Neuropilin-1 Mediates Neutrophil Elastase Uptake And Antigen Cross-Presentation In Breast Cancer Cells

We previously showed that soluble neutrophil elastase (NE) is rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer (BrCa). NE uptake led to cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapeutic target in hematologic malignancies. The mechanism of NE uptake, however, remains unknown. Here, we demonstrated that NE internalization was temperature- and time-sensitive, dose-saturation was observed, and uptake was partially blocked by chlorpromazine (CPZ) and wortmannin, supporting a receptor-mediated process and suggesting a role for clathrin- and PI3K-dependent mechanisms. To identify a candidate NE receptor, we performed mass spectrometry (MS) of proteins after anti-NE co-immunoprecipitation of NE-pulsed MDA-MB-231 BrCa cells and we identified neuropilin-1 (NRP1) as an NE co-associated protein. Binding of NE to NRP1 was confirmed by ELISA, and peptide epitope mapping studies indicated NE bound via it consensus sequence RRXR, which is also known to bind the b1b2 domain of NRP1. NE bound to NRP1 with high affinity (Kd=38.7 nM) as measured with biolayer interferometry. To confirm the role of NRP1 in cellular uptake of NE, we transfected MDA-MB-231 BrCa cells with siRNA or shRNA against NRP1 and showed that NRP1 knockdown resulted in a 2-fold decrease in NE uptake as determined with flow cytometry. Similarly, blocking with an NRP1-neutralizing antibody decreased NE uptake by 60% vs. isotype control antibody, an effect that was also observed in other breast cancer cell lines that expressed NRP1. Conversely, transient expression of NRP1 in the NRP1-deficient T47D BrCa cell line was sufficient to induce uptake of NE. Importantly, knockdown of NRP1 expression in MDA-231 cells also prevented PR1 cross-presentation as determined with the anti-PR1/HLA-A2 monoclonal antibody 8F4. This was confirmed by the loss of susceptibility of NE-pulsed MDA-MB-231 cells to lysis by PR1-specific cytotoxic T cells following knockdown of NRP1 expression. Our data support a novel function of NRP1 in the uptake and cross-presentation of neutrophil-derived proteins by non-hematopoietic cancer cells. Because 8F4 mediates killing of PR1/HLA-A2+ leukemia and NRP1 is broadly expressed on many tumors, our results suggest a role for immunotherapy strategies that target NE-derived peptides on NRP1+ tumors. Citation Format: Celine Kerros, Satyendra C. Tripathi, Dongxing Zha, Sergeeva Anna, Haley L. Peters, Hiroyuki Katayama, Pariya Sukhumalchandra, Kathryn R. Cox, Alexander A. Perakis, Lisa S. St John, Gheath Alatrash, Elizabeth A. Mittendorf, Karen C. Dwyer, Samir M. Hanash, Jeffrey J. Molldrem. Neuropilin-1 mediates neutrophil elastase uptake and antigen cross-presentation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3984. doi:10.1158/1538-7445.AM2017-3984