Supplementary Figure S8: (A) PK data for AZD9668, when administered by intraperitoneal (I.P.) injection at a concentration of 100mg/kg. N=3 mice were used per timepoint, and the t1/2 of the drug was calculated to 2.16 hours. (B) Specificity of AZD9668 to inhibit NE activity was confirmed in vitro using a NE-specific substrate, under indicated doses of AZD9668. AZD5069 (CXCR2 antagonist) was used as a negative control. (C) Whole body weights of PyMT NE+/+ mice treated with either Vehicle (black) or 100mg/kg AZD9668 (pink) every day, BID for 60 consecutive days. (D, E) Western blot analysis done on endpoint tumors, to confirm NE activity in (D) PyMT NE+/+ and PyMT NE-/- mice and (E) PyMT NE+/+ mice treated with either Vehicle or 100mg/kg AZD9668, as described in Figure 5 of main text.
ARTICLE ABSTRACT
Metastatic disease remains the leading cause of death due to cancer, yet the mechanism(s) of metastasis and its timely detection remain to be elucidated. Neutrophil elastase (NE), a serine protease secreted by neutrophils, is a crucial mediator of chronic inflammation and tumor progression. In this study, we used the PyMT model (NE+/+ and NE−/−) of breast cancer to interrogate the tumor-intrinsic and -extrinsic mechanisms by which NE can promote metastasis. Our results showed that genetic ablation of NE significantly reduced lung metastasis and improved metastasis-free survival. RNA-sequencing analysis of primary tumors indicated differential regulation of tumor-intrinsic actin cytoskeleton signaling pathways by NE. These NE-regulated pathways are critical for cell-to-cell contact and motility and consistent with the delay in metastasis in NE−/− mice. To evaluate whether pharmacologic inhibition of NE inhibited pulmonary metastasis and phenotypically mimicked PyMT NE−/− mice, we utilized AZD9668, a clinically available and specific NE inhibitor. We found AZD9668 treated PyMT-NE+/+ mice showed significantly reduced lung metastases, improved recurrence-free, metastasis-free and overall survival, and their tumors showed similar molecular alterations as those observed in PyMT-NE−/− tumors. Finally, we identified a NE-specific signature that predicts recurrence and metastasis in patients with breast cancer. Collectively, our studies suggest that genetic ablation and pharmacologic inhibition of NE reduces metastasis and extends survival of mouse models of breast cancer, providing rationale to examine NE inhibitors as a treatment strategy for the clinical management of patients with metastatic breast cancer.
