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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 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).
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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).

Mentions: To further explore the mechanisms by which EIF5A2 regulates MMP-2 activity, we evaluated the effects of EIF5A2 knockdown or over-expression on the activation of signal transduction pathways. The MAPK signaling pathway regulates MMP2 expression depending on cellular context, and is mediated by p38 MAPK, ERK1/2, and JNK/c-Jun [14-17]. In agreement with previous findings, our study showed that the levels of phosphorylated ERK1/2 and p38 MAPK were dramatically decreased in both EIF5A2-silenced PLC8024 and Huh7 cells, but increased in EIF5A2-overexpressing SMMC7721 cells (Fig. 6A). Interestingly, phosphorylated JNK and c-Jun were also significantly decreased in EIF5A2-silenced PLC8024 cells, but only slight changes were observed in EIF5A2-silenced Huh7 and SMMC7721 cells ectopically expressing EIF5A2 (Fig. 6A). However, phosphorylated Akt levels remained unchanged in regardless of EIF5A2 expression. To further explore the role of p38 MAPK and ERK1/2 in the induction of MMP-2 activity by EIF5A2, siRNAs targeting to p38 MAPK and ERK1/2 were transfected into the EIF5A2-expressing SMMC7721 cells. MMP-2 activity in the EIF5A2-transfected SMMC7721 cells was shown to be decreased by siRNAs against p38 MAPK but not ERK1/2 (Fig. 6B). To examine whether the c-Jun participates in the induction of MMP-2 activity by EIF5A2, luciferase reporter vector containing the wild-type MMP-2 promoter and a deletion mutation of AP-1 binding site within the MMP-2 promoter (ΔAP-1-MMP-2) were transiently co-transfected with pRL-TK into PLC8024-NC and sh#3 cells or co-transfected with vector or EIF5A2 over-expression vector into SMMC7721 cells [18]. In contrast to the wild-type promoter reporter, the luciferase activity of ΔAP-1-MMP-2 was unchanged between PLC8024-NC and sh#3 cells (Fig. 6C). However, in SMMC7721 cells, EIF5A2 over-expression enhanced luciferase activity driven by the deletion mutant promoter (Fig. 6C). Moreover, the deletion of the AP-1 binding site significantly reduced reporter activity in both cell lines, which indicates that AP-1 is an important regulatory factor for MMP-2 activity (Fig. 6C). Taken together, these results indicated that the stimulation of MMP-2 by the EIF5A2 signaling pathway depended on specific cellular contexts. To further explore whether EIF5A2 regulates MMP-2 expression primarily at the transcriptional level, the MMP-2 3′-UTR (untranslated region) was cloned into the pMIR-REPORT luciferase reporter vector. Dual-luciferase reporter analysis showed that EIF5A2 silencing did not alter the activity of the MMP2 3′-UTR luciferase reporter, indicating that EIF5A2 may regulate MMP-2 gene expression by enhancing binding of transcription factors binding such as AP-1 at the MMP-2 promoter (Supplementary Fig. 4). However, the roles of other potential transcriptional factors in the induction of MMP2 by EIF5A2 still warrant further evaluation.


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 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).
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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).
Mentions: To further explore the mechanisms by which EIF5A2 regulates MMP-2 activity, we evaluated the effects of EIF5A2 knockdown or over-expression on the activation of signal transduction pathways. The MAPK signaling pathway regulates MMP2 expression depending on cellular context, and is mediated by p38 MAPK, ERK1/2, and JNK/c-Jun [14-17]. In agreement with previous findings, our study showed that the levels of phosphorylated ERK1/2 and p38 MAPK were dramatically decreased in both EIF5A2-silenced PLC8024 and Huh7 cells, but increased in EIF5A2-overexpressing SMMC7721 cells (Fig. 6A). Interestingly, phosphorylated JNK and c-Jun were also significantly decreased in EIF5A2-silenced PLC8024 cells, but only slight changes were observed in EIF5A2-silenced Huh7 and SMMC7721 cells ectopically expressing EIF5A2 (Fig. 6A). However, phosphorylated Akt levels remained unchanged in regardless of EIF5A2 expression. To further explore the role of p38 MAPK and ERK1/2 in the induction of MMP-2 activity by EIF5A2, siRNAs targeting to p38 MAPK and ERK1/2 were transfected into the EIF5A2-expressing SMMC7721 cells. MMP-2 activity in the EIF5A2-transfected SMMC7721 cells was shown to be decreased by siRNAs against p38 MAPK but not ERK1/2 (Fig. 6B). To examine whether the c-Jun participates in the induction of MMP-2 activity by EIF5A2, luciferase reporter vector containing the wild-type MMP-2 promoter and a deletion mutation of AP-1 binding site within the MMP-2 promoter (ΔAP-1-MMP-2) were transiently co-transfected with pRL-TK into PLC8024-NC and sh#3 cells or co-transfected with vector or EIF5A2 over-expression vector into SMMC7721 cells [18]. In contrast to the wild-type promoter reporter, the luciferase activity of ΔAP-1-MMP-2 was unchanged between PLC8024-NC and sh#3 cells (Fig. 6C). However, in SMMC7721 cells, EIF5A2 over-expression enhanced luciferase activity driven by the deletion mutant promoter (Fig. 6C). Moreover, the deletion of the AP-1 binding site significantly reduced reporter activity in both cell lines, which indicates that AP-1 is an important regulatory factor for MMP-2 activity (Fig. 6C). Taken together, these results indicated that the stimulation of MMP-2 by the EIF5A2 signaling pathway depended on specific cellular contexts. To further explore whether EIF5A2 regulates MMP-2 expression primarily at the transcriptional level, the MMP-2 3′-UTR (untranslated region) was cloned into the pMIR-REPORT luciferase reporter vector. Dual-luciferase reporter analysis showed that EIF5A2 silencing did not alter the activity of the MMP2 3′-UTR luciferase reporter, indicating that EIF5A2 may regulate MMP-2 gene expression by enhancing binding of transcription factors binding such as AP-1 at the MMP-2 promoter (Supplementary Fig. 4). However, the roles of other potential transcriptional factors in the induction of MMP2 by EIF5A2 still warrant further evaluation.

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