Role and Mechanism of HER2 Self-Association in Cancer

描述（由申请人提供）：在一系列癌症中发现了HER2（一种受体酪氨酸激酶（RTK））的过表达，最值得注意的是30%的乳腺癌和10%的卵巢癌。受体水平升高与组成型受体活化、细胞转化和临床环境中患者的较差存活相关。已知RTK的活化涉及二聚化。HER2具有有效的胞质激酶结构域，但本身不能结合任何配体，与其他RTK异二聚化，优先与激酶缺陷但配体结合的HER3异二聚化。异二聚体的优选配体是调蛋白。然而，在升高的水平下，全长和膜结合的HER2可以独立地自缔合配体。配体非依赖性自缔合相对于配体依赖性异源二聚化总体增加的相对贡献是HER2过表达癌症的一个未解决的问题。我们工作的一个关键目标是在分子水平上剖析这两个反应。为此，我们将鉴定并突变HER2的自缔合界面。我们最近发现，无催化活性的HER3及其可溶性细胞外结构域（ECD）在低浓度下自缔合，这是一种被配体结合破坏的反应。我们将使用HER3的强自缔合及其与HER2的高同源性来鉴定两种受体中的自缔合位点。这种鉴定依赖于通过3D聚类分析以及替代诱变和选择方法的计算预测。我们将应用FRET和DHFR片段互补，以获得在完整的细胞受体协会的程度的直接测量。我们将使用这些方法来确认在HER3和HER2中鉴定的区域，并评估HER2的浓度依赖性受体自缔合和组成性信号传导与肿瘤形成增强之间的相关性。此外，我们有证据表明，HER3的自我关联可能会调节heregulin信号转导。我们将评估HER3自我缔合的可能调节作用及其对HER2的配体依赖性和非依赖性信号传导的影响。更好地理解特定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，有助于理解和改善赫赛汀等药物的作用，并确定特定和可测量的相关事件作为药物开发的靶点。

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.