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Michael Klagsbrun - One of the best experts on this subject based on the ideXlab platform.
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Inflammation and Lymphedema Are Exacerbated and Prolonged by Neuropilin 2 Deficiency.
The American journal of pathology, 2016Co-Authors: Patrick Mucka, Rosalyn M Adam, Elena Geretti, Michael Klagsbrun, Nicholas Levonyak, Bernadette M.m. Zwaans, Irit Adini, Diane R. BielenbergAbstract:The vasculature influences the progression and resolution of tissue inflammation. Capillaries express vascular endothelial growth factor (VEGF) receptors, including neuropilins (NRPs), which regulate interstitial fluid flow. NRP2, a receptor of VEGFA and semaphorin (SEMA) 3F ligands, is expressed in the vascular and lymphatic endothelia. Previous studies have demonstrated that blocking VEGF receptor 2 attenuates VEGFA-induced vascular permeability. The inhibition of NRP2 was hypothesized to decrease vascular permeability as well. Unexpectedly, massive tissue swelling and edema were observed in NRP2 −/− mice compared with wild-type littermates after delayed-type hypersensitivity reactions. Vascular permeability was twofold greater in inflamed blood vessels in NRP2-deficient mice compared to those in NRP2-intact littermates. The addition of exogenous SEMA3F protein inhibited vascular permeability in Balb/cJ mice, suggesting that the loss of endogenous Sema3F activity in the NRP2-deficient mice was responsible for the enhanced vessel leakage. Functional lymphatic capillaries are necessary for draining excess fluid after inflammation; however, NRP2 -mutant mice lacked superficial lymphatic capillaries, leading to 2.5-fold greater fluid retention and severe lymphedema after inflammation. In conclusion, NRP2 deficiency increased blood vessel permeability and decreased lymphatic vessel drainage during inflammation, highlighting the importance of the NRP2/SEMA3F pathway in the modulation of tissue swelling and resolution of postinflammatory edema.
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hypoxia induces tumor and endothelial cell migration in a semaphorin 3f and vegf dependent manner via transcriptional repression of their common receptor neuropilin 2
Cell Adhesion & Migration, 2011Co-Authors: Silvia Coma, Akio Shimizu, Michael KlagsbrunAbstract:Neuropilin-2 (NRP2) is a receptor expressed by tumor cells and endothelial cells (EC) that binds both semaphorin 3F (SEMA3F), a potent inhibitor of tumor angiogenesis and metastasis, and vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. It was found that glioblastoma and melanoma cells repressed NRP2 expression when maintained under hypoxic conditions and after treatment with the hypoxia-mimetic agent desferrioxamine (DFO), at both the mRNA and protein levels. Silencing of HIF1-α, the hypoxia-induced subunit of the hypoxia inducible factor (HIF), abrogated DFO-induced NRP2 repression. Conversely, ectopic expression of HIF1-α directly repressed NRP2 promoter activity and expression. NRP2 is the sole receptor for SEMA3F. Loss of NRP2 expression in tumor cells inhibited SEMA3F-dependent activities, such as inactivation of RhoA, depolymerization of F-actin, and inhibition of tumor cell migration. On the other hand, loss of NRP2 expression in tumor cells increased VEGF prote...
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Hypoxia induces tumor and endothelial cell migration in a semaphorin 3F- and VEGF-dependent manner via transcriptional repression of their common receptor neuropilin 2.
Cell adhesion & migration, 2011Co-Authors: Silvia Coma, Akio Shimizu, Michael KlagsbrunAbstract:Neuropilin-2 (NRP2) is a receptor expressed by tumor cells and endothelial cells (EC) that binds both semaphorin 3F (SEMA3F), a potent inhibitor of tumor angiogenesis and metastasis, and vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. It was found that glioblastoma and melanoma cells repressed NRP2 expression when maintained under hypoxic conditions and after treatment with the hypoxia-mimetic agent desferrioxamine (DFO), at both the mRNA and protein levels. Silencing of HIF1-α, the hypoxia-induced subunit of the hypoxia inducible factor (HIF), abrogated DFO-induced NRP2 repression. Conversely, ectopic expression of HIF1-α directly repressed NRP2 promoter activity and expression. NRP2 is the sole receptor for SEMA3F. Loss of NRP2 expression in tumor cells inhibited SEMA3F-dependent activities, such as inactivation of RhoA, depolymerization of F-actin, and inhibition of tumor cell migration. On the other hand, loss of NRP2 expression in tumor cells increased VEGF protein levels in conditioned media, with no effects on VEGF mRNA levels. This increase in VEGF protein levels promoted paracrine activation of EC, including VEGF receptor-2 phosphorylation, and activation of downstream signaling proteins such as p44/42 MAPK and p38 MAPK. In addition, the elevated VEGF levels induced EC migration and sprouting, two key steps of tumor angiogenesis in vivo. It was concluded that hypoxia regulates VEGF and SEMA3F activities through transcriptional repression of their common receptor NRP2, providing a novel mechanism by which hypoxia induces tumor angiogenesis, growth and metastasis.
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A mutated soluble neuropilin-2 B domain antagonizes vascular endothelial growth factor bioactivity and inhibits tumor progression.
Molecular cancer research : MCR, 2010Co-Authors: Elena Geretti, Akio Shimizu, Laurens A. Van Meeteren, Andrew C. Dudley, Lena Claesson-welsh, Michael KlagsbrunAbstract:Neuropilins (NRP1 and NRP2) are co-receptors for vascular endothelial growth factor (VEGF) and mediate angiogenesis and tumor progression. VEGF binds to the NRP1 and NRP2 B domains. Previously, it was shown that mutagenesis of the soluble NRP2 B domain (MutB-NRP2) increased affinity to VEGF by 8-fold. Here, we show that MutB-NRP2 inhibited 125I-VEGF binding to NRP1, NRP2 and VEGFR-2. It antagonized VEGF-induced VEGFR-2/NRP2 complex formation and inhibited VEGF-induced activation of AKT, a mediator of cell survival, without affecting activation of VEGFR-2. In 3D embryoid bodies (EB), a model of VEGF-induced angiogenesis, MutB-NRP2 inhibited VEGF-induced sprouting. When overexpressed in human melanoma cells, MutB-NRP2 inhibited tumor growth compared to control tumors. Avastin (Bevacizumab), a monoclonal antibody to VEGF, inhibited VEGF interactions with VEGFR-2, but not with NRPs. The combination of MutB-NRP2 and Avastin resulted in an enhanced inhibition of human melanoma tumor growth compared to MutB-NRP2 treatment only or Avastin treatment only. In conclusion, these results indicate that MutB-NRP2 is a novel antagonist of VEGF bioactivity and tumor progression.
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site directed mutagenesis in the b neuropilin 2 domain selectively enhances its affinity to vegf165 but not to semaphorin 3f
Journal of Biological Chemistry, 2007Co-Authors: Elena Geretti, Akio Shimizu, Peter Kurschat, Michael KlagsbrunAbstract:Abstract Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nm, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280–290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.
Arthur M. Mercurio - One of the best experts on this subject based on the ideXlab platform.
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Abstract 1785: Domain-specific antibodies to Neuropilin 2 implicate VEGF-C and not Semaphorin 3F in breast cancer stem cell function
Experimental and Molecular Therapeutics, 2020Co-Authors: Leslie A. Nangle, Hira Lal Goel, Luke Burman, Kristina Hamel, Nathaniel Bloom, Arthur M. MercurioAbstract:Introduction: There is a strong body of evidence indicating that the expression of Neuropilin-2 (NRP2) is enriched in breast cancer stem cells (CSCs) and that NRP2 signaling is critical for breast CSC function and resistance development. For this reason, the rationale for targeting NRP2 as a therapeutic strategy is compelling and timely. A major limitation that has hampered the development of this therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that block NRP2 signalling. aTyr Pharma has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of breast cancer. A significant advance made by aTyr is that through specific domain reactivity, they have demonstrated differential effects on ligand blocking, receptor homo and heterodimerization and functional activity. Importantly a subset of such antibodies show differential activity in the mammosphere assay of triple negative breast cancer. Results: Flow cytometry was used to assess the specificity of the in-house anti-NRP2 mAbs to NRP2 using A549 wild type versus NRP2 knockout clonal cells. The in-house anti-NRP2 mAbs bound to A549 wild-type cells while showing little or no binding to the NRP2 knockout clonal cells, and exhibited significantly superior specificity, and sensitivity compared to existing commercially available antibodies. Displacement studies demonstrated that the tested anti-NRP2 mAbs showed different capabilities in blocking of VEGF-C or SEMA-3F binding to Expi293-hNRP2 cells, and were categorized as blockers (>90% inhibition), partial blockers (30-90% inhibition), or non-blockers (no obvious inhibition). To further extend the assessment of the biological activity of the anti-NRP2 antibodies, their activity was assessed in a receptor dimerization assay. Vectors encoding a split luciferase pBiT1.1 and pBiT2.1, and a cell permeable substrate, were obtained from Promega corporation. The complete extracellular domain and transmembrane helices of NRP2, FLT4 (VEGFR3), KDR (VEGFR2) and plexin A1 (PLXNA1) were cloned into the vectors and screened for optimal orientation, following established methods. Tested antibodies were able to impair respective VEGF and SEMA3 induced dimerization of receptor pairs NRP2/FLT4, NRP2/KDR and NRP2/PLXNA1. Surprisingly select antibodies show extremely specific and non-obvious functional differentiation. Direct functional assessment of a subset of these antibodies on breast CSCs revealed that the VEGF blocking, but not the SEMA3 blocking anti-NRP2 amAbs had the ability to inhibit serial passage mammosphere formation, an indicator of self-renewal potential. Conclusions: A series of domain specific antibodies to NRP2 have been developed and characterized. These antibodies show differential binding to specific domains of NRP2, can inhibit either VEGF-C or Sema3F binding to NRP2, and differentially effect receptor dimerization. The use of these antibodies enabled us to implicate VEGF-C/NRP2 signaling but not SEMA-3F/NRP2 signaling in the function of breast CSCs. Citation Format: Leslie A. Nangle, Luke Burman, Hira L. Goel, Zhiwen Xu, Kristina Hamel, Nathaniel Bloom, Arthur M. Mercurio. Domain-specific antibodies to Neuropilin 2 implicate VEGF-C and not Semaphorin 3F in breast cancer stem cell function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1785.
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The VEGF receptor neuropilin 2 promotes homologous recombination by stimulating YAP/TAZ-mediated Rad51 expression.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Ameer L. Elaimy, Hira Lal Goel, John J. Amante, Lihua Julie Zhu, Mengdie Wang, Charlotte S. Walmsley, Thomas J. Fitzgerald, Arthur M. MercurioAbstract:Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF–NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF–NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF–NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ–TEAD transcriptional target. We also discovered that VEGF–NRP2–YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF–NRP2 or YAP/TAZ. These findings reveal roles for VEGF–NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF–NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.
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Neuropilin 2: Novel Biomarker and Therapeutic Target for Aggressive Prostate Cancer
2015Co-Authors: Arthur M. MercurioAbstract:Abstract : The focus of this proposal is Neuropilin-2 (NRP2), a VEGF receptor that is not expressed in normal prostate but is expressed in prostate cancer and correlates with Gleason grade. We demonstrated that PTEN deletion induces NRP2 expression and propose that NRP2 contributes to the function of prostate cancer stem cells and tumor formation. Recently, we obtained rigorous genetic evidence implicating NRP2 in the formation of prostate tumors, and in the genesis and function of prostate cancer stem cells. We also discovered that NRP2 facilitates the expression of Bmi-1, a transcriptional repressor, and that NRP2 suppresses the IGF-1 receptor (IGF-1R) by a mechanism that involves transcriptional repression by Bmi-1. We have obtained preliminary evidence that targeting NRP2 directly on tumor cells in combination with IGF-1R inhibition is effective treating aggressive prostate carcinoma and pursuing this possibility more rigorously. Recently, we generated a model of resistance to therapy that reinforces the importance of VEGF/NRP2 signaling to the function of prostate cancer stem cells and the approach of targeting this pathway for therapy.
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Structural Basis for VEGF-C Binding to Neuropilin-2 and Sequestration by a Soluble Splice Form.
Structure (London England : 1993), 2015Co-Authors: Matthew W. Parker, Hira Lal Goel, Arthur M. Mercurio, Andrew Linkugel, Craig W. Vander KooiAbstract:Vascular endothelial growth factor C (VEGF-C) is a potent lymphangiogenic cytokine that signals via the coordinated action of two cell surface receptors, Neuropilin-2 (NRP2) and VEGFR-3. Diseases associated with both loss and gain of VEGF-C function, lymphedema and cancer, respectively, motivate studies of VEGF-C/NRP2 binding and inhibition. Here, we demonstrate that VEGF-C binding to NRP2 is regulated by C-terminal proteolytic maturation. The structure of the VEGF-C C terminus in complex with the ligand binding domains of NRP2 demonstrates that a cryptic NRP2 binding motif is released upon proteolysis, allowing specific engagement with the b1 domain of NRP2. Based on the identified structural requirements for NRP2 binding to VEGF-C, we hypothesized that the endogenous secreted splice form of NRP2, s9NRP2, may function as a selective inhibitor of VEGF-C. We find that s9NRP2 forms a stable dimer that potently inhibits VEGF-C/NRP2 binding and cellular signaling. These data provide critical insight into VEGF-C/NRP2 binding and inhibition.
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Neuropilin-2 regulates α6β1 integrin in the formation of focal adhesions and signaling
Journal of Cell Science, 2012Co-Authors: Hira Lal Goel, Bryan M. Pursell, Clive Standley, Kevin E. Fogarty, Arthur M. MercurioAbstract:The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2high and NRP2low populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2high population adhered more avidly to laminin and expressed high levels of the α6β1 integrin than the NRP2low population. The NRP2high population formed numerous focal adhesions on laminin that were not seen in the NRP2low population. These results were substantiated using breast carcinoma cell lines that express NRP2 and α6β1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and α6β1 integrin was detected by co-immunoprecipitation. NRP2 is necessary for focal adhesion formation on laminin and for the association of α6β1 integrin with the cytoskeleton. NRP2 also facilitates α6β1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin. The mechanism by which NRP2 regulates the interaction of α6β1 integrin with laminin to form focal adhesions involves PKC activation. Together, our data reveal a new VEGF–NRP2 signaling pathway that activates the α6β1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer.
Takeshi Kawauchi - One of the best experts on this subject based on the ideXlab platform.
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the coup tfii neuropilin 2 is a molecular switch steering diencephalon derived gabaergic neurons in the developing mouse brain
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Shigeaki Kanatani, Takao Honda, Michihiko Aramaki, Kanehiro Hayashi, Ken Ichiro Kubo, Mami Ishida, Daisuke H Tanaka, Takeshi KawauchiAbstract:The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)–induced expression of Neuropilin-2 (NRP2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and NRP2. The present findings show that suppression of COUP-TFII/NRP2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/NRP2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/NRP2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.
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The COUP-TFII/neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Shigeaki Kanatani, Takao Honda, Michihiko Aramaki, Kanehiro Hayashi, Ken Ichiro Kubo, Mami Ishida, Daisuke H Tanaka, Takeshi Kawauchi, Katsutoshi Sekine, Sayaka KusuzawaAbstract:The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)–induced expression of Neuropilin-2 (NRP2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and NRP2. The present findings show that suppression of COUP-TFII/NRP2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/NRP2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/NRP2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.
Gan-feng Xie - One of the best experts on this subject based on the ideXlab platform.
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Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis
Cell & bioscience, 2020Co-Authors: Xi Luo, Qiu-xia Shu, Can Chen, Yu-zhu Gong, Xiao-long Zhao, Gan-feng XieAbstract:Angiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified. Dataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis. NRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients. Vascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.
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Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis
2020Co-Authors: Xi Luo, Qiu-xia Shu, Can Chen, Yu-zhu Gong, Xiao-long Zhao, Gan-feng XieAbstract:Abstract Background: Angiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified.Methods: Dataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis.Results: NRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients.Conclusion: Vascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.
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Vascular NRP2 triggers PNET angiogenesis via activating SSH1-cofilin axis
2020Co-Authors: Xi Luo, Qiu-xia Shu, Can Chen, Yu-zhu Gong, Xiao-long Zhao, Gan-feng XieAbstract:Abstract Background Angiogenesis is a critical step in pancreatic neuroendocrine tumour (PNET) growth and may be a selective target for PNET therapy. However, PNET is robustly resistant to current antiangiogenic therapies which primarily target the VEGFR pathway. Thus, PNET angiogenesis mechanism was urgently to be clarified. Methods Dataset analysis was used to identify PNET angiogenesis related genes. Immunohistochemistry was performed to determine relation among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assay were investigated to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2 inducing angiogenesis was detected by mutant construction, Western blot, and immunofluorescence assay. In vivo, mice model was performed to evaluate the effect of NRP2 antibody, and clinical data was recruited to verify association between NRP2 and patients prognosis. Results NRP2, a VEGFR2 co-receptor, positively correlated with vascularity rather than with VEGFR2 in PNET tissues. NRP2 promoted PNET cell medium-treated HUVEC cell migration via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Mechanistically, cofilin phosphatase slingshot-1 (SSH1) was highly expressed involved in NRP2-activating cofilin. Silencing SSH1 rescued NRP2-activated HUVEC cell migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumour growth. High NRP2 expression was associated with poor prognosis in PNET patients. Conclusion Vascular NRP2 promotes PNET angiogenesis via activating SSH1/cofilin/actin axis. Our findings demonstrate NRP2 to be an important regulator of angiogenesis and potential therapeutic target of anti-angiogenesis therapy for PNET.
Bang H. Hoang - One of the best experts on this subject based on the ideXlab platform.
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Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma
Molecular cancer, 2015Co-Authors: Yi Guo, Kapjun Kim, Peter Mcqueen, Samia Ghaffar, Alexander B. Christ, Carol Lin, Ramez N. Eskander, Bang H. HoangAbstract:Neuropilin 2 (NRP2) isa multi-functional co-receptor to many receptors, including VEGF receptor, c-Met and others. NRP2 has recently been implicated in tumor angiogenesis, growth, and metastasis of many other cancers. However, its role in osteosarcoma remains poorly understood. NRP2 was overexpressed in osteosarcoma cell lines and tissues, and associated with poor survival of osteosarcoma patients. Knockdown of NRP2 expression by short-hairpin (Sh) RNA resulted in reduced tumor growth, metastasis, and blood vessel formation of osteosarcoma. Knockdown of NRP2 expression by ShRNA also inhibited the recruitment of HUVEC cells to osteosarcoma cells. Inhibition of Wnt signaling by overexpression of secreted Wnt antagonists soluble LRP5, Frzb, and WIF1 markedly down-regulated mRNA and protein expression of NRP2 in osteosarcoma cell lines. Regulation of NRP2 receptor expression may represent a novel approach for treatment of osteosarcoma through retarding osteosarcoma growth, metastasis and blood vessel formation. In addition, down-regulation of NRP2 expression can be achieved by expression of secreted Wnt antagonists.
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Abstract 4414: Neuropilin2 is a novel target gene of Wnt signaling pathway regulating angiogenesis, metastasis and growth of osteosarcoma
Molecular and Cellular Biology, 2014Co-Authors: Yi Guo, Carol Lin, Cheng-fong Chen, Bang H. HoangAbstract:Objective: Multivascularity of osteosarcoma (OS) rendered the anti-angiogenesis is a promising therapeutics. Neuropilin 2 (NRP2), a coreceptor of vascular endothelial growth factor (VEGF), has been implicated in tumor angiogenesis, growth, and metastasis of many cancers. Its act in osteosarcoma still remains poorly understood. In order to explore the role of NRP2 in modulating tumor growth, angiogenesis, invasion, and lung metastasis of osteosarcoma, we conduct the current study. Methods: Cell culture, RNA interference, stable transfection, real time PCR, western blot, motility and invasion assay, in vivo tumorigenesis model, tube formation assay, ChIP. Results: By microtissue array assay, the expression of NRP2 is implicated in metastasis and poor prognosis of osteosarcoma patients. The in vitro data showed NRP2 knockdown has an inhibitory effect on OS tumor cell growth, both anchoragedependent and anchorageindependent way. Xenograft model showed silencing of NRP2 expression in OS cells has an extraordinary inhibitory effect. Both migration and invasion were significantly impaired in two NRP2 knockdown OS cell lines in vitro. Again, a lung metastasis mice model was used to verify the antimetastasis effect after NRP2 silencing and the results showed metastasis was inhibited. The blood vessels and capillaries in the xenograft samples were evaluated by immunohistochemistry staining for CD31. The capillaries in NRP2 knockdown tumors were rarely seen compared with the intact capillaries in control tumors. In order to explore the mechanism underlying the anti angiogenesis effect, we conducted the coculture of tumor cells and HUVEC cells in both contact and noncontact way. The ability of recruiting HUVEC cells by OS cells were dramatically inhibited by NRP2 knockdown. Also NRP2 silencing caused the tumor cells to behave less involved in tube formation. ChIP assay revealed TCF4, wnt effector, bound all the five binding sites in NRP2 promoter region. Conclusion: We demonstrated that NRP2 plays an important role in modulating tumor growth, angiogenesis, invasion, and lung metastasis of osteosarcoma. Citation Format: Tao Ji, Yi Guo, Carol Lin, Cheng-Fong Chen, Xiaolin Zi, Bang H. Hoang. Neuropilin2 is a novel target gene of Wnt signaling pathway regulating angiogenesis, metastasis and growth of osteosarcoma. [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 4414. doi:10.1158/1538-7445.AM2014-4414
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Abstract 440: Wnt signaling regulates neuropilin-2 (NRP2) expression and contributes to cancer cell invasiveness in castration-resistant prostate cancer (CRPC)
Tumor Biology, 2012Co-Authors: Shuman Liu, Yi Guo, Zhengyu Jia, Zheng Sun, Micheal Lilly, Dan Mercola, Bang H. HoangAbstract:Progression to CRPC represents a major challenge for treatment of advanced PCa. However, underlying mechanisms of this process remain largely unknown. We found that androgen deprivation in mice bearing PCa patient-derived tumors and in PCa LNCaP cells resulted in increased expression of wnt transcriptional factors: TCF4 and LEF1, as well as c-myc and NRP2. Using GenBank and MatInspector software, we identified five potential binding sites for TCF4 in the 5′ flanking promoter region spanning from –3891 to +108 base pairs of the NRP2 gene. Treatment of LNCaP cells with Wnt3a and Wnt 5a conditioned medium increased NRP2 mRNA expression; and inhibition of Wnt signaling by overexpression of secreted Wnt antagonists (WIF1 and Frzb/sFRP3) in two CRPC cell lines: C4-2B and PC3 down-regulated the expression of NRP2 in both in vitro cell cultures and in in vivo xenograft tumors. Chromatin immunoprecipitation/real-time PCR a! nalysis revealed that both WIF1 and Frzb/sFRP3 expression decreased the in vivo binding of TCF4 and beta-catenin to the NRP2 promoter at five predicted sites. Knock-down of NRP2 by short-hairpin RNA in CRPC cell lines markedly reduced cellular invasiveness and migration capacity. Furthermore, we analyzed gene microarray data sets from the UCI SPECS project. The total of 204 samples from 82 patients include 66 tumors, 65 tumor-adjacent stromas, 28 far stromas and 45 normal prostate samples. The mRNA expression levels of the genes of interest were normalized to the average expression levels of these genes from the normal prostate samples. We showed that the percentage of samples with WIF1 down-regulation progressively increased from far stroma to adjacent stroma and tumors. Strikingly, the levels of NRP2 mRNA are up-regulated in all tested adjacent stroma and most of tumor samples. Furthermore, the expression levels of NRP2 in far and adjacent stroma and tumors are inversely related to WIF1 mRNA levels (correlation coefficients are –0.6918, –0. 2349 and –0.5931; Ps Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 440. doi:1538-7445.AM2012-440
