Axin-induced Wnt signaling in mouse embryogenesis and Wnt-related cancers

描述(由申请人提供)：规范的Wnt信号通路对于大多数器官系统的发育和成人干细胞种群的维持都是必需的。异常信号可以在几个组织中驱动肿瘤的形成。Axin蛋白被认为是Wnt途径的负调控蛋白，因为它们在破坏复合体中的功能，在没有Wnt配体的情况下阻止信号传递。令人惊讶的是，通过遗传或药物方法稳定Axin蛋白会导致e8.5小鼠胚胎后部的信号增加，而Wnt信号在头部减少，这表明Wnt信号是以组织特异性的方式调节的。这项拟议研究的目标是了解Axin1和Axin2的积极和消极作用是如何在发育过程中受到组织特异性调控的。具体地说，这项研究的目的是描述在原始条纹中控制组织特异性的Axin刺激的Wnt信号的机制，鉴定Axin刺激的Wnt信号的生化基础，并检查稳定的Axin2是否促进肠道和乳腺的肿瘤发生。利用遗传学和基于细胞的方法，将识别在原始条纹中建立轴蛋白刺激的Wnt信号的蛋白质。高水平WNT的作用 我们将研究在小鼠胚胎中表达的配体、核心信号成分的表达、该途径潜在调节物的遗传相互作用，以及在这个时间和位置表达的交叉信号通路，以确定它们对Axin刺激的Wnt信号的贡献。这些数据将被用来概括Axin诱导的ES细胞中的Wnt信号。为了便于含Axin复合体的生化分析和含Axin复合体的细胞定位成像，将产生一只表达来自rosa26基因的有条件标记的、稳定的Axin1的小鼠。对同时参与Axin抑制和Axin诱导的Wnt信号的ES细胞或组织中含有Axin的复合体的免疫纯化将通过Western印迹来比较核心通路组件的结合。将进行质谱分析，以确定独特的蛋白质或翻译后修饰是否会对稳定的Axin产生不同的信号结果。最后，纸巾 在成人中，将评估Wnt信号对稳定的Axin2的反应增加，集中在由Wnt控制的干细胞利基维持的种群。此外，还将评估稳定的Axin2对两种WNT相关癌症模型--结直肠癌的Apcmin模型和乳腺癌的MMTV-WNT1模型的影响，重点是肿瘤的发生率、发病或侵袭性，以确定某些肿瘤是否由于稳定的Axin蛋白而加重，解决稳定Axin药物在癌症治疗中的作用。

DESCRIPTION (provided by applicant): The canonical Wnt signaling pathway is required for the development of most organ systems and for the maintenance of stem cell populations in the adult. Aberrant signaling can drive the formation of tumors in several tissues. Axin proteins are considered negative regulators of the Wnt pathway due to their function in the destruction complex, which prevents signaling in the absence of Wnt ligand. Surprisingly, stabilization of Axin proteins by either genetic or pharmacological methods leads to an increase in signaling in the posterior of the e8.5 mouse embryo, whereas Wnt signaling is decreased in the head, suggesting that Wnt signaling is regulated in a tissue-specific manner. The goals of the proposed research are to understand how the positive and negative roles of Axin1 and Axin2 are tissue-specifically regulated during development. Specifically, the aims of this research are the characterization of the mechanisms controlling tissue-specific, Axin-stimulated Wnt signaling in the primitive streak, identification of the biochemical basis of Axin-stimulated Wnt signaling, and examination of whether stabilized Axin2 promotes tumorigenesis in the intestine and mammary gland. Using genetics and cell-based approaches, the proteins that establish Axin-stimulated Wnt signaling in the primitive streak will be identified. The role of high levels of Wnt ligand, the expression of core signaling components, the genetic interaction of potential modulators of the pathway, and intersecting signaling pathways expressed at this time and location in the mouse embryo will be examined for their contribution to Axin-stimulated Wnt signaling. These data will be used to recapitulate Axin-induced Wnt signaling in ES cells. To facilitate biochemical analysis of Axin-containing complexes and imaging of the cellular localization of Axin- containing complexes, a mouse that expresses a conditional tagged, stabilized Axin1 from the Rosa26 locus will be generated. Immunopurification of Axin-containing complexes from ES cells or tissues engaged in both Axin-inhibited and Axin-induced Wnt signaling will be assessed by Western blot to compare the binding of core pathway components. Mass spectrometry will be performed to identify whether unique proteins or post- translational modifications lead to different signaling outcomes in response to stabilized Axin. Finally, tissues in adults will be evaluated for increased Wnt signaling in response to stabilized Axin2, concentrating on populations maintained by Wnt-controlled stem cell niches. Additionally, the effect of stabilized Axin2 on two models of Wnt-related cancer, the Apcmin model of colorectal cancer and the MMTV-Wnt1 model of breast cancer, will be assessed, focusing on the incidence, onset, or invasiveness of the tumors that arise to determine whether some tumors increase in severity due to stabilized Axin proteins, addressing the utility of Axin-stabilizing drugs in cancer treatment.