Crizotinib, a multitarget tyrosine kinase inhibitor, is the standard first-line drug used for the clinical treatment of locally advanced or metastatic ALK-positive non-small cell lung cancer. However, the liver injury induced by crizotinib is a clinical problem that needs to be solved urgently. Therefore, the mechanism underlying crizotinib-induced liver injury must be elucidated to identify prevention and treatment methods. By establishing the mouse and cell models of crizotinib-induced liver injury, we found that crizotinib induced apoptosis in mouse liver tissue, L02 cells, and HepG2 cells. After treatment with crizotinib, the N6-methyladenosine (m6A) reading protein YTHDF3 was aberrantly downregulated in mouse liver tissue and L02 cells. RNA sequencing, m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq), and MeRIP-qPCR were performed to identify the target gene of Lcn2. The upregulation of LCN2 was detected in both in vitro and in vivo models. Genetic inhibition of Lcn2 resulted in a reduction in the incidence of liver cell apoptosis induced by crizotinib. Additionally, knocking down YTHDF3 increased the mRNA stability and expression level of LCN2, whereas the overexpression of YTHDF3 inhibited the expression of LCN2 and apoptosis induced by crizotinib in L02 cells. Further mechanistic studies revealed a potential association between the YTHDF3 protein and Lcn2 mRNA and that YTHDF3 may affect the stability of Lcn2 mRNA in an m6A-dependent manner. Our findings revealed that the m6A reading protein YTHDF3-LCN2-apoptosis axis plays a critical role in mediating the hepatotoxicity of crizotinib, which provides potential intervention approaches for alleviating crizotinib-induced liver injury.

Tumor heterogeneity and the suppressive microenvironment are key challenges that limit the effectiveness of cancer treatment. In this study, we systematically elucidated the molecular characteristics and mechanisms underlying the suppressive immune microenvironment using a combination of single-cell RNA sequencing, spatial transcriptomics, and metabolomics for a series of human esophageal squamous cell carcinoma and matched non-tumor tissues. We found that COL17A1+ epithelial cells exhibited greater malignancy, characterized by the accumulation of triglycerides and phosphocholine. We also identified a tumor-specific POSTN+ fibroblast subgroup. We identified a unique epithelial-fibroblast niche with low infiltration of effector immune cells and substantial lipid enrichment, composed of POSTN+ fibroblasts and COL17A1+ epithelial cells, in which their crosstalk contributed to tumor progression. We confirmed that the INHBA/TP63 axis played a key role in mediating the regulation of COL17A1+ tumor cells by POSTN+ fibroblasts. Our findings provide new insights into the characteristics of the tumor microenvironment and the crosstalk between tumor cells and fibroblasts, offering valuable multi-omics data for elucidating tumor progression mechanisms.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have produced durable complete responses, but the eventual development of acquired resistance presents a major challenge in the treatment of non-small cell lung cancer (NSCLC). N7-methylguanosine (m7G), a prevalent post-transcriptional modification within RNA, plays regulatory roles in RNA stability, expression dynamics, and functional diversity. Despite these insights, the contribution of m7G methylation to EGFR-TKIs resistance remains poorly characterized. Here, we demonstrate that internal m7G modifications of mRNA and their associated methyltransferase complex, methyltransferase-like 1 (METTL1)/WD repeat domain 4 (WDR4), are significantly elevated in NSCLC specimens, which correlates with therapeutic resistance. Functional assays confirmed that METTL1/WDR4 enhances gefitinib resistance in both cellular and animal models through internal RNA m7G methyltransferase activity in NSCLC. Mechanistically, m7G MeRIP-seq combined with RNA-seq identified sodium channel and clathrin linker 1 (SCLT1) as the m7G target of METTL1/WDR4. METTL1/WDR4 knockdown led to decreased methylation level and mRNA stability of the SCLT1 transcript. Importantly, overexpression of wild-type METTL1, but not its catalytically inactive mutant, restored mRNA stability. Furthermore, METTL1/WDR4-mediated m7G modification of SCLT1 regulates gefitinib resistance by activating the NF-κB signaling. Our findings reveal the crucial role of aberrant mRNA internal m7G modification in EGFR-TKIs resistance, suggesting that targeting the METTL1/WDR4-SCLT1-NF-κB axis holds a promising therapeutic potential for overcoming EGFR-TKIs resistance.

BackgroundRNA 5-methylcytosine (m5C) plays an important role in the progression of hepatocellular carcinoma (HCC). Dysregulation of ferroptosis is closely associated with HCC. However, the effect of the epigenetic mRNA m5C modification on ferroptosis in HCC remains unclear.MethodsIn this study, ferroptosis was evaluated by detecting lipid reactive oxygen species (lipid ROS), ferrous ion and 4-hydroxynonenal (4-HNE) in xenograft mouse model, diethylnitrosamine (DEN)-initiated HCC model and so forth. The regulatory mechanisms of YBX1 in mRNA translation were elucidated using RNA sequencing, ribosome sequencing, RNA immunoprecipitation (RIP)-sequencing, bisulphite sequencing and immunoprecipitation (IP)-mass spectrometry assays. Dual-luciferase reporter, RIP-qPCR, Co-IP, RNA pulldown and methylated RNA immunoprecipitation (MeRIP)-quantitative polymerase chain reaction (qPCR) assays were performed to validate the mechanism of YBX1 in regulating mRNA translation by m5C modification.ResultsHere, we found that YBX1 promoted the translation of Ring Finger Protein 115 (RNF115) mRNA through m5C modification, thereby inhibiting ferroptosis and promoting HCC development. Moreover, RNF115 was identified as an E3 ubiquitin ligase for dihydroorotate dehydrogenase (DHODH), promoting Lys27 (K27) ubiquitination and inhibiting its autophagic degradation to counteract ferroptosis. In addition, YBX1 bound to the m5C modification sites of RNF115 3 '-untranslated region (UTR) and interacted with Eukaryotic Translation Initiation Factor 4A1 (EIF4A1) to bridge the 5 '-UTR regions, promoting mRNA circularisation and translation, while NOP2/Sun RNA methyltransferase 2 (NSUN2) was identified as responsible for m5C modification of RNF115 mRNA in HCC.ConclusionsThe current work revealed that YBX1 promoted RNF115 mRNA translation in an m5C-dependent manner, thereby regulating DHODH ubiquitination and expression to suppress ferroptosis. This research sheds light on the mechanism of YBX1 in m5C-modified mRNAs translation and ferroptosis, highlighting its promise as a biomarker for prognosis and a target for therapy in HCC.Key points YBX1 inhibits ferroptosis in HCC by regulating the RNF115-DHODH axis. RNF115, an E3 ligase, mediates K27 ubiquitination and autophagic degradation of DHODH. YBX1 binds to the m5C sites of RNF115 mRNA 3 '-UTR and interacts with EIF4A1 to bridge the 5 '-UTR, promoting mRNA circularisation and translation. High expression of YBX1/RNF115 predicts the poor overall survival in HCC.

5-methylcytosine (m5C) is a prevalent RNA modification that has various impacts on mRNA fate. Here, we generated 30 single-base resolution RNA m5C methylomes and revealed the dynamic nature of m5C of heart, muscle, lung, esophagus, stomach, pancreas, colon, jejunum, and rectum from 7 adult human individuals using RNA-BisSeq. Based on clustering analysis, the heart and muscle formed one cluster, while the remaining tissues constituted another cluster. Intriguingly, we observed a discrepancy pattern between m5C levels and gene expression in these tissues when comparing the m5C methylome and transcriptome. Moreover, we identified differences in NSUN2-mediated m5C modifications between esophageal paracancerous tissues and healthy individual tissues. Notably, NSUN2 was found to interact with PLXNA1 mRNA, and silencing NSUN2 in esophageal squamous cell carcinoma (ESCC) cells resulted in the downregulation of PLXNA1 expression through an m5C-mediated mechanism. Overall, our study provides valuable insights into the m5C profile and the relationship between the methylome and transcriptome in human tissues, highlighting the potential role of m5C modification as an epitranscriptomic biomarker.

Hepatocellular carcinoma (HCC) is one of the primary forms of liver cancer and is currently the sixth most prevalent malignancy worldwide. In addition to surgical interventions, effective drug treatment is essential for treating HCC. With an increasing number of therapeutic drugs for liver cancer undergoing clinical studies, the therapeutic strategies for advanced HCC are more diverse than ever, leading to improved prospects for HCC patients. Molecular targeted drugs and immunotherapies have become crucial treatment options for HCC. Treatment programs include single-agent molecular-targeted drugs, immunotherapies, combinations of immunotherapies with molecular-targeted drugs, and dual immune checkpoint inhibitors. However, further exploration is necessary to determine the optimal pharmacological treatment regimens, and the development of new effective drugs is urgently needed. This review provides an overview of the current globally approved drugs for liver cancer, as well as the latest advances in ongoing clinical research and drug therapies. Additionally, the review offers an outlook and discussion on the prospects for the development of drug therapy approaches for HCC.

Background: Gastric cancer stem cells (GCSCs) are important tumour cells involved in tumourigenesis and gastric cancer development. However, their clinical value remains unclear due to the limitations of the available technologies. This study aims to explore the clinical significance of GCSCs, their connection to the tumour microenvironment, and their underlying molecular mechanisms. Methods: Stem-like tumour cells were identified by mining single-cell transcriptomic data from multiple samples. Integrated analysis of single-cell and bulk transcriptome data was performed to analyse the role of stem-like tumour cells in predicting clinical outcomes by introducing the intermediate variable mRNA stemness degree (SD). Consensus clustering analysis was performed to develop an SD-related molecular classification strategy to assess the clinical characteristics in gastric cancer. A prognostic model was constructed using a customized approach that comprehensively considered SD-related gene signatures based on an artificial neural network. Results: By analysing single-cell data and validating immunofluorescence results, we identified a PCLAF+ stem-like tumour cell population in GC. By calculating SD, we observed that PCLAF+ stem-like tumour cells were associated with poor prognosis and certain clinical features. The SD was negatively correlated with the abundance of most immune cell types. Furthermore, we proposed an SD-related classification method and prognostic model. In addition, the customised prognostic model can be used to predict whether a patient respond to PD-1/PD-L1 immunotherapy. Conclusion: We identified a cluster of stem-like cells and elucidated their clinical significance, highlighting the possibility of their use as immunotherapeutic targets.

Background RNA m(5)C methylation has been extensively implicated in the occurrence and development of tumors. As the main methyltransferase, NSUN2 plays a crucial regulatory role across diverse tumor types. However, the precise impact of NSUN2-mediated m(5)C modification on breast cancer (BC) remains unclear. Our study aims to elucidate the molecular mechanism underlying how NSUN2 regulates the target gene HGH1 (also known as FAM203) through m(5)C modification, thereby promoting BC progression. Additionally, this study targets at preliminarily clarifying the biological roles of NSUN2 and HGH1 in BC. Methods Tumor and adjacent tissues from 5 BC patients were collected, and the m(5)C modification target HGH1 in BC was screened through RNA sequencing (RNA-seq) and single-base resolution m(5)C methylation sequencing (RNA-BisSeq). Methylation RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR) confirmed that the methylation molecules NSUN2 and YBX1 specifically recognized and bound to HGH1 through m(5)C modification. In addition, proteomics, co-immunoprecipitation (co-IP), and Ribosome sequencing (Ribo-Seq) were used to explore the biological role of HGH1 in BC. Results As the main m(5)C methylation molecule, NSUN2 is abnormally overexpressed in BC and increases the overall level of RNA m(5)C. Knocking down NSUN2 can inhibit BC progression in vitro or in vivo. Combined RNA-seq and RNA-BisSeq analysis identified HGH1 as a potential target of abnormal m(5)C modifications. We clarified the mechanism by which NSUN2 regulates HGH1 expression through m(5)C modification, a process that involves interactions with the YBX1 protein, which collectively impacts mRNA stability and protein synthesis. Furthermore, this study is the first to reveal the binding interaction between HGH1 and the translation elongation factor EEF2, providing a comprehensive understanding of its ability to regulate transcript translation efficiency and protein synthesis in BC cells. Conclusions This study preliminarily clarifies the regulatory role of the NSUN2-YBX1-m(5)C-HGH1 axis from post-transcriptional modification to protein translation, revealing the key role of abnormal RNA m(5)C modification in BC and suggesting that HGH1 may be a new epigenetic biomarker and potential therapeutic target for BC.

BACKGROUND:RNA 5-methylcytosine (m5C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m5C modification in tumor drug resistance remain unclear.METHODS:The correlation between RNA m5C methylation, m5C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m5C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays.RESULTS:RNA m5C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and m5C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m5C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m5C reader Y-box binding protein 1 (YBX1).CONCLUSIONS:Our study reveals a critical function of aberrant RNA m5C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

RNA modifications affect many biological processes and physiological diseases. The 5-methylcytosine (m5C) modification regulates the progression of multiple tumors. However, its characteristics and functions in hepatocellular carcinoma (HCC) remain largely unknown. Here, we found that HCC tissues had a higher m5C methylation level than the adjacent normal tissues. Transcriptome analysis revealed that the hypermethylated genes mainly participated in the phosphokinase signaling pathways, such as the Ras and PI3K-Akt pathways. The m5C methyltransferase NSUN2 was highly expressed in HCC tissues. Interestingly, the expression of many genes was positively correlated with the expression of NSUN2, including GRB2, RNF115, AATF, ADAM15, RTN3, and HDGF. Real-time PCR assays further revealed that the expression of the mRNAs of GRB2, RNF115, and AATF decreased significantly with the down-regulation of NSUN2 expression in HCC cells. Furthermore, NSUN2 could regulate the cellular sensitivity of HCC cells to sorafenib via modulating the Ras signaling pathway. Moreover, knocking down NSUN2 caused cell cycle arrest. Taken together, our study demonstrates the vital role of NSUN2 in the progression of HCC.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) positively affect the initial control of non-small cell lung cancer (NSCLC). Rapidly acquired resistance to EGFR-TKIs is a major hurdle in successful treatment. However, the mechanisms that control the resistance of EGFR-TKIs remain largely unknown. RNA structures have widespread and crucial functions in many biological regulations; however, the functions of RNA structures in regulating cancer drug resistance remain unclear. Here, the psoralen analysis of RNA interactions and structures (PARIS) method is used to establish the higher-order RNA structure maps of EGFR-TKIs-resistant and -sensitive cells of NSCLC. Our results show that RNA structural regions are enriched in untranslated regions (UTRs) and correlate with translation efficiency (TE). Moreover, yrdC N 6 -threonylcarbamoyltransferase domain containing (YRDC) promotes resistance to EGFR-TKIs. RNA structure formation in YRDC 3′ UTR suppresses embryonic lethal abnormal vision-like 1 (ELAVL1) binding, leading to EGFR-TKI sensitivity by impairing YRDC translation. A potential therapeutic strategy for cancer treatment is provided using antisense oligonucleotide (ASO) to perturb the interaction between RNA and protein. Our study reveals an unprecedented mechanism through which the RNA structure switch modulates EGFR-TKI resistance by controlling YRDC mRNA translation in an ELAVL1-dependent manner.

AbstractBackgroundCancer cell–specific variation and circulating tumour DNA (ctDNA) methylation are promising biomarkers for non‐invasive cancer detection and molecular classification. Nevertheless, the applications of ctDNA to the early detection and screening of cancer remain highly challenging due to the scarcity of cancer cell–specific ctDNA, the low signal‐to‐noise ratio of DNA variation, and the lack of non‐locus‐specific DNA methylation technologies.MethodsWe enrolled three cohorts of breast cancer (BC) patients from two hospitals in China (BC: n = 123; healthy controls: n = 40). We developed a ctDNA whole‐genome bisulfite sequencing technology employing robust trace ctDNA capture from up to 200 μL plasma, mini‐input (1 ng) library preparation, unbiased genome‐wide coverage and comprehensive computational methods.ResultsA diagnostic signature comprising 15 ctDNA methylation markers exhibited high accuracy in the early (area under the curve [AUC] of 0.967) and advanced (AUC of 0.971) BC stages in multicentre patient cohorts. Furthermore, we revealed a ctDNA methylation signature that discriminates estrogen receptor status (Training set: AUC of 0.984 and Test set: AUC of 0.780). Different cancer types, including hepatocellular carcinoma and lung cancer, could also be well distinguished.ConclusionsOur study provides a toolset to generate unbiased whole‐genome ctDNA methylomes with a minimal amount of plasma to develop highly specific and sensitive biomarkers for the early diagnosis and molecular subtyping of cancer.

背景与目的 诱导性多能干细胞(induced pluripotent stem cells,iPSCs)和胚胎干细胞(embryonic stem cells,ESCs)具有许多共同特征,包括相似的形态、基因表达和体外分化谱.然而,iPSCs的基因组稳定性远低于ESCs.在本研究中,我们研究了iPSCs中DNA损伤修复的改变是否为其具有更大诱变倾向的原因.方法 将小鼠iPSCs、ESCs和胚胎成纤维细胞暴露于电离辐射(4 Gy),导致双链DNA断裂.照射4 h后使用全基因组重测序评估DNA损伤修复的保真度.我们还分析了分别源自iPSCs或ESCs的小鼠的基因组稳定性.结果 照射后,与胚胎干细胞和胚胎成纤维细胞相比,iPSCs具有较低的DNA损伤修复能力,有更多的体细胞突变和短片段插入缺失.iPSCs有更多的非同源末端连接DNA修复和更少的同源重组DNA修复.源自iPSCs的小鼠比ESCs小鼠以及C57对照小鼠的DNA损伤修复能力更低.结论 本研究结果部分表明,iPSCs的低基因组稳定性及其在体内的高致瘤性是由DNA损伤修复的低保真度所致.

Aim: Cancer stem cells (CSCs) drive triple-negative breast cancer recurrence via their properties of self-renewal, invasiveness and radio/chemotherapy resistance. This study examined how CSCs might sustain these properties. Materials & methods: Transcriptomes, DNA methylomes and histone modifications were compared between CSCs and non CSCs. Results: Transcriptome analysis revealed several pathways that were activated in CSCs, whereas cell cycle regulation pathways were inhibited. Cell development and signaling genes were differentially methylated, with histone methylation analysis suggesting distinct H3K4me2 and H3K27me3 enrichment profiles. An integrated analysis revealed several tumor suppressor genes downregulated in CSCs. Conclusion: Differential activation of various signaling pathways and genes contributes to the tumor-promoting properties of CSCs. Therapeutic targets identified in the analysis may contribute to improving treatment options for patients.

Histone methylation is a kind of important epigenetic modification which occurs on the lysine residue or arginine residue of histone tails（Zhang and Reinberg,2001）.It takes part in multiple biological processes,including gene expression,genomic stability,stem cell maturity,genetic imprinting,mitosis and development（Fischle et al.,2005）.Abnormal histone methylation pattern may

Background Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) share many common features, including similar morphology, gene expression and in vitro differentiation profiles. However, genomic stability is much lower in iPSCs than in ESCs. In the current study, we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis. Methods Mouse iPSCs, ESCs and embryonic fibroblasts were exposed to ionizing radiation (4 Gy) to introduce double-strand DNA breaks. At 4 h later, fidelity of DNA damage repair was assessed using whole-genome re-sequencing. We also analyzed genomic stability in mice derived from iPSCs versus ESCs. Results In comparison to ESCs and embryonic fibroblasts, iPSCs had lower DNA damage repair capacity, more somatic mutations and short indels after irradiation. iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair. Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice. Conclusions The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due, at least in part, to low fidelity of DNA damage repair.

>DNA methylation plays an essential role in mammalian development[1].However,how DNA methylation is inherited between generations and if there is family-specific DNA methylation pattern remains to be elucidated[2].In this study,we collect male blood samples including a big pedigree of the descendants of an ancient Chinese empire and samples from different haplogroups to study their whole genome DNA methylation pattern.We find 115 male family-specific methylation sites from three families.The difference of whole genome DNA methylation pattern correlates with