Background: Neuropilins (NRPs) are cell surface co-receptors known to enhance signaling from multiple ligands, including growth factors and their cognate receptors, such as HGF/MET. We have previously shown that the novel “b” isoform of NRP2 (NRP2b) is specifically associated with aggressive NSCLC and is required for TGFβ-induced EMT. We also found that NRP2b promotes AKT activation, drug resistance and metastatic spread, while the canonical NRP2a isoform inhibits these processes. However, the mechanisms supporting these differential functions remain obscure.

Results: Live cell imaging revealed that NRP2b expression led to slow internalization/rapid surface recycling of HGF-bound MET compared to NRP2a, which promoted rapid internalization and little/no recycling. Co-immunoprecipitation experiments showed that GSK3β interacted robustly with NRP2b, but only weakly with NRP2a. Our previous work demonstrated analogous differential recruitment of PTEN, but with reversed preferences; i.e., robust binding to NRP2a, but weak binding to NRP2b. Importantly, GSK3β inhibition blocked HGF-dependent migration of lung cancer cells expressing NRP2b, but not in cells expressing NRP2a. Given that PTEN is an established target of GSK3β, and that modification by GSK3β leads to its proteasome-mediated degradation, PTEN levels were examined in cells expressing individual NRP2 isoforms. PTEN was significantly reduced by expression of NRP2b, but not by NRP2a. The reduction of PTEN in the presence of NRP2b was blocked by inhibition of GSK3β, and by proteasome inhibition with MG132. Despite lower PTEN levels, phosphorylation of the GSK3β target site on Thr366 was specifically increased by NRP2b. Co-immunoprecipitation of GSK3β with NRP2b deletion mutants identified a C-terminal 15 amino acid peptide as the site of interaction. Mutants without this sequence failed to recruit GSK3β and failed to reduce PTEN levels. In silico modeling of this peptide suggested an amphipathic alpha-helical structure with a high affinity docking site (ΔG = -45 kcal) on the atomic structure of GSK3β.

Conclusion: Our results suggest that NRP2b promotes receptor signaling to AKT by differential endosomal trafficking and by recruitment of GSK3β. This kinase will phosphorylate and destabilize active PTEN recruited into the complex by NRP2a, which heterodimerizes with NRP2b. Increased AKT signaling following PTEN loss is likely responsible for the pro-tumorigenic properties of NRP2b, including resistance to targeted agents and metastatic spread. This novel mechanism for NRP2b function suggests that targeting its interaction with GSK3β might be a fruitful therapeutic strategy to reduce drug resistance and/or metastatic spread in lung cancer.

Citation Format: Anastasios Dimou, Cecile Nasarre, Monika Gooz, Rose Pagano, Yuri Peterson, Kent Armeson, Harry A. Drabkin, Patrick Nasarre, Chadrick E. Denlinger, Robert M. Gemmill. NRP2b promotes migration, drug resistance and AKT activation in lung cancers by recruiting GSK3â to phosphorylate and destabilize PTEN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2624.