DDX3 is a DEAD box RNA helicase with diverse biological functions. Using colony formation assay, our results revealed that DDX3 inhibited the colony formation ability of various tumor cells, and this inhibition might be due to a reduced growth rate caused by DDX3. Additionally, we identified p21 waf1/cip1 , a cyclin-dependent kinase inhibitor, as a target gene of DDX3, and the up-regulation of p21 waf1/cip1 expression accounted for the colony-suppressing activity of DDX3. Moreover, DDX3 exerted its transactivation function on p21 waf1/cip1 promoter through an ATPase-dependent but helicase-independent mechanism, and the four Sp1 sites located within the À123 to À63 region, relative to the transcription start site of p21 waf1/cip1 promoter, were essential for the response to DDX3. Furthermore, DDX3 interacted and cooperated with Sp1 to up-regulate the promoter activity of p21 waf1/cip1 . To determine the relevance of DDX3 in clinical cancers, the expression profile of DDX3 in various tumors was also examined. A declined expression of DDX3 mRNA and protein was found in f58% to 73% of hepatoma specimens, which led to the reduction of p21 waf1/cip1 expression in a manner independent of p53 status. Additionally, an alteration of subcellular localization from nuclei to cytoplasm was also observed in >70% of cutaneous squamous cell carcinoma samples. Because DDX3 exhibits tumor suppressor functions, such as a growth-suppressive property and transcriptional activation of the p21 waf1/cip1 promoter, and is inactivated through down-regulation of gene expression or alteration of subcellular localization in tumor cells, all these features together suggest that DDX3 might be a candidate tumor suppressor. (Cancer Res 2006; 66(13): 6579-88)

DDX3 is a human RNA helicase with plethoric functions. Our previous studies have indicated that DDX3 is a transcriptional regulator and functions as a tumor suppressor. In this study, we use a bicistronic reporter to demonstrate that DDX3 specifically represses cap-dependent translation but enhances hepatitis C virus internal ribosome entry site-mediated translation in vivo in a helicase activity-independent manner. To elucidate how DDX3 modulates translation, we identified translation initiation factor eukaryotic initiation factor 4E (eIF4E) as a DDX3-binding partner. Interestingly, DDX3 utilizes a consensus eIF4E-binding sequence YIPPHLR to interact with the functionally important dorsal surface of eIF4E in a similar manner to other eIF4E-binding proteins. Furthermore, cap affinity chromatography analysis suggests that DDX3 traps eIF4E in a translationally inactive complex by blocking interaction with eIF4G. Point mutations within the consensus eIF4E-binding motif in DDX3 impair its ability to bind eIF4E and result in a loss of DDX3's regulatory effects on translation. All these features together indicate that DDX3 is a new member of the eIF4E inhibitory proteins involved in translation initiation regulation. Most importantly, this DDX3-mediated translation regulation also confers the tumor suppressor function on DDX3. Altogether, this study demonstrates regulatory roles and action mechanisms for DDX3 in translation, cell growth and likely viral replication.

Upon environmental insults, SGs (stress granules) aid cell survival by serving as sites of translational silencing. RNA helicase DDX3 was reported to associate with SGs. However, its role in SG physiology remains undefined. We have demonstrated previously that DDX3 acts as an eIF4E (eukaryotic initiation factor 4E)-inhibitory protein to suppress translation. In the present study, we indentified the SG marker PABP1 [poly(A)-binding protein 1] as another direct interaction partner of DDX3. We established various stimuli as novel stressors that direct DDX3 with eIF4E and PABP1 into SGs, but not to processing bodies. Interestingly, down-regulation of DDX3 interfered with SG assembly, led to nuclear accumulation of PABP1 and reduced cell viability following stress. Conversely, supplementation with a shRNA (short hairpin RNA)-resistant DDX3 restored SG formation, the translocation of PABP1 into SGs and cell survival. Notably, the SG-inducing capacity of DDX3 is independent of its ATPase and helicase activities, but mapped to the eIF4E-binding region. Moreover, the eIF4E-binding-defective mutant DDX3 was impaired in its SG-inducing ability and protective effect on cell survival under adverse conditions. All together, the present study has characterized DDX3 as a pivotal SG-nucleating factor and illustrates co-ordinative roles for DDX3, eIF4E and PABP1 in integrating environmental stress with translational regulation.

Long noncoding RNAs (lncRNAs) have been implicated in hypoxia/HIF-1-associated cancer progression through largely unknown mechanisms. Here we identify MIR31HG as a hypoxia-inducible lncRNA and therefore we name it LncHIFCAR (long noncoding HIF-1α co-activating RNA); we describe its oncogenic role as a HIF-1α co-activator that regulates the HIF-1 transcriptional network, crucial for cancer development. Extensive analyses of clinical data indicate LncHIFCAR level is substantially upregulated in oral carcinoma, significantly associated with poor clinical outcomes and representing an independent prognostic predictor. Overexpression of LncHIFCAR induces pseudo-hypoxic gene signature, whereas knockdown of LncHIFCAR impairs the hypoxia-induced HIF-1α transactivation, sphere-forming ability, metabolic shift and metastatic potential in vitro and in vivo. Mechanistically, LncHIFCAR forms a complex with HIF-1α via direct binding and facilitates the recruitment of HIF-1α and p300 cofactor to the target promoters. Our results uncover an lncRNA-mediated mechanism for HIF-1 activation and establish the clinical values of LncHIFCAR in prognosis and potential therapeutic strategy for oral carcinoma.