DDX3 regulates cancer immune surveillance via 3' UTR-mediated cell-surface expression of PD-L1.

Programmed death-1 (PD-1)/PD ligand-1 (PD-L1)-mediated immune escape contributes to cancer development and has been targeted as an anti-cancer strategy. Here, we show that inhibition of the RNA helicase DDX3 increased CD8+ T cell infiltration in syngeneic oral squamous cell carcinoma tumors. DDX3 knockdown compromised interferon-γ-induced PD-L1 expression and, in particular, reduced the level of cell-surface PD-L1. DDX3 promoted surface PD-L1 expression by recruiting the adaptor protein 2 (AP2) complex to the 3' UTR of PD-L1 mRNA. DDX3 depletion or 3' UTR truncation increased the binding of the coatomer protein complexes to PD-L1, leading to its intracellular accumulation. Therefore, this 3' UTR-dependent mechanism may counteract cellular negative effects on surface trafficking of PD-L1. Finally, pharmaceutic disruption of DDX3's interaction with AP2 reduced surface PD-L1 expression, supporting that the DDX3-AP2 pathway routes PD-L1 to the cell surface. Targeting DDX3 to modulate surface trafficking of immune checkpoint proteins may provide a potential strategy for cancer immunotherapy.

DDX3 modulates the tumor microenvironment via its role in endoplasmic reticulum-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.

DDX3 Activates CBC-eIF3-Mediated Translation of uORF-Containing Oncogenic mRNAs to Promote Metastasis in HNSCC.

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.

DDX3 Modulates Neurite Development via Translationally Activating an RNA Regulon Involved in Rac1 Activation.

UNLABELLED: The RNA helicase DDX3 is a component of neuronal granules, and its gene mutations are linked to intellectual disability (ID). Here we demonstrate that DDX3 depletion in neurons impairs neurite development by downregulating Rac1 level and activation. Moreover, DDX3 activates the translation of functionally coherent mRNAs involved in Rac1 activation including Rac1 Among the DDX3 regulon, Prkaca encodes the catalytic subunit of PKA, a potential activator of Rac1 in neurons. DDX3-modulated PKAcα and Rac1 expression tunes the strength of PKA-Rac1 signaling and thereby contributes to neurite outgrowth and dendritic spine formation. Inhibition of DDX3 activity or expression in neonatal mice impaired dendritic outgrowth and spine formation of hippocampal neurons, echoing neuronal deficits underling DDX3 mutation-associated ID. Finally, we provide evidence that DDX3 activates local protein synthesis through a 5' UTR-dependent mechanism in neurons. The novel DDX3 regulon may conduct a spatial and temporal control of Rac1 signaling to regulate neurite development. SIGNIFICANCE STATEMENT: DDX3X mutations are linked to intellectual disability (ID). We provide first evidence that DDX3 is required for neurite outgrowth and dendritic spine formation in vitro and in vivo We identified a DDX3 regulon constituting functionally cohesive mRNAs involved in Rac1 signaling, which contributes to DDX3-modulated neurite development. Inhibition or ablation of DDX3 in vivo shortened neurite lengths and impaired dendritic spine formation in hippocampal neurons, reflecting the prevalence of ID-associated DDX3X mutations in the helicase domain. Mechanistically, DDX3 activates local protein synthesis of mRNAs sharing similar 5' UTR structures and therefore controls Rac1 signaling strength in neurites.

RBM4 promotes neuronal differentiation and neurite outgrowth by modulating Numb isoform expression.

RBM4 participates in cell differentiation by regulating tissue-specific alternative pre-mRNA splicing. RBM4 also has been implicated in neurogenesis in the mouse embryonic brain. Using mouse embryonal carcinoma P19 cells as a neural differentiation model, we observed a temporal correlation between RBM4 expression and a change in splicing isoforms of Numb, a cell-fate determination gene. Knockdown of RBM4 affected the inclusion/exclusion of exons 3 and 9 of Numb in P19 cells. RBM4-deficient embryonic mouse brain also exhibited aberrant splicing of Numb pre-mRNA. Using a splicing reporter minigene assay, we demonstrated that RBM4 promoted exon 3 inclusion and exon 9 exclusion. Moreover, we found that RBM4 depletion reduced the expression of the proneural gene Mash1, and such reduction was reversed by an RBM4-induced Numb isoform containing exon 3 but lacking exon 9. Accordingly, induction of ectopic RBM4 expression in neuronal progenitor cells increased Mash1 expression and promoted cell differentiation. Finally, we found that RBM4 was also essential for neurite outgrowth from cortical neurons in vitro. Neurite outgrowth defects of RBM4-depleted neurons were rescued by RBM4-induced exon 9-lacking Numb isoforms. Therefore our findings indicate that RBM4 modulates exon selection of Numb to generate isoforms that promote neuronal cell differentiation and neurite outgrowth.

hnRNP Q regulates Cdc42-mediated neuronal morphogenesis.

The RNA-binding protein hnRNP Q has been implicated in neuronal mRNA metabolism. Here, we show that knockdown of hnRNP Q increased neurite complexity in cultured rat cortical neurons and induced filopodium formation in mouse neuroblastoma cells. Reexpression of hnRNP Q1 in hnRNP Q-depleted cells abrogated the morphological changes of neurites, indicating a specific role for hnRNP Q1 in neuronal morphogenesis. A search for mRNA targets of hnRNP Q1 identified functionally coherent sets of mRNAs encoding factors involved in cellular signaling or cytoskeletal regulation and determined its preferred binding sequences. We demonstrated that hnRNP Q1 bound to a set of identified mRNAs encoding the components of the actin nucleation-promoting Cdc42/N-WASP/Arp2/3 complex and was in part colocalized with Cdc42 mRNA in granules. Using subcellular fractionation and immunofluorescence, we showed that knockdown of hnRNP Q reduced the level of some of those mRNAs in neurites and redistributed their encoded proteins from neurite tips to soma to different extents. Overexpression of dominant negative mutants of Cdc42 or N-WASP compromised hnRNP Q depletion-induced neurite complexity. Together, our results suggest that hnRNP Q1 may participate in localization of mRNAs encoding Cdc42 signaling factors in neurites, and thereby may regulate actin dynamics and control neuronal morphogenesis.