Results Prevalence of RPL 39 A 14 V Mutation in Metaplastic Breast Cancer

Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer. Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39. Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio 1⁄4 0.71, 95% confidence interval 1⁄4 0.55 to 0.91, P1⁄4 .006) and iNOS expression (P1⁄4 .003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor N-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P1⁄4 .04 and P1⁄4 .02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1. Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer. Metaplastic breast carcinoma is a rare form of breast cancer, accounting for 0.2% to 5% of all breast cancers (1). However, it is one of the most chemotherapy-refractory breast cancer subtypes and is associated with a higher rate of recurrence and poorer overall survival (OS) (2). Metaplastic breast cancers exhibit diversity in their carcinomatous and sarcomatous features, heterologous elements, and histologic and stromal appearance (2–15). This diversity makes it a difficult disease to treat. Metaplastic breast cancers remain a therapeutic dilemma primarily due to our limited understanding of its pathogenesis. Treatment guidelines for metaplastic breast cancer are yet to be established, and currently treatment is similar to basal-like triple-negative breast cancers (TNBCs) (16). Metaplastic breast A R T IC LE Received: January 12, 2016; Revised: August 3, 2016; Accepted: November 2, 2016 © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. 1 of 10 JNCI J Natl Cancer Inst (2017) 109(6): djw292 doi: 10.1093/jnci/djw292 First published online January 1, 2017 Article Downloaded from https://academic.oup.com/jnci/article-abstract/109/6/djw292/2758642 by IUPUI University Library user on 05 January 2018 cancers express several markers associated with basal-like cancers (eg, epidermal growth factor receptor and cytokeratin 5/6); however, unlike basal-like carcinomas, they are typically chemoresistant (17–19). Three-year survival rates are approximately 40% for node-positive patients compared with approximately 70% for basal-like TNBCs (15). Node-positive patients have poorer clinical outcome, with recurrence patterns tending to favor pulmonary metastasis (2). Using integrated genomics and proteomics approaches, metaplastic breast cancers were found to be closely related to the claudin-low breast cancer subtype (20). Claudin-low breast cancer is characterized by the loss of cell-cell adhesion genes (5). Compared with other breast cancer subtypes, metaplastic breast cancers and claudinlow breast cancers are enriched for stem cell–like and epithelial-to-mesenchymal transition (EMT) characteristics (20,21), which partially contribute to their chemoresistance. A better understanding of the molecular mechanisms underlying metaplastic breast cancer pathogenesis is essential to the discovery of novel therapeutic targets for treatment. We recently identified a novel cancer gene, ribosomal protein L39 (RPL39), responsible for stem cell self-renewal, treatment resistance, and lung metastasis in TNBC (22). Mechanistically, RPL39 increased inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production (22). Furthermore, the RPL39 A14V mutation was found to be a predictor of early distant metastatic relapse to the lung and worse OS. Although RPL39 plays a critical role in TNBC, the function of this protein in other breast cancer subtypes is currently unknown. As metaplastic breast cancers display stem-like and EMT features, we investigated the role and mechanism of action of RPL39 in this breast cancer subtype using patient samples and in vitro and in vivo models.