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Molecular Medicine

, Volume 17, Issue 1–2, pp 103–112 | Cite as

Inhibition of Sonic Hedgehog and Notch Pathways Enhances Sensitivity of CD133+ Glioma Stem Cells to Temozolomide Therapy

  • Ilya V. Ulasov
  • Suvobroto Nandi
  • Mahua Dey
  • Adam M. Sonabend
  • Maciej S. Lesniak
Research Article

Abstract

Malignant gliomas are currently treated with temozolomide (TMZ), but often exhibit resistance to this agent. CD133+ cancer stem cells, a population believed to contribute to the tumor’s chemoresistance, bear the activation of Notch and Sonic hedgehog (SHH) pathways. In this study, we examined whether inhibition of both pathways enhances the efficacy of TMZ monotherapy in the context of glioma stem cells. Transcriptional analysis of Notch and SHH pathways in CD133+-enriched glioma cell populations showed the activity of these pathways. CD133+ cells were less susceptible to TMZ treatment than the unsorted glioma counterparts. Interestingly, Notch and SHH pathway transcriptional activity in CD133+ glioma cells was further enhanced by TMZ exposure, which led to NOTCH 1, NCOR2, and GLI1 upregulation (6.64-, 3.73-, and 2.79-fold, respectively) and CFLAR downregulation (4.22-fold). The therapeutic effect of TMZ was enhanced by Notch and SHH pathway pharmacological antagonism with GSI-1 and cyclopamine. More importantly, simultaneous treatment involving TMZ with both of these compounds led to a significant increase in CD133+ glioma cytotoxicity than treatment with any of these agents alone (P < 0.05). In conclusion, CD133+ glioma cells overexpress genes involved in Notch and SHH pathways. These pathways contribute to the chemoresistant phenotype of CD133+ glioma cells, as their antagonism leads to an additive effect when used in combination with TMZ.

Notes

Acknowledgments

This work was supported by the National Cancer Institute (R01-CA122930, R01-CA138587, R21-CA135728), the National Institute of Neurological Disorders and Stroke (K08-NS046430), The Alliance for Cancer Gene Therapy Young Investigator Award and the American Cancer Society (RSG-07-276-01-MGO). We are thankful to the Flow Core Facility (the University of Chicago) for help with separation of tumor cells.

Supplementary material

10020_2011_1701103_MOESM1_ESM.pdf (620 kb)
Supplementary material, approximately 421 KB.

References

  1. 1.
    Hegi ME, et al. (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N. Engl. J. Med. 352:997–1003.CrossRefGoogle Scholar
  2. 2.
    Stupp R, et al. (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352:987–96.CrossRefGoogle Scholar
  3. 3.
    Branch P, Aquilina G, Bignami M, Karran P. (1993) Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature 362:652–54.CrossRefGoogle Scholar
  4. 4.
    Dosch J, Christmann M, Kaina B. (1998) Mismatch G-T binding activity and MSH2 expression is quantitatively related to sensitivity of cells to methylating agents. Carcinogenesis 19:567–73.CrossRefGoogle Scholar
  5. 5.
    Yip S, et al. (2009) MSH6 mutations arise in glioblastomas during temozolomide therapy and mediate temozolomide resistance. Clin. Cancer Res. 15:4622–9.CrossRefGoogle Scholar
  6. 6.
    Chua C, et al. (2008) Characterization of a side population of astrocytoma cells in response to temozolomide. J. Neurosurg. 109:856–66.CrossRefGoogle Scholar
  7. 7.
    Bleau AM, et al. (2009) PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell. 4:226–35.CrossRefGoogle Scholar
  8. 8.
    Bao S, et al. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:756–60.CrossRefGoogle Scholar
  9. 9.
    Pollard SM, et al. (2009) Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell. 4:568–80.CrossRefGoogle Scholar
  10. 10.
    Palos TP, Zheng S, Howard BD. (1999) Wnt signaling induces GLT-1 expression in rat C6 glioma cells. J. Neurochem. 73:1012–23.CrossRefGoogle Scholar
  11. 11.
    Sareddy GR, Challa S, Panigrahi M, Babu PP. (2009) Wnt/beta-catenin/Tcf signaling pathway activation in malignant progression of rat gliomas induced by transplacental N-ethyl-N-nitrosourea exposure. Neurochem. Res. 34:1278–88.CrossRefGoogle Scholar
  12. 12.
    Shou J, Ali-Osman F, Multani AS, Pathak S, Fedi P, Srivenugopal KS. (2002) Human Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its overexpression sensitizes brain tumor cells to apoptosis following alkylation damage of DNA. Oncogene 21:878–89.CrossRefGoogle Scholar
  13. 13.
    Dahmane N, et al. (2001) The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development 128:5201–12.PubMedGoogle Scholar
  14. 14.
    Ehtesham M, et al. (2007) Ligand-dependent activation of the hedgehog pathway in glioma progenitor cells. Oncogene 26:5752–61.CrossRefGoogle Scholar
  15. 15.
    Clement V, Sanchez P, de Tribolet N, Radovanovic I, Ruiz i Altaba A. (2007) HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr. Biol. 17:165–72.CrossRefGoogle Scholar
  16. 16.
    Artavanis-Tsakonas S, Rand MD, Lake RJ. (1999) Notch signaling: cell fate control and signal integration in development. Science 284:770–76.CrossRefGoogle Scholar
  17. 17.
    Miele L, Osborne B. (1999) Arbiter of differentiation and death: Notch signaling meets apoptosis. J Cell Physiol 181:393–409.CrossRefGoogle Scholar
  18. 18.
    Purow BW, et al. (2005) Expression of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma cell survival and proliferation. Cancer Res. 65:2353–63.CrossRefGoogle Scholar
  19. 19.
    Zhang XP, et al. (2008) Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells. Mol. Cell. Biochem. 307:101–8.CrossRefGoogle Scholar
  20. 20.
    Lee J, et al. (2006) Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell. 9:391–403.CrossRefGoogle Scholar
  21. 21.
    Bao S, et al. (2008) Targeting cancer stem cells through L1CAM suppresses glioma growth. Cancer Res. 68:6043–8.CrossRefGoogle Scholar
  22. 22.
    Wang J, et al. (2008) c-Myc is required for maintenance of glioma cancer stem cells. PLoS One 3:e3769.CrossRefGoogle Scholar
  23. 23.
    Sodsai P, Hirankarn N, Avihingsanon Y, Palaga T. (2008) Defects in Notch1 upregulation upon activation of T cells from patients with systemic lupus erythematosus are related to lupus disease activity. Lupus 17:645–53.CrossRefGoogle Scholar
  24. 24.
    Karlsson C, et al. (2007) Differentiation of human mesenchymal stem cells and articular chondrocytes: analysis of chondrogenic potential and expression pattern of differentiation-related transcription factors. J. Orthop. Res. 25:152–63.CrossRefGoogle Scholar
  25. 25.
    Platet N, et al. (2007) Influence of oxygen tension on CD133 phenotype in human glioma cell cultures. Cancer Lett. 258:286–90.CrossRefGoogle Scholar
  26. 26.
    Kwong J, Lo KW, To KF, Teo PM, Johnson PJ, Huang DP. (2002) Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. Clin. Cancer Res. 8:131–7.PubMedGoogle Scholar
  27. 27.
    Meloni AR, et al. (2006) Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2. Mol. Cell. Biol. 26:7550–60.CrossRefGoogle Scholar
  28. 28.
    Chou TC. (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 70:440–6.CrossRefGoogle Scholar
  29. 29.
    Joo KM, Nam DH. (2009) Prospective identification of cancer stem cells with the surface antigen CD133. Methods Mol. Biol. 568:57–71.CrossRefGoogle Scholar
  30. 30.
    Wu A, et al. (2008) Persistence of CD133+ cells in human and mouse glioma cell lines: detailed characterization of GL261 glioma cells with cancer stem cell-like properties. Stem Cells Dev. 17:173–84.CrossRefGoogle Scholar
  31. 31.
    Murat A, et al. (2008) Stem cell-related “self-renewal” signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma. J. Clin. Oncol. 26:3015–24.CrossRefGoogle Scholar
  32. 32.
    Liu G, et al. (2006) Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol. Cancer 5:67.CrossRefGoogle Scholar
  33. 33.
    Chen JK, Taipale J, Cooper MK, Beachy PA. (2002) Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 16:2743–8.CrossRefGoogle Scholar
  34. 34.
    Chen JK, Taipale J, Young KE, Maiti T, Beachy PA. (2002) Small molecule modulation of Smoothened activity. Proc. Natl. Acad. Sci. U. S. A. 99:14071–6.CrossRefGoogle Scholar
  35. 35.
    Taipale J, et al. (2000) Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406:1005–9.CrossRefGoogle Scholar
  36. 36.
    Fan X, et al. (2010) Notch pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts. Stem Cells 28:5–16.PubMedPubMedCentralGoogle Scholar
  37. 37.
    Tammam J, et al. (2009) Down-regulation of the Notch pathway mediated by a gamma-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia. Br. J. Pharmacol. 158:1183–1195.CrossRefGoogle Scholar
  38. 38.
    Chou TC, Talalay P. (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22:27–55.CrossRefGoogle Scholar
  39. 39.
    Weng AP, et al. (2003) Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol. Cell. Biol. 23:655–64.CrossRefGoogle Scholar
  40. 40.
    Bar EE, et al. (2007) Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma. Stem Cells 25:2524–33.CrossRefGoogle Scholar
  41. 41.
    Kanamori M, et al. (2007) Contribution of Notch signaling activation to human glioblastoma multiforme. J. Neurosurg. 106:417–27.CrossRefGoogle Scholar
  42. 42.
    Shih AH, Holland EC. (2006) Notch signaling enhances nestin expression in gliomas. Neoplasia 8:1072–82.CrossRefGoogle Scholar

Copyright information

© The Feinstein Institute for Medical Research 2011

Authors and Affiliations

  • Ilya V. Ulasov
    • 1
  • Suvobroto Nandi
    • 1
  • Mahua Dey
    • 1
  • Adam M. Sonabend
    • 1
  • Maciej S. Lesniak
    • 1
  1. 1.The Brain Tumor CenterThe University of ChicagoChicagoUSA

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