Autocrine signaling through Ras regulates cell survival activity in human glioma cells: a potential crosstalk between Ras and PI3K-Akt pathway

K Sakata, MD,a, S Kato, MD,a, JC Fox,MD,c, M Shigemori,MD,b, M Morimatsu,MD,a.

a Department of Pathology, b Department of Neurosurgery, Kurume University, School of Medicine, Kurume, Fukuoka-ken Japan, c Merck & Co. Blue Bell, PA

AIM: Autocrine fibroblast growth factor (FGF) signaling mediates an uncontrollable growth of human gliomas. We investigated the downstream signaling of FGF receptor through Ras on the cell survival activity. METHODS: U251MG human glioma cells were infected with adenovirus vectors expressing dominant negative type I FGF receptor (DNFR), constitutive active Ras (RasL61), or dominant negative Ras (RasN17). Proliferation was assessed by hand counting and BrdU incorporation. Apoptosis was detected with in situ TUNEL staining. Expression of transgenes and Akt and MAPK activities were measured by Western blotting. RESULTS: DNFR prevented glioma cell accumulation with apoptosis, which was alleviated by exogenous EGF (can activate Ras in dependent of FGFR) but not bFGF. RasL61 prevented and RasN17 enhanced DNFR-induced apoptosis. In U251 cells, cell survival signaling through Akt was constitutively active, which may be dependent on autocrine signaling and dysfunction of PTEN, a tumor suppresser gene limiting PI3K activity. DNFR dose-dependently inhibited Akt-activity and this inhibition was recovered by RasL61, whereas RasN17 inhibited Akt activity. Wortmannin (PI3K inhibitor) inhibited Akt activity and mildly promoted apoptosis. RasL61 prevented the down-regulation of Akt activity and apoptosis induced by wortmannin. RasN17 plus wortmannin strongly inhibited Akt activity and promoted marked apoptosis. PD98059 (MEK inhibitor) inhibited auto-growth of U251 cells without affecting apoptosis. CONCLUSIONS: Our data suggested that cell survival activity of human gliomas is largely depend on a crosstalk between Ras and PI3K-Akt pathway. Ras may play a central role in the tumor-promoting autocrine signaling expected as a potential target for molecular-based therapeutics.

KEY WORDS: brain tumor, signal transduction, apoptosis, adenovirus vector.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Apoptosis - Molecular Mechanisms.

This presentation received an honorable mention in our poster contest and was recognized with the Symposium Presidents' Award for Scientific Excellence.