Gliomas are the most common adult primary brain tumors, and the most malignant form, glioblastoma multiforme, is invariably fatal. The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is altered in most glioblastoma multiforme. PTEN, an important negative regulator of the PI3K-Akt pathway, is also commonly mutated in glioma, leading to constitutive activation of Akt. One ultimate consequence is phosphorylation and inactivation of FOXO forkhead transcription factors that regulate genes involved in apoptosis, cell cycle arrest, nutrient availability, DNA repair, stress, and angiogenesis. We tested the ability of a mutant FOXO1 factor that is not subject to Akt phosphorylation to overcome dysregulated PI3K-Akt signaling in two PTEN-null glioma cell lines, U87 and U251. Adenovirus-mediated gene transfer of the mutant FOXO1 successfully restored cell cycle arrest and induced cell death in vitro and prolonged survival in vivo in xenograft models of human glioma (33% survival at 1 year of animals bearing U251 tumors). However, U87 were much more resistant than U251 to mutant FOXO1-induced death, showing evidence of increased nuclear export and Aktindependent phosphorylation of FOXO1 at S249. A cyclindependent kinase 2 inhibitor decreased phosphorylation of S249 and rendered U87 cells significantly more susceptible to mutant FOXO1-induced death. Our results indicate that targeting FOXO1, which is at the convergence point of several growth factor receptor tyrosine kinase pathways, can effectively induce glioma cell death and inhibit tumor growth. They also highlight the importance of Akt-independent phosphorylation events in the nuclear export of FOXO1. [Cancer Res 2009;69(13):5433-40]
Background: Cell migration plays a key role in tumor invasion and metastasis. Deleted in colorectal cancer (DCC) and the unc5 homologues are receptors for secreted netrins that regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Altered netrin and netrin receptor expression has been reported in aggressive tumors, including glioblastoma. Here, we investigated the involvement of netrin and netrin receptors in glioma cell migration and tumor formation using rat C6 glioma, a counterpart to human glioblastoma multiforme. Methods: We used Western blot analysis and RT-PCR to characterize netrin and netrin receptor expression in rat C6 glioma. C6 motility was assessed using a transfilter chemotaxis assay. To study the effect of ectopic DCC expression on C6 glioma cells, we determined the in vivo growth of three different cell populations after intracerebral implantation in rats. Three-dimensional reconstruction of tumors was performed to calculate tumor volume. Results: We determined that rat C6 glioma cells express netrin-1, netrin-3, and the netrin receptor unc5B, but not DCC.Using transfilter migration assays we demonstrated that C6 cells migrate away from a source of netrin-1, consistent with chemorepulsion signaled by Unc5B. In the absence of a gradient of netrin, disrupting netrin protein secreted by these cells reduced C6 cell motility, suggesting that autocrine netrin promotes C6 cell motility in this assay. Ectopic expression of DCC reduced the rate of migration in the absence of any gradient, and reduced the rate of directional cell migration up a gradient of laminin-1. Intracerebral implantation of glioma cells engineered to express full-length DCC (pCEP4-DCC) resulted in reduced tumor volumes and sharp borders between tumor and brain tissue, compared to tumors arising from parental or pCEP4 vector-transfected cells. Conclusions: These results provide evidence that netrin and Unc5B may promote cell motility by weakening interactions between tumor cells and that loss of DCC promotes growth and invasion of rat glioma.
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