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Post-Transcriptional Down-Regulation of Atoh1/ Math1 by Bone Morphogenic Proteins Suppresses Medulloblastoma Development

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Post-Transcriptional Down-Regulation of Atoh1/ Math1 by Bone Morphogenic Proteins Suppresses Medulloblastoma Development Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION induce differentiation of GNPs by binding to their recep- Post-transcriptional tors, BMPR1a, BMPR1b, and BMPR2, to activate down-regulation of Atoh1/ Smad1,5,8 phosphorylation and gene regulation and to trigger the transcription of two basic helix–loop–helix Math1 by bone morphogenic (bHLH) proteins, Id1 and Id2 (Angley et al. 2003; Rios et proteins suppresses al. 2004). Here, we demonstrate that BMPs similarly block the proliferation of MB cells in vitro and in vivo medulloblastoma development and provide evidence that down-regulation of the Shh- induced transcription factor, Atoh1, is required for these Haotian Zhao,1,3 Olivier Ayrault,1,3 effects. Frederique Zindy,1 Jee-Hae Kim,2 and Martine F. Roussel1,4 Results and Discussion BMPs antagonize Shh-dependent proliferation and 1Departments of Genetics and Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, induce differentiation of GNPs and GNP-like MB cells USA; 2Laboratory of Developmental Neurobiology, Primary GNPs isolated from postnatal day 7 (P7) mouse Rockefeller University, New York, New York 10021, USA cerebella, a time at which their proliferation is maximal, were enriched by equilibrium Percoll density gradient Bone morphogenic proteins 2 and 4 (BMP2 and BMP4) centrifugation and cultured in vitro (Uziel et al. 2005). inhibit proliferation and induce differentiation of cer- Treatment of GNPs with recombinant human BMP2 or ebellar granule neuron progenitors (GNPs) and primary BMP4 in the presence of Shh reduced their incorporation GNP-like medulloblastoma (MB) cells. This occurs of BrdU so that after3dofBMPtreatment, only ∼5% of through rapid proteasome-mediated degradation of GNPs remained in cycle (Fig. 1A; Supplemental Fig. 1A). Math1 (Atoh1), a transcription factor expressed in pro- Inhibition of proliferation was confirmed by analysis of liferating GNPs. Ectopic expression of Atoh1, but not of the cells’ DNA content and mimicked effects of Shh Sonic hedgehog (Shh)-regulated Gli1 or Mycn, cancels withdrawal or of the cells’ response to cyclopamine, an inhibitor of Shh signaling (Fig. 1B). these BMP-mediated effects and restores Shh-dependent To evaluate whether BMPs also inhibit proliferation of proliferation of GNPs and MB cells in vitro and in vivo. tumor cells, we isolated and cultured primary GNP-like Genes regulating the BMP signaling pathway are down- tumor cells from MBs arising in predisposed Cdkn2c−/−, regulated in mouse MBs. Thus, BMPs are potent inhibi- Trp53Fl/Fl, Nes-cre+, and Cdkn2c−/−, Ptch1+/− mice (Uziel tors of MB and should be considered as novel therapeutic et al. 2005; Zindy et al. 2007). Because these tumors ex- agents. press a constitutively activated Shh signaling pathway (Lee et al. 2003, Zindy et al. 2007), they no longer depend Supplemental material is available at http://www.genesdev.org. on Shh addition to the culture medium to proliferate. Received November 21, 2007; revised version accepted The number of MB cells approximately doubled after 72 January 16, 2008. h of culture but did not expand in number when treated either with BMP4 or cyclopamine (Fig. 1C; Supplemental Fig. 1B). Like primary GNPs, only 8% of GNP-like tumor Balancing the proliferation, migration, and differentia- cells remained in S phase after 72 h of culture in the tion of cerebellar granule neuron progenitors (GNPs) is presence of BMP2, BMP4, BMP7, or cyclopamine (Fig. essential for proper development of the cerebellum and 1D; Supplemental Fig. 1C, left panel). FACS analysis of for suppression of medulloblastoma (MB), the most com- propidium iodide-stained cells indicated that they had mon malignant pediatric brain tumor. In the external arrested in G1 phase with a 2N DNA content, but unlike germinal layer (EGL) of the developing postnatal cerebel- previous reports (Hallahan et al. 2003), Annexin V stain- lum, proliferation of GNPs is stimulated through the ing of tumor cells did not demonstrate increased apopto- Sonic hedgehog (Shh) signaling pathway (Wallace 1999; sis (Supplemental Fig. 1C, right panel). Immunostaining Wechsler-Reya and Scott 2003), which is deregulated by of GNPs (Fig. 1E) and GNP-like tumor cells (Fig. 1F) mutations in ∼30% of human MB (Dahmane et al. 2001; treated for 72 h with BMP2 or BMP4 revealed increased Kenney et al. 2003; Oliver et al. 2003; Marino 2005). expression of Tag1 (Cntn2) (Fig. 1E [panels b,c vs. a], F Bone morphogenic proteins (BMPs), a subgroup of the [panel b vs. a]), Class III ␤-tubulin/Tuj1 (Tubb1) (Fig. 1E transforming growth factor-␤ (Tgfb1) superfamily, also [panels h,i vs. g], F [panel f vs. e]), NeuN (Neuna60) and play critical roles in fate determination, patterning, dif- NF200 (Nefh) (Supplemental Fig. 1D) and Cdkn1b ferentiation, and cell survival during cerebellar develop- (p27Kip1) (Supplemental Fig. 1E), several markers of neu- ment (Angley et al. 2003; Rios et al. 2004; Fogarty et al. ronal differentiation. Thus, primary GNPs and MB cells 2005). BMPs antagonize Shh-dependent proliferation and exit the division cycle and differentiate in response to BMP treatment without evidence of apoptosis. Although basic fibroblast growth factor (bFGF) was previously [Keywords: Medulloblastoma; Sonic hedgehog; bone morphogenic pro- shown to block Shh-dependent proliferation in GNPs tein; Atoh1/Math1; Mycn] and MB cells (Fogarty et al. 2007), bFGF did not mimic 3 These authors contributed equally to this work. the effects of BMPs under our conditions of cell purifi- 4Corresponding author. E-MAIL [email protected]; FAX (901) 495-2381. cation and culture. Comparison of gene expression pro- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1636408. files of GNPs purified from P6 cerebella of wild-type and 722 GENES & DEVELOPMENT 22:722–727 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press BMPs suppress medulloblastoma modestly reduced in cells treated for 24 h with Shh in the presence of BMPs (Fig. 2A). However, after 72-h treat- ment with Shh and BMPs, Mycn, Ccnd1, and Cdk2 pro- tein levels, together with Gli1, Gli2, and Mycn mRNAs, were markedly diminished (Fig. 2B; Supplemental Fig. 2A) (Alvarez-Rodriguez et al. 2007). In turn, the levels of three transcription factors—Neurod1, Zic1, and Pax6— expressed in granule neurons (Aruga et al. 1998; Miyata et al. 1999; Yamasaki et al. 2001) were unchanged after 24 h (Fig. 2A), while after3dofBMPtreatment, Neurod1 and Zic1 levels were slightly decreased (Fig. 2B). Again, Cntn2 expression was increased after 72 h of BMP treat- ment as cells ceased proliferating (Fig. 1E). Thus, while Shh and Bmp signaling converge in regulating the cell division cycle, they do so in a different manner. During cerebellar development, the bHLH transcrip- tion factor Atoh1 is detected in proliferating GNPs, but not in their post-mitotic derivatives (Akazawa et al. 1995; Ben-Arie et al. 1996, 1997; Lumpkin et al. 2003; Machold and Fishell 2005). In Atoh1-null mice, the specification of GNPs is intact and granule neuron iden- tity is maintained, but proliferation of GNPs and their subsequent differentiation and migration are compro- Figure 1. BMP induces cell cycle arrest and differentiation of GNPs mised (Ben-Arie et al. 1997; Gazit et al. 2004). Unlike the and GNP-like MB cells. (A) GNPs were treated for 24, 48, and 72 h many other regulators whose alterations in expression in the presence of Shh (black bars), Shh and BMP2 (gray bars), or Shh temporally mirrored the rate of BMP-induced cell cycle and BMP4 (diagonal shaded bars). Cell proliferation was measured withdrawal, Atoh1 protein expression became undetect- by BrdU incorporation (n = 3). (B) GNPs were treated for 72 h with- out Shh (blank bar), with Shh (black bar), with Shh and BMP4 (di- able as early as 24 h after BMP treatment (Fig. 2A) and agonal shaded bar), or with Shh and cyclopamine (vertical shaded remained down-regulated after 72 h in the presence of bar). Cell proliferation was determined by FACS analysis of DNA BMP (Fig. 2B) as confirmed by immunofluorescence content. The percentage of cells in S phase is shown. (C) Prolifera- staining (Supplemental Fig. 2B). tion of tumor cells grown in the absence (Blank, —Ⅵ—, n =5)or Like primary GNPs, BMP-treated MB cells showed presence (—ᮡ—, n = 5) of BMP4 or cyclopamine (—ᮢ—, n = 5) for 72 h was assessed by counting cell number. (D) GNP-like tumor comparably increased levels of phosphorylated Smad1,5,8 cells were treated without (black bars) or with (diagonal shaded bars) and ID2 (Fig. 2C), and ID1 (data not shown), indicating BMP4 or with cyclopamine (blank bars) for 48 and 72 h. The per- that this signaling pathway remained intact in tumor centage of cells in S phase was determined as in B. (E) Expression of Cntn2 and Tubb1 in GNPs after 72-h treatment with Shh (panels a,d,g,j), Shh and BMP2 (panels b,e,h,k), or Shh and BMP4 (panels c,f,i,l) was determined by immunofluorescence. Red fluorescent staining indicates the expression of Cntn2 (panels a–c) or Tubb1 (panels g–i). (Panels d–f,j–l) DAPI staining was used to confirm cell viability. (F) Tumor cells treated for 72 h without (panels a,c,e,g)or with (panels b,d,f,h) BMP4 were stained with antibodies to Cntn2 (panels a,b) or Tubb1 (panels e,f). (Panels c,d,g,h) DAPI staining con- firmed cell viability. Error bars indicate SEM and asterisks indicate P < 0.05 (Student’s t-test). tumor-prone mice (Supplemental Fig. 1F, panel a) with those of MBs arising in genetically predisposed mice (Supplemental Fig. 1F, panel b) revealed that many effec- tors of BMP signaling were down-regulated in MBs, sug- gesting that BMP signaling might normally play a role in tumor suppression.
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