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• W3198909712 abstract "•DDX3-AREG axis promotes cancer progression through microenvironment remodeling•DDX3 activates AREG translation via binding to its 3′ UTR•DDX3 interacts with the signal recognition particle (SRP)•DDX3-SRP-mediated mRNA recruitment assists ER-associated translation Using antibody arrays, we found that the RNA helicase DDX3 modulates the expression of secreted signaling factors in oral squamous cell carcinoma (OSCC) cells. Ribo-seq analysis confirmed amphiregulin (AREG) as a translational target of DDX3. AREG exerts important biological functions in cancer, including promoting cell migration and paracrine effects of OSCC cells and reprogramming the tumor microenvironment (TME) of OSCC in mice. DDX3-mediated translational control of AREG involves its 3′-untranslated region. Proteomics identified the signal recognition particle (SRP) as an unprecedented interacting partner of DDX3. DDX3 and SRP54 were located near the endoplasmic reticulum, regulated the expression of a common set of secreted factors, and were essential for targeting AREG mRNA to membrane-bound polyribosomes. Finally, OSCC-associated mutant DDX3 increased the expression of AREG, emphasizing the role of DDX3 in tumor progression via SRP-dependent, endoplasmic reticulum-associated translation. Therefore, pharmacological targeting of DDX3 may inhibit the tumor-promoting functions of the TME. Using antibody arrays, we found that the RNA helicase DDX3 modulates the expression of secreted signaling factors in oral squamous cell carcinoma (OSCC) cells. Ribo-seq analysis confirmed amphiregulin (AREG) as a translational target of DDX3. AREG exerts important biological functions in cancer, including promoting cell migration and paracrine effects of OSCC cells and reprogramming the tumor microenvironment (TME) of OSCC in mice. DDX3-mediated translational control of AREG involves its 3′-untranslated region. Proteomics identified the signal recognition particle (SRP) as an unprecedented interacting partner of DDX3. DDX3 and SRP54 were located near the endoplasmic reticulum, regulated the expression of a common set of secreted factors, and were essential for targeting AREG mRNA to membrane-bound polyribosomes. Finally, OSCC-associated mutant DDX3 increased the expression of AREG, emphasizing the role of DDX3 in tumor progression via SRP-dependent, endoplasmic reticulum-associated translation. Therefore, pharmacological targeting of DDX3 may inhibit the tumor-promoting functions of the TME. Head and neck cancers encompass a heterogeneous group of tumors in the upper aerodigestive tract; the main type is oral squamous cell carcinomas (OSCCs), which arise primarily in the oral cavity (Peltanova et al., 2019Peltanova B. Raudenska M. Masarik M. Effect of tumor microenvironment on pathogenesis of the head and neck squamous cell carcinoma: a systematic review.Mol. Cancer. 2019; 18: 63Crossref PubMed Scopus (101) Google Scholar). OSCC has a propensity to metastasize through lymphatics to regional lymph nodes. Cancer cells establish a myriad of interactions with components of their microenvironment, including vascular endothelial cells, infiltrating immune cells, and fibroblasts. The tumor microenvironment (TME) greatly influences cancer cell growth and invasion, immunogenicity, and even drug resistance. Via a complex network that includes secreted growth factors/cytokines and the extracellular matrix, cancer cells modify their stromal neighbors, which in turn impact tumor growth, metastasis, and response to therapy. Changes in the TME and immune surveillance represent a crucial hallmark of various types of cancer including OSCC (Eckert et al., 2016Eckert A.W. Wickenhauser C. Salins P.C. Kappler M. Bukur J. Seliger B. Clinical relevance of the tumor microenvironment and immune escape of oral squamous cell carcinoma.J. Transl. Med. 2016; 14: 85Crossref PubMed Scopus (52) Google Scholar). Understanding how cancer cells produce factors that exert autocrine and paracrine effects on cancer progression is important for the development of targeted therapies. Cancer cells have an increased demand for mRNA translational control to augment global or selective protein expression for rapid cell growth and quick adaption to environmental stresses (Robichaud et al., 2018Robichaud N. Sonenberg N. Ruggero D. Schneider R.J. Translational control in cancer.Cold Spring Harb Perspect. Biol. 2018; 11: a032896Google Scholar). A number of the DEAD/H-box RNA helicases including DDX3 contribute to translation control in cancer cells (Sharma and Jankowsky, 2014Sharma D. Jankowsky E. The Ded1/DDX3 subfamily of DEAD-box RNA helicases.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 343-360Crossref PubMed Scopus (82) Google Scholar). By means of its RNA helicase activity, DDX3 promotes the translation of mRNAs containing a long or structured 5′ untranslated region (UTR), as is the case for many oncogene and or chemokine transcripts (Lai et al., 2008Lai M.C. Lee Y.H. Tarn W.Y. The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control.Mol. Biol. Cell. 2008; 19: 3847-3858Crossref PubMed Scopus (151) Google Scholar; Soto-Rifo et al., 2012Soto-Rifo R. Rubilar P.S. Limousin T. de Breyne S. Decimo D. Ohlmann T. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs.EMBO J. 2012; 31: 3745-3756Crossref PubMed Scopus (154) Google Scholar). Accordingly, depletion of DDX3 results in cell cycle arrest and reduced migration capacity of cancer cells and impairs the migration and phagocytosis of macrophages (Chen et al., 2015Chen H.H. Yu H.I. Cho W.C. Tarn W.Y. DDX3 modulates cell adhesion and motility and cancer cell metastasis via Rac1-mediated signaling pathway.Oncogene. 2015; 34: 2790-2800Crossref PubMed Scopus (78) Google Scholar; Ku et al., 2019Ku Y.C. Lai M.H. Lo C.C. Cheng Y.C. Qiu J.T. Tarn W.Y. Lai M.C. DDX3 participates in translational control of inflammation induced by infections and injuries.Mol. Cell Biol. 2019; 39: e00285-18Crossref PubMed Scopus (10) Google Scholar; Lai et al., 2010Lai M.C. Chang W.C. Shieh S.Y. Tarn W.Y. DDX3 regulates cell growth through translational control of cyclin E1.Mol. Cell Biol. 2010; 30: 5444-5453Crossref PubMed Scopus (107) Google Scholar). DDX3 also participates in internal ribosome entry site-mediated translation of several viral and cellular mRNAs (Geissler et al., 2012Geissler R. Golbik R.P. Behrens S.E. The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes.Nucl. Acids Res. 2012; 40: 4998-5011Crossref PubMed Scopus (53) Google Scholar; Han et al., 2020Han S. Sun S. Li P. Liu Q. Zhang Z. Dong H. Sun M. Wu W. Wang X. Guo H. Ribosomal protein L13 promotes IRES-driven translation of foot-and-mouth disease virus in a helicase DDX3-dependent manner.J. Virol. 2020; 94 (e01679-19)Crossref Scopus (14) Google Scholar; Phung et al., 2019Phung B. Ciesla M. Sanna A. Guzzi N. Beneventi G. Cao Thi Ngoc P. Lauss M. Cabrita R. Cordero E. Bosch A. et al.The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma.Cell Rep. 2019; 27: 3573-3586.e7Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Via this mechanism, DDX3 promotes the translation of microphthalmia-associated transcription factor (MITF) in melanoma cells (Phung et al., 2019Phung B. Ciesla M. Sanna A. Guzzi N. Beneventi G. Cao Thi Ngoc P. Lauss M. Cabrita R. Cordero E. Bosch A. et al.The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma.Cell Rep. 2019; 27: 3573-3586.e7Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Moreover, our previous report revealed that DDX3 promotes the translation of a set of stress response oncogenic factors, including activating transcription factor 4 (ATF4), by counteracting the suppressive effect of upstream open reading frames and hence increases cancer cell invasion. Moreover, DDX3 can facilitate the assembly of functional 80S ribosomes independently of its ATPase activity (Geissler et al., 2012Geissler R. Golbik R.P. Behrens S.E. The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes.Nucl. Acids Res. 2012; 40: 4998-5011Crossref PubMed Scopus (53) Google Scholar). Besides its role in translational control, DDX3 also participates in transcriptional activation and cellular signaling (Sharma and Jankowsky, 2014Sharma D. Jankowsky E. The Ded1/DDX3 subfamily of DEAD-box RNA helicases.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 343-360Crossref PubMed Scopus (82) Google Scholar). Genetic mutations or dysregulation of DDX3 has been found in various types of cancer (Bol et al., 2015Bol G.M. Xie M. Raman V. DDX3, a potential target for cancer treatment.Mol. Cancer. 2015; 14: 188Crossref PubMed Scopus (72) Google Scholar). We previously reported that upregulation of DDX3 is associated with poor survival of patients with head and neck squamous cell carcinoma (HNSCC) (Chen et al., 2018Chen H.H. Yu H.I. Yang M.H. Tarn W.Y. DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in HNSCC.Cancer Res. 2018; 78: 4512-4523Crossref PubMed Scopus (29) Google Scholar). DDX3 is detected in the nucleus of normal cells but localizes predominantly in the cytoplasm of OSCC cells. Cytoplasmic localization of DDX3 implies a high demand of DDX3-mediated translational control in cancer cells. DDX3 is essential for the expression of ATF4 in OSCC cells, which promotes cell migration and invasion (Chen et al., 2018Chen H.H. Yu H.I. Yang M.H. Tarn W.Y. DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in HNSCC.Cancer Res. 2018; 78: 4512-4523Crossref PubMed Scopus (29) Google Scholar). In this study, we first observed that DDX3 was essential for the paracrine activity of OSCC cells. This finding promoted us to investigate whether DDX3 may regulate secretory pathways via translational control. There is a role for DDX3 in promoting the translation of mRNAs that contain suppressive upstream open reading frames (Chen et al., 2018Chen H.H. Yu H.I. Yang M.H. Tarn W.Y. DDX3 activates CBC-eIF3-Mediated translation of uORF-containing oncogenic mRNAs to promote metastasis in HNSCC.Cancer Res. 2018; 78: 4512-4523Crossref PubMed Scopus (29) Google Scholar). Hence, a high level of DDX3 in cancer cells increases the expression of the stress response protein activating transcription factor 4 (ATF4) and thereby promotes metastasis. Here, we report that DDX3 regulates the translation of mRNAs encoding secreted signaling factors. Secreted and membrane proteins are synthesized by endoplasmic reticulum (ER)-bound ribosomes and subsequently pass through the secretory pathway to their final destinations. During translation, the signal recognition particle (SRP) recognizes their N-terminal signal peptide emerging from the ribosome and leads ribosome-bound mRNAs to the translocation complex at the ER surface (Zhang and Shan, 2014Zhang X. Shan S.O. Fidelity of cotranslational protein targeting by the signal recognition particle.Annu. Rev. Biophys. 2014; 43: 381-408Crossref PubMed Scopus (40) Google Scholar). The nascent polypeptide is subsequently translocated into the ER lumen through the translocon. Signal peptide-independent pathways for targeting transcripts to the ER have also been identified (Chartron et al., 2016Chartron J.W. Hunt K.C. Frydman J. Cotranslational signal-independent SRP preloading during membrane targeting.Nature. 2016; 536: 224-228Crossref PubMed Scopus (80) Google Scholar). ER membrane-associated RNA-binding proteins (RBPs), such as yeast She2p and mammalian p180 and AEG-1, can anchor those transcripts to the ER (Cui et al., 2012Cui X.A. Zhang H. Palazzo A.F. p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum.PLoS Biol. 2012; 10: e1001336Crossref PubMed Scopus (73) Google Scholar; Genz et al., 2013Genz C. Fundakowski J. Hermesh O. Schmid M. Jansen R.P. Association of the yeast RNA-binding protein She2p with the tubular endoplasmic reticulum depends on membrane curvature.J. Biol. Chem. 2013; 288: 32384-32393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar; Hsu et al., 2018Hsu J.C. Reid D.W. Hoffman A.M. Sarkar D. Nicchitta C.V. Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.RNA. 2018; 24: 688-703Crossref PubMed Scopus (19) Google Scholar), but how these RBPs select mRNAs is unclear. A type of secretion-enhancing cis-regulatory targeting element has been identified in yeast secreted/membrane protein-encoding mRNAs; such elements may stabilize target mRNAs and facilitate their translation and subsequent translocation and secretion (Cohen-Zontag et al., 2019Cohen-Zontag O. Baez C. Lim L.Q.J. Olender T. Schirman D. Dahary D. Pilpel Y. Gerst J.E. A secretion-enhancing cis regulatory targeting element (SECReTE) involved in mRNA localization and protein synthesis.PLoS Genet. 2019; 15: e1008248Crossref PubMed Scopus (7) Google Scholar). A recent study found that the ER-associated RBP, TIS11B, forms reticular granules that enrich the transcripts containing AU-rich elements in the 3′ UTR. HuR binds to such elements and recruits an effector protein for efficient transport of nascent membrane proteins through the secretory pathway to the plasma membrane (Berkovits and Mayr, 2015Berkovits B.D. Mayr C. Alternative 3' UTRs act as scaffolds to regulate membrane protein localization.Nature. 2015; 522: 363-367Crossref PubMed Scopus (207) Google Scholar; Ma and Mayr, 2018Ma W. Mayr C. A membraneless organelle associated with the endoplasmic reticulum enables 3'UTR-mediated protein-protein interactions.Cell. 2018; 175: 1492-1506.e19Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The details of how different RBPs modulate the translation of different sets of ER-targeted mRNAs also remain to be investigated. In this study, we began to investigate the role of DDX3 in regulating secretory pathways in OSCC. We then found that DDX3 regulated the translation of secreted signaling factors and further explored the underlying mechanism and biological significance. We have previously reported that DDX3 is essential for cell migration and invasion of SAS cells (an aggressive OSCC cell line), but whether it can influence the autocrine and paracrine signaling activity has not been characterized. In this study, we initially observed that SAS cells exhibited morphological alteration when cultured in SAS cell-derived conditioned medium (CM) (Figure 1A). However, such a change was not observed when using CM from SAS cells that had been transfected with DDX3 targeting siRNA (Figure 1A). This result indicated that DDX3 was essential for the paracrine signaling effect of SAS cells. To explore whether DDX3 modulates the expression of secreted signaling factors, we first used antibody arrays to examine the changes in protein levels of 60 cytokines/growth factors upon DDX3 depletion. In DDX3-knockdown SAS cells, the signals for 15 spots were decreased by ≥ 2-fold, whereas one spot was increased (Figure 1B). Meanwhile, we performed ribosome profiling (Ribo-seq) in DDX3-depleted SAS cells to determine whether DDX3 regulates the translation of any secreted factor identified above (Figure 1C). RNA sequencing (RNA-seq) revealed that knockdown of DDX3 altered the expression level of ∼2.5% of all identified transcripts. To reveal translational targets of DDX3, Ribo-seq read counts were normalized to those of RNA-seq for each gene. Hence, the translation of ∼4% of identified transcripts could potentially be affected by DDX3. Among the factors identified in the above antibody arrays, we confirmed that the translation of amphiregulin (AREG) was prominently downregulated upon DDX3 depletion (Figure 1D), whereas many other factors had very low read counts, so we were unable to discern whether their translation is modulated by DDX3. Nevertheless, except for AREG, Ribo-seq also revealed additional growth factors or cytokines as the translational targets of DDX3, including neuregulin-1 (NRG1), endothelin-1 (EDN1), etc. (Table S1). We focused on AREG because it was identified in both antibody arrays and Ribo-seq analysis and has been considered a marker for poor prognosis of patients with HNSCC (Figure 1E) according to The Human Protein Atlas database. Using immunoblotting and ELISA, we confirmed that DDX3 knockdown reduced AREG expression in the cell lysate and culture medium of several OSCC cells including SAS cells (Figures 1F and 1G). Moreover, immunoblotting also confirmed that DDX3 regulated the expression of several other growth factors and immunoregulatory factors (Figure S1B), indicating an unprecedented role for DDX3 in regulating the expression of secreted signaling factors. Next, we investigated whether DDX3-regulated AREG expression has any functional effect on OSCC. Although AREG promotes cell proliferation and migration in various cancers (Berasain and Avila, 2014Berasain C. Avila M.A. Amphiregulin.Semin. Cell Dev. Biol. 2014; 28: 31-41Crossref PubMed Scopus (134) Google Scholar; Busser et al., 2011Busser B. Sancey L. Brambilla E. Coll J.L. Hurbin A. The multiple roles of amphiregulin in human cancer.Biochim. Biophys. Acta. 2011; 1816: 119-131PubMed Google Scholar), overexpression of AREG in SAS cells, however, had no effect on cell proliferation (Figure S2A). Perhaps, the level of endogenous AREG was sufficient for optimal proliferation of SAS cells. Nevertheless, AREG overexpression induced mesenchymal cell-like membrane protrusions in SAS cells (Figure 2A), as observed in Figure 1A, whereas knockdown of AREG using short hairpin (sh) RNA abolished such a morphological change (Figure S2B). Recombinant AREG (rAREG) treatment also induced mesenchymal cell-like morphology (Figure S2C) and upregulated mesenchymal proteins, including vimentin (VIM), SLUG (SNAI2), actin alpha 2 (ATCA2), and fibroblast activation protein (FAP) in SAS cells and three other OSCC cell lines (GNM, HSC3, and OECM1) (Figure S2D). Moreover, we observed that CM derived from SAS cells could promote cell migration, whereas CM from DDX3-depleted SAS cells lost such an activity (Figure 2B, CM/shC and CM/shD). Supplement of rAREG into DDX3-depleted SAS CM restored cell migration (Figure 2B, CM/shD + rAREG). Therefore, DDX3-regulated AREG exerts an autocrine activity in promoting cell migration. Next, we evaluated whether DDX3-regulated AREG has a paracrine role. To analyze angiogenesis, we characterized capillary-like tube formation ability of human endothelial EA.hy926 cells. As above, we evaluated CM from control (CM/shC) or DDX3-depleted SAS cells (CM/shD) and AREG-supplemented CM from DDX3-depleted cells (CM/shD + AREG). SAS cell CM efficiently induced tube formation of human endothelial EA.hy926 cells, whereas knockdown of DDX3 abolished this activity (Figure 2C). Moreover, AREG antibody-treated CM also inhibited tube formation of EA.hy926 cells (Figure S2E), suggesting that AREG exerts an angiogenic effect through a paracrine pathway. rAREG restored the angiogenesis-promoting activity of CM derived from DDX3 knockdown cells (Figure 2C). Therefore, the DDX3-AREG axis likely contributes to tumor-induced angiogenesis. Tumor-associated macrophages are abundant tumor-infiltrating immune cells in the TME and contribute to lymph node metastases and poor prognosis of OSCC (Weber et al., 2016Weber M. Iliopoulos C. Moebius P. Buttner-Herold M. Amann K. Ries J. Preidl R. Neukam F.W. Wehrhan F. Prognostic significance of macrophage polarization in early stage oral squamous cell carcinomas.Oral Oncol. 2016; 52: 75-84Crossref PubMed Google Scholar). We first examined whether DDX3 modulates macrophage differentiation of human monocytic THP-1 cells. SAS cell CM induced the expression of macrophage marker genes (IL1B, IL23A, MAF, and VEGFA) in THP-1 cells (Figure 2D, CM/shC); knockdown of DDX3 or AREG reduced this activity by ∼50–80% (CM/shD and CM/shAREG). However, the observation that rAREG was insufficient to induce macrophage gene expression indicated that AREG-induced factors in SAS cells rather than AREG itself promoted macrophage differentiation (Figure S2F). Because M2-polarized tumor-associated macrophages in general promote tumorigenesis, we evaluated whether the DDX3-AREG pathway promotes M2 differentiation. We first observed that SAS cell CM could induce M2 marker (CD163, IL10, and MRC1) expression in phorbol ester-primed THP-1 cells in a DDX3-dependent manner (Figure S2G). On the other hand, phorbol ester-primed THP-1 cells expressed a higher level of M1 markers (HLADR, IL12B, and IL18) in DDX3 knockdown SAS CM than that of control CM (Figure S2H). Because M2 macrophages have angiogenic potential, we then functionally examined whether SAS CM-primed THP-1 cells could induce angiogenesis (Figure 2E, diagram). Indeed, CM of the aforementioned primed THP-1 cells efficiently induced tubularization of EA.hy926 cells, whereas the CM of THP-1 that had been cultured in DDX3-depleted SAS CM was incapable of doing so (Figure 2E). This result implied that the DDX3-AREG axis can potentiate THP-1 differentiation into M2 macrophages. Next, we investigated whether the DDX3-AREG axis influences the TME in vivo using an allograft model. We evaluated several mouse OSCC cell lines, which were established from carcinogen-treated transgenic K14-EGFP-miR-211 C57BL/6 mice (Chen et al., 2019bChen Y.F. Liu C.J. Lin L.H. Chou C.H. Yeh L.Y. Lin S.C. Chang K.W. Establishing of mouse oral carcinoma cell lines derived from transgenic mice and their use as syngeneic tumorigenesis models.BMC Cancer. 2019; 19: 281Crossref PubMed Scopus (10) Google Scholar). MOC-L1 was selected because this cell line was found to highly express AREG and potently activate macrophage differentiation (Figures S3A and S3B). Knockdown of DDX3 in MOC-L1 cells reduced both proliferation and migration (Figures S3C and S3D) and attenuated the ability of CM to induce tube formation and macrophage gene expression, as observed in DDX3-depleted SAS cells (Figures S3E and S3F). Next, DDX3-depleted MOC-L1 cells or control cells were transplanted into C57BL/6 mice (Figure 3A). Efficient depletion of DDX3 as well as AREG in siDDX3-transfected MOC-L1 tumors was reproducibly observed even at two weeks after transfection (Figure 3B). Control MOC-L1 tumors were larger in average (Figure 3C, siD#1) and had visible blood vessels compared with DDX3 knockdown tumors (Figure 3D). Administration of DDX3 knockown-MOC-L1 tumors with recombinant mouse AREG (rAREGm) restored the vasculature and tumor size (Figures 3C and 3D, siD#1 + rAREGm). Cell growth assays indicated that the DDX3-AREG axis did not directly affect cell proliferation in vitro (Figures S3G–S3I). Using periodic acid-Schiff to stain blood vessel, we confirmed the above observation on angiogenesis (Figure 3D, PAS). Immunofluorescence staining for the macrophage marker F4/80 revealed that control MOC-L1 tumors had a higher degree of macrophage infiltration than did DDX3-depleted tumors, and rAREGm treatment increased the intensity of macrophage staining in DDX3-depleted tumors (Figure 3D, F4/80). In contrast, immunostaining for the T-cell activation marker granzyme B indicated that infiltration of cytotoxic immune cells was exaggerated in DDX3-depleted tumors compared with the control, and rAREGm treatment attenuated such infiltration (Figure 3D, Granzyme B). This result indicated that the DDX3-AREG axis promoted macrophage infiltration and suppressed cytotoxic immunosurveillance in OSCC. Together, DDX3 exerts several tumor-promoting activities by regulating the expression of AREG (Figure 3E). The above result confirmed the important biological effect of the DDX3-AREG axis in tumor progression and microenvironmental remodeling. Next, we investigated the mechanism underlying how DDX3 may regulate the translation of AREG. We constructed humanized Renilla luciferase reporters containing either the 5′ or 3′ UTR or both UTRs of AREG (Figure 4A). After normalization with the control firefly luciferase, we observed that the 5′ UTR and 3’ UTR, respectively, suppressed and enhanced reporter translation (Figure 4B, siC). Knockdown of DDX3 decreased the translation of all the reporters without affecting their mRNA levels, indicating that both the 5′ and -3′ UTRs of AREG exert DDX3-dependent translation control (Figures 4B and S4A). RNAfold analysis revealed that the 5′ UTR forms extensive stem-loop structures (Figure S4B). Therefore, we tested the effect of wild-type and ATP hydrolysis (K230E) or RNA unwinding (S382L)-defective DDX3 (Yedavalli et al., 2004Yedavalli V.S. Neuveut C. Chi Y.H. Kleiman L. Jeang K.T. Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function.Cell. 2004; 119: 381-392Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) in the translation of the 5′ UTR reporter in DDX3-depleted SAS cells. The result showed that only the wild type but not mutant DDX3 could moderately restore 5′ UTR-mediated reporter expression likely via the translational control (Figures 4C and S4C, 5′), indicating its activity in resolving secondary structures of the 5′ UTR. To our surprise, those two mutants were able to restore 3′ UTR reporter translation without affecting reporter mRNA level (Figures 4C and S4C, 3′) and AREG expression (Figure 4D). To further evaluate whether these two activities are dispensable for 3′ UTR-mediated translation, we tested the double mutant (S382L; K230E). This mutant rescued AREG expression in DDX3-depleted cells (Figure 4D, lane 5), emphasizing that DDX3 participates in 3′ UTR-mediated translation in an RNA helicase-independent manner. Therefore, DDX3 may regulate AREG mRNA translation via at least two distinct mechanisms (Figure 4E). This unprecedented and intriguing result prompted us to investigate the underlying mechanism. The above result that DDX3 may regulate the translation of AREG via 3′ UTR-mediated translation control prompted us to identify its interacting partners that participate in the expression of secreted proteins. We performed immunoprecipitation of endogenous DDX3 from SAS cells and analyzed the coprecipitates using mass spectrometry. After RNase digestion, coprecipitates were fractionated by electrophoresis. Thirteen visible bands were subjected to mass spectrometry analysis (Figure S5A). In total, 444 proteins were identified, and the 10 non-redundant proteins with the highest scores for each band are listed in Table S2. Gene Ontology analysis revealed enrichment of DDX3-interacting proteins in translation initiation and SRP-dependent cotranslational protein targeting to the membrane (Table S3). Using the technique “stable isotope labeling by amino acids in cell culture” followed by Gene Ontology analysis, we identified a similar set of DDX3-interacting partners (Table S4). We knocked down 22 candidate genes that are involved in mRNA processing and translation using shRNA and observed that depletion of SRP components (SRP9 and 68) reduced the translation of the AREG 5’+3′ UTR reporter to a level comparable to that of DDX3 knockdown (Figure 5A). Further examination revealed that depletion of either SRP component compromised the translation but not mRNA level of the 3′ UTR reporter (Figures 5B and S5B), whereas it had no effect on the 5′ UTR reporter (Figure S5C). All these SRPs were also required for the expression of endogenous AREG (Figure 5C). We deduced that DDX3 acts in conjunction with the SRP in 3′ UTR-mediated translation control. In light of SRP54 as a key SRP factor (Wild et al., 2019Wild K. Juaire K.D. Soni K. Shanmuganathan V. Hendricks A. Segnitz B. Beckmann R. Sinning I. Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions.Nucl. Acids Res. 2019; 47: 3184-3196Crossref PubMed Scopus (11) Google Scholar), we evaluated its interaction with DDX3 and role in AREG expression. Immunoprecipitation of DDX3 or SRP54 from the SAS cell lysates followed by immunoblotting consistently revealed RNA-independent interaction between DDX3 and SRP54 (Figure 5D). Both of them also interacted with ribosomal protein RPL13, indicating their engagement in mRNA translation (Figure 5D). Indirect immunofluorescence revealed that endogenous DDX3 distributed to the regions surrounding the ER protein GRP94 and was located in proximity to SRP54 (Figures 5E and 5F), further supporting a role for DDX3 in ER-associated translation. Depletion of SRP54 also decreased the expression of DDX3-regulated secreted factors that we examined but had no effect on macrophage migration inhibitory factor (MIF) or E-cadherin (Figure 5G). Therefore, DDX3 likely controls the expression of secreted proteins in conjunction with the SRP via ER-associated translation. We hypothesized that DDX3 recruits the SRP to the 3′ UTR of mRNAs encoding secreted proteins and facilitates their translation at the ER membrane. To test this hypothesis, we performed a pull-down assay using biotinylated AREG 3′ UTR. The result showed that DDX3 and SRP54/68 interacted with the AREG 3′ UTR (Figure 6A, lane 9). Depletion of DDX3 diminished the association of the SRP proteins with the AREG 3′ UTR (lane 10). Interestingly, depletion of either SRP protein also reduced the binding of DDX3 to the AREG 3′ UTR (lanes 11, 12), suggesting their interdependent association with target mRNAs. Using immunoprecipitation coupled with quantitative reverse transcription-PCR, we confirmed the association of DDX3 and SRP proteins with endogenous AREG mRNA in SAS cells (Figure 6B). Next, we evaluated whether DDX3 and/or SRP54/68 are essential for anchoring AREG mRNA on the membrane. Depletion of either factor re" @default.
• W3198909712 created "2021-09-13" @default.
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• W3198909712 date "2021-09-01" @default.
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• W3198909712 title "DDX3 modulates the tumor microenvironment via its role in endoplasmic reticulum-associated translation" @default.
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