Virchows Archiv

, Volume 471, Issue 1, pp 23–30 | Cite as

High neuronatin (NNAT) expression is associated with poor outcome in breast cancer

  • Norbert Nass
  • Sarah Walter
  • Dörthe Jechorek
  • Christine Weissenborn
  • Atanas Ignatov
  • Johannes Haybaeck
  • Saadettin Sel
  • Thomas Kalinski
Original Article


Neuronatin (NNAT) is a proteolipid involved in cation homeostasis especially in the developing brain. Its expression has been associated with the progression of lung cancer, glioblastoma, and neuroblastoma as well as glucose induced apoptosis in pancreatic cells. We performed a retrospective study of 148 breast cancer specimens for NNAT expression by immunohistochemistry to evaluate this protein as a prognostic marker for breast cancer. We found a high NNAT immunoreactivity score (by multivariate cox regression) to be an independent prognostic marker for relapse-free (hazard ratio HR = 3.55, p = 0.002) and overall survival (HR = 6.29, p < 0.001). However, NNAT expression was not associated with classical parameters such as hormone receptor expression (p = 0.86) or lymph node metastasis (p = 0.83). Additional independent risk factors in this study population were tumor size (≤2 cm; overall survival: HR = 0.36, p = 0.023; relapse-free survival: HR = 0.26, p < 0.01) and blood vessel infiltration (overall survival: HR = 0.34 p < 0.01). NNAT expression determined by immunohistochemistry might therefore become a helpful additional biomarker to identify high-risk breast cancer patients.


Breast cancer Neuronatin Immunohistochemistry Prognosis 



Parts of this study have been supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) to T.K. (KA2663/3-1). Professional assistance of our immunohistological laboratory, especially Carola Kügler, Claudia Miethke, and Nadine Wiest, is greatly appreciated.

Compliance with ethical standards


This study was funded by a grant of the Deutsche Forschungsgemeinschaft (DFG) to T.K. (KA2663/3-1).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Evans HK, Wylie AA, Murphy SK, Jirtle RL (2001) The neuronatin gene resides in a “micro-imprinted” domain on human chromosome 20q11.2. Genomics 77:99–104. doi: 10.1006/geno.2001.6612 CrossRefPubMedGoogle Scholar
  2. 2.
    John RM, Aparicio SA, Ainscough JF et al (2001) Imprinted expression of neuronatin from modified BAC transgenes reveals regulation by distinct and distant enhancers. Dev Biol 236:387–399. doi: 10.1006/dbio.2001.0327 CrossRefPubMedGoogle Scholar
  3. 3.
    Wijnholds J, Chowdhury K, Wehr R, Gruss P (1995) Segment-specific expression of the neuronatin gene during early hindbrain development. Dev Biol 171:73–84. doi: 10.1006/dbio.1995.1261 CrossRefPubMedGoogle Scholar
  4. 4.
    Sel S, Patzel E, Poggi L et al (2017) Temporal and spatial expression pattern of Nnat during mouse eye development. Gene Expr Patterns 23–24:7–12. doi: 10.1016/j.gep.2016.12.002 CrossRefPubMedGoogle Scholar
  5. 5.
    Usui H, Morii K, Tanaka R et al (1997) cDNA cloning and mRNA expression analysis of the human neuronatin. High level expression in human pituitary gland and pituitary adenomas. J Mol Neurosci MN 9:55–60. doi: 10.1007/BF02789395 CrossRefPubMedGoogle Scholar
  6. 6.
    Joseph R, Dou D, Tsang W (1994) Molecular cloning of a novel mRNA (neuronatin) that is highly expressed in neonatal mammalian brain. Biochem Biophys Res Commun 201:1227–1234CrossRefPubMedGoogle Scholar
  7. 7.
    Joseph RM (2014) Neuronatin gene: imprinted and misfolded: studies in Lafora disease, diabetes and cancer may implicate NNAT-aggregates as a common downstream participant in neuronal loss. Genomics 103:183–188. doi: 10.1016/j.ygeno.2013.12.001 CrossRefPubMedGoogle Scholar
  8. 8.
    Siu I-M, Bai R, Gallia GL et al (2008) Coexpression of neuronatin splice forms promotes medulloblastoma growth. Neuro-Oncology 10:716–724. doi: 10.1215/15228517-2008-038 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Pitale PM, Howse W, Gorbatyuk M (2017) Neuronatin protein in health and disease. J cell Physiol 232:477–48.1. doi: 10.1002/jcp.25498 CrossRefPubMedGoogle Scholar
  10. 10.
    Chu K, Tsai M-J (2005) Neuronatin, a downstream target of BETA2/NeuroD1 in the pancreas, is involved in glucose-mediated insulin secretion. Diabetes 54:1064–1073CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Joe MK, Lee HJ, Suh YH et al (2008) Crucial roles of neuronatin in insulin secretion and high glucose-induced apoptosis in pancreatic beta-cells. Cell Signal 20:907–915. doi: 10.1016/j.cellsig.2008.01.005 CrossRefPubMedGoogle Scholar
  12. 12.
    Mzhavia N, Yu S, Ikeda S et al (2008) Neuronatin: a new inflammation gene expressed on the aortic endothelium of diabetic mice. Diabetes 57:2774–2783. doi: 10.2337/db07-1746 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Suh YH, Kim WH, Moon C et al (2005) Ectopic expression of Neuronatin potentiates adipogenesis through enhanced phosphorylation of cAMP-response element-binding protein in 3T3-L1 cells. Biochem Biophys Res Commun 337:481–489. doi: 10.1016/j.bbrc.2005.09.078 CrossRefPubMedGoogle Scholar
  14. 14.
    Xu DS, Yang C, Proescholdt M et al (2012) Neuronatin in a subset of glioblastoma multiforme tumor progenitor cells is associated with increased cell proliferation and shorter patient survival. PLoS One 7:e37811. doi: 10.1371/journal.pone.0037811 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Renner M, Wolf T, Meyer H et al (2013) Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas. Genome Biol 14:r137. doi: 10.1186/gb-2013-14-12-r137 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Uchihara T, Okubo C, Tanaka R et al (2007) Neuronatin expression and its clinicopathological significance in pulmonary non-small cell carcinoma. J Thorac Oncol Off Publ Int Assoc Study Lung Cancer 2:796–801. doi: 10.1097/JTO.0b013e318145af5e Google Scholar
  17. 17.
    Okubo C, Minami Y, Tanaka R et al (2006) Analysis of differentially expressed genes in neuroendocrine carcinomas of the lung. J Thorac Oncol 1:780–786. doi: 10.1016/S1556-0864(15)30406-8 CrossRefPubMedGoogle Scholar
  18. 18.
    Kuerbitz SJ, Pahys J, Wilson A et al (2002) Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia. Carcinogenesis 23:559–564CrossRefPubMedGoogle Scholar
  19. 19.
    Revill K, Dudley KJ, Clayton RN et al (2009) Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma. Endocr Relat Cancer 16:537–548. doi: 10.1677/ERC-09-0008 CrossRefPubMedGoogle Scholar
  20. 20.
    Ryu S, McDonnell K, Choi H et al (2013) Suppression of miRNA-708 by polycomb group promotes metastases by calcium-induced cell migration. Cancer Cell 23:63–76. doi: 10.1016/j.ccr.2012.11.019 CrossRefPubMedGoogle Scholar
  21. 21.
    Lin H-H, Bell E, Uwanogho D et al (2010) Neuronatin promotes neural lineage in ESCs via Ca(2+) signaling. Stem Cells Dayt Ohio 28:1950–1960. doi: 10.1002/stem.530 CrossRefGoogle Scholar
  22. 22.
    Ignatov A, Ignatov T, Weissenborn C et al (2011) G-protein-coupled estrogen receptor GPR30 and tamoxifen resistance in breast cancer. Breast Cancer Res Treat 128:457–466. doi: 10.1007/s10549-011-1584-1 CrossRefPubMedGoogle Scholar
  23. 23.
    Duffy MJ, Harbeck N, Nap M et al (2017) Clinical use of biomarkers in breast cancer: updated guidelines from the European Group on Tumor Markers (EGTM). Eur J Cancer Oxf Engl 75:284–298. doi: 10.1016/j.ejca.2017.01.017 CrossRefGoogle Scholar
  24. 24.
    Haybittle JL, Blamey RW, Elston CW et al (1982) A prognostic index in primary breast cancer. Br J Cancer 45:361–366CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Todd JH, Dowle C, Williams MR et al (1987) Confirmation of a prognostic index in primary breast cancer. Br J Cancer 56:489–492CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Galea MH, Blamey RW, Elston CE, Ellis IO (1992) The Nottingham prognostic index in primary breast cancer. Breast Cancer Res Treat 22:207–219CrossRefPubMedGoogle Scholar
  27. 27.
    Denkert C, Budczies J, von Minckwitz G et al (2015) Strategies for developing Ki-67 as a useful biomarker in breast cancer. Breast Edinb Scotl 24(Suppl 2):S67–S72. doi: 10.1016/j.breast.2015.07.017 CrossRefGoogle Scholar
  28. 28.
    Buus R, Sestak I, Kronenwett R et al (2016) Comparison of EndoPredict and EPclin with oncotype DX recurrence score for prediction of risk of distant recurrence after endocrine therapy. J Natl Cancer Inst. doi: 10.1093/jnci/djw149
  29. 29.
    Ignatov T, Weißenborn C, Poehlmann A et al (2013) GPER-1 expression decreases during breast cancer tumorigenesis. Cancer Investig 31:309–315. doi: 10.3109/07357907.2013.789901 CrossRefGoogle Scholar
  30. 30.
    Ji Y, Han Z, Shao L, Zhao Y (2016) Ultrasound-targeted microbubble destruction of calcium channel subunit α 1D siRNA inhibits breast cancer via G protein-coupled receptor 30. Oncol Rep 36:1886–1892. doi: 10.3892/or.2016.5031 PubMedPubMedCentralGoogle Scholar
  31. 31.
    Hernández-Bedolla MA, González-Domínguez E, Zavala-Barrera C et al (2016) Calcium-sensing-receptor (CaSR) controls IL-6 secretion in metastatic breast cancer MDA-MB-231 cells by a dual mechanism revealed by agonist and inverse-agonist modulators. Mol Cell Endocrinol 436:159–168. doi: 10.1016/j.mce.2016.07.038 CrossRefPubMedGoogle Scholar
  32. 32.
    Urtreger AJ, Kazanietz MG, Bal de Kier Joffé ED (2012) Contribution of individual PKC isoforms to breast cancer progression. IUBMB Life 64:18–26. doi: 10.1002/iub.574 CrossRefPubMedGoogle Scholar
  33. 33.
    Emberley ED, Murphy LC, Watson PH (2004) S100A7 and the progression of breast cancer. Breast Cancer Res 6:153–159. doi: 10.1186/bcr816 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of PathologyOtto von Guericke University MagdeburgMagdeburgGermany
  2. 2.Department of Obstetrics and GynecologyOtto von Guericke University MagdeburgMagdeburgGermany
  3. 3.Department of OphthalmologyUniversity of HeidelbergHeidelbergGermany

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