Tumor Biology

, Volume 32, Issue 1, pp 113–127 | Cite as

Expression and epigenetic modulation of sonic hedgehog-GLI1 pathway genes in neuroblastoma cell lines and tumors

  • Mehdi H. Shahi
  • Paula Schiapparelli
  • Mohammad Afzal
  • Subrata Sinha
  • Juan A. Rey
  • Javier S. Castresana
Research Article


It is well known that sonic hedgehog signaling pathway plays a vital role during early embryonic development. It is also responsible for stem cell renewal and development of several cancers like colorectal and breast carcinoma and major brain tumors as medulloblastoma and glioblastoma. The role of sonic hedgehog signaling in the development of neuroblastoma has not been thoroughly investigated. In this study, we attempted to determine the expression of Bmi-1 stem cell marker and of Shh pathway downstream target genes glioma-associated oncogene homolog 1 (GLI1), protein patched homolog 1 (PTCH1), Cyclin D2, plakoglobin (γ catenin), NK2 homeobox 2 (NKX2.2), paired box gene 6 (PAX6), secreted frizzled-related protein 1 (SFRP1), and hedgehog interacting protein (HHIP) in 11 neuroblastoma cell lines and 41 neuroblastoma samples. Also, inhibition of the pathway was performed genetically by GLI1 knockdown siRNA or chemically by cyclopamine. After inhibition, low transcript expression was detected in downstream target genes like PTCH1, in the cell lines. We further preformed promoter methylation studies of Cyclin D2, PTCH1, HHIP, and SFRP1 genes by melting curve analysis-based methylation assay (MCA-Meth) and methylation-specific PCR (MSP). Results revealed no methylation in Cyclin D2 gene promoter in neuroblastoma samples or in cell lines; one cell line (MHH-NB-11) showed PTCH1 methylation; 3/11 (27%) cell lines and 9/41 (22%) neuroblastoma samples showed HHIP methylation; and 3/11 (27%) cell lines and 11/41 (27%) samples showed SFRP1 methylation. Taken together, our results suggest the possibility of two levels of control of the sonic hedgehog signaling pathway: transcriptional and epigenetic, which might offer new therapeutic possibilities to modulate the pathway and try to suppress tumor growth.


Sonic hedgehog Neuroblastoma Methylation Cyclopamine GLI1 



Dulbeco’s modified Eagle’s medium




Hedgehog interacting protein


Melting Curve Analysis-based methylation assay


Methylation-specific PCR


Quantitative (real-time) reverse transcribed-PCR


Secreted frizzled-related protein 1


Sonic hedgehog



Authors are grateful to Laura Stokes for help with editing the manuscript, to Dr. Paula Lázcoz for assisting during culture of neuroblastoma cell lines, and to CIMA, Pamplona, Spain for providing FACS facility. M.H. Shahi was a fellow of AECI (Agencia Española de Cooperación Internacional), Madrid, Spain. J.S. Castresana expresses his gratitude to the Asociación Española de Pediatría, Madrid, for the VIII Premio Nutribén de Investigación Pediátrica. This research was supported in part by grants from the Departmento de Salud del Gobierno de Navarra (9/07), Caja Navarra (08/13912), and Fundación Universitaria de Navarra, Pamplona; Fondo de Investigación Sanitaria (PI081849), and Fundación Mapfre Medicina, Madrid.


  1. 1.
    Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 2003;3:203–16.CrossRefPubMedGoogle Scholar
  2. 2.
    Ruiz i Altaba A. The works of GLI and the power of hedgehog. Nat Cell Biol. 1999;1:E147–8.CrossRefPubMedGoogle Scholar
  3. 3.
    Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001;15:3059–87.CrossRefPubMedGoogle Scholar
  4. 4.
    Ding Q, Fukami S, Meng X, Nishizaki Y, Zhang X, Sasaki H, et al. Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of GLI1. Curr Biol. 1999;9:1119–22.CrossRefPubMedGoogle Scholar
  5. 5.
    Dai P, Akimaru H, Tanaka Y, Maekawa T, Nakafuku M, Ishii S. Sonic hedgehog-induced activation of the gli1 promoter is mediated by GLI3. J Biol Chem. 1999;274:8143–52.CrossRefPubMedGoogle Scholar
  6. 6.
    Ruiz i Altaba A. Gli proteins encode context-dependent positive and negative functions: implications for development and disease. Development. 1999;126:3205–16.PubMedGoogle Scholar
  7. 7.
    Kinzler KW, Bigner SH, Bigner DD, Trent JM, Law ML, O’Brien SJ, et al. Identification of an amplified, highly expressed gene in a human glioma. Science. 1987;236:70–3.CrossRefPubMedGoogle Scholar
  8. 8.
    Yoon JW, Kita Y, Frank DJ, Majewski RR, Konicek BA, Nobrega MA, et al. Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation. J Biol Chem. 2002;277:5548–55.CrossRefPubMedGoogle Scholar
  9. 9.
    Chuang PT, McMahon AP. Vertebrate hedgehog signalling modulated by induction of a hedgehog-binding protein. Nature. 1999;397:617–21.CrossRefPubMedGoogle Scholar
  10. 10.
    Katoh Y, Katoh M. Hedgehog signaling pathway and gastrointestinal stem cell signaling network. Int J Mol Med. 2006;18:1019–23.PubMedGoogle Scholar
  11. 11.
    Martin ST, Sato N, Dhara S, Chang R, Hustinx SR, Abe T, et al. Aberrant methylation of the Human hedgehog interacting protein (HHIP) gene in pancreatic neoplasms. Cancer Biol Ther. 2005;4:728–33.CrossRefPubMedGoogle Scholar
  12. 12.
    Olsen CL, Hsu PP, Glienke J, Rubanyi GM, Brooks AR. Hedgehog-interacting protein is highly expressed in endothelial cells but down-regulated during angiogenesis and in several human tumors. BMC Cancer. 2004;4:43.CrossRefPubMedGoogle Scholar
  13. 13.
    Finch PW, He X, Kelley MJ, Uren A, Schaudies RP, Popescu NC, et al. Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action. Proc Natl Acad Sci USA. 1997;94:6770–5.CrossRefPubMedGoogle Scholar
  14. 14.
    Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, Weijenberg MP, et al. A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet. 2002;31:141–9.CrossRefPubMedGoogle Scholar
  15. 15.
    Ingram WJ, Wicking CA, Grimmond SM, Forrest AR, Wainwright BJ. Novel genes regulated by sonic hedgehog in pluripotent mesenchymal cells. Oncogene. 2002;21:8196–205.CrossRefPubMedGoogle Scholar
  16. 16.
    Shih YL, Shyu RY, Hsieh CB, Lai HC, Liu KY, Chu TY, et al. Promoter methylation of the secreted frizzled-related protein 1 gene SFRP1 is frequent in hepatocellular carcinoma. Cancer. 2006;107:579–90.CrossRefPubMedGoogle Scholar
  17. 17.
    Cheng YY, Yu J, Wong YP, Man EP, To KF, Jin VX, et al. Frequent epigenetic inactivation of secreted frizzled-related protein 2 (SFRP2) by promoter methylation in human gastric cancer. Br J Cancer. 2007;97:895–901.PubMedGoogle Scholar
  18. 18.
    Stoehr R, Wissmann C, Suzuki H, Knuechel R, Krieg RC, Klopocki E, et al. Deletions of chromosome 8p and loss of SFRP1 expression are progression markers of papillary bladder cancer. Lab Invest. 2004;84:465–78.CrossRefPubMedGoogle Scholar
  19. 19.
    Joesting MS, Perrin S, Elenbaas B, Fawell SE, Rubin JS, Franco OE, et al. Identification of SFRP1 as a candidate mediator of stromal-to-epithelial signaling in prostate cancer. Cancer Res. 2005;65:10423–30.CrossRefPubMedGoogle Scholar
  20. 20.
    Zhang YW, Miao YF, Yi J, Geng J, Wang R, Chen LB. Transcriptional inactivation of secreted frizzled-related protein 1 by promoter hypermethylation as a potential biomarker for non-small cell lung cancer. Neoplasma. 2010;57:228–33.CrossRefPubMedGoogle Scholar
  21. 21.
    Lee Y, Miller HL, Jensen P, Hernan R, Connelly M, Wetmore C, et al. A molecular fingerprint for medulloblastoma. Cancer Res. 2003;63:5428–37.PubMedGoogle Scholar
  22. 22.
    Briscoe J, Sussel L, Serup P, Hartigan-O’Connor D, Jessell TM, Rubenstein JL, et al. Homeobox gene Nkx2.2 and specification of neuronal identity by graded sonic hedgehog signalling. Nature. 1999;398:622–7.CrossRefPubMedGoogle Scholar
  23. 23.
    Colin C, Virard I, Baeza N, Tchoghandjian A, Fernandez C, Bouvier C, et al. Relevance of combinatorial profiles of intermediate filaments and transcription factors for glioma histogenesis. Neuropathol Appl Neurobiol. 2007;33:431–9.CrossRefPubMedGoogle Scholar
  24. 24.
    Stuart JJ, Brown SJ, Beeman RW, Denell RE. The Tribolium homeotic gene abdominal is homologous to abdominal-A of the drosophila bithorax complex. Development. 1993;117:233–43.PubMedGoogle Scholar
  25. 25.
    Salem CE, Markl ID, Bender CM, Gonzales FA, Jones PA, Liang G. PAX6 methylation and ectopic expression in human tumor cells. Int J Cancer. 2000;87:179–85.CrossRefPubMedGoogle Scholar
  26. 26.
    Mansouri A, Hallonet M, Gruss P. Pax genes and their roles in cell differentiation and development. Curr Opin Cell Biol. 1996;8:851–7.CrossRefPubMedGoogle Scholar
  27. 27.
    Mayes DA, Hu Y, Teng Y, Siegel E, Wu X, Panda K, et al. PAX6 suppresses the invasiveness of glioblastoma cells and the expression of the matrix metalloproteinase-2 gene. Cancer Res. 2006;66:9809–17.CrossRefPubMedGoogle Scholar
  28. 28.
    Zhou YH, Wu X, Tan F, Shi YX, Glass T, Liu TJ, et al. PAX6 suppresses growth of human glioblastoma cells. J Neurooncol. 2005;71:223–9.CrossRefPubMedGoogle Scholar
  29. 29.
    Kopper L, Hajdu M. Tumor stem cells. Pathol Oncol Res. 2004;10:69–73.CrossRefPubMedGoogle Scholar
  30. 30.
    Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, et al. Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci USA. 2003;100:15178–83.CrossRefPubMedGoogle Scholar
  31. 31.
    Leung C, Lingbeek M, Shakhova O, Liu J, Tanger E, Saremaslani P, et al. Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas. Nature. 2004;428:337–41.CrossRefPubMedGoogle Scholar
  32. 32.
    Li DW, Tang HM, Fan JW, Yan DW, Zhou CZ, Li SX, et al. Expression level of bmi-1 oncoprotein is associated with progression and prognosis in colon cancer. J Cancer Res Clin Oncol. 2010;136:997–1006.CrossRefPubMedGoogle Scholar
  33. 33.
    Singh AK, Lockett MA, Bradley JD. Predictors of radiation-induced esophageal toxicity in patients with non-small-cell lung cancer treated with three-dimensional conformal radiotherapy. Int J Radiat Oncol Biol Phys. 2003;55:337–41.CrossRefPubMedGoogle Scholar
  34. 34.
    Sasaki M, Ikeda H, Itatsu K, Yamaguchi J, Sawada S, Minato H, et al. The overexpression of polycomb group proteins Bmi1 and EZH2 is associated with the progression and aggressive biological behavior of hepatocellular carcinoma. Lab Invest. 2008;88:873–82.CrossRefPubMedGoogle Scholar
  35. 35.
    Mihic-Probst D, Kuster A, Kilgus S, Bode-Lesniewska B, Ingold-Heppner B, Leung C, et al. Consistent expression of the stem cell renewal factor BMI-1 in primary and metastatic melanoma. Int J Cancer. 2007;121:1764–70.CrossRefPubMedGoogle Scholar
  36. 36.
    Reinisch CM, Uthman A, Erovic BM, Pammer J. Expression of BMI-1 in normal skin and inflammatory and neoplastic skin lesions. J Cutan Pathol. 2007;34:174–80.CrossRefPubMedGoogle Scholar
  37. 37.
    Molofsky AV, He S, Bydon M, Morrison SJ, Pardal R. Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16ink4a and p19Arf senescence pathways. Genes Dev. 2005;19:1432–7.CrossRefPubMedGoogle Scholar
  38. 38.
    Meyyappan M, Wong H, Hull C, Riabowol KT. Increased expression of cyclin d2 during multiple states of growth arrest in primary and established cells. Mol Cell Biol. 1998;18:3163–72.PubMedGoogle Scholar
  39. 39.
    Barber RD, Harmer DW, Coleman RA, Clark BJ. Gapdh as a housekeeping gene: analysis of gapdh mrna expression in a panel of 72 human tissues. Physiol Genomics. 2005;21:389–95.CrossRefPubMedGoogle Scholar
  40. 40.
    Agren M, Kogerman P, Kleman MI, Wessling M, Toftgard R. Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional GLI-binding site. Gene. 2004;330:101–14.CrossRefPubMedGoogle Scholar
  41. 41.
    Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996;93:9821–6.CrossRefPubMedGoogle Scholar
  42. 42.
    Evron E, Umbricht CB, Korz D, Raman V, Loeb DM, Niranjan B, et al. Loss of cyclin d2 expression in the majority of breast cancers is associated with promoter hypermethylation. Cancer Res. 2001;61:2782–7.PubMedGoogle Scholar
  43. 43.
    Shahi MH, Lorente A, Castresana JS. Hedgehog signalling in medulloblastoma, glioblastoma and neuroblastoma. Oncol Rep. 2008;19:681–8.PubMedGoogle Scholar
  44. 44.
    Brooks AR, Shiffman D, Chan CS, Brooks EE, Milner PG. Functional analysis of the human cyclin d2 and cyclin d3 promoters. J Biol Chem. 1996;271:9090–9.CrossRefPubMedGoogle Scholar
  45. 45.
    Katoh Y, Katoh M. Identification and characterization of DISP3 gene in silico. Int J Oncol. 2005;26:551–6.PubMedGoogle Scholar
  46. 46.
    Flora A, Klisch TJ, Schuster G, Zoghbi HY. Deletion of Atoh1 disrupts sonic hedgehog signaling in the developing cerebellum and prevents medulloblastoma. Science. 2009;326:1424–7.CrossRefPubMedGoogle Scholar
  47. 47.
    Ayrault O, Zhao H, Zindy F, Qu C, Sherr CJ, Roussel MF. Atoh1 inhibits neuronal differentiation and collaborates with gli1 to generate medulloblastoma-initiating cells. Cancer Res. 2010;70:5618–27.CrossRefPubMedGoogle Scholar
  48. 48.
    Mao L, Xia YP, Zhou YN, Dai RL, Yang X, Duan SJ, et al. A critical role of sonic hedgehog signaling in maintaining the tumorigenicity of neuroblastoma cells. Cancer Sci. 2009;100:1848–55.CrossRefPubMedGoogle Scholar
  49. 49.
    Gry M, Rimini R, Stromberg S, Asplund A, Ponten F, Uhlen M, et al. Correlations between RNA and protein expression profiles in 23 human cell lines. BMC Genomics. 2009;10:365.CrossRefPubMedGoogle Scholar
  50. 50.
    Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503–11.CrossRefPubMedGoogle Scholar
  51. 51.
    Biedler JL, Roffler-Tarlov S, Schachner M, Freedman LS. Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res. 1978;38:3751–7.PubMedGoogle Scholar
  52. 52.
    Biedler JL, Spengler BA. Metaphase chromosome anomaly: association with drug resistance and cell-specific products. Science. 1976;191:185–7.CrossRefPubMedGoogle Scholar
  53. 53.
    Biedler JL, Spengler BA. A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cell lives in culture. J Natl Cancer Inst. 1976;57:683–95.PubMedGoogle Scholar
  54. 54.
    Marini P, MacLeod RA, Treuner C, Bruchelt G, Bohm W, Wolburg H, et al. Sima, a new neuroblastoma cell line combining poor prognostic cytogenetic markers with high adrenergic differentiation. Cancer Genet Cytogenet. 1999;112:161–4.CrossRefPubMedGoogle Scholar
  55. 55.
    Pritchard JI, Olson JM. Methylation of PTCH1, the Patched-1 gene, in a panel of primary medulloblastomas. Cancer Genet Cytogenet. 2008;180:47–50.CrossRefPubMedGoogle Scholar
  56. 56.
    Wolf I, Bose S, Desmond JC, Lin BT, Williamson EA, Karlan BY, et al. Unmasking of epigenetically silenced genes reveals DNA promoter methylation and reduced expression of PTCH in breast cancer. Breast Cancer Res Treat. 2007;105:139–55.CrossRefPubMedGoogle Scholar
  57. 57.
    Diccianni MB, Omura-Minamisawa M, Batova A, Le T, Bridgeman L, Yu AL. Frequent deregulation of p16 and the p16/g1 cell cycle-regulatory pathway in neuroblastoma. Int J Cancer. 1999;80:145–54.CrossRefPubMedGoogle Scholar
  58. 58.
    Bullions LC, Levine AJ. The role of beta-catenin in cell adhesion, signal transduction, and cancer. Curr Opin Oncol. 1998;10:81–7.CrossRefPubMedGoogle Scholar
  59. 59.
    Amitay R, Nass D, Meitar D, Goldberg I, Davidson B, Trakhtenbrot L, et al. Reduced expression of plakoglobin correlates with adverse outcome in patients with neuroblastoma. Am J Pathol. 2001;159:43–9.PubMedGoogle Scholar
  60. 60.
    Ericson J, Rashbass P, Schedl A, Brenner-Morton S, Kawakami A, van Heyningen V, et al. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell. 1997;90:169–80.CrossRefPubMedGoogle Scholar
  61. 61.
    Vokes SA, Ji H, McCuine S, Tenzen T, Giles S, Zhong S, et al. Genomic characterization of GLI-activator targets in sonic hedgehog-mediated neural patterning. Development. 2007;134:1977–89.CrossRefPubMedGoogle Scholar
  62. 62.
    Briscoe J, Pierani A, Jessell TM, Ericson J. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell. 2000;101:435–45.CrossRefPubMedGoogle Scholar
  63. 63.
    Caldwell GM, Jones C, Gensberg K, Jan S, Hardy RG, Byrd P, et al. The Wnt antagonist SFRP1 in colorectal tumorigenesis. Cancer Res. 2004;64:883–8.CrossRefPubMedGoogle Scholar
  64. 64.
    Bak M, Hansen C, Friis Henriksen K, Tommerup N. The Human hedgehog-interacting protein gene: structure and chromosome mapping to 4q31.21--> q31.3. Cytogenet Cell Genet. 2001;92:300–3.CrossRefPubMedGoogle Scholar
  65. 65.
    Cui H, Hu B, Li T, Ma J, Alam G, Gunning WT, et al. Bmi-1 is essential for the tumorigenicity of neuroblastoma cells. Am J Pathol. 2007;170:1370–8.CrossRefPubMedGoogle Scholar
  66. 66.
    Mao L, Xia YP, Zhou YN, Dai RL, Yang X, Wang YJ, et al. Activation of sonic hedgehog signaling pathway in olfactory neuroblastoma. Oncology. 2009;77:231–43.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2010

Authors and Affiliations

  • Mehdi H. Shahi
    • 1
    • 2
    • 3
  • Paula Schiapparelli
    • 1
  • Mohammad Afzal
    • 2
  • Subrata Sinha
    • 3
  • Juan A. Rey
    • 4
  • Javier S. Castresana
    • 1
    • 5
  1. 1.Brain Tumor Biology Unit-CIFAUniversity of Navarra School of SciencesPamplonaSpain
  2. 2.Department of ZoologyAligarh Muslim UniversityAligarhIndia
  3. 3.Department of BiochemistryAll India Institute of Medical SciencesNew DelhiIndia
  4. 4.Research UnitLa Paz University HospitalMadridSpain
  5. 5.Unidad de Biología de Tumores Cerebrales, CIFAUniversidad de NavarraPamplonaSpain

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