The Role of Lyn in Glioma Progression and Migration

描述（由申请人提供）：我们先前已经表明，连接的整联蛋白avp 3和PDGFr之间的合作促进胶质母细胞瘤细胞的运动性。在这里，我们建议分析与这种反应相关的信号机制。我们的初步数据暗示细胞Src家族成员，林恩：（i）林恩的活性在胶质母细胞瘤中更高（IV级）肿瘤活检，与间变性星形细胞瘤相比（“）林恩通过胶质母细胞瘤细胞上连接的整联蛋白α tvp 3和PDGFr的协同作用而特异性活化，尽管Fyn是在这些细胞中表达的主要细胞Src家族成员;和（iii）林恩对于促进与连接的整联蛋白α ν β 3和PDGFr之间的合作相关的迁移是必需的。我的合作研究者Dan Flynn博士最近表明，细胞Src家族成员的N-末端可以决定信号的特异性，从而在功能上区分细胞Src家族成员（c-Src和c-Yes）。因此，我们假设林恩的N-末端决定了区分林恩和Fyn的信号传导的特异性，并且在胶质瘤肿瘤活检样品中体内发现的林恩水平升高促进了这些肿瘤的运动性/侵袭性特征，从而促进了胶质瘤的进展。我们还发现，胶质母细胞瘤细胞上的连接的整合素avp 3和PDGFr的合作导致粘着斑激酶（FAK）和HEF 1（CAS家族成员）的磷酸化增加。因此，我们假设林恩通过FAK/HEF 1信号传导机制促进胶质母细胞瘤肿瘤的运动/侵袭特征。在此，我们将使用稳定转染林恩、林恩突变体和林恩/Fyn嵌合体，以及siRNA技术：（1）确定林恩中通过连接的整合素ctvp 3和PDGFr的协同作用特异性激活其所必需的结构域;（2）确定林恩是否是体内恶性胶质细胞迁移/侵袭所必需的，以及林恩的氨基末端（SH 4-独特-SH 3-SH 2或SH 4-独特结构域）是该效应所必需的;和3）确定FAK和下游效应物HEF 1是否是粘附玻连蛋白的细胞迁移所必需的。（连接的整联蛋白avp 3）和PDGF刺激的胶质母细胞瘤细胞，以及用林恩转染的SYF小鼠胚胎成纤维细胞。结果将是普遍感兴趣的，即，神经胶质瘤也可能是脑肿瘤以外的其他肿瘤，因为增加的细胞Src家族成员活性也可能促进非神经胶质瘤肿瘤的进展和转移。

DESCRIPTION (provided by applicant): We have shown previously that cooperation between ligated integrin avp3 and the PDGFr promotes the motility of glioblastoma cells. Here, we propose to analyze the signaling mechanisms associated with this response. Our preliminary data implicate the cellular Src family member, Lyn: (i) The activity of Lyn is higher in glioblastoma (Grade IV) tumor biopsies, as compared to anaplastic astrocytoma (Grade III) tumor biopsies and normal brain; (") Lyn is specifically activated by the cooperation of ligated integrin otvp3 and the PDGFr on glioblastoma cells, although Fyn is the predominant cellular Src family member expressed in these cells; and (Hi) Lyn is necessary for the promotion of migration associated with the cooperation between ligated integrin avp3 and the PDGFr. My coinvestigator, Dr. Dan Flynn, has shown recently that the N-terminus of a cellular Src family member can dictate specificity in signaling that functionally differentiates cellular Src family members (c-Src and c-Yes). Thus, we hypothesize that the N-terminus of Lyn dictates the specificity in signaling that differentiates Lyn from Fyn and that the elevated levels of Lyn found in vivo in glioma tumor biopsy samples promote the motility/invasion characteristics of these tumors, thereby contributing to slioma progression. We also find that the cooperation of ligated integrin avp3 and the PDGFr on glioblastoma cells results in increased phosphorylation of focal adhesion kinase (FAK) and of HEF1, a CAS family member. Thus, we hypothesize that Lyn promotes the motility/invasion characteristic of the glioblastoma tumors through a FAK/HEF1 signaling mechanism. Here, we will use stable transfection with Lyn, Lyn mutants, and Lyn/Fyn chimeras, as well as siRNA technology to: (1) Determine the domains in Lyn that are necessary for its specific activation by the cooperation of ligated integrin ctvp3 and the PDGFr; (2) Determine whether Lyn is necessary for malignant glial cell migration/invasion in vivo, and whether the amino-terminus of Lyn (SH4-Unique-SH3-SH2 or SH4-Unique domains) is required for this effect; and 3) Determine whether FAK and the downstream effector, HEF1, are necessary for migration of vitronectin-adherent (ligated integrin avp3) and PDGF-stimulated glioblastoma cells, and SYF mouse embryo fibroblasts transfected with Lyn. The results will be of general interest, i.e., tumors other than brain tumors, as increased cellular Src family member activity also likely promotes the progression and metastasis of non-glioma tumors.