The Role of Lyn in Glioma Progression and Migration

描述(申请人提供)：我们之前已经证明，连接的整合素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。我们的初步数据提示：(I)Lyn在胶质母细胞瘤(IV级)肿瘤活检组织中的活性高于间变性星形细胞瘤(III级)肿瘤活检组织和正常脑组织；(“)Lyn通过连接的整合素otvp3和PDGFR在胶质母细胞瘤细胞上特异性地激活，尽管Fyn是这些细胞表达的主要细胞Src家族成员；并且(Hi)Lyn对于促进与连接的整合素avp3和PDGFR之间的合作相关的迁移是必需的。我的合作者Dan Flynn博士最近表明，细胞Src家族成员的N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，并且在胶质瘤活组织标本中体内发现的高水平的LYN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协同作用导致粘着斑激酶(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技术来：(1)确定Lyn中通过连接的整合素ctwp3和PDGFR的协作而特异性激活Lyn所必需的结构域；(2)确定Lyn是否是恶性胶质细胞在体内迁移/侵袭所必需的，以及这种作用是否需要Lyn的氨基末端(SH4-唯一-SH3-SH2或SH4-唯一域)；3)确定FAK及其下游效应分子HEF1是否对玻璃连结蛋白黏附分子(连接整合素avp3)和PDGF刺激的胶质母细胞瘤细胞以及转导Lyn的SYF小鼠胚胎成纤维细胞的迁移是必需的。这一结果将引起人们的普遍关注，即脑肿瘤以外的其他肿瘤，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.