Targeting Cilia Initiation & Maintenance in SHH-Medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Of the four molecular subtypes, Sonic Hedgehog Medulloblastoma (SHH-MB) involves aberrant growth signaling between SHH mitogen and its receptors on primary cilium in cerebellar granule cells. We hypothesized that in mouse models genetically engineered to induce SHH-MB, additional genetic modifications dysregulating primary cilium would inhibit the cancer phenotype, either decreasing severity or preventing it altogether. We established breeding cages with conditional knockouts of Gpr161 (GPR161 normally represses SHH signaling, knockout promotes SHH-MB), knockouts of Ptch1 (a SHH receptor with more aggressive tumors upon haploinsufficiency), and Pcm1 knockouts (centriolar satellite protein critical for primary cilia stability). Our methods included: PCR and gel electrophoresis to assess genotype, BrdU injections, cerebellum dissection, cryo-sectioning, antibody staining, and immunofluorescence imaging using confocal microscopy. We immunostained cerebellar tissues from various genotypes with cell cycle (CyclinD1), primary cilia (ARL13B), mitotic (pHH3), and proliferative markers (BrdU). We found that mice with Gpr161 or Ptch1 deletion develop SHH-MB and "persistent" external granular layers with increased cilia density versus controls. There is also a large presence of BrdU (-) and CyclinD1 (-) cells with cilia, begging the question of where in the cell cycle these cells belong. Heterozygous deletion of Pcm1 (+/ko) does not appear to diminish cancer phenotype, as a Ptch1 +/ko; Pcm1 +/ko mouse demonstrated SHH-MB and increased cilia density. A full knockout of Pcm1, however, does seem to rescue the cancer phenotype, as a Gpr161 f/f; Pcm1 ko/ko; Nestin-Cre mouse did not have any tumor, and it had low cilia density. We demonstrate that in mice with SHH-MB-predisposing genetic mutations, additional mutation of proteins regulating stability of primary cilia might prevent cancer phenotype. These results could open an avenue for investigating therapeutic inhibition or downregulation of cerebellar primary cilia in human patients with genetic predisposition for SHH-MB.