Lab-Cell Biology and Signaling

Abstract

Japan Glioblastoma (GBM), one of the most frequently occurring malignancies in the central nervous system, still has a very poor prognosis. To improve the prognosis of GBM patients, various attempts have been made. Immunotherapy targeting Wilms' tumor 1 (WT1) has proved to be effective in GBM (Izumoto et al. 2008). However, the functional roles of WT1 in GBM have not been intensively studied. In this study, we aim to examine the functional roles of WT1 in GBM. We established WT1 shRNA knocked down GBM cell lines (U87 and U251) and examined the functional roles in vitro and in vivo. In cell-proliferation assays, we plated 3 × 10 4 cells in 12-well plates. On day 4 the numbers of the proliferated cells were (39 + 7.8) ×10 4 cells in the U87 control shRNA, U87 WT1 shRNA, U251 control shRNA, and U251 WT1 shRNA, respectively (P , 0.05). Furthermore, an Annexin V apoptosis assay showed the numbers of the apoptotic cells per 30 times magnified microscopic field were 2.5 + 0.6, 15.3 + 5.7, 3.0 + 1.7, and 9.0 + 1.0 cells in the U87 control shRNA, U87 WT1 shRNA, U251 control shRNA, and U251 WT1 shRNA, respectively (P , 0.05). In the in vivo experiment, U87 control shRNA and U87 WT1 shRNA cells were intracranially injected into newborn pups of immunodeficient mice. At day 30, all of the mice transplanted with U87MG control shRNA developed GBM, whereas none of the mice transplanted with U87MG WT1 shRNA developed GBM. These results suggest that WT1 is involved in GBM cell proliferation, apoptosis, and tumor formation. Malignant gliomas are highly invasive and chemoresistant brain tumors with extremely poor prognosis. Glioma invasion is strongly associated with the resistance of these tumors to therapy, but the mechanisms that underlie this association are poorly understood. Targeting soluble factors triggering invasion and resistance could substantially affect the difficult-to-reach, infiltrative glioma cells that are a major source of recurrence. Fibulin-3, a matrix protein absent in normal brain tissue but upregulated in gliomas, promotes tumor invasion by unknown mechanisms. We show here that fibulin-3 is a novel soluble activator of Notch signaling that antagonizes DLL3, an autocrine inhibitor of Notch, and promotes tumor-cell survival and invasion in a Notch-dependent manner. Using a strategy for inducible knockdown, we demonstrate that controlled downregulation of fibulin-3 reduces Notch signaling and leads to increased apoptosis, reduced self-renewal of glioblastoma-initiating cells, and impaired growth and dispersion of intracranial tumors. Finally, we show that fibulin-3 expression correlates with expression levels of Notch-dependent genes (Hes1, Hes5) and is a marker of Notch activation in clinical gliomas. These results underscore a major role of the tumor extracellular matrix in regulating glioma invasion and resistance to apoptosis via activation of the key Notch pathway. More importantly, this is the first description of a noncanonical, soluble activator of Notch in a cancer model and a demonstration of how Not...