The p53 inhibitor MDM2 facilitates Sonic Hedgehog-mediated tumorigenesis and influences cerebellar foliation

Disruption of cerebellar granular neuronal precursor (GNP) maturation can result in defects in motor coordination and learning, or in medulloblastoma, the most common childhood brain tumor. The Sonic Hedgehog (Shh) pathway is important for GNP proliferation; however, the factors regulating the extent and timing of GNP proliferation, as well as GNP differentiation and migration are poorly understood. The p53 tumor suppressor has been shown to negatively regulate the activity of the Shh effector, Gli1, in neural stem cells; however, the contribution of p53 to the regulation of Shh signaling in GNPs during cerebellar development has not been determined. Here, we exploited a hypomorphic allele of Mdm2 (Mdm2(puro)), which encodes a critical negative regulator of p53, to alter the level of wild-type MDM2 and p53 in vivo. We report that mice with reduced levels of MDM2 and increased levels of p53 have small cerebella with shortened folia, reminiscent of deficient Shh signaling. Indeed, Shh signaling in Mdm2-deficient GNPs is attenuated, concomitant with decreased expression of the Shh transducers, Gli1 and Gli2. We also find that Shh stimulation of GNPs promotes MDM2 accumulation and enhances phosphorylation at serine 166, a modification known to increase MDM2-p53 binding. Significantly, loss of MDM2 in Ptch1(+/-) mice, a model for Shh-mediated human medulloblastoma, impedes cerebellar tumorigenesis. Together, these results place MDM2 at a major nexus between the p53 and Shh signaling pathways in GNPs, with key roles in cerebellar development, GNP survival, cerebellar foliation, and MB tumorigenesis.     

Sonic Hedgehog-Induced Histone Deacetylase Activation Is Required for Cerebellar Granule Precursor Hyperplasia in Medulloblastoma

Medulloblastoma, the most common pediatric brain tumor, is thought to arise from deregulated proliferation of cerebellar granule precursor (CGP) cells. Sonic hedgehog (Shh) is the primary mitogen that regulates proliferation of CGP cells during the early stages of postnatal cerebellum development. Aberrant activation of Shh signaling during this time has been associated with hyperplasia of CGP cells and eventually may lead to the development of medulloblastoma. The molecular targets of Shh signaling involved in medulloblastoma formation are still not well-understood. Here, we show that Shh regulates sustained activation of histone deacetylases (HDACs) and that this activity is required for continued proliferation of CGP cells. Suppression of HDAC activity not only blocked the Shh-induced CGP proliferation in primary cell cultures, but also ameliorated aberrant CGP proliferation at the external germinal layer (EGL) in a medulloblastoma mouse model. Increased levels of mRNA and protein of several HDAC family members were found in medulloblastoma compared to wild type cerebellum suggesting that HDAC activity is required for the survival/progression of tumor cells. The identification of a role of HDACs in the early steps of medulloblastoma formation suggests there may be a therapeutic potential for HDAC inhibitors in this disease.     

Disruption of the PACAP gene promotes medulloblastoma in ptc1 mutant mice

Hedgehog (Hh) proteins and cAMP-dependent protein kinase A (PKA) generally play opposing roles in developmental patterning events. Humans and mice heterozygous for mutations in the sonic hedgehog (Shh) receptor gene patched-1 (ptc1) have an increased incidence of certain types of cancer, including medulloblastoma (MB), a highly aggressive tumor of the cerebellum. Despite the importance of PKA in Hh signaling, little is known about how PKA activity is regulated in the context of Hh signaling, or the consequences of improper regulation. One molecule that can influence PKA activity is pituitary adenylyl cyclase-activating peptide (PACAP), which has been shown to regulate cerebellar granule precursor proliferation in vitro, a cell population thought to give rise to MB. To test for a PACAP/Hh interaction in the initiation or propagation of these tumors, we introduced a PACAP mutation into ptc1 mutant mice. Deletion of a single copy of PACAP increased MB incidence approximate 2.5-fold, to 66%, thereby demonstrating that PACAP exerts a powerful inhibitory action on the induction, growth or survival of these tumors. Tumors from PACAP/ptc1 mutant mice retained PACAP receptor gene expression, and exhibited superinduction of Hh target genes compared to those from ptc1+/- mice. Moreover, PACAP inhibited proliferation of cell lines derived from tumors in a PKA-dependent manner, and inhibited expression of the Hh target gene gli1. The results provide genetic evidence that PACAP acts as a physiological factor that regulates the pathogenesis of Hh pathway-associated MB tumors.     

Hedgehog and PI-3 kinase signaling converge on Nmyc1 to promote cell cycle progression in cerebellar neuronal precursors

Neuronal precursor cells in the developing cerebellum require activity of the sonic hedgehog (Shh) and phosphoinositide-3-kinase (PI3K) pathways for growth and survival. Synergy between the Shh and PI3K signaling pathways are implicated in the cerebellar tumor medulloblastoma. Here, we describe a mechanism through which these disparate signaling pathways cooperate to promote proliferation of cerebellar granule neuron precursors. Shh signaling drives expression of mRNA encoding the Nmyc1 oncoprotein (previously N-myc), which is essential for expansion of cerebellar granule neuron precursors. The PI3K pathway stabilizes Nmyc1 protein via inhibition of GSK3-dependent Nmyc1 phosphorylation and degradation. The effects of PI3K activity on Nmyc1 stabilization are mimicked by insulin-like growth factor, a PI3K agonist with roles in central nervous system precursor growth and tumorigenesis. These findings indicate that Shh and PI3K signaling pathways converge on N-Myc to regulate neuronal precursor cell cycle progression. Furthermore, they provide a rationale for therapeutic targeting of PI3K signaling in medulloblastoma.     

Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors

Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. We report that sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G(1) cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors.     

Dual Phosphorylation of suppressor of fused (Sufu) by PKA and GSK3beta regulates its stability and localization in the primary cilium

Suppressor of fused (Sufu) is an essential negative regulator of the sonic hedgehog (Shh) pathway, but little is known about how Sufu itself is normally regulated. Here, we report that Sufu is phosphorylated at Ser-342 and Ser-346 by GSK3β and cAMP-dependent protein kinase A (PKA), respectively, and phosphorylation at this dual site stabilizes Sufu against Shh signaling-induced degradation. We further show that localization of Sufu in the primary cilium is induced by Shh signaling and is required for the turnover of both phosphorylated and total Sufu. Perturbing Sufu phosphorylation with PKA inhibitors or replacing Ser-346 with alanine reduced the stay and replacing Ser-342 and Ser-346 with aspartic acid prolonged the stay of Sufu in the cilia. Finally, ciliary localization of Gli2/3 also required Smo and was similarly influenced by perturbations of PKA activity or mutations at the dual Sufu phosphorylation site. Thus, Shh likely induced trafficking of phospho-Sufu into the primary cilium in a complex with Gli2/3, and dephosphorylation triggered a retrograde export, allowing Sufu to be degraded by the ubiquitin-proteasome system.