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. 2019 May 14;38(1):196.
doi: 10.1186/s13046-019-1178-z.

Eukaryotic Initiation Factor 4A2 Promotes Experimental Metastasis and Oxaliplatin Resistance in Colorectal Cancer

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Free PMC article

Eukaryotic Initiation Factor 4A2 Promotes Experimental Metastasis and Oxaliplatin Resistance in Colorectal Cancer

Zhan-Hong Chen et al. J Exp Clin Cancer Res. .
Free PMC article

Abstract

Background: Deregulation of protein translation control is a hallmark of cancers. Eukaryotic initiation factor 4A2 (EIF4A2) is required for mRNA binding to ribosome and plays an important role in translation initiation. However, little is known about its functions in colorectal cancer (CRC).

Methods: Analysis of CRC transcriptome data from TCGA identified that EIF4A2 was associated with poor prognosis. Immunohistochemistry study of EIF4A2 was carried out in 297 paired colorectal tumor and adjacent normal tissue samples. In vitro and in vivo cell-biological assays were performed to study the biological functions of EIF4A2 on experimental metastasis and sensitivity to oxaliplatin treatment. Bioinformatic prediction, chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay were carried out to unveil the transcription factor of EIF4A2 regulation.

Results: EIF4A2 Expression is significantly higher in colorectal tumors. Multivariate analysis suggests EIF4A2 as an independent predictor of overall, disease-free and progression-free survival. Dysfunction of EIF4A2 by genetic knock-down or small-molecule inhibitor silvestrol dramatically inhibited CRC invasion and migration, sphere formation and enhanced sensitivity to oxaliplatin treatment in vitro and in vivo. Notably, EIF4A2 knock-down also suppressed lung metastasis in vivo. qRT-PCR and immunoblotting analyses identified c-Myc as a downstream target and effector of EIF4A2. ChIP and dual-luciferase reporter assays validated the bioinformatical prediction of ZNF143 as a specific transcription factor of EIF4A2.

Conclusions: EIF4A2 promotes experimental metastasis and oxaliplatin resistance in CRC. Silvestrol inhibits tumor growth and has synergistic effects with oxaliplatin to induce apoptosis in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models.

Keywords: Colorectal cancer; Eukaryotic initiation factor 4A2 (EIF4A2); PDX; Silvestrol; ZNF143.

Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the ethics committee of the Sun Yat-sen university cancer center (IRB number: GZR2017–057) and written informed consents were obtained from all patients. Our animal study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (L102012017004Y).

Consent for publication

All authors have agreed to publish this manuscript.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Expression of EIF4A2 is high in colorectal tumors and predicts poor survival of patients. a Data analysis of TCGA identified high EIF4A2 level was associated with poor prognosis in CRC patients. b, c The EIF4A2 mRNA level was significantly higher in CRC tissues than that of normal tissues in Notterman dataset and C-skyzypczak dataset from Oncomine (https://www.oncomine.com). d The EIF4A2 level was significantly higher in 152 human CRC tissues that of 72 normal tissues hospitalized in SYSUCC. e Representative images show low or high expression of EIF4A2 in CRC tumor tissues compared to adjacent normal tissues analyzed by IHC. The EIF4A2 expression was significantly higher in tumor tissues (**, P < 0.01). f The overall survival curve of 297 CRC patients with low and high expression of EIF4A2 were generated using the Kaplan-Meier method (log-rank test). g The disease-free survival curve of 245 CRC patients underwent curative surgery with low and high expression of EIF4A2 were generated using the Kaplan-Meier method (log-rank test)
Fig. 2
Fig. 2
Knocking-down EIF4A2 inhibits migration and invasion of CRC cells. a, b, c, d Knocking-down EIF4A2 significantly inhibited transwell migration and invasion in DLD1 and HCT116 cells. e Knocking-down EIF4A2 significantly reduced sphere formation in DLD1 and HCT116 cells. f Knocking-down EIF4A2 significantly reduced a panel of stemness-associated genes (NANOG, OCT-4, BMI-1, NOTCH-1, ALDH1 and SMO), surface antigens associated with cancer stem cells (CD24, CD44, CD105 and CD133) and cell surface transporter genes (ABCC2 and ABCG2) related to drug resistance in DLD1 and HCT116 cells. *, P < 0.05; **, P < 0.01 versus the control
Fig. 3
Fig. 3
Knocking-down EIF4A2 inhibits metastasis formation in the lung. a, b Knocking-down EIF4A2 significantly reduced numbers of mice with lung metastasis (a) and metastatic nodules (b). c Representative images of HE staining showed metastatic nodules. d RT-qPCR arrays were used to screen 84 metastasis-related genes and showed that C-MYC, KRAS and MTA1 mRNAs significantly reduced after knocking-down EIF4A2. e Western blots showed that c-Myc significantly decreased after knocking-down EIF4A2 stably, but MTA1 and KRAS remained unchanged. *, P < 0.05; **, P < 0.01 versus the control
Fig. 4
Fig. 4
Knocking-down EIF4A2 improves sensitivity of CRC cells to oxaliplatin. a, b IHC was performed to quantify the expression of EIF4A2 in 74 advanced CRC patients receiving oxaliplatin-based regimens as the first-line chemotherapy. Time to progression and overall survival were generated by the Kaplan-Meier method (log-rank test). c High EIF4A2 expression indicated poor response to oxaliplatin-based regimens. 36.8% of CRC patients with high EIF4A2 expression were evaluated as PD, while 11.1% of CRC patients with low EIF4A2 expression were PD. d EIF4A2 and C-MYC mRNA levels significantly increased in HCT116/OXA cells. e Western blots showed that EIF4A2 obviously increased in HCT116/OXA cells. f The half-maximal inhibitory concentration (IC50) of oxaliplatin significantly decreased in HCT116 and DLD1 cells stably knocking-down EIF4A2. g The apoptosis rate induced by oxaliplatin in HCT116 and DLD1 cells with EIF4A2 knockdown was significantly higher. h, i, k The shEIF4A2–1 stably transduced cells treated with oxaliplatin showed the most significant reduction in tumor weight and volume. j, l IHC staining showed that the number of KI67-positive cells decreased most significantly in the shEIF4A2–1 stably transduced cells treated with oxaliplatin. *, P < 0.05; **, P < 0.01 versus the control
Fig. 5
Fig. 5
EIF4A inhibitor Silvestrol inhibits tumor growth, sphere formation and induces apoptosis when combined with oxaliplatin in CRC cells. a MTT assays showed Viability of the DLD1 and HCT116 cells treated with silvestrol of different concentrations. b Images (left) and quantification (right) of the indicated cells treated with silvestrol in colony formation assays. c Quantification of cell apoptosis of the indicated cells treated with silvestrol in Annexin-V/propidium iodide (PI) assays. d Representative images (left) and quantification (right) of sphere formation of the indicated cells treated with silvestrol. *, P < 0.05; **, P < 0.01 versus the control
Fig. 6
Fig. 6
Silvestrol (SIL) inhibits tumor growth and has synergistic effects with oxaliplatin (OXA) in CDX and PDX. a, b Mice planted with indicated cancer cells or PDX were treated as indicated. The group treated with SIL and OXA together showed the most significant decrease in tumor volume and weight in the CDX (a) and PDX (b) models. c The mice treated with SIL + OXA combination treatment showed the most significant decrease of Ki67-positive cells in IHC staining. d The mice treated with SIL + OXA combination treatment showed the most significant increase of apoptotic cells in TUNEL assays. *, P < 0.05; **, P < 0.01 versus the control
Fig. 7
Fig. 7
Transcription of EIF4A2 is regulated by ZNF143. a Western blot analyses showed that EIF4A2 were significantly decreased after siZNF143–2 transfection in the indicated cells. b Level of ZNF143 was significantly higher in tumors than in normal tissues of TGCA CRC dataset. c A positive correlation was found between the mRNA levels of ZNF143 and EIF4A2. d Western blot analyses showed that c-Myc were significantly decreased after siZNF143–2 transfection in the indicated cells. e ChIP assays confirmed that ZNF143 could bind to the EIF4A2 promoter in DLD1 and HCT116 cells. Quantification of immunoprecipitated DNA was shown by RT-qPCR. f Luciferase assays showed that ZNF143 overexpression increased the luciferase activities driven by EIF4A2 promoter, while co-transfected point-mutated EIF4A2 promoter showed no response. g EIF4A2 promotes experimental metastasis and oxaliplatin resistance in CRC. ZNF143 is transcription factor of EIF4A2. Silvestrol inhibits tumor growth, invasion, migration, cancer stemness and improves oxaliplatin resistance. *, P < 0.05; **, P < 0.01 versus the control

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