A proteomics approach to isolating neuropilin-dependent α5 integrin trafficking pathways: neuropilin 1 and 2 co-traffic α5 integrin through endosomal p120RasGAP to promote polarised fibronectin fibrillogenesis in endothelial cells.

Integrin trafficking to and from membrane adhesions is a crucial mechanism that dictates many aspects of a cell's behaviour, including motility, polarisation, and invasion. In endothelial cells (ECs), the intracellular traffic of α5 integrin is regulated by both neuropilin 1 (NRP1) and neuropilin 2 (NRP2), yet the redundancies in function between these co-receptors remain unclear. Moreover, the endocytic complexes that participate in NRP-directed traffic remain poorly annotated. Here we identify an important role for the GTPase-activating protein p120RasGAP in ECs, promoting the recycling of α5 integrin from early endosomes. Mechanistically, p120RasGAP enables transit of endocytosed α5 integrin-NRP1-NRP2 complexes to Rab11+ recycling endosomes, promoting cell polarisation and fibronectin (FN) fibrillogenesis. Silencing of both NRP receptors, or p120RasGAP, resulted in the accumulation of α5 integrin in early endosomes, a loss of α5 integrin from surface adhesions, and attenuated EC polarisation. Endothelial-specific deletion of both NRP1 and NRP2 in the postnatal retina recapitulated our in vitro findings, severely impairing FN fibrillogenesis and polarised sprouting. Our data assign an essential role for p120RasGAP during integrin traffic in ECs and support a hypothesis that NRP receptors co-traffic internalised cargoes. Importantly, we utilise comparative proteomics analyses to isolate a comprehensive map of NRP1-dependent and NRP2-dependent α5 integrin interactions in ECs.

Neuropilin 2 and soluble neuropilin 2 in neuroendocrine neoplasms.

Neuropilin 2 (NRP2), a transmembrane non-tyrosine kinase receptor, has been described as a potential critical player in the tumourigenesis of several solid cancers and particularly in neuroendocrine neoplasms (NENs). A soluble form of NRP2 (sNRP2) has been previously described and corresponds to a truncated splice isoform. Its prognostic value has never been studied in NEN. NRP2 expression was studied by immunochemistry on tissue microarrays (n = 437) and on circulating tumour cells (CTCs, n = 5 patients with neuroendocrine carcinoma, NEC). We described the levels of sNRP2 in 229 patients with NEN using the ELISA method to identify the factors associated with sNRP2 levels and to evaluate its prognostic role; 90 blood donors represented the healthy control group. NRP2 was found in 97% of neuroendocrine tumours (396/410) and in 74% of NEC (20/27). NRP2 was also expressed in CTC of all the studied patients. The receiver operating characteristic (ROC) analysis showed that sNRP2 had a weak capacity to discriminate between NEN patients and healthy controls (area under curve (AUC) = 0.601, P = 0.053). Abnormal sNRP2 levels were associated with inflammatory syndrome, bone and peritoneal metastases, and abnormal chromogranin A levels. Patients with high sNRP2 levels (sNRP2Q3-Q4) had significantly poorer overall survival in multivariate analysis (HR 0.16, 95% CI (0.04-0.67), P = 0.015). In conclusion, the present study found that sNRP2 and NRP2 could represent a new prognostic biomarker and a therapeutic target, respectively, particularly in aggressive NEN.

Tretinoin (2,4-difluoro-phenyl) triazole activates proapoptotic protein expression and targets NRP2 protein to inhibit esophageal carcinoma cell growth.

The present study investigated the effect of tretinoin (2,4-difluoro-phenyl) triazole (TDFPT) on the growth and proliferation of Kyse-270 and EC9706 esophageal carcinoma cells and explored the underlying mechanism. The results demonstrated that TDFPT treatment of Kyse-270 and EC9706 cells led to a dose-dependent reduction in cell proliferation. Colony formation was significantly (p < .05) reduced in Kyse-270 and EC9706 cells on treatment with various concentrations of TDFPT. In TDFPT-treated Kyse-270 and EC9706 cells, the expression of Bcl-2 protein showed a remarkable decrease, whereas the level of Bax protein was found to be higher compared with the control cells. Cell invasion showed a prominent decrease in Kyse-270 and EC9706 cells on treatment with TDFPT. Treatment with TDFPT led to a prominent suppression in the expression of MMP-9 and NRP2 in Kyse-270 and EC9706 cells. In silico studies using the AutoDock Vina and discovery studio software revealed that various confirmations of TDFPT bind to NRP2 protein with the affinity ranging from -8.6 to -6.1 kcal/mol. It was found that the TDFPT interacts with NRP2 protein by binding to alanine (ALA A:295), proline (PRO A:306), glutamine (GLN A:307), and isoleucine (ILE A:293) amino acid residues. In summary, TDFPT exposure suppresses esophageal carcinoma cell proliferation, inhibits colony formation ability, and activates apoptotic pathway. Thus, TDFPT acts as an effective antiproliferative agent for esophageal carcinoma cells and needs to be investigated further as chemotherapeutic molecule.

Association of plasma NRP2 and VEGF-C levels with prostate cancer disease severity.

BACKGROUND: Neuropilin 2 (NRP2) expression in tissue is an independent prognostic factor for aggressive prostate cancer. Since the NRP2 pathway activation is thought to occur in part through secondary resistance, quantification of NRP2 in initial tissue biopsy specimens collected at diagnosis may have limited utility in identifying patients at highest risk for morbidity and mortality. Given that metastatic tissue is only occasionally obtained for analysis, there is a need for development of a plasma biomarker indicative of NRP2 pathway activation to potentially inform prostate cancer prognosis. Therefore, we investigated if plasma levels of NRP2 or vascular endothelial growth factor C (VEGF-C), a known soluble ligand of NRP2, are prognostic for prostate cancer. We hypothesized that plasma NRP2 and VEGF-C would be associated with more advanced disease or relapsed disease. METHODS: NRP2 and VEGF-C levels were quantified by enzyme-linked immunoassay in plasma samples obtained from 145 prostate cancer patients in an opportunistic biobank. These patients were either (1) newly diagnosed (N = 28), (2) in remission (N = 56), or (3) relapsed disease (N = 61). Plasma samples from 15 adult males without known malignancy served as a comparator cohort. Statistical analysis was performed to investigate the association of plasma NRP2/VEGF-C with patient outcomes, adjusting for age, race, prostate-specific antigen (PSA), Gleason score, and tumor stage at diagnosis. RESULTS: Neither NRP2 nor VEGF-C levels were significantly different in cancer patients compared to noncancer controls. We observed no clear association between plasma NRP2 and disease severity. Increased plasma VEGF-C was significantly associated with disease remission and correlated with Stage I/II and intermediate-risk Gleason score. Neither NRP2 nor VEGF-C correlated with PSA level. CONCLUSIONS: Although tissue NRP2 expression correlates with severe disease, this was not observed for plasma NRP2. Plasma NRP2 levels did not correlate with disease severity or relapse. VEGF-C was highest in patients in remission and with less severe disease. Future investigation is needed to identify noninvasive methods to assess tumor NRP2 status.

Deficiency of inflammation-sensing protein neuropilin-2 in myeloid-derived macrophages exacerbates colitis via NF-κB activation.

Neuropilin-2 (NRP2) is a multifunctional protein engaged in the regulation of angiogenesis, lymphangiogenesis, axon guidance, and tumor metastasis, but its function in colitis remains unclear. Here, we found that NRP2 was an inflammation-sensing protein rapidly and dramatically induced in myeloid cells, especially in macrophages, under inflammatory contexts. NRP2 deficiency in myeloid cells exacerbated dextran sulfate sodium salt-induced experimental colitis by promoting polarization of M1 macrophages and colon injury. Mechanistically, NRP2 could be induced via NF-κB activation by TNF-α in macrophages, but exerted an inhibitory effect on NF-κB signaling, forming a negative feedback loop with NF-κB to sense and alleviate inflammation. Deletion of NRP2 in macrophages broke this negative feedback circuit, leading to NF-κB overactivation, inflammatory exacerbation, and more severe colitis. Collectively, these findings reveal inflammation restriction as a role for NRP2 in macrophages under inflammation contexts and suggest that NRP2 in macrophages may relieve inflammation in inflammatory bowel disease. © 2023 The Pathological Society of Great Britain and Ireland.

Expression of placental growth factor, neuropilin-1, and neuropilin-2 in primary pterygium tissue.

PURPOSE: The aim of this study was to evaluate the expression of placental growth factor (PLGF), neuropilin-1 (NP-1), and neuropilin-2 (NP-2) molecules in primary pterygium tissue compared with normal conjunctival tissue. METHODS: The records of 42 patients who underwent excision surgery with autografts for primary pterygium (pterygium group) and 20 patients who underwent conjunctival nevus excision surgery (control group) in the same period were reviewed retrospectively. The samples obtained from the pterygium tissues in the pterygium group and the clean conjunctival tissues adjacent to the nevus in the control group were collected from the archive. Immunohistochemical stains of the primary antibodies-1/100 diluted PLGF, NP-1, and NP-2 (Abcam Cambridge Science Park, UK)-were applied to all groups. Staining intensities and the percentage of positive cells in epithelial, endothelial, stromal, and inflammatory cells were analyzed by an experienced pathologist. RESULTS: The positivity rates of PLGF and NP-2 expression in epithelial, endothelial, stromal, and inflammatory cells were found to be higher in the pterygium group than in the control group (PLGF: p < 0.001, p < 0.001, p = 0.001, and p < 0.001, respectively; NP-2: p < 0.001 for all). Staining intensities for PLGF and NP-2 were higher in the pterygium group than in the control group (PLGF: p < 0.001, p < 0.001, p = 0.005, and p < 0.001, respectively; NP-2: p < 0.001, p < 0.001, p = 0.001, and p < 0.001, respectively). However, no significant differences were found in any cell type in terms of NP-1 expression positivity rates (p = 0.730, p = 0.121, p = 0.524, and p = 0.624, respectively) or staining intensity (p = 0.716, p = 0.147, p = 0.147, and p = 0.780, respectively). CONCLUSION: PLGF and NP-2 levels were found to be higher in pterygium tissue, while there was no difference in NP-1. These results indicate the possible roles of NP-2 and PLGF in primary pterygium.

Development and Characterization of a Novel Neuropilin-2 Antibody for Immunohistochemical Staining of Cancer and Sarcoidosis Tissue Samples.

Neuropilin-2 (NRP2) is a cell surface receptor that plays key roles in lymphangiogenesis, but also in pathophysiological conditions such as cancer and inflammation. NRP2 targeting by efzofitimod, a novel immunomodulatory molecule, is currently being tested for the treatment of pulmonary sarcoidosis. To date, no anti-NRP2 antibodies are available for companion diagnostics. Here we describe the development and characterization of a novel NRP2 antibody. Using a variety of research techniques, that is, enzyme-linked immunoassay, Western blot, biolayer interferometry, and immunohistochemistry, we demonstrate that our antibody detects all major NRP2 isoforms and does not cross-react with NRP1. Using this antibody, we show high NRP2 expression in granulomas from sarcoidosis patient skin and lung biopsies. Our novel anti-NRP2 antibody could prove to be a useful clinical tool for sarcoidosis and other indications where NRP2 has been implicated. Clinical Trial Registration: clinicaltrials.gov NCT05415137.

CircPI4KA Overexpression Enhances Carcinogenesis and Glycolysis Metabolism in Papillary Thyroid Carcinoma by Causing the miR-1287-5p-Mediated NRP2 Expression Elevation.

Circular RNAs (circRNAs) are implicated in regulating the pathogenesis of papillary thyroid carcinoma (PTC). Herein, we aimed to investigate how circRNA phosphatidylinositol 4-kinase IIIα (circPI4KA, hsa_circ_0062389) functioned as an oncogene in PTC. CircPI4KA, microRNA-1287-5p (miR-1287-5p) and Neuropilin-2 (NRP2) level detection were completed by reverse transcription-quantitative polymerase chain reaction assay. Cell proliferation was assessed through Cell Counting Kit-8 assay, colony formation assay, and EdU assay. Transwell assay was used for detecting migration and invasion abilities. Cell migration was also determined by wound healing assay. Cell apoptosis was assessed using flow cytometry assay. The protein examination was performed using western blot. Glycolysis was evaluated via commercial kits. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted for target analysis. The role of circPI4KA in vivo was explored and analyzed via tumor xenograft assay. CircPI4KA was significantly upregulated in PTC tissues and cells. Knockdown of circPI4KA suppressed proliferation, migration, invasion, glycolysis, and induced apoptosis of PTC cells. CircPI4KA interacted with miR-1287-5p in PTC cells. The antitumor function of circPI4KA downregulation was reversed by inhibition of miR-1287-5p. The miR-1287-5p directly targeted NRP2, and circPI4KA elevated the NRP2 expression by sponging miR-1287-5p. PTC progression was impeded by miR-1287-5p via targeting NRP2. Silencing circPI4KA inhibited tumor growth in vivo through the miR-1287-5p/NRP2 axis. The collective results revealed that circPI4KA induced the upregulation of NRP2 via sponging miR-1287-5p, thus acting as a carcinogenic factor in PTC.

Neuropilin 2 in osteoblasts regulates trabecular bone mass in male mice.

Introduction: Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global Nrp2 knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts. Methods: We first examined the in vitro effect of the calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on Nrp2 transcription in osteoblasts. We next generated mice with a conditional deletion of Nrp2 in the osteoblast cell lineage under control of the paired related homeobox 1 promoter and mice with a conditional Nrp2 knockdown in osteoclasts under control of the Lysozyme promoter. Mice were examined under basal conditions or after treatment with either the bone anabolic vitamin D3 analog WY 1048 or with 1,25(OH)2D3. Results and discussion: We show that Nrp2 expression is induced by 1,25(OH)2D3 in osteoblasts and is associated with enrichment of the vitamin D receptor in an intronic region of the Nrp2 gene. In male mice, conditional deletion of Nrp2 in osteoblast precursors and mature osteoblasts recapitulated the bone phenotype of global Nrp2 knockout mice, with a reduced cortical cross-sectional tissue area and lower trabecular bone content. However, female mice with reduced osteoblastic Nrp2 expression display a reduced cross-sectional tissue area but have a normal trabecular bone mass. Treatment with the vitamin D3 analog WY 1048 (0.4 µg/kg/d, 14 days, ip) resulted in a similar increase in bone mass in both genotypes and genders. Deleting Nrp2 from the osteoclast lineage did not result in a bone phenotype, even though in vitro osteoclastogenesis of hematopoietic cells derived from mutant mice was significantly increased. Moreover, treatment with a high dose of 1,25(OH)2D3 (0.5 µg/kg/d, 6 days, ip), to induce osteoclast-mediated bone resorption, resulted in a similar reduction in trabecular and cortical bone mass. In conclusion, osteoblastic Nrp2 expression is suggested to regulate bone homeostasis in a sex-specific manner.

Therapeutic blocking of VEGF binding to neuropilin-2 diminishes PD-L1 expression to activate antitumor immunity in prostate cancer.

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti-PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2, VEGFA, and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2High PD-L1High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.

Inhibition of VEGF binding to neuropilin-2 enhances chemosensitivity and inhibits metastasis in triple-negative breast cancer.

Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody.

Neuropilin-1 Knockout and Rescue Confirms Its Role to Promote Metastasis in MDA-MB-231 Breast Cancer Cells.

Breast cancer (BC) metastasis remains a leading cause of female mortality. Neuropilin-1 (NRP-1) is a glycoprotein receptor that plays ligand-dependent roles in BC. Clinical studies indicate its correlation with metastatic disease; however, its functional role in BC metastasis remains uncertain. CRISPR-Cas9 was used to knockout the NRP-1 gene in MDA-MB-231 BC cells, and the effects on metastasis were determined using an orthotopic mouse engraftment model. NRP-1 expression in knockout cells was rescued using a recombinant cDNA with a silent mutation in the sgRNA target-adjacent PAM sequence. Differentially expressed genes between NRP-1 knockout and control cells were determined using whole-transcriptome sequencing and validated using real-time PCR. NRP-1KO cells showed a pronounced reduction in the metastasis to the lungs. KEGG pathway analysis of the transcriptome data revealed that PI3K and ECM receptor interactions were among the top altered pathways in the NRP-1KO cells. In addition, reduction in metastasis enhancers proteins, Integrin-ß3 and Tenascin-C, and genes CCL20 and FN1 and upregulation of metastasis suppressor genes, ACVRL and GPX3 in NRP-1KO were detected. These findings provide evidence for a functional role for NRP-1 in BC metastasis, supporting further exploration of NRP-1 and the identified genes as targets in treating metastatic BC.

Palmitoylation regulates neuropilin-2 localization and function in cortical neurons and conveys specificity to semaphorin signaling via palmitoyl acyltransferases.

Secreted semaphorin 3F (Sema3F) and semaphorin 3A (Sema3A) exhibit remarkably distinct effects on deep layer excitatory cortical pyramidal neurons; Sema3F mediates dendritic spine pruning, whereas Sema3A promotes the elaboration of basal dendrites. Sema3F and Sema3A signal through distinct holoreceptors that include neuropilin-2 (Nrp2)/plexinA3 (PlexA3) and neuropilin-1 (Nrp1)/PlexA4, respectively. We find that Nrp2 and Nrp1 are S-palmitoylated in cortical neurons and that palmitoylation of select Nrp2 cysteines is required for its proper subcellular localization, cell surface clustering, and also for Sema3F/Nrp2-dependent dendritic spine pruning in cortical neurons, both in vitro and in vivo. Moreover, we show that the palmitoyl acyltransferase ZDHHC15 is required for Nrp2 palmitoylation and Sema3F/Nrp2-dependent dendritic spine pruning, but it is dispensable for Nrp1 palmitoylation and Sema3A/Nrp1-dependent basal dendritic elaboration. Therefore, palmitoyl acyltransferase-substrate specificity is essential for establishing compartmentalized neuronal structure and functional responses to extrinsic guidance cues.

Adipose-derived mesenchymal stem cells-sourced exosomal microRNA-7846-3p suppresses proliferation and pro-angiogenic role of keloid fibroblasts by suppressing neuropilin 2.

BACKGROUND: Exosomes (Exos) and their contained microRNAs (miRNAs) have been emergingly recognized as key regulators in spanning biological processes, including proliferation and angiogenesis. AIM OF THE STUDY: This work investigates the function of Exos derived from adipose-derived mesenchymal stem cells (adMSCs) in viability of keloid fibroblasts (KFs). METHODS: Abnormally expressed miRNAs in keloid tissues were screened using the GEO dataset GSE113620. Meanwhile, miRNAs enriched in adMSC-Exos were predicted by bioinformatics system. Exos were extracted from acquired adMSCs and identified, which were co-incubated with KFs. Uptake of Exos by KFs was examined by fluorescence staining. Viability, proliferation, and apoptosis of KFs were analyzed by CCK-8, EdU labeling, and TUNEL assays. Conditioned medium of KFs was collected to stimulate angiogenesis of human umbilical vein endothelial cells (HUVECs). Binding between miR-7846-3p and neuropilin 2 (NRP2) was validated by luciferase assay. Protein levels of NRP2 and the Hedgehog pathway molecules were analyzed by western blot analysis. RESULTS: miR-7846-3p was predicted as an exosomal miRNA aberrantly expressed in keloids. AdMSC-Exos reduced viability, proliferation, and apoptosis resistance of KFs, and they blocked the angiogenesis of HUVECs. miR-7846-3p targeted NRP2 mRNA. miR-7846-3p upregulation in KFs suppressed NRP2 expression and reduced the expression of Hedgehog pathway molecules SHH, SMO, and GLI1. Either miR-7846-3p inhibition in Exos or NRP2 overexpression in KFs blocked the effects of Exos and restored the viability, proliferation, and pro-angiogenic role of KFs. CONCLUSION: This work unravels that adMSC-Exos-derived miR-7846-3p suppresses NRP2 and inactivates the Hedgehog signaling to reduce proliferation and pro-angiogenic role of KFs.

Neuropilin-2 Inhibits Drug Resistance and Progression of Melanoma Involving the MiR-331-3p Regulated Cascade.

BACKGROUND: MicroRNAs (miRs) are small noncoding RNAs that are crucial in the development and progression of tumours. Melanoma is an aggressive form of skin cancer and is resistant to most of the chemotherapeutic agents. However, the role of miRs in melanoma remains poorly studied. OBJECTIVE: The work aimed to demonstrate that miR-331-3p is downregulated in melanoma against the benign melanocytic nevi. METHODS: RT-PCR analysis was performed for the expression of proteins; cell proliferation and wound healing assays were carried out. Flow cytometry study was conducted for cell cycle analysis; colony formation assay was performed by soft agar method. For developing a tumour xenograft model, nu/nu mice were selected. RESULTS: Up-regulation of miR-331-3p in melanoma cells decreased cell proliferation, cell migration, and also drug resistance. Over-expression of miR-331-3p resulted in suppression of NRP2 and up-regulation of E-cadherin levels. Moreover, the levels of MDR1, ABCG-2, and ABCG-5 were decreased. However, the knockdown of NRP2 demonstrated similar effects as that of miR- 331-3p overexpression in tumour cells. Overexpression of miR-331-3p caused significant inhibition of tumour growth and its metastasis in mice model of melanoma, which was associated with depletion of NRP2 protein and increased expression of E-cadherin. However, the effects of miR- 331-3p on the migration, cell proliferation, and self-renewal were overturned by the upregulation of NRP2, which also resulted in the inhibition of E-cadherin and overexpression of MDR-1, ABCG-2, and ABCG-5. CONCLUSION: The findings point out the key role of miR-331-3p in the progression and drug resistance of melanoma involving NRP2.

CPSF4 promotes tumor-initiating phenotype by enhancing VEGF/NRP2/TAZ signaling in lung cancer.

Lung cancer is the leading cause of malignant tumor-related deaths worldwide. The presence of tumor-initiating cells in lung cancer leads to tumor recurrence, metastasis, and resistance to conventional treatment. Cleavage and polyadenylation specificity factor 4 (CPSF4) activation in tumor cells contributes to the poor prognosis of lung cancer. However, the precise biological functions and molecular mechanisms of CPSF4 in the regulation of tumor-initiating cells remain unclear. We demonstrated that CPSF4 promotes tumor-initiating phenotype and confers chemoresistance to paclitaxel both in vitro and in vivo. Mechanistically, we showed that CPSF4 binds to the promoters of vascular endothelial growth factor (VEGF) and neuropilin-2 (NRP2) and activated their transcription. In addition, we showed that CPSF4/VEGF/NRP2-mediated tumor-initiating phenotype and chemoresistance through TAZ induction. Furthermore, analysis of clinical data revealed that lung cancer patients with high CPSF4 expression exhibit high expression levels of VEGF, NRP2, and TAZ and that expression of these proteins are positively correlated with poor prognosis. Importantly, selective inhibition of VEGF, NRP2, or TAZ markedly suppressed CPSF4-mediated tumor-initiating phenotype and chemoresistance. Our findings reveal the mechanism of CPSF4 modulating tumor-initiating phenotype and chemoresistance in lung cancer and indicate that the CPSF4-VEGF-NRP2-TAZ signaling pathway may be a prognosis marker and therapeutic target in lung cancer.

IKKß increases neuropilin-2 and promotes the inhibitory function of CD9+ Bregs to control allergic diseases.

Regulatory B cells (Bregs) potently suppress immune disorders, including allergic contact hypersensitivity (CHS). IKKß overactivation is prominent in various inflammatory diseases. However, its effect on Bregs has not been defined. This study is to investigate the new regulator and inhibitory mechanism of Bregs. IkkßC46A transgenic mice with a Cys46 mutation, resulting in increased IKKß activation, were employed for analysis. IL-10-competent CD9+ Bregs were expanded in IkkßC46A mice and B cell specific-IkkßC46A mutation mice. IkkßC46A mutant CD9+ Bregs had stronger suppressive effects on CD4+ and CD8+ T cells in vitro and CHS responses in vivo. The inhibitory CD9+ Bregs from IkkßC46A mice were characterized by upregulated Neuropilin 2 (Nrp2) and IL-10 in comparison with that of Ikkßwt mice. Interestingly, increased expression of Nrp2 was observed in CD9+ Bregs compared with that of CD9- B cells in wild-type mice. The suppressive activity of wild-type CD9+ Bregs in vitro was attenuated by inhibition of Nrp2 on Bregs or silencing its ligand Sema3f on CD4+ T cells. Our findings delineate a distinct role of IKKß activation in enhancing Bregs to disturb the immune balance. It identifies Nrp2 as a novel regulatory molecule of Bregs that partly contributes to B cell-mediated immune tolerance.

The activation of TGF-ß signaling promotes cell migration and invasion of ectopic endometrium by targeting NRP2.

Endometriosis is a gynecological disorder seriously affecting the health and life of women of reproductive age. Neuropilin 2 (NRP2) has been indicated to display a high level in ectopic endometrium. Nevertheless, the specific function of NRP2 in endometriosis is unanswered. RT-qPCR was utilized to detect expression of NRP2 and SMAD family member 2 (SMAD2) in endometrial tissues or endometrial stromal cells (ESCs). Protein levels of transforming growth factor beta (TGF-ß) signaling-associated markers and epithelial-mesenchymal transition (EMT)-related markers were examined by western blotting. Transwell assays were utilized for detecting the impact of NRP2 on ectopic ESC phenotypes. ChIP and luciferase reporter assays were performed for identification of the relationship between NRP2 and SMAD2. In this study, NRP2 was overexpressed in ectopic endometria in comparison to eutopic endometria. Depletion of NRP2 restrained ectopic ESC migration, invasiveness and EMT. TGF-ß signaling-mediated activation of SMAD2 transcriptionally upregulated NRP2 expression in ectopic ESCs. TGF-ß treatment could rescue NRP2 silencing-induced suppressive impact on the behaviors of ectopic ESCs. Overall, the activation of TGF-ß signaling contributes to the migration and invasiveness of ectopic ESCs by targeting NRP2.

Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility.

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

Neuropilin-2 promotes lineage plasticity and progression to neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC), a lethal subset of prostate cancer, is characterized by loss of AR signaling and resulting resistance to AR-targeted therapy during neuroendocrine transdifferentiation, for which the molecular mechanisms remain unclear. Here, we report that neuropilin 2 (NRP2) is upregulated in both de novo and therapy-induced NEPC, which induces neuroendocrine markers, neuroendocrine cell morphology, and NEPC cell aggressive behavior. NRP2 silencing restricted NEPC tumor xenograft growth. Mechanistically, NRP2 engages in reciprocal crosstalk with AR, where NRP2 is transcriptionally inhibited by AR, and in turn suppresses AR signaling by downregulating the AR transcriptional program and confers resistance to enzalutamide. Moreover, NRP2 physically interacts with VEGFR2 through the intracellular SEA domain to activate STAT3 phosphorylation and subsequently SOX2, thus driving NEPC differentiation and growth. Collectively, these results characterize NRP2 as a driver of NEPC and suggest NRP2 as a potential therapeutic target in NEPC.