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.
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