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. 2014 Aug 27;9(8):e106032.
doi: 10.1371/journal.pone.0106032. eCollection 2014.

Retinoblastoma binding protein 2 (RBP2) promotes HIF-1α-VEGF-induced angiogenesis of non-small cell lung cancer via the Akt pathway

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Retinoblastoma binding protein 2 (RBP2) promotes HIF-1α-VEGF-induced angiogenesis of non-small cell lung cancer via the Akt pathway

Lei Qi et al. PLoS One. .

Abstract

Background: Pathological angiogenesis plays an essential role in tumor aggressiveness and leads to unfavorable prognosis. The aim of this study is to detect the potential role of Retinoblastoma binding protein 2 (RBP2) in the tumor angiogenesis of non-small cell lung cancer (NSCLC).

Methods: Immunohistochemical staining was used to detect the expression of RBP2, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and CD34. Two pairs of siRNA sequences and pcDNA3-HA-RBP2 were used to down-regulate and up-regulate RBP2 expression in H1975 and SK-MES-1 cells. An endothelial cell tube formation assay, VEGF enzyme-linked immunosorbent assay, real-time PCR and western blotting were performed to detect the potential mechanisms mediated by RBP2 in tumor angiogenesis.

Results: Of the 102 stage I NSCLC specimens analyzed, high RBP2 protein expression is closely associated with tumor size (P = 0.030), high HIF-1α expression (P = 0.028), high VEGF expression (P = 0.048), increased tumor angiogenesis (P = 0.033) and poor prognosis (P = 0.037); high MVD was associated with high HIF-1α expression (P = 0.034), high VEGF expression (P = 0.001) and poor prognosis (P = 0.040). Multivariate analysis indicated that RBP2 had an independent influence on the survival of patients with stage I NSCLC (P = 0.044). By modulating the expression of RBP2, our findings suggested that RBP2 protein depletion decreased HUVECs tube formation by down-regulating VEGF in a conditioned medium. RBP2 stimulated the up-regulation of VEGF, which was dependent on HIF-1α, and activated the HIF-1α via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Moreover, VEGF increased the activation of Akt regulated by RBP2.

Conclusions: The RBP2 protein may stimulate HIF-1α expression via the activation of the PI3K/Akt signaling pathway under normoxia and then stimulate VEGF expression. These findings indicate that RBP2 may play a critical role in tumor angiogenesis and serve as an attractive therapeutic target against tumor aggressiveness for early-stage NSCLC patients.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Immunohistochemical staining of the RBP2 protein and microvessels using the streptavidin–peroxidase method (magnification ×400).
(A) High RBP2 protein expression in squamous cell cancer; (B) high RBP2 protein expression in adenocarcinoma; (C) negative RBP2 protein expression in NSCLC; (D) high HIF-1α expression in squamous cell cancer; (E) high HIF-1α protein expression in adenocarcinoma; (F) high VEGF expression in squamous cell cancer; (G) high VEGF in adenocarcinoma; (H) high CD34 expression in squamous cell cancer; (I) high CD34 expression in adenocarcinoma.
Figure 2
Figure 2. Correlation between RBP2 expression and MVD, and Kaplan–Meier curves of overall survival stratified according to RBP2 protein and MVD.
(A) Correlation between RBP2 expression and MVD for stage I NSCLC. NSCLC with high RBP2 protein expression showed significantly higher intratumoral MVD than that with low RBP2 protein expression (P = 0.033, Mann-Whitney U test). (B) Kaplan–Meier curves of overall survival demonstrated a poor 5-year overall survival rate in patients with RBP2 protein overexpression (53.8% versus 72.0%, P = 0.037). (C) Kaplan–Meier curves of overall survival demonstrated a poor 5-year overall survival rate in patients with high MVD (52.2% versus 71.4%, P = 0.040).
Figure 3
Figure 3. Expression of RBP2 in human NSCLC cell lines and the transfection efficiency of siRNA and pcDNA3-HA-RBP2.
(A) RBP2 is overexpressed in human NSCLC cell lines SK-MES-1, A549, SPCA-1 and H1975 compared to the human bronchial epithelial cell line BEAS2B cells. (B) Effects of RBP2 siRNA1, RBP2 siRNA2 and pcDNA3-HA-RBP2 on the expression of the RBP2 protein.
Figure 4
Figure 4. Down-regulation of the RBP2 protein decreased the tube formation by HUVECs induced by conditioned medium.
Tube formation assay: (A) control-siRNA H1975 cells; (B) RBP2-siRNA1 H1975 cells; (C) RBP2-siRNA2 H1975 cells. (D) Quantitative analysis of the tube formation by HUVECs induced by conditioned medium; (E) Down-regulation of the RBP2 protein decreased the expression levels of VEGF in conditioned media. (F) The tube formation induced by the conditioned medium of control siRNA H1975 cells was blocked by the VEGFR inhibitor (sunitinib malate, 2.5 µM). (G) The reduced tube formation induced by the conditioned medium of RBP2-siRNA2 H1975 cells was rescued by adding VEGF-165 (2 ng/ml).
Figure 5
Figure 5. RBP2 stimulates the mRNA and protein expression of HIF-1α and VEGF.
(A) RBP2 up-regulated the HIF-1α protein in a time-dependent manner under normoxic conditions. (B) Depletion of RBP2 decreased the expression of HIF-1α and VEGF in H1975 cells. (C) Up-regulation of RBP2 increased the expression of HIF-1α and VEGF in SK-MES-1 cells. (D) Real-time RT-PCR showed that the mRNA expression levels of HIF-1α and VEGF were significantly decreased in RBP2-depleted H1975 cells compared to control cells. (E) Real-time RT-PCR showed that the mRNA expression levels of HIF-1α and VEGF were significantly increased in RBP2-overexpressing SK-MES-1 cells.
Figure 6
Figure 6. RBP2 induction of VEGF is dependent on HIF-1α.
(A) Depletion of HIF-1α with an siRNA specific against HIF-1α in RBP2-overexpressing SK-MES-1 cells led to the down-regulation of VEGF compared with the scramble non-specific control siRNA. (B) Up-regulation of HIF-1α expression in RBP2-siRNA2 H1975 cells led to the up-regulation of VEGF.
Figure 7
Figure 7. RBP2 activates HIF-1α via the PI3K/Akt signaling pathway.
(A) Silencing RBP2 expression in H1975 cells significantly decreased the phosphorylation of Akt, and the forced expression of RBP2 in SK-MES-1 cells increased the activity of Akt. (B) When Akt was constitutively activated in RBP2-siRNA2 H1975, the expression of HIF-1α and VEGF were increased compared to the control. The PI3K/Akt inhibitor LY294002 significantly inhibited the expression of HIF-1α and VEGF in pcDNA3-HA-RBP2 SK-MES-1 cells. (C) Westernblots showing the time course of Akt phosphorylation in RBP2-siRNA2 H1975 cells due to VEGF-165 (25 ng/mL). (D) In the presence of recombinant human VEGF-165 stimulation, the activation of Akt was increased in RBP2-siRNA2 H1975 cells and RBP2-overexpressing SK-MES-1 cells (25 ng/mL, 30 minutes).

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The project was supported by the National Natural Science Foundation of China (No. 30571844), the Science and Technology Development Foundation of Shandong Province (No. 2009GG10002007), and the National Natural Science Foundation of Shandong Province (No. ZR2009CM090). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.