A subset of medulloblastomas, the most common brain tumor in children, is hypothesized to originate from granule neuron precursors (GNPs) in which the sonic hedgehog (SHH) pathway is over-activated. MXD3, a basic helix-look-helix zipper transcription factor of the MAD family, has been reported to be upregulated during postnatal cerebellar development and to promote GNP proliferation and MYCN expression. Mxd3 is upregulated in mouse models of medulloblastoma as well as in human medulloblastomas. Therefore, we hypothesize that MXD3 plays a role in the cellular events that lead to medulloblastoma biogenesis. In agreement with its proliferative role in GNPs, MXD3 knock-down in DAOY cells resulted in decreased proliferation. Sustained overexpression of MXD3 resulted in decreased cell numbers due to increased apoptosis and cell cycle arrest. Structure-function analysis revealed that the Sin3 interacting domain, the basic domain, and binding to E-boxes are essential for this activity. Microarray-based expression analysis indicated up-regulation of 84 genes and down-regulation of 47 genes. Potential direct MXD3 target genes were identified by ChIP-chip. Our results suggest that MXD3 is necessary for DAOY medulloblastoma cell proliferation. However, increased level and/or duration of MXD3 expression ultimately reduces cell numbers via increased cell death and cell cycle arrest.
References
[1]
Polkinghorn WR, Tarbell NJ (2007) Medulloblastoma: tumorigenesis, current clinical paradigm, and efforts to improve risk stratification. Nat Clin Pract Oncol 4: 295–304.
[2]
Wechsler-Reya R, Scott MP (2001) The developmental biology of brain tumors. Annu Rev Neurosci 24: 385–428.
[3]
Wechsler-Reya RJ, Scott MP (1999) Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22: 103–114.
[4]
Oliver TG, Grasfeder LL, Carroll AL, Kaiser C, Gillingham CL, et al. (2003) Transcriptional profiling of the Sonic hedgehog response: a critical role for N-myc in proliferation of neuronal precursors. Proc Natl Acad Sci U S A 100: 7331–7336.
[5]
Eisenman RNE (2006) The Myc/Max/Mad Transcription Factor Network. Berlin: Springer.
[6]
Cole MD, Nikiforov MA (2006) Transcriptional activation by the Myc oncoprotein. Curr Top Microbiol Immunol 302: 33–50.
[7]
Ayer DE, Lawrence QA, Eisenman RN (1995) Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3. Cell 80: 767–776.
[8]
Foley KP, McArthur GA, Queva C, Hurlin PJ, Soriano P, et al. (1998) Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation. Embo J 17: 774–785.
[9]
Schreiber-Agus N, Meng Y, Hoang T, Hou H, Chen K, et al. (1998) Role of Mxi1 in ageing organ systems and the regulation of normal and neoplastic growth. Nature 393: 483–487.
[10]
Queva C, McArthur GA, Iritani BM, Eisenman RN (2001) Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis. Mol Cell Biol 21: 703–712.
[11]
Hurlin PJ, Queva C, Koskinen PJ, Steingrimsson E, Ayer DE, et al. (1995) Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation. Embo J 14: 5646–5659.
[12]
Queva C, Hurlin PJ, Foley KP, Eisenman RN (1998) Sequential expression of the MAD family of transcriptional repressors during differentiation and development. Oncogene 16: 967–977.
[13]
Fox EJ, Wright SC (2001) S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells. Biochem J 359: 361–367.
[14]
Diaz E, Ge Y, Yang YH, Loh KC, Serafini TA, et al. (2002) Molecular analysis of gene expression in the developing pontocerebellar projection system. Neuron 36: 417–434.
[15]
Yun JS, Rust JM, Ishimaru T, Diaz E (2007) A novel role of the Mad family member Mad3 in cerebellar granule neuron precursor proliferation. Mol Cell Biol 27: 8178–8189.
[16]
Gore Y, Lantner F, Hart G, Shachar I (2010) Mad3 negatively regulates B cell differentiation in the spleen by inducing Id2 expression. Mol Biol Cell 21: 1864–1871.
[17]
Goodrich LV, Milenkovic L, Higgins KM, Scott MP (1997) Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277: 1109–1113.
[18]
Barisone GA, Yun JS, Diaz E (2008) From cerebellar proliferation to tumorigenesis: new insights into the role of Mad3. Cell Cycle 7: 423–427.
[19]
Nair SK, Burley SK (2006) Structural aspects of interactions within the Myc/Max/Mad network. Curr Top Microbiol Immunol 302: 123–143.
[20]
Rottmann S, Luscher B (2006) The Mad side of the Max network: antagonizing the function of Myc and more. Curr Top Microbiol Immunol 302: 63–122.
[21]
Nair SK, Burley SK (2003) X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors. Cell 112: 193–205.
[22]
Fisher F, Crouch DH, Jayaraman PS, Clark W, Gillespie DA, et al. (1993) Transcription activation by Myc and Max: flanking sequences target activation to a subset of CACGTG motifs in vivo. EMBO J 12: 5075–5082.
[23]
Crouch DH, Fisher F, Clark W, Jayaraman PS, Goding CR, et al. (1993) Gene-regulatory properties of Myc helix-loop-helix/leucine zipper mutants: Max-dependent DNA binding and transcriptional activation in yeast correlates with transforming capacity. Oncogene 8: 1849–1855.
[24]
Komashko VM, Acevedo LG, Squazzo SL, Iyengar SS, Rabinovich A, et al. (2008) Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells. Genome Res 18: 521–532.
[25]
Rabinovich A, Jin VX, Rabinovich R, Xu X, Farnham PJ (2008) E2F in vivo binding specificity: comparison of consensus versus nonconsensus binding sites. Genome Res 18: 1763–1777.
[26]
Krig SR, Jin VX, Bieda MC, O’Geen H, Yaswen P, et al. (2007) Identification of genes directly regulated by the oncogene ZNF217 using chromatin immunoprecipitation (ChIP)-chip assays. J Biol Chem 282: 9703–9712.
[27]
Messeguer X, Escudero R, Farre D, Nunez O, Martinez J, et al. (2002) PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics 18: 333–334.
[28]
Farre D, Roset R, Huerta M, Adsuara JE, Rosello L, et al. (2003) Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nucleic Acids Res 31: 3651–3653.
[29]
Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, et al. (2003) DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4: P3.
[30]
Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44–57.
[31]
Migheli A, Piva R, Casolino S, Atzori C, Dlouhy SR, et al. (1999) A cell cycle alteration precedes apoptosis of granule cell precursors in the weaver mouse cerebellum. Am J Pathol 155: 365–373.
[32]
Meyer N, Kim SS, Penn LZ (2006) The Oscar-worthy role of Myc in apoptosis. Semin Cancer Biol 16: 275–287.
[33]
Boone DN, Qi Y, Li Z, Hann SR (2011) Egr1 mediates p53-independent c-Myc-induced apoptosis via a noncanonical ARF-dependent transcriptional mechanism. Proc Natl Acad Sci U S A 108: 632–637.
[34]
Orian A, van Steensel B, Delrow J, Bussemaker HJ, Li L, et al. (2003) Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev 17: 1101–1114.
[35]
Steiger D, Furrer M, Schwinkendorf D, Gallant P (2008) Max-independent functions of Myc in Drosophila melanogaster. Nat Genet 40: 1084–1091.
[36]
O’Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ (2006) Comparison of sample preparation methods for ChIP-chip assays. Biotechniques 41: 577–580.
[37]
Barrio S, Gallardo M, Albizua E, Jimenez A, Rapado I, et al. (2011) Epigenomic profiling in polycythaemia vera and essential thrombocythaemia shows low levels of aberrant DNA methylation. J Clin Pathol.
[38]
Wolyniec K, Wotton S, Kilbey A, Jenkins A, Terry A, et al. (2009) RUNX1 and its fusion oncoprotein derivative, RUNX1-ETO, induce senescence-like growth arrest independently of replicative stress. Oncogene 28: 2502–2512.
[39]
Nasir A, Helm J, Turner L, Chen DT, Strosberg J, et al. (2011) RUNX1T1: a novel predictor of liver metastasis in primary pancreatic endocrine neoplasms. Pancreas 40: 627–633.
[40]
Piya S, Moon AR, Song PI, Hiscott J, Lin R, et al. (2011) Suppression of IRF4 by IRF1, 3, and 7 in Noxa Expression is a necessary event for IFN–mediated Tumor Elimination{gamma}. Mol Cancer Res.
[41]
Krieg AJ, Hammond EM, Giaccia AJ (2006) Functional analysis of p53 binding under differential stresses. Mol Cell Biol 26: 7030–7045.
[42]
Amin MA, Matsunaga S, Ma N, Takata H, Yokoyama M, et al. (2007) Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells. Biochem Biophys Res Commun 360: 320–326.
[43]
Koh CM, Gurel B, Sutcliffe S, Aryee MJ, Schultz D, et al. (2011) Alterations in nucleolar structure and gene expression programs in prostatic neoplasia are driven by the MYC oncogene. Am J Pathol 178: 1824–1834.
[44]
Sibella-Arguelles C (2001) The proliferation of human T lymphoblastic cells induced by 5-HT1B receptors activation is regulated by 5-HT-moduline. C R Acad Sci III 324: 365–372.
[45]
Stewart A, Guan H, Yang K (2010) BMP-3 promotes mesenchymal stem cell proliferation through the TGF-beta/activin signaling pathway. J Cell Physiol 223: 658–666.
[46]
Chen XR, Wang JW, Li X, Zhang H, Ye ZY (2010) Role of BMP3 in progression of gastric carcinoma in Chinese people. World J Gastroenterol 16: 1409–1413.
[47]
Shan J, Zhao W, Gu W (2009) Suppression of cancer cell growth by promoting cyclin D1 degradation. Mol Cell 36: 469–476.
[48]
Allende-Vega N, Sparks A, Lane DP, Saville MK (2010) MdmX is a substrate for the deubiquitinating enzyme USP2a. Oncogene 29: 432–441.
[49]
Collins VP (1995) Gene amplification in human gliomas. Glia 15: 289–296.
[50]
McCabe MG, Ichimura K, Liu L, Plant K, Backlund LM, et al. (2006) High-resolution array-based comparative genomic hybridization of medulloblastomas and supratentorial primitive neuroectodermal tumors. J Neuropathol Exp Neurol 65: 549–561.
