Abstract 2351: Characterization of Neutrophil elastase uptake in breast cancer: implications for immunotherapy
Authors: Kerros Celine , Tripathi Satyendra C. , Philips Anne V. , Al-Atrash Gheath , Ruisaard Kathryn E. , Dwyer Karen C. , Mittendorf Elizabeth A. , Hanash Samir , Molldrem Jeffrey J. 
Published: August 2015

Abstract
Neutrophil Elastase (NE)_a serine protease released by neutrophils in the tumor microenvironment_induces invasion and metastasis. We demonstrated that while NE is not endogenously expressed in breast cancer cells, breast cancer cells (BrCA) take up exogenous NE derived from tumor-associated neutrophils. NE uptake results in the expression of the HLA-A2-restricted peptides CCNE1 and PR1, derived from cyclin E and NE, respectively, on the surface of triple-negative (TN) BrCA cells (MDA-MB-231). Expression of these peptides/HLA-A2 molecules induces BrCA cell susceptibility to cytolysis by CCNE1- and PR1-specific cytotoxic T lymphocytes (CTLs), and to 8F4, a monoclonal antibody that binds specifically to the PR1/HLA-A2 complex. We hypothesize that NE uptake is a receptor-mediated process that results in cross-presentation of NE-derived peptides on HLA molecules of BrCA cells.
In this study, we found that NE uptake in BrCA is specific, time and dose-dependent, and become saturated, suggesting a receptor-mediated uptake mechanism. We showed that MDA-MB-231 did not take up cathepsin G, a related serine protease, suggesting specificity of NE receptor uptake. NE internalization was partially blocked by chlorpromazine and by wortmannin, implicating clathrin-dependent uptake and PI3Kinase-dependence, respectively. Confocal microscopy showed that NE was co-localized with the early endosome marker, EEA-1, as early as 10 minutes after uptake. Flow cytometry indicated that surface-bound NE on MDA-MB-231 cells decreased after five minutes, and Western blot showed a simultaneous decrease of MAPKinases phosphorylation (p-Erk, p-p38) and loss of IRS-1 signaling. Inhibition of NE enzyme activity by serine proteases inhibitors, such as elafin or PMSF, potentiated NE uptake in BrCA cells, indicating that enzyme activity is not required for uptake. Conclusion: The results support a novel mechanism of rapid receptor-mediated uptake of soluble exogenous NE by TN BrCA. NE uptake is efficient, PI3 kinase-dependent, sensitive to clathrin inhibition, and associated with down-regulation of Erk phosphorylation and IRS-1. In addition, uptake does not require NE enzyme activity. Following uptake, NE co-localizes to an early endosomal compartment, an organelle associated with HLA class I peptide loading. Since we previously linked NE uptake to CCNE1 and PR1 peptide presentation on MDA-MB-231 cells, which enhanced BrCA susceptibility to immunotherapies that target CCNE1 and PR1, our results here strongly suggest receptor-mediated NE uptake that could regulate HLA class I peptide presentation. Understanding the mechanisms that regulate NE uptake and peptide presentation on the surface of BrCA will help us develop new strategies to enhance peptide presentation by BrCA, potentially improving the response of BrCA and other tumor types to antigen-specific immunotherapies.
Citation Format: Celine Kerros, Satyendra C. Tripathi, Anne V. Philips, Gheath Al-Atrash, Kathryn E. Ruisaard, Karen C. Dwyer, Elizabeth A. Mittendorf, Samir Hanash, Jeffrey J. Molldrem. Characterization of Neutrophil elastase uptake in breast cancer: implications for immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2351. doi:10.1158/1538-7445.AM2015-2351