Lung cancer is the most common cause of cancer death in men

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1 Research Volume 9 Issue 5 - June 26, 2009 [ ] Neuropilin 1 and Lung Cancer Progression: Potential Role of Targeting Neuropilin 1 as an Antitumor Strategy Yuh-Ling Chen 1,*, Tse-Ming Hong 2, and Pan-Chyr Yang 3,4 1 Institute of Oral Medicine, College of Medicine, National Cheng Kung University 2 Graduate Institute of Clinical Medicine, College of Medicine, National Cheng Kung University 3 Institute of Biomedical Sciences, Academia Sinica 4 Department of Internal Medicine National Taiwan University Hospital, Taipei, Taiwan yuhling@mail.ncku.edu.tw Clin. Cancer Res. 13: (2007) Lung cancer is the most common cause of cancer death in men worldwide and the second most common in women. Lung cancer is often classified into two major types: small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Around 80% of lung cancer patients present with non-small-cell lung carcinoma (NSCLC). Metastasis is the major cause of death in lung cancer patients. It is a complicated process involving cell proliferation, invasion of the basement membrane and stroma, angiogenesis, and distant spread that is controlled by metastasis-enhancing and metastasissuppressing genes. Neuropilin 1 (NRP1) was known as an axonal extension and angiogenic mediators during embryonic development. NRP1 is a transmembrane glycoprotein and a coreceptor for two extracellular ligands, semaphorins/ collapsins, and vascular endothelial growth factor. In a previous study, we identified by cdna microarray that NRP1 expression is positively correlated with the invasion ability of cancer cells. In this study, we investigated the role of NRP1 as an enhancer for cancer invasion, metastasis, and angiogenesis and its signaling pathways, prognostic significance, and therapeutic implications. In this article we first found the correlation of the clinical prognostic significance of human non-smallcell lung carcinoma (NSCLC) with NRP1 expression and reported NPR1 is an independent predictor of metastasis and poor survival in NSCLC patients. Real-time quantitative RT-PCR was used to determine the number of NRP1 transcripts in lung cancer tissues from 60 patients with NSCLC. Patients with high NRP1 expression had shorter disease-free and overall survival compared with low NRP1-expression patients (Fig. 1). 1 of 5

2 Fig. 1 NRP1 mrna expression correlates with cancer relapse and survival in NSCLC patients. Second, we found that NRP1 functions as a cancer invasion and angiogenesis enhancer. To knock down NRP1 expression, two individual sirnas directed against the NRP1 gene were transfected into NRP1- positive lung cancer cells CL1-5. Significant suppression of NRP1 expression was achieved by sirna-1 and sirna-2 (Fig. 2A). Both NRP1 sirnas decreased the invasion ability of CL1-5 cells in a dosedependent manner compared with the nonsilencing sirna control (Fig. 2B). Mice injected with NRP1- silenced cells (CL1-5/shNRP1) developed significantly fewer pulmonary metastatic nodules than those with control lung cancer cells (CL1-5/shLuc) (Fig. 3). Fig. 2 Suppression of NRP1expression inhibited the invasion ability of CL1-5 cells. 2 of 5

3 Fig. 3 Mice injected with CL1-5/shNRP1 cells developed significantly fewer pulmonary metastatic nodules than those with CL1-5/shLuc cells. To identify whether any new signature motif can bind and inhibit NRP1-mediated invasion, we used mammalian cell expressed NRP1 proteins as bait to screen a random cyclic 7-mer peptide library for NRP1-binding peptides. The two most potent peptides (cyclic 7-mer peptides, DG1, and DG2) were selected and chemically synthesized for further analysis of NRP1 inhibition. DG1 or DG2 peptides inhibited CL1-5 cell invasion in a dose-dependent manner (Fig. 4A) and specifically inhibited VEGF165- induced phosphorylation of VEGFR2 (Fig. 4B). To understand whether DG1 can reduce angiogenesis or tumorigenesis, in vivo angiogenesis and xenograft tumor assays were done. DG1 inhibited tumor angiogenesis in vivo (Fig. 4C). The tumor angiogenesis activity of DG1-treated CL1-5 cells decreased significantly by 3-fold compared with the untreated tumor cells. Fig. 4 Cyclic 7-mer peptides DG1 and DG2 bind NRP1 and inhibit CL1-5 invasion and angiogenesis in vivo. A. The effect of peptides on CL1-5 cells invasive activity. B. The selected peptides reduce phosphorylation of VEGFR2. C. The effect of peptides on tumor angiogenesis in vivo. Immunohistochemical staining of the Matrigel plug sections with an anti-cd31 antibody showed a significant decrease in CD31-positive vessels in plugs containing DG1 peptide compared with mock- 3 of 5

4 treated plugs. We next investigated the effect of DG1 on tumorigenicity in vivo using the xenograft tumor assay. DG-1 treatment significantly reduced tumor volume in mice 21 days after the inoculation of the CL1-5 cells, compared with the tumor volume without DG-1 treatment (Fig. 5). Fig. 5 The effect of peptides on tumorigenesis in vivo. Taken together, our results indicate that NRP1 is an enhancer of cancer invasion and angiogenesis and is an independent predictor of cancer relapse and poor survival in NSCLC patients. Suppression of NRP1 signaling inhibits cancer invasion, tumorigenesis, angiogenesis, and in vivo metastasis. NRP1 may be a potential new therapeutic target in NSCLC. Two potent synthetic anti-nrp1 peptides (DG1 and DG2), which can block NRP1 signaling pathways, inhibit tumorigenesis, cancer invasion, and angiogenesis, were identified (Fig. 6). Fig. 6 Summary diagram showing that VEGF165 can bind to NRP1 and trigger the NRP1/VEGFR2/PI3K/ Akt signaling pathways and result in tumor angiogenesis, cancer cell invasion, and tumorigenesis. The synthetic peptides DG1/DG2 can specifically block this signaling pathway and may have therapeutic potential. 4 of 5

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