Abstract 264: Neutrophil elastase (NE) inhibition enhances metastasis free survival by altering regulation of tumor intrinsic cytoskeletal and cellular adhesion pathways in murine breast cancer models
Authors: Lulla Amriti R. , Akli Said , Warma Lucas D. , Fowlkes Natalie W. , Hunt Kelly K. , Rao Xiayu , Wang Jing , Watowich Stephanie S. , Keyomarsi Khandan 
Published: June 2022

Abstract
Background: Neutrophil elastase (NE), a serine protease exclusively secreted in neutrophils, is a crucial mediator of chronic inflammation and tumor progression. Studies from our group reveal that NE may be a prognostic marker of metastasis. In a cohort of 192 breast cancer patients (58% ER/PR+ve, 20%-HER-2 +ve and 22% TNBC), our results show that higher infiltration of NE-positive Tumor Associated Neutrophils (TANs), is associated with a decrease in recurrence free survival (hazard ratio=3.4, 95% CI, 1.1-5.5), regardless of breast cancer subtype Further, we observe that the 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, we have identified the tumor-intrinsic and -extrinsic mechanisms by which NE promotes metastasis.
Methods: The role of NE in breast cancer metastasis was assessed using Elane+/+ and Elane-/- mice in FVB/NJ genetic background, bearing PyMT tumors (orthotopic and spontaneous) respectively. These mice develop lung metastasis in 80-90% of the tumor-bearing mice within 1-3 months of primary tumor initiation. To assess metastasis-free survival (MFS), mice received orthotopic engraftment of PyMT tumors, followed by resection and subsequent monitoring for metastasis. The efficacy of the NE inhibitor AZD9668 was assessed in Elane+/+ mouse models by treating the mice with 100mg/kg B.I.D treatments.
Preliminary results: Genetic ablation of NE (Elane-/-) in the PyMT models, reduced lung metastasis by ~90% (Lung metastatic index =21.8 vs. 2.7, respectively; p=0.0044). Survival studies in FVB Elane-/- showed a MFS benefit of 345 days compared to controls, which succumbed to metastasis-related death in 46 days post primary tumor resection. RNA-sequencing analysis of PyMT- Elane+/+ and Elane-/- tumors showed differential regulation of tumor intrinsic actin cytoskeleton and integrin signaling pathways between the two genetic backgrounds. These NE-regulated pathways are critical for cell-to-cell contact and tissue integrity, explaining the delay in metastasis in the Elane-/- mice. 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) and significantly reduced instances of primary tumor recurrence.
Conclusions: Collectively, our studies suggest that genetic and pharmacological ablation of NE reduces metastasis and extends MFS in in vivo models of breast cancer. 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 R. Lulla, Said Akli, Lucas D. Warma, Natalie W. Fowlkes, Kelly K. Hunt, Xiayu Rao, Jing Wang, Stephanie S. Watowich, Khandan Keyomarsi. Neutrophil elastase (NE) inhibition enhances metastasis free survival by altering regulation of tumor intrinsic cytoskeletal and cellular adhesion pathways in murine breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 264.