Immunity has evolved to balance the destructive nature of inflammation with wound healing to overcome trauma, infection, environmental insults, and rogue malignant cells. The inflammatory response is marked by overlapping phases of initiation, resolution, and post-resolution remodeling. However, the disruption of these events can lead to prolonged tissue damage and organ dysfunction, resulting long-term disease states. Macrophages are the archetypic phagocytes present within all tissues and are important contributors to these processes. Pleiotropic and highly plastic in their responses, macrophages support tissue homeostasis, repair, and regeneration, all while balancing immunologic self-tolerance with the clearance of noxious stimuli, pathogens, and malignant threats. Neuropilin-2 (Nrp2), a promiscuous co-receptor for growth factors, semaphorins, and integrins, has increasingly been recognized for its unique role in tissue homeostasis and immune regulation. Notably, recent studies have begun to elucidate the role of Nrp2 in both non-hematopoietic cells and macrophages with cardiothoracic disease. Herein, we describe the unique role of Nrp2 in diseases of the heart and lung, with an emphasis on Nrp2 in macrophages, and explore the potential to target Nrp2 as a therapeutic intervention.

Abstract The epithelial–mesenchymal transition (EMT) and its reversal, mesenchymal–epithelial transition (MET), are fundamental processes involved in tumor cell invasion and metastasis. SEMA3F is a secreted semaphorin and tumor suppressor downregulated by TGF-β1 and ZEB1-induced EMT. Here, we report that neuropilin (NRP)-2, the high-affinity receptor for SEMA3F and a coreceptor for certain growth factors, is upregulated during TGF-β1–driven EMT in lung cancer cells. Mechanistically, NRP2 upregulation was TβRI dependent and SMAD independent, occurring mainly at a posttranscriptional level involving increased association of mRNA with polyribosomes. Extracellular signal—regulated kinase (ERK) and AKT inhibition blocked NRP2 upregulation, whereas RNA interference-mediated attenuation of ZEB1 reduced steady-state NRP2 levels. In addition, NRP2 attenuation inhibited TGF-β1–driven morphologic transformation, migration/invasion, ERK activation, growth suppression, and changes in gene expression. In a mouse xenograft model of lung cancer, NRP2 attenuation also inhibited locally invasive features of the tumor and reversed TGF-β1–mediated growth inhibition. In support of these results, human lung cancer specimens with the highest NRP2 expression were predominantly E-cadherin negative. Furthermore, the presence of NRP2 staining strengthened the association of E-cadherin loss with high-grade tumors. Together, our results demonstrate that NRP2 contributes significantly to TGF-β1–induced EMT in lung cancer. Cancer Res; 73(23); 7111–21. ©2013 AACR.

PDF file - 97K, shRNA target sequences (S1); Primers used for quantitative RT-PCR (S2).

Supplementary Data from The TRC8 Ubiquitin Ligase Is Sterol Regulated and Interacts with Lipid and Protein Biosynthetic Pathways

PDF file - 1864K, NRP2 is stably up-regulated by TGFβ in lung cancer cells without increasing the halflife of mRNA or protein (S1); NRP2 up-regulation is blocked by inhibition of TβRI, AKT, ERK and ZEB1, but not by expression of inhibitory SMAD7 (S2); NRP2 knockdown blunts TGFβ-mediated transcriptional responses, ERK and AKT phosphorylation but does not influence SMAD activation (S3); NRP2 knockdown blocks cell scattering induced by TGFβ (S4); Immunohistochemical detection of NRP2 and cytokeratin in TGFβ exposed xenograft tumors with NRP2 or control knockdowns (S5); E-cadherin and NRP2 immunostaining of samples from a tumor microarray (S6); Predicted miRNA target sites in the 3' UTR of NRP2 and up-regulated NRP2 protein levels in hnRNP E1 knockdown cells (S7).

Supplementary Figure 1, Table 1, Methods and Materials from Semaphorin SEMA3F Affects Multiple Signaling Pathways in Lung Cancer Cells

Tumor-associated macrophages (TAMs) exert profound influence over breast cancer progression, promoting immunosuppression, angiogenesis, and metastasis. Neuropilin-2 (NRP2), consisting of the NRP2a and NRP2b isoforms, is a co-receptor for heparin-binding growth factors including VEGF-C and Class 3 Semaphorins. Selective upregulation in response to environmental stimuli and independent signaling pathways endow the NRP2 isoforms with unique functionality, with NRP2b promoting increased Akt signaling via receptor tyrosine kinases including VEGFRs, MET, and PDGFR. Although NRP2 has been shown to regulate macrophage/TAM biology, the role of the individual NRP2a/NRP2b isoforms in TAMs has yet to be evaluated. Using transcriptional profiling and spectral flow cytometry, we show that NRP2 isoform expression was significantly higher in TAMs from murine mammary tumors. NRP2a/NRP2b levels in human breast cancer metastasis were dependent upon the anatomic location of the tumor and significantly correlated with TAM infiltration in both primary and metastatic breast cancers. We define distinct phenotypes of NRP2 isoform-expressing TAMs in mouse models of breast cancer and within malignant pleural effusions from breast cancer patients which were exclusive of neuropilin-1 expression. Genetic depletion of either NRP2 isoform in macrophages resulted in a dramatic reduction of LPS-induced IL-10 production, defects in phagosomal processing of apoptotic breast cancer cells, and increase in cancer cell migration following co-culture. By contrast, depletion of NRP2b, but not NRP2a, inhibited production of IL-6. These results suggest that NRP2 isoforms regulate both shared and unique functionality in macrophages and are associated with distinct TAM subsets in breast cancer.

Objective: Innate and acquired resistance is the principle factor limiting the efficacy of tyrosine kinase inhibitors in lung cancer. We have observed a dramatic upregulation of the cell surface co-receptor neuropilin-2b in lung cancers clinically treated with tyrosine kinase inhibitors correlating with acquired resistance. We hypothesize that neuropilin-2b plays a functional role in acquired tyrosine kinase inhibitor resistance. Methods: Non-small cell lung cancer proliferation and survival were determined during chronic tyrosine kinase inhibitor exposure in the presence or absence of neuropilin-2b knock-down. Interactions of neuropilin- 2a and neuropilin-2b isoforms with PTEN and GSK3b were assessed by immunoprecipitation. Neuropilin-2a and neuropilin-2b mutants deleted for their cytoplasmic domains were used to identify regions responsible for neuropilin-2b-GSK3b interaction. Because GSK3b is known to phosphorylate and degrade PTEN, phospho-PTEN and total PTEN levels were assessed after transfection of neuropilin-2a and neuropilin-2b wild-type and mutant constructs. Results: Non-small cell lung cancer cells chronically treated with gefitinib or osimertinib developed drug resistance and exhibited logarithmic growth in the presence of endothelial growth factor receptor tyrosine kinase inhibitors. However, neuropilin-2b knockdown cells remained sensitive to gefitinib. Likewise, neuropilin-2b knockdown suppressed and neuropilin-2a knockdown enhanced cellular migration. Acquired drug resistance and cell migration correlated with neuropilin-2b-dependent AKT activation with the intermediate step of GSK3bdependent PTEN degradation. A specific binding site for GSK3b on the cytoplasmic domain of neuropilin-2b was identified with truncated protein constructs and computer modeling. Conclusions: Neuropilin-2b facilitates non-small cell lung cancer resistance to tyrosine kinase inhibitors, and this biological effect relates to AKT activation. Neuropilin-2b GSK3b interactions appear to be essential for PTEN degradation and AKT activation in lung cancer cells. Disruption of the neuropilin-2b GSK3b interaction may represent a novel treatment strategy to preserve sensitivity to tyrosine kinase inhibitors in non-small cell lung cancer.

BackgroundTumor-associated macrophages (TAMs) exert profound influence over breast cancer progression, promoting immunosuppression, angiogenesis, and metastasis.1 Neuropilin-2 (NRP2), consisting of NRP2a and NRP2b isoforms, is a co-receptor for heparin-binding growth factors including VEGF-C and Class 3 Semaphorins. Selective upregulation in response to environmental stimuli and independent signaling pathways endow the NRP2 isoforms with unique functionality.2 3 We have shown that the two isoforms of NRP2 endow opposing functionality to tumor cells due to distinct signaling pathways, with NRP2b promoting metastatic behavior.3 Although NRPs have been shown to regulate macrophage/TAM biology, the role of NRP2 isoforms in TAM functionality has yet to be evaluated.MethodsTo assess the contribution of NRP2 isoforms to macrophage biology, conditional NRP2a and NRP2b knockout mice and stable shRNA knockdown of NRP2a or NRP2b in Raw264.7 macrophages were generated. Phagocytosis, lysosomal processing of phagocytosed cargo, cytokine production, and influence on tumor cell migration were assessed in vitro using NRP2 isoform knockdown macrophages. NRP2 isoform expression was evaluated on TAMs from murine 4T1 and EO771 mammary carcinoma models using spectral cytometry and single-cell qPCR. NRP2 isoforms and approximated immune composition were evaluated in paired primary tumors and distant metastasis using RNAseq in a cohort of 99 breast cancer patients. High-dimensional myeloid phenotyping was performed on malignant pleural effusions (MPEs) from breast cancer patients or effusions of benign origin using 33-color spectral cytometry and unbiased computational analysis.ResultsNRP2 isoform expression was significantly increased in TAMs from murine tumors compared to macrophages from healthy mammary glands. NRP2 isoforms in human primary and metastatic breast cancer were strongly correlated with one another and positively correlated with increased TAMs. Distinct phenotypes of NRP2 isoform-expressing TAMs in were present in 4T1 and EO771 mouse breast cancers and within MPEs from breast cancer patients which were associated with high levels of activation and potential response to a hypoxic tumor niche. Genetic depletion of either NRP2 isoform resulted in dramatic reduction of LPS-induced IL-10 production, defects in phagosomal processing of apoptotic breast cancer cells, and increase in cancer cell migration following co-culture. By contrast, inhibition of IL-6 production was specific in NRP2b knockdown cells while phagocytic uptake of labeled particulates was inhibited only by NPR2a knockdown.ConclusionsThese results demonstrate that NRP2 isoforms regulate both shared and distinct functionality in macrophages and that NRP2 isoform expression identifies unique TAM subsets in breast cancer.AcknowledgementsThis work was supported by awards from the Susan G. Komen Foundation (CCR15329745), U.S. Department of Defense (W81XWH1910650), and American Lung Association/Thoracic Surgery Foundation to ACS. RD was supported by funding from a Department of Veteran's Affairs Career Development Award (CX001771-01A2) and the University of Pittsburgh's Dean Faculty Advancement Award. ESY was supported by the NCI of the NIH under R03 CA245774. MTL was supported by the NCI of the NIH under awards R01CA181450 and R01CA206012 as well as ITTC/UPMCE. RD and ACS were further supported by funding from the Department of Cardiothoracic Surgery.ReferencesWilliams CB, Yeh ES, Soloff AC. Tumor-associated macrophages: unwitting accomplices in breast cancer malignancy. Npj Breast Cancer [Internet]. Breast Cancer Research Foundation/Macmillan Publishers Limited; 2016;2:15025. Available from: http://dx.doi.org/10.1038/npjbcancer.2015.252.Nasarre P, Gemmill RM, Potiron VA, Roche J, Lu X, Barón AE, et al. Neuropilin-2 is upregulated in lung cancer cells during TGF-β1–Induced epithelial–mesenchymal transition. Cancer Res [Internet] 2013;73:7111 LP–7121. Available from: http://cancerres.aacrjournals.org/content/73/23/7111.abstract3.Gemmill RM, Nasarre P, Nair-Menon J, Cappuzzo F, Landi L, D'Incecco A, et al. The neuropilin 2 isoform NRP2b uniquely supports TGFβ-mediated progression in lung cancer. Sci Signal [Internet] 2017;10. Available from: http://stke.sciencemag.org/content/10/462/eaag0528.abstractEthics ApprovalThe study was approved by the University of Pittsburgh's Institutional Review Board approval number CR19120172-005.

Abstract Introduction: Neuropilin-2b (NRP2b) expression is associated with pro-tumorigenic properties of NSCLCs. We previously reported that NRP2b promotes metastasis and drug resistance, while the canonical NRP2a isoform is inhibitory. Our recent work indicates that the NRP2b cytoplasmic domain recruits GSK3β to phosphorylate and promote degradation of co-recruited PTEN, thereby enhancing AKT activity leading to enhanced survival, migration and drug resistance. We identified a 15-amino acid motif near the NRP2b C-terminus required for interaction with GSK3β. Herein, we describe the anticancer effect of peptides and drug-like macrocycles designed to disrupt this interaction. Methods: We used the cytoplasmic sequence of NRP2b for decoy peptides designed to disrupt the interaction with GSK3β. Control peptides were synthesized with alanine replacements for three amino acids suggested as crucial for GSK3β recruitment. All peptides were N-terminally myristoylated to promote association with and flipping to the inner leaflet of the plasma membrane. A library of over 42,000 drug-like macrocycles was screened in silico for compounds predicted to fit and compete with the NRP2b:GSK3β binding interface. Decoy peptides and macrocycles were assessed for anti-NRP2b activity using assays for migration and drug-tolerant persister cells. Transwell migration assays were performed using a Neuro Probe Reusable Multiwell Chemotaxis Chamber. Persister cell assays were performed with EGFR mutant PC9 and HCC827 cells in the presence of EGFR inhibitors (gefitinib or osimertinib), followed by drug withdrawal and colony formation assays. Results: As previously reported, knockdown of NRP2b inhibited, while knockdown of NRP2a enhanced, migration of lung cancer cell lines. Importantly, emergence of drug-tolerant persister cells was similarly inhibited by knockdown of NRP2b and enhanced by knockdown of NRP2a. Wild type, but not control, peptides abrogated the pro-tumorigenic effects of NRP2a knockdown on both migration and persister cell formation with IC50s of ~250 nM. The most effective macrocycles, M3 and M7, inhibited NRP2b-dependent migration with IC50s of 1-3 µM. Both macrocycles also inhibited generation of persister cell colonies from shNRP2a-PC-9 cells. However, only M3 was effective at blocking persisters from shNRP2a-HCC827 cells. Conclusions: NRP2b:GSK3β interaction is a therapeutic target in NSCLC affecting migration/invasion and persister colony formation associated with the emergence of resistance to EGFR inhibitors. This work justifies further development of these peptides and drug-like macrocycles as therapeutic modalities in lung cancer. Citation Format: Cecile Nasarre, Yuri K. Peterson, Patrick Nasarre, Anastasios Dimou, Kent E. Armeson, Harry A. Drabkin, Nancy Demore, Chadrick E. Denlinger, Robert M. Gemmill. Inhibition of the NRP2b:GSK3β binding interaction with peptides and macrocycles exerts anticancer effects in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2994.

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.

Abstract Introduction Neuropilins are neural guidance molecules which contribute to tissue development. We have shown that the two isoforms of neuropilin-2 endow opposing functionality to tumor cells due to distinct signaling pathways, with Nrp2b promoting metastatic behavior. Due to the role of macrophages (Mθ) in organogenesis and metastasis, we examine the role of Nrp2 isoforms in these cells. Methods Stable shRNA knockdown of Nrp2a or Nrp2b in Raw264.7 Mθ were generated. Phagocytosis, cytokine production, and migration were assessed in knockdowns in response to stimuli (TGFβ, HGF, VEGF, IL-10, IFNγ, LPS, β-glucan). Nrp2 isoforms in Mθ from mouse mammary tissue or EO771-induced mammary tumors were measured by FACS and RT-PCR. Mθ were phenotyped via FACS for wound-healing or inflammatory markers. Single-cell (sc)qPCR for a 96 gene panel examining components of signaling pathways, autophagy, metabolism, and pro/anti-tumor responses was performed on 576 CD11b+F4/80+ TAMs FACS-sorted from EO771 tumors. Results Nrp2b expression was significantly upregulated in TAMs compared to Mθ of the blood, spleen, or mammary tissues. 56% of the scqPCR transcripts analyzed were significantly altered in Nrp2bHigh vs. Nrp2bLow TAMs, and computation analysis (PCA/tSNE) revealed two distinct TAM subsets enriched for Nrp2b. Nrp2b+ Raw264.7 cells showed decreased ability to phagocytose tumor cells, but increased rates of division and migration in response to growth factors compared to Nrp2a+ counterparts. Conclusions We demonstrate, for the first time, that the principle neuropilin-2 isoforms are present in Mθ, regulate unique functionality, and that Nrp2b+ TAMs are both upregulated in mammary tumors and represent a phenotypically unique subtype.

We report that CD47 was upregulated in different EMT-activated human breast cancer cells versus epithelial MCF7 cells. Overexpression of SNAI1 or ZEB1 in epithelial MCF7 cells activated EMT and upregulated CD47 while siRNA-mediated targeting of SNAI1 or ZEB1 in mesenchymal MDA-MB-231 cells reversed EMT and strongly decreased CD47. Mechanistically, SNAI1 and ZEB1 upregulated CD47 by binding directly to E-boxes in the human CD47 promoter. TCGA and METABRIC data sets from breast cancer patients revealed that CD47 correlated with SNAI1 and Vimentin. At functional level, different EMT-activated breast cancer cells were less efficiently phagocytosed by macrophages vs. MCF7 cells. The phagocytosis of EMT-activated cells was rescued by using CD47 blocking antibody or by genetic targeting of SNAI1, ZEB1 or CD47. These results provide a rationale for an innovative preclinical combination immunotherapy based on PD-1/PD-L1 and CD47 blockade along with EMT inhibitors in patients with highly aggressive, mesenchymal, and metastatic breast cancer.

New distributional records, new larval host records, various collecting notes, and observations are reported for the North American species of the tribe Agallissini LeConte, 1873 (Coleoptera: Cerambycidae: Cerambycinae): Agallissus lepturoides (Duponchel Chevrolat, 1841), Osmopleura chamaeropis (Horn, 1893), and Zagymnus clerinus (LeConte, 1873). The species are illustrated and distribution maps are provided.

Cancer is a disease of dysregulated growth and differentiation, along with the ability of the cancer cell to invade, metastasize and escape immune surveillance. Altered transcription factor activity from mutations, gene fusions, copy number alterations and growth factor signaling contributes substantially to the malignant phenotype. In addition, transcriptional deregulation has increasingly been shown to result from alterations in epigenetic modifying genes. In part, epigenetic regulation includes DNA methylation, as well as the covalent modification of histones by druggable targets that control chromatin accessibility. One epigenetic modifier, EZH2, is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2) that methylates lysine 27 of histone H3 (H3K27) associated with gene silencing. Of note, the activities of PRC2 and the nucleosome remodeling complex, SWI/SNF, are antagonistic and mutations in the SWI/SNF subunits, BRG1 and ARID1a, result in EZH2 dependency and sensitivity to EZH2 inhibitors. Mutations of BAP1, seen frequently in mesothelioma, have also been associated with EZH2 inhibitor sensitivity. In lung cancer and other malignant diseases, the upregulation of EZH2 is often a poor prognostic factor. However, in some cellular contexts, loss of EZH2/PRC2 function has also been linked to disease progression. Thus, understanding the biology and regulation of EZH2 is critical for the rational use of EZH2 inhibitors.

Abstract Neuropilins (NRPs) 1 and 2 are highly-related receptors for class 3 semaphorins, and interact with heparin-binding growth factors and their receptors (e.g. HGF-MET, VEGF-VEGFR2 etc.). We previously reported that NRP2 is upregulated by TGFβ and is required for its pro-tumorigenic activity. Here, we show that this upregulation preferentially involves NRP2b, a largely uninvestigated isoform encoding a divergent, yet highly-conserved C-terminus. Importantly, using a panel of lung cancer cell lines and orthotopic metastasis model, NRP2b promoted migration, invasion, metastasis and tumorsphere formation, whereas the prototype receptor, NRP2a had opposite effects. TGFβ-mediated resistance to gefitinib in EGFR mutated tumors also was dependent on NRP2b expression. In addition, NRP2b, but not NRP2a, linked MET activation by HGF to AKT phosphorylation. Mechanistically, in co-immunoprecipitation assays, we found that NRP2a robustly interacted with PTEN, while the interaction with NRP2b was weak. At the clinical level, NRP2b expression was commonly upregulated in patient lung cancer samples and this upregulation was a significant poor-prognostic factor. Collectively, these data indicate that NRP2b plays an important role in lung cancer invasion, metastasis and EGFR inhibitor resistance. Furthermore, isoform-specific interactions between NRP2 and PTEN may be responsible for the observed differences in MET signaling in response to HGF. Citation Format: Anastasios Dimou, Patrick Nasarre, Joyce Nair-Menon, Federico Cappuzzo, Lorenza Landi, Armida D'Incecco, Hidetaka Uramato, Takeshi Yoshida, Eric Haura, Monica Gooz, Kent Armeson, Robert Gemmill, Harry Drabkin. NRP2b, a unique isoform of NRP2, promotes aggressive lung cancer phenotypes [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 4853. doi:10.1158/1538-7445.AM2017-4853

Neuropilins (NRP1 and NRP2) are co-receptors for heparin-binding growth factors and class 3 semaphorins. Different isoforms of NRP1 and NRP2 are produced by alternative splicing. We found that in non-small cell lung cancer (NSCLC) cell lines, transforming growth factor-β (TGFβ) signaling preferentially increased the abundance of NRP2b. NRP2b and NRP2a differ only in their carboxyl-terminal regions. Although the presence of NRP2b inhibited cultured cell proliferation and primary tumor growth, NRP2b enhanced cellular migration, invasion into Matrigel, and tumorsphere formation in cultured cells in response to TGFβ signaling and promoted metastasis in xenograft mouse models. These effects of overexpressed NRP2b contrast with the effects of overexpressed NRP2a. Hepatocyte growth factor (HGF)-induced phosphorylation of the kinase AKT was specifically promoted by NRP2b, whereas inhibiting the HGF receptor MET attenuated NRP2b-dependent cell migration. Unlike NRP2a, NRP2b did not bind the PDZ domain scaffolding protein GAIP carboxyl terminus-interacting protein (GIPC1) and only weakly recruited phosphatase and tensin homolog (PTEN), potentially explaining the difference between NRP2b-mediated and NRP2a-mediated effects. Analysis of NSCLC patient tumors showed that NRP2b abundance correlated with that of the immune cell checkpoint receptor ligand PD-L1 as well as with epithelial-to-mesenchymal transition (EMT) phenotypes in the tumors, acquired resistance to epidermal growth factor receptor (EGFR) inhibitors, disease progression, and poor survival in patients. NRP2b knockdown attenuated the acquisition of resistance to the EGFR inhibitor gefitinib in cultured NSCLC cells. Thus, in NSCLC, NRP2b contributed to the oncogenic response to TGFβ and correlated with tumor progression in patients.