Abstract 2025: Nicotine promotes lung metastasis in triple negative breast cancer through paracrine signaling in the tumor microenvironment

Triple-negative breast cancer (TNBC) is responsible for a majority of the mortalities through aggressive metastatic spread, which accounts for a poor 5-year survival. Despite progress in the development of drugs that efficiently target cancer cells, treatments for TNBC are often ineffective. Increasing evidence from epidemiologic studies indicate that cigarette smoking is a major risk factor of TNBC with increased metastatic burden and recurrence. Nicotine, the major addictive component of cigarettes, is not carcinogenic but can promotes cancer progression. However, how nicotine promotes breast cancer lung metastasis is not well understood. To address this question, we orthotopically implanted 4T1 cells (TNBC) into Balb/c mice followed by i.p. injection of nicotine and measured tumor growth and metastatic progression. We found that nicotine increased the growth of primary tumor by 10-fold while it promoted the lung metastasis burden by 100-fold in vivo. Similar results were obtained in experimental metastasis. Immunohistochemical analysis of the primary and lung metastasis tissues showed predominant infiltration of neutrophils in lung metastasis lesions. This observation was further validated by depleting neutrophils using anti-Ly6G antibody, which showed significant decrease in lung metastasis burden in vivo. Intriguingly, infiltrated neutrophils in the lung metastasis tissues selectively overexpressed N2-polarization phenotype. Furthermore, in vitro treatment of primary neutrophils with nicotine induced them into N2 phenotype in a Stat3-dependent manner. To explore further the functional contribution of neutrophils to metastasis, we cultured MDA-MB-231 and MCF10CA (TNBC) cells in primary neutrophils conditioned medium (CM) prior treated with or without nicotine. We found that both MDA-MB-231 and MCF10CA cells displayed epithelial phenotype with loss of mesenchymal markers and gain of epithelial markers in nicotine-induced primary neutrophils CM. These results strongly suggest that nicotine-induced polarization of N2-neutrophils plays a critical role in regulating cancer cell plasticity. We then examined secretory factor(s) from nicotine-treated primary neutrophils using a customized qPCR array system. We found that secretion of LCN2 was significantly augmented in nicotine treated primary neutrophils conditioned medium. In addition, clinical data of stage-II breast cancer patients also showed high LCN2 serum level in smokers than non-smokers. Collectively, our results suggest that nicotine promotes N2 polarization of neutrophils thereby supporting MET transition in a paracrine manner via secreting LCN2 that facilitate metastatic colonization of TNBC cells. Citation Format: Abhishek Tyagi, Sambad Sharma, Kerui Wu, Shih-Ying Wu, Fei Xing, Yin Liu, Dan Zhao, Ravindra Pramod Deshpande, Yin-Yuan MO, Kounosuke Watabe. Nicotine promotes lung metastasis in triple negative breast cancer through paracrine signaling in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2025.