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Supplementary Data from DDX3 Activates CBC-eIF3–Mediated Translation of uORF-Containing Oncogenic mRNAs to Promote Metastasis in HNSCC

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posted on 2023-03-31, 02:02 authored by Hung-Hsi Chen, Hsin-I Yu, Muh-Hwa Yang, Woan-Yuh Tarn

Figure S1. DDX3 expression is correlated with cell invasive activity and ATF4 expression. Figure S2. Microarray-identified genes are enriched for pro-metastatic genes and ATF4 targets, and DDX3 is required for migration and ATF4 expression in all of the HNSCC cell lines examined. Figure S3. DDX3 and ATF4 are required for EMT gene expression. Figure S4. DDX3 does not regulate ATF4 protein stability. Figure S5. DDX3 regulate ATF4 translation independent of ER Stress. Figure S6. The DDX3/CBC/eIF3 complex directly activates the translation of uORF-containing mRNAs in vitro and in vivo. Figure S7. RT-qPCR analysis of luciferase mRNAs in the in vivo translation assay. Figure S8. The CBC and eIF3 complexes are involved in the translation of uORF-containing mRNAs. Table S1. List of sense shRNA or siRNA sequences Table S2. Univariate and multivariate cox regression analyses in HNSCC patient samples. Table S3. List of genes that showed more than two-fold changes upon DDX3 knockdown. Table S4. GO terms enriched in DDX3 knockdown-upregulated or -downregulated genes analyzed by DAVID software. Table S5. List of upstream transcription factors potentially involved in DDX3-regulated genes predicted by IPA software. Table S6. Percentages of siDDX3-downregulated, DDX3-upregulated or non-target genes overlapping with uTIS or non-uTIS genes from TIS-db or with uORF-containing genes from uORFdb. Table S7. Percentages of CBP20 CLIPs or eIF3 CLIPs overlapping with DDX3 CLIPs or eIF4A3 CLIPs. Table S8. Percentages of DDX3 CLIPs, CBP20 CLIPs, eIF3 CLIPs or eIF4A3 CLIPs overlapping with uTIS or non-uTIS genes from TIS-db.

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Institute of Biomedical Sciences, Academia Sinica

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ARTICLE ABSTRACT

Mutated or dysregulated DDX3 participates in the progression and metastasis of cancer via its multiple roles in regulating gene expression and cellular signaling. Here, we show that the high expression levels of DDX3 in head and neck squamous cell carcinoma (HNSCC) correlate with lymph node metastasis and poor prognosis and demonstrate that DDX3 is essential for the proliferation, invasion, and metastasis of oral squamous cell carcinoma (OSCC) cells. Microarray analyses revealed that DDX3 is required for the expression of a set of pro-metastatic genes, including ATF4-modulated genes in an aggressive OSCC cell line. DDX3 activated translation of ATF4 and a set of its downstream targets, all of which contain upstream open reading frames (uORF). DDX3 promoted translation of these targets, likely by skipping the inhibitory uORF. DDX3 specifically enhanced the association of the cap-binding complex (CBC) with uORF-containing mRNAs and facilitated recruitment of the eukaryotic initiation factor 3 (eIF3). CBC and certain eIF3 subunits contributed to the expression of metastatic-related gene expression. Taken together, our results indicate a role for the novel DDX3–CBC–eIF3 translational complex in promoting metastasis.Significance: The discovery of DDX3-mediated expression of oncogenic uORF-containing genes expands knowledge on translational control mechanisms and provides potential targets for cancer therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4512/F1.large.jpg Cancer Res; 78(16); 4512–23. ©2018 AACR.