Image1_Identification and validation of molecular subtype and prognostic signature for lung adenocarcinoma based on neutrophil extracellular traps.JPEG

Background: Neutrophil Extracellular Traps (NETs) are fibrous networks made of DNA-histone complexes and proteins protruded from activated neutrophils. Accumulating evidences have highlighted the vital role of NETs in tumor progression and diffusion. However, limited systematic studies regarding the role of NETs in LUAD have been performed.Methods: Differentially expressed NETs-related genes and their mutation landscape were identified with TCGA database. Consensus clustering analysis was performed to determine the NETs-related subtypes of LUAD. LASSO algorithm was employed to construct a prognostic signature. Moreover, GSE30219 and GSE31210 were used as independent validation. We also constructed a lncRNA-miRNA-mRNA regulatory axis with several miRNA and lncRNA databases.Results: Consensus clustering identified two NETs-related clusters in LUAD. High NETs score was correlated with a favorable overall survival, abundant immune cell infiltration, and high activity of immune response signal pathways. Six NET-related genes (G0S2, KCNJ15, S100A12, AKT2, CTSG, and HMGB1) with significant prognostic value were screened to develop a prognostic signature. LUAD patients with low-risk had a significantly favorable overall survival both in the training set and validation set. Moreover, NETs-related risk score and clinical stage could act as an independent prognostic factor for LUAD patients. Significant correlation was obtained between risk score and tumor immune microenvironment. We also identified lncRNA BCYRN1/miR-3664-5p/CTSG regulatory axis that may be involved in the progression of LUAD.Conclusion: We developed two molecular subtypes and a prognostic signature for LUAD based on NETs-related genes. This stratification could provide more evidences for estimating the prognosis and immunotherapy of LAUD patients.</p

Table1_Identification and validation of molecular subtype and prognostic signature for lung adenocarcinoma based on neutrophil extracellular traps.DOCX

Background: Neutrophil Extracellular Traps (NETs) are fibrous networks made of DNA-histone complexes and proteins protruded from activated neutrophils. Accumulating evidences have highlighted the vital role of NETs in tumor progression and diffusion. However, limited systematic studies regarding the role of NETs in LUAD have been performed.Methods: Differentially expressed NETs-related genes and their mutation landscape were identified with TCGA database. Consensus clustering analysis was performed to determine the NETs-related subtypes of LUAD. LASSO algorithm was employed to construct a prognostic signature. Moreover, GSE30219 and GSE31210 were used as independent validation. We also constructed a lncRNA-miRNA-mRNA regulatory axis with several miRNA and lncRNA databases.Results: Consensus clustering identified two NETs-related clusters in LUAD. High NETs score was correlated with a favorable overall survival, abundant immune cell infiltration, and high activity of immune response signal pathways. Six NET-related genes (G0S2, KCNJ15, S100A12, AKT2, CTSG, and HMGB1) with significant prognostic value were screened to develop a prognostic signature. LUAD patients with low-risk had a significantly favorable overall survival both in the training set and validation set. Moreover, NETs-related risk score and clinical stage could act as an independent prognostic factor for LUAD patients. Significant correlation was obtained between risk score and tumor immune microenvironment. We also identified lncRNA BCYRN1/miR-3664-5p/CTSG regulatory axis that may be involved in the progression of LUAD.Conclusion: We developed two molecular subtypes and a prognostic signature for LUAD based on NETs-related genes. This stratification could provide more evidences for estimating the prognosis and immunotherapy of LAUD patients.</p

Image2_Identification and validation of molecular subtype and prognostic signature for lung adenocarcinoma based on neutrophil extracellular traps.JPEG

Background: Neutrophil Extracellular Traps (NETs) are fibrous networks made of DNA-histone complexes and proteins protruded from activated neutrophils. Accumulating evidences have highlighted the vital role of NETs in tumor progression and diffusion. However, limited systematic studies regarding the role of NETs in LUAD have been performed.Methods: Differentially expressed NETs-related genes and their mutation landscape were identified with TCGA database. Consensus clustering analysis was performed to determine the NETs-related subtypes of LUAD. LASSO algorithm was employed to construct a prognostic signature. Moreover, GSE30219 and GSE31210 were used as independent validation. We also constructed a lncRNA-miRNA-mRNA regulatory axis with several miRNA and lncRNA databases.Results: Consensus clustering identified two NETs-related clusters in LUAD. High NETs score was correlated with a favorable overall survival, abundant immune cell infiltration, and high activity of immune response signal pathways. Six NET-related genes (G0S2, KCNJ15, S100A12, AKT2, CTSG, and HMGB1) with significant prognostic value were screened to develop a prognostic signature. LUAD patients with low-risk had a significantly favorable overall survival both in the training set and validation set. Moreover, NETs-related risk score and clinical stage could act as an independent prognostic factor for LUAD patients. Significant correlation was obtained between risk score and tumor immune microenvironment. We also identified lncRNA BCYRN1/miR-3664-5p/CTSG regulatory axis that may be involved in the progression of LUAD.Conclusion: We developed two molecular subtypes and a prognostic signature for LUAD based on NETs-related genes. This stratification could provide more evidences for estimating the prognosis and immunotherapy of LAUD patients.</p

Image3_Identification and validation of molecular subtype and prognostic signature for lung adenocarcinoma based on neutrophil extracellular traps.JPEG

Background: Neutrophil Extracellular Traps (NETs) are fibrous networks made of DNA-histone complexes and proteins protruded from activated neutrophils. Accumulating evidences have highlighted the vital role of NETs in tumor progression and diffusion. However, limited systematic studies regarding the role of NETs in LUAD have been performed.Methods: Differentially expressed NETs-related genes and their mutation landscape were identified with TCGA database. Consensus clustering analysis was performed to determine the NETs-related subtypes of LUAD. LASSO algorithm was employed to construct a prognostic signature. Moreover, GSE30219 and GSE31210 were used as independent validation. We also constructed a lncRNA-miRNA-mRNA regulatory axis with several miRNA and lncRNA databases.Results: Consensus clustering identified two NETs-related clusters in LUAD. High NETs score was correlated with a favorable overall survival, abundant immune cell infiltration, and high activity of immune response signal pathways. Six NET-related genes (G0S2, KCNJ15, S100A12, AKT2, CTSG, and HMGB1) with significant prognostic value were screened to develop a prognostic signature. LUAD patients with low-risk had a significantly favorable overall survival both in the training set and validation set. Moreover, NETs-related risk score and clinical stage could act as an independent prognostic factor for LUAD patients. Significant correlation was obtained between risk score and tumor immune microenvironment. We also identified lncRNA BCYRN1/miR-3664-5p/CTSG regulatory axis that may be involved in the progression of LUAD.Conclusion: We developed two molecular subtypes and a prognostic signature for LUAD based on NETs-related genes. This stratification could provide more evidences for estimating the prognosis and immunotherapy of LAUD patients.</p

NSK-01105, a Novel Sorafenib Derivative, Inhibits Human Prostate Tumor Growth via Suppression of VEGFR2/EGFR-Mediated Angiogenesis

<div><p>The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i> models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited <i>ex vivo</i> angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities.</p></div

NSK-01105 inhibited VEGF-induced migration of HUVECs.

<p>(a, b) NSK-01105 inhibited HUVECs migration by wound-healing assay. Cells were starved to inactivate cell proliferation and then wounded by pipette tips. Stimulated with VEGF, large numbers of HUVECs migrated into the clear area, whereas, as potent as sorafenib, NSK-01105 significantly inhibited the VEGF-induced migration at 0.5, 1 and 2.5 µM. Migration distances were calculated by IPP software. (c, d) NSK-01105 inhibited HUVECs migration by transwell cell migration assay. Cells were cultured in serum-free medium along with the indicated concentrations of VEGF, NSK-01105 and/or sorafenib in the upper chambers and the lower chambers were filled with 10% FBS medium. Stimulated with VEGF, large numbers of HUVECs migrated into the lower surface of the polycarbonate, whereas NSK-01105 and sorafenib significantly inhibited the VEGF-induced migration. Cells were counted under a microscope. Columns, mean; bars, SD (n = 4). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls, <i>P</i><0.05.</p

The effect of NSK-01105 and sorafenib in prostate cancer xenograft models.

<p>*, <i>p</i><0.05, compared with control. Significant difference was calculated by one-way analysis of variance.</p><p>The effect of NSK-01105 and sorafenib in prostate cancer xenograft models.</p

NSK-01105 inhibited VEGF-induced cell viability in HUVECs.

<p>(a) Chemical structures of the NSK-01105 and Sorafenib. (b) NSK-01105 inhibited the VEGF-induced viability of endothelial cells. Stimulated with VEGF (10 ng/mL), HUVECs showed a high rate of viability. VEGF-induced viability of HUVECs was significantly inhibited by NSK-01105 and sorafenib at concentrations of 5, 10 and 20 µM for 24 h. Columns, mean; bars, SD (n = 6). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls,<i>P</i><0.05.</p

NSK-01105 inhibited the activation of VEGFR2-mediated signaling pathways in HUVECs.

<p>VEGF stimulated VEGFR-2 autophosphorylation in HUVECs. (a) NSK-01105 and sorafenib suppressed the activation of VEGFR2 and its downstream key kinases FAK and eNOS at 5 µM in HUVECs. (b) The ratios of the optical density between target molecules and β-actin. The optical density was quantified by IPP software. Columns, mean; bars, SD (n = 3). *, compared with vehicle controls, <i>P</i><0.05; #, compared with VEGF controls, <i>P</i><0.05.</p

NSK-01105 inhibited VEGF secretion and the activation of VEGFR2-mediated signaling pathways in prostate cancer cells.

<p>(a) NSK-01105 inhibited VEGF secretion in prostate cancer cells. VEGF levels were estimated by ELISA method. NSK-01105 and sorafenib suppressed VEGF secretion in both cell lines at concentrations of 2.5, 5 and 10 µM. Columns, mean; bars, SD (n = 3). *, compared with vehicle controls, <i>P</i><0.05. (b) NSK-01105 and sorafenib suppressed the activation of VEGFR2 and its downstream key kinases FAK and eNOS at 5 µM in both prostate cancer cells. NSK-01105 suppressed the phosphorylation of EGFR, while sorafenib had little effect on EGFR activation at 5 µM in both prostate cancer cell lines. (c) The ratios of the optical density between target molecules and β-actin. The optical density was quantified by IPP software. Columns, mean; bars, SD (n = 3). *, compared with vehicle controls, P <0.05.</p