Clinicopathological significance of lipocalin 2 nuclear expression in invasive breast cancer

  • Sasagu Kurozumi
  • Sami Alsaeed
  • Nnamdi Orah
  • Islam M. Miligy
  • Chitra Joseph
  • Abrar Aljohani
  • Michael S. Toss
  • Takaaki Fujii
  • Ken Shirabe
  • Andrew R. Green
  • Mohammed A. Aleskandarany
  • Emad A. RakhaEmail author
Preclinical study



The epithelial–mesenchymal transition (EMT) plays a key role in breast cancer progression and metastasis. Lipocalin 2 (LCN2) is involved in the regulation of EMT. The aim of this study was to investigate the clinicopathological significance of LCN2 expression in breast cancer.


The expression of LCN2 protein was immunohistochemically assessed in two well-characterised annotated cohorts of breast cancer (discovery cohort, n = 612; validation cohort, n = 1363). The relationship of LCN2 expression and subcellular location with the clinicopathological factors and outcomes of patients was analysed.


Absent or reduced nuclear LCN2 expression was associated with features of aggressive behaviour, including high histological grade, high Nottingham Prognostic Index, high Ki67 labelling index, hormone receptor negativity and human epidermal growth factor receptor 2 positivity. The high cytoplasmic expression of LCN2 was correlated with lymph node positivity. The nuclear downregulation of LCN2 was correlated with the overexpression of EMT associated proteins (N-cadherin and Twist-related protein 2) and basal biomarkers (cytokeratin 5/6 and epidermal growth factor receptor). Unlike the cytoplasmic expression of LCN2, the loss of nuclear expression was a significant predictor of poor outcome. The combinatorial expression tumours with high cytoplasmic and low nuclear expression were associated with the worst prognosis.


Tumour cell expression of LCN2 plays a role in breast cancer progression with loss of its nuclear expression which is associated with aggressive features and poor outcome. Further functional analysis is warranted to confirm the relationship between the subcellular localisation LCN2 and behaviour of breast cancer.


Invasive breast cancer Lipocalin 2 Epithelial–mesenchymal transition N-cadherin Basal type 



The authors thank the Breast Cancer Now Tissue Bank and the University of Nottingham and Pathological Society of Great Britain and Ireland.


This research was funded by the Pathological Society Trainees Small Grant Application (No: 2201) and the University of Nottingham (Nottingham Life Cycle 6).

Compliance with ethical standards

Conflicts of interest

KS has received research grants from CHUGAI Pharmaceutical Co., Ltd. and Ono Pharmaceutical Co., Ltd. The remaining authors declare that they have no conflicts of interest.

Research involving human participants

This study was approved by the Nottingham Research Ethics Committee 2 (Reference title: Development of a molecular genetic classification of breast cancer). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

Informed consent

Informed consent was obtained from all participants in the study.

Supplementary material

10549_2019_5488_MOESM1_ESM.tif (124 kb)
Supplementary material 1 (TIFF 123 kb)
10549_2019_5488_MOESM2_ESM.tif (82 kb)
Supplementary material 2 (TIFF 82 kb)
10549_2019_5488_MOESM3_ESM.tif (106 kb)
Supplementary material 3 (TIFF 106 kb)
10549_2019_5488_MOESM4_ESM.docx (23 kb)
Supplementary material 4 (DOCX 22 kb)
10549_2019_5488_MOESM5_ESM.docx (21 kb)
Supplementary material 5 (DOCX 20 kb)


  1. 1.
    Cancer Research UK (2019) Breast cancer statistics/Statistics by cancer type/Cancer Statistics for the UK. Accessed 04 June 2019
  2. 2.
    Cheng G, Sun X, Wang J et al (2014) HIC1 silencing in triple-negative breast cancer drives progression through misregulation of LCN2. Cancer Res 74:862–872PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Wang HH, Wu MM, Chan MW et al (2014) Long-term low-dose exposure of human urothelial cells to sodium arsenite activates lipocalin-2 via promoter hypomethylation. Arch Toxicol 88:1549–1559PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Ören B, Urosevic J, Mertens C et al (2016) Tumour stroma-derived lipocalin-2 promotes breast cancer metastasis. J Pathol 239:274–285PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Yang J, McNeish B, Butterfield C et al (2013) Lipocalin 2 is a novel regulator of angiogenesis in human breast cancer. FASEB J 27:45–50PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Liao CJ, Li PT, Lee YC et al (2013) Lipocalin 2 induces the epithelial-mesenchymal transition in stressed endometrial epithelial cells: possible correlation with endometriosis development in a mouse model. Reproduction 147:179–187PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Kim SL, Lee ST, Min IS et al (2017) Lipocalin 2 negatively regulates cell proliferation and epithelial to mesenchymal transition through changing metabolic gene expression in colorectal cancer. Cancer Sci 108:2176–2186PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Wang YP, Yu GR, Lee MJ et al (2013) Lipocalin-2 negatively modulates the epithelial-to-mesenchymal transition in hepatocellular carcinoma through the epidermal growth factor (TGF-beta1)/Lcn2/Twist1 pathway. Hepatology 58:1349–1361PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Fernández CA, Yan L, Louis G et al (2005) The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients. Clin Cancer Res 11:5390–5395PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Leung L, Radulovich N, Zhu CQ et al (2012) Lipocalin2 promotes invasion, tumorigenicity and gemcitabine resistance in pancreatic ductal adenocarcinoma. PLoS ONE 7(10):e46677PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Feng M, Feng J, Chen W et al (2016) Lipocalin2 suppresses metastasis of colorectal cancer by attenuating NF-κB-dependent activation of snail and epithelial mesenchymal transition. Mol Cancer 15:77PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Tung MC, Hsieh SC, Yang SF et al (2013) Knockdown of lipocalin-2 suppresses the growth and invasion of prostate cancer cells. Prostate 73:1281–1290PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Leng X, Ding T, Lin H et al (2009) Inhibition of lipocalin 2 impairs breast tumorigenesis and metastasis. Cancer Res 69:8579–8584PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Shi H, Gu Y, Yang J et al (2008) Lipocalin 2 promotes lung metastasis of murine breast cancer cells. J Exp Clin Cancer Res 27:83PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Uhlen M, Zhang C, Lee S (2017) A pathology atlas of the human cancer transcriptome. Science 357:2507CrossRefGoogle Scholar
  16. 16.
    Aleskandarany MA, Abduljabbar R, Ashankyty I et al (2016) Prognostic significance of androgen receptor expression in invasive breast cancer: transcriptomic and protein expression analysis. Breast Cancer Res Treat 159:215–227PubMedCrossRefGoogle Scholar
  17. 17.
    Rakha EA, Agarwal D, Green AR et al (2017) Prognostic stratification of oestrogen receptor-positive HER2-negative lymph node-negative class of breast cancer. Histopathology 70:622–631PubMedCrossRefGoogle Scholar
  18. 18.
    Rakha EA, Elsheikh SE, Aleskandarany MA et al (2009) Triple-negative breast cancer: distinguishing between basal and nonbasal subtypes. Clin Cancer Res 15:2302–2310PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Green AR, Powe DG, Rakha EA et al (2013) Identification of key clinical phenotypes of breast cancer using a reduced panel of protein biomarkers. Br J Cancer 109:1886–1894PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Rakha EA, Soria D, Green AR et al (2014) Nottingham Prognostic Index Plus (NPI+): a modern clinical decision making tool in breast cancer. Br J Cancer 110:1688–1697PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Aleskandarany MA, Green AR, Ashankyty I et al (2016) Impact of intratumoural heterogeneity on the assessment of Ki67 expression in breast cancer. Breast Cancer Res Treat 158:287–295PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Muftah AA, Aleskandarany MA, Al-Kaabi MM et al (2017) Ki67 expression in invasive breast cancer: the use of tissue microarrays compared with whole tissue sections. Breast Cancer Res Treat 164:341–348PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Kurozumi S, Joseph C, Sonbul S et al (2018) Clinicopathological and prognostic significance of Ras association and pleckstrin homology domains 1 (RAPH1) in breast cancer. Breast Cancer Res Treat 172(1):61–68PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Kurozumi S, Joseph C, Sonbul S et al (2018) Clinical and biological roles of Kelch-like family member 7 in breast cancer: a marker of poor prognosis. Breast Cancer Res Treat 170:525–533PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Kurozumi S, Joseph C, Raafat S et al (2019) Utility of ankyrin 3 as a prognostic marker in androgen-receptor-positive breast cancer. Breast Cancer Res Treat 176:63–73PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    McCarty KS Jr, Miller LS et al (1985) Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch Pathol Lab Med 109:716–721PubMedPubMedCentralGoogle Scholar
  27. 27.
    Detre S, Saclani Jotti G, Dowsett MA (1995) “Quickscore” method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas. J Clin Pathol 48:876–878PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Wu Y, Sarkissyan M, Vadgama JV (2016) Epithelial-mesenchymal transition and breast cancer. J Clin Med 5:E13PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Lehmann BD, Bauer JA, Chen X et al (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121:2750–2767PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Masuda H, Baggerly KA, Wang Y et al (2013) Differential response to neoadjuvant chemotherapy among 7 triple-negative breast cancer molecular subtypes. Clin Cancer Res 19:5533–5540CrossRefGoogle Scholar
  31. 31.
    Nagi C, Guttman M, Jaffer S et al (2005) N-cadherin expression in breast cancer: correlation with an aggressive histologic variant–invasive micropapillary carcinoma. Breast Cancer Res Treat 94:225–235PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Aleskandarany MA, Sonbul S, Surridge R et al (2017) Rho-GTPase activating-protein 18: a biomarker associated with good prognosis in invasive breast cancer. Br J Cancer 117:1176–1184PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Fang X, Cai Y, Liu J et al (2011) Twist2 contributes to breast cancer progression by promoting an epithelial-mesenchymal transition and cancer stem-like cell self-renewal. Oncogene 30:4707–4720PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Hanai J, Mammoto T, Seth P et al (2005) Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells. J Biol Chem 280:13641–13647PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Yang J, Bielenberg DR, Rodig SJ et al (2009) Lipocalin 2 promotes breast cancer progression. Proc Natl Acad Sci USA 106:3913–3918PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Sørlie T, Perou CM, Tibshirani R et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98:10869–10874PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Perou CM, Sørlie T, Eisen MB et al (2000) Molecular portraits of human breast tumours. Nature 406:747–752PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Nielsen TO, Hsu FD, Jensen K et al (2004) Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10:5367–5374PubMedCrossRefGoogle Scholar
  39. 39.
    Kurozumi S, Yamaguchi Y, Matsumoto H et al (2018) Comparing protein and mRNA expressions of the human epidermal growth factor receptor family in estrogen receptor-positive breast cancer. Med Mol Morphol 52:90–98PubMedCrossRefGoogle Scholar
  40. 40.
    Green AR, Soria D, Stephen J et al (2016) Nottingham Prognostic Index Plus: validation of a clinical decision making tool in breast cancer in an independent series. J Pathol Clin Res 2:32–40PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Martin M, Holmes FA, Ejlertsen B et al (2017) ExteNET Study Group (2017) Neratinib after trastuzumab-based adjuvant therapy in HER2-positive breast cancer (ExteNET): 5-year analysis of a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol 18:1688–1700PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Baselga J, Cortés J, Kim SB et al (2012) CLEOPATRA Study Group (2012) Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. N Engl J Med 366:109–119PubMedCrossRefPubMedCentralGoogle Scholar
  43. 43.
    Geyer CE, Forster J, Lindquist D et al (2006) Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 355:2733–2743PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Lee EK, Kim HJ, Lee KJ et al (2011) Inhibition of the proliferation and invasion of hepatocellular carcinoma cells by lipocalin 2 through blockade of JNK and PI3 K/Akt signaling. Int J Oncol 38:325–333PubMedPubMedCentralGoogle Scholar
  45. 45.
    Leng X, Lin H, Ding T et al (2008) Lipocalin 2 is required for BCR-ABL-induced tumorigenesis. Oncogene 27:6110–6119PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Bauer M, Eickhoff JC, Gould MN et al (2008) (NGAL) is a predictor of poor prognosis in human primary breast cancer. Breast Cancer Res Treat 108:389–397PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Wenners AS, Mehta K, Loibl S et al (2012) Neutrophil gelatinase-associated lipocalin (NGAL) predicts response to neoadjuvant chemotherapy and clinical outcome in primary human breast cancer. PLoS ONE 7:e45826PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Rodvold JJ, Mahadevan NR, Zanetti M (2012) Lipocalin 2 in cancer: when good immunity goes bad. Cancer Lett 316:132–138PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sasagu Kurozumi
    • 1
    • 2
  • Sami Alsaeed
    • 1
  • Nnamdi Orah
    • 1
  • Islam M. Miligy
    • 1
  • Chitra Joseph
    • 1
  • Abrar Aljohani
    • 1
  • Michael S. Toss
    • 1
  • Takaaki Fujii
    • 2
  • Ken Shirabe
    • 2
  • Andrew R. Green
    • 1
  • Mohammed A. Aleskandarany
    • 1
  • Emad A. Rakha
    • 1
    • 3
    Email author
  1. 1.Division of Cancer and Stem Cells, School of Medicine, Nottingham Breast Cancer Research CentreUniversity of NottinghamNottinghamUK
  2. 2.Department of General Surgical ScienceGunma University Graduate School of MedicineGunmaJapan
  3. 3.Department of Histopathology, Division of Cancer and Stem Cells, School of MedicineThe University of Nottingham and Nottingham University Hospitals NHS Trust, Nottingham City HospitalNottinghamUK

Personalised recommendations