INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors.

METHODS: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models.

RESULTS: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.

CONCLUSIONS: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

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Published in Breast Cancer Research, v. 17, article 25, p. 1-16.

© 2015 Granados-Principal et al.; licensee BioMed Central.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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s13058-015-0527-x-s1.tif (1012 kB)
L-NAME, 1400 W, and L-NMMA (micromolar range) on proliferation, migration, and mammosphere formation of TNBC cell lines.

s13058-015-0527-x-s2.tif (6275 kB)
Representative images of mammospheres in MDA-MB-231 cells treated with iNOS inhibitors.

s13058-015-0527-x-s3.tif (6361 kB)
Representative images of mammospheres in SUM159 cells treated with iNOS inhibitors.

s13058-015-0527-x-s4.tiff (2723 kB)
Migration and Western blot of NOS isoforms, EMT transcription factors, and hypoxia in TNBC cell lines treated with iNOS inhibitors.

s13058-015-0527-x-s5.tiff (2322 kB)
NOS2 knockdown decreases cell proliferation, migration, mammosphere formation, spliced XBP1, and Smad2/3 signaling.

s13058-015-0527-x-s6.tiff (3604 kB)
In vivoeffects of L-NMMA in SUM159 xenografts.

s13058-015-0527-x-s7.tiff (909 kB)
L-NMMA levels in plasma and tumor tissue.

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