Neuropilin-1 Expression Research Articles

Prognostic value and in vitro biological relevance of Neuropilin 1 and Neuropilin 2 in osteosarcoma.

Neoadjuvant chemotherapy in osteosarcoma increased the long-term survival of patients with localized disease considerably but metastasizing osteosarcoma remained largely treatment resistant. Neuropilins, transmembrane glycoproteins, are important receptors for VEGF dependent hyper-vascularization in tumor angiogenesis and their aberrant expression promotes tumorigenesis and metastasis in many solid tumors. Our analysis of Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2) immunostaining in a tissue microarray of 66 osteosarcoma patients identified NRP2 as an indicator of poor overall, metastasis-free and progression free survival while NRP1 had no predictive value. Patients with tumors that expressed NRP2 in the absence of NRP1 had a significantly worse prognosis than NRP1(-)/NRP2(-), NRP1(+) or NRP1(+)/NRP2(+) tumors. Moreover, patients with overt metastases and with NRP2-positive primary tumors had a significantly shorter survival rate than patients with metastases but NRP2-negative tumors. Furthermore, the expression of both NRP1 and NRP2 in osteosarcoma cell lines correlated to a variable degree with the metastatic potential of the respective cell line. To address the functional relevance of Neuropilins for VEGF signaling we used shRNA mediated down-regulation and blocking antibodies of NRP1 and NRP2 in the metastatic 143B and HuO9-M132 cell lines. In 143B cells, VEGFA signaling monitored by AKT phosphorylation was more inhibited by blocking of NRP1, whereas in HuO9-M132 cells NRP2 blocking was more effective indicating that NRP1 and NRP2 can substitute each other in the functional interaction with VEGFR1. Altogether, these data point to NRP2 as a powerful prognostic marker in osteosarcoma and together with NRP1 as a novel target for tumor-suppressive therapy.

Prognostic impact of neuropilin-1 expression in children with B-lineage acute lymphoblastic leukemia: Retraction

Prognostic impact of neuropilin-1 expression in children with B-lineage acute lymphoblastic leukemia: Retraction

Expression of VEGFR2 and NRP-1 in non-small cell lung cancer and their clinical significance.

Vascular-targeted therapy is gradually becoming more appealing for patients with lung cancer. It is unclear whether vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) can be biomarkers for clinical treatment. We aimed to investigate the expression levels of VEGFR2 and NRP-1 in human non-small cell lung cancer (NSCLC) and their clinical significance by observing patient prognosis. VEGFR2 and NRP-1 were assessed by immunohistochemistry (IHC) in 40 patients with NSCLC and in 10 patients with benign lesions of lung; kinase insert domain receptor (KDR) and NRP-1 copy number gain (CNG) was assessed by fluorescence in situ hybridization (FISH). The distributions of overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method and compared between groups by log-rank test. Rates of positive immunostaining for VEGFR2 and NRP-1 were 58% and 55%, respectively. KDR and NRP-1 CNG (+) were detected in 32.5% and 30% of tumors, respectively. Levels of both VEGFR2 and NRP-1 in lung tumors were significantly different than in the control tissue (χ(2)=11.22, P=0.001; χ(2)=9.82, P=0.001, respectively); similar results were obtained using CNGs (χ(2)=4.39, P=0.036; χ(2)=3.95, P=0.046, respectively). Statistically significant correlations were observed with histological grade, clinical TNM stage and the lymph node status (P<0.05), but not age, gender or pathology type (P>0.05). VEGFR2 showed a strong correlation with NRP-1 (Rs=0.68, P=0.00); similar results were observed with KDR and NRP-1 CNG (Rs=0.32, P=0.04). Significant differences in OS and PFS were observed between the groups with higher VEGFR2 and NRP-1 and those with lower expression (P<0.05). According to these data, VEGFR2 and NRP-1 are highly expressed in NSCLC. We can conclude that they play a key role in NSCLC occurrence, development and metastasis and are associated with patient prognosis (P<0.05 for OS and PFS). This information will be beneficial for clinical anti-angiogenic treatment in NSCLC.

Prognostic Impact of Neuropilin-1 Expression in Egyptian Children with B-lineage Acute Lymphoblastic Leukemia.

BackgroundNeuropilins are transmembrane glycoproteins that act as receptors for vascular endothelial growth factors and are involved in the process of tumor angiogenesis.ObjectiveThe aim of this work was to study the prognostic value of Neuropilin-1 (NRP-1) expression in Egyptian children with B-lineage acute lymphoblastic leukemia (ALL).Patients and methodsThis study was conducted on fifty children with newly diagnosed B-lineage ALL, admitted to Oncology Unit, Pediatric Department, Tanta University Hospitals in the period from August 2010 to March 2014. This series included 32 males and 18 females with ages ranging from 3–17 years and a mean value of 9 ± 3.5 years. Twenty healthy children, age and sex matched, were also included in this study as a control group. For all patients, the following examens were done: Bone marrow aspiration, cytochemistry, immunophenotyping and estimation of Neuropilin-1 expression on blast cells by flow cytometry.ResultsThe present study revealed highly significant differences in Neuropilin-1 expression between B-lineage ALL lymphoblasts and control lymphocytes. A significant higher Neuropilin-1 expression was found in pre-B ALL (74.04%) compared with early pre-B (23.55%). Neuropilin-1 positive expression was associated with significantly higher white blood cells count (Mean = 69.3±18.53 ×103/mm3 versus 32.5±11.64 ×103/mm3 and p=0.003), bone marrow blasts percentage (Mean=76.12±21.4 % versus 41.2±19.71% and p= 0.003), serum lactate dehydrogenase levels (Mean=1992.2 ± 58.6 unit/L versus 955.1± 234.7 unit/L and p=0.001) at diagnosis compared with negative Neuropilin-1 expression. The levels of Neuropilin-1 on BM blasts at diagnosis were higher in patients who subsequently relapsed (Mean=53.8 ± 27.1) or later died (Mean=81.51 ± 9.94) during the period of follow-up compared to those who achieved and maintained complete remission (Mean=18.17 ± 10.4) with p value of 0.001. Furthermore, patients with higher Neuropilin-1 expression had significantly shorter overall survival (Median 27.99 months and p= 0.0133) and disease-free survival (Median=10.23 months and p= 0.0002) than patients with low Neuropilin-1 expression (Median disease-free survival was 38.7 months).ConclusionOur findings suggest that Neuropilin-1 is a poor prognosis factor in children with B-lineage ALL and so we recommend the inclusion of Neuropilin-1 as a prognostic marker in children with B-lineage ALL. Its presence at high levels suggests a poor prognosis, and the necessity of intensive therapeutic intervention.

Neuropilin-2 mediates lymphangiogenesis of colorectal carcinoma via a VEGFC/VEGFR3 independent signaling

Lymphangiogenesis critically contributes to the lymphatic metastasis of colorectal carcinomas (CRCs), but the underlying mechanism of CRC lymphangiogenesis remains largely elusive. We have previously demonstrated that Semaphorin-3F (SEMA3F) is critically involved in CRC metastasis, and the receptor of SEMA3F, neuropilin-2 (NRP2), originally described as an axon guiding chemorepulsant implicated in nerve development, has been suggested in promoting lymphangiogenesis via acting as an obligate co-receptor of VEGFR3 cooperatively enhancing the activity of VEGF-C. Our present study revealed that in colorectal carcinomas, NRP2 expression levels of tumor-associated lymphatic endothelial cells (LECs) are significantly correlated with the density of tumor lymphatic vessels. In vitro, activation of NRP2 in LECs substantially facilitates their migration, sprouting, and tubulogenesis capacity via regulating the rearrangement of cytoskeleton polarity. In vivo model further showed that in the xenografts generated from SEMA3F knockdown CRC cells, NRP2 is substantially activated in tumor-associated LECs, resulting in a significantly increased tumor lymphangiogenesis. Further evidence demonstrated that CRC cell induces the activation of NRP2 in LECs to promote tumor lymphangiogenesis via integrinα9β1/FAK/Erk pathway independent VEGF-C/VEGFR3 signaling. Our study for the first time revealed the novel molecular mechanism of NRP2-mediated-lymphangiogenesis in CRCs, suggesting NRP2 as a potential therapeutic target in preventing lymphatic metastasis of CRCs.

Prognostic significance of VEGF-C, semaphorin 3F, and neuropilin-2 expression in oral squamous cell carcinomas and their relationship with lymphangiogenesis.

Neuropilin-2 (NRP2), a receptor for vascular endothelial growth factor C (VEGF-C) and semaphorin 3 F (SEMA3F), is a possible regulator of tumor progression and angiogenesis. However, little evidence of a correlation between NRP2 expression and lymphangiogenesis has been reported. SEMA3F might suppress lymphangiogenesis by competing with VEGF-C for binding to NRP2. We evaluated lymphatic vessel density (LVD), lymphatic vessel invasion (LVI), the expression levels of VEGF-C, SEMA3F, and NRP2 by immunohistochemistry in 80 cases of oral squamous cell carcinomas (OSCCs). In these tumors, the expression of NRP2 was positively associated with T stage classification, lymph node metastasis, LVD, LVI, and the expression of VEGF-C. In contrast, low expression of SEMA3F was significantly related to poor differentiation and higher incidence of lymph node metastasis. Patients expressing high levels of VEGF-C or NRP2, or low levels of SEMA3F had a higher risk of recurrence and shorter overall survival. Multivariate analysis showed that VEGF-C and NRP2 were independent prognostic markers for overall survival. Results indicate SEMA3F is an inhibitor of tumor progression and provide evidence for an association between NRP2 and VEGF-C, lymphangiogenesis, lymph node metastasis. This suggests the prognostic significance and potential therapeutic value of the VEGF-C/SEMA3F/NRP2 axis for OSCCs.

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.

Novel expression of Neuropilin 1 on human tumor-infiltrating lymphocytes in colorectal cancer liver metastases

Objectives: Neuropilin 1 (NRP1) is a transmembrane protein with diverse roles in physiological and pathological settings. NRP1 expression has been reported on T cells in inflammatory microenvironments and in secondary lymphoid tissue. Tumor-infiltrating lymphocytes (TILs) play an important role in cancer prognosis. In this study, we investigated NRP1 expression on TILs and peripheral blood mononuclear cells (PBMCs) from colorectal cancer liver metastases (LI/CRC).Methods: TILs from LI/CRC and PBMCs from healthy donors and patients were analyzed for expression of NRP1, in addition to other Treg-related markers. PBMCs were co-cultured in vitro with tumor tissue and analyzed for NRP1 expression.Results: We report for the first time that NRP1 is highly expressed on CD3+CD4+ TILs compared to PBMCs. NRP1 expression correlated closely with CD25 expression in TILs. NRP1 was expressed on both Helios+ and Helios– FoxP3-expressing Tregs and on a FoxP3–Helios– T cell subset. It was also induced on PBMCs following in vitro co-culture with tumor tissue.Conclusions: NRP1 is upregulated on TILs and can be induced on PBMCs by tumor tissue. Further studies are warranted to define the function of NRP1 on human TILs. As a therapeutic target, NRP1 may allow selective targeting of TIL subsets including suppressive Tregs.

Expression and significance of Neuropilin-1 and CD4+ CD25+ Treg in human gliomas

Objective To investigate the expression and significance of Neuropilin-1 (NRP-1) mRNA and CD4 + CD25 + Treg in human glioma tissues.Methods The specimens of 41 patients with gliomas,including 11 cases of grade Ⅰ,12 grade Ⅱ,8 grade Ⅲ and 8 grade Ⅳ,were studied in the present study.11 cases of normal brain tissues from patients performed decompression operation resulting from cerebral hernia were selected as the control group.The expression of NRP-1 mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) and the contents of CD4 + CD25 + Treg by flow cytometer in the glioma tissues and normal brain tissues.Results NRP-1 mRNA and CD4 + CD25 + Treg were expressed in different degrees in normal brain tissues and gliomas.The expression level in tumor tissues was higher than that in the normal brain tissues.The expression was related to the pathological grades of gliomas.The NRP-1 expression was significantly positively related with the CD4 + CD25 + Treg expression (P ＜ 0.05).Conslusions The mRNA expression levels of NRP-1 and CD4 + CD25 + Treg were significantly higher in human gliomas which were positively associated with the grades of gliomas.NRP-1 and CD4 + CD25 + Treg might be related to the immunological tolerance in tumor microenvironment of human gliomas. Key words: Glioma; Neuropilin-1 ; CD4+ CD25+ Regulatory cells

Neuropilin-1 expression is induced on tolerant self-reactive CD8+ T cells but is dispensable for the tolerant phenotype.

Establishing peripheral CD8+ T cell tolerance is vital to avoid immune mediated destruction of healthy self-tissues. However, it also poses a major impediment to tumor immunity since tumors are derived from self-tissue and often induce T cell tolerance and dysfunction. Thus, understanding the mechanisms that regulate T cell tolerance versus immunity has important implications for human health. Signals received from the tissue environment largely dictate whether responding T cells become activated or tolerant. For example, induced expression and subsequent ligation of negative regulatory receptors on the surface of self-reactive CD8+ T cells are integral in the induction of tolerance. We utilized a murine model of T cell tolerance to more completely define the molecules involved in this process. We discovered that, in addition to other known regulatory receptors, tolerant self-reactive CD8+ T cells distinctly expressed the surface receptor neuropilin-1 (Nrp1). Nrp1 was highly induced in response to self-antigen, but only modestly when the same antigen was encountered under immune conditions, suggesting a possible mechanistic link to T cell tolerance. We also observed a similar Nrp1 expression profile on human tumor infiltrating CD4+ and CD8+ T cells. Despite high expression on tolerant CD8+ T cells, our studies revealed that Nrp1 had no detectable role in the tolerant phenotype. Specifically, Nrp1-deficient T cells displayed the same functional defects as wild-type self-reactive T cells, lacking in vivo cytolytic potential, IFNγ production, and antitumor responses. While reporting mostly negative data, our findings have therapeutic implications, as Nrp1 is now being targeted for human cancer therapy in clinical trials, but the precise molecular pathways and immune cells being engaged during treatment remain incompletely defined.

Peritumoral Neuropilin-1 and VEGF receptor-2 expression increases time to recurrence in hepatocellular carcinoma patients undergoing curative hepatectomy.

To determined Neuropilin-1 (NRP-1) and vascular endothelial growth factor receptor 2 (VEGFR-2) expression in the tumoral and peritumoral tissues of 214 treatment-naïve HCC patients and its correlation with overall survival (OS) and time to recurrence (TTR). NRP-1 and VEGFR-2 expression were examined by tissue microarray and peritumoral hypoxia by pimonidazole staining and angiogenesis by microvessel density (MVD). OS and TTR were evaluated by Kaplan-Meier analysis and log-rank test. Peritumoral NRP-1 and VEGFR-2 expression were significantly higher than that of the tumoral tissue (p < 0.001 for both), and high peritumoral expression of both factors was negatively associated with tumor size (p < 0.001 for both). Patients with high peritumoral expression of both proteins had the longest median OS (>94.0 months) and TTR (>84.0 months). The multivariate Cox proportional hazards analysis revealed that patients with high peritumoral expression of both NRP-1 and VEGFR-2 were more than 4 times less likely to have recurrence (p = 0.004) and more than 10 times likely to survive (p < 0.001). Peritumoral NRP-1 and VEGFR-2 expression is associated with prolonged TTR and extended OS of HCC patients and both may be useful as predictors of surgical outcome of HCC patients and explored as potential therapeutic targets.

Upregulation of CRMP4, a new prostate cancer metastasis suppressor gene, inhibits tumor growth in a nude mouse intratibial injection model

Prostate cancer, the most commonly diagnosed male cancer in North America, has a high incidence of bone metastasis. Our previous study showed collapsin response mediator protein 4 (CRMP4) gene inhibited prostate cancer migration and invasion. In this study, we investigated whether overexpression of CRMP4 gene in prostate cancer cells inhibit tumor bone metastasis. The stable prostate cancer cells overexpressing the CRMP4 gene were constructed using lentivirus infection. Prostate cancer bone metastasis nude mouse model was built though orthotopic prostate implantation, intracardiac injection and intratibial injection with CRMP4 overexpress and control cancer cells. Small animal PET/CT scanning results showed no difference of bone metastatic capacity in orthotopic and intracardiac injection models between CRMP4 overexpression and control group, while CRMP4 overexpression inhibited tumor growth in the intratibial injection model. Moreover, our invitro study showed CRMP4 overexpression downregulates the Neuropilin1 (NRP1) expression and upregulate the Noggin expression. Immunohistochemical staining of the hind limbs of intratibial injection model was confirmed with cytological experiments. Taken together, our research indicated CRMP4 inhibits prostate cancer cells growth in the nude mouse bone microenvironment and this effect may relate with regulation of NRP1 and Noggin expression.

Abstract IA17: Targeting placental growth factor/ neuropilin-1 pathway inhibits growth and spread of medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common pediatric brain tumor and a heterogeneous disease. Several studies have characterized tumor genetic heterogeneity with the hope of identifying targets for personalized therapy. In fact, inhibitors of Sonic Hedgehog (Shh) - a developmental pathway aberrantly activated in a subset of MBs - are currently being evaluated. Unfortunately, mutations and potential developmental adverse effects in the pediatric population can limit these therapies. We propose targeting the tumor stroma as an efficacious alternative treatment option. In particular, we aim to identify and target crucial cancer cell-stromal cell crosstalk signaling that sustains tumor growth and spread. In this context, we have evaluated the effects of blocking signaling of Placental Growth Factor (PIGF) - a member of the vascular endothelial growth factor (VEGF) family - that is dispensable in development but relevant during disease, in the growth and progression of MB. Here, we show the expression of PIGF and its receptor Neuropilin-1 (NRP-1) across primary tumors; dissect the mechanisms that regulate PIGF secretion and downstream signaling; and demonstrate the dramatic efficacy of specific anti-PIGF and anti-NRP-1 blocking antibodies in three MB models in vivo. Methods: Expression of PIGF and NRP-1 was analyzed in a cohort of 32 surgical samples of pediatric MBs by immunohistochemistry, array-comparative genomic hybridization, and deep-gene sequencing. The correlation between NRP-1 expression and 5-year overall survival was evaluated retrospectively in an independent, clinically annotated cohort of 42 MBs. Human orthotopic xenograft (D283-Med; D341-Med) and spontaneous (Smo/Smo) established tumors were treated with anti-PIGF (Thrombogenics or Genentech) or anti-NRP-1 (Genentech) antibodies. Tumor growth, progression and response to therapy was followed by 1) clinical evaluation of posterior fossa symptoms and body weight; 2) whole-body luminescence and blood GLuc levels; 3) small animal MRI; or 4) Optical Frequency Domain Imaging (OFDI). Results: We report that: 1) PIGF is expressed by more than 90% of primary pediatric MBs, regardless of their genetic subtype or histological classification; and high expression of NRP-1 correlates with poor overall survival of patients; 2) MB cells release Shh ligands that stimulate paracrine production of stromal PIGF by cerebellar granule neurons; 3) PIGF signals through NRP-1 - and independent of vascular endothelial growth factor receptor 1 (VEGFR1) - to activate the Erk/MEK pathway and sustain tumor survival; 4) Blockade of PIGF or NRP-1 with specific antibodies causes dramatic regression of MB, decreases spinal metastatic burden, and prolongs survival in in vivo orthotopic and spontaneous mouse models. Conclusion: This work identifies PIGF as the first common target across MB subgroups and provides insight into the critical role of tumor-stroma interactions. The study demonstrates that targeting PIGF/NRP-1 may provide a safe and efficient treatment option for pediatric MBs and lays the ground for evaluation of anti-PIGF therapy in clinical trials. Reference: M. Snuderl et al, Cell, 152(2):1065-76 (2013). Citation Format: Matija Snurdel, Ana Batista, Nathaniel Kirkpatrick, Carmen Almodovar, Lars Riedemann, Teresa Peterson, Napoleone Ferrara, Michael Klagsbrun, Dan G. Duda, Dai Fukumura, Lei Xu, Peter Carmeliet, Rakesh Jain. Targeting placental growth factor/ neuropilin-1 pathway inhibits growth and spread of medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA17.

Abstract 4414: Neuropilin2 is a novel target gene of Wnt signaling pathway regulating angiogenesis, metastasis and growth of osteosarcoma

Abstract 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

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

Abstract 153: High neuropilin-1 expression on monocytes is positively associated with trastuzumab-mediated antibody-dependent cellular cytotoxicity of the HER2-overexpressing breast cancer cell line

Abstract Purpose Neuropilin-1 (Nrp1) was initially characterized as a guide for migrating cells and axons in developing nervous systems and is essential for the precise formation of neurons and vasculature. However, it was recently reported to also play an important role in the immune system. This study aimed to investigate the role of Nrp1 on monocytes in trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) targeting the HER2-positive human breast cancer cell line SKBR3. In addition, we evaluated the gene expression profile of monocytes expressing high and low levels of Nrp1. Methods Peripheral blood mononuclear cells from healthy volunteers were prepared using BD Vacutainer CPT Cell Preparation Tubes. The cells were stained with fluorescein isothiocyanate-conjugated anti-CD14 and allophycocyanin-conjugated anti-Nrp1 antibodies, and Nrp1high and Nrp1low monocyte subsets were sorted using FACSAria. These cells were used as effector cells in a conventional ADCC assay with SKBR3 as target cells and trastuzumab as the antibody. Target cell cytotoxicity was measured using the LDH cytotoxic test kit. The effector cell to target cell ratios of 20:1 and 10:1 were used. Gene expression analysis was performed by Affymetrix GeneChip microarrays. All study protocols were approved by the Ethics Committee for Clinical Research, Kyoto University Hospital, Kyoto, Japan (authorization number G424). Results The LDH cytotoxic test revealed significantly higher target cancer cell cytotoxicity in sorted Nrp1high monocytes than in Nrp1low monocytes (75% vs. 45%, p&amp;lt;0.01). Trastuzumab-mediated ADCC was significantly suppressed by Nrp1 neutralization antibodies in a dose-dependent manner (Control arm 14.3%, Treatment arm 6%; p&amp;lt;0.01). Gene expression analysis revealed that 308 genes were up-regulated in the Nrp1high population, including monocyte-specific chemokine receptor and hematopoietic transcriptional factor. Conclusion These results suggest that determination of Nrp1 expression can be used to classify monocytes as populations with and without cellular cytotoxic activity. Analysis of Nrp1 expression levels in monocytes can therefore be used to identify HER2-positive breast cancer patients for whom trastuzumab therapy will be less effective. Citation Format: Kosuke Kawaguchi, Eiji Suzuki, Masao Kawashima, Masakazu Toi. High neuropilin-1 expression on monocytes is positively associated with trastuzumab-mediated antibody-dependent cellular cytotoxicity of the HER2-overexpressing breast cancer cell line. [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 153. doi:10.1158/1538-7445.AM2014-153

Neuropilin-1 enforces extracellular matrix signalling via ABL1 to promote angiogenesis.

Neuropilin-1 (NRP1), together with neuropilin-2, belongs to the neuropilin family. Neuropilins are transmembrane proteins essential for vascular and neural development and act as co-receptors for secreted signalling molecules of the class 3 semaphorin and vascular endothelial growth factor A (VEGF-A) families. NRP1 promotes VEGF-A signal in blood vascular endothelium and semaphorin signal in lymphatic endothelium, by forming complexes with its co-receptors. Mouse mutant studies established that NRP1 expression is essential during development because mice lacking NRP1 expression die embryonically and show severe neuronal and cardiovascular defects. Even though the contribution of NRP1 to vascular development has been mainly ascribed to its function as a VEGF-A receptor, recent evidence suggests that NRP1 contributes to angiogenesis through VEGF-independent mechanisms. In the present paper, we provide an overview of NRP1 functions in the vasculature and discuss current knowledge of NRP1-dependent signalling in the endothelium.

Protein kinase LKB1 promotes RAB7-mediated neuropilin-1 degradation to inhibit angiogenesis.

After internalization, transmembrane receptors (TMRs) are typically recycled back to the cell surface or targeted for degradation. Efficient TMR trafficking is critical for regulation of several processes, including signal transduction pathways, development, and disease. Here, we determined that trafficking of the angiogenic receptor neuropilin-1 (NRP-1) is abrogated by the liver kinase B1 (LKB1), a serine-threonine kinase of the calcium calmodulin family. We found that aberrant NRP-1 expression in tumor cells from patients with lung adenocarcinoma is associated with decreased levels of LKB1. In cultured lung cells, LKB1 accentuated formation of a complex between NRP-1 and RAB7 in late endosomes. LKB1 specifically bound GTP-bound RAB7, but not a dominant-negative GDP-bound form of RAB7, promoting rapid transfer and lysosome degradation of NRP-1. siRNA-mediated depletion of RAB7 disrupted the transfer of NRP-1 to the lysosome, resulting in recovery of the receptor as well as increased tumor growth and angiogenesis. Together, our findings indicate that LKB1 functions as a RAB7 effector and suppresses angiogenesis by promoting the cellular trafficking of NRP-1 from RAB7 vesicles to the lysosome for degradation. Furthermore, these data suggest that LKB1 and NRP-1 have potential as therapeutic targets for limiting tumorigenesis.

NRP-1 expression is strongly associated with the progression of pituitary adenomas.

The purpose of the present study was to identify a predictive marker associated with tumor progression or recurrence. We investigated the expression of p53, Ki-67, Bax, Bcl-2, vascular endothelial growth factor (VEGF)-A, VEGFR-1, VEGFR-2 and neuropilin-1 (NRP-1) in pituitary adenomas (PAs) with/without tumor progression during follow-up periods. We compared the expression of these molecules in primary and recurrent specimens to identify a predictive marker associated with tumor progression. Nineteen patients had no progression for more than 5-years of follow-up. Nine patients had tumor progression within 5years of their first transsphenoidal surgery (TSS) surgery and underwent re-TSS for treating progression of adenoma. Tumor size was larger and involvement of the cavernous sinus was more frequent in the progression group than these variables in the no progression group. A strong association was observed between NRP-1 expression and tumor progression. No significant risk for developing tumor progression was associated with Ki-67, p53, Bax, Bcl-2, VEGFR-1, VEGFR-2, or VEGF-A expression. Four of nine patients showed strong NRP-1 immunoreactivity in progression specimens. Negative NRP-1 immunoreactivity in the initial specimens was converted into strong positivity in the progression specimens of five patients. NRP-1 could be a relevant PA marker of progression and could be a potential target for medical therapy.

SPECT and near-infrared fluorescence imaging of breast cancer with a neuropilin-1-targeting peptide.

Breast cancer is the most common malignant cancer and is the leading cause of cancer death among females. Molecular imaging is a promising approach for the early detection and staging of breast cancer as well as for assessing therapeutic responses. Tumor-targeting peptides are effective targeting vehicles for molecular imaging. Here, we identified a breast cancer-targeting peptide CLKADKAKC (CK3) contains a cryptic C-end rule motif that may mediate its binding to neuropilin-1 (NRP-1), an attractive therapeutic target which expression was associated with poor outcome of the patients with breast cancer. Phage CK3 bound to NRP-1-positive breast cancer cells, which could be inhibited by peptide CK3 in a dose-dependent manner or by knock-down NRP-1 expression. Consistently, NRP-1 overexpression in cells increased the binding of phage CK3. Furthermore, peptide CK3 co-localized with NRP-1. Importantly, unlike previously reported NRP-1-targeting peptides with exposed C-end rule motifs, peptide CK3 did not penetrate into lungs and heart in vivo, which could make it more clinically applicable. Single-photon emission CT (SPECT) and near-infrared fluorescence (NIRF) imaging showed enrichment of peptide CK3 to the xenograft tumors in nude mice. In conclusion, as a novel NRP-1-targeting peptide, peptide CK3 could be used for breast cancer molecular imaging, which may represent a new avenue for breast cancer diagnostics, staging and assessments of therapeutic response.