Skip to main content

Advertisement

SpringerLink
Log in

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

  • Research Article
  • Published:
Tumor Biology

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

DMEM:

Dulbeco’s modified Eagle’s medium

Hh:

Hedgehog

HHIP:

Hedgehog interacting protein

MCA-Meth:

Melting Curve Analysis-based methylation assay

MSP:

Methylation-specific PCR

qRT-PCR:

Quantitative (real-time) reverse transcribed-PCR

SFRP1:

Secreted frizzled-related protein 1

SHH:

Sonic hedgehog

References

  1. Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 2003;3:203–16.

    Article  CAS  PubMed  Google Scholar 

  2. Ruiz i Altaba A. The works of GLI and the power of hedgehog. Nat Cell Biol. 1999;1:E147–8.

    Article  CAS  PubMed  Google Scholar 

  3. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001;15:3059–87.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  6. Ruiz i Altaba A. Gli proteins encode context-dependent positive and negative functions: implications for development and disease. Development. 1999;126:3205–16.

    PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  9. Chuang PT, McMahon AP. Vertebrate hedgehog signalling modulated by induction of a hedgehog-binding protein. Nature. 1999;397:617–21.

    Article  CAS  PubMed  Google Scholar 

  10. Katoh Y, Katoh M. Hedgehog signaling pathway and gastrointestinal stem cell signaling network. Int J Mol Med. 2006;18:1019–23.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  29. Kopper L, Hajdu M. Tumor stem cells. Pathol Oncol Res. 2004;10:69–73.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  43. Shahi MH, Lorente A, Castresana JS. Hedgehog signalling in medulloblastoma, glioblastoma and neuroblastoma. Oncol Rep. 2008;19:681–8.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  45. Katoh Y, Katoh M. Identification and characterization of DISP3 gene in silico. Int J Oncol. 2005;26:551–6.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  52. Biedler JL, Spengler BA. Metaphase chromosome anomaly: association with drug resistance and cell-specific products. Science. 1976;191:185–7.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  58. Bullions LC, Levine AJ. The role of beta-catenin in cell adhesion, signal transduction, and cancer. Curr Opin Oncol. 1998;10:81–7.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  1. Brain Tumor Biology Unit-CIFA, University of Navarra School of Sciences, Pamplona, Spain

    Mehdi H. Shahi, Paula Schiapparelli & Javier S. Castresana

  2. Department of Zoology, Aligarh Muslim University, Aligarh, India

    Mehdi H. Shahi & Mohammad Afzal

  3. Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India

    Mehdi H. Shahi & Subrata Sinha

  4. Research Unit, La Paz University Hospital, Madrid, Spain

    Juan A. Rey

  5. Unidad de Biología de Tumores Cerebrales, CIFA, Universidad de Navarra, Irunlarrea 1, 31008, Pamplona, Spain

    Javier S. Castresana

Authors
  1. Mehdi H. Shahi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paula Schiapparelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohammad Afzal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Subrata Sinha
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan A. Rey
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Javier S. Castresana
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Javier S. Castresana.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shahi, M.H., Schiapparelli, P., Afzal, M. et al. Expression and epigenetic modulation of sonic hedgehog-GLI1 pathway genes in neuroblastoma cell lines and tumors. Tumor Biol. 32, 113–127 (2011). https://doi.org/10.1007/s13277-010-0105-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-010-0105-x

Keywords

Search

Navigation