AbstractBackgroundHedgehog (HH) signaling is critical for the expansion of granule neuron precursors (GNPs) within the external granular layer (EGL) during cerebellar development. Aberrant HH signaling within GNPs is thought to give rise to medulloblastoma (MB) - the most commonly-observed form of malignant pediatric brain tumor. Evidence in both invertebrates and vertebrates indicates that cyclic AMP-dependent protein kinase A (PKA) antagonizes HH signalling. Receptors specific for the neuropeptide pituitary adenylyl cyclase activating polypeptide (PACAP, gene name ADCYAP1) are expressed in GNPs. PACAP has been shown to protect GNPs from apoptosisin vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1double mutant mice exhibit an increased incidence of MB compared toptch1mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis.MethodsPrimary MB tumorsphere cultures were prepared from thirteenptch1+/-/p53+/-double mutant mice and treated with the smoothened (SMO) agonist purmorphamine, the SMO antagonist SANT-1, the neuropeptide PACAP, the PKA activator forskolin, and the PKA inhibitor H89. Gene expression ofgli1and [3H]-thymidine incorporation were assessed to determine drug effects on HH pathway activity and proliferation, respectively. PKA activity was determined in cell extracts by Western blotting using a phospho-PKA substrate antibody.ResultsPrimary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts.Gli1gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation ofgli1gene expression by PACAP was reversed by the PKA inhibitor H89, which also blocked PKA activation. Treatment of tumorsphere cultures with PACAP, forskolin, and SANT-1 for 24 or 48 hours reduced proliferation.ConclusionsPrimary tumorspheres derived fromptch1+/-/p53+/-mice exhibit constitutive HH pathway activity. PACAP antagonizes HH signalling in these cells in a manner blocked by the PKA antagonist H89. PACAP and pharmacological activation of PKA also inhibited proliferation. Our data suggests that regulation of HH signaling by PACAP/PKA signaling may provide an alternative to SMO inhibition for the treatment of MB.