RESUMO
BACKGROUND: A better understanding of ductal carcinoma in situ (DCIS) is urgently needed to identify these preinvasive lesions as distinct clinical entities. Semaphorin 3F (SEMA3F) is a soluble axonal guidance molecule, and its coreceptors Neuropilin 1 (NRP1) and NRP2 are strongly expressed in invasive epithelial BC cells. METHODS: We utilized two cell line models to represent the progression from a healthy state to the mild-aggressive or ductal carcinoma in situ (DCIS) stage and, ultimately, to invasive cell lines. Additionally, we employed in vivo models and conducted analyses on patient databases to ensure the translational relevance of our results. RESULTS: We revealed SEMA3F as a promoter of invasion during the DCIS-to-invasive ductal carcinoma transition in breast cancer (BC) through the action of NRP1 and NRP2. In epithelial cells, SEMA3F activates epithelialmesenchymal transition, whereas it promotes extracellular matrix degradation and basal membrane and myoepithelial cell layer breakdown. CONCLUSIONS: Together with our patient database data, these proof-of-concept results reveal new SEMA3F-mediated mechanisms occurring in the most common preinvasive BC lesion, DCIS, and represent potent and direct activation of its transition to invasion. Moreover, and of clinical and therapeutic relevance, the effects of SEMA3F can be blocked directly through its coreceptors, thus preventing invasion and keeping DCIS lesions in the preinvasive state.
Assuntos
Neoplasias da Mama , Carcinoma Intraductal não Infiltrante , Invasividade Neoplásica , Proteínas do Tecido Nervoso , Neuropilina-1 , Neuropilina-2 , Humanos , Neuropilina-1/metabolismo , Neuropilina-1/genética , Feminino , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neuropilina-2/metabolismo , Neuropilina-2/genética , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Intraductal não Infiltrante/genética , Linhagem Celular Tumoral , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Transição Epitelial-Mesenquimal/genética , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/genética , Regulação Neoplásica da Expressão Gênica , Transdução de SinaisRESUMO
BACKGROUND: Osteoporosis (OP) is a high-incidence bone disease that is prone to osteoporotic fractures (OF), so it has attracted widespread attention. AIM: This study investigated the specific expression and role of miR-331 in patients with OP and OF. The findings have profound implications for the clinical prevention and treatment of these conditions. METHODS: The study included 60 OP patients, 46 OF patients, and 40 healthy controls. The expression level of miR-331-3p was detected using RT-qPCR. BMP2 was used to stimulate differentiation in MC3T3-E1 cells. After induction, the expression activity of osteogenic differentiation-related gene markers was detected using RT-qPCR. The target gene analysis was conducted using a luciferase reporter assay. RESULTS: The levels of miR-331-3p were significantly elevated, while NRP2 levels were significantly reduced in OF patients. Post-surgery, miR-331-3p levels decreased over time. MiR-331-3p was found to negatively regulate the luciferase activity of NPR2 in MC3T3-E1 cells. Furthermore, overexpression of miR-331-3p inhibited cell proliferation and decreased the levels of osteoblast differentiation markers. CONCLUSION: The up-regulation of miR-331-3p can promote OP and might also encourage the occurrence of OF by regulating NRP2. However, this needs further verification.
Assuntos
MicroRNAs , Neuropilina-2 , Osteoporose , Fraturas por Osteoporose , MicroRNAs/genética , Humanos , Fraturas por Osteoporose/genética , Fraturas por Osteoporose/metabolismo , Feminino , Osteoporose/genética , Osteoporose/metabolismo , Camundongos , Animais , Pessoa de Meia-Idade , Idoso , Neuropilina-2/genética , Neuropilina-2/metabolismo , Masculino , Diferenciação Celular/genética , Osteoblastos/metabolismo , Proliferação de Células/genética , Osteogênese/genética , Osteogênese/fisiologia , Expressão Gênica/genética , Regulação para CimaRESUMO
Lujo virus (LUJV) is a human pathogen that was the cause of a deadly hemorrhagic fever outbreak in Africa. LUJV is a divergent member of the Arenaviridae with some similarities to both the "Old World" and "New World" serogroups, but it uses a cell-entry receptor, neuropilin-2 (NRP2), that is distinct from the receptors of OW and NW viruses. Though the receptor binding domain of LUJV has been characterized structurally, the overall organization of the trimeric spike complex and how NRP2 is recognized in this context were unknown. Here, we present the structure of the membrane-embedded LUJV spike complex determined by cryo-electron microscopy. Analysis of the structure suggested that a single NRP2 molecule is bound at the apex of the trimeric spike and that multiple subunits of the trimer contact the receptor. The binding of NRP2 involves an intriguing arginine-methionine interaction, which we analyzed using quantum mechanical modeling methods. We compare the LUJV spike structure with the only other available structure of a complete arenaviral spike, which is the Lassa virus. The similarities and differences between them shed light on Arenavirus evolution, inform vaccine design, and provide information that will be useful in combating future Arenavirus outbreaks.
Assuntos
Microscopia Crioeletrônica , Neuropilina-2 , Humanos , Neuropilina-2/metabolismo , Neuropilina-2/química , Modelos Moleculares , Ligação ProteicaRESUMO
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.
Assuntos
Endossomos , Células Endoteliais , Fibronectinas , Integrina alfa5 , Neuropilina-1 , Neuropilina-2 , Proteômica , Proteína p120 Ativadora de GTPase , Animais , Camundongos , Endossomos/metabolismo , Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Integrina alfa5/metabolismo , Integrina alfa5/genética , Integrinas , Neuropilina-1/metabolismo , Neuropilina-1/genética , Neuropilina-2/metabolismo , Neuropilina-2/genética , Proteína p120 Ativadora de GTPase/metabolismo , Proteína p120 Ativadora de GTPase/genética , Transporte Proteico , Proteômica/métodosRESUMO
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.
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Biomarcadores Tumorais , Tumores Neuroendócrinos , Neuropilina-2 , Humanos , Feminino , Neuropilina-2/metabolismo , Neuropilina-2/genética , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/sangue , Idoso , Adulto , Biomarcadores Tumorais/metabolismo , Prognóstico , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Idoso de 80 Anos ou mais , Adulto JovemRESUMO
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.
Assuntos
Túnica Conjuntiva , Nevo , Pterígio , Neoplasias Cutâneas , Humanos , Túnica Conjuntiva/anormalidades , Neuropilina-1 , Neuropilina-2 , Fator de Crescimento Placentário , Pterígio/diagnóstico , Pterígio/cirurgia , Estudos RetrospectivosRESUMO
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.
Assuntos
Colite , NF-kappa B , Humanos , Animais , Camundongos , NF-kappa B/metabolismo , Neuropilina-2/genética , Neuropilina-2/metabolismo , Colite/patologia , Inflamação/patologia , Macrófagos/patologia , Sulfato de Dextrana/toxicidade , Sulfato de Dextrana/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
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.
Assuntos
Neoplasias da Próstata , Fator C de Crescimento do Endotélio Vascular , Adulto , Humanos , Masculino , Recidiva Local de Neoplasia , Neuropilina-2/metabolismo , Antígeno Prostático Específico , Neoplasias da Próstata/patologiaRESUMO
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.
Assuntos
Carcinoma , Neoplasias Esofágicas , Humanos , Neuropilina-2/uso terapêutico , Proteínas Reguladoras de Apoptose , Tretinoína/farmacologia , Tretinoína/uso terapêutico , Triazóis/farmacologia , Linhagem Celular Tumoral , Neoplasias Esofágicas/metabolismo , Proliferação de Células , ApoptoseRESUMO
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.
Assuntos
Neoplasias , Sarcoidose , Humanos , Neuropilina-2/metabolismo , Anticorpos Monoclonais , Neoplasias/diagnóstico , Imuno-Histoquímica , Sarcoidose/diagnósticoRESUMO
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.
Assuntos
MicroRNAs , Neuropilina-2 , RNA Circular , Neoplasias da Glândula Tireoide , Humanos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Glicólise/genética , MicroRNAs/genética , Neuropilina-2/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , RNA Circular/genéticaRESUMO
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.
Assuntos
Osso Esponjoso , Neuropilina-2 , Osteoblastos , Animais , Feminino , Masculino , Camundongos , Colecalciferol , Estudos Transversais , Neuropilina-2/genética , Fator A de Crescimento do Endotélio Vascular , CalcitriolRESUMO
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.
Assuntos
Neoplasias , Transdução de Sinais , Animais , Feminino , Camundongos , Células MDA-MB-231 , Neuropilina-1/genética , Neuropilina-1/metabolismo , Neuropilina-2 , Transdução de Sinais/genética , HumanosRESUMO
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.
Assuntos
Neoplasias da Próstata , Fator A de Crescimento do Endotélio Vascular , Masculino , Animais , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neuropilina-2/genética , Neuropilina-2/metabolismo , Transdução de Sinais , Antígeno B7-H1/genética , Neoplasias da Próstata/metabolismoRESUMO
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.
Assuntos
Neuropilina-2 , Neoplasias de Mama Triplo Negativas , Humanos , Neuropilina-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ligação Proteica , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Neuropilina-1/metabolismoRESUMO
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.
Assuntos
Queloide , Células-Tronco Mesenquimais , MicroRNAs , Humanos , Proliferação de Células/genética , Células Endoteliais , Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Queloide/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Neuropilina-2/genética , Neuropilina-2/metabolismoRESUMO
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.
Assuntos
Semaforinas , Semaforinas/metabolismo , Semaforina-3A/metabolismo , Neuropilina-2/genética , Neuropilina-2/metabolismo , Lipoilação , Neurônios/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismoRESUMO
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.
Assuntos
Melanoma , MicroRNAs , Animais , Camundongos , Humanos , Neuropilina-2/genética , Neuropilina-2/metabolismo , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Movimento Celular/genética , Resistência a Medicamentos , Caderinas/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão GênicaRESUMO
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.
Assuntos
Carcinogênese , Fator de Especificidade de Clivagem e Poliadenilação , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , Neuropilina-2 , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Fator A de Crescimento do Endotélio Vascular , Humanos , Linhagem Celular Tumoral , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neuropilina-2/genética , Fenótipo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Carcinogênese/genéticaRESUMO
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.