Abstract B02: Sonic hedgehog signaling in neuronal migration and tumorigenesis

Background: Neuronal progenitors in the brain are born in sites known as germinal zones (GZ) where they undergo proliferation and differentiation before migrating to their final positions at a distant site. Developmental cues that control this transition are tightly regulated to ensure proper brain morphogenesis as dysregulation leads to brain malformations and cancer. A major challenge in the field of developmental neurobiology is to delineate the molecular underpinnings that execute this developmental program. We use the cerebellum as a model, where granule neuron progenitors (GNP) are born in the outer layers of the external germinal layer (EGL) where they subsequently undergo differentiation and polarization before migrating radially to their final destination in the internal granule layer (IGL). GNPs with hyperactivated sonic hedgehog (Shh) signaling fail to initiate these processes continue to proliferate within the EGL eventually giving rise to Shh-driven medulloblastoma (MB). Migration away from the EGL requires Par3, a component of the Par polarity complex, which functions to recruit JamC, a junctional adhesion molecule, to the membrane to initiate cell-cell contacts vital for radial migration. Siah2, an E3 ubiquitin ligase highly expressed in GNPs, targets Par3 for degradation hence preventing their exit from the EGL while its subsequent downregulation in mature CGNs spurs GZ exit. How GNPs control the levels of Siah2 is unknown. Results: We show that Shh signaling in GNPs maintain Siah2 protein expression in a Ras-Erk dependent pathway. Levels of active Ras sampled from purified GNPs show a decrease from the post-natal days (P), P4 to P15, which is directly correlated to Siah2 protein levels. Interestingly, these protein levels remain high in Ptc +/- P18 -/- MB cells. Among the four subgroups of MB sampled in the Pediatric Cancer Genome Project, Shh-MB represents the subgroup with the highest expression of Siah2 transcript. In line with these findings, are functional studies that show that the arrested migration in GNPs due to activation of Shh signaling or Ras gain of function is rescued by exogenous Par3, Ras knockdown or inhibition of Siah2. To understand the cross-talk between Shh and Ras signaling, we examined where Ras is active in GNPs. We find using a live cell imaging probe that active Ras localizes to the primary cilium, the organelle that transduces Shh signals. Immunofluorescence studies also confirm the presence of phosphorylated Mek (pMek1/2) at the base of the primary cilium. Intriguingly, GNPs treated with smoothened agonist (SAG) maintains large primary cilia, which can be decreased in the presence of Ras-Erk inhibitors, while the differentiated CGNs lose this structure. Using SIM imaging techniques and 3D reconstruction we show that the volume of pMek1/2 at the base of the primary cilia increases in GNPs treated with SAG compared to vehicle control and this increase can be inhibited by Ras-Erk inhibitors, suggesting that Ras pathway activity is required for ciliogenesis or cilia-maintenance in GNPs. Conclusion: We propose that Shh signaling prevents GNPs from exiting the EGL by maintaining the expression of Siah2 in a Ras-Erk dependent pathway. Differentiated CGNs retract the primary cilia, hence becoming insensitive to Shh allowing them to downregulate Siah2 to initiate GZ exit. Aberrant Shh signaling disrupts this developmental program by maintaining GNPs in the germinal zone which we postulate to be the initial step in Shh-MB tumorigenesis. Citation Format: Taren Ong, David J. Solecki. Sonic hedgehog signaling in neuronal migration and tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B02.