Non-canonical Wnt/Dishevelled signaling and cancer cell malignancy

描述(申请人提供)：这项研究的总体目标是研究非典型Wnt信号通过DVL蛋白发挥作用的机制，并测试该途径调节癌细胞侵袭并与恶性乳腺癌相关的假说。规范的Wnt/？-catenin通路参与了许多癌症的发生，但最近的证据表明，非规范的、非规范的Wnt信号与恶性肿瘤的后期进展有关。该途径在细胞的极性和形态发生中也起着至关重要的作用。WNT诱导的DVL的磷酸化为非典型的Wnt信号提供了可靠的检测方法，也为分子解剖非典型途径及其在恶性肿瘤中的功能提供了一个起点。DVL磷酸化在Wnt信号中的功能意义将通过测试DVL突变体的特性来研究。我们还将测试非规范Wnt信号抑制ER阴性乳腺癌细胞侵袭的假设。这将通过细胞迁移和侵袭的体外分析以及信号分析来实现。DVL磷酸化在细胞迁移中的作用将被探索，我们将确定在这种情况下激活的非典范Wnt信号的下游成分。鉴于DVL蛋白在介导和调节Wnt信号中的关键作用，将利用共聚焦显微镜全面分析DVL在非规范Wnt信号作用下亚细胞分布的变化。将采用蛋白质组学的方法来确定DVL与之相互作用的蛋白质中的关键变化，这是非规范Wnt信号的结果。此外，还将产生转基因小鼠，其中非规范的Wnt信号将以受调控的方式在乳腺中被激活。将确定这种信号对乳腺发育的影响，并将小鼠与现有的乳腺癌小鼠模型杂交，直接测试非典型Wnt信号在肿瘤向浸润性和转移性癌症进展中的后果。由于有广泛的证据表明Wnt在癌症中异常表达，而且大多数Wnt蛋白可以引发非典型信号，因此这种鲜为人知的非典型Wnt信号通路可能具有广泛的医学意义。对所涉及的分子机制的表征将为出于治疗目的操纵该途径提供机会。

DESCRIPTION (provided by applicant): The overall objectives of this research are to investigate mechanisms of non-canonical Wnt signaling acting via Dishevelled (Dvl) protein, and to test the hypothesis that this pathway regulates cancer cell invasion and is relevant to malignant breast cancer. The canonical Wnt/?-catenin pathway contributes to the initiation of many cancers but recent evidence indicates that ?-catenin-independent, or non-canonical, Wnt signaling is relevant to later stages of malignant progression. This pathway also plays crucial roles in cell polarity and morphogenesis. Wnt-induced phosphorylation of Dvl provides a reliable assay for non-canonical Wnt signaling and a starting point for molecular dissection of the non-canonical pathway and its functions in malignancy. The functional significance of Dvl phosphorylation in Wnt signaling will be investigated by testing the properties of Dvl mutants that are resistant to Wnt-induced phosphorylation. We will also test the hypothesis that non-canonical Wnt signaling inhibits the invasion of ER-negative breast cancer cells. This will be achieved by in vitro assays of cell migration and invasion, in conjunction with signaling assays. The role of Dvl phosphorylation in cell migration will be explored, and we will identify downstream components of non-canonical Wnt signaling that are activated in this context. In view of the key role of Dvl protein in mediating and modulating Wnt signaling, a comprehensive analysis of changes in the subcellular distribution of Dvl in response to non-canonical Wnt signals will be performed by confocal microscopy. A proteomics approach will be taken to identify key changes in the proteins with which Dvl interacts as a result of non-canonical Wnt signals. In addition, transgenic mice will be generated in which non-canonical Wnt signaling will be activated in the mammary gland in a regulated manner. Effects of this signaling on mammary development will be determined and the mice will be crossed with existing mouse models of breast cancer to test directly the consequences of non-canonical Wnt signaling in tumor progression towards invasive and metastatic cancer. Since there is extensive evidence of aberrant Wnt expression in cancer, and most Wnt proteins can elicit non-canonical signals, it is likely that this poorly understood pathway of non-canonical Wnt signaling may be of widespread medical significance. Characterization of the molecular mechanisms involved will provide opportunities for manipulating the pathway for therapeutic purposes.