title: Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures.
abstract: BACKGROUND
  Hedgehog (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 apoptosis in vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1 double mutant mice exhibit an increased incidence of MB compared to ptch1 mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis.
  
  
  METHODS
  Primary MB tumorsphere cultures were prepared from thirteen ptch1+/-/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 of gli1 and [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.
  
  
  RESULTS
  Primary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts. Gli1 gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation of gli1 gene 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.
  
  
  CONCLUSIONS
  Primary tumorspheres derived from ptch1+/-/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.
authors.affiliation: Semel Institute/Department of Psychiatry and Biobehavioral Sciences, David Geffen, School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 USA.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Cohen
authors.forename: Daniel Z
authors.initials: DZ
authors.lastname: Resnick
authors.forename: Pawel
authors.initials: P
authors.lastname: Niewiadomski
authors.forename: Hongmei
authors.initials: H
authors.lastname: Dong
authors.forename: Linda M
authors.initials: LM
authors.lastname: Liau
authors.forename: James A
authors.initials: JA
authors.lastname: Waschek
mesh_terms: D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002528:Cerebellar Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D020536:Enzyme Activators / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D053823:Hedgehog Proteins / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D008527:Medulloblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D009025:Morpholines / Q000494:pharmacology; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010879:Piperazines / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000072086:Smoothened Receptor; D018874:Spheroids, Cellular; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D000071676:Zinc Finger Protein GLI1
chemical_list: C495402:Adcyap1 protein, mouse; C495504:Adcyap1r1 protein, mouse; D020536:Enzyme Activators; C498520:Gli1 protein, mouse; D053823:Hedgehog Proteins; D007546:Isoquinolines; D051741:Kruppel-Like Transcription Factors; D009025:Morpholines; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010879:Piperazines; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D047428:Protein Kinase Inhibitors; C000606240:Ptch1 protein, mouse; D011687:Purines; D011720:Pyrazoles; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; C468919:SANT-1 compound; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D013449:Sulfonamides; D016159:Tumor Suppressor Protein p53; D000071676:Zinc Finger Protein GLI1; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C470893:purmorphamine