Neuropilin-1 Expression Research Articles

MicroRNA-320 targeting neuropilin 1 inhibits proliferation and migration of vascular smooth muscle cells and neointimal formation.

This study shows that microRNA-320 (miR-320) is associated with many important cell functions, including cell differentiation, proliferation, migration, and apoptosis. However, the role of miR-320 in vascular smooth muscle cells (VSMCs) and proliferative vascular diseases is still completely unclear. In our study, we found that the expression of miR-320 in human VSMCs after PDGF stimulation was significantly down-regulated in time- and dose-dependent manner. Function analyses identified that miR-320 could inhibit the proliferation and migration of VSMCs in both basal and PDGF-stimulated conditions. Furthermore, Neuropilin 1 (NRP1) was demonstrated as a direct target of miR-320 in Luciferase reporter assays and miR-320 overexpression inhibited the expression of NRP1 with or without PDGF treatment. Finally, miR-320 was markedly decreased in mice carotid arteries after ligated injury, while the restoration of miR-320 via Ad-miR-320 attenuated neointimal hyperplasia by declining the NRP1 expression. The results confirmed that miR-320 regulated proliferation and migration of VSMCs and neointimal formation by targeting NRP1. These novel findings implied that the regulation of NRP1 expression by miR-320 has important significance in the early diagnosis and treatment of proliferation vascular diseases.

Increased Expression of TGF-β and IFN-γ in Peripheral Blood Mononuclear Cells (PBMCs) Cultured in Conditioned Medium (CM) of K562 Cell Culture.

In the present study, we investigated the effects of conditioned media (CM) collected from the cancer cell lines (K562, MCF-7, and HeLa) on peripheral blood mononuclear cells (PBMCs) isolated from the healthy human blood. The soluble factors in the CM are probably responsible for the differential mRNA expressions of Foxp3, Helios, Neuropilin- 1 (NRP-1), and glycoprotein A repetitions predominant (GARP), along with IFN-γ and TGF-β in PBMCs cultured with cancer cells CM. The PBMCs cultured with CM of K562 showed increased expression of Foxp3, Helios, NRP-1, GARP, IFN-γ, and TGF-β compared to PBMCs cultured with CM of MCF-7 and HeLa cells. In addition, the intracellular staining on PBMCs cultured with CM from cell lines were also evaluated for CD4, CD25, Foxp3, Helios, and NRP-1 by multicolor flow cytometry. The expression of CD4+CD25+Foxp3+, CD4+Helios+Foxp3+ and CD+NRP-1+Foxp3+ showed retarded cell population compared to control PBMCs. Our data suggest that soluble factors in CM of cancer cells may trigger the immune response in PBMCs resulting in a systematic response. Further research could lead to the identification of specific soluble factors that are involved in trafficking of cells into the immune cascades, which could be a safe and promising strategy for targeting human cancers.

The Role of the Tumor Microenvironment in Neuropilin 1-Induced Radiation Resistance in Lung Cancer Cells.

Background: Neuropilin 1 (NRP1) is a pleiotropic receptor which can interact with multiple ligands and their receptors. It plays an important role in the process of axonal growth, angiogenesis, tumor metastasis and radiation resistance in endothelial cells and some tumor cells. Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis, and has received considerable attention in recent years.Material and Methods: In this study, A549 lung cancer cell lines with different NRP1 expression levels were constructed in vitro, a two-dimensional (2D), three-dimensional (3D) co-culture system and tumor-bearing model was established in SCID mice. Western blot, qRT-PCR, immunofluorescence, cytometric bead array and flow cytometry were used to investigate the effect of the tumor microenvironment in NRP1-induced lung cancer cell radiation resistance.Results: In 2D or 3D co-culture system, NRP1 could be regulated inflammatory factors such as TNF, IL-6 IL-8 and IL-17 and the related chemokines MCP-1, IP-10 and RANTES in the tumor microenvironment, which in turn induced radiation resistance in lung cancer cells. In addition, different expression levels of NRP1 in 2D, 3D culture systems and tumor-bearing models were able to significantly regulate cell phenotype, proliferative capacity, epithelial-mesenchymal transition (EMT) and the radiation resistance of A549 cells.Conclusion: Our results verified that NRP1, inflammatory factors, chemokines and related signaling pathways, which affect the transformation of related cell components and thus lung cancer cell immune tolerance and migratory ability, all play an important role in radiation resistance.

Altered expression of regulatory molecules in the skin of psoriasis.

The presence of T regulatory cells (Tregs) is highly required in normal skin in order to maintain immune tolerance to commensal microbes and to prevent the development of immune-mediated inflammation. Psoriasis is a chronic inflammatory skin disease in which effector T cells, namely, Th17 and their relevant pro-inflammatory cytokines are increased in peripheral blood as well as in the inflamed skin. The status of Tregs in psoriatic skin is continuously studied. In this case, CD4 + CD25high T cells and other regulatory cytokines such as IL-35 are demonstrated to be significantly decreased. Aiming to better characterize Tregs in psoriatic skin and to establish the finding of their abnormal balance, we assessed the expression of semaphorin3A and neuropilin-1 (both reported as biomarkers of Tregs). Semaphorin3A and neuropilin-1 expressing Tregs were found to be significantly decreased in psoriatic skin when compared to normal skin. These findings were supported by demonstrating the downregulation of IL-10 expression in psoriatic skin. Our findings suggest that semaphoring3A may turn to be a new promising therapeutic approach in the process of improving Treg function in psoriasis.

High Neuropilin-1 Expression in Acute Myeloid Leukemia Is Mainly Associated with Monocytic Subtypes and Extramedullary Invasion

Introduction: Neuropilins are single-pass transmembrane glycoproteins acting as receptors for the vascular endothelial growth factor (VEGF) family that mediates angiogenesis and vascular development. In Acute Myeloid leukemia (AML), Neuropilin-1 high expression was found to contribute in disease evolution.Aim of Work: To evaluate Neuropilin-1 (NRP-1) expression in AML patients especially monocytic subtypes and assess its role in disease progression and extramedullary invasion (EMI).Patients and Methods: We measured NRP-1 expression level in 50 denovo AML patients who presented to the National Cancer institute, Cairo university. Expression was assessed by Real time quantitative PCR (RQ-PCR) using normalized copy number equation (2 -ΔΔCT) in relation to normal healthy controls. AML patients were characterized according to their Bone marrow and peripheral blood morphology, karyotype, immunophenotyping and FLT3 internal tandem duplication (FLT3-ITD) by polymerase chain reaction (PCR). Patients were classified into 2 groups according to median NRP-1 expression into low ≤60 and high >60-fold expression.Results: 33 males (66%) and 17 females (34%) with a median age of 34 years (18-73) were included. Cytogenetic risk categories were low in 12 (24%), intermediate in 34 (68%) and high in 4 (8%) patients. No correlation was found between high NRP-1 and cytogenetic risk classes or FLT3-ITD.Out of 26 patients showing high NRP-1 expression; 19 (73%) were <40 years vs. 7(27%) >40 years (p=0.049) and 24 (92%) showed BM blasts >50% vs. 2 (8%) with < 50% BM blasts (p<0.001). A positive correlation was found between NRP-1 Fold -change and BM blasts (r=0.653, p=0.004). Out of 19 monocytic FAB subtypes (M4, M5), high NRP-1 expression was expressed in 16 (84%) vs. 10/31 (32%) patients presenting with other FAB subtypes (p=0.042). Out of 26 patients showing high NRP-1 expression, extramedullary invasion (EMI) was found in 23/26 (88%) vs. 3/26 (12%) patients only with no EMI (p=0.001).Conclusion: Neuropilin-1 is useful as a prognostic marker of extramedullary invasion for AML patients especially in monocytic subtypes . Its role in disease progression and severity is still to be evaluated in our patients.Key Words: Neuropilin-1, AML, extramedullary invasion DisclosuresNo relevant conflicts of interest to declare.

The role of the hypoxia-Nrp-1 axis in the activation of M2-like tumor-associated macrophages in the tumor microenvironment of cervical cancer.

To explore the mechanisms through which hypoxic tumor microenvironment (TME) modulates the transition of tumor-associated macrophages (TAMs). The migration ability of RAW264.7 macrophages was determined by transwell assay. Flow cytometric, western blot and immunofluorescence analyses of CD206 further validated the M2 polarization of macrophages. Immunofluorescence, western blot and qRT-PCR were performed to detect the expression of neuropilin-1 (Nrp-1) and carbonic anhydrase IX (CAIX). An intermittent hypobaric hypoxia (IH) animal model was established to evaluate the role of hypoxia in activating M2-like TAMs in vivo. We also used immunohistochemistry to analyze the association between CAIX, CD163+ macrophages and Nrp-1 in a series of 72 human cervical cancer specimens. We found that the hypoxic cervical TME educated the recruited macrophages to transform into the M2 phenotype. Nrp-1 expression was significantly increased in hypoxia-primed cervical cancer cells. Blocking Nrp-1 expression prevented hypoxic cells from recruiting and polarizing macrophages towards the M2 phenotype. Hypoxia exposure significantly increased the expression of Nrp-1 as well as the infiltration of macrophages in vivo. Consistently, immunochemical staining in serial tissue sections of cervical cancer revealed upregulated levels of Nrp-1 in CAIX-positive hypoxic regions along with a concurrent significant elevation of M2 macrophages. Nrp-1 and M2-like TAMs were related to the malignant properties of cervical cancer, such as the FIGO stage and lymph node metastasis. Nrp-1 plays critical roles in hypoxic TME-induced activation and pro-tumoral effects of TAMs in cervical cancer. Interfering with Nrp-1 may be a potential therapeutic strategy in treating cervical cancer.

Abstract 16777: Augmentation of Spinal Cord Pre-Collateralization to Prevent Paraplegia After Aortic Surgery

Introduction: Spinal cord ischemia from complex aortic surgery can lead to paraplegia. Spinal cord is supplied by a rich collateral network of segmental arteries. Emerging data demonstrate that patients with staged aortic repairs suffer fewer spinal cord injuries than those with a single operation. Hypothesis: Paraplegia can be induced in a swine model by ligation of segmental spinal arteries. Induction of spinal cord pre-collateralization by staging the spinal segmental arterial coverage can minimize the risk of paraplegia. Methods: Twenty-four female Yorkshire pigs (50-70kg) underwent abdominal exposure of the aorta. Lumbar aortic group underwent ligation of the lumbar segmental arteries (5-7) and median sacral trunk. Thoracic aortic coverage consisted of stent graft placement from the left subclavian to celiac artery. Full coverage un-staged group underwent a simultaneous lumbar and thoracic coverage. Similar procedure was performed one week apart in staged full coverage group. Fluorescent microspheres were injected to evaluate regional spinal cord flow distribution. Protein expression in the spinal cord was analyzed using Western Blot. Results: Reproducible paresis was demonstrated in full coverage animals. This was associated with decreased viable spinal cord neurons. In the staged full coverage, complete motor recovery occurred within 24h of the second stage. Neurologic recovery was associated with higher levels of Neuropilin-1. Regional blood flow was quantified by microsphere density in various segments of spinal cord using laser confocal microscopy. Conclusions: A reproducible model of spinal cord injury can be carried out in swine by disrupting spinal cord segmental blood flow. This injury can be ameliorated by partial or staged coverage of the segmental arteries. Induction of regional spinal cord pre-collateralization can be demonstrated using fluorescent microspheres. This may be dependent on Neuropilin-1 expression in the tissue.

Expression of neuropilin-1 is linked to glioma associated microglia and macrophages and correlates with unfavorable prognosis in high grade gliomas.

High grade gliomas, including glioblastoma (GB), are devastating malignancies with very poor prognosis. Over the course of the last decade, there has been a failure to develop new treatments for GB. Reasons for this failure include the lack of validation of novel molecular targets, which are often characterized in animal models and directly transposed to human trials. Here we build on our previous findings, which describe how the multi-functional co-receptor Neuropilin-1 (NRP1) signals through glioma associated microglia/macrophages (GAMS) to promote murine glioma, and investigate NRP1 expression in human glioma. Clinical and gene expression data were obtained via The Cancer Genome Atlas (TCGA), and analyzed using R statistical software. Additionally, CIBERSORT in silico deconvolution was used to determine fractions of immune cell sub-populations within the gene expression datasets. We find that NRP1 expression is correlated with poor prognosis, glioma grade, and associates with the mesenchymal GB subtype. In human GB, NRP1 expression is highly correlated with markers of monocytes/macrophages, as well as genes that contribute to the pro-tumorigenic phenotype of these cells.

Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies.

Cancer cell dependence on activated oncogenes is therapeutically targeted, but acquired resistance is virtually unavoidable. Here we show that the treatment of addicted melanoma cells with BRAF inhibitors, and of breast cancer cells with HER2-targeted drugs, led to an adaptive rise in neuropilin-1 (NRP1) expression, which is crucial for the onset of acquired resistance to therapy. Moreover, NRP1 levels dictated the efficacy of MET oncogene inhibitors in addicted stomach and lung carcinoma cells. Mechanistically, NRP1 induced a JNK-dependent signaling cascade leading to the upregulation of alternative effector kinases EGFR or IGF1R, which in turn sustained cancer cell growth and mediated acquired resistance to BRAF, HER2, or MET inhibitors. Notably, the combination with NRP1-interfering molecules improved the efficacy of oncogene-targeted drugs and prevented or even reversed the onset of resistance in cancer cells and tumor models. Our study provides the rationale for targeting the NRP1-dependent upregulation of tyrosine kinases, which are responsible for loss of responsiveness to oncogene-targeted therapies.

Abstract A23: Neuropilin-1 expression on regulatory T cells in ovarian cancer

Abstract Introduction: Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that suppress autoimmune responses, but also prevent clearance of tumors and chronic viral infections. In ovarian cancer, a higher frequency of tumor-infiltrating Treg is associated with poor prognosis, but insights into the mechanisms governing the survival and suppressive activity of Treg in this context are limited. Our group has recently described a signaling axis through neurophilin-1 (NRP1) on Treg in murine models of cancer that promotes the survival and suppressive function of Treg. Genetic knockout of NRP1 on murine Treg leads to reduced tumor growth and increased survival, underscoring the importance of NRP1+ Treg in suppressing antitumor immunity. Given the importance of NRP1+ Treg in murine cancer models, we sought to explore the expression pattern of NRP1 on tumor-infiltrating Treg from patients with ovarian cancer. Materials and Methods: Ovarian tumors were obtained through the health sciences tissue bank at the University of Pittsburgh, and ovarian ascites fluid was obtained through Magee Women’s Research Institute. Peripheral blood was obtained from healthy donors through the Red Cross. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, and tumor-infiltrating lymphocytes were isolated by first mechanically disrupting the tumor, followed by treatment for 15 minutes at 37°C with 50 ug/mL Liberase DL. For flow cytometric analysis of Treg, cells from blood and tumors were first stained for surface markers, followed by live/dead discrimination, fixation and permeabilization, and staining for intracellular markers. Cells were analyzed on an LSR Fortessa at the Hillman Cancer Center Flow Core. For functional assays, Treg were selected from ascites fluid and assayed for their ability to suppressive naïve CD8+ T cells ex vivo. Wilcoxon rank sum tests were used to assess differences between groups, and a two-sided alpha less than 5% was considered significant. Results: A total of 12 healthy donors and 20 ovarian cancer patients were studied. Intracellular and surface NRP1 expression was assessed by flow cytometry on CD4+CD25+FOXP3+ Treg from healthy donor peripheral blood and tumor-infiltrating lymphocytes from either ovarian cancer tumors or ascites fluid from patients with ovarian cancer. Intracellular NRP1 was expressed on a median of 1.8% (interquartile range [IQR]: 0.69% to 4.8%) of Treg from healthy donors compared with a median of 66% (IQR: 28% to 90%; p&amp;lt;0.001) and 82% (IQR: 38% to 99%; p=0.004) of Treg from ovarian tumor-infiltrating lymphocytes (TIL) and ascites, respectively. Surface NRP1 was detected on 0.1% (IQR: 0% to 1%) of Treg in PBMC from healthy donors, compared with 11.1% (IQR: 1.1% to 60%; p=0.01) on ovarian TIL and 82% (IQR: 14% to 82%; p=0.0015) on ascites. Comparing expression of intracellular NRP1 on Treg from N=2 benign ovarian tissues versus N=14 malignant ovarian tumors, there was a trend towards increased NRP1 expression (median 9.3% versus 54.5%, respectively; p=0.044). Finally, Treg isolated from ascites fluid were capable of suppressing CD8+ T cells ex vivo proliferation in a dose-dependent manner. Conclusions and Future Directions: Both intracellular and surface NRP1 are expressed more frequently on tumor-infiltrating Treg and Treg from ascites fluid compared to Treg from healthy donor peripheral blood. Although the number of patients currently included in this study is small, there was also evidence that intracellular NRP1 expression on Treg was higher on malignant compared with benign tissue. Additionally, Treg from ascites fluid were capable of suppressing CD8 cells ex vivo. Future studies will evaluate whether blockade of NRP1 on human Treg limits either the survival or suppressive capacity of Treg. Note: This abstract was not presented at the conference. Citation Format: Anthony R. Cillo, Tullia Bruno, Francesmary Modugno, Robert Edwards, Dario Vignali. Neuropilin-1 expression on regulatory T cells in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A23.

Neuropilin-1 (NRP-1) upregulated by IL-6/STAT3 signaling contributes to invasion in pancreatic neuroendocrine neoplasms

Although the upregulation of Neuropilin-1 (NRP-1) is associated with many solid tumors, its role in pancreatic neuroendocrine neoplasms (pNEN) has not been well elucidated. The aim of this study was to investigate the role of NRP-1 in improving treatment and determining the prognosis of pNEN. In this study, the expression of NRP-1 in pNEN tissue samples and pNEN cell line BON1 was analyzed by Western blot, polymerase chain reaction (PCR) and immunocytochemistry upon exposure to interleukin-6 (IL-6). Additionally, pNEN cell line BON1 was transfected with small interfering RNAs against NRP-1 or signal transducer and activator of transcription 3 (STAT3) and assessed by in vitro invasion assays. The expression of NRP-1 in pNEN tissues was markedly increased compared with adjacent normal pancreatic tissues. High NRP-1 expression was strongly correlated with tumor grades (P = .026), lymph node metastasis (P = .025), and tumor-node-metastasis stages (P = .012). Furthermore, NRP-1 downregulation notably inhibited the metastatic capacity of pNEN cells, and STAT3 knockdown was found to downregulate the expression of NRP-1. BON1 cells upregulated NRP-1 expression upon stimulation with IL-6. This was accompanied by activation/phosphorylation of the AKT and STAT3 signaling pathways. Western blot of extracts of human pNENs confirmed increased NRP-1 expression, as well as AKT/STAT3 phosphorylation in tissue of pNENs with elevated expression levels of IL-6. In conclusion, our findings suggest that NRP-1 is upregulated in pNEN and is correlated with the metastatic capacity of pNEN cells, potentially via interaction with the IL-6/STAT3 signaling pathway.

Neuropilin-1 expression is associated with lymph node metastasis in breast cancer tissues.

PurposeNeuropilin-1 (NRP1) as an isoform-specific receptor for vascular endothelial growth factor and placenta growth factor in endothelial cells has been demonstrated to be expressed in breast cancer cells where it plays functional roles in cell survival, invasion, and migration. We hypothesized that an expression of NRP1 in breast cancer tissues is associated with clinicopathological data of patients and expression of the tumor suppressor miR-206.Patients and methodsWe evaluated the expression of NRP1 in 48 invasive ductal carcinomas of the breast and their corresponding adjacent noncancerous tissues (ANCTs) by means of real-time polymerase chain reaction. We also extracted data on miR-206 gene expression from the same cohort of patients to evaluate the correlation between expression levels of miR-206 and NRP1. In addition, we quantified NRP1 protein levels using the enzyme-linked immunosorbent assay technique.ResultsNo significant difference was found in NRP1 expression between tumoral tissues and ANCTs. We also assessed the associations between expression levels of NRP1 and clinicopathological data of patients and found no significant associations between NRP1 transcript levels and any characteristic. However, NRP1 protein concentrations were significantly higher in patients with lymph node involvement compared with those without lymph node involvement. No correlation was found between NRP1 and miR-206 expression levels.ConclusionNRP1 protein levels might be an indicator of metastasis potential in breast cancer. Future studies are needed to confirm these results in larger cohorts of patients.

Abstract 2117: Role of Neuropilin 2 in orchestrating the functions of osteoclasts in promoting prostate cancer bone metastasis

Abstract Background: Bone metastasis is one of the major clinical concerns that causes skeletal-related malignancies and increased mortality. Bone is one of the preferred sites for metastatic prostate cancer. The metastatic prostate cancer cells interact with bone cells (osteoblasts and osteoclasts), resulting in an imbalance in the bone homeostasis leading to increased activation of osteoblasts over osteoclasts. Our preliminary data indicated a non-tyrosine kinase receptor Neuropilin 2 (NRP2) is expressed in osteoclasts (OC) induced by PCa cells and acts as a negative regulator of osteoclasts' function. We hypothesize that PCa-induced NRP2 expression in OC is necessary for low osteolytic activity and thus favors an osteoblastic lesion in PCa bone metastasis. Methods: Mouse OC precursors were isolated from bone marrow of C57BL/6 mice as well as transgenic CSF1R-cre; NRP2 Flox/Flox inducible mice where addition of 4-hydroxytamoxifen depletes NRP2 specifically from the myeloid cells. Differentiation of OCs was conducted under the conditions of RANKL and M-CSF and in conditioned medium (CM) collected from PCa cell line LNCaP C4-2B (promotes high osteoblastic and low osteoclastic activity) and PC3 (predominantly osteoclastic activity) to mimic the conditions in normal bone and PCa bone metastasis. NRP2 expression at protein and mRNA was evaluated. TRAP staining and pit formation were conducted to confirm the differentiation and function of OCs. Results and Discussion: We observed an increase in NRP2 expression in OCs induced by RANKL and M-CSF and in PC3 and LNCaP C4-2B CM simultaneously. TRAP staining and activity confirmed the differentiation of OCs under these conditions. Interestingly, depletion of NRP2 and treatment either in RANKL and M-CSF or LNCaP C4-2B CM exhibited a drastic increase in osteoclast differentiation and function. mRNA analysis revealed an increase in expression of osteoclastic genes following NRP2 depletion in RANKL and M-CSF and LNCaP C4-2B CM. However, NRP2-depleted OC precursors when treated with PC3 CM showed no change in osteoclastogenesis. These findings advocate a role of NRP2 in inhibiting osteoclastic activity in PCa bone metastasis and that osteolytic PCa evades NRP2 inhibition. Using in vitro and transgenic mice, we will elucidate the molecular mechanisms through which NRP2 regulates osteoclast differentiation and function in normal bone and in LNCaP C4-2B CM. We will also address how PC3 CM-induced OCs escapes the inhibition of NRP2. Together, this approach will elucidate the role of NRP2 axis on OCs in promoting PCa-induced bone metastasis and will aid in determining whether NRP2 axis can be a therapeutic target. Citation Format: Navatha Shree Polavaram, Arup Bag, Samikshan Dutta, Kaustubh Datta. Role of Neuropilin 2 in orchestrating the functions of osteoclasts in promoting prostate cancer bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2117.

Abstract 2595: Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer

Abstract The treatment paradigm for late stage non-small cell lung carcinoma (NSCLC) has shifted towards molecular targeted therapy like epidermal growth factor receptor (EGFR), one of the most studied targets. Tyrosine kinase inhibitors (TKIs) against EGFR have proven to be effective, however TKI resistance is common due to the acquisition of the T790M mutation in the EGFR kinase domain. There are currently very few studies suggesting the role of VEGFR in mediating erlotinib resistance. We suggest that over-expression of vascular endothelial growth factor receptors (VEGFRs) in tumors may create an autocrine loop that may mediate TKI resistance. The purpose of this study is to investigate the VEGF/VEGFR autocrine loop as a potential mechanism of TKI resistance via alternative signaling. H2170 NSCLC parental cells were obtained from ATCC. The resistant cells were obtained by treating parental cells with increasing concentrations of erlotinib. DNA sequencing was performed to exclude the presence of the T790M mutation. Quantitative real-time PCR was carried out for target genes coding for VEGF, VEGFR-2, and neuropilin-1 (NP-1). Fluorescence-activated cell sorting (FACS) analysis and immunofluorescence (IF) analysis were performed with antibodies against VEGFR-2 and NP-1. The viability of resistant cells was assessed after being treated with increasing concentrations of a VEGF inhibitor (VEGF neutralizing antibody) in medium with or without erlotinib using a cell proliferation assay (MTT). Thirty paraffin embedded tumor biopsies were immunostained (IHC) with an antibody against VEGFR-2. On the transcriptional level, real-time PCR revealed a 13.1, 3.4 and a 2.9 fold increase in gene expression for VEGF, VEGFR-2 and NP-1 respectively in the resistant cells compared to the parental cells. When looking at membrane bound receptors, FACS analysis showed a 2.1 and a 3.8 fold increase in the expression of VEGFR and NP-1 in resistant cells, respectively. IF studies correlated with the FACS results, showing significantly higher expression of both VEGFR-2 and NP-1 in the resistant cells. MTT assays demonstrated that the VEGF inhibitor was able to decrease cell death from 10 to 80% (0.1-10ug/ml) as evidenced by decreasing viability of resistant cells treated with increasing concentrations of VEGF inhibitor compared to the untreated control. Kaplan-Meier analysis revealed shorter median survival with high vs low expression of total VEGFR-2 expression (7mo vs 25mo, p=0.05) in NSCLC tumors in patients diagnosed with late stage NSCLC. Our results suggest that by up-regulating the VEGF/VEGFR autocrine system as an alternative signaling pathway to promote proliferation, tumor cells may bypass the inhibitory effect of TKI's on the EGFR pathway, leading to resistance. Furthermore, IHC demonstrated the presence of high expression of VEGFR-2 in tumors, suggesting its potential as a prognostic biomarker for lung cancer. Citation Format: Chike Osude, Leo Lin, Brad Foster, Neelu Puri. Role of VEGF/VEGFR autocrine in mediating EGFR TKI resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2595.

The expression of selected molecular markers of immune tolerance in psoriatic patients

Psoriasis is a chronic autoinflammatory disease whose underlying molecular mechanisms remain unclear. The disease is mediated by the cells and molecules of both the innate and adaptive immune systems. Some T cell surface molecules, including neuropilin-1 (NRP1), programmed death 1 (PD-1) and the human leukocyte antigen G (HLA-G), are known to play a role in the maintenance of immune tolerance. The aim of this study was to investigate HLA-G, NRP1 and programmed cell death gene (PDCD1) mRNA expression in psoriatic patients. The study included 72 psoriatic patients and 35 healthy individuals. Twentyone patients (29.17%) suffered from concomitant psoriatic arthritis. The mRNA expression of HLA-G, NRP1, and PDCD1 were determined using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The severity of skin lesions was assessed by means of the Psoriasis Area and Severity Index (PASI), Body Surface Area (BSA), the Patient Global Assessment (PGA), and the Dermatology Life Quality Index (DLQI). The median value of the PASI was 11.5, and of BSA was 15.8%. The expressions of NRP1 and PDCD1, but not HLA-G, were significantly lower in psoriatic patients in comparison with the control group. The expression of HLA-G, NRP1 and PDCD1 were not significantly different in the psoriatic arthritis and psoriasis vulgaris patients. The results of this study suggest that the molecular markers of immune tolerance, i.e., HLA-G, NRP1, and PD-1, may be involved in the immune response in psoriatic patients.

Long non‑coding RNA 00152 functions as a competing endogenous RNA to regulate NRP1 expression by sponging with miRNA‑206 in colorectal cancer.

Colorectal cancer (CRC) is the third most common type of cancer; however, the molecular mechanisms underlying colorectal tumor metastasis and growth remain elusive. Recently, accumulating evidence has indicated that long non‑coding RNAs (lncRNAs) play a critical role in CRC progression and metastasis; however, the biological role and clinical significance of lncRNA 00152 (lnc00152) in CRC remains largely unknown. Thus, in this study, lnc00152 expression was measured in 80human CRC tissue samples, 40non‑cancerous tissue samples, and 3CRC cell lines (SW480, SW620 and LoVo) using RT‑qPCR. We examined the effects of lnc00152 on CRC cells following transfection with lnc00152 overexpression plasmid or respective siRNA invitro and invivo. Luciferase assays revealed the mechanism driving competitive endogenous RNA (ceRNA). We identified that lnc00152 was aberrantly overexpressed in colorectal tumors and cancer cells and that lnc00152 was modulated by miRNA‑206. lnc00152 overexpression enhanced the proliferative and invasive ability of CRC cells invitro, promoted tumor growth invivo, and was associated with the shorter overall survival of patients with CRC. In addition, lnc00152 overexpression promoted epithelial-mesenchymal transition(EMT) and increased neuropilin‑1 (NRP1) expression in the CRC cells. By contrast, lnc00152 silencing exerted a counteractive effect. Collectively, these findings demonstrate the critical role of lnc00152 in tumor growth and progression in CRC, and identify a novel therapeutic target associated with CRC development and progression.

Role of the NRP-1-mediated VEGFR2-independent pathway on radiation sensitivity of non-small cell lung cancer cells.

To determine if inhibiting neuropilin-1 (NRP-1) affects the radiosensitivity of NSCLC cells through a vascular endothelial growth factor receptor 2 (VEGFR2)-independent pathway, and to assess the underlying mechanisms. The expression of VEGFR2, NRP-1, related signaling molecules, abelson murine leukemia viral oncogene homolog 1 (ABL-1), and RAD51 were determined by RT-PCR and Western blotting, respectively. Radiosensitivity was assessed using the colony-forming assay, and the cell apoptosis were analyzed by flow cytometry. We selected two cell lines with high expression levels of VEGFR2, including Calu-1 cells that have high NRP-1 expression, and H358 cells that have low NRP-1 expression. Upon inhibition of p-VEGFR2 by apatinib in Calu-1 cells, the expression of NRP-1 protein and other related proteins in the pathway was still high. Upon NRP-1 siRNA treatment, the expression of both NRP-1 and RAD51 decreased (p < 0.01; p < 0.05). Upon ABL-1 siRNA treatment, the expression of NRP-1 was increased and the expression of RAD51 was unchanged. Calu-1 cells treated with NRP-1 siRNA exhibited significantly higher apoptosis and radiation sensitivity in radiation therapy compared to Calu-1 cells treated with apatinib alone (p < 0.01; p < 0.01). The apoptosis and radiation sensitivity in H358 cells with NRP-1 overexpression was similar to the control group regardless of VEGFR2 inhibition. We demonstrated that when VEGFR2 was inhibited, NRP-1 appeared to regulate RAD51 expression through the VEGFR2-independent ABL-1 pathway, consequently regulating radiation sensitivity. In addition, the combined inhibition of VEGFR2 and NRP-1 appears to sensitize cancer cells to radiation.

NRP1 knockdown promotes the migration and invasion of human neuroblastoma-derived SK‑N‑AS cells via the activation of β1integrin expression.

Neuropilin1 (NRP1) is a transmembrane glycoprotein, which regulates many aspects of cellular function by functioning as co-receptor of various ligands. Recent studies have suggested that NRP1 promotes tumorigenesis, not only by activating the growth of tumor vessels, but also by activating the growth or migration of tumor cells themselves. The present study was performed to elucidate the roles of NRP1 in the development and/or progression of neuroblastoma(NB). In contrast to previous observations in various types of cancer, the analysis of public datasets indicated that lower levels of NRP1 expression were significantly associated with a shorter survival period of patients with NB. Consistent with this finding, wound-healing assay and Matrigel invasion assay revealed that NB cells in which NRP1 was knocked down exhibited increased migratory and invasive abilities. Further analyses indicated that β1integrin expression was markedly increased in NB cells in which NRP1 was knocked down, and NB cells in which β1integrin was knocked down exhibited decreased migratory and invasive abilities. The results presented herein indicate that NRP1 exerts tumor suppressive effects in NB, at least in part by regulating the expression of β1integrin.

Neuropilin-2 regulates airway inflammatory responses to inhaled lipopolysaccharide.

Neuropilins are multifunctional receptors that play important roles in immune regulation. Neuropilin-2 (NRP2) is expressed in the lungs, but whether it regulates airway immune responses is unknown. Here, we report that Nrp2 is weakly expressed by alveolar macrophages (AMs) in the steady state but is dramatically upregulated following in vivo lipopolysaccharide (LPS) inhalation. Ex vivo treatment of human AMs with LPS also increased NRP2 mRNA expression and cell-surface display of NRP2 protein. LPS-induced Nrp2 expression in AMs was dependent upon the myeloid differentiation primary response 88 signaling pathway and the transcription factor NF-κB. In addition to upregulating display of NRP2 on the cell membrane, inhaled LPS also triggered AMs to release soluble NRP2 into the airways. Finally, myeloid-specific ablation of NRP2 resulted in increased expression of the chemokine (C-C motif) ligand 2 ( Ccl2) in the lungs and prolonged leukocyte infiltration in the airways following LPS inhalation. These findings suggest that NRP2 expression by AMs regulates LPS-induced inflammatory cell recruitment to the airways and reveal a novel role for NRP2 during innate immune responses in the lungs.

Neuropilin-1 expression in adipose tissue macrophages protects against obesity and metabolic syndrome

Obesity gives rise to metabolic complications by mechanisms that are poorly understood. Although chronic inflammatory signaling in adipose tissue is typically associated with metabolic deficiencies linked to excessive weight gain, we identified a subset of neuropilin-1 (NRP1)-expressing myeloid cells that accumulate in adipose tissue and protect against obesity and metabolic syndrome. Ablation of NRP1 in macrophages compromised lipid uptake in these cells, which reduced substrates for fatty acid β-oxidation and shifted energy metabolism of these macrophages toward a more inflammatory glycolytic metabolism. Conditional deletion of NRP1 in LysM Cre-expressing cells leads to inadequate adipose vascularization, accelerated weight gain, and reduced insulin sensitivity even independent of weight gain. Transfer of NRP1+ hematopoietic cells improved glucose homeostasis, resulting in the reversal of a prediabetic phenotype. Our findings suggest a pivotal role for adipose tissue-resident NRP1+-expressing macrophages in driving healthy weight gain and maintaining glucose tolerance.