Inhibition Of Neuropilin-1 Research Articles

Dual effects of targeting neuropilin-1 in lenvatinib-resistant hepatocellular carcinoma: inhibition of tumor growth and angiogenesis.

In the era of immunotherapy, lenvatinib (LEN) still holds an important position in the sequential treatment of advanced hepatocellular carcinoma (HCC). However, the sustained therapeutic effect of LEN is not sufficient, and there is a need to address the development of resistance. Neuropilin-1 (NRP1) is known to act as a coreceptor for epidermal growth factor receptor (EGFR), Met, and vascular endothelial growth factor receptor 2 (VEGFR2), which have been reported to be involved in LEN resistance. In this study, we used cell culture and in vivo xenograft models to evaluate the contribution of NRP1 in the acquisition of LEN resistance in HCC as well as the potential of NRP1 as a therapeutic target. LEN resistance increased EGF/EGFR and hepatocyte growth factor (HGF)/Met signaling in liver cancer cells and VEGFA/VEGFR2 and HGF/Met signaling in vascular endothelial cells, thereby promoting cell proliferation, cell migration, and angiogenesis. We found that activation of NRP1 is essential for the enhancement of these signaling. In addition, NRP1 inhibition combined with LEN therapy synergistically improved the antitumor effects against LEN-resistant HCC, indicating that NRP1 is an attractive therapeutic target.NEW & NOTEWORTHY We demonstrated that neuropilin-1 (NRP1) was an essential coreceptor mediating the activation of multiple signaling pathways in the acquisition of resistance to lenvatinib (LEN) in HCC. The addition of NRP1 inhibition to LEN had a synergistic antitumor effect on LEN-resistant HCC in culture and in vivo xenograft models.

NRP1 antagonism as a novel therapeutic target in nasal polyps of patients with chronic rhinosinusitis.

Neuropilin-1 (NRP1) is expressed on the surface epithelium of respiratory tract and immune cells, demonstrating its possible function in regulating the immune response in airway disease. However, its role in patient with chronic rhinosinusitis (CRS) remains unknown. This study aimed to elucidate the role of NRP1 in CRS with nasal polyps (CRSwNP). Sinonasal biopsy specimens were immunohistochemically stained to investigate NRP1 expression. Double immunofluorescence, immunoblotting, and real-time polymerase chain reaction were performed to evaluate NRP1 in primary human nasal epithelial cells (hNECs). An NRP1 inhibitor was administered to a murine nasal polyp (NP) model. NRP1 was highly expressed in the epithelium in patients with CRSwNP compared to nasal tissue from controls and CRS without NP patients. NRP1 and vascular endothelial growth factor were upregulated in hNECs under hypoxia. Treatment with NRP1 inhibitor (EG00229) reduced the secretion of interleukin (IL)-1β, IL-6, IL-8, and IL-33 cytokines, as well as inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2 in hNECs. We found that NRP1 was highly expressed in the airway epithelium in the murine NP model. The group treated with the NRP1 inhibitor had significantly fewer nasal polypoid lesions and reduced accumulations of immune cells. These findings reveal that NRP1 is upregulated in CRS and NP epithelium, and the inhibition of NRP1 may lead to a reduction in NP growth and immune cell infiltration. Our results suggest that NRP1 inhibition could be a novel possibility for treating nasal polyposis.

Low Expression of the NRP1 Gene Is Associated with Shorter Overall Survival in Patients with Sonic Hedgehog and Group 3 Medulloblastoma.

Medulloblastoma (MB) is the most common type of malignant pediatric brain tumor. Neuropilin-1 (NRP1), encoded by the NRP1 gene, is a transmembrane glycoprotein overexpressed in several types of cancer. Previous studies indicate that NRP1 inhibition displays antitumor effects in MB models and higher NRP1 levels are associated with poorer prognosis in MB patients. Here, we used a large MB tumor dataset to examine NRP1 gene expression in different molecular subgroups and subtypes of MB. We found overall widespread NRP1 expression across MB samples. Tumors in the sonic hedgehog (SHH) subgroup showed significantly higher NRP1 transcript levels in comparison with Group 3 and Group 4 tumors, with SHH samples belonging to the α, β, Δ, and γ subtypes. When all MB subgroups were combined, lower NRP1 expression was associated with significantly shorter patient overall survival (OS). Further analysis showed that low NRP1 was related to poorer OS, specifically in MB subgroups SHH and Group 3 MB. Our findings indicate that patients with SHH and Group 3 tumors that show lower expression of NRP1 in MB have a worse prognosis, which highlights the need for subgroup-specific investigation of the NRP1 role in MB.

Inhibition of neuropilin-1 enhances the therapeutic effects of lenvatinib in suppressing cholangiocarcinoma cells via the c-Met pathway

Neuropilin-1 (NRP-1) participates in the progression of cholangiocarcinoma (CCA) and lenvatinib is an approved tyrosine kinase inhibitor treating several other cancers. Our exploratory study reveals that the inhibitory activities of lenvatinib largely rely on the expression levels of NRP-1 in CCA cells, leading to the present study that aims to investigate whether inhibition of NRP-1 could enhance the effects of lenvatinib in suppressing CCA. By using stable transfected CCA cells depleted of NRP-1 and EG00229, a specific NRP-1 inhibitor, we examined cell proliferation, cell cycle distribution and apoptosis, and detected the expression of key molecules and the involvement of the c-Met pathway. Xenograft mouse models were employed to verify the in vitro results. NRP-1 depletion and EG00229 strengthened lenvatinib in inhibiting the proliferation and promoting the apoptosis of CCA cells, and their additive or synergistic effects were confirmed in animal models. Mechanistically, lenvatinib induced the activation of the c-Met pathway, while either NRP-1 depletion or EG00229 inhibited this activation, which could be stimulated by its ligand hepatocyte growth factor. NRP-1 inhibition resulted in a significant alteration in the expression/activation of downstream pathways and molecules, which are key factors regulating cell proliferation and apoptosis. In conclusion, the present results indicate that the inhibition of NRP-1 enhances the efficacy of lenvatinib via the c-Met pathway, and warrant further studies on the pharmacological utility of EG00229, particularly, in the combination of lenvatinib as a promising adjunct therapeutic strategy for combating CCA.

Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

Ionizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 – mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell – specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A – NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A – NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

Contribution of Neuropilin-1 in Radiation-Survived Subclones of NSCLC Cell Line H1299.

Non-small cell lung cancer (NSCLC) is an aggressive lung cancer accounting for approximately 85% of all lung cancer patients. For the patients with Stages IIIA, IIIB, and IIIC, the 5-year survival is low though with the combination with radiotherapy and chemotherapy. In addition, the occurrence of tumor cells (repopulated tumors) that survive irradiation remains a challenge. In our previous report, we subcloned the radiation-surviving tumor cells (IR cells) using the human NSCLC cell line, H1299, and found that the expression of neuropilin-1 (NRP-1) was upregulated in IR cells by the microarray analysis. Here, we investigated the contribution of neuropilin-1 to changes in the characteristics of IR cells. Although there were no differences in angiogenic activity in the tube formation assay between parental and IR cells, the cell motility was increased in IR cells compared to parental cells in the cell migration assay. This enhanced cell motility was suppressed by pretreatment with anti-NRP-1 antibody. Although further studies are necessary to identify other molecules associated with NRP-1, the increase in cellular motility in IR cells might be due to the contribution of NRP-1. Inhibition of NRP-1 would help control tumor malignancy in radiation-surviving NSCLC.

Abstract 526: Novel mechanisms of Neuropilin-1 inhibition result in improved tumor growth inhibition in vivo

Abstract Background Cygnal Therapeutics, pioneer in exoneuronal biology, has identified the neuronal protein, Neuropilin-1 (NRP1) as a critical regulator of tumor growth and anti-tumor immunity. NRP1 is a co-receptor that complexes with diverse ligands and their cognate receptors. As such, it plays a role in multiple different biological processes, including axon guidance and angiogenesis. NRP1 contains two CUB domains (a1 and a2) involved in binding the ligand Semaphorin3A (SEMA3A), two Factor V/VIII domains (b1 and b2) involved in VEGF ligand binding and one MAM domain (c domain). While functional antibodies with anti-tumor activity have been generated against the SEMA3A and VEGF binding domains, targeting other domains of NRP1 has not been well-studied. For example, the c-domain of NRP1 has been implicated in the dimerization of NRP1, a prerequisite for functionality. Thus c-domain blockers might inhibit multiple signaling pathways required for tumor growth. Alternatively, binders targeting the interface of two different domains might allow for simultaneous blocking of multiple ligands. We therefore hypothesized that these novel binders to alternative regions of NRP1 such as the b1/b2 interface and the c-domain would have improved and significant anti-tumor activity. Methods Antibodies with diverse binding characteristics were tested for in vivo anti-tumor activity in multiple syngeneic models, including anti-PD1 non-responsive models. Ability of these of the anti-NRP1 antibodies to elicit an adaptive immune response was evaluated. Results Novel binders to alternative regions of NRP1 such as domain interfaces and the c-domain resulted in significant tumor growth inhibition in multiple syngeneic models. Additionally, the NRP1 antagonists induced a robust CD8 T cell response, indicating immune-stimulatory role of these antibodies. Conclusions Cygnal has developed fully human monoclonal antibodies against previously untargeted domains of NRP1. These novel domain binders show remarkable inhibition of tumor growth in multiple syngeneic models and offer a novel means of therapeutic intervention in patients. Citation Format: Shalini Sethumadhavan, Eric Zhu, Aaron Fulgham, Mandana Abbassi, Ameya Apte, Tiffany Liao, Chengfeng Merriman, Katherine Molloy, Caitlin Stein, Amber Hanna, James Geoghegan, Bianka Prinz, Hongyue Dai, Jonathan Hurov, Pearl Huang, Daniel Blom. Novel mechanisms of Neuropilin-1 inhibition result in improved tumor growth inhibition in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 526.

Impaired AGO2/miR-185-3p/NRP1 axis promotes colorectal cancer metastasis

Increasing evidence suggests that global downregulation of miRNA expression is a hallmark of human cancer, potentially due to defects in the miRNA processing machinery. In this study, we found that the protein expression of Argonaute 2 (AGO2), a key regulator of miRNA processing, was downregulated in colorectal cancer (CRC) tissues, which was also consistent with the findings of the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Furthermore, the correlation between the levels of AGO2 and epithelial-mesenchymal transition (EMT) markers (E-cadherin and vimentin) indicated that reduced levels of AGO2 promoted EMT in CRC. Low expression of AGO2 was an indicator of a poor prognosis among CRC patients. Knockdown of AGO2 in CRC cells promoted migration, invasion and metastasis formation in vitro and in vivo but had no influence on proliferation. To provide detailed insight into the regulatory roles of AGO2, we performed integrated transcriptomic, quantitative proteomic and microRNA sequencing (miRNA-seq) analyses of AGO2 knockdown cells and the corresponding wild-type cells and identified neuropilin 1 (NRP1) as a new substrate of AGO2 via miR-185-3p. Our data provided evidence that knockdown of AGO2 resulted in a reduction of miR-185-3p expression, leading to the upregulation of the expression of NRP1, which is a direct target of miR-185-3p, and elevated CRC cell metastatic capacity. Inhibition of NRP1 or treatment with a miR-185-3p mimic successfully rescued the phenotypes of impaired AGO2, which suggested that therapeutically targeting the AGO2/miR-185-3p/NRP1 axis may be a potential treatment approach for CRC.

Effects of MLL5 and HOXA regulated by NRP1 on radioresistance in A549.

Radiotherapy is widely used in the management of lung cancer, and physicians are aware that the effect of radiotherapy is dependent on radiosensitivity. Although a series of blockers and activators targeting molecules related to radioresistance have been developed as radiation sensitizers, compensatory mechanisms or drug resistance limits their clinical efficacy. The identification of a key molecule related to lung cancer cell radioresistance or an effective molecular target is a challenging but important problem in radiation oncology. A previous study found that neuropilin 1 (NRP1) is related to radioresistance in A549 cells and is associated with VEGF, PI3K-Akt, MAPK-ERK, P38, NF-κβ and TGF-β. Inhibition of NRP1 can increase the radiosensitivity of A549 cells. Therefore, NRP1 may be a molecular target for radiotherapy-sensitizing drugs in lung cancer. The present study investigated the key downstream genes of NRP1, verified their regulation and clarified their roles in regulating lung cancer radioresistance. NRP1 positively regulated the downstream homeobox genes (HOXs) HOXA6, HOXA9 and mixed lineage leukaemia 5 (MLL5) in addition to MLL5-regulated HOXA6 and HOXA9, but these genes did not regulate NRP1. MLL5, HOXA6 and HOXA9 levels were decreased in tumour tissues and positively correlated with NRP1. All of these genes were induced by ionizing radiation in vivo and in vitro. NRP1 expression was significantly lower in squamous cell carcinoma compared with that in adenocarcinoma, and lymph node metastasis occurred more often in patients with lung cancer with high MLL5 and NRP1 expression compared with patients with low MLL5 and NRP1 expression. Collectively, these data confirmed that NRP1 is associated with MLL5 and regulates radioresistance through HOXA6 and HOXA9.

MiR-200c sensitizes Olaparib-resistant ovarian cancer cells by targeting Neuropilin 1

BackgroundOvarian cancer (OC) is the most lethal gynecological malignancy and the second leading cause of cancer-related death in women. Treatment with PARP inhibitors (PARPi), such as Olaparib, has been recently introduced for OC patients, but resistance may occur and underlying mechanisms are still poorly understood. The aim of this study is to identify target genes within the tumor cells that might cause resistance to Olaparib. We focused on Neuropilin 1 (NRP1), a transmembrane receptor expressed in OC and correlated with poor survival, which has been also proposed as a key molecule in OC multidrug resistance.MethodsUsing three OC cell lines (UWB, UWB-BRCA and SKOV3) as model systems, we evaluated the biological and molecular effects of Olaparib on OC cell growth, cell cycle, DNA damage and apoptosis/autophagy induction, through MTT and colony forming assays, flow cytometry, immunofluorescence and Western blot analyses. We evaluated NRP1 expression in OC specimens and cell lines by Western blot and qRT-PCR, and used RNA interference to selectively inhibit NRP1. To identify miR-200c as a regulator of NRP1, we used miRNA target prediction algorithms and Pearsons’ correlation analysis in biopsies from OC patients. Then, we used a stable transfection approach to overexpress miR-200c in Olaparib-resistant cells.ResultsWe observed that NRP1 is expressed at high levels in resistant cells (SKOV3) and is upmodulated in partially sensitive cells (UWB-BRCA) upon prolonged Olaparib treatment, leading to poor drug response. Our results show that the selective inhibition of NRP1 is able to overcome Olaparib resistance in SKOV3 cells. Moreover, we demonstrated that miR-200c can target NRP1 in OC cells, causing its downmodulation, and that miR-200c overexpression is a valid approach to restore Olaparib sensitivity in OC resistant cells.ConclusionsThese data demonstrate that miR-200c significantly enhanced the anti-cancer efficacy of Olaparib in drug-resistant OC cells. Thus, the combination of Olaparib with miRNA-based therapy may represent a promising treatment for drug resistant OC, and our data may help in designing novel precision medicine trials for optimizing the clinical use of PARPi.

Dysfunction of MiR-148a-NRP1 Functional Axis Suppresses Osteogenic Differentiation of Periodontal Ligament Stem Cells Under Inflammatory Microenvironment.

Periodontitis is a chronic inflammatory disease that can lead to the loss of periodontal bone tissue. The osteogenic potential of periodontal ligament stem cells (PDLSCs) is significantly decreased in periodontitis microenvironment. However, the mechanism is still unclear. We used Porphyromonas gingivalis lipopolysaccharide (LPS) as a stimulator of PDLSCs to mimic the periodontal inflammatory environment. The mineralization capability was restrained in LPS-stimulated PDLSCs, and the level of miR-148a increased, while the level of Neuropilin 1 (NRP1) decreased. Downregulation of miR-148a could reverse the osteogenesis deficiency of PDLSCs under LPS treatment. In addition, the expression of miR-148a in PDLSCs was negatively correlated with the expression of NRP1. Furthermore, overexpression of NRP1 upregulated the osteogenesis ability of LPS-stimulated PDLSCs, while inhibition of NRP1 eliminated the stimulative effect of miR-148a inhibitor on osteogenic differentiation. These data illustrated that the inflammatory environment mimicked by LPS inhibits osteogenesis by upregulation of miR-148a and subsequent downregulation of NRP1. We also found, compared to healthy periodontal tissues, miR-148a level increased, while NRP1 level decreased in periodontitis tissues. These two phenomena also exist in PDLSCs that come from the upper two types of tissues. To summarize, the decline of osteogenic potential of PDLSCs under inflammatory condition of periodontitis is related to miR-148a/NRP1 functional axis. This study may provide a novel strategy in the molecular aspect for the therapy of periodontitis.

NRP1 regulates HMGB1 in vascular endothelial cells under high homocysteine condition

Endothelial inflammation plays an important role in hyperhomocysteinemia (HHcy)-associated vascular diseases. High mobility group box 1 (HMGB1) is a pro-inflammatory danger molecule produced by endothelial cells. However, whether HMGB1 is involved in vascular endothelial inflammation of HHcy is poorly understood. Neuropilin-1 (NRP1) mediates inflammatory response and activates mitogen-activated protein kinases (MAPKs) pathway that has been reported to be involved in regulation of HMGB1. The aim of this study was to determine the alteration of HMGB1 in HHcy, and the role of NRP1 in regulation of endothelial HMGB1 under high homocysteine (Hcy) condition. In the present study, we first observed that the plasma level of HMGB1 was elevated in HHcy patients and an experimental rat model, and increased HMGB1 was also observed in the thoracic aorta of an HHcy rat model. HMGB1 was induced by Hcy accompanied with upregulated NRP1 in vascular endothelial cells. Overexpression of NRP1 promoted expression and secretion of HMGB1 and endothelial inflammation; knockdown of NRP1 inhibited HMGB1 and endothelial inflammation induced by Hcy, which partially regulated through p38 MAPK pathway. Furthermore, NRP1 inhibitor ATWLPPR reduced plasma HMGB1 level and expression of HMGB1 in the thoracic aorta of HHcy rats. In conclusion, our data suggested that Hcy requires NRP1 to regulate expression and secretion of HMGB1. The present study provides the evidence for inhibition of NRP1 and HMGB1 to be the novel therapeutic targets of vascular endothelial inflammation in HHcy in the future. NEW & NOTEWORTHY This study shows for the first time to our knowledge that the plasma level of high mobility group box 1 (HMGB1) is elevated in hyperhomocysteinemia (HHcy) patients, and homocysteine promotes expression and secretion of HMGB1 partially regulated by neuropilin-1 in endothelial cells, which is involved in endothelial inflammation. Most importantly, these new findings will provide a potential therapeutic strategy for vascular endothelial inflammation in HHcy.

Genetic status of KRAS modulates the role of Neuropilin-1 in tumorigenesis

Neuropilin-1 (NRP1), a non–tyrosine kinase receptor, is overexpressed in many cancers including pancreatic and lung cancers. Inhibition of NRP1 expression, however, has differing pro-tumor vs. anti-tumor effects, depending on the cancer types. To understand the differential role of NRP1 in tumorigenesis process, we utilized cells from two different cancer types, pancreatic and lung, each containing either wild type KRAS (KRASwt) or mutant KRAS (KRASmt). Inhibition of NRP1 expression by shRNA in both pancreatic and lung cancer cells containing dominant active KRASmt caused increased cell viability and tumor growth. On the contrary, inhibition of NRP1, in the tumor cells containing KRASwt showed decreased tumor growth. Importantly, concurrent inhibition of KRASmt and NRP1 in the tumor cells reverses the increased viability and leads to tumor inhibition. We found that NRP1 shRNA expressing KRASmt tumor cells caused increased cell viability by decreasing SMAD2 phosphorylation. Our findings demonstrate that the effects of NRP1 knockdown in cancer cells are dependent on the genetic status of KRAS.

Abstract 4908: Neuropilin-1 is up-regulated in the adaptive response of prostate tumors to androgen targeted therapies and is prognostic of metastatic progression and patient mortality

Abstract Aims: Androgen-targeted therapies (ATTs) are the mainstay treatment for metastatic prostate cancer (PCa). However, ATTs promote adaptation of tumour cells and lead to castration resistant disease (CRPC). We have recently identified the cell surface receptor, Neuropilin-1 (NRP1) as increased during EMT and in CRPC. However, the role of NRP1 in the prostate epithelium is poorly understood. This study aims to determine whether the inhibition of NRP1 will be a feasible therapeutic strategy for blocking PCa metastasis and therapy resistance. Methods: qPCR and western blotting were used to assess NRP1 expression in PCa cell lines. NRP1 expression in CRPC was assessed using a murine LNCaP xenograft model of castration. NRP1 was knocked down with shRNA sequences from the pLKO.1 lentiviral construct. For metastasis assays, PC3 cells were microinjected into the zebrafish yolk sac and metastatic dissemination imaged 5 days later. NRP1 expression in radical prostatectomy (RP) samples from Mayo Clinic (545 patients) and Johns Hopkins Medical Institutions (JHMI; 188 patients) cohorts was quantified by Affymetrix exon arrays and multivariable analysis performed. Wound scratch migration and invasion assays were performed with the WoundMaker™ tool and IncuCyte™ FLR imaging systems. Results: NRP1 levels were elevated in humanCRPC xenografts, metastatic and castrate resistant clinical PCa samples (p <0.0001), and PCa cell lines. NRP1 suppression significantly reduced metastasis of human xenografts in zebrafish and the migratory and invasive behaviour of metastatic PCa cells (p=0.0002). Multivariable analysis identified NRP1 as a significant independent prognostic indicator of metastasis and prostate cancer specific mortality in two large clinical cohorts (Mayo Clinic and JHMI; p=0.008/0.048 and 0.013/0,034 respectively). We show that NRP1 knockdown promotes E-Cadherin expression and loss of vimentin in mesenchymal PCa cells. Conclusion: These results will provide the preclinical data necessary to rationalise the use of anti-NRP1 directed adjuvant therapies for clinical use in PCa patients receiving ATTs, and will pave the way for larger scale preclinical and clinical trials in the PCa setting. Citation Format: Marianna Volpert, Brian Tse, Ellca Ratther, Nataly Stylianou, Mannan Nouri, Melanie Lehman, Stephen McPherson, Mani Roshan-Moniri, Mandeep Takhar, Nicholas Erho, Mohamed Alshalafa, Elai Davicioni, Robert Jenkins, Ashley Ross, Jeffrey Karnes, Robert Den, Ladan Fazli, Martin Gleave, Elizabeth Williams, Paul Rennie, Ralph Buttyan, Pamela Russell, Colleen Nelson, Brett Hollier. Neuropilin-1 is up-regulated in the adaptive response of prostate tumors to androgen targeted therapies and is prognostic of metastatic progression and patient mortality [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 4908. doi:10.1158/1538-7445.AM2017-4908

Role of NRP-1 in VEGF-VEGFR2-Independent Tumorigenesis.

Recent studies suggest that neuropilin-1 (NRP-1) promotes angiogenesis mainly via VEGF and its receptors. It promotes tumorigenesis via formation of the NRP-1/ VEGF (vascular endothelial growth factor)/VEGFR2 (vascular endothelial growth factor receptor 2) complex. In addition to VEGF and its receptors, NRP-1 also binds with other growth factors such as platelet-derived growth factor (PDGF) and platelet-derived growth factor receptor (PDGFR). PDGF plays important roles in cellular proliferation and, in particular, blood vessel formation. Moreover, recent studies show that NRP-1 promotes angiogenesis via the NRP-1-ABL pathway, but independent of VEGF-VEGFR2. RAD51 is a protein involved in the signaling pathways of NRP1-ABL and PDGF(R), the expression of which is positively associated with cell radioresistance and chemoresistance. NRP-1 activates the signaling pathways of ABL and PDGF(R) to upregulate RAD51, which induces resistance to radiotherapy and chemotherapy in cancer cells. Furthermore, NRP-1 activates the tumor microenvironment by binding with fibronectin and activating ABL, thereby promoting tumor growth. Inhibition of NRP-1 may overcome the limitations of individually inhibiting the VEGF-VEGFR2 pathway in cancer therapy and provide new ideas for cancer treatment. Therefore, we review the role of NRP-1 in VEGF-VEGFR2-independent tumorigenesis.

Neuropilin 1 expression correlates with the Radio-resistance of human non-small-cell lung cancer cells.

The purpose of this study was to determine the correlation between over-expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio-sensitivity of non-small cell lung carcinoma (NSCLC) cells. 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V-Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X-ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF-κB. Finally, to examine the effect of shNRP1 on proliferation and radio-sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1-A549) showed a significant reduction in colony-forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA-mediated NRP1 inhibition also significantly enhanced the radio-sensitivity of NSCLC cells both in vitro and in vivo. The over-expression of NRP1 was correlated with growth, survival and radio-resistance of NSCLC cells via the VEGF-PI3K- NF-κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio-sensitization of NSCLC.

Calreticulin promotes migration and invasion of esophageal cancer cells by upregulating neuropilin-1 expression via STAT5A.

We previously revealed that the calreticulin (CRT) gene is a candidate oncogene promoting cell migration and invasion and that neuropilin-1 (NRP1) is a possible effector downstream of CRT in esophageal squamous carcinoma cells. This study aims to explore the mechanisms underlying the migration and invasion of esophageal cancer cells regulated by CRT through NRP1. Quantitative reverse-transcription polymerase chain reaction, Western blot analysis, chromatin immunoprecipitation, and reporter gene assays were used to investigate the relationship between CRT and NRP1. In vitro and in vivo assays were carried out to evaluate the effects of NRP1 on malignant phenotypes of ESCC cells and tumor metastasis in NOD/SCID mice. Immunohistochemistry was performed to analyze the expression of CRT and NRP1 in esophageal squamous cell carcinomas (ESCC). Knockdown of CRT decreased the expression of NRP1. Inhibition of NRP1 reduced ESCC cell motility in vitro and experimental metastasis in vivo. Ectopic expression of NRP1 rescued the defects of cell migration and invasion in CRT-shRNA cells. CRT depletion inhibited STAT5A phosphorylation at the Y694 site via a CaMKII-independent pathway. Moreover, STAT5A directly regulated NRP1 transcription. Knockdown of CRT or NRP1 led to a downregulation of MMP2, MMP9, and FAK. Notably, positive correlation was found between CRT and NRP1 expression in ESCC tissues (P = 5.87 × 10(-5)). CRT and NRP1 coexpression was significantly associated with lymph node metastasis (P = 0.025). Our findings suggest that NRP1 is a critical downstream effector of CRT in promoting cell migration and invasion, which might contribute to the metastasis of ESCC.

Abstract 26: MUC1 enhances neuropilin-1 signaling in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) has the worst prognosis of all cancers and is the 4th leading cause of cancer-related deaths in the United States. Mucin1 (MUC1) is a transmembrane glycoprotein over-expressed in more than 60% of PDA and its expression correlates with high metastases and poor prognosis. In PDA, there is a correlation between blood vessel density, tumor levels of VEGF, and disease progression. We have recently discovered a novel association between MUC1 and neuropilin-1 (NRP-1) expression. Neuropilin-1 (NRP-1) is a co-receptor for VEGF165 and blockade of NRP-1-VEGF165 interaction has been shown to inhibit angiogenesis and tumor growth in several other cancer types. Thus, we hypothesize that MUC1 induces a pro-angiogenic tumor microenvironment by increasing levels of VEGF and NRP-1, thus enhancing disease progression and metastases. We have generated Muc1-expressing KC and Muc1-null KCKO mouse PDA cell lines for this study. Compared to KCKO, KC cells secrete more VEGF and express higher levels of NRP-1 as well as VEGF receptors (1/2/3). Although the cell culture supernatants from KC do not alter the NRP-1 level on the endothelial line 2H11 cells, they significantly promote the 2H11 cell tube formation. Consistent with the in vitro results, tumor lysates from KC-bearing mice have higher levels of NRP-1 and VEGFR2 and its phosphorylations at Tyr residues 951, 996, and 1175. In human PDA cell lines, inhibition of MUC1 expression by specific siRNA reduced NRP-1 level while overexpression of full-length MUC1 increases NRP-1 expression. This suggests a direct regulation of NRP-1 by MUC1. Furthermore, the over-expression of MUC1 in BxPC3 human PDA cell line induces strong angiogenesis and promotes tumor metastasis in a zebrafish tumor xenograft model. Finally, we report that inhibition of NRP-1 by its specific blocking peptide suppresses BxPC3.MUC1 tumor growth in vivo. Taking together, MUC1 in PDA cells can enhance their own NRP-1 levels and promote the VEGF-VEGFR-NRP-1 signaling, which may favor the creation of a pro-angiogenic microenvironment for tumor growth and metastases. Our findings highlight the potential for MUC1 targeted blockade of VEGF-NRP-1 interaction in PDA by conjugating the NRP-1 inhibitory agents to a MUC1 targeting antibody. Citation Format: Ru Zhou, Jennifer Curry, Priyanka Grover, Lopamudra Das Roy, TinChung Leung, Pinku Mukherjee. MUC1 enhances neuropilin-1 signaling in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 26. doi:10.1158/1538-7445.AM2014-26

Preserved vascular integrity and enhanced survival following neuropilin-1 inhibition in a mouse model of CD8 T cell-initiated CNS vascular permeability

BackgroundAltered permeability of the blood–brain barrier (BBB) is a feature of numerous neurological conditions including multiple sclerosis, cerebral malaria, viral hemorrhagic fevers and acute hemorrhagic leukoencephalitis. Our laboratory has developed a murine model of CD8 T cell-initiated central nervous system (CNS) vascular permeability in which vascular endothelial growth factor (VEGF) signaling plays a prominent role in BBB disruption.FindingsIn this study, we addressed the hypothesis that in vivo blockade of VEGF signal transduction through administration of peptide (ATWLPPR) to inhibit neuropilin-1 (NRP-1) would have a therapeutic effect following induction of CD8 T cell-initiated BBB disruption. We report that inhibition of NRP-1, a co-receptor that enhances VEGFR2 (flk-1) receptor activation, decreases vascular permeability, brain hemorrhage, and mortality in this model of CD8 T cell-initiated BBB disruption. We also examine the expression pattern of VEGFR2 (flk-1) and VEGFR1 (flt-1) mRNA expression during a time course of this condition. We find that viral infection of the brain leads to increased expression of flk-1 mRNA. In addition, flk-1 and flt-1 expression levels decrease in the striatum and hippocampus in later time points following induction of CD8 T cell-mediated BBB disruption.ConclusionThis study demonstrates that NRP-1 is a potential therapeutic target in neuro-inflammatory diseases involving BBB disruption and brain hemorrhage. Additionally, the reduction in VEGF receptors subsequent to BBB disruption could be involved in compensatory negative feedback as an attempt to reduce vascular permeability.

Defective Retinal Vascular Endothelial Cell Development As a Consequence of Impaired Integrin αVβ8-Mediated Activation of Transforming Growth Factor-β

Deletions of the genes encoding the integrin αVβ8 (Itgav, Itgb8) have been shown to result in abnormal vascular development in the CNS, including prenatal and perinatal hemorrhage. Other work has indicated that a major function of this integrin in vivo is to promote TGFβ activation. In this paper, we show that Itgb8 mRNA is strongly expressed in murine Müller glia and retinal ganglion cells, but not astrocytes. We further show that Itgb8 deletion in the entire retina severely perturbs development of the murine retinal vasculature, elevating vascular branch point density and vascular coverage in the superficial vascular plexus, while severely impairing formation of the deep vascular plexus. The stability of the mutant vasculature is also impaired as assessed by the presence of hemorrhage and vascular basal lamina sleeves lacking endothelial cells. Specific deletion of Itgb8 in Müller glia and neurons, but not deletion in astrocytes, recapitulates the phenotype observed following Itgb8 in the entire retina. Consistent with αVβ8's role in TGFβ1 activation, we show that retinal deletion of Tgfb1 results in very similar retinal vascular abnormalities. The vascular deficits appear to reflect impaired TGFβ signaling in vascular endothelial cells because retinal deletion of Itgb8 reduces phospho-SMAD3 in endothelial cells and endothelial cell-specific deletion of the TGFβRII gene recapitulates the major deficits observed in the Itgb8 and TGFβ1 mutants. Of special interest, the retinal vascular phenotypes observed in each mutant are remarkably similar to those of others following inhibition of neuropilin-1, a receptor previously implicated in TGFβ activation and signaling.