This work identifies a hierarchical signaling axis driven by the RNA methyltransferase NSUN2 that fuels LUAD progression and establishes the NSUN2-E2F1-RAD54L axis as a critical driver of LUAD and a promising target for therapeutic intervention.
Abstract
Lung adenocarcinoma (LUAD) is a leading cause of cancer-related mortality, often driven by the hyperactivation of cell cycle and DNA repair pathways. While genomic alterations in these pathways are well-documented, the role of RNA epigenetics in orchestrating these oncogenic signals remains poorly understood. Here, we identify a hierarchical signaling axis driven by the RNA methyltransferase NSUN2 that fuels LUAD progression. We demonstrate that NSUN2 is frequently amplified and overexpressed in LUAD, where it correlates with advanced disease stage and poor patient survival. Mechanistically, NSUN2 catalyzes 5-methylcytosine (m5C) modification of the E2F1 3'-untranslated region (3'-UTR). This modification, recognized by the reader protein YBX1, enhances the translational efficiency of E2F1 transcripts. The resulting accumulation of E2F1 transcriptionally activates the DNA translocase RAD54L, which we redefine as a proactive oncogenic effector essential for cell proliferation, invasion, and in vivo tumorigenesis. Integrated multi-omics and rescue experiments confirm that the NSUN2-mediated translational induction of E2F1 is a prerequisite for RAD54L-driven malignancy. Our findings uncover a novel epitranscriptomic-transcriptional regulatory cascade, establishing the NSUN2-E2F1-RAD54L axis as a critical driver of LUAD and a promising target for therapeutic intervention.
Summary Lung adenocarcinoma (LUAD) remains a leading cause of cancer-related death, underscoring the need for an improved molecular understanding. This study investigated the regulatory mechanism of the long non-coding RNA deoxyguanosine kinase antisense RNA 1 (DGUOK-AS1) in LUAD. DGUOK-AS1 was significantly upregulated in LUAD cells and serum samples, and its elevated expression showed a preliminary association with LUAD. Functional experiments demonstrated that DGUOK-AS1 promoted LUAD proliferation and migration both in vitro and in vivo, partly by acting as a competing endogenous RNA for miR-2467-5p to modulate PRMT5 expression. Mechanistically, RNA-binding motif protein 15 (RBM15) enhanced DGUOK-AS1 stability through m6A modification, which in turn enabled heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) binding in an m6A-dependent manner via its RNA recognition motif 3 (RRM3) domain, promoting degradation. RBM15 knockdown attenuated the malignant phenotype through the miR-2467-5p/PRMT5 axis. These findings reveal an m6A-dependent mechanism governing DGUOK-AS1 stability and provide insights into its contribution to LUAD progression.
Menghao Yang, Jiaen Wu, Youjie Li et al.· iScience· 0 citations
Flap endonuclease 1 (FEN1), a key enzyme in DNA metabolism, exhibits oncogenic properties in various cancers. However, its functional role and underlying mechanisms in lung adenocarcinoma (LUAD) progression and tumor immunity remain poorly defined. Here, we demonstrate that FEN1 is significantly upregulated in LUAD tissues and serves as an independent prognostic factor for poor survival. Genetic depletion of FEN1 suppressed LUAD cell proliferation by inducing G0/G1 cell cycle arrest and cellular senescence, while significantly impairing migration and invasion capabilities. Mechanistically, integrated proteomic and molecular analyses revealed that FEN1 knockdown disrupts the carboxyl-terminal subunit of mucin 1 (MUC1-C)/PI3K/AKT autoregulatory loop, a critical driver of oncogenic signaling. Furthermore, FEN1 downregulation inhibited the MUC1-C/p65/ programmed death-ligand 1 (PD-L1) signaling axis, thereby alleviating tumor-mediated immunosuppression. This was evidenced by enhanced antitumor immunity, characterized by the local expansion of CD4
+
T cells and restored effector function of CD8
+
T cells, both in vitro and in vivo. Mechanistically, independent of its nuclease activity, FEN1 interacts with the N-terminus of eukaryotic translation initiation factor 4A3 (EIF4A3) to facilitate its binding to MUC1-C mRNA. Specifically, FEN1 is required for EIF4A3-mediated maintenance of both the transcript stability and translation efficiency of MUC1-C. Clinically, FEN1, MUC1-C, and PD-L1 are coordinately upregulated in LUAD tissues, and their co-expression predicts poor patient survival irrespective of EGFR mutation status. Our findings establish the FEN1/EIF4A3/MUC1-C axis as a novel mechanism driving LUAD progression by concurrently regulating intrinsic malignancy and extrinsic immune evasion, presenting FEN1 targeting as a promising dual-hit therapeutic strategy.
Min Wu, Ben-Meng Wu, Xue-Bing Yan et al.· Cell Death & Disease· 0 citations
BACKGROUND
Osteosarcoma (OS) represents a common primary malignant bone tumor associated with unfavorable clinical outcomes. Growing evidence underscores the crucial involvement of N6-methyladenosine (m6A) modifications in tumor development, but the specific mechanisms underlying the m6A regulatory network in OS remain to be elucidated.
METHODS
Potential key target genes in OS were identified through bioinformatic analyses, followed by the characterization of m6A-related regulatory proteins, specifically, writer and reader proteins, which showed significant associations with these targets. To elucidate the mechanistic role of m6A methylation in regulating UHRF1 expression, a series of in vitro assays were conducted. These included RNA pull-down, MeRIP-PCR, dot blot, dual-luciferase reporter assays, and RNA stability assays, which collectively confirmed the interaction between m6A regulatory proteins and UHRF1 mRNA. For functional investigations, OS cell lines (U2OS, Saos2, and 143B) with gene silencing or overexpression were established, and the role of UHRF1 in cellular proliferation, migration, and invasion was assessed using CCK-8 assays, Transwell migration and invasion assays, flow cytometry, and wound healing assays. In addition, GSH/GSSG ratio, Fe2+ concentration, and ROS levels were measured using commercial assay kits to explore ferroptosis-related functional mechanisms. To validate the in vivo relevance of our findings, a xenograft mouse model was established. Finally, functional rescue experiments were performed to mechanistically confirm the critical role of the ZCCHC4-UHRF1-CDO1 regulatory axis in OS progression.
RESULTS
ZCCHC4, functioning as an m6A methyltransferase, enhances the stability of UHRF1 mRNA by catalyzing its m6A modification, thereby promoting increased expression of UHRF1. In parallel, IGF2BP3, an established m6A reader protein, specifically recognizes and binds to the m6A-modified sites on UHRF1 mRNA, further stabilizing the transcript and modulating its downstream biological functions. METTL3/METTL14 knockdown experiments ruled out the contribution of classical m6A methyltransferases, confirming that ZCCHC4 is the primary methyltransferase for UHRF1. Dual-luciferase assays and bisulfite sequencing revealed that UHRF1 suppresses CDO1 transcription by inducing high methylation of its promoter, thereby reducing ROS/Fe2+ levels and increasing GSH, which in turn blocks ferroptosis. In U2OS, Saos2, and 143B cells, silencing UHRF1 or ZCCHC4 inhibited proliferation, migration, and invasion while activating ferroptosis. Overexpression of UHRF1 had the opposite effect. In vivo models confirmed that UHRF1 silencing inhibited tumor growth. Furthermore, UHRF1 overexpression partially reversed the phenotypes induced by ZCCHC4 knockdown.
CONCLUSION
In summary, this study reveals for the first time the complete molecular mechanism by which ZCCHC4-mediated UHRF1 m6A methylation promotes OS progression through epigenetic suppression of CDO1 transcription and inhibition of ferroptosis. This regulatory axis (ZCCHC4-IGF2BP3-UHRF1-CDO1-ferroptosis) provides multiple therapeutic targets for OS and lays a solid foundation for the future development of anticancer strategies based on the regulation of ferroptosis, while also offering a promising pathway for clinical translation.
Kai Song, Ju Liu, Bowen Han et al.· Bone· 0 citations
Background: Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, with resistance to immunotherapy posing a major clinical challenge. Natural killer (NK) cells exhibit impaired infiltration and cytotoxicity in HCC; however, the mechanisms underlying NK cell-mediated immune evasion are still poorly understood. This study investigated how NOP2/Sun RNA methyltransferase 2 (NSUN2), a 5-methylcytosine (m5C) RNA methyltransferase, induces metabolic reprogramming and immunosuppression to drive HCC progression. Methods: We conducted a genome-wide CRISPR screen in HCC cells cocultured with NK cells. To delineate the downstream mechanisms, we integrated profiling of the m5C epitranscriptome, transcriptome, and chromatin landscape with metabolic characterization. The impact of NSUN2 on histone lactylation and programmed cell death 1 ligand 1 (PD-L1) transcription was further investigated. Functional assays in vitro and in vivo using syngeneic murine models and pharmacological inhibition validated these findings. Clinical relevance was assessed using patient tissues, The Cancer Genome Atlas dataset, and immunotherapy cohorts. Results: Genome-wide CRISPR screening in HCC cell–NK cell coculture models identified NSUN2 as a key suppressor of NK cell-mediated cytotoxicity. Mechanistically, NSUN2-mediated RNA m5C modification enhanced the messenger RNA stability and expression of glycolytic enzymes, including enolase 1 (ENO1), pyruvate kinase M1/2 (PKM), and lactate dehydrogenase A (LDHA), thereby increasing lactate production. Accumulated lactate promoted histone H3 lysine 18 lactylation (H3K18la), which enhanced chromatin accessibility at the CD274 (encoding PD-L1) promoter and recruited signal transducer and activator of transcription 3 (STAT3) to drive PD-L1 expression, ultimately inhibiting NK cell-mediated cytotoxicity. Clinically, high NSUN2 expression was associated with elevated PD-L1 levels, poor prognosis, and immunotherapy resistance in patients with HCC. In vivo, NSUN2 knockout increased NK cell infiltration and suppressed tumor growth, while the STAT3 inhibitor TTI-101 combined with anti-PD-L1 therapy enhanced NK cell cytotoxicity and inhibited HCC progression. Conclusions: Our data demonstrated that NSUN2 drove immune evasion in HCC by coupling m5C-dependent glycolytic reprogramming with H3K18la-mediated epigenetic activation of PD-L1. These findings suggest that NSUN2 could represent a critical nexus between m5C RNA methylation and immunosuppression, providing a therapeutic rationale for combination immunotherapy in HCC.
Sheng-Wei Mao, Yuan-Yuan Fang, Jun Gao et al.· Cancer Communications· 0 citations
Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have markedly improved outcomes in EGFR-mutant lung adenocarcinoma (LUAD), yet acquired resistance to the third-generation inhibitor osimertinib remains inevitable. Although metabolic reprogramming is increasingly recognized as a driver of therapeutic resistance, the epigenetic consequences of lactate accumulation and their functional relevance in osimertinib resistance are poorly understood. Here we report that osimertinib-resistant LUAD cells exhibit enhanced glycolytic flux, increased intracellular lactate levels, and elevated histone H4 lysine 8 lactylation (H4K8la). Integrative transcriptomic analysis revealed significant enrichment of cell-cycle–associated pathways in resistant tumors, despite downregulation of canonical Cyclin B1 and Cyclin D1. Among the upregulated genes, GAS2L3 emerged as a prominent candidate. Genome-wide CUT&Tag profiling demonstrated enrichment of H4K8la at the GAS2L3 promoter, which was confirmed by ChIP–qPCR. Functionally, GAS2L3 overexpression accelerated S/G2–M progression and promoted proliferation under drug pressure, whereas its silencing induced cell-cycle arrest and restored osimertinib sensitivity both in vitro and in xenograft models. Notably, resistant cells displayed elevated P53 expression without induction of its canonical effector P21, indicating checkpoint uncoupling and a noncanonical mode of cell-cycle regulation. Mechanistically, glycolysis inhibition reduced H4K8la and GAS2L3 expression and partially re-sensitized resistant cells, while exogenous lactate restored H4K8la levels and resistance phenotypes, establishing a metabolically driven epigenetic circuit. Clinically, high GAS2L3 expression was associated with shorter progression-free survival in osimertinib-treated patients. Collectively, our findings identify a glycolysis–H4K8la–GAS2L3 axis that drives noncanonical cell-cycle reprogramming independently of classical Cyclin–CDK activation, thereby promoting acquired osimertinib resistance. These results uncover a metabolism-epigenetics–cell cycle interface that may represent a therapeutic vulnerability in EGFR-mutant LUAD.
Shu-Man Zhen, Xiaohui Bai, Shutang Liu et al.· Cell Death Discovery· 0 citations
It is found that the hyperlactate microenvironment induced by abnormal glycolysis in WT significantly upregulates the level of histone H3K18 lactylation (H3K18la), which promotes WT cell proliferation and migration.
Yanping Wang, Hongjie Gao, Bifei Zhang et al.· Advancement of science· 0 citations
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