Medulloblastoma (MB) is a malignant pediatric brain tumor that is thought to arise in many cases from transformation of granule neuron precursors (GNPs) within the external granular layer (EGL) of the developing cerebellum reviewed in . Medulloblastoma is classified into the following subtypes based on their phenotypic  and genetic characteristics : classic, large cell, anaplastic, desmoplastic and MB with extensive nodularity. Analyses of human desmoplastic MB samples revealed altered activity of, and/or mutations in, molecules of the sonic hedgehog (HH) signaling pathway including patched-1 (ptch1) and glioma-associated oncogene homolog 1 (gli-1) [1, 3–5]. HH signaling is pivotal in the regulation of CNS patterning and development [6–8] stem cell self-renewal  and MB pathogenesis [4, 5, 10], and has also been implicated in a variety of additional human cancers including lung, pancreatic, and prostate reviewed in . Mutations in ptch1 (the gene that codes for the Patched-1 12 pass transmembrane protein, a negative regulator of the HH signaling cascade), are observed with Gorlin's (Basal Cell Nevus) Syndrome - an autosomal hereditary disease characterized by the development of MB, basal cell carcinomas, and rhabdomyosarcoma [12, 13]. Ptch1
-/- mice die in utero while ptch1
mice typically survive but develop cerebellar tumors closely resembling MB in about 15% of the cases .
Our present understanding of the HH signaling cascade suggests that binding of the hedgehog ligands to PTCH-1 de-represses smoothened (SMO), allowing for the cascade of events leading to the activation of cubitus interruptus (Ci) in flies and the Gli transcription factors in vertebrates reviewed in [14, 17]. The Gli transcription factors bind to the promoters of several genes considered to be HH targets (including gli1 and ptch1) [16–19]. Presently, molecules that act directly on components of the canonical HH pathway, including several that target SMO, are in various stages of clinical trials . On the other hand, the protein kinase A (PKA) signalling pathway is known to antagonize hedgehog (HH) signaling in both invertebrates  and vertebrates [22, 23], suggesting an alternative approach to blocking overactive HH activity. Nonetheless, little is know about the significance of the PKA/HH interaction in the pathogenesis of MB and other tumors.
PACAP is a 38-amino-acid peptide originally identified on the basis of its ability to induce production of cAMP in pituitary cells . PACAP regulates a variety of biological functions including neuroblast proliferation [25, 26], and neuroblast survival . In the developing brain PACAP binds to, and signals through, the G-protein coupled receptor PAC1, increasing cAMP production and PKA activity . PAC1 is also coupled to other signaling cascades in some cells, including phospholipase C (PLC) , phosphatidylinositol 3-Kinase (PI3-K) , src , and the MAP kinase pathways [32–34].
Gene transcripts for the HH targets genes ptch1 and gli1 are expressed within the EGL in the developing rat and mouse cerebella [26, 35], indicating that the HH pathway is active in these cells. PAC1 gene transcripts are also present in the EGL , suggesting that the PACAP and HH pathways might interact within the proliferating cells in this layer. Moreover, the robust proliferative action of SHH on cultured GNPs was completely blocked by either treatment with PACAP or pharmacological induction of cAMP/PKA . The potential significance of a PACAP/HH interaction in MB was recently demonstrated. First, PAC1 gene transcripts were found to be abundantly expressed in MB preneoplastic lesions in ptch1 mutant mice . Second, deletion of a single copy of PACAP in ptch1
mutant mice was found to increase the incidence of MB approximately 2.5 fold . Taken together, these data suggest that PACAP may regulate HH signaling in MB pathogenesis. In this work we investigate the interaction between HH and PACAP/PKA signaling in murine primary tumorspheres derived from MB tumors arising in ptch1
double mutant mice. Our data suggests that regulation of HH signaling by PACAP/PKA may provide a novel alternative to SMO inhibition for the treatment of medulloblastoma (MB).