Ang-2 promotes lung cancer metastasis by increasing epithelial-mesenchymal transition

Abstract

Lung cancer is the most common malignant tumor with increasing angiopoietin-2 (Ang-2) and a high rate of metastasis. However, the mechanism of Ang-2 enhancing tumor proliferation and facilitating metastasis remains to be clarified. In this study, Ang-2 expression and its gene transcription on effects of biological behaviors and epithelial-mesenchymal transition (EMT) were investigated in lung cancers. Total incidence of Ang-2 expression in the cancerous tissues was up to 91.8 % (112 of 122) with significantly higher (χ²=103.753, P²=7.883, P=0.005), differentiation degree (χ²=4.554, P=0.033), tumor node metastasis (TNM) staging (χ²=5.039, P=0.025), and 5-year survival rate (χ² =11.220, P²=18.881, P²=0.81, P=0.776) or III & IV (χ²=1.845, P=0.174). Over-expression of Ang-2 or Ang-2 mRNA in lung A549 and NCI-H1975 cells were identified among different cell lines. When silencing Ang-2 in A549 cells with specific shRNA-1 transfection, the cell proliferation was significantly inhibited in a time-dependent manner, with up-regulating E-cadherin, down-regulating Vimentin, Twist, and Snail expression, and decreasing invasion and metastasis of cancer cell abilities, suggesting that Ang-2 promote tumor metastasis through increasing EMT, and it could be a potential target for lung cancer therapy.

Keywords: Ang-2; EMT; RNA interference; lung cancer; prognosis.

Figure 1. Ang-2 expression and cellular distribution by immunohistochemistryAng-2 expression in the cancerous-and their paracancerous-tissues of 122 patients with lung cancers were analyzed by immunohistochemistry with anti-human Ang-2 antibody (ab155106, Abcam, USA)

(A1 and A2), the stronger straining of Ang-2 expression in lung adenocarcinoma tissues, with deeper brown staining particles mainly localized in the cytoplasm and cell membranes; (B1 and B2), the light straining of Ang-2 expression in their surrounding tissues; a1&b1, the original magnification × 40; a2&b2, the original magnification × 400.

Figure 2. Kaplan-Meier survival curves of Ang-2 overexpressionThe survival curves of high Ang-2 expression in patients with lung cancer were made by the Kaplan-Meier method

(A), the overall survival curve of high Ang-2 expression in patients with lung cancer; (B), the overall survival curve of high Ang-2 expression in patients with lung cancer at TNM stage I; (C), the overall survival curve of high Ang-2 expression in patients with lung cancer at stage II; and (D), the overall survival curve of high Ang-2 expression in patinets with lung cancer at stage III-IV, respectively.

Figure 3. Ang-2 expression, gene transcription and shRNA suppression Ang-2 expression and gene transcription in lung Beas-2B cell, lung cancer (SPC-A-1, NCI-1650, A549, and NCI-1975) cell lines were analyzed at protein- by Western blotting or at mRNA-level by qRT-PCR

(A), the Ang-2 expressions in different cells at protein level with β-actin as control; (B), the ratios from Ang-2 to β-actin in different cells at protein level; (C), the ratios from Ang-2 to GAPDH expression in different cells at mRNA level; (D), the phase-contrast image (X100 magnification) with the Ang-2-shRNA1 successfully transfected into A549 cells at 24 h; (E), the fluorescence image (X100 magnification) with the Ang-2-shRNA1 successfully transfected into A549 cells; (F), the different alterations of Ang-2 expression with β-actin as loading control at 48 h; (G), the ratios from Ang-2 to β-actin protein in different cell lines; and (H), the similar alterations of Ang-2 normalized to GAPDH in different cell lines at mRNA level. Ang-2, angiopoietin-2; Ang-2 Exp., angiopoietin-2 protein expression; shRNA, the Ang-2-shRNA transfection group; NC, the negative control group; and Control, the blank control group. ^*P<0.05. ^**P<0.001.

Figure 4. Silencing Ang-2 on effect of biological behaviors and EMT of cells

The possibilities of cell proliferation, migration, invasion and EMT of the A549 cells were altered after the most effective Ang-2-shRNA-1 transfection. (A & B), the cell proliferation ability was examined using CCK-8 at 450 nm. (C), the cell patterns (×200 magnification) with the crystal violet staining solution: c1, the cells in the shRNA group; c2, the cells in the NC group, and c3, the cells in the control group; (D), the cell migration abilities were presented as total number of cells that migrated to the bottom chamber without or with the transwell-precoated matrigel, as calculated at 6 random fields; (E), the cellinvasion abilities were presented as total number of cells that migrated to the bottom chamber without or with the transwell-precoated matrigel, as calculated at 6 random fields; (F), the EMT-related epithelial indicators and mesenchymal markers after silencing Ang-2 transcritpion of A549 cells were analyzed by the Western blotting with β-actin as loading control; and (G), the relative ratio from each protein to β-actin (n=3) compared with the control group. Ang-2, angiopoietin-2; shRNA, the Ang-2-shRNA transfection group; NC, the negative control group; and Control, the blank control group; ^*P<0.05. ^**P<0.001.

Figure 5. Possibility mechanism of tumor derived Ang-2 up-regulating EMT and facilitating lung cancer metastasis

Ang-2 has been implicated in mediating inflammatory processes, and upregulated in multiple inflammation-related tumors or signaling pathway. With lung cancer volume increasing, intratumoral hypoxia in the absence of vascularization resulted in tumor cells to express Ang-2 inducing angiogenesis for tumor growth and increasing vascular permeability. Overexpression of Ang-2 facilitating proliferation, invasion, metastasis, and EMT of lung cancer cells that could be a new mechanism insight into tumor metastasis by inhibiting E-cadherin and upregulating VIM, Twist and Snail signaling. Ang, angiopoietin; Ang-2, angiopoietin-2; CaMK, calcium/calmodulin-dependent protein kinase; EMT, epithelial-mesenchymal transition; ENO1, enolase 1; EPO, erythropoietin; ET1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Glut1, glucose transporter 1; HIF-1α, hypoxia-inducible factor-1α; HK1, hexokinase 1; HRE, hypoxia-response element; IGF2, insulin-like growth factor 2; IP3, inositol 1,4,5-trisphate; mTOR, mechanistic target of rapamycin kinase; NOS2, nitric oxide synthase 2; Oncogenes, ras, c-myc etc; PLC-γ, phospholipase C gamma; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; RTK, receptor tyrosine kinase; TGFA, transforming growth factor alpha; tumor suppressor genes, pVHL, P₅₃, PTEN, etc. VEGF, vascular endothelial growth factors; VHL, von Hippel-Lidau; VIM, Vimentin.