ANGPTL2 promotes VEGF-A synthesis in human lung cancer and facilitates lymphangiogenesis

Figure 1. Adipokine levels in lung cancer tissue and normal healthy samples. (A–M) Adipokine mRNA expression in human lung cancer tissue and adjacent normal tissue was analyzed in records from the TCGA database. *p < 0.05 compared with normal tissue.

Figure 2. Levels of ANGPTL2 expression correlate with clinicopathologic features of lung adenocarcinoma tissue infiltrated by lymphatic vessels. (A, B) ANGPTL2 expression in human lung cancer tissue and adjacent normal tissue samples was analyzed by IHC staining. (C) The association between ANGPTL2 expression and regional lymph node metastasis was analyzed in samples from the TCGA database. (D) Associations between ANGPTL2 expression and overall survival rates of lung cancer patients were analyzed using the Kaplan-Meier Plotter database. (E, F) LYVE-1 expression in human lung cancer tissue and adjacent normal tissue samples was analyzed by IHC staining. (G, H) Data obtained from the GEO database (GDS4402/1431848_at) were analyzed for ANGPTL2 expression in primary lung tumor tissue and lung cancer with lymph node metastasis tissue samples. *p < 0.05 compared with normal tissue.

Figure 3. ANGPTL2 levels correlate with lung cancer cell migratory activity and facilitate LEC tube formation and migration. (A) The migratory ability of human lung cancer cell lines (CL1-0, CL1-5 and A549) was measured by the Transwell assay. (B, C) ANGPTL2 mRNA and protein expression in lung cancer cell lines was examined by Western blot (n=3) and qPCR. (D) Cells were transfected with ANGPTL2 cDNA or shRNA, then ANGPTL2 expression was measured by Western blot (n=3). *p < 0.05 compared with CL1-0 cells. (E–J) Cells were transfected with ANGPTL2 cDNA or shRNA. The CM was collected and applied to the LECs. LEC tube formation and migration was examined. *p < 0.05 compared with controls.

Figure 4. ANGPTL2 promotes VEGF-A-dependent LEC tube formation and migration. (A–E) Associations between ANGPTL2 expression and lymphangiogenic factors (VEGF-A, VEGF-C, FIGF, HGF and FGF2) were analyzed using data from the TCGA database. (F–H) CL1-0 cells were transfected with ANGPTL2 cDNA; CL1-5 and A549 cells were transfected with ANGPTL2 shRNA. Levels of mRNA expression were examined by qPCR. *p < 0.05 compared with controls. (I, J) CL1-0 cells were transfected with ANGPTL2 cDNA. The CM was collected and applied to the LECs with VEGF-A or VEGF-C antibody. LEC tube formation and migration was examined. (K–N) Cells were transfected with ANGPTL2 shRNA. The CM was collected and applied to the LECs with VEGF-A. LEC tube formation and migration was examined. *p < 0.05 compared with CL1-0 CM; #p < 0.05 compared with Control; #p < 0.05 compared with CL1-5 or A549 with ANGPTL2 shRNA CM.

Figure 5. ANGPTL2 increases VEGF-A synthesis in lung cancer cells via the integrin α5β1 receptor, p38 and NF-κB signaling. (A–C) Cells were transfected with ANGPTL2 cDNA, then stimulated with LILRB2 and integrin α5β1 siRNA or SB203580 and PDTC; VEGF-A expression was measured by Western blot (n=3). Quantitative data of the protein level were obtained using ImageJ software. Densitometric analysis of protein expression was normalized to β-actin. (D, E) Cells were transfected with ANGPTL2 cDNA, then stimulated with integrin α5β1 siRNA and SB203580; p38 and NF-κB activation was measured by Western blot (n=3) and NF-κB luciferase activity. Quantitative data of the protein level were obtained using ImageJ software. Densitometric analysis of protein expression was normalized to β-actin. *p < 0.05 compared with CL1-0; #p < 0.05 compared with Control.

Figure 6. ANGPTL2 promotes tumor growth and lymphangiogenesis in vivo. (A–C) CL1-0 and CL1-5 cells were subcutaneously injected into the right flanks of BALB/c-nu mice. Four weeks later, the mice were sacrificed and the tumors were excised and weighed (n=5). (D) Tumor sections were immunostained using ANGPTL2, LYVE-1 and VEGF-A antibodies. *p < 0.05 compared with controls.

Figure 7. Schematic diagram summarizes the mechanisms by which ANGPTL2 facilitates lymphangiogenesis in human lung cancer cells. ANGPTL2 increases VEGF-A production and subsequently facilitates LEC lymphangiogenesis in human lung cancer cells via the integrin α5β1 receptor, p38 and NF-κB signaling.