Dissecting the mechanism of Wnt signal transduction using chemical probes

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。包括b-catenin、APC和Axin在内的Wnt信号核心成分的突变不适当地激活了结肠癌和肝癌中的Wnt信号。许多进一步的癌症显示出不适当的Wnt途径激活的迹象(例如。在50%的乳腺癌中稳定核型b-连环蛋白)，尽管导致途径激活的机制尚不清楚。类似的不确定性围绕着在其他疾病中导致致病性Wnt途径放松调控的机制(S)。为了开发针对Wnt途径的类药物小分子，我们建立了一个由多个小组组成的联盟，这些小组的专业知识从药物化学、Wnt信号转导和细胞生物学到结肠癌中Wnt信号失控的小鼠模型和转移疾病的临床试验。在之前的工作中，我们在基于细胞的实验中筛选了70,000个小分子类药物化合物的文库，以寻找WNT/TCF依赖的转录抑制物。在一系列去卷积分析之后，我们确定了四种小分子抑制物，它们阻止了肿瘤细胞的生长，似乎作用于Wnt信号转导级联中的不同点。利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抑制活性的一个可能的分子靶点。FET基因家族的成员已被证明与转录因子融合，是一系列人类肉瘤(如尤文斯肉瘤中的EWS)的主要启动癌基因，但EWS基因家族成员与Wnt信号转导之间的联系很少。作为案例学习的一部分，我们现在建议在Wnt信号的背景下研究这些化合物抑制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信号的分子过程，以及抗WNT信号药物改变EWS/WNT功能的机制。在生化水平上，我们将研究FET家族成员和Wnt信号通路组件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和Wnt信号蛋白b-catenin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。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信号组件的表达的能力。这两种技术在实验室中都得到了很好的证实，初步数据表明，EWS和FUS表达的RNAi减少降低了Wnt/TCF依赖的信号转导。从长远来看，缺失分析将被用来确定EWS家族成员在Wnt途径和药物结合中所需的功能区域，从而确定药物作用的机制。这项工作的成果将是对EWS家族成员在WNT信号中的作用的分子理解。这项工作将直接促进一系列可能的抗癌药物的开发，并将产生关于WNT-EWS途径相互作用的基础生物学的高质量信息，这些信息将有助于信号转导领域。

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.