Sequential activation of Snail1 and N-Myc modulates sonic hedgehog-induced transformation of neural cells

Abstract

Activation of the Sonic hedgehog (Shh) pathway and increased expression of Gli1 play an important role in proliferation and transformation of granule cell progenitors (GCP) in the developing cerebellum. Medulloblastomas arising from cerebellar GCPs are frequently driven by Shh pathway-activating mutations; however, molecular mechanisms of Shh pathway dysregulation and transformation of neural progenitors remain poorly defined. We report that the transcription factor and oncogene Snail1 (Sna1) is directly induced by Shh pathway activity in GCPs, murine medulloblastomas, and human medulloblastoma cells. Enforced expression of Sna1 was sufficient to induce GCPs and medulloblastoma cell proliferation in the absence of Shh/Gli1 exposure. In addition, enforced expression of Sna1 increased transformation of medulloblastoma cells in vitro and in vivo. Analysis of potential Sna1 targets in neural cells revealed a novel Sna1 target, N-Myc, a transcription factor known to play a role in Shh-mediated GCP proliferation and medulloblastoma formation. We found that Sna1 directly induced transcription of N-Myc in human medulloblastoma cells and that depletion of N-Myc ablated the Sna1-induced proliferation and transformation. Taken together, these results provide further insight into the mechanism of Shh-induced transformation of neural progenitor cells and suggest that induction of Sna1 may serve to amplify the oncogenic potential of Shh pathway activation through N-Myc induction.

Figure 1. Shh pathway activation induces Sna1 expression in neural progenitor cells

A. Top, qRT-PCR shows Sna1 mRNA is increased 6.7-fold in the cerebella of P6 Ptc^+/− mice versus WT siblings. Bottom, RT-PCR shows Sna1 mRNA is increased in the cerebella of P6 and adult Ptc^+/− mice versus WT siblings. B. Left, Sna1 mRNA was induced 8.51-fold in hippocampal NSCs in the presence of elevated Gli1, but not Gli1^ZFD. Right, Sna1 mRNA was induced in GCPs expressing Gli1. C. Western blot analyses shows Sna1 protein was increased in both WT and Gli1 null cells exposed to Shh ligand for 24 hours. D. Western blot analysis shows that Sna1 and N-Myc protein were increased in GCPs exposed to Shh ligand alone, but not in the presence of cycloheximide (CHX).

Figure 2. Shh pathway activation induces Sna1 in medulloblastoma

Protein (A) and cDNA (B) samples were prepared from the tumors and contiguous normal cerebella of four tumor-bearing ND2:SmoA1 mice. A. Western blot analysis shows that three tumors (T) show increased Sna1 expression as compared to contiguous cerebellum (C). B. qRT-PCR analyses show that the tumor with highly elevated Gli1 also shows an increase in Sna1 mRNA, however Sna1 mRNA is not increased when Gli is only slightly induced or not induced. C. qRT-PCR analyses of mRNA expression in tumor stem-like cell lines developed from Ptc^+/− medulloblastomas show that the cell line with elevated Gli1 expression (5808) also has high Sna1 mRNA expression. Sna1 is not detected in the cell line with low Gli1 expression (4895) or in WT CBSCs.

Figure 3. Shh pathway activation induces Sna1 in human medulloblastoma cells

A and B. qRT-PCR analysis shows that Sna1 mRNA expression is robustly increased by exposure to Shh ligand in ONS76 (A) and Daoy cells (B) after 8 hours as compared toGli1 mRNA expression. C. A ChIP assay performed on Daoy cells shows that Gli1 can bind to the promoter of Sna1 between 1217 and 1709 bases upstream of transcription start. D. ChIP performed on cells treated with cyclopamine shows a reduction of Gli1 occupancy on the Sna1 promoter as compared to vehicle control. E. Daoy cells transfected with the wild-type Sna1 promoter (WT) driving luciferase expression show a 1383% increase in promoter induction in by Gli1 as compared to control cells. Mutation of putative GBEs in the region of Gli11 binding prevents (sites −1685, −1449 and −1333) or reduces (site −1297) Sna1 promoter induction in the presence of Gli1.

Figure 4. Sna1 promotes GCP proliferation

A. Phosphorylated histone H3 (pH3) and (B) Ki67 immunoreactivity is increased approximately 200% in GCPs infected with adenovirus expressing Sna1 versus uninfected controls. C. Ki67 immunoreactivity is increased approximately 276% in GCPs treated with Shh versus vehicle control.

Figure 5. Sna1 promotes medulloblastoma cell proliferation

A. EdU incorporation by ONS76 and Daoy cells increased by 231% and 41%, respectively in Sna1-transfected cells versus controls. B. Proliferation of Daoy cells decreased by 15% in cells transfected with shRNA against Sna1 (shSna1) versus cells transfected with scrambled shRNA (Scr.; p=0.0027) as quantified by EdU incorporation; this decrease is rescued by re-expression of Sna1 (p=0.0002).

Figure 6. Sna1 induces N-Myc in neural cells

A. Western blot analyses revealed increased expression of N-Myc in medulloblastoma cells and GCPs expressing Sna1. β-catenin is induced by Sna1 only in Daoy cells. CyclinD1 was unaffected by Sna1. B. RT-PCR shows increased expression of N-Myc in medulloblastoma cells expressing Sna1. C. A ChIP assay revealed that Sna1 binds to the N-Myc promoter between positions −1651 and −932. D. Daoy cells transfected with the wild-type N-Myc promoter (WT) driving luciferase expression show a 258% increase in promoter induction by Sna1 as compared to the control. Mutation of E-box sequences in the region of Sna1 binding prevents N-Myc promoter by Sna1. E. Daoy cells transfected with shRNA against Sna1 show a 75% reduction in N-Myc promoter activity compared to scrambled shRNA.

Figure 7. Sna1 increases transformation of medulloblastoma cells, which is abolished by N-Myc depletion

A. Daoy cells expressing Sna1 formed twice the number of colonies than did cells expressing vector alone. B. Daoy colony formation was reduced with depletion of Sna1 with Sna1-specific shRNA (shSna1). C. Subcutaneous xenograft tumors derived from Sna1-expressing Daoy cells were larger upon resection than control (GFP) tumors. D. EdU incorporation by ONS76 cells shows increased proliferation in cells expressing Sna1 (p<0.0001) was abolished in cells also transfected with shN-Myc (p=0.002); this is rescued by re-expression of N-Myc (p=0.0009). E. The contact-independent growth advantage of Sna1-expressing Daoy (p=0.03) and ONS76 cells (p=0.001) was abolished with N-Myc shRNA (shN-Myc; p=0.04 and p=0.014, respectively).