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

Wang FW, Cai MY, Mai SJ, Chen JW, Bai HY, Li Y, Liao YJ, Li CP, Tian XP, Kung HF, Guan XY, Xie D - Oncotarget (2014)

Bottom Line: In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine; Sun Yat-sen University Cancer Center, Guangzhou, China. These authors contributed equally to this work.

ABSTRACT

Unlabelled: 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.

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Related in: MedlinePlus

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.
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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.

Mentions: Blood vessels are comprised of basement membranes (BMs), pericytes and vascular endothelial cells. Vascular BM components are required for the initiation and resolution of angiogenesis [11]. Since type IV collagen, a substrate for MMP-2 and the main component of the vascular BM, is crucial for BM stability and assembly [12, 13]. We hypothesized that EIF5A2 may also affect tumor blood vessel wall remodeling or vessel structure via regulation of MMP-2 activity. Collagen IV and CD34 expression were examined by immunofluorescent staining of xenograft tissue sections. Compared with the control group, tumor micro-vessels in xenografts from EIF5A2 knockdown tumor cells exhibit more continuous and smooth wall, more regular endothelial lining, and morphology similar to normal vasculature (Fig. 4A). Similarly, vessel walls in EIF5A2 and MMP2 low expression human HCC tissue sections were more continuous (Fig. 4B, Supplementary Fig. 3). Transmission electron microscopy further revealed that the vessels in the EIF5A2 knockdown group were more likely to have continuous BMs, well differentiated endothelial cells, and same polarity in their endothelial linings (Fig. 4C). These data suggest that EIF5A2 ablation could induce formation of integrated, continuous vascular walls and normalized endothelial cells lining. Moreover, transmission electron microscopy indicated that the micro-vessels in EIF5A2 knockdown group displayed larger lumens and thinner walls which may enhance blood perfusion and facilitate nutrient and oxygen exchange.


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

Wang FW, Cai MY, Mai SJ, Chen JW, Bai HY, Li Y, Liao YJ, Li CP, Tian XP, Kung HF, Guan XY, Xie D - Oncotarget (2014)

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.
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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.
Mentions: Blood vessels are comprised of basement membranes (BMs), pericytes and vascular endothelial cells. Vascular BM components are required for the initiation and resolution of angiogenesis [11]. Since type IV collagen, a substrate for MMP-2 and the main component of the vascular BM, is crucial for BM stability and assembly [12, 13]. We hypothesized that EIF5A2 may also affect tumor blood vessel wall remodeling or vessel structure via regulation of MMP-2 activity. Collagen IV and CD34 expression were examined by immunofluorescent staining of xenograft tissue sections. Compared with the control group, tumor micro-vessels in xenografts from EIF5A2 knockdown tumor cells exhibit more continuous and smooth wall, more regular endothelial lining, and morphology similar to normal vasculature (Fig. 4A). Similarly, vessel walls in EIF5A2 and MMP2 low expression human HCC tissue sections were more continuous (Fig. 4B, Supplementary Fig. 3). Transmission electron microscopy further revealed that the vessels in the EIF5A2 knockdown group were more likely to have continuous BMs, well differentiated endothelial cells, and same polarity in their endothelial linings (Fig. 4C). These data suggest that EIF5A2 ablation could induce formation of integrated, continuous vascular walls and normalized endothelial cells lining. Moreover, transmission electron microscopy indicated that the micro-vessels in EIF5A2 knockdown group displayed larger lumens and thinner walls which may enhance blood perfusion and facilitate nutrient and oxygen exchange.

Bottom Line: In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine; Sun Yat-sen University Cancer Center, Guangzhou, China. These authors contributed equally to this work.

ABSTRACT

Unlabelled: 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.

Show MeSH
Related in: MedlinePlus