DDX18 Facilitates the Tumorigenesis of Lung Adenocarcinoma by Promoting Cell Cycle Progression through the Upregulation of CDK4.

Lung adenocarcinoma (LUAD) is the most prevalent and aggressive subtype of lung cancer, exhibiting a dismal prognosis with a five-year survival rate below 5%. DEAD-box RNA helicase 18 (DDX18, gene symbol DDX18), a crucial regulator of RNA metabolism, has been implicated in various cellular processes, including cell cycle control and tumorigenesis. However, its role in LUAD pathogenesis remains elusive. This study demonstrates the significant upregulation of DDX18 in LUAD tissues and its association with poor patient survival (from public databases). Functional in vivo and in vitro assays revealed that DDX18 knockdown potently suppresses LUAD progression. RNA sequencing and chromatin immunoprecipitation experiments identified cyclin-dependent kinase 4 (CDK4), a cell cycle regulator, as a direct transcriptional target of DDX18. Notably, DDX18 depletion induced G1 cell cycle arrest, while its overexpression promoted cell cycle progression even in normal lung cells. Interestingly, while the oncogenic protein c-Myc bound to the DDX18 promoter, it did not influence its expression. Collectively, these findings establish DDX18 as a potential oncogene in LUAD, functioning through the CDK4-mediated cell cycle pathway. DDX18 may represent a promising therapeutic target for LUAD intervention.

Liriodendrin stimulates proliferation and milk protein synthesis of mammary epithelial cells via the PI3K-DDX18 signaling.

Liriodendrin is a lignan compound that is involved in a wide variety of physiological functions, however it is unknown whether liriodendrin plays an important role in milk production in the mammary glands. In this study, we explored the role and molecular mechanism of Liriodendrin in milk synthesis of mammary epithelial cells (MECs). Bovine MECs were treated with liriodendrin (0, 0.45, 0.9, 1.35, 1.8, and 2.25 mM) for 24 h. Liriodendrin dose-dependently increased cell number, cell cycle transition, and milk protein synthesis, as well as Cyclin D1 and mTOR phosphorylation, with the maximal effects observed at a dose of 1.35 mM. Liriodendrin increased the expression of DDX18, which mediated liriodendrin stimulation of Cyclin D1 and mTOR mRNA expression. PI3K inhibition and DDX18 knockdown experiments further confirmed that liriodendrin regulates the mRNA expression of Cyclin D1 and mTOR via the PI3K-DDX18 signaling. Mouse feeding experiment showed that liriodendrin dose-dependently promotes ß-casein and DDX18 expression in mouse mammary gland. In this study, DDX18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein. These findings reveal that liriodendrin stimulates proliferation and milk protein synthesis of MECs via the PI3K-DDX18 signaling.

DNA 6mA demethylase ALKBH1 regulates DDX18 expression to promote proliferation of human head and neck squamous cell carcinoma.

PURPOSE: Human head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Currently, surgical resection plus a combination of chemotherapy and radiotherapy is the standard treatment for HNSCC, and the 5-year survival rate of patients with HNSCC remains very low because of the higher incidence of metastasis with consequent recurrence. Here, we aimed to investigate the potential role of DNA N6-methyladenine (6mA) demethylase ALKBH1 in tumor cell proliferation in HNSCC. METHODS: The expression of ALKBH1 in 10 pairs of HNSCC/normal tissues and 3 HNSCC cell lines were measured by qRT‒PCR and western blotting. Colony formation, flow cytometry, patient-derived HNSCC organoid assays were used to assess the role of ALKBH1 in HNSCC cell proliferation in cell lines and human HNSCC patients. MeDIP-seq, RNA sequencing, Dot blotting and western blotting were used to evaluate the regulatory effect of ALKBH1 on the expression of DEAD-box RNA helicase DDX18. A dual-luciferase reporter assay was used to assess the putative effect of DNA 6mA levels on DDX18 transcription. RESULTS: ALKBH1 was highly expressed in HNSCC cells and patient tissues. Functional experiments revealed that ALKBH1 knockdown in SCC9, SCC25, and CAL27 cells inhibited their proliferation in vitro. Using patient-derived HNSCC organoid assay, we found that knockdown of ALKBH1 inhibited the proliferation and colony formation of HNSCC patients-derived organoids. Moreover, we found that ALKBH1 can enhance DDX18 expression by erasing DNA 6mA level and regulating its promoter activity. ALKBH1 deficiency blocked tumor cell proliferation by inhibiting DDX18 expression. Exogenous overexpression of DDX18 rescued the cell proliferation arrest caused by ALKBH1 knockdown. CONCLUSION: Our data reveal the important role of ALKBH1 in regulating proliferation of HNSCC.

DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

R loops occur frequently in genomes and contribute to fundamental biological processes at multiple levels. Consequently, understanding the molecular and cellular biology of R loops has become an emerging area of research. Here, it is shown that poly(ADP-ribose) polymerase-1 (PARP-1) can mediate the association of DDX18, a putative RNA helicase, with R loops thereby modulating R-loop homeostasis in endogenous R-loop-prone and DNA lesion regions. DDX18 depletion results in aberrant endogenous R-loop accumulation, which leads to DNA-replication defects. In addition, DDX18 depletion renders cells more sensitive to DNA-damaging agents and reduces RPA32 and RAD51 foci formation in response to irradiation. Notably, DDX18 depletion leads to γH2AX accumulation and genome instability, and RNase H1 overexpression rescues all the DNA-repair defects caused by DDX18 depletion. Taken together, these studies uncover a function of DDX18 in R-loop-mediated events and suggest a role for PARP-1 in mediating the binding of specific DDX-family proteins with R loops in cells.

DEAD-Box Helicase 18 Counteracts PRC2 to Safeguard Ribosomal DNA in Pluripotency Regulation.

Embryonic stem cells (ESCs) exhibit high levels of ribosomal RNA (rRNA) transcription and ribosome biogenesis. Here, we reveal an unexpected role for an essential DEAD-box helicase, DDX18, in antagonizing the polycomb repressive complex 2 (PRC2) to prevent deposition of the repressive H3K27me3 mark onto rDNA in pluripotent cells. DDX18 binds and sequesters PRC2 in the outer layer of the nucleolus and counteracts PRC2 complex formation in vivo and in vitro. DDX18 knockdown leads to increased occupancy of PRC2 and H3K27me3 at rDNA loci, accompanied by drastically decreased rRNA transcription and reduced ribosomal protein expression and translation. Auxin-induced rapid degradation of DDX18 enhances PRC2 binding at rDNA. The inhibition of PRC2 partially rescues the effects of DDX18 depletion on rRNA transcription and ESC self-renewal. These results demonstrate a critical role for DDX18 in safeguarding the chromatin and transcriptional integrity of rDNA by counteracting the epigenetic silencing machinery to promote pluripotency.

Cellular DEAD-box RNA helicase 18 (DDX18) Promotes the PRRSV Replication via Interaction with Virus nsp2 and nsp10.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an aetiological agent that can lead to reproductive failure and respiratory diseases of pigs. The replication and pathogenesis of PRRSV, although poorly understood, has been associated with the host factors. DDX18 is a member of DEAD-box RNA helicases (DDXs) family which were proved to participate in viral replication. Previously, we found the DDX18 interacts with both nsp2 and nsp10 of PRRSV by Co-Immunoprecipitation (Co-IP). In the present study, we demonstrated the interactions of DDX18 with nsp2 and nsp10, and located DDX18's binding regions as the N-terminus of nsp2 and both the N-terminus and C-terminus of nsp10. The expression of the nsp2 or nsp10 in MARC-145 cells and primary PAM cells redistributed DDX18 from the nucleus to the cytoplasm, and promoted the viral replication, but silencing of the DDX18 gene in MARC-145 cells down-regulated the replication of PRRSV. These findings proved that the cellular RNA helicase DDX18 plays a role in the replication of PRRSV, and provides insights into the understanding of PRRSV replication.