DDX56 modulates post-transcriptional Wnt signaling through miRNAs and is associated with early recurrence in squamous cell lung carcinoma.

BACKGROUND: Early recurrence is a major obstacle to prolonged postoperative survival in squamous cell lung carcinoma (SqCLC). The molecular mechanisms underlying early SqCLC recurrence remain unclear, and effective prognostic biomarkers for predicting early recurrence are needed. METHODS: We analyzed primary tumor samples of 20 SqCLC patients using quantitative proteomics to identify differentially-expressed proteins in patients who experienced early versus late disease recurrence. The expression and prognostic significance of DDX56 was evaluated using a SqCLC tumor tissue microarray and further verified using different online databases. We performed in vitro and in vivo experiments to obtain detailed molecular insight into the functional role of DDX56 in SqCLC. RESULTS: We found that DDX56 exhibited increased expression in tumors of patients who experienced early versus late disease recurrence. Increased DDX56 expression in SqCLC tumors was subsequently confirmed as an independent prognostic factor of poor recurrence-free survival in independent SqCLC cohorts. Functionally, DDX56 promotes SqCLC cell growth and migration in vitro, and xenograft tumor progression in vivo. Mechanistically, DDX56 post-transcriptionally promotes expression of multiple Wnt signaling pathway-related genes, including CTNNB1, WNT2B, and represses a subset of miRNAs, including miR-378a-3p, a known suppressor of Wnt signaling. Detailed analysis revealed that DDX56 facilitated degradation of primary miR-378a, leading to down-regulation of mature miR-378a-3p and thus derepression of the target gene WNT2B. CONCLUSION: We identified DDX56 as a novel independent prognostic biomarker that exerts its oncogenic effects through miRNA-mediated post-transcriptional regulation of Wnt signaling genes to promote early SqCLC recurrence. DDX56 may assist in identifying SqCLC patients at increased risk of early recurrence and who could benefit from Wnt signaling-targeted therapies.

DDX56 inhibits type I interferon by disrupting assembly of IRF3-IPO5 to inhibit IRF3 nucleus import.

Transcription factor IRF3-mediated type I interferon induction plays a role in antiviral innate immunity. However, mechanisms for the control and regulation of IRF3 nuclear import remain largely unknown. We have identified DEAD box polypeptide 56 (DDX56) as a negative regulator of virus-triggered IFN-ß induction. Overexpression of DDX56 suppressed nuclear translocation of IRF3 via disrupting the IRF3-IOP5 interaction, whereas knockdown or knockout of DDX56 had the opposite effect. In addition, the interaction between DDX56 and IRF3 increased during viral infection. We further found that the D166 site of DDX56 was essential for inhibiting IRF3 import into the nucleus. Our findings suggest that DDX56 regulates antiviral innate immunity by inhibiting the nuclear translocation of IRF3, revealing a novel mechanism of the DDX56-mediated innate antiviral response.This article has an associated First Person interview with the first author of the paper.

Oncogenic splicing abnormalities induced by DEAD-Box Helicase 56 amplification in colorectal cancer.

Alternative splicing, regulated by DEAD-Box Helicase (DDX) families, plays an important role in cancer. However, the relationship between the DDX family and cancer has not been fully elucidated. In the present study, we identified a candidate oncogene DDX56 on Ch.7p by a bioinformatics approach using The Cancer Genome Atlas (TCGA) dataset of colorectal cancer (CRC). DDX56 expression was measured by RT-qPCR and immunochemical staining in 108 CRC patients. Clinicopathological and survival analyses were carried out using three CRC datasets. Biological roles of DDX56 were explored by gene set enrichment analysis (GSEA), and cell proliferation in vitro and in vivo, cell cycle assays, and using DDX56-knockdown or overexpressed CRC cells. RNA sequencing was carried out to elucidate the effect of DDX56 on mRNA splicing. We found that DDX56 expression was positively correlated with the amplification of DDX56 and was upregulated in CRC cells. High DDX56 expression was associated with lymphatic invasion and distant metastasis and was an independent poor prognostic factor. In vitro analysis, in vivo analysis and GSEA showed that DDX56 promoted proliferation ability through regulating the cell cycle. DDX56 knockdown reduced intron retention and tumor suppressor WEE1 expression, which functions as a G2-M DNA damage checkpoint. We have identified DDX56 as a novel oncogene and prognostic biomarker of CRC that promotes alternative splicing of WEE1.

Identification and Validation in a Novel Classification of Helicase Patterns for the Prediction of Tumor Proliferation and Prognosis.

Background: Helicases have been classified as a class of enzymes that determine the stability of the cellular genome. There is growing evidence that helicases can help tumor cells resist drug killing by repairing Deoxyribose Nucleic Acid (DNA) or stabilizing transcription, which may contribute to the understanding of drug resistance. Currently, identifying cancer biomarkers among helicases and stratifying patients according to helicase activity will be able to guide treatment well. Methods: We clustered 371 hepatocellular carcinoma (HCC) patients from The Cancer Genome Atlas (TCGA) by consensus clustering based on helicase expression profiles to identify potential molecular subtypes. The Multiscale Embedded Gene Co-Expression Network Analysis (MEGENA) algorithm was used to find core differential gene modules between different molecular subtypes, and single-cell analysis was utlized to explore the potential function of hub gene. Immunohistochemical (IHC) staining was used to verify the diagnostic value of DDX56 and its ability to reflect the proliferation efficiency of cancer cells. Results: We identified two subtypes associated with helicase. High helicase subtype was associated with poor clinical outcome, massive M0 macrophage infiltration, and highly active cell proliferation features. In addition, we identified a new biomarker, DDX56, which has not been reported in HCC, was highly expressed in HCC tissues, associated with poor prognosis, and was also shown to be associated with high cell proliferative activity. Conclusion: In conclusion, based on helicase expression profiles, we have developed a new classification system for HCC, which is a proliferation-related system, and has clinical significance in evaluating prognosis and treating HCC patients, including immunotherapy and chemotherapy. In addition, we identified a new biomarker, DDX 56, which is overexpressed in HCC tissues, predicts a poor prognosis and is a validated index of tumor cell proliferation.

DDX56 antagonizes IFN-ß production to enhance EMCV replication by inhibiting IRF3 nuclear translocation.

DEAD (Asp-Glu-Ala-Asp)-box RNA helicases (DDX) play important roles in viral infection, either as cytosolic viral nucleic acids sensors or as essential host factors for viral replication. In this study, we identified DDX56 as a positive regulator for encephalomyocarditis virus (EMCV) replication. EMCV infection promotes DDX56 expression via its viral proteins, VP3 and 3C. We showed that DDX56 overexpression promotes EMCV replication whereas its loss dampened EMCV replication. Consequently, knockdown of DDX56 increases type I interferon (IFN) expression during EMCV infection. We also showed that DDX56 interrupts IFN regulatory factor 3 (IRF3) phosphorylation and its nucleus translocation by directly targeting KPNA3 and KPNA4 in an EMCV-triggered MDA5 signaling activation cascade leading to the blockade of IFN-ß production. Overall, we showed that DDX56 is a novel negative regulator of EMCV-mediated IFN-ß responses and that DDX56 plays a critical role in EMCV replication. These findings reveal a novel strategy for EMCV to utilize a host factor to evade the host innate immune response and provide us new insight into the function of DDX56.

Pan-cancer analysis identifies DDX56 as a prognostic biomarker associated with immune infiltration and drug sensitivity.

DDX56, a member of the RNA helicase family, is upregulated in colon adenocarcinoma, lung squamous cell carcinoma, and osteosarcoma. However, the relationships between DDX56 and other tumors are not clear, and the molecular mechanism of its action is not fully understood. Here, we explore the biological functions of DDX56 in 31 solid tumors and clarify that DDX56 can promote oncogenesis and progression in multiple tumor types based on multi-omics data. Bioinformatics analysis revealed that the cancer-promoting effects of DDX56 were achieved by facilitating tumor cell proliferation, inhibiting apoptosis, inducing drug resistance, and influencing immune cell infiltration. Furthermore, we found that copy number alterations and low DNA methylation of DDX56 were likely to be related to aberrantly high DDX56 expression. Our results suggest that DDX56 is a potential pan-cancer biomarker that could be used to predict survival and response to therapy, as well as a potential novel therapeutic target. We validated some of our results and illustrated their reliability using CRISPR Screens data. In conclusion, our results clarify the role of DDX56 in the occurrence and development of multiple cancers and provide insight into the molecular mechanisms involved in the process of pathogenesis, indicating a direction for future research on DDX56 in cancers.

DDX56 inhibits PRV replication through regulation of IFN-ß signaling pathway by targeting cGAS.

Pseudorabies virus (PRV) is an agent of Aujeszky's disease, and causes great economic losses to pig farming. Re-outburst of pseudorabies implies that new control measures are urgently needed. We show here that DDX56 possesses the ability to inhibit PRV replication in vitro, which may be an important factor for PRV infection. Overexpression of DDX56 inhibited PRV genomic DNA transcription and lower titers of PRV infection in PK15 cells, whereas down-regulation of the DDX56 expression had a promotion role on virus replication. Further study demonstrated that DDX56 exerted its proliferation-inhibitory effects of PRV through up-regulating cGAS-STING-induced IFN-ß expression. Moreover, we found that DDX56 could promote cGAS expression and direct interaction also existed between DDX56 and cGAS. Based on this, DDX56-regulated IFN-ß pathway may be targeted at cGAS. To verify this, down-regulated cGAS expression in DDX56 over-expression cells was performed. Results indicated that knockdown of cGAS expression could abrogate the inhibition role of DDX56 on PRV proliferation and weaken the effect of DDX56 on IFN-ß expression. In addition, DDX56 played a promotion role in IRF3 phosphorylation and nucleus translocation. Altogether, our results highlight DDX56's antiviral role in PRV infection, and our findings contribute to a better understanding of host factors controlling PRV replication.

DEAD-box helicase 56 functions as an oncogene promote cell proliferation and invasion in gastric cancer via the FOXO1/p21 Cip1/c-Myc signaling pathway.

DEAD-box helicase (DDX) family exerts a critical effect on cancer initiation and progression through alternative splicing, transcription and ribosome biogenesis. Increasing evidence has demonstrated that DEAD-box helicase 56 (DDX56) is over-expressed in several cancers, which plays an oncogenic role. Till the present, the impact of DDX56 on gastric cancer (GC) remains unclear. We conducted high-throughput sequencing (RNA-seq) to demonstrate aberrant DDX56 levels within 10 GC and matched non-carcinoma tissue samples. DDX56 levels were detected through qRT-PCR, western blotting (WB) and immunochemical staining in GC patients. We conducted gain- and loss-of-function studies to examine DDX56's biological role in GC development. In vitro, we carried out 5­Ethynyl­2­deoxyuridine (EdU), scratch, Transwell, and flow cytometry (FCM) assays for detecting GC cell growth, invasion, migration and apoptosis. Additionally, gene set enrichment analysis (GSEA), WB assay, and Encyclopedia of RNA Interactomes (ENCORI) were carried out for analyzing DDX56-regulated downstream genes and signaling pathways. In vivo, tumor xenograft experiment was performed for investigating how DDX56 affected GC development within BALB/c nude mice. Functionally, DDX56 knockdown arrested cell cycle at G1 phase, invasion and migration of AGS and MKN28 cells, and enhanced their apoptosis. Ectopic DDX56 expression enhanced the cell growth, migration and invasion, and inhibited apoptosis. Knockdown of DDX56 suppressed GC growth in the tumor models of BALB/c nude mice. Mechanistically, DDX56 post-transcriptionally suppressed FOXO1/p21 Cip1 protein expression, which could activate its downstream cyclin E1/CDK2/c-Myc signaling pathways. This sheds lights on the GC pathogenic mechanism and offers a potential anti-cancer therapeutic target.

The DEAD-box helicase DDX56 is a conserved stemness regulator in normal and cancer stem cells.

Across the animal kingdom, adult tissue homeostasis is regulated by adult stem cell activity, which is commonly dysregulated in human cancers. However, identifying key regulators of stem cells in the milieu of thousands of genes dysregulated in a given cancer is challenging. Here, using a comparative genomics approach between planarian adult stem cells and patient-derived glioblastoma stem cells (GSCs), we identify and demonstrate the role of DEAD-box helicase DDX56 in regulating aspects of stemness in four stem cell systems: planarians, mouse neural stem cells, human GSCs, and a fly model of glioblastoma. In a human GSC line, DDX56 localizes to the nucleolus, and using planarians, when DDX56 is lost, stem cells dysregulate expression of ribosomal RNAs and lose nucleolar integrity prior to stem cell death. Together, a comparative genomic approach can be used to uncover conserved stemness regulators that are functional in both normal and cancer stem cells.

Ddx56 maintains proliferation of mouse embryonic stem cells via ribosome assembly and interaction with the Oct4/Sox2 complex.

BACKGROUND: Embryonic stem cells (ESCs) are important source of clinical stem cells for therapy, so dissecting the functional gene regulatory network involved in their self-renewal and proliferation is an urgent task. We previously reported that Ddx56 interacts with the core transcriptional factor Oct4 by mass spectrometry analysis in ESCs. However, the exact function of Ddx56 in ESCs remains unclear. METHODS: We investigated the role of Ddx56 in mouse ESCs (mESCs) through both gain- and loss-of-function strategies. The effect of Ddx56 on mESCs was determined based on morphological changes, involvement in the network of pluripotency markers (Nanog, Oct4, Sox2), and altered lineage marker expression. In addition, the role of Ddx56 in mESCs was evaluated by polysome fractionation, qRT-PCR, and co-immunoprecipitation (co-IP). Finally, RNA sequencing was applied to explore potential network regulation by Ddx56 in mESCs. RESULT: We found that Ddx56 participated in ribosome assembly, as knockout or RNAi knockdown of Ddx56 led to ribosome dysfunction and cell lethality. Surprisingly, exogenous expression of C-terminal domain truncated Ddx56 (Ddx56 ΔC-ter) did not affect ribosome assembly, but decreased mESC proliferation by downregulation of proliferation-related genes and cell cycle changing. In terms of mechanism, Ddx56 interacted with the Oct4 and Sox2 complex by binding to Sox2, whereas Ddx56 ΔC-ter showed weaker interaction with Sox2 and led to retardation of mESC proliferation. CONCLUSIONS: Ddx56 maintains ESC proliferation by conventional regulation of ribosome assembly and interaction with the Oct4 and Sox2 complex.

Integrated Analysis of DEAD-Box Helicase 56: A Potential Oncogene in Osteosarcoma.

Background: Osteosarcoma is a solid tumor common in the musculoskeletal system. The DEAD-box helicase (DDX) families play an important role in tumor genesis and proliferation. Objective:To screen potential molecular targets in osteosarcoma and elucidate its relationship with DDX56. Methods: We employed the Gene Expression Omnibus and The Cancer Genome Atlas datasets for preliminary screening. DDX56 expression was measured by RT-qPCR in three osteosarcoma cell lines. Biological roles of DDX56 were explored by Gene ontology, Kyoto Encyclopedia of Genes and Genomes and Ingenuity Pathway Analysis. Cell proliferation, cycle, and apoptosis assays were performed using Lentivirus™ knockdown technique. Results: It was found that DDX56 expression was regularly upregulated in osteosarcoma tissue and cell lines, while DDX56 knockdown inhibited cell proliferation and promoted cell apoptosis. Conclusions: The findings suggest DDX56 as a potential therapeutic target for the treatment of osteosarcoma.

DDX56 cooperates with FMDV 3A to enhance FMDV replication by inhibiting the phosphorylation of IRF3.

The components of foot-and-mouth disease virus (FMDV) interact with host cellular proteins to promote self-replication and evade the host immune response. Previous studies have shown that FMDV 3A, 2C and 2B proteins interact with host cellular proteins involved in FMDV replication. However, whether the other host proteins have an impact on FMDV replication is less understood. In this study, we identified DDX56 as a positive regulator of FMDV replication. DDX56 overexpression increased FMDV replication, whereas DDX56 knockdown had the opposite effect. DDX56 interacted and cooperated with FMDV 3A to inhibit the type I interferon by reducing the phosphorylation of IRF3. Moreover, the D166 site of DDX56 played a role in increasing FMDV replication and cooperating with FMDV 3A to inhibit the phosphorylation of IRF3. Additionally, knockdown of DDX56 or FMDV 3A results also showed that DDX56 cooperated with FMDV 3A to inhibit the phosphorylation of IRF3. These results suggest that the interaction between FMDV 3A protein and the host protein DDX56 is critical for FMDV replication.

The nucleolar helicase DDX56 redistributes to West Nile virus assembly sites.

Flaviviruses, including the human pathogen, West Nile virus (WNV), are known to co-opt many host factors for their replication and propagation. To this end, we previously reported that the nucleolar DEAD-box RNA helicase, DDX56, is important for production of infectious WNV virions. In this study, we show that WNV infection results in relocalization of DDX56 from nucleoli to virus assembly sites on the endoplasmic reticululm (ER), an observation that is consistent with a role for DDX56 in WNV virion assembly. Super-resolution microscopy revealed that capsid and DDX56 localized to the same subcompartment of the ER, however, unexpectedly, stable interaction between these two proteins was only detected in the nucleus. Together, these data suggest that DDX56 relocalizes to the site of virus assembly during WNV infection and that its interaction with WNV capsid in the cytoplasm may occur transiently during virion morphogenesis.