Effect of the combination of retinoic acid and rapamycin on cerebellar granule cell and medulloblastoma proliferation

Abstract

2077 Background: Cerebellar granule cell precursors (CGCPs) are neuroblasts that proliferate in early postnatal life and may become transformed, giving rise to medulloblastoma. The proliferation of CGCPs is driven by mitogenic signals including Sonic Hedgehog (SHH), and growth factors that activate the protein mTOR. Dysregulation of these intercellular signals can promote medulloblastoma formation. We propose that microenvironmental signals that down-regulate the response of CGCPs to mitogens may inhibit medulloblastoma growth. Retinoic acid (RA) is an endogenous signaling molecule with potent anti-neoplastic effects. We investigated whether SHH, mTOR, and RA signaling pathways interact to regulate CGCP and medulloblastoma proliferation. Methods: We measured proliferation in cultured CGCP explants and the CGCP-derived murine medulloblastoma cell line PZp53 using quantitative phosphohistone-H3 immunocytochemistry. We examined the effects of adding to culture medium SHH, the mTOR inhibitor rapamycin, and all trans-RA (ATRA) in specific combinations. We compared CGCPs from wild type animals to CGCPs from mice with constitutive mTOR activation due to TSC2 mutation. Results: A minimum concentration of 1uM ATRA inhibited SHH-driven CGCP proliferation measurably but incompletely, while 10uM ATRA caused widespread necrosis. CGCPs from TSC2 mutant animals, in which mTOR was constitutively active, were 50% less effected by 1uM ATRA than wild type CGCPs. PZp53 medulloblastoma cells were relatively resistant to ATRA, tolerating 10uM ATRA with reduced but persistent proliferation. 10nM rapamycin decreased but did not eliminate PZp53 proliferation. The combination of rapamycin and ATRA, however, acted synergistically, suppressing proliferation >90%. This suppression persisted at 10-fold lower drug concentrations. Conclusions: CGCPs and CGCP-derived medulloblastoma cells integrate signals transduced by SHH, mTOR, and RA pathways. These signaling pathways can be manipulated by pharmacologic agents in combinations that confer dramatically enhanced antineoplastic effect. We are investigating the molecular basis of the synergy of rapamycin and ATRA. We plan to test the combination in xenografts and ultimately in patients with medulloblastoma. No significant financial relationships to disclose.