Abstract 831: Discovery of a novel soluble Neuropilin-2 isoform with antiangiogenic and antitumorigenic activity

Abstract

Abstract Upregulation of the non-tyrosine kinase receptor neuropilin-2 (NRP2) is described for several tumor entities and correlates with disease progression. Two membrane-anchored isoforms are transcribed from the NRP2 gene that only differ in their terminal exons to encode transmembrane stretches and short cytoplasmic tails, respectively. While NRP2a was shown to support tumor cell proliferation, NRP2b facilitates tumor cell migration, resulting in enhanced metastatic spreading. A third, soluble NRP2 isoform encoding only aminoterminal protein domains exists as dimeric protein and functions in sequestration of the VEGF-C ligand. By using a next-generation sequencing approach to enrich for NRP2 terminal exons, we identified NRP2-Mo83, a novel NRP2 isoform, from a high expressing tumor cell line, and verified secreted NRP2-Mo83 protein by mass spectrometry in cell supernatants. Preliminary mass spectrometric data give an indication of the presence of soluble NRP2-Mo83 protein in human serum samples.NRP2-Mo83 mRNA is expressed at variable quantities in vitro in a panel of bladder carcinoma cell lines, but is downregulated in a small cohort of macrodissected urothelial carcinoma samples. Transcript abundances of a NRP2 transcript classified as non-coding with high sequence similarity to the NRP2-Mo83 3’ terminus confirm the observed downregulation in the TCGA bladder carcinoma cohort.Preliminary data indicate that recombinant NRP2-Mo83 protein exerts a dose-dependent inhibitory effect on tumor cell growth in vitro in all tested bladder carcinoma cell lines in the presence of serum. However, a fraction of cell lines does not respond to NRP2-Mo83 under serum-starved conditions. Proliferation of human endothelial cells (HUVEC) is inhibited in a dose-dependent manner by recombinant NRP2-Mo83 protein in vitro. In vivo, NRP2-Mo83 reveals an anti-angiogenic effect on the chicken chorioallantoic membrane.Recombinant NRP2-Mo83 is secreted by HEK293 cells. Enrichment of endogenous and purification of recombinant NRP2-Mo83 protein is achieved by exploiting its capacity to bind heparin. In contrast to the described s9NRP2 isoform, NRP2-Mo83 is largely monomeric in solution, as verified by size exclusion chromatography. We identified N and O glycosylation as well as the capacity for polysialation as posttranslational modifications of recombinant NRP2-Mo83 protein, similar to membrane anchored NRP2 isoforms. NanoDSF experiments indicate that calcium ions stabilize NRP2-Mo83. We present NRP2-Mo83 as a novel NRP2 splice isoform, validated its expression at the mRNA and protein level. NRP2-Mo83 appears down-regulated during bladder tumorigenesis, congruent with its function in bladder tumor growth inhibition and its anti-angiogenic properties in vitro and in vivo. The identification of NRP2-Mo83 in human serum may suggest a suppressive function even under non-pathological conditions. Citation Format: Thomas Mayr, Paul L. Dix, Sayed-Mohammad Hasheminasab, Chuanpit Hampel, Marc Sylvester, Niels Schneberger, Sarah Foerster, Gregor Hagelueken, Samikshan Dutta, Glen Kristiansen, Regine Schneider-Stock, Kaustubh Datta, Michael H. Muders. Discovery of a novel soluble Neuropilin-2 isoform with antiangiogenic and antitumorigenic activity [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 831.