m6A RNA methylation

m6A is the most abundant and widely studied post-transcriptional RNA modification and refers to methylation at the sixth position of the adenylate nucleotide [7]. As early as the 1970s, m6A was recognized as a major modification in eukaryotic cell messenger RNA (mRNA) and other non-coding RNA. m6A has demonstrated regulatory roles in protein production by influencing mRNA stability, translation, splicing, and processing, as well as embryonic development and cell differentiation [8, 9]. The insertion of m6A is a dynamically reversible process, and relies on three types of methylation regulators: writers (methyltransferases), erasers (demethylases), and readers (binding proteins), collectively termed the writing-erasing-reading (WER) system (summarized in Table 1). Writers catalyze the installation of m6A and consist of methyltransferase-like protein 3 (METTL3), METTL14, and their accessory proteins RNA-binding motif protein 15 (RBM15), zinc finger CCCH-type containing 13 (ZC3H13), Wilms Tumor 1 associated protein (WTAP), and KIAA1429. Erasers remove the m6A modifications by demethylation and include AlkB homolog 5 (ALKBH5) and FTO-alpha-ketoglutarate-dependent dioxygenase (FTO) [10, 11]. Readers recognize m6A modifications on RNA and include insulin-like growth factor 2 mRNA binding proteins (IGF2BP), heterogeneous nuclear ribonucleoprotein (hnRNP), and members of the YTH family [12, 13]. The IGF2BP family consists of four distinct C-terminal KH domains, responsible for identifying m6A modifications, and two N-terminal RNA recognition motifs (RRMs) [14], that together promote the stability and translation of mRNA downstream target genes [15]. IGF2BP3, a member of the IGF2BP family, functions as a transcriptional regulator with implications for proliferation, invasion, and chemotherapy resistance of tumor cells [16, 17]. Elevated expression of IGF2BP3 has been consistently associated with poorer prognosis in lung cancer patients [18]. At present, evidence suggests that m6A modification regulates a series of malignant biological behaviors, and targeting m6A and associated regulatory proteins may be a promising method to prevent tumor progression [19]. However, more studies are needed to fully elucidate the complex mechanism of m6A modification in the regulation of the initiation and development of cancer.

Table 1

Regulatory proteins of RNA modification

Modification	Structure	Writer	Eraser	Reader
m6A		RBM15 ZC3H13 METTL3,14 WTAP KIAA1429	ALKBH5 FTO	IGF2BP hnRNP YTHDF1-3 YTHDC1-2
m5C		DNMT2 NSUN1-7	TET2	ALYREF YBX1
m7G		METTL1 WDR4	–	–
m1A		TRMT6 TRMT61A TRMT61B TRMT10C NML	FTO ALKBH1 ALKBH3 ALKBH7	YTHDF1 YTHDF2 YTHDF3 YTHDC1
ac4C		NAT10	–	–

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m5C RNA methylation

m5C is another common RNA modification that involves the addition of a methyl group to the carbon-5 position of the cytosine base in RNA. m5c modifications can exist on mRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), and long non-coding RNA (lncRNA), and are widely distributed across archaea, prokaryotes, and eukaryotes [20]. m5C participates in multiple stages of RNA metabolism, including splicing, stabilization, translation, maturation, and decay [21]. m5C expression relies on writers, such as NOP2/Sun domain (NSUN) RNA methyltransferase and DNA methyltransferase 2 (DNMT2), to catalyze m5C methylation [22, 23], while erasers, including tet methylcytosine dioxygenase 2 (TET2), demethylates m5C [24]. RNA binding protein ALY/REF export factor (ALYREF) recognizes m5C modifications on mRNA to promote mRNA nuclear output [22], while Y-box binding protein 1 (YBX1) binds directly to m5C modifications to stabilize mRNA [25]. In recent years, abnormal m5C modification and dysregulation of writers, erasers, and readers have played significant roles in the development and progression of various cancers, such as bladder, stomach, hepatocellular carcinoma (HCC), colorectal, prostate, breast, and lung cancer [23, 26]. However, further research is needed to identify the specific target genes and signal pathways that regulate m5C.

m7G RNA methylation

m7G is a highly conserved and prevalent post-transcriptional modification that plays a crucial role in regulating gene expression. In mRNA, m7G is primarily catalyzed by METTL1, a common methyltransferase, and forms a complex with WD repeat domain 4 (WDR4) [27]. In eukaryotes and a few archaea, m7G methylation at the mRNA 5ʹ cap is mediated by RNMT and RNMT-activating miniprotein (RAM) enzyme complex, that stabilize and protect the modified mRNA against exonuclization [28]. m7G influences various stages of mRNA processing and metabolism, including transcriptional stabilization, nuclear export, pre-mRNA splicing, translation, and polyadenylation [29, 30]. Additionally, m7G modifications have been shown to regulate the function of other RNA species, including rRNA and tRNA. The BUD23/TRMT112 methyltransferase complex catalyzes the formation of m7G at the G1639 site of 18s rRNA and induces 18s rRNA precursor biogenesis [31]. Dysregulation of m7G regulation can lead to aberrant m7G modification, promoting the development and progression of cancer.

m1A RNA methylation

In 1961, it was discovered that m1A modified the first nitrogen atom of RNA adenosine in mRNA, tRNA, rRNA, and lncRNA [32]. Although m1A modification is less common than m6A, both types of modifications share similar enzymes. It was found that nitrogen atoms could be transferred from m1A to m6A after Dimroth rearrangement in alkaline environments [33]. This suggests that RNA regulation may be dynamically alternating between m1A and m6A under certain conditions. m1A is controlled by methyltransferases (tRNA methyltransferase 6 (TRMT6), TRMT10C, TRMT61A, TRMT61B, and NML), demethylases (ALKBH1, ALKBH3, ALKBH7 and FTO), and readers (YTH N6-methyladenosine RNA binding protein 1–3 (YTHDF1-3) and YTHDC1). m1A regulates RNA stability and the initiation and extension of translation by modifying mRNA, tRNA, and rRNA [34]. Under physiological conditions, the methyl group in m1A carries a positive electrostatic charge which disrupts the base pairing with uracil [35]. Abnormal expression of m1A modifications may impact the biological function of RNAs and their potential interaction with partner proteins. Studies have found that aberrant m1A expression can contribute to a variety of diseases, including cardiovascular disease, lung disease, and Alzheimer's disease, as well as upregulate tumor cell proliferation, invasion, metabolism, and apoptosis [36–38].

ac4C RNA acetylation

N4-acetylcytidine (ac4C) is a highly conserved RNA acetylation modification found in eukaryotic and prokaryotic tRNAs, rRNAs, and mRNAs [39]. It plays a dominant role in the expression of genetic information and has been associated with a variety of conditions [40]. Previous studies have suggested that ac4C increases gene expression by regulating mRNA stability and translation efficiency, ensuring the accurate reading of codons and maintaining stability in tRNA [41]. Findings indicate that the location of the ac4c modification influences the function of RNA molecules. Insertion at helix 34 and 45 in rRNA was shown to promote self-biosynthesis and ribosome 40S subunit assembly and improve translation accuracy by enhancing mRNA stability [42, 43]. Conversely, ac4C expressed in the coding sequence (CDS) promoted translation extension, while insertion in the 5ʹ-untranslated region (5ʹ-UTR) inhibited the initiation of translation [44]. There is increasing evidence to suggest that ac4C plays an important role in the development, progression, and metastasis of cancer. The acetyltransferase NAT10, in conjunction with the adaptor protein THUMPD1 or attachment box C/D snoRNAs, is the only known protein that catalyzes the formation of ac4C [45]. Elevated NAT10 expression has been identified in various cancers across multiple organ systems, such as hepatocellular carcinoma, gastric, colon, pancreatic, cervical, bladder, esophageal, and lung cancer, with high levels significantly associated with increased malignant and aggressive characteristics of tumors [39].

RNA modifications in NSCLC

Lung cancer is the primary cause of cancer-related death worldwide [1, 46]. NSCLC is the dominant histopathological subtype of lung cancer [47], accounting for approximately 85% of lung cancers [48]. RNAs have been implicated in ineffective treatment strategies and the emergence of drug resistance, two major factors contributing to NSCLC tumor progression and poor prognosis. Therefore, in-depth research into the underlying mechanisms of NSCLC development and progression is critical to identify potential novel therapeutic targets.

m6A modification in NSCLC

m6A-dependent glycolysis in LUAD progression

Glycolysis is the primary pathway by which cells obtain energy in a low-oxygen environment. However, cancer cells exploit this process by reprogramming their metabolism to adapt to a hypoxic tumor microenvironment, a hallmark of malignancy [49, 50]. This metabolic shift increases glucose uptake, glycolysis, and lactic acid production, providing a continuous source of energy that fuels cancer cell proliferation, invasion, and migration [51]. Previous studies have linked m6A-dependent glycolysis to the progression of gastric, colorectal, and lung cancer [52, 53]. Ma et al. [54] recently reported significantly higher m6A levels in LUAD tumor tissue compared to adjacent healthy tissue. Further studies showed that m6A levels in LUAD tumor tissues were modulated by an upregulation of METTL3 and a downregulation of ALKBH5. LUAD mouse models demonstrated that the combination of increased METTL3 and decreased ALKBH5 had a strong tumor-promoting effect. Similarly, patients exhibiting simultaneous upregulation of METTL3 and downregulation of ALKBH5 had shorter survival times compared to patients expressing either molecule alone. The m6A modification reader YTHDF1 also plays a vital role in this process. High METTL3 and low ALKBH5 levels enhanced m6A methylation at position 359 A, facilitating the binding of ENO1 to YTHDF1 and enhancing ENO1 translation. ENO1 is a key enzyme involved in the catalyzation of 2-phosphoglycerate to produce phosphoenolpyruvate, promoting cytoplasmic glycolysis and promotion of LUAD progression (Fig. 1C). Therefore, targeting the m6A-dependant glycolytic pathway, particularly the ENO1-YTHDF1 complex, may be an effective therapeutic strategy for LUAD management.

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Fig. 1

Molecular mechanism of m6A modification involved in progression of NSCLC. A HNRNPA2B1 is increased in NSCLC and regulates lncRNA MEG3 through recognition of m6A, contributing to NSCLC tumorigenesis. B The m6A reader IGF2BP3 recognizes the m6A modification on MCM5 mRNA and increases MCM5 expression, thereby promoting the metastasis of LUAD cells. C Upregulation of the m6A writer METTL3, downregulation of m6A eraser ALKBH5, and upregulation of m6A reader YTHDF1 promote ENO1 mRNA translation by mediating m6A modification, thereby increasing cytoplasmic glycolysis and LUAD progression. D The IGF2BP3-COX6B2 axis mediates TKI resistance in NSCLC by promoting OXPHOS

Angiogenesis in NSCLC

Angiogenesis is the formation of new blood vessels driven by vascular endothelial growth factor (VEGF) and is an essential component for tumor growth and metastasis [57]. Zhang et al. [58] recently revealed that m6A modification, mediated by high METTL3 expression or low ALKBH5 expression, accelerated lung cancer angiogenesis by promoting the translation of VEGF-A (Fig. 2B). Further research found that m6A-mediated VEGF-A translation was initiated by methylation of the internal ribosome entry site (IRES) at the VEGF-A mRNA 5ʹ-UTR site, leading to the recruitment of the YTHDC2/eIF4GI complex. In addition, TRPM7, regulated by METTL3 and IGF2BP2, has been associated with the proliferation, migration, and angiogenesis of NSCLC. Another molecular factor that is also regulated by METTL3 and IGF2BP2 and contributes to angiogenesis and metastasis is lncRNA deoxyguanosine kinase antisense RNA 1 (DGUOK-AS1). Feng et al. [59] showed that DGUOK-AS1 silencing reduced the stability of TRPM7 by modulating METTL3/IGF2BP2-mediated m6A modification, inhibiting proliferation, migration, and angiogenesis of NSCLC cells (Fig. 2A). This was the first time an association between DGUOK-AS1 and TRPM7 was found. Fang et al. [60] also reported that IGF2BP2 was highly expressed in LUAD tumor cells and was responsible for upregulating FLT4 expression by mediating m6A modification of FLT4 mRNA. Elevated FLT4 expression promoted angiogenesis and metastasis of LUAD by activating the PI3K–AKT pathway (Fig. 2C). Therefore, the IGF2BP2–FLT4–PI3K–AKT signaling pathway may play a key role in lung cancer progression and function as a prognostic marker, potentially highlighting an alternative research direction for NSCLC targeting drugs.

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Fig. 2

m6A regulatory protein affects lung cancer angiogenesis by regulating the expression of related proteins. A High expression of m6A writer METTL3 and reader IGF2BP2 influence lung cancer angiogenesis and metastasis by modulating TRPM7 expression. B High expression of METTL3 or low expression of ALKBH5 accelerates the angiogenesis of lung cancer by increasing the expression of VEGF-A. C The expression of IGF2BP2 increases in LUAD tumor cells and modulates the expression of FLT4 by mediating m6A modification. This promotes the angiogenesis and metastasis of lung adenocarcinoma by activating the PI3K–AKT pathway

m6A modification mediates ferroptosis in NSCLC

Ferroptosis is an iron-dependent mode of regulated cell death that inhibits cancer growth, characterized by an increase in internal reactive oxygen species (ROS) and lipid peroxidation [61]. Ferroptosis is regulated primarily by glutathione peroxidase 4 (GPX4), as well as m6A regulatory proteins, such as METTL3, IGF2BP3, FTO, and YTHDF2 [62–65]. Recent studies have shown that exosomal miR-4443 regulated the expression of anti-ferroptosis factor 1 (FSP1) in an m6A-dependent manner via METTL3 [62]. High expression of miR-4443 has been shown to negatively regulate METTL3, resulting in increased FSP1 levels, which in turn inhibited cisplatin-induced ferroptosis and promoted cisplatin resistance in NSCLC (Fig. 3B). In addition, Xu et al. [63] also found that high expression of IGF2BP3 in LUAD enhanced mRNA stability and expression level of various anti-ferroptosis factors, such as GPX4, SLC3A2, ACSL3, and FTH1, which inhibited ferroptosis and promoted LUAD progression (Fig. 3A). These findings highlight the role of m6A and associated regulatory proteins in modulating ferroptosis in NSCLC. Targeting proteins like METTL3 or IGF2BP3 may serve as a new therapeutic strategy for LUAD.

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Fig. 3

Mechanisms of ferroptosis mediated by m6A and m5C modifications in NSCLC. A High expression of IGF2BP3 in LUAD enhances the mRNA stability and expression level of anti-ferroptosis factor through m6A modification, inhibiting iron ferroptosis and promoting LUAD progression. B Exosome miR-4443 negatively regulates METTL3, promoting the expression of FSP1 and regulating iron death by regulating m6A modification. C m5C writer NSUN2 is highly expressed in NSCLC tumor cells, and m5C modification is mediated in the NRF2 mRNA 5ʹUTR region recognized by m5C reader YBX1. The stability of NRF2 mRNA is enhanced, leading to the upregulation of NRF2 expression and inhibiting ferroptosis

m5C modification in NSCLC

Development of EGFR-TKIs drug resistance in patients with EGFR mutant NSCLC

EGFR-targeted therapy is the latest first-line treatment for patients with advanced NSCLC who have developed EGFR-activated mutations [73]. However, due to the inherent resistance to EGFR-TKIs, approximately 30% of patients have poor treatment response and poor recovery [74]. The development of the EGFR-mutant genotype is a significant challenge in the treatment of NSCLC, leading to poor prognosis and reduced treatment efficacy [75, 76]. Currently, known risk factors for EGFR-TKIs acquired resistance are secondary T790M mutation, MET amplification, HER2 amplification, and transformation into SCLC. However, little has been reported regarding the role and mechanism of RNA m5C modifications. Wang et al. [77] has suggested that RNA m5C methylation modification in NSCLC tumor drug resistance was mediated by the NSUN2-YBX1-QSOX1 axis (Fig. 4C). NSUN2 expression levels were elevated in NSCLC, and overexpression of NSUN2 modulated QSOX1 protein levels in an MFC-YBX1-dependent manner. The expression of QSOX1 was correlated with endogenous gefitinib resistance. Therefore, the NSUN2-YBX1-QSOX1 axis may act as a potential candidate biomarker for prognosis and therapeutic target in NSCLC patients with intrinsic resistance to EGFR-TKIs.

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Fig. 4

Molecular mechanisms of m5C modification involved in the progression of NSCLC. A YBX1 recognizes m5C methylation in PFKFB4 mRNA and induces an increase in PFKFB4 expression, thus promoting the progression of LUSC. B NSUN2 enhances the stability of downstream target PIK3R2 mRNA by regulating m5C modification, thereby activating the PI3K–AKT signal and promoting the malignant progression of LUAD. C The NSUN2-YBX1-QSOX1 axis mediates TKI resistance in NSCLC patients. D LINC02159 binds to ALYREF and activates downstream Hippo and Wnt/β-catenin pathways by mediating m5C to upregulate YAP1 expression, contributing to carcinogenesis

m7G modification in lung cancer

It has been reported that m7G regulatory proteins are upregulated and promote tumor progression in several cancers, including prostate cancer, bladder cancer, liver cancer, and lung cancer. However, the association between m7G regulatory proteins and tumor progression in LUAD patients has not been fully elucidated. Research has shown that a methyltransferase complex consisting of WDR4 and METTL1 catalyzes eukaryotic tRNA m7G modification, promoting the translation of cell cycle-related mRNAs and the growth and invasion of lung cancer [88]. In vitro analysis corroborated the association between the complex and cancer development, finding that knockdown of the WDR4-METTL1 complex slowed the progression of lung cancer cells. In addition, the m7G-related gene, NUDT4, promoted the proliferation of cancer cells in lung adenocarcinoma. While the specific mechanism underlying the anti-proliferative effects is not fully understood, NUDT4 may act as a novel target to inhibit the proliferation of tumor cells [89]. In addition, an m7G-Riskscore of LUAD was generated that incorporated m7G genetic alterations in the tumor microenvironment, such as CAND1, RRM2, and SLC2A1, to predict the overall survival (OS) of patients [90]. Analysis revealed that patients exhibiting a higher m7G-Riskscore had shorter OS, and m7G-Riskscore was highly correlated with clinical characteristics and prognosis of tumors, potentially highlighting its utility as an independent prognostic factor. Other studies have found that METTL1 promoted the formation of m7G methylation on VEGF-A mRNA, leading to an increase in VEGF-A translation, which in turn promoted tumor angiogenesis [91]. These findings highlight the role of m7G and associated regulatory proteins in the formation and progression of cancer and possible targets for therapy. However, the mechanisms underlying LUAD progression require further investigation.

m1A modification and lung cancer

ALKBH3, a member of the AlkB family and a demethylase targeting m1A, has been found to be involved in lung cancer tumorigenesis. Tasaki et al. [92] found that ALKBH3 is highly expressed in lung adenocarcinoma cells, with a significant negative correlation between expression profile and relapse-free survival (RFS). Further studies showed that ALKBH3 promoted the growth of lung adenocarcinoma cells by accelerating the G1/S transition and driving cellular proliferation. ALKBH3 gene silencing using siRNA transfection effectively elicited cellular senescence and inhibition of lung adenocarcinoma cell growth by inducing expression of p21/p27, highlighting the specific role of ALKBH3 in lung cancer and its therapeutic potential [92]. In addition, Zhou et al. [93] developed a comprehensive scoring system, termed Writer-Score, that integrated four different types of RNA modifications to evaluate the effect of neoadjuvant immunotherapy in patients with NSCLC. Subsequent research discovered a strong correlation between Writer-Score and the expression level of PD-L1. Primarily used to predict the prognosis of NSCLC patients who receive neoadjuvant PD-L1 inhibitors, the Writer-Score demonstrated high prognostic efficacy in NSCLC patients receiving neoadjuvant immunotherapy [93]. These findings highlight the potential utility of targeting m1A-associated regulatory proteins to mitigate the progression of lung cancer.

Not applicable.