotinylated small molecule analogues of one series of active compounds, we have identified the FET oncogene family as a probable molecular target of the Wnt-inhibitory activity. Members of the FET gene family has been shown to be fused to transcription factors and are the primary initiating oncogene in a range of human sarcomas (Eg EWS in Ewings Sarcoma), but few links have previously been established between EWS gene family members and Wnt signaling. As part of the CASE studentship, we now propose to study the mechanism by which the compounds inhibit EWS family function in the context of the Wnt signaling. This work will involve overlapping biochemical and molecular approaches to identify the molecular process by which the FET family regulates Wnt signaling and the mechanism by which the anti-Wnt signaling drug alters EWS/Wnt function. At the biochemical level, we will study the physical interaction between FET family members and components of the Wnt signalling pathway, since published reports suggest a direct interaction between the FET family member, Fus and the Wnt signaling proteins, b-catenin and the TCF. At the cell biological level, we will study how alterations to FET family member expression alter Wnt signaling. This work will rely on our ability to alter the expression of EWS and Wnt signaling components through RNAi gene depletion and cDNA gene overexpression. Both of these techniques are well established within the laboratory and preliminary data has shown that RNAi reduction of EWS and Fus expression lowers Wnt/TCF-dependent signaling. In the longer-term, deletion analysis will be used to identify regions of the EWS family members that are required for function in the Wnt pathway and drug binding leading to the identification of the mechanism of drug action. The output of the work will be a molecular understanding of the role of EWS family members in Wnt signaling. This work will directly contribute to the development of a series of putative anti-cancer drugs and will generate high quality information on the basic biology of Wnt-EWS pathway interactions that will contribute to the field of signal transduction.

Wnt 配体信号通路被认为是癌症、心脏病、关节炎、精神分裂症和阿尔茨海默病等疾病的关键治疗靶点。 “核心”Wnt 信号传导成分（包括 b-连环蛋白、APC 和 Axin）的突变会不适当地激活结肠癌和肝癌中的 Wnt 信号传导。许多其他癌症显示出不适当的 Wnt 通路激活的迹象（例如，50% 的乳腺癌中存在稳定的核 b-连环蛋白），尽管导致通路激活的机制仍不清楚。类似的不确定性还存在于其他疾病中导致致病性 Wnt 通路失调的机制上。为了开发针对 Wnt 通路的药物样小分子，我们建立了一个由多个小组组成的联盟，这些小组拥有从药物化学、Wnt 信号转导和细胞生物学到结肠癌中 Wnt 信号失调的小鼠模型和转移性疾病临床试验的专业知识。在之前的工作中，我们在基于细胞的 Wnt/TCF 依赖性转录抑制剂检测中筛选了 70,000 种小分子药物样化合物的库。经过一系列反卷积分析，我们鉴定出了 4 种小分子抑制剂，它们可以阻止肿瘤细胞生长，并且似乎在 Wnt 信号转导级联中的不同点发挥作用。使用一系列活性化合物的生物素化小分子类似物，我们已经确定 FET 癌基因家族是 Wnt 抑制活性的可能分子靶标。 FET 基因家族的成员已被证明与转录因子融合，并且是一系列人类肉瘤（例如尤文肉瘤中的 EWS）中的主要起始癌基因，但之前很少在 EWS 基因家族成员和 Wnt 信号传导之间建立联系。作为 CASE 学生资助的一部分，我们现在提议研究这些化合物在 Wnt 信号传导背景下抑制 EWS 家族功能的机制。这项工作将涉及重叠的生化和分子方法，以确定 FET 家族调节 Wnt 信号传导的分子过程以及抗 Wnt 信号传导药物改变 EWS/Wnt 功能的机制。在生化水平上，我们将研究 FET 家族成员与 Wnt 信号通路成分之间的物理相互作用，因为已发表的报告表明 FET 家族成员 Fus 与 Wnt 信号蛋白、b-连环蛋白和 TCF 之间存在直接相互作用。在细胞生物学水平上，我们将研究 FET 家族成员表达的改变如何改变 Wnt 信号传导。这项工作将依赖于我们通过 RNAi 基因缺失和 cDNA 基因过表达来改变 EWS 和 Wnt 信号成分表达的能力。这两种技术均已在实验室内得到很好的应用，初步数据表明 RNAi 减少 EWS 和 Fus 表达可降低 Wnt/TCF 依赖性信号传导。从长远来看，缺失分析将用于识别 Wnt 通路功能和药物结合所需的 EWS 家族成员区域，从而确定药物作用机制。这项工作的成果将是对 EWS 家族成员在 Wnt 信号传导中的作用的分子理解。这项工作将直接有助于一系列假定的抗癌药物的开发，并将产生有关 Wnt-EWS 通路相互作用的基础生物学的高质量信息，这将有助于信号转导领域。