The Role of Lyn in Glioma Progression and Migration

描述（由申请人提供）：我们之前已经证明，结扎整合素avp3和PDGFr之间的合作促进了胶质母细胞瘤细胞的运动。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Lyn:(i)与间变性星形细胞瘤（III级）肿瘤活检和正常大脑相比，Lyn在胶质母细胞瘤（IV级）肿瘤活检中的活性更高；（“）尽管Fyn是胶质母细胞瘤细胞中主要表达的细胞Src家族成员，但Lyn被连接的整合素otvp3和PDGFr在胶质母细胞瘤细胞上的合作特异性激活；（Hi） Lyn是促进迁移所必需的，这与连接整合素avp3和PDGFr之间的合作有关。我的合作研究者Dan Flynn博士最近表明，细胞Src家族成员的n端可以在功能上区分细胞Src家族成员（c-Src和c-Yes）的信号传导中决定特异性。因此，我们假设Lyn的n端决定了区分Lyn和Fyn的信号的特异性，并且在胶质瘤活检样本中发现的体内Lyn水平升高促进了这些肿瘤的运动/侵袭特征，从而促进了胶质瘤的进展。我们还发现，连接的整合素avp3和PDGFr在胶质母细胞瘤细胞上的合作导致局灶黏着激酶（FAK）和CAS家族成员HEF1的磷酸化增加。因此，我们假设Lyn通过FAK/HEF1信号机制促进胶质母细胞瘤肿瘤的运动/侵袭特性。在这里，我们将使用Lyn、Lyn突变体和Lyn/Fyn嵌合体的稳定转染，以及siRNA技术：(1)通过连接整合素ctvp3和PDGFr的合作，确定Lyn中特异性激活所必需的结构域；(2)确定体内恶性胶质细胞迁移/侵袭是否需要Lyn，是否需要Lyn的氨基末端（SH4-Unique- sh3 - sh2或SH4-Unique结构域）来实现这一作用；3)确定FAK和下游效应物HEF1在体外连接素粘附（结扎整合素avp3）和pdgf刺激的胶质母细胞瘤细胞以及转染Lyn的SYF小鼠胚胎成纤维细胞的迁移中是否必要。该结果将引起除脑肿瘤以外的其他肿瘤的普遍关注，因为细胞Src家族成员活性的增加也可能促进非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.