Poor prognosis of patients with triple-negative breast cancer can be stratified by RANK and RANKL dual expression
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As clinical studies have correlated RANK expression levels with survival in breast cancer, and that RANK signaling is dependent on its cognate ligand RANKL, we hypothesized that dual protein expression further stratifies the poor outcome in TNBC.
RANK mRNA and protein expression was evaluated in TNBC using genomic databases, cell lines and in a tissue microarray of curated primary tumor samples derived from 87 patients with TNBC. RANK expression was evaluated either by Mann–Whitney U test on log-normalized gene expression data or by Student’s t test on FACS data. Analysis of RANK and RANKL immunostaining was calculated by H-score, and correlations to clinical factors performed using χ 2 or Fisher’s exact test. Associations with RFS and OS were assessed using univariate and multivariate Cox proportional hazard models. Survival estimates were generated using the Kaplan–Meier method.
In three distinct datasets spanning 684 samples, RANK mRNA expression was higher in primary tumors derived from TNBC patients than from those with other molecular subtypes (P < 0.01). Cell surface-localized RANK protein was consistently higher in TNBC cell lines (P = 0.037). In clinical samples, TNBC patients that expressed both RANK and RANKL proteins had significantly worse RFS (P = 0.0032) and OS (P = 0.004) than patients with RANK-positive, RANKL-negative tumors. RANKL was an independent, poor prognostic factor for RFS (P = 0.04) and OS (P = 0.01) in multivariate analysis in samples that expressed both RANK and RANKL.
RANK and RANKL co-expression is associated with poor RFS and OS in patients with TNBC.
KeywordsTriple-negative breast cancer RANK RANKL Relapse-free survival Overall survival
Fluorescence-activated cell sorting
Human epidermal growth factor receptor 2
Receptor activator of nuclear factor kappa B
Triple-negative breast cancer
We thank Stephanie Deming for providing editorial support on behalf of Scientific Publications at MD Anderson Cancer Center, and Albert Rhee for providing medical writing support on behalf of Amgen, Inc.
Amgen, Inc. funded the study and supported the authors in the development of the study design, and in the collection, analysis, and interpretation of data. MER, TF, DB, HM, BLE, WCD, and NU contributed to the study conception and design; MER, TF, DB, BLE, SK, XW, JR, WW, GH, DT, WCD, and NU contributed to the collection, and analysis of data; MER, TF, DB, BLE, HM, BJE, SK, XW, JMR, WAW, GNH, DT, WCD, and NTU contributed to the interpretation of the data and development of the manuscript. Medical writing support was provided by Amgen, Inc. The decision to submit was taken jointly by all authors and Amgen, Inc. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Funding support for this study was provided by Amgen, Inc. Technical support for generation of flow cytometry data was provided by The University of Texas MD Anderson Cancer Center Flow Cytometry and Cellular Imaging Core Facility, funded by the National Cancer Institute Cancer Support Grant P30CA16672.
Compliance with ethical standards
Conflict of interest
Author NU is principal investigator of the above-mentioned study of denosumab (NCT01952054), which is supported by Amgen, Inc. Authors WCD and DB are former employees and shareholders of Amgen, Inc. Authors MER, TF, SK, HM, XW, JMR, WAW, BJE, GNH, DT, BLE declare no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional 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.
This study adhered to ethical guidelines at MD Anderson Cancer Center and the IRB study protocol (LAB05-0530).
- 10.Azim HA Jr, Peccatori FA, Brohee S, Branstetter D, Loi S, Viale G, Piccart M, Dougall WC, Pruneri G, Sotiriou C (2015) RANK-ligand (RANKL) expression in young breast cancer patients and during pregnancy. Breast Cancer Res 17:24. doi: 10.1186/s13058-015-0538-7 CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, Hanada R, Joshi PA, Aliprantis A, Glimcher L, Pasparakis M, Khokha R, Ormandy CJ, Widschwendter M, Schett G, Penninger JM (2010) Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature 468(7320):98–102. doi: 10.1038/nature09387 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Palafox M, Ferrer I, Pellegrini P, Vila S, Hernandez-Ortega S, Urruticoechea A, Climent F, Soler MT, Munoz P, Vinals F, Tometsko M, Branstetter D, Dougall WC, Gonzalez-Suarez E (2012) RANK induces epithelial-mesenchymal transition and stemness in human mammary epithelial cells and promotes tumorigenesis and metastasis. Cancer Res 72(11):2879–2888. doi: 10.1158/0008-5472.CAN-12-0044 CrossRefPubMedGoogle Scholar
- 16.Pfitzner BM, Branstetter D, Loibl S, Denkert C, Lederer B, Schmitt WD, Dombrowski F, Werner M, Rudiger T, Dougall WC, von Minckwitz G (2014) RANK expression as a prognostic and predictive marker in breast cancer. Breast Cancer Res Treat 145(2):307–315. doi: 10.1007/s10549-014-2955-1 CrossRefPubMedGoogle Scholar
- 17.Hein A, Bayer CM, Schrauder MG, Haberle L, Heusinger K, Strick R, Ruebner M, Lux MP, Renner SP, Schulz-Wendtland R, Ekici AB, Hartmann A, Beckmann MW, Fasching PA (2014) Polymorphisms in the RANK/RANKL genes and their effect on bone specific prognosis in breast cancer patients. Biomed Res Int 2014:842452. doi: 10.1155/2014/842452 CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Zhang L, Teng Y, Zhang Y, Liu J, Xu L, Qu J, Hou K, Yang X, Liu Y, Qu X (2012) Receptor activator for nuclear factor kappa B expression predicts poor prognosis in breast cancer patients with bone metastasis but not in patients with visceral metastasis. J Clin Pathol 65(1):36–40. doi: 10.1136/jclinpath-2011-200312 CrossRefPubMedGoogle Scholar
- 21.Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M, Hicks DG, Lester S, Love R, Mangu PB, McShane L, Miller K, Osborne CK, Paik S, Perlmutter J, Rhodes A, Sasano H, Schwartz JN, Sweep FC, Taube S, Torlakovic EE, Valenstein P, Viale G, Visscher D, Wheeler T, Williams RB, Wittliff JL, Wolff AC (2010) American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 28(16):2784–2795. doi: 10.1200/JCO.2009.25.6529 CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, Allred DC, Bartlett JM, Bilous M, Fitzgibbons P, Hanna W, Jenkins RB, Mangu PB, Paik S, Perez EA, Press MF, Spears PA, Vance GH, Viale G, Hayes DF, American Society of Clinical O, College of American P (2013) 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 31(31):3997–4013. doi: 10.1200/JCO.2013.50.9984 CrossRefPubMedGoogle Scholar
- 27.Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121(7):2750–2767. doi: 10.1172/JCI45014 CrossRefPubMedPubMedCentralGoogle Scholar
- 28.Zheng Y, Chow SO, Boernert K, Basel D, Mikuscheva A, Kim S, Fong-Yee C, Trivedi T, Buttgereit F, Sutherland RL, Dunstan CR, Zhou H, Seibel MJ (2014) Direct crosstalk between cancer and osteoblast lineage cells fuels metastatic growth in bone via auto-amplification of IL-6 and RANKL signaling pathways. J Bone Miner Res 29(9):1938–1949. doi: 10.1002/jbmr.2231 CrossRefPubMedGoogle Scholar
- 32.Holland PM, Miller R, Jones J, Douangpanya H, Piasecki J, Roudier M, Dougall WC (2010) Combined therapy with the RANKL inhibitor RANK-Fc and rhApo2L/TRAIL/dulanermin reduces bone lesions and skeletal tumor burden in a model of breast cancer skeletal metastasis. Cancer Biol Ther 9(7):539–550CrossRefPubMedGoogle Scholar
- 33.Yoldi G, Pellegrini P, Trinidad EM, Cordero A, Gomez-Miragaya J, Serra-Musach J, Dougall WC, Munoz P, Pujana MA, Planelles L, Gonzalez-Suarez E (2016) RANK signaling blockade reduces breast cancer recurrence by inducing tumor cell differentiation. Cancer Res 76(19):5857–5869. doi: 10.1158/0008-5472.CAN-15-2745 CrossRefPubMedGoogle Scholar