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Open Access Research article

Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures

Joseph R Cohen12, Daniel Z Resnick13, Pawel Niewiadomski14, Hongmei Dong1, Linda M Liau56 and James A Waschek15*

Author Affiliations

1 Semel Institute/Department of Psychiatry and Biobehavioral Sciences, David Geffen, School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 USA

2 Graduate Neuroscience Interdepartmental Program, University of California at Los Angeles, Los Angeles, CA 90095 USA

3 University of California at Berkeley, Berkeley, CA 94720 USA

4 Division of Oncology, Stanford University School of Medicine, Palo Alto, CA 94305 USA

5 Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 USA

6 Department of Neurosurgery, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 USA

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BMC Cancer 2010, 10:676  doi:10.1186/1471-2407-10-676

Published: 9 December 2010

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.