Ablation of EIF5A2 induces tumor vasculature remodeling and improves tumor response to chemotherapy via regulation of matrix metalloproteinase 2 expression

Abstract

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with poor clinical outcome. Our previous work has shown that eukaryotic initiation factor 5A2 (EIF5A2) over-expression enhances HCC cell metastasis. In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients. Both in vitro and in vivo assays indicated that ablation of endogenous EIF5A2 inhibited tumor angiogenesis by reducing matrix metalloproteinase 2 (MMP-2) expression. Given that MMP-2 degrades collagen IV, a main component of the vascular basement membrane (BM), we subsequently investigated the effect of EIF5A2 on tumor vasculature remodeling using complementary approaches, including fluorescent immunostaining, transmission electron microscopy, tumor perfusion assays and tumor hypoxia assays. Taken together, our results indicate that EIF5A2 silencing increases tumor vessel wall continuity, increases blood perfusion and improves tumor oxygenation. Additionally, we found that ablation of EIF5A2 enhanced the chemosensitivity of HCC cells to 5-Fluorouracil (5-FU). Finally, we demonstrated that EIF5A2 might exert these functions by enhancing MMP-2 activity via activation of p38 MAPK and JNK/c-Jun pathways.

Conclusion: This study highlights an important role of EIF5A2 in HCC tumor vessel remodeling and indicates that EIF5A2 represents a potential therapeutic target in the treatment of HCC.

Figure 1. EIF5A2 downregulation represses tumor angiogenesis

(A, B) Immunoblot analysis of indicated proteins in HCC cell lines. (C) EIF5A2 ablation inhibited tube formation of HUVECs induced by HCC cells. Representative images (100x) and quantification of HUVECs cultured on Matrigel-coated plates with 75% conditioned medium from the indicated cell transfected with NC or shRNA-EIF5A2. (D) EIF5A2 ablation inhibited tumor angiogenesis in a xenograft model. Sections of tumor tissues derived from PLC8024-NC or PLC8024sh#2 or sh#3 xenografts were stained for EIF5A2 and mouse CD34 by IHC. The three most intensely vascularized areas were evaluated under light microscopy. Representative images (100x) and the quantitative MVD measurement of two groups are presented (^*, P<0.05).

Figure 2. MMP-2 is required for EIF5A2-induced angiogenesis in HCC

(A) Changes in mRNA expression of the indicated genes in two EIF5A2-suppressed HCC cell lines were assessed by real-time qPCR. (B) Analysis of MMP-2 activity in TCM by gelatin zymography. TCM was collected from tumor cells transfected with NC or shRNA-EIF5A2 or with control vector or eIF5A2 over-expression vector. Recombinant human MMP2 (rHuMMP2) as the positive control. (C) Analysis of MMP-2 expression in xenograft tumor tissue sections. (D) Inhibition of MMP-2 (final concentration of anti-MMP2 antibody: 6ug/ml) repressed the pro-angiogenic effect of EIF5A2 over-expression in SMMC7721 cells. (E) Re-expression of MMP-2 in EIF5A2 knockdown PLC8024 cells restored the pro-angiogenic effect of EIF5A2.

Figure 3. Association among expression of EIF5A2, MMP-2, MVD and its prognostic significance in HCC patients

(A) Representative images of IHC staining for EIF5A2, MMP-2 and CD34 in two HCC cases (100x). EIF5A2 expression positively correlated with MMP-2 expression and tumor MVD. (B) X-tile analysis was employed to determine the cut-point for EIF5A2 expression in HCC. The cut-point (H score=150) highlighted by the black/white circle in the horizontal axis (left panel) was demonstrated on a histogram of the cohort (middle panel), and a Kaplan-Meier plot (right panel).

Figure 4. EIF5A2 ablation increases micro-vessel wall continuity and promotes normal endothelial cell lining and differentiation

(A) Xenograft tumor sections stained for CD34 (green) and collagen IV (red) revealed more continuous BMs in EIF5A2- ablation cells compared to control cells. (B) Representative images of human HCC tissue sections stained for CD34 and collagen IV by IF (400x). Twenty cases (10 with high EIF5A2 and MMP-2 expression and 10 with low EIF5A2 and MMP-2 expression), were analyzed for tumor vessel wall continuity by IF. Increased continuity of vessel walls was seen in all cases with low EIF5A2 and MMP-2 expression. For A and B, the three most intensely vascularized areas were evaluated under immunofluorescence microscopy. (C) Transmission electron microscopy of vessels in tumors derived from PLC8024 cells transfected with NC or sh#3. Left column, arrowheads indicate red blood cell. Compared with NC group, endothelial cells in the EIF5A2 knockdown group were lined with more continuous BMs (right column, arrowhead). For the NC group, left column, original magnification 5800x; right column, 13500x. For sh#3 group, left column, original magnification 2400x; right column, 13500x.

Figure 5. EIF5A2 ablation increases tumor blood perfusion, reduces tumor hypoxia and enhances sensitivity to chemotherapy

(A) Representative images of Doppler laser imaging of intratumoral blood flow. Left side: Doppler imaging; Right side: the corresponding position of the Doppler imaging on the mice. The mean value of the blood flow is the mean number of the blood flow of six tumors in one group mice. Bar graphs show quantification of corresponding perfusions. P=0.011. (B) Increased tumor vessel perfusion and reduced vessel leakiness in the PLC8024-sh#3 group following injection with Texas Red-conjugated Dextran (red) and FITC-conjugated Lectin (green). White arrowheads on merged images indicated sites of leakage. (C) Reduced staining for pimonidazole in tumors derived from PLC8024 cells transfected with NC or sh#3. Arrowheads indicated necrosis regions. Cells peripheral to necrotic areas were stained most intensely, indicating severe hypoxia. (D) EIF5A2 silencing sensitizes xenografts to 5-fluorouracil. Representative images of xenograft tumors induced by PLC8024-NC or PLC8024-sh#3 in nude mice. Left column, before treatment; middle column, 5 weeks after treatment with 5-FU (intraperitoneal injection, 40mg/kg body weight, once weekly for 5 weeks); right column, representative tumors excised from mice after 5 weeks of treatment. E. Tumor growth curves (N=6 mice per group); tumor volumes were measured on a weekly basis.

Figure 6. EIF5A2 induces MMP-2 transcription by activating p38 MAPK and JNK/c-Jun pathways

(A) Immunoblot for indicated mediators of MAPK pathway signaling in cells transfected with NC or sh#3 or control vector or EIF5A2 over-expression vector. (B) MMP-2 activity in TCM examined by gelatin zymography. TCM was collected from cells transfected with NC or si-RNA targeting p38 MAPK or ERK1/2. (C) Analysis of luciferase activity using MMP-2 promoter luciferase reporter vector in indicated cells. Basic: pGL3-basic; WT: wild type promoter; ΔAP-1: deletion of AP-1 binding site (**, P<0.01; ^*, P<0.05, T test).