Tumor Biology

, Volume 37, Issue 5, pp 6935–6942 | Cite as

Aberrant expression of Notch1, HES1, and DTX1 genes in glioblastoma formalin-fixed paraffin-embedded tissues

  • Rajeswari Narayanappa
  • Pritilata Rout
  • Madhuri G. S. Aithal
  • Ashis Kumar Chand
Original Article


Glioblastoma is the most common malignant brain tumor accounting for more than 54 % of all gliomas. Despite aggressive treatments, median survival remains less than 1 year. This might be due to the unavailability of effective molecular diagnostic markers and targeted therapy. Thus, it is essential to discover molecular mechanisms underlying disease by identifying dysregulated pathways involved in tumorigenesis. Notch signaling is one such pathway which plays an important role in determining cell fates. Since it is found to play a critical role in many cancers, we investigated the role of Notch genes in glioblastoma with an aim to identify biomarkers that can improve diagnosis. Using real-time PCR, we assessed the expression of Notch genes including receptors (Notch1, Notch2, Notch3, and Notch4), ligands (JAG1, JAG2, and DLL3), downstream targets (HES1 and HEY2), regulator Deltex1 (DTX1), inhibitor NUMB along with transcriptional co-activator MAML1, and a component of gamma-secretase complex APH1A in 15 formalin-fixed paraffin-embedded (FFPE) patient samples. Relative quantification was done by the 2−ΔΔCt method; the data are presented as fold change in gene expression normalized to an internal control gene and relative to the calibrator. The data revealed aberrant expression of Notch genes in glioblastoma compared to normal brain. More than 85 % of samples showed high Notch1 (P = 0.0397) gene expression and low HES1 (P = 0.011) and DTX1 (P = 0.0001) gene expression. Our results clearly show aberrant expression of Notch genes in glioblastoma which can be used as putative biomarkers together with histopathological observation to improve diagnosis, therapeutic strategies, and patient prognosis.


Gene expression Notch genes Glioblastoma Biomarker Real-time PCR 



This study was funded by the Department of Science and Technology, New Delhi, India (grant number SR/FT/LS-170/2008). We thank the Brain Bank, NIMHANS, Bangalore, India, for providing the clinical samples.

Compliance with ethical standards

Conflicts of interest


Ethical approval

For this type of study, formal consent is not required.


  1. 1.
    Ostrom QT, Gittleman H, Liao P, et al. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007-2011. Neuro Oncol. 2014;16 Suppl 4:iv1–63.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Mischel PS, Vinters HV. Neuropathology and molecular pathogenesis of primary brain tumors. Brain Tumor Immunother. 2001;3–45.Google Scholar
  3. 3.
    Godard S, Getz G, Delorenzi M, et al. Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes. Cancer Res. 2003;63:6613–25.PubMedGoogle Scholar
  4. 4.
    Nutt CL, Mani DR, Betensky RA, et al. Gene expression-based classification of malignant gliomas correlates better with survival than histological classification. Cancer Res. 2003;63:1602–7.PubMedGoogle Scholar
  5. 5.
    Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell. 2009;137:216–33.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Furukawa T, Mukherjee S, Bao ZZ, et al. rax, Hes1, and Notch1 promote the formation of Müller glia by postnatal retinal progenitor cells. Neuron. 2000;26:383–94.CrossRefPubMedGoogle Scholar
  7. 7.
    Gaiano N, Fishell G. The role of Notch in promoting glial and neural stem cell fates. Annu Rev Neurosci. 2002;25:471–90.CrossRefPubMedGoogle Scholar
  8. 8.
    Li HS, Wang D, Shen Q, et al. Inactivation of Numb and Numblike in embryonic dorsal forebrain impairs neurogenesis and disrupts cortical morphogenesis. Neuron. 2003;40:1105–18.CrossRefPubMedGoogle Scholar
  9. 9.
    Rusanescu G, Mao J. Notch3 is necessary for neuronal differentiation and maturation in the adult spinal cord. J Cell Mol Med. 2014;18:2103–16.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Wilhelmsson U, Faiz M, de Pablo Y, et al. Astrocytes negatively regulate neurogenesis through the Jagged1-mediated Notch pathway. Stem Cells. 2012;30:2320–9.CrossRefPubMedGoogle Scholar
  11. 11.
    Presente A, Andres A, Nye JS. Requirement of Notch in adulthood for neurological function and longevity. Neuroreport. 2001;12:3321–5.CrossRefPubMedGoogle Scholar
  12. 12.
    Axelson H. Notch signaling and cancer: emerging complexity. Semin Cancer Biol. 2004;14:317–9.CrossRefPubMedGoogle Scholar
  13. 13.
    Hansson EM, Lendahl U, Chapman G. Notch signaling in development and disease. Semin Cancer Biol. 2004;14:320–8.CrossRefPubMedGoogle Scholar
  14. 14.
    Lobry C, Oh P, Aifantis I. Oncogenic and tumor suppressor functions of Notch in cancer: it’s NOTCH what you think. J Exp Med. 2011;208:1931–5.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Rahman MT, Nakayama K, Rahman M, et al. Notch3 overexpression as potential therapeutic target in advanced stage chemoresistant ovarian cancer. Am J Clin Pathol. 2012;138:535–44.CrossRefPubMedGoogle Scholar
  16. 16.
    Cui H, Kong Y, Xu M, et al. Notch3 functions as a tumor suppressor by controlling cellular senescence. Cancer Res. 2013;73:3451–9.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Baumgart A, Mazur PK, Anton M, et al. Opposing role of Notch1 and Notch2 in a KrasG12D-driven murine non-small cell lung cancer model. Oncogene. 2015;34:578–88.CrossRefPubMedGoogle Scholar
  18. 18.
    Purow BW, Haque RM, Noel MW, et al. Expression of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma cell survival and proliferation. Cancer Res. 2005;65:2353–63.CrossRefPubMedGoogle Scholar
  19. 19.
    Zhang XP, Zheng G, Zou L, et al. Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells. Mol Cell Biochem. 2008;307:101–8.CrossRefPubMedGoogle Scholar
  20. 20.
    Chen J, Kesari S, Rooney C, et al. Inhibition of notch signaling blocks growth of glioblastoma cell lines and tumor neurospheres. Genes Cancer. 2010;1:822–35.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hovinga KE, Shimizu F, Wang R, et al. Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate. Stem Cells. 2010;28:1019–29.CrossRefPubMedGoogle Scholar
  22. 22.
    Ridgway J, Zhang G, Wu Y, et al. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature. 2006;444:1083–7.CrossRefPubMedGoogle Scholar
  23. 23.
    Adepoju O, Wong A, Kitajewski A, et al. Expression of HES and HEY genes in infantile hemangiomas. Vasc Cell. 2011;3:19.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Viatour P, Ehmer U, Saddic LA, et al. Notch signaling inhibits hepatocellular carcinoma following inactivation of the RB pathway. J Exp Med. 2011;208:1963–76.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kizys MM, Cardoso MG, Lindsey SC, et al. Optimizing nucleic acid extraction from thyroid fine-needle aspiration cells in stained slides, formalin-fixed/paraffin-embedded tissues, and long-term stored blood samples. Arq Bras Endocrinol Metabol. 2012;56:618–26.CrossRefPubMedGoogle Scholar
  26. 26.
    Aithal MGS, Rajeswari N. Validation of housekeeping genes for gene expression analysis in glioblastoma using quantitative real-time polymerase chain reaction. Brain Tumor Res Treat. 2015;3:24–9.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Bolós V, Mira E, Martínez-Poveda B, et al. Notch activation stimulates migration of breast cancer cells and promotes tumor growth. Breast Cancer Res. 2013;15:R54.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Guo D, Li C, Teng Q, et al. Notch1 overexpression promotes cell growth and tumor angiogenesis in myeloma. Neoplasma. 2013;60:33–40.CrossRefPubMedGoogle Scholar
  29. 29.
    Natarajan S, Li Y, Miller EE, et al. Notch1-induced brain tumor models the sonic hedgehog subgroup of human medulloblastoma. Cancer Res. 2013;73:5381–90.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Ahn S, Hyeon J, Park CK. Notch1 and Notch4 are markers for poor prognosis of hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int. 2013;12:286–94.CrossRefPubMedGoogle Scholar
  31. 31.
    Yousif NG, Sadiq AM, Yousif MG, et al. Notch1 ligand signaling pathway activated in cervical cancer: poor prognosis with high-level JAG1/Notch1. Arch Gynecol Obstet. 2015. doi: 10.1007/s00404-015-3694-1.PubMedGoogle Scholar
  32. 32.
    Sasnauskienė A, Jonušienė V, Krikštaponienė A, et al. NOTCH1, NOTCH3, NOTCH4, and JAG2 protein levels in human endometrial cancer. Medicina (Kaunas). 2014;50:14–8.CrossRefGoogle Scholar
  33. 33.
    Ye QF, Zhang YC, Peng XQ, et al. Silencing Notch-1 induces apoptosis and increases the chemosensitivity of prostate cancer cells to docetaxel through Bcl-2 and Bax. Oncol Lett. 2012;3:879–84.PubMedPubMedCentralGoogle Scholar
  34. 34.
    Wei G, Chang Y, Zheng J, et al. Notch1 silencing inhibits proliferation and invasion in SGC-7901 gastric cancer cells. Mol Med Rep. 2014;9:1153–8.PubMedGoogle Scholar
  35. 35.
    Chen AC, Guo LY, Ostaszewski BL, et al. Aph-1 associates directly with full-length and C-terminal fragments of gamma-secretase substrates. J Biol Chem. 2010;285:11378–91.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Bae SK, Bessho Y, Hojo M, et al. The bHLH gene Hes6, an inhibitor of Hes1, promotes neuronal differentiation. Development. 2000;127:2933–43.PubMedGoogle Scholar
  37. 37.
    Gibert JM, Simpson P. Evolution of cis-regulation of the proneural genes. Int J Dev Biol. 2003;47:643–51.PubMedGoogle Scholar
  38. 38.
    Luo B, Aster JC, Hasserjian RP, et al. Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor. Mol Cell Biol. 1997;17:6057–67.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Wu L, Griffin JD. Modulation of Notch signaling by mastermind-like (MAML) transcriptional co-activators and their involvement in tumorigenesis. Semin Cancer Biol. 2004;14:348–56.CrossRefPubMedGoogle Scholar
  40. 40.
    Zhang P, Yang Y, Nolo R, et al. Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness. Oncogene. 2010;29:2916–26.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Izon DJ, Aster JC, He Y, et al. Deltex1 redirects lymphoid progenitors to the B cell lineage by antagonizing Notch1. Immunity. 2002;16:231–43.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Rajeswari Narayanappa
    • 1
    • 2
  • Pritilata Rout
    • 3
  • Madhuri G. S. Aithal
    • 1
  • Ashis Kumar Chand
    • 4
  1. 1.Department of BiotechnologyDayananda Sagar College of EngineeringBangaloreIndia
  2. 2.Division of Molecular MedicineSt. John’s Research InstituteBangaloreIndia
  3. 3.Department of PathologySt. John’s Medical CollegeBangaloreIndia
  4. 4.Department of NeurosurgerySt. John’s Medical CollegeBangaloreIndia

Personalised recommendations