Abstract P3-10-02: Neutrophil elastase as a therapeutic target to inhibit metastasis in breast cancer
Authors: Lulla Amriti Rajender , Akli Said , Chrisikos Taylor T , Pina Marc A. , Zhou Yifan , Li Haiyan , Watowich Stephanie S , Keyomarsi Khandan 
Published: March 2020

Background: Chronic inflammation, a hallmark of cancer, is associated with poor prognosis in human various malignancies. Predominantly, tumor associated neutrophils (TANs) and myeloid-derived suppressor cells (MDSCs) are immune cells well characterized to promote inflammation, support tumor growth and metastasis, by secreting chemokines, serine proteases and reactive oxygen species. Increased TANs and MDSCs are predictive markers of both tumor progression and metastasis, suggesting that their inhibition may provide viable anti-tumor strategies. Neutrophil elastase (NE), a serine protease exclusively secreted by TANs and MDSCs, is a crucial mediator of inflammation and tumor progression. Recent preliminary studies from our group suggests that genetic deletion of Elane (encoding NE) inhibits lung metastasis in in vivo models of breast cancer. Yet the precise mechanism(s) by which NE promotes tumorigenesis and metastasis of breast cancer remains to be elucidated. To address this gap in knowledge, the current focus of our work is to identify the tumor-intrinsic and -extrinsic mechanisms by which NE promotes metastasis.

Methods: To determine the prognostic significance of NE, we analyzed 192 breast cancer patients (58% ER/PR+ve, 20%-HER-2 +ve and 22% TNBC) for infiltration of TANs. To examine the role of NE in breast cancer metastasis, we have generated Elane+/+ and Elane-/- mice in BALB/c and FVB/N genetic backgrounds, bearing 4T1 and PyMT tumors (orthotopic and spontaneous) respectively. Each of these mouse models develop lung metastasis in 80-90% in tumor-bearing mice within 1-3 months of primary tumor initiation.

Results: Immunohistochemical staining of NE, a marker of TANs shows that higher infiltration of NE-positive TANs is associated with recurrence free survival with a hazard ratio of 3.4 (95% CI, 1.1-5.5). Genetic ablation of NE (Elane −/−) in the PyMT and 4T1 models strongly inhibits lung metastasis. Specifically, the number of lung foci was reduced by ~90% in PyMT Elane−/− mice compared to controls (21.8 vs. 2.7, respectively; p=0.0044). Similarly, ~50% decrease in the number of lung foci was observed in the 4T1 Elane−/− mice compared to control (7.8 vs. 3.4, respectively; p=0.0281). Comparison of inflammatory factors in the tumor microenvironment revealed that Csf3 (G-CSF), Cxcr2 (CXCR2) and Cxcl1 (CXCL1/KC) were significantly elevated in tumors from PyMT/4T1 Elane+/+ mice vs. PyMT/4T1 Elane-/- animals. Mechanistically, our result implies NE-dependent recruitment of immunosuppressive subsets (CXCL1 and related chemokines), thus sustaining inflammation at the primary tumor site. NE- dependent secretion of CXCL1 can further enrich for cancer stem-like cells (CSCs) via CXCR2 signaling, thus aiding metastasis. Lastly, we interrogated the efficacy reversible NE inhibitor AZD9668 in Elane+/+ mouse models. 100mg/kg daily treatment of AZD9668 reduced lung metastasis in PyMT mice by 94% compared to vehicle-treated mice (0.49+/- 0.21% vs. 0.03+/-0.01%; p=0.05).

Conclusions: Collectively, our studies suggest that genetic and pharmacological ablation of NE reduces metastasis in in vivo models of breast cancer. NE inhibitors thus far have only been clinically tested in COPD patients and their efficacy in breast cancer remains to be evaluated. Our preclinical studies presented here are likely to provide the much needed rationale for the use of this class of NE inhibitors as a viable treatment strategy for the metastatic breast cancer.

Citation Format: Amriti Rajender Lulla, Said Akli, Taylor T Chrisikos, Marc A. Pina, Yifan Zhou, Haiyan Li, Stephanie S Watowich, Khandan Keyomarsi. Neutrophil elastase as a therapeutic target to inhibit metastasis in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-02.