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RESEARCH COMMUNICATION
1 Departments of Genetics and Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA; 2 Laboratory of Developmental Neurobiology, Rockefeller University, New York, New York 10021, USA
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Abstract |
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[Keywords: Medulloblastoma; Sonic hedgehog; bone morphogenic protein; Atoh1/Math1; Mycn]]
Received November 21, 2007; revised version accepted January 16, 2008.
; Wechsler-Reya and Scott 2003), which is deregulated by mutations in 
30% of human MB (Dahmane et al. 2001; Kenney et al. 2003; Oliver et al. 2003; Marino 2005). Bone morphogenic proteins (BMPs), a subgroup of the transforming growth factor-β (Tgfb1) superfamily, also play critical roles in fate determination, patterning, differentiation, and cell survival during cerebellar development (Angley et al. 2003; Rios et al. 2004; Fogarty et al. 2005). BMPs antagonize Shh-dependent proliferation and induce differentiation of GNPs by binding to their receptors, BMPR1a, BMPR1b, and BMPR2, to activate Smad1,5,8 phosphorylation and gene regulation and to trigger the transcription of two basic helix–loop–helix (bHLH) proteins, Id1 and Id2 (Angley et al. 2003; Rios et al. 2004). Here, we demonstrate that BMPs similarly block the proliferation of MB cells in vitro and in vivo and provide evidence that down-regulation of the Shh-induced transcription factor, Atoh1, is required for these effects. |
Results and Discussion |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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Primary GNPs isolated from postnatal day 7 (P7) mouse cerebella, a time at which their proliferation is maximal, were enriched by equilibrium Percoll density gradient centrifugation and cultured in vitro (Uziel et al. 2005). Treatment of GNPs with recombinant human BMP2 or BMP4 in the presence of Shh reduced their incorporation of BrdU so that after 3 d of BMP treatment, only 5% of GNPs remained in cycle (Fig. 1A; Supplemental Fig. 1A). Inhibition of proliferation was confirmed by analysis of the cells’ DNA content and mimicked effects of Shh withdrawal or of the cells’ response to cyclopamine, an inhibitor of Shh signaling (Fig. 1B).
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; Zindy et al. 2007). Because these tumors express a constitutively activated Shh signaling pathway (Lee et al. 2003, Zindy et al. 2007), they no longer depend on Shh addition to the culture medium to proliferate. The number of MB cells approximately doubled after 72 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 cells remained in S phase after 72 h of culture in the presence of BMP2, BMP4, BMP7, or cyclopamine (Fig. 1D; Supplemental Fig. 1C, left panel). FACS analysis of propidium iodide-stained cells indicated that they had arrested in G1 phase with a 2N DNA content, but unlike previous reports (Hallahan et al. 2003), Annexin V staining of tumor cells did not demonstrate increased apoptosis (Supplemental Fig. 1C, right panel). Immunostaining of GNPs (Fig. 1E) and GNP-like tumor cells (Fig. 1F) treated for 72 h with BMP2 or BMP4 revealed increased expression of Tag1 (Cntn2) (Fig. 1E [panels b,c vs. a], F [panel b vs. a]), Class III β-tubulin/Tuj1 (Tubb1) (Fig. 1E [panels h,i vs. g], F [panel f vs. e]), NeuN (Neuna60) and NF200 (Nefh) (Supplemental Fig. 1D) and Cdkn1b (p27Kip1) (Supplemental Fig. 1E), several markers of neuronal differentiation. Thus, primary GNPs and MB cells exit the division cycle and differentiate in response to BMP treatment without evidence of apoptosis. Although basic fibroblast growth factor (bFGF) was previously shown to block Shh-dependent proliferation in GNPs and MB cells (Fogarty et al. 2007), bFGF did not mimic the effects of BMPs under our conditions of cell purification and culture. Comparison of gene expression profiles of GNPs purified from P6 cerebella of wild-type and 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 effectors of BMP signaling were down-regulated in MBs, suggesting that BMP signaling might normally play a role in tumor suppression.
BMP treatment leads to rapid down-regulation of Atoh1 protein
When immunoblotting (Fig. 2A,B) and quantitative RT–PCR (q-RT–PCR) (Supplemental Fig. 2A) were used to survey gene expression in GNPs treated with Shh alone or together with BMP, Smad1,5,8 phosphorylation, and protein levels of Id1 and Id2, were greatly increased after BMP treatment, but not by Shh alone (Fig. 2A,B). Conversely, expression of Shh-responsive targets, Gli1, Mycn, and Ccnd1 (cyclin D1), was induced by Shh (Kenney and Rowitch 2000; Kenney et al. 2003; Corrales et al. 2004), but their protein levels were either unchanged or modestly reduced in cells treated for 24 h with Shh in the presence of BMPs (Fig. 2A). However, after 72-h treatment with Shh and BMPs, Mycn, Ccnd1, and Cdk2 protein 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 after 3 d of BMP treatment, Neurod1 and Zic1 levels were slightly decreased (Fig. 2B). Again, Cntn2 expression was increased after 72 h of BMP treatment 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.
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; 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 identity is maintained, but proliferation of GNPs and their subsequent differentiation and migration are compromised (Ben-Arie et al. 1997; Gazit et al. 2004). Unlike the many other regulators whose alterations in expression temporally mirrored the rate of BMP-induced cell cycle withdrawal, Atoh1 protein expression became undetectable as early as 24 h after BMP treatment (Fig. 2A) and remained down-regulated after 72 h in the presence of BMP (Fig. 2B) as confirmed by immunofluorescence staining (Supplemental Fig. 2B). Like primary GNPs, BMP-treated MB cells showed comparably increased levels of phosphorylated Smad1,5,8 and ID2 (Fig. 2C), and ID1 (data not shown), indicating that this signaling pathway remained intact in tumor cells. Atoh1 expression was maintained in cultured MB cells during at least 3 d of culture consistent with constitutive activation of the Shh signaling pathway. The relative levels of Atoh1 mRNA were also higher in MBs than in primary GNPs (Supplemental Fig. 1F, bottom lane, panel b vs. a). Yet, Atoh1 protein levels decreased rapidly within 12 h and became undetectable by 24 h after BMP addition (Fig. 2C). Whereas cyclopamine treatment down-regulated Mycn expression within 12 h, it did not reduce Atoh1 protein levels as quickly (Fig. 2C). Conversely, BMP treatment did not affect the levels of Mycn within the first 24 h of culture but reduced the levels of Mycn and Cdk2 only after 3 d, concomitant with the exit of the tumor cells from the cell division cycle and their differentiation (Fig. 2C). Thus, as in normal GNPs, activation of BMP signaling in tumor cells resulted in rapid disappearance of Atoh1 protein without affecting Shh activity.
BMP-dependent Atoh1 protein down-regulation occurs via a post-transcriptional mechanism
Atoh1 protein levels were maintained when proliferating GNPs were cultured with Shh but decreased rapidly in its absence (Fig. 3A). Atoh1 protein and mRNA levels were similarly reduced when GNPs, cultured in the presence of Shh, were treated with cyclopamine (Supplemental Fig. 2C,D, respectively), again highlighting the fact that Atoh1 expression in proliferating GNPs depends on Shh pathway activation (Berman et al. 2002; Kenney et al. 2003). However, Atoh1 protein levels were no longer detected after only 12 h of BMP treatment (Fig. 3B). In contrast, even after 18 h of BMP4 exposure, Atoh1 RNA levels, as well as those of Gli1 and Gli2, remained similar to those quantified in GNPs treated with Shh alone (Fig. 3C). Similarly, BMP treatment of MB cells did not affect Atoh1 RNA levels (data not shown). Thus, BMP treatment appeared to trigger a rapid loss of Atoh1 protein expression via a post-transcriptional mechanism.
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In GNPs and GNP-like MB cells treated with BMP, the levels of Id1 and Id2 were greatly increased (Fig. 2A–C) raising the possibility that they might also affect Atoh1 protein turnover by competing with Atoh1 for binding to Tcfe2a. Interestingly, when Id1 (data not shown) or Id2 was transfected together with Atoh1 and Tcfe2a into 293T cells, the levels of stabilized Atoh1 protein were significantly reduced (Fig. 3E, panel d). Thus, by shifting Atoh1 from transcriptionally active complexes containing Tcfe2a to inactive Id-containing complexes, BMP treatment may also enhance Atoh1 protein turnover. In this respect, Atoh1 regulation may mirror that of Mash1 (Ascl1), another bHLH protein expressed during CNS development (Shou et al. 1999; Vinals et al. 2004). Like Ascl1, Ser 193 phosphorylation of Atoh1 is expected to regulate its binding to Tcfe2a and its stability. These findings suggested that Atoh1 down-regulation was likely to be required for cell cycle exit in response to BMP treatment. Indeed, ectopic overexpression of Atoh1, but not of two Atoh1 DNA-binding mutants (E165G and R158G) or the Shh-responsive proteins Mycn and Gli1, abolished the BMP-induced cell cycle arrest of primary GNPs (Fig. 3F). Thus, down-regulation of Atoh1 expression preceded BMP-induced cell cycle arrest, whereas expression of functional Atoh1 protein maintained Shh-mediated proliferation of GNPs treated with BMP.
BMPs inhibit tumor growth in vivo
To directly assess whether BMP inhibits MB development in vivo, GNP-like tumor cells purified from the aforementioned murine MBs were infected with retroviral vectors coexpressing human BMP4 and green fluorescent protein (GFP), or GFP alone, and injected subcutaneously into the flanks of athymic mice. Allografts in the flank of recipient mice derived from 5 x 105 tumor cells infected with the control vector grew rapidly into large tumors containing many GFP-positive cells (37.8% ± 14.1, n = 8) quantified by flow cytometric analysis of dissociated tumor cells (Fig. 4A [panel a], B). Typically, tumors were palpable 1 wk after injection and were harvested 2–4 wk later. In contrast, allografts from tumor cells infected with the vector coexpressing BMP4 and GFP were much smaller and almost devoid of GFP-positive cells (2.4% ± 1.51, n = 8) (Fig. 4A [panel b], B). All allografts maintained pathological and molecular features of the original MB (Supplemental Fig. 3). Because the efficiency of retroviral infection of donor cells typically ranged from 40% to 60%, the small tumors lacking GFP expression that arose in BMP-infected tumor cells might have originated from uninfected tumor cells. Indeed, tumor cells infected with control virus and sorted for GFP expression grew back quickly, but no tumors developed when cells infected with virus coexpressing BMP4 and GFP were first sorted for GFP expression prior to injection (Fig. 4A, panel d). Importantly, tumors failed to grow when cells were pretreated with BMP4 for 3 d in vitro before injection, whereas allografts grew back quickly from untreated tumor cells (Supplemental Fig. 3). Because cultured GNPs overexpressing Atoh1 were resistant to BMP-induced effects, we assessed whether enforced expression of Atoh1 in tumor cells would render them resistant to BMP treatment. Tumor cells purified from spontaneously derived MBs from Cdkn2c–/–, Ptch1+/– mice were infected with retroviral vectors expressing Atoh1 and GFP or GFP alone, and were transplanted into the flanks of immunocompromised recipient mice. Allografts from tumor cells infected with GFP alone grew back with the same time of onset and growth rate as noninfected tumor cells, and retained the same pathological features of the original MB (data not shown). Remarkably, allografts from MB cells infected with viruses expressing Atoh1 and GFP were brightly fluorescent (Fig. 4A, panel c), and virtually all tumor cells purified from these transplants overexpressed Atoh1 (98% ± 1.15, n = 3) (Fig. 4B). GNP-like tumor cells isolated from both types of allografts were treated with BMP4 or cyclopamine for 72 h in culture. As expected, cyclopamine effectively blocked the proliferation of tumor cells from both types of transplants (Fig. 4C), indicating that tumor cells depend on the constitutively activated Shh pathway activity for their proliferation. BMP4 treatment of GNP-like tumor cells that expressed GFP alone significantly reduced the proportion of cells in S phase (Fig. 4C), but in contrast, failed to do so in tumor cells overexpressing Atoh1 (Fig. 4C).
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; Romer et al. 2004; Sanchez and Ruiz i Altalba 2005). BMP2 induces apoptosis in human MB cells (Hallahan et al. 2003), while BMP2 and BMP4 suppress human glioblastoma as well (Piccirillo et al. 2007). Although BMP and cyclopamine each arrest the proliferation of MB cells, they elicit unique responses, raising the possibility that BMP agonists and SHH antagonists might be therapeutically combined to reduce the potential deleterious effect of either agent. Indeed, we found that BMP and cyclopamine acted additively to suppress proliferation and to induce differentiation of GNP-like MB cells. While BMP4 or cyclopamine alone functioned optimally at 100 ng/mL or 10 µM, respectively, to arrest tumor cell proliferation, a similar level of inhibition was achieved with a combination of only 2.5 µM cyclopamine and 25 ng/mL BMP4 (Fig. 4D). Nonetheless, caution should be exercised in evaluating the effectiveness and safety of such compounds, particularly in the treatment of pediatric malignancies, since compounds of this type may well have adverse effects on normal developmental processes. |
Materials and methods |
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Mice (Cdkn2c–/–, Trp53Fl/Fl, Nes-cre+) and (Cdkn2c–/–, Ptch1+/–) were derived and maintained as described (Uziel et al. 2005; Zindy et al. 2007). Immunocompromised athymic mice (CD1; Jackson Laboratory) were used as allograft recipients.
Cell culture
Purification of GNPs and GNP-like tumor cells was performed as described (Zindy et al. 2007). GNPs were maintained in Neurobasal medium containing B27 supplement, 2 mM glutamine, 100 U/mL penicillin/streptomycin (all from Invitrogen), 0.45% D-glucose, 1x SPITE medium supplement, and 1x linoleic acid–oleic acid (all from Sigma-Aldrich). GNP-like tumor cells were grown in medium containing N2 supplement and 4 mg/mL bovine serum albumin (Invitrogen) instead of SPITE and linoleic acid–oleic acid. Human recombinant BMP2, BMP4, or BMP7 (R&D) were used at 100 ng/mL and cyclopamine (LC Laboratories) was used at 10 µM. 293T cells were maintained in Dulbecco’s Modified Eagle’s Medium with 10% fetal calf serum, and 100 U/mL penicillin/streptomycin. DNA expression constructs were transfected using Lipofectamine 2000 (Invitrogen). Where indicated, MG-132 (Calbiochem) was used at 10 µM and cycloheximide (Sigma-Aldrich) was used at 100 µg/mL.
DNA constructs, retrovirus production, and infection
Human BMP2/4 hybrid cDNA was generated as described previously (Peng et al. 2001). Generation of point mutations (E165G and R158G) in the Atoh1 DNA-binding domain was performed by PCR site-directed mutagenesis. cDNAs encoding a BMP2/4 hybrid, wild type, or mutant Atoh1, Gli1, Mycn, Id1, Id2, and Tcfe2a were cloned into an MSCV-IRES-GFP vector, verified by DNA sequencing, and used to generate retroviruses as described (Zindy et al. 2007). Enriched GNPs and GNP-like tumor cells were infected during the preplating stage.
Other analytical procedures
Immunoblotting and immunohistochemistry were performed (Zindy et al. 2007) using antibodies raised against Cdk2, Ccnd1, Id1, Id2, Neurod1, and β-actin (Santa Cruz Biotechnology); phosphorylated Smad1,5,8 (Cell Signaling); Zic1 and Gli1 (Rockland); Mycn (Calbiochem); Atoh1 (Developmental Studies Hybridoma Bank, DSHB); Pax6 and Tubb1 (Covance); glial fibrillary acidic protein (GFAP); and synaptophysin (DAKO). For immunofluorescence, cells fixed on slides were blocked with 3% normal goat serum and then incubated with primary antibodies raised against Atoh1 and Tubb1 (Covance); Cntn2 (DSHB); Cdkn1b (Beckman Dickinson); BrdU (Santa Cruz Biotechnology); Neuna60 (Millipore); Nefh (Sigma-Aldrich); and GFP (Invitrogen) (Uziel et al. 2005). Cultured GNPs were incubated with 10 µM BrdU (BD Biosciences) for 1.5 h and stained with antibodies to BrdU as described (Uziel et al. 2005). BrdU-positive nuclei within at least 600 DAPI- or GFP-labeled cells were counted under a fluorescent microscope, and the percentage of BrdU-positive cells within the total number of DAPI- or GFP-labeled stained nuclei are indicated. Each treatment was repeated independently at least three times. Q-RT–PCR on RNA extracted from cultured cells was performed with primers and probes described previously (Lee et al. 2003; Zindy et al. 2007).
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Footnotes |
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E-MAIL martine.roussel{at}stjude.org; FAX (901) 495-2381.
Supplemental material is available at http://www.genesdev.org.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1636408.
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  M. R. Grimmer and W. A. Weiss BMPs oppose Math1 in cerebellar development and in medulloblastoma Genes & Dev., March 15, 2008; 22(6): 693 - 699. [Full Text] [PDF]
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—, n = 5) or presence (—
—, n = 5) of BMP4 or cyclopamine (—
—, n = 5) for 72 h was assessed by counting cell number. (D) GNP-like tumor cells were treated without (black bars) or with (diagonal shaded bars) BMP4 or with cyclopamine (blank bars) for 48 and 72 h. The percentage 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 confirmed cell viability. Error bars indicate SEM and asterisks indicate P < 0.05 (Student’s t-test).
