Objective: Medulloblastoma is the most common malignant brain tumor in children. Survival rates are below 50% in patients with metastatic tumors, and close to 0% in those who relapse with metastatic tumors compared to greater than 80% in patients without tumor metastasis. We previously showed that overexpression of the platelet-derived growth factor receptor (PDGFR) correlates with metastatic medulloblastoma. Upon binding to the ligand PDGF, PDGFR, a receptor tyrosine kinase (RTK), undergoes autophosphorylation which in turn initiates signal transduction through the RAS/MAPK and PI3K-AKT pathways leading to cell proliferation, migration, survival and gene transcription.

Methods: To determine whether PDGFR signaling is critical to these processes in medulloblastoma cells, we inhibited the PDGFR expression and activity in Daoy human medulloblastoma cells using three separate methods: 1) small interfering RNA (siRNA) to PDGFR; 2) RTK inhibition of PDGFR with Gleevec; and 3) anti-PDGF neutralizing antibodies, and measured the resultant changes in gene expression and survival following PDGF stimulation. These changes were compared to baseline serum-depleted cells, with and without PDGFR inhibition, stimulated with PDGF.

Results: PDGFR-active cells stimulated with PDGF showed enhanced survival and proliferation and exhibited gene expression changes at 24h, as measured by Affymetrix microarray profiling, that were specific to the subtype of ligand (PDGFAA or PDGFBB) used for stimulation. PDGFBB, which activates both the alpha and the beta receptor subtypes, was more potent than the PDGFAA, which only activates the alpha receptor subtype of PDGFR. PDGFBB induced gene expression encoding effectors that primarily regulate signal transduction and cell-cycle control, while PDGFAA enduced gene expression encoding effectors that predominantly affect chemotaxis. Following Gleevec treatment, both PDGFAA and BB up-regulated expression of PI3K and induced Akt phosphorylation in a portion of surviving cells, suggesting a bypass pathway to overcome the effect of PDGFR tyrosine kinase inhibition. PDGF neutralizing antibodies and transient siRNA transfection abrogated the gene expression changes and subsequent survival, indicating that this effect is dependent on the presence of PDGFR and its interaction with PDGF.

Conclusion: These results suggest that PDGFR is critical for medulloblastoma survival through a signal transduction mechanism that promotes pro-survival gene transcription. In addition, physical interaction of the receptor in the membrane seems to exert a significant effect on PDGFR-induced gene expression because only siRNA silencing was able to abrogate this PDGFR-induced gene expression. Thus, a fully intact PDGFR appears to be important for maintaining signal transduction, gene expression, survival and migration in medulloblastoma cells.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA