Role and Mechanism of HER2 Self-Association in Cancer

描述（由申请人提供）：HER2，一种酪氨酸激酶受体（RTK）的过表达在一系列癌症中被发现，最明显的是，30%的乳腺癌和10%的卵巢癌。该受体水平升高与组成型受体激活、细胞转化和临床患者较差的生存率有关。已知rtk的激活涉及二聚化。HER2具有强大的细胞质激酶结构域，但自身不能结合任何配体，它与其他rtk异二聚，优先与激酶缺陷但与配体结合的HER3结合。异二聚体的首选配体是调节蛋白。然而，在高水平时，全长和膜结合的HER2可以独立地自结合配体。对于HER2过表达的癌症来说，不依赖于配体的自我结合与依赖于配体的异源二聚化的总体增加的相对贡献是一个尚未解决的问题。我们工作的一个关键目标是在分子水平上剖析这两种反应。为此，我们将识别并突变HER2的自关联接口。我们最近发现，催化活性不高的HER3及其可溶性细胞外结构域（ECD）在低浓度下自结合，该反应被配体结合破坏。我们将利用HER3的强自结合及其与HER2的高度同源性来确定这两种受体的自结合位点。这种鉴定依赖于三维聚类分析的计算预测以及替代诱变和选择方法。我们将应用FRET和DHFR片段互补来获得完整细胞中受体关联程度的直接测量。我们将使用这些方法来确认HER3和HER2中已识别的区域，并评估HER2的浓度依赖性受体自关联与组成信号和肿瘤形成增强之间的相关性。此外，我们有证据表明HER3自结合可能调节heregulin信号传导。我们将评估HER3自结合可能的调节作用及其对HER2依赖配体和独立信号传导的影响。更好地了解特异性受体在高表达水平下的相互作用将增强我们对HER2细胞转化的理解，有助于理解和改进赫赛汀等药物的作用，并确定特异性和可测量的关联事件作为药物开发的靶标。

DESCRIPTION (provided by applicant): Overexpression of HER2, a receptor tyrosine kinase (RTK) is found in a series of cancers, most notably, 30% of breast cancers and 10% of ovarian cancers. Elevated levels of the receptor are associated with constitutive receptor activation, cellular transformation, and poorer survival of patients in a clinical setting. Activation of RTKs is known to involve dimerization. HER2, which has a potent cytoplasmic kinase domain but fails to bind any ligand by itself, heterodimerizes with other RTKs, preferentially with the kinase-deficient but ligand-binding HER3. The preferred ligand for the heterodimer is heregulin. However, at elevated levels, full-length and membrane-bound HER2 can self-associate ligand independently. The relative contribution of ligand-independent self-association versus an overall increase in ligand-dependent heterodimerization is an unresolved question for HER2 overexpressing cancers. A key objective of our work is to dissect those two reactions on a molecular level. To this end, we will identify and mutate the self association interface of HER2. We found recently that the catalytically inactive HER3, as well as its soluble extracellular domain (ECD), self-associates at low concentrations, a reaction that is disrupted by ligand binding. We will use the strong self-association of HER3 and its high homology with HER2 to identify self association sites in both receptors. This identification relies on computational predictions by 3D cluster analysis as well as alternative mutagenesis and selection approaches. We will apply FRET and DHFR fragment complementation to obtain direct measurements of the extent of receptor association in intact cells. We will use these methods to confirm the identified regions in HER3 and HER2 and evaluate the correlation between the concentration-dependent receptor self-association of HER2 and constitutive signaling and the enhancement of tumor formation. In addition we have evidence that HER3 self-association may modulate heregulin signaling. We will evaluate the possible regulatory role of HER3 self-association and its impact on ligand-dependent and independent signaling by HER2. A better understanding of the specific receptor interactions at elevated expression levels will enhance our understanding of cellular transformation by HER2, help understand and improve the action of drugs like Herceptin, and identify specific and measurable association events as targets for drug development.