BACKGROUND: Neuropilins (NRPs) participate in many processes related to cancer development such as angiogenesis, lymphangiogenesis and metastasis. Although endometrial cancer is one of the most common gynecological cancers, it has not been studied in terms of NRPs expression.
OBJECTIVE: The aim of this study was to investigate the potential utility of NRPs as important factors in the diagnosis and treatment of endometrial cancer.
METHODS: Our study consisted of 45 women diagnosed with endometrial cancer at the following degrees of histological differentiation: G1, 17; G2, 15; G3, 13 cases. The control group included 15 women without neoplastic changes. The immunohistochemical reactions were evaluated using light microscopy.
RESULTS: We did not detect the expression of NRP-1 and NRP-2 in the control group. NRP-1 expression was found exclusively in cancer cells. It was higher in G2 and G3 and reached about 190% of G1. NRP-2 expression was observed in the endothelium and was similar across all three cancer grades. In cancer cells, NRP-2 expression increased with the degree of histological differentiation.
CONCLUSION: NRP1 and NRP2 are candidates for complementary diagnostic molecular markers and promising new targets for molecular, personalized anticancer therapies.

Castration-resistant prostate cancer (CRPC) lacks effective treatment, and studies have shown that PARPi inhibitors, such as Olaparib, are somewhat effective; however, the efficacy of Olaparib in CRPC still needs to be further improved. Nitrogen permease regulator-like 2 (NPRL2) is reported to be a tumor suppressor candidate gene and is closely related to the DNA repair pathway, which can affect the sensitivity of many chemotherapeutic drugs. However, there is no research on whether NPRL2 is associated with sensitivity to Olaparib. Hence, in the present study, we examined the NPRL2 expression levels in several PCa cell lines (LNCaP, PC3, and enzalutamide-resistant LNCaP, named LNPER) by Western blot. In addition, we investigated the role of NPRL2 expression and silencing in cell proliferation and in the regulation of ataxia telangiectasia mutated (ATM), which can mediate DNA repair and sensitivity to Olaparib. Furthermore, we performed in vitro and in vivo experiments to determine the mechanism of action of NPRL2 in adjusting Olaparib sensitivity. Our findings demonstrated that the NPRL2 expression level was upregulated in PCa cells, especially CRPC cells. NPRL2 overexpression promoted growth and resistance to Olaparib, and NPRL2 silencing inhibited proliferation, enhanced sensitivity to Olaparib, and increased CRRL2 expression in PCa cells. In addition, the Olaparib-induced growth delay in NPRL2-silenced PC3 tumors in mice correlated with ATM signaling downregulation, an apoptosis increase and migration/invasion suppression. Our results indicate that NPRL2 silencing enhances sensitivity to Olaparib treatment in CRPC and that NPRL2 may serve as a potential therapeutic target and predict resistance to Olaparib in CRPC.

OBJECTIVE: Cervical cancer is one of the most common malignant tumors. Our previous results showed that long non-coding RNA (lncRNA) XLOC_006390 plays an important role in cervical cancer. In this study, we have explored the mechanism of action of lncRNA XLOC_006390.
METHODS: LncRNA XLOC_006390 was proposed to exercise its function as a competing endogenous RNA (ceRNA), and its potential targeted miRNAs was predicted through the database LncBase Predicted v.2. Two miRNAs, miR-331-3p, and miR-338-3p, were chosen for the study. Expression of miRNAs and lncRNA in cervical cancer cells and tissues was detected by reverse transcription polymerase chain reaction. To determine the correlation, silencing of XLOC_006390, over-expression of miR-331-3p, and miR-338-3p was performed in SiHa and Caski cell lines, respectively.
RESULTS: Based on the interactive effect between miRNA and lncRNA, miR-331-3p and miR-338-3p were significantly downregulated in cervical cancer cells and tissues, and their expression levels were negatively related to that of lncRNA. Our results also showed that the expression of miR-331-3p target gene
CONCLUSION: Taken together, our study demonstrated that XLOC_006390 may serve as a ceRNA and reversely regulates the expression of miR-331-3p and miR-338-3p, thus facilitating cervical cancer tumorigenesis and metastasis.

Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB.

Several aryl hydrocarbon receptor (AhR)-active pharmaceuticals were screened as inhibitors of pancreatic cancer cell invasion and identified two compounds, omeprazole, that inhibited invasion. Inhibition of highly invasive Panc1 cell invasion by omeprazole involves an AhR-dependent non-genomic pathway, and omeprazole-mediated inhibition of Panc1 cell invasion was dependent on Jun-N-terminal kinase (JNK) and mitogen-activated kinase kinase 7 (MKK7). The failure of omeprazole to induce nuclear translocation of the AhR was not due to overexpression of cytosolic AhR partner proteins Hsp90 or XAP2, and results of DNA sequencing show that the AhR expressed in Panc1 cells was not mutated. Results of RNAseq studies indicate that omeprazole induced an AhR-dependent downregulation of several pro-invasion factors including activated leukocyte cell adhesion molecule (ALCAM), long chain fatty acid CoA-synthase (CSL4), stathmin 3 (STMN3) and neuropillin 2 (NRP2), and the specific functions of these genes are currently being investigated.

The objective of this study was to explore the role of HOTAIR in the development of cervical cancer, as well as its downstream signaling pathway. We conducted computational analysis, luciferase assay to explore downstream of HOTAIR and miR-331-3p. Real-time PCR and Western blot were carried out to detect the relationship among E7, HOTAIR, miR-331-3p, NRP2, and P53. Finally, MTT assay and flow cytometry analysis were performed to validate the effect of E7 and miR-331-3p on cell apoptosis and proliferation. NRP2 was identified as a virtual target gene of miR-331-3p with a binding site of miR-331-3p, and HOTAIR was directly sponged to miR-331-3p, miR-331-3p reduced luciferase activity of wild-type of NRP2 3'UTR and HOTAIR, but not those of mutant NRP2 3'UTR and HOTAIR. MiR-331-3p down-regulated NRP2 and E7 expression levels, and further promoted cell apoptosis, while inhibited cell proliferation. Cell transfected with HPV16 E7 displayed lower levels of HOTAIR, NRP2 and P53, a higher level of miR-331-3p, over-expression of E7 further repressed cell apoptosis, while improved cell proliferation compared with control. Normal HPV (+) group exhibited a higher miR-331-3p, and lower mRNA levels of HOTAIR and NRP2 than HPV (-) group. According to the result of IHC (immunohistochemistry), we found that NRP2 protein was highly expressed in HPV (-) group compared to that in HPV (+) group. E7-HOTAIR-miR-331-3p-NRP2-E7 formed a regulatory loop, and could be involved in the pathogenesis of cervical cancer.

BACKGROUND: Malignant melanoma of the skin can metastasize through blood vessels and lymphatics. The primary tumor develops a vascular microenvironment characterized by abnormal blood vessels and lymphatics and a physicochemical microenvironment characterized by low oxygen tension, regions with hypoxic tissue, and high interstitial fluid pressure (IFP). This study aimed at identifying relationships between the metastatic route of melanomas and characteristic features of the microvascular and physicochemical microenvironments of the primary tumor.
METHODS: Two patient-derived xenograft (PDX) models (E-13, N-15) and four cell line-derived xenografts (CDX) models (C-10, D-12, R-18, T-22) of human melanoma were included in the study. Tumors were transplanted to an orthotopic site in BALB/c-nu/nu mice, and when the tumors had grown to a volume of 500-600 mm
RESULTS: C-10, D-12, and E-13 tumors disseminated primarily by the hematogenous route and developed pulmonary metastases. These tumors showed high angiogenic activity and high expression of the F3 gene as well as ANGPT2 and TIE1, genes encoding proteins of the angiopoietin-tie system. N-15, R-18, and T-22 tumors disseminated mainly by the lymphogenous route and developed metastases in draining lymph nodes. These tumors had highly elevated IFP and showed high expression of NRP2, a gene encoding neuropilin-2.
CONCLUSION: The primary metastatic route of orthotopic human melanoma xenografts and the development of lung and lymph node metastases are influenced significantly by the microvascular and physicochemical microenvironments of the primary tumor.

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) enables epithelial cancer cells to acquire mesenchymal features and contributes to metastasis and resistance to treatment. This process involves epigenetic reprogramming for gene expression. We explored global histone modifications during TGF-β1-induced EMT in two non-small cell lung cancer (NSCLC) cell lines and tested different epigenetic treatment to modulate or partially reverse EMT.
RESULTS: Loss of classical epithelial markers and gain of mesenchymal markers were verified in A549 and H358 cell lines during TGF-β1-induced EMT. In addition, we noticed increased expression of the axonal guidance protein semaphorin 3C (SEMA3C) and PD-L1 (programmed death-ligand 1) involved in the inhibition of the immune system, suggesting that both SEMA3C and PD-L1 could be the new markers of TGF-β1-induced EMT. H3K79me3 and H2BK120me1 were decreased in A549 and H358 cell lines after a 48-h TGF-β1 treatment, as well as H2BK120ac in A549 cells. However, decreased H3K79me3 was not associated with expression of the histone methyltransferase DOT1L. Furthermore, H3K79me3 was decreased in tumors compared in normal tissues and not associated with cell proliferation. Associations of histone deacetylase inhibitor (SAHA) with DOT1L inhibitors (EPZ5676 or SGC0946) or BET bromodomain inhibitor (PFI-1) were efficient to partially reverse TGF-β1 effects by decreasing expression of PD-L1, SEMA3C, and its receptor neuropilin-2 (NRP2) and by increasing epithelial markers such as E-cadherin.
CONCLUSION: Histone methylation was modified during EMT, and combination of epigenetic compounds with conventional or targeted chemotherapy might contribute to reduce metastasis and to enhance clinical responses.

Mounting evidence indicates that hotspot p53 mutant proteins often possess gain-of-function property in promoting cell mobility and tumor metastasis. However, the molecular mechanisms are not totally understood. In this study, we demonstrate that the hotspot mutation, p53-R273H, promotes cell migration, invasion in vitro and tumor metastasis in vivo. p53-R273H significantly represses expression of DLX2, a homeobox protein involved in cell proliferation and pattern formation. We show that p53-R273H-mediated DLX2 repression leads to upregulation of Neuropilin-2 (NRP2), a multifunctional co-receptor involved in tumor initiation, growth, survival and metastasis. p53-R273H-induced cell mobility is effectively suppressed by DLX2 expression. Furthermore, knockdown of NRP2 significantly inhibits p53-R273H-induced tumor metastasis in xenograft mouse model. Together, these results reveal an important role for DLX2-NRP2 in p53-R273H-induced cell mobility and tumor metastasis.

The publication of the human genome sequence provided direction in the search for novel diagnostic and therapeutic methods for the treatment of human diseases. The aim of the present study was to investigate the hypothesis that the expression profile of genes involved in the regulation of angiogenesis may be a marker in endometrial cancer that facilitates the diagnosis and prognosis of patients, as well as the identification of novel therapeutic targets. The current study included 36 patients with grade (G) 1 to 3 endometrial cancer, and a control group of patients consisting of females that qualified for the removal of the uterus. Out of these, 28 samples (control, 3; G1, 7; G2, 12; and G3, 6) were selected for microarray analysis. Molecular analysis of the endometrial samples involved the extraction of total RNA, purification of the obtained extracts and subsequent analysis of the gene expression profiles using an oligonucleotide microarray technique (GeneChip® Human Genome U133A plates). The results indicated that the mRNA expression profile of genes involved in the regulation of angiogenesis varies depending on the degree of histological differentiation of endometrial adenocarcinoma. Similar results were obtained from descriptive statistics characterizing the expression profile of 691 mRNAs associated with the regulation of angiogenesis in the groups of patients with endometrial adenocarcinoma. In addition, the results of the present study indicated that neuropilin2 (NRP2) may serve an important role in the activity of endothelial cells, and may affect vascular endothelial growth factor, and potentially plexins and integrins via regulation of their functions. An understanding of how these proteins interact remains to be determined; however, elucidating these interactions may provide an explanation for the mechanisms underlying angiogenesis. In conclusion, the results of the present study suggest that NRP2 may be a valuable target for investigation in future pharmacological studies involving angiogenesis in endometrial cancer.

Neuropilin 2 (Nrp2) plays an important role in regulating lymphangiogenesis. Nrp2 expression in early tongue cancer was investigated to predict lymph node metastasis and the long-term prognosis. The relationships between clinicopathological variables of cT1-T2N0 tongue squamous cell carcinoma (SCC) and overexpression of Nrp2, vascular endothelial growth factor C (VEGFC), vascular endothelial growth factor receptor 3 (VEGFR3), and semaphorin 3F (Sema3F) were analyzed. Expression levels were compared using oral SCC cell lines. The Nrp2 gene was silenced to determine the impact of Nrp2. Cytoplasmic Nrp2 overexpression predicted regional metastasis with sensitivity and specificity of 90.3% and 42.1%, respectively. Cytoplasmic Nrp2 overexpression (P<0.001) and VEGFC overexpression (P=0.006) were significantly related to regional metastasis (Student t-test). However, only cytoplasmic Nrp2 overexpression was an independent prognostic factor for both disease-free survival (DFS; P=0.008) and overall survival (OS; P=0.016) (Cox regression); the risk of recurrence was 12-times higher (P=0.015) and risk of mortality was 8-times higher (P=0.016). Co-localization of Nrp2 and VEGFC was greater within the cytoplasm of aggressive cell lines (HN12 and RCa-T). Nrp2 plays a role in tumourigenesis; VEGFC supplementation cannot rescue the biological function of Nrp2 in Nrp2-depleted cell lines. Cytoplasmic Nrp2 overexpression is associated with decreased OS and DFS. Cytoplasmic Nrp2 overexpression may be a reliable diagnostic and prognostic marker for early tongue SCC.

Neuropilins (NRP1 and NRP2) are co-receptors for heparin-binding growth factors and class 3 semaphorins. Different isoforms of NRP1 and NRP2 are produced by alternative splicing. We found that in non-small cell lung cancer (NSCLC) cell lines, transforming growth factor-β (TGFβ) signaling preferentially increased the abundance of NRP2b. NRP2b and NRP2a differ only in their carboxyl-terminal regions. Although the presence of NRP2b inhibited cultured cell proliferation and primary tumor growth, NRP2b enhanced cellular migration, invasion into Matrigel, and tumorsphere formation in cultured cells in response to TGFβ signaling and promoted metastasis in xenograft mouse models. These effects of overexpressed NRP2b contrast with the effects of overexpressed NRP2a. Hepatocyte growth factor (HGF)-induced phosphorylation of the kinase AKT was specifically promoted by NRP2b, whereas inhibiting the HGF receptor MET attenuated NRP2b-dependent cell migration. Unlike NRP2a, NRP2b did not bind the PDZ domain scaffolding protein GAIP carboxyl terminus-interacting protein (GIPC1) and only weakly recruited phosphatase and tensin homolog (PTEN), potentially explaining the difference between NRP2b-mediated and NRP2a-mediated effects. Analysis of NSCLC patient tumors showed that NRP2b abundance correlated with that of the immune cell checkpoint receptor ligand PD-L1 as well as with epithelial-to-mesenchymal transition (EMT) phenotypes in the tumors, acquired resistance to epidermal growth factor receptor (EGFR) inhibitors, disease progression, and poor survival in patients. NRP2b knockdown attenuated the acquisition of resistance to the EGFR inhibitor gefitinib in cultured NSCLC cells. Thus, in NSCLC, NRP2b contributed to the oncogenic response to TGFβ and correlated with tumor progression in patients.

We have previously shown that Rab17, a small GTPase associated with epithelial polarity, is specifically suppressed by ERK2 (also known as MAPK1) signalling to promote an invasive phenotype. However, the mechanisms through which Rab17 loss permits invasiveness, and the endosomal cargoes that are responsible for mediating this, are unknown. Using quantitative mass spectrometry-based proteomics, we have found that knockdown of Rab17 leads to a highly selective reduction in the cellular levels of a v-SNARE (Vamp8). Moreover, proteomics and immunofluorescence indicate that Vamp8 is associated with Rab17 at late endosomes. Reduced levels of Vamp8 promote transition between ductal carcinoma

Lymph node (LN) metastasis has been suggested as a major prognostic factor for oral cancer. Knockdown of the growth factors and receptors involved in these metastatic mechanisms could significantly reduce LN metastasis and improve the survival of oral cancer patients after treatment. The present study, therefore, aimed to evaluate the expression levels of the following growth factors and receptors in squamous cell carcinoma (SCC) of the tongue: the vascular endothelial growth factor (VEGF)‑C and VEGF‑D, which bind to the cell surface tyrosine kinase receptor VEGF receptor‑3 (VEGFR‑3); C‑C motif chemokine receptor 7 (CCR7); neuropilin (NRP)1 and NRP2; and semaphorin 3E (SEMA3E). Furthermore, we assessed microvessel density (MVD) and lymphatic vessel density (LVD) to demonstrate the correlation between these factors and regional LN metastasis, with respect to the clinicopathological features. Finally, we analyzed the correlation between these proteins and overall or disease‑free survival, in order to demonstrate their prognostic value. Univariate analysis revealed a significant association between LN metastasis and the expression levels of VEGF‑C, VEGFR‑3, CCR7, NRP1, and SEMA3E, as well as LVD, in SCC cells. In contrast, multivariate analysis identified associations between LN metastasis and NRP1 expression, as well as between LN metastasis and LVD; however, no correlation was found between LN metastasis and the expression levels of the other proteins. The expression levels of VEGF‑C, VEGFR‑3, NRP1, and SEMA3E, as well as LVD, were correlated with disease‑free survival time. These results indicate that LN metastasis is associated with poor survival in SCC. This study suggests that NRP1 expression and LVD are independent factors that are likely to predict the risk of LN metastasis in SCC of the tongue, whereas the expression of VEGF‑C, VEGFR‑3, CCR7, and SEMA3E are non‑independent predictive factors.

BACKGROUND: Renal cell carcinoma (RCC) is a seventh ranked malignancy with poor prognosis. RCC is lethal at metastatic stage as it does not respond to conventional systemic treatments, and there is an urgent need to find out promising novel biomarkers for effective treatment. The goal of this study was to evaluate the biomarkers that can be potential therapeutic target and predict effective inhibitors to treat the metastatic stage of RCC.
METHODS: We conducted transcriptomic profiling to identify differentially expressed genes associated with RCC. Molecular pathway analysis was done to identify the canonical pathways and their role in RCC. Tissue microarrays (TMA) based immunohistochemical stains were used to validate the protein expression of cyclinD1 (CCND1) and were scored semi-quantitatively from 0 to 3+ on the basis of absence or presence of staining intensity in the tumor cell. Statistical analysis determined the association of CCND1 expression with RCC. Molecular docking analyses were performed to check the potential of two natural inhibitors, rutin and curcumin to bind CCND1.
RESULTS: We detected 1490 significantly expressed genes (1034, upregulated and 456, downregulated) in RCC using cutoff fold change 2 and p value < 0.05. Hes-related family bHLH transcription factor with YRPW motif 1 (HEY1), neuropilin 2 (NRP2), lymphoid enhancer-binding factor 1 (LEF1), and histone cluster 1 H3h (HIST1H3H) were most upregulated while aldolase B, fructose-bisphosphate (ALDOB), solute carrier family 12 (SLC12A1), calbindin 1 (CALB1) were the most down regulated genes in our dataset. Functional analysis revealed Wnt/β-catenin signaling as the significantly activated canonical pathway (z score = 2.53) involving cyclin D1 (CCND1). CCND1 was overexpressed in transcriptomic studies (FC = 2.26, p value = 0.0047) and TMA results also showed the positive expression of CCND1 in 53 % (73/139) of RCC cases. The ligands - rutin and curcumin bounded with CCND1 with good affinity.
CONCLUSION: CCND1 was one of the important upregulated gene identified in microarray and validated by TMA. Docking study showed that CCND1 may act as a potential therapeutic target and its inhibition could focus on the migratory, invasive, and metastatic potential of RCC. Further in vivo and in vitro molecular studies are needed to investigate the therapeutic target potential of CCND1 for RCC treatment.

Aberrant expression of microRNAs (miRNAs) is involved in the development and progression of various types of cancers. In this study, we investigated the role of miR-331-3p in cell proliferation and the expression of keratinocyte differentiation markers of uterine cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) are putative target molecules that regulate the human papillomavirus (HPV) related oncoproteins E6 and E7. Cell proliferation in the human cervical cancer cell lines SKG-II, HCS-2, and HeLa was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cellular apoptosis was measured using the TdT-mediated dUTP nick end labeling (TUNEL) and Annexin V assays. Quantitative RT-PCR was used to measure the messenger RNA (mRNA) expression of the NRP2, E6, E7, p63, and involucrin (IVL) genes. A functional assay for cell growth was performed using cell cycle analyses. Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M phase arrest and apoptosis in SKG-II, HCS-2 and HeLa cells. The luciferase reporter assay of the NRP2 3'-untranslated region revealed the direct regulation of NRP2 by miR-331-3p. Gene expression analyses using quantitative RT-PCR in SKG-II, HCS-2, and HeLa cells overexpressing miR-331-3p or suppressing NRP2 revealed down-regulation of E6, E7, and p63 mRNA and up-regulation of IVL mRNA. Moreover, miR-331-3p overexpression was suppressed NRP2 expression in protein level. We showed that miR-331-3p and NRP2 were key effectors of cell proliferation by regulating the cell cycle, apoptosis. NRP-2 also regulates the expression of E6/E7 and keratinocyte differentiation markers. Our findings suggest that miR-331-3p has an important role in regulating cervical cancer cell proliferation, and that miR-331-3p may contribute to keratinocyte differentiation through NRP2 suppression. miR-331-3p and NRP2 may contribute to anti-cancer effects.

Oesophageal squamous cell carcinoma (ESCC) is the most common histological subtype of oesophageal cancer. The disease is particularly prevalent in southern China. The incidence of the disease is on the rise and its overall survival rate remains dismal. Identification and characterization of better molecular markers for early detection and therapeutic targeting are urgently needed. Here, we report levels of transmembrane and soluble neuropilin-2 (NRP2) to be significantly up-regulated in ESCC, and to correlate positively with advanced tumour stage, lymph node metastasis, less favourable R category and worse overall patient survival. NRP2 up-regulation in ESCC was in part a result of gene amplification at chromosome 2q. NRP2 overexpression promoted clonogenicity, angiogenesis and metastasis in ESCC in vitro, while NRP2 silencing by lentiviral knockdown or neutralizing antibody resulted in a contrary effect. This observation was extended in vivo in animal models of subcutaneous tumourigenicity and tail vein metastasis. Mechanistically, overexpression of NRP2 induced expression of ERK MAP kinase and the transcription factor ETV4, leading to enhanced MMP-2 and MMP-9 activity and, as a consequence, suppression of E-cadherin. In summary, NRP2 promotes tumourigenesis and metastasis in ESCC through deregulation of ERK-MAPK-ETV4-MMP-E-cadherin signalling. NRP2 represents a potential diagnostic or prognostic biomarker and therapeutic target for ESCC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumor cell interactions with their microenvironment, and neighboring endothelial cells in particular, are critical for tumor cell survival and the metastatic process. Within the spectrum of tumors, melanomas are notorious for their ability to metastasize at a relatively early stage of development; however, little is known about the molecular pathways mediating this process. We recently performed a screen to assess critical mediators of melanoma metastasis by evaluating melanoma-endothelial cell communication. Neuropilin-2 (NRP2), a cell surface receptor involved in angiogenesis and axonal guidance, was found to be an important mediator of melanoma-endothelial cell cross-talk in these studies. Here we seek to further define the role of NRP2 in melanoma growth and progression. We use stable gene silencing of NRP2 in melanomas from varying stages of tumor progression to define the role of NRP2 in melanoma growth, migration, invasion, and metastasis. We found that NRP2 gene silencing in metastatic melanoma cell lines inhibited tumor cell growth in vitro; furthermore, knockdown of NRP2 expression in the metastatic melanoma cell line 1205Lu significantly inhibited in-vivo tumor growth and metastasis. We conclude that NRP2 plays an important role in mediating melanoma growth and metastasis and suggest that targeting this cell surface molecule may represent a significant therapeutic strategy for patients diagnosed with aggressive forms of melanoma.

The nervous system is now recognized to be a relevant component of the tumor microenvironment. Receptors for neuropeptides and neurotransmitters have been identified in breast cancer. However, very little is known about the role of neurogenes in regulating breast cancer progression. Our purpose was to identify neurogenes associated with breast cancer tumorigenesis with a potential to be used as biomarker and/or targets for treatment. We used three databases of human genes: GeneGo, GeneCards and Eugenes to generate a list of 1266 relevant neurogenes. Then we used bioinformatics tools to interrogate two published breast cancer databases SAGE and MicMa (n=96) and generated a list of 7 neurogenes that are differentially express among breast cancer subtypes. The clinical potential was further investigated using the GOBO database (n=1881). We identified 6 neurogenes that are differentially expressed among breast cancer subtypes and whose expression correlates with prognosis. Histamine receptor1 (HRH1), neuropilin2 (NRP2), ephrin-B1 (EFNB1), neural growth factor receptor (NGFR) and amyloid precursor protein (APP) were differentially overexpressed in basal and HER2-enriched tumor samples and syntaxin 1A (STX1A) was overexpressed in HER2-enriched and luminal B tumors. Analysis of HRH1, NRP2, and STX1A expression using the GOBO database showed that their expression significantly correlated with a shorter overall survival (p < 0.0001) and distant metastasis-free survival (p < 0.0001). In contrast, elevated co-expression of NGFR, EFNB1 and APP was associated with longer overall (p < 0.0001) and metastasis-free survival (p < 0.0001). We propose that HRH1, NRP2, and STX1A can be used as prognostic biomarkers and therapeutic targets for basal and HER2-enriched breast cancer subtypes.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. The epithelial to mesenchymal transition (EMT) describes the transformation of well-differentiated epithelial cells to a de-differentiated phenotype and plays a central role in the invasion and intrahepatic metastasis of HCC cells. Modulation of the transforming growth factor-β (TGF-β) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. The meta-analysis of a panel of EMT gene expression studies revealed that neuropilin 2 (NRP2) is significantly upregulated in cells that have undergone EMT induced by TGF-β. In this study we assessed the functional role of NRP2 in epithelial and mesenchymal-like HCC cells and focused on the molecular interplay between NRP2 and TGF-β/Smad signaling.
METHODS: NRP2 expression was analyzed in human HCC cell lines and tissue arrays comprising 133 HCC samples. Cell migration was examined by wound healing and Transwell assays in the presence and absence of siRNA against NRP2. NRP2 and TGF-β signaling were analyzed by Western blotting and confocal immunofluorescence microscopy.
RESULTS: We show that NRP2 is particularly expressed in HCC cell lines with a dedifferentiated, mesenchymal-like phenotype. NRP2 expression is upregulated by the canonical TGF-β/Smad signaling while NRP2 expression has no impact on TGF-β signaling in HCC cells. Reduced expression of NRP2 by knock-down or inhibition of TGF-β signaling resulted in diminished cell migration independently of each other, suggesting that NRP2 fails to collaborate with TGF-β signaling in cell movement. In accordance with these data, elevated levels of NRP2 correlated with a higher tumor grade and less differentiation in a large collection of human HCC specimens.
CONCLUSIONS: These data suggest that NRP2 associates with a less differentiated, mesenchymal-like HCC phenotype and that NRP2 plays an important role in tumor cell migration upon TGF-β-dependent HCC progression.

Breast cancer is a heterogeneous disease, having multiple subtypes with different malignant phenotypes. The triple-negative breast cancer, or basal breast cancer, is highly aggressive, metastatic, and difficult to treat. Previously, we identified that key molecules (IL6, CSF2, CCL5, VEGFA, and VEGFC) secreted by tumor cells and stromal cells in basal breast cancer can promote metastasis. It remains to assess whether these molecules function similarly in other subtypes of breast cancer. Here, we characterize the relative gene expression of the five secreted molecules and their associated receptors (GP130, GMRA, GMRB, CCR5, VEGFR2, NRP1, VEGFR3, NRP2) in the basal, HER2 (human epidermal growth factor receptor 2) positive, luminal A, and luminal B subtypes using high throughput data from tumor samples in The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). IL6 and CCL5 gene expression are basal breast cancer specific, whereas high gene expression of GP130 was observed in luminal A/B. VEGFA/C and CSF2 mRNA are overexpressed in HER2 positive breast cancer, with VEGFA and CSF2 also overexpressed in basal breast cancer. Further study of the specific protein function of these factors within their associated cancer subtypes may yield personalized biomarkers and treatment modalities.

Head and neck squamous cell carcinomas (HNSCC) often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. HNSCCs are remarkably lymphangiogenic and represent a clear example of a cancer that utilizes the lymphatic vasculature for malignant dissemination; however, the molecular mechanisms underlying lymphangiogenesis in HNSCC is still poorly understood. Of interest, we found that an axon guidance molecule, Semaphorin 3F (SEMA3F), is among the top 1% underexpressed genes in HNSCC, and that genomic loss of SEMA3F correlates with increased metastasis and decreased survival. SEMA3F acts on its coreceptors, plexins and neuropilins, among which neuropilin-2 (NRP2) is highly expressed in lymphatic endothelial cells (LEC) but not in oral epithelium and most HNSCCs. We show that recombinant SEMA3F promotes LEC collapse and potently inhibits lymphangiogenesis in vivo. By reconstituting all possible plexin and neuropilin combinations, we found that SEMA3F acts through multiple receptors, but predominantly requires NRP2 to signal in LECs. Using orthotopic HNSCC metastasis mouse models, we provide direct evidence that SEMA3F re-expression diminishes lymphangiogenesis and lymph node metastasis. Furthermore, analysis of a large tissue collection revealed that SEMA3F is progressively lost during HNSCC progression, concomitant with increased tumor lymphangiogenesis. SEMA3F is localized to 3p21, an early and frequently deleted locus in HNSCC and many other prevalent human malignancies. Thus, SEMA3F may represent an antilymphangiogenic metastasis suppressor gene widely lost during cancer progression, hence serving as a prognostic biomarker and an attractive target for therapeutic intervention to halt metastasis.

BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.

INTRODUCTION: Triple-negative breast cancers, particularly the claudin-low subtype, are highly aggressive and exhibit increased tumor-initiating cell (TIC) characteristics. In this study, we demonstrate that vascular endothelial growth factor C (VEGF-C) is highly expressed in the claudin-low breast cancer subtype and also that it mediates tumor progression, not only through its role in lymphangiogenesis but also through regulating TIC characteristics and the response to reactive oxygen species (ROS).
METHODS: VEGF C expression was examined in breast cancer subtypes, and a VEGF C expression signature was derived. VEGF C expression and/or its associated signature was correlated with TIC and chemoresistance signatures. In vitro and in vivo assays were performed to determine whether VEGF-C expression alters TIC characteristics and the response of breast cancer cells to chemotherapy and oxidative stress. Array analysis was used to identify a downstream effector of VEGF-C, superoxide dismutase 3 (Sod3), which was tested for its involvement in VEGF-C-mediated resistance to oxidative stress and enhancement of in vivo metastasis. The VEGF-C-associated receptor neuropilin 2 (Nrp2) was knocked down to determine whether it is required for the observed effects of VEGF-C. Expression of VEGF C and Sod3 was assessed in human breast cancers.
RESULTS: VEGF C is highly expressed in claudin-low breast cancers, and VEGF C and the VEGF C signature are associated with TIC-related gene signatures. VEGF-C-knockdown in mammary carcinoma cells decreases TIC properties in vitro and in vivo, sensitizing cells to oxidative stress and chemotherapy. We identified Sod3 as a target of VEGF-C in breast cancer cells by demonstrating that it is required for VEGF-C-mediated cell survival in response to oxidative stress and for VEGF-C-mediated metastasis. We demonstrate that Nrp2 is the VEGF-C-associated receptor that mediates alterations in Sod3 expression and the response of tumor cells to oxidative stress. We show that VEGF C and Sod3 are positively associated in human breast cancer.
CONCLUSIONS: We describe a novel mechanism by which VEGF-C contributes to metastasis via its ability to enhance TIC-associated characteristics, particularly the response to ROS. We identified Sod3 as a critical mediator of VEGF-C-induced metastasis, and we provide evidence that the VEGF-C-Sod3 axis plays a role in human breast cancers.

Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.

Altered microRNA (miRNA) expression is a hallmark of many cancer types. The combined analysis of miRNA and messenger RNA (mRNA) expression profiles is crucial to identifying links between deregulated miRNAs and oncogenic pathways. Therefore, we investigated the small non-coding (snc) transcriptomes of nine clear cell renal cell carcinomas (ccRCCs) and adjacent normal tissues for alterations in miRNA expression using a publicly available small RNA-Sequencing (sRNA-Seq) raw-dataset. We constructed a network of deregulated miRNAs and a set of differentially expressed genes publicly available from an independent study to in silico determine miRNAs that contribute to clear cell renal cell carcinogenesis. From a total of 1,672 sncRNAs, 61 were differentially expressed across all ccRCC tissue samples. Several with known implications in ccRCC development, like the upregulated miR-21-5p, miR-142-5p, as well as the downregulated miR-106a-5p, miR-135a-5p, or miR-206. Additionally, novel promising candidates like miR-3065, which i.a. targets NRP2 and FLT1, were detected in this study. Interaction network analysis revealed pivotal roles for miR-106a-5p, whose loss might contribute to the upregulation of 49 target mRNAs, miR-135a-5p (32 targets), miR-206 (28 targets), miR-363-3p (22 targets), and miR-216b (13 targets). Among these targets are the angiogenesis, metastasis, and motility promoting oncogenes c-MET, VEGFA, NRP2, and FLT1, the latter two coding for VEGFA receptors.

Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity.

Although a number of growth factors and receptors are known to control tumor angiogenesis, relatively little is known about the mechanism by which these factors influence the directional endothelial cell migration required for cancer microvessel formation. Recently, it has been shown that the focal adhesion protein paxillin is required for directional migration of fibroblasts in vitro. Here, we show that paxillin knockdown enhances endothelial cell migration in vitro and stimulates angiogenesis during normal development and in response to tumor angiogenic factors in vivo. Paxillin produces these effects by decreasing expression of neuropilin 2 (NRP2). Moreover, soluble factors secreted by tumors that stimulate vascular ingrowth, including vascular endothelial growth factor (VEGF), also decrease endothelial cell expression of paxillin and NRP2, and overexpression of NRP2 reverses these effects. These results suggest that the VEGF-paxillin-NRP2 pathway could represent a new therapeutic target for cancer and other angiogenesis-related diseases.

BACKGROUND: It is currently not possible to predict the metastatic potential of early-stage melanoma lesions by histological examination alone; however, a significant number of thin melanomas will progress over time to advanced disease. Molecular biomarkers that could identify patients with melanoma at high risk at the time of original diagnosis would contribute significantly to improved patient outcomes and increased survival. Neuropilin-2 (NRP2), a cell surface receptor involved in tumour-associated angiogenesis and lymphangiogenesis, has recently been shown to be expressed in melanoma.
OBJECTIVES: To evaluate the potential value of NRP2 gene transcript levels as biomarkers for malignant melanoma progression.
METHODS: We measured NRP2 gene expression in a panel of formalin-fixed paraffin-embedded tissue specimens consisting of naevi, primary melanomas and metastatic melanomas using quantitative reverse transcriptase-polymerase chain reaction technique.
RESULTS: NRP2 levels are clearly segregated among the groups of naevi, primary and metastatic melanoma samples with a statistical trend towards increasing NRP2 gene expression correlating with disease progression. Logistic regression analysis reveals that the probability of malignant progression increases with elevated levels of NRP2 (odds ratio of 2·60 with confidence interval 1·29-5·21). Within the group of primary melanomas, there is a positive correlation (r = 0·823) between NRP2 expression and Breslow depth. This correlation was validated in an independent sample set of patients with melanoma.
CONCLUSIONS: This preliminary study strongly supports the significance of NRP2 as a useful biomarker for malignant progression of melanoma, which may be useful for early identification of patients with melanoma at high risk.

Neuropilins (NRPs) are co-receptors for class 3 semaphorins and for members of the vascular endothelial growth factor (VEGF) family of angiogenic cytokines. Genetic analysis of the role of NRPs in mice shows that NRP1 is essential for embryonic neuronal pathfinding and cardiovascular development, mediated via semaphorins and VEGF, respectively, while NRP2 has a more restricted role in neuronal patterning and lymphangiogenesis. NRPs are thought to mediate functional responses, most importantly cell migration, as a result of complex formation with other receptors, such as plexins in the case of semaphorins and the VEGF receptor, VEGFR2, resulting in enhanced signalling via some intracellular pathways. Recent findings indicate that NRPs may have important biological roles in other physiological and disease-related processes. In particular, NRPs are highly expressed in diverse tumour cell lines and human neoplasms and have been implicated in several biological processes regulating tumour growth in vivo, suggesting that NRP1 may be a future therapeutic target in cancer.