Characterization Of Neutrophil Elastase Receptor In Breast Cancer: Implication For Immunotherapy

Neutrophil Elastase (NE), a serine protease released by tumor-associated neutrophils in the tumor microenvironment induces invasion and metastasis. We have demonstrated that NE is not endogenously expressed in breast cancer (BrCA), but is taken up by BrCA cells. NE uptake results in the expression of the HLA-A2-bound 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 peptide/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. Here, we found that NE uptake is specific, time- and dose-dependent, and saturable, 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 receptor uptake. NE internalization was partially blocked by chlorpromazine and by wortmannin, suggesting clathrin-dependent uptake and PI3Kinase-dependence, respectively. Confocal microscopy showed that NE was colocalized with the early endosome marker, EEA-1, as early as 10 min after uptake. Flow cytometry indicated that surface-bound NE on MDA-MB-231 cells decreased after 5 minutes, and both flow cytometry and Western blot showed a simultaneous decrease of phospho-Erk and loss of IRS-1 signaling. Inhibition of NE enzyme activity by elafin or PMSF potentiated NE uptake in BrCA cells, thus 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 colocalizes to an early endosomal compartment, an organelle associated with peptide loading of MHC-I molecules for expression of the cell surface. We previously showed that the NE-derived peptide PR1 is cross-presented on MDA-MB-231 cells after NE uptake, leading to susceptibility to immunotherapies that target PR1/HLA-A2. Taken together, our results demonstrate receptor-mediated NE uptake, which is a potentially novel paradigm that could vastly expand the number of tumor-associated antigens that could be targeted on BrCA. An understanding of the mechanism that mediates NE uptake will help us develop strategies to increase expression of immunogenic peptides on cancer cells and to guide the design of targeted immunotherapies against CCNE1 and PR1 as new clinical therapies for BrCA. Citation Format: Celine Kerros, Satyendra C Tripathi, Anne V Philips, Gheath Al-Atrash, Kathryn E Ruisaard, Karen C Dwyer, Elizabeth A Mittendorf, Samir M Hanash, Jeffrey J Molldrem. Characterization of neutrophil elastase receptor in breast cancer: Implication for immunotherapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-09.