Kallikrein-related peptidase 6 (KLK6) expression differentiates tumor subtypes and predicts clinical outcome in breast cancer patients

  • Christoforos Haritos
  • Kleita Michaelidou
  • Konstantinos Mavridis
  • Ioannis Missitzis
  • Alexandros Ardavanis
  • John Griniatsos
  • Andreas Scorilas
Original Article
  • 23 Downloads

Abstract

Novel molecular markers that address the heterogeneity of breast cancer (BC) and provide meaningful prognostic information for BC patients are needed. Kallikrein-related peptidase 6 (KLK6) is aberrantly expressed and functionally implicated in BC and, like other members of the KLK family, may prove a useful molecular tool for clinical management. Our objective was to assess, for the first time, the clinical relevance of KLK6 mRNA expression in BC. Total RNA was isolated from 165 breast tumors, as well as 100 adjacent non-cancerous tumor specimens. After cDNA synthesis, and following quality control, quantitative real-time PCR for KLK6 expression analysis took place. Receiver operating characteristic curves were constructed in order to assess the ability of KLK6 mRNA expression levels to differentiate between molecular BC subtypes. Survival analyses, using DFS as endpoint, were performed at the univariate and multivariate levels. Publicly available BC databases and online survival analysis tools were used to validate our findings. A significant downregulation of KLK6 mRNA expression was observed in BC tissue sections compared to the non-cancerous component (P < 0.001). The expression of KLK6 is positively associated with tumor grade (P = 0.038) and is overexpressed in TNBC and HER2-positive tumors (P < 0.001). Aberrant KLK6 expression predicts the clinical outcome of BC patients in terms of DFS, independently of currently used prognostic markers (HR = 7.11, 95% CI = 1.19–42.45). The differential expression of KLK6 and its association with unfavorable outcome in BC patients was validated via in silico analyses. Although an independent external cohort is necessary to confirm our findings, we proved for the first time that KLK6 can provide independent prognostic information for BC patients.

Keywords

KLK6 Kallikreins KLKs Serine proteases Biological tumor marker Prognostic biomarker 

Abbreviations

AUC

Area under the curve

BC

Breast cancer

CI

Confidence interval

Ct

Threshold cycle

DFS

Disease-free survival

DMFS

Distant metastasis-free survival

ECM

Extracellular matrix

EGFR

Epidermal growth factor receptor

EMT

Epithelial to mesenchymal transition

ER

Estrogen receptor

HER2

Human epidermal growth factor receptor 2

HPRT1

Hypoxanthine phosphoribosyltransferase 1

IHC

Immunohistochemistry

KLK

Kallikrein-related peptidase

PARs

Protease-activated receptors

PR

Progesterone receptor

ROC

Receiver operating characteristic

RQ units

Relative quantification units

rs

Spearman correlation coefficient

RSSPC

Robust single sample predictor classification

RT-qPCR

Quantitative real-time PCR

SSPs

Single sample predictors

TNBC

Triple-negative breast cancer

TNM

Tumor–node–metastasis

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

10238_2018_487_MOESM1_ESM.tif (9.5 mb)
Quality control of the developed qPCR assay for quantification of KLK6 expression. Dissociation curves of (A) HPRT1 and (B) KLK6 amplicons. (C) Corresponding 3.0% w/v agarose gel electrophoresis of the RT-qPCR products of randomly selected breast tissue samples. (D) Standard curves for HPRT1 and KLK6, constructed using serial dilutions of calibrator cDNA, covering several orders of magnitude. M: molecular weight marker; PC: positive control, NC: negative control. (TIFF 9682 kb)
10238_2018_487_MOESM2_ESM.doc (54 kb)
Supplementary material 2 (DOC 53 kb)

References

  1. 1.
    Stefanini AC, da Cunha BR, Henrique T, Tajara EH. Involvement of Kallikrein-related peptidases in normal and pathologic processes. Dis Mark. 2015;2015:946572.  https://doi.org/10.1155/2015/946572.Google Scholar
  2. 2.
    Michaelidou K, Kladi-Skandali A, Scorilas A. Kallikreins as biomarkers in human malignancies. In: Preedy VR, Patel VB, editors. Biomarkers in cancer. Dordrecht: Springer; 2015. p. 135–65.CrossRefGoogle Scholar
  3. 3.
    Schmitt M, Magdolen V, Yang F, et al. Emerging clinical importance of the cancer biomarkers kallikrein-related peptidases (KLK) in female and male reproductive organ malignancies. Radiol Oncol. 2013;47(4):319–29.  https://doi.org/10.2478/raon-2013-0053.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Scorilas A, Mavridis K. Predictions for the future of kallikrein-related peptidases in molecular diagnostics. Expert Rev Mol Diagn. 2014;14(6):713–22.  https://doi.org/10.1586/14737159.2014.928207.CrossRefPubMedGoogle Scholar
  5. 5.
    Kryza T, Silva ML, Loessner D, Heuze-Vourc’h N, Clements JA. The kallikrein-related peptidase family: dysregulation and functions during cancer progression. Biochimie. 2016;122:283–99.  https://doi.org/10.1016/j.biochi.2015.09.002.CrossRefPubMedGoogle Scholar
  6. 6.
    Schmitt M, Dorn J, Kiechle M, Diamandis EP, Luo L. Clinical relevance of kallikrein-related peptidases in breast cancer. Berlin, Boston: DE GRUYTER; 2012. p. 111–44.Google Scholar
  7. 7.
    Bayani J, Diamandis EP. The physiology and pathobiology of human kallikrein-related peptidase 6 (KLK6). Clin Chem Lab Med. 2011;50(2):211–33.  https://doi.org/10.1515/CCLM.2011.750.PubMedGoogle Scholar
  8. 8.
    Pampalakis G, Prosnikli E, Agalioti T, Vlahou A, Zoumpourlis V, Sotiropoulou G. A tumor-protective role for human kallikrein-related peptidase 6 in breast cancer mediated by inhibition of epithelial-to-mesenchymal transition. Cancer Res. 2009;69(9):3779–87.  https://doi.org/10.1158/0008-5472.CAN-08-1976.CrossRefPubMedGoogle Scholar
  9. 9.
    Sidiropoulos KG, Ding Q, Pampalakis G, et al. KLK6-regulated miRNA networks activate oncogenic pathways in breast cancer subtypes. Mol Oncol. 2016;10(7):993–1007.  https://doi.org/10.1016/j.molonc.2016.03.008.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ehrenfeld P, Manso L, Pavicic MF, et al. Bioregulation of kallikrein-related peptidases 6, 10 and 11 by the kinin B(1) receptor in breast cancer cells. Anticancer Res. 2014;34(12):6925–38.PubMedGoogle Scholar
  11. 11.
    Ghosh MC, Grass L, Soosaipillai A, Sotiropoulou G, Diamandis EP. Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells. Tumour Biol. 2004;25(4):193–9.  https://doi.org/10.1159/000081102.CrossRefPubMedGoogle Scholar
  12. 12.
    Klucky B, Mueller R, Vogt I, et al. Kallikrein 6 induces E-cadherin shedding and promotes cell proliferation, migration, and invasion. Cancer Res. 2007;67(17):8198–206.  https://doi.org/10.1158/0008-5472.CAN-07-0607.CrossRefPubMedGoogle Scholar
  13. 13.
    Scarisbrick IA, Epstein B, Cloud BA, et al. Functional role of kallikrein 6 in regulating immune cell survival. PLoS ONE. 2011;6(3):e18376.  https://doi.org/10.1371/journal.pone.0018376.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Drucker KL, Paulsen AR, Giannini C, et al. Clinical significance and novel mechanism of action of kallikrein 6 in glioblastoma. Neuro Oncol. 2013;15(3):305–18.  https://doi.org/10.1093/neuonc/nos313.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Michel N, Heuze-Vourc’h N, Lavergne E, et al. Growth and survival of lung cancer cells: regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor. Biol Chem. 2014;395(9):1015–25.  https://doi.org/10.1515/hsz-2014-0124.CrossRefPubMedGoogle Scholar
  16. 16.
    McShane LM, Altman DG, Sauerbrei W, et al. Reporting recommendations for tumor marker prognostic studies (REMARK). Breast Cancer Res Treat. 2006;100(2):229–35.  https://doi.org/10.1007/s10549-006-9242-8.CrossRefPubMedGoogle Scholar
  17. 17.
    Michaelidou K, Ardavanis A, Scorilas A. Clinical relevance of the deregulated kallikrein-related peptidase 8 mRNA expression in breast cancer: a novel independent indicator of disease-free survival. Breast Cancer Res Treat. 2015;152(2):323–36.  https://doi.org/10.1007/s10549-015-3470-8.CrossRefPubMedGoogle Scholar
  18. 18.
    Detre S, Saclani Jotti G, Dowsett M. A “quickscore” method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas. J Clin Pathol. 1995;48(9):876–8.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997–4013.  https://doi.org/10.1200/JCO.2013.50.9984.CrossRefPubMedGoogle Scholar
  20. 20.
    Goldhirsch A, Wood WC, Coates AS, et al. Strategies for subtypes–dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol. 2011;22(8):1736–47.  https://doi.org/10.1093/annonc/mdr304.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Feeley LP, Mulligan AM, Pinnaduwage D, Bull SB, Andrulis IL. Distinguishing luminal breast cancer subtypes by Ki67, progesterone receptor or TP53 status provides prognostic information. Mod Pathol. 2014;27(4):554–61.  https://doi.org/10.1038/modpathol.2013.153.CrossRefPubMedGoogle Scholar
  22. 22.
    Gill S, Sargent D. End points for adjuvant therapy trials: has the time come to accept disease-free survival as a surrogate end point for overall survival? Oncologist. 2006;11(6):624–9.  https://doi.org/10.1634/theoncologist.11-6-624.CrossRefPubMedGoogle Scholar
  23. 23.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8.  https://doi.org/10.1006/meth.2001.1262.CrossRefPubMedGoogle Scholar
  24. 24.
    Camp RL, Dolled-Filhart M, Rimm DL. X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res. 2004;10(21):7252–9.  https://doi.org/10.1158/1078-0432.CCR-04-0713.CrossRefPubMedGoogle Scholar
  25. 25.
    Rhodes DR, Yu J, Shanker K, et al. ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia. 2004;6(1):1–6.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Jezequel P, Campone M, Gouraud W, et al. bc-GenExMiner: an easy-to-use online platform for gene prognostic analyses in breast cancer. Breast Cancer Res Treat. 2012;131(3):765–75.  https://doi.org/10.1007/s10549-011-1457-7.CrossRefPubMedGoogle Scholar
  27. 27.
    Jezequel P, Frenel JS, Campion L, et al. bc-GenExMiner 3.0: new mining module computes breast cancer gene expression correlation analyses. Database (Oxford). 2013;2013:bas060.  https://doi.org/10.1093/database/bas060.CrossRefGoogle Scholar
  28. 28.
    Gyorffy B, Lanczky A, Eklund AC, et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat. 2010;123(3):725–31.  https://doi.org/10.1007/s10549-009-0674-9.CrossRefPubMedGoogle Scholar
  29. 29.
    Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends–an update. Cancer Epidemiol Biomark Prev. 2016;25(1):16–27.  https://doi.org/10.1158/1055-9965.EPI-15-0578.CrossRefGoogle Scholar
  30. 30.
    Zardavas D, Irrthum A, Swanton C, Piccart M. Clinical management of breast cancer heterogeneity. Nat Rev Clin Oncol. 2015;12(7):381–94.  https://doi.org/10.1038/nrclinonc.2015.73.CrossRefPubMedGoogle Scholar
  31. 31.
    Koren S, Bentires-Alj M. Breast tumor heterogeneity: source of fitness, hurdle for therapy. Mol Cell. 2015;60(4):537–46.  https://doi.org/10.1016/j.molcel.2015.10.031.CrossRefPubMedGoogle Scholar
  32. 32.
    Ellsworth RE, Blackburn HL, Shriver CD, Soon-Shiong P, Ellsworth DL. Molecular heterogeneity in breast cancer: state of the science and implications for patient care. Semin Cell Dev Biol. 2017;64:65–72.  https://doi.org/10.1016/j.semcdb.2016.08.025.CrossRefPubMedGoogle Scholar
  33. 33.
    Haynes B, Sarma A, Nangia-Makker P, Shekhar MP. Breast cancer complexity: implications of intratumoral heterogeneity in clinical management. Cancer Metastasis Rev. 2017.  https://doi.org/10.1007/s10555-017-9684-y.PubMedGoogle Scholar
  34. 34.
    Yousef GM, Yacoub GM, Polymeris ME, Popalis C, Soosaipillai A, Diamandis EP. Kallikrein gene downregulation in breast cancer. Br J Cancer. 2004;90(1):167–72.  https://doi.org/10.1038/sj.bjc.6601451.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Mange A, Dimitrakopoulos L, Soosaipillai A, Coopman P, Diamandis EP, Solassol J. An integrated cell line-based discovery strategy identified follistatin and kallikrein 6 as serum biomarker candidates of breast carcinoma. J Proteom. 2016;142:114–21.  https://doi.org/10.1016/j.jprot.2016.04.050.CrossRefGoogle Scholar
  36. 36.
    Schrader CH, Kolb M, Zaoui K, et al. Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients. Mol Cancer. 2015;14:107.  https://doi.org/10.1186/s12943-015-0381-6.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Petraki C, Dubinski W, Scorilas A, et al. Evaluation and prognostic significance of human tissue kallikrein-related peptidase 6 (KLK6) in colorectal cancer. Pathol Res Pract. 2012;208(2):104–8.  https://doi.org/10.1016/j.prp.2011.12.010.CrossRefPubMedGoogle Scholar
  38. 38.
    Ahmed N, Dorn J, Napieralski R, et al. Clinical relevance of kallikrein-related peptidase 6 (KLK6) and 8 (KLK8) mRNA expression in advanced serous ovarian cancer. Biol Chem. 2016;397(12):1265–76.  https://doi.org/10.1515/hsz-2016-0177.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kim TW, Lee SJ, Kim JT, et al. Kallikrein-related peptidase 6 induces chemotherapeutic resistance by attenuating auranofin-induced cell death through activation of autophagy in gastric cancer. Oncotarget. 2016;7(51):85332–48.  https://doi.org/10.18632/oncotarget.13352.PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.The Breast ClinicSaint Savvas Anticancer HospitalAthensGreece
  2. 2.Department of Biochemistry and Molecular BiologyNational and Kapodistrian University of AthensAthensGreece
  3. 3.First Department of OncologySaint Savvas Anticancer HospitalAthensGreece
  4. 4.First Department of Surgery, Medical SchoolNational and Kapodistrian University of Athens, Laiko HospitalAthensGreece

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