guanosine-diphosphate and Glioblastoma

Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-β2/Transportin (Kapβ2). SiRNA-directed Kapβ2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments.

Topics: Active Transport, Cell Nucleus; beta Karyopherins; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Central Nervous System Neoplasms; Cytosol; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glioblastoma; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Molecular Weight; ran GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Thionucleotides; Transfection

A mutant epidermal growth factor receptor (DeltaEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG. DeltaEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated DeltaEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.DeltaEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of DeltaEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Antibodies, Monoclonal; Cell Division; Cell Line; DNA, Neoplasm; ErbB Receptors; Glioblastoma; GRB2 Adaptor Protein; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Immunoglobulin G; Mice; Mice, Nude; Mutagenesis, Site-Directed; Phosphoproteins; Point Mutation; Proteins; ras Proteins; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Deletion; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured