2.1 N⁶-methyladenosine

Discovered in 1974, m⁶A is the predominant RNA modification in eukaryotes.³² Its broad influence manifests in biological growth, development, and cancer.^{33, 34} m6A plays a crucial role in influencing RNA stability, transport, splicing, and translation, affecting overall RNA expression.^{13, 35} m⁶A modification involves the catalysis of RNA methyltransferase (the “writer”), the removal of demethylase (the “eraser”), and interaction with m⁶A-binding protein (the “reader”). The writer includes various proteins,³⁶ such as methyltransferase-like 3 (METTL3),²⁰ methyltransferase-like 14 (METTL14),³⁷ Wilms’ tumor 1 associated protein (WTAP), zinc finger CCCH-type containing 13 (ZC3H13), RNA-binding motif protein 15 (RBM15), vir-like m⁶A methyltransferase associated, zinc finger protein 217 (ZFP217), and Hakai E3 ubiquitin–protein ligase (HAKAI).³⁸ The primary m⁶A writer complex includes METTL3 as the catalytic subunit, METTL14 as the stabilizer, and WTAP as the regulator. This complex binds to messenger RNA (mRNA) and increases the methylation of adenosine residues.^{38, 39}

In various cancer tissues, the METTL3 and METTL14 complexes play diverse roles, functioning as both oncogenes and tumor suppressors.^40-42 m⁶A demethylases, including fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), can dynamically, rapidly, and signal-dependently influence the function of m⁶A modifications.

FTO, the first demethylase discovered, has high-efficiency oxidative demethylation activity.⁴³ FTO was initially discovered due to its role in regulating obesity and diabetes.⁴⁴ FTO-mediated RNA m⁶A demethylation is not specific. FTO binds multiple RNA species, including mRNA, small nuclear RNA (snRNA), and tRNA, and can demethylate internal m⁶A and cap N6,2′-O-dimethyladenosine (m⁶A_m) in mRNA, internal m⁶A in U6 RNA, internal and cap m⁶A_m in snRNAs, and m¹A in tRNA.⁴⁵ The FTO gene is a multifaceted regulator, orchestrating a complex array of cellular processes.⁴⁶ It functions by demethylating the m⁶A modification on cyclin D1 mRNA. This demethylation leads to the degradation of cyclin D1 mRNA, which impairs the progression of the G1 cell cycle phase and, ultimately, causes a decline in cancer cell proliferation.⁴⁷ Furthermore, FTO is instrumental in regulating apoptosis, serving as a critical arbiter of cell survival and programmed cell death.⁴⁸ The gene's influence extends to cellular motility, where it governs migration.⁴⁸ These diverse roles underscore FTO's significance as a molecular nexus in cancer biology, with far-reaching implications for both tumor development and potential therapeutic intervention strategies.

Meanwhile, ALKBH5 can demethylate m⁶A sequences in single-stranded RNA (ssRNA). ALKBH5 has been employed in anti-PD-1 immunotherapy to regulate lactic acid levels in the tumor microenvironment (TME) and manage the accumulation of Tregs and myeloid-derived suppressor cells (MDSCs). ALKBH5 knockdown in a 4T1 mouse tumor model improved immunotherapy efficacy and mouse survival.⁴⁹

The identified m⁶A readers include the YTH domain family (YTHDF)1/2/3, YTH domain-containing protein 1/2 (YTHDC1/2), heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1), heterogeneous nuclear ribonucleoprotein C (HNRNPC), heterogeneous nuclear ribonucleoprotein G (HNRNPG), eukaryotic translation initiation factor 3 (eIF3), staphylococcal nuclease domain 1 (SND1), and insulin-like growth factor 2 mRNA-binding protein 1–3 (IGF2BP1–3). HNRNPA2B1 is a key regulator of proliferation, migration, and invasion in oral squamous cell carcinoma (OSCC), influencing tumor growth and metastatic potential. Its role in these processes makes it a significant therapeutic target and a potential marker for disease prognosis.⁵⁰ YTHDF2, an m⁶A reader, is pivotal in many cellular processes, including but not limited to migration, invasion, metastasis, proliferation, apoptosis, cell cycle regulation, viability, adhesion, differentiation, and inflammation, across various neoplastic diseases.^51-55 YTHDF2 exhibits differential affinities for mRNAs with m⁶A modifications; the C-terminus specifically targets these modified mRNAs, while the N-terminus directs the YTHDF2–mRNA complex to the cellular RNA decay checkpoint for accelerated degradation. YTHDF2's recognition of m⁶A modifications in mRNAs is essential for regulating mRNA translation and stability.⁵⁶

2.2 5-Methylcytosine

The m⁵C modification, which is catalyzed by S-adenosylmethionine (SAM) at the carbon-5 position of cytosine in RNA, is common in both mRNA and noncoding RNA (ncRNA). Sulfite sequencing has identified m⁵C within the coding regions of mRNA, especially near its translation initiation site.⁵⁷ Another study discovered that m⁵C modification is also present in the untranslated region (UTR) of mRNA transcripts.⁵⁸ m⁵C significantly influences many biological processes, such as cell proliferation, differentiation, migration, and apoptosis.^{59, 60}

m⁵C modifications are primarily facilitated by writer proteins (methyltransferases), eraser proteins (demethylases), and reader proteins (binding proteins). m⁵C methyltransferases include NOP2/Sun RNA Methyltransferase Family (NSUN) methyltransferase and DNA methyltransferase 2 (DNMT2),^61-63 which transfer a methyl group from adenosylmethionine to cytosine.

NSUN2 is a crucial RNA methyltransferase that introduces m⁵C into RNA. It methylates the majority of expressed tRNAs, as well as other ncRNAs and certain mRNAs.^{15, 64} In addition, NSUN2 functions as a direct glucose sensor, promoting tumorigenesis and resistance to immunotherapy by sustaining 3′ repair exonuclease 2 (TREX2) expression, which inactivates the cyclic GMP–AMP synthase (cGAS)/STING pathway in response to glucose activation.⁶⁵ In colorectal cancer (CRC), NSUN2 reprograms glucose metabolism m⁵C-dependently, increasing lactate production. Lactate accumulation in CRC cells, in turn, activates NSUN2 transcription through histone H3K18 lactylation (H3K18la) and induces NSUN2 lactylation at Lys356. This positive feedback loop of metabolic reprogramming promotes CRC progression. NSUN2 also plays a pivotal role in the progression of HCC.⁶⁷

The principal demethylases for m⁵C belong to the ten-eleven translocation (TET) enzyme family. Quantitative analysis via liquid chromatography–tandem mass spectrometry (LC–MS/MS/MS) indicates that TET overexpression can significantly increase the level of 5-hydroxymethylcytosine in human embryonic kidney 293T cells.^{68, 69} m⁵C readers can recognize and bind to m⁵C sites on RNA to enact biological functions. Two-dimensional liquid chromatography (2D-LC) analysis of m⁵C-modified RNA identified two m⁵C mRNA readers: ALY/REF export factor (ALYREF) and Y-box-binding protein 1 (YBX1).^{70, 71}

Studies have shown that ALYREF can recognize and bind to m⁵C sites in mRNA, facilitating its export from the nucleus to the cytoplasm. The overexpression of ALYREF increases bladder cancer (BCa) cell proliferation by promoting pyruvate kinase M2 (PKM2)-mediated glycolysis.²⁸ Similarly, YBX1 binds to m⁵C, regulating its presence in both coding and ncRNAs and influencing rRNA maturation.⁷²

2.3 N¹-methyladenosine

m¹A is an ancient RNA modification found in bacteria, archaea, and eukaryotes. It involves three components: a writer, an eraser, and a reader.

The writer complex mainly comprises various methyltransferases, including the tRNA methyltransferase (TRMT)6–TRMT61A complex (for mRNA and mitochondrial tRNA),⁷³ TRMT61B (for mitochondrial tRNA and rRNA),⁷⁴ TRMT10B (for tRNA), TRMT10C (for mitochondrial tRNA and mRNA),⁷⁵ and nucleomethylin (NML, also known as RRP8, for rRNA).^{76, 77} As a modification that occurs after transcription, m¹A plays a crucial role in RNA stability by affecting base pairing.⁷⁸

The m¹A erasers include demethylases, such as AlkB homolog 1 (ALKBH1), AlkB homolog 3 (ALKBH3), AlkB homolog 7 (ALKBH7), and FTO, that can remove methyl groups.⁷⁹ The enzymes FTO, ALKBH1, and ALKBH7 specifically target tRNA, while ALKBH3 acts on both tRNA and mRNA.^{80, 81} Although these m¹A erasers share some functional similarities with m⁶A erasers, proteins that specifically recognize m¹A in RNA have not yet been identified. The reader comprises various binding proteins, including YTHDF1, YTHDF2, YTHDF3, and YTHDC1.⁸²

The m¹A modification is essential in shaping the immune microenvironment and adds to the complexity of the TME. Changes in m¹A modification patterns have been detected in several cancers, including ovarian and colon cancer, HCC, and OSCC; these changes are linked to poorer prognoses.^{83, 84}

The m¹A methyl group, which carries a positive charge, affects RNA base pairing, altering the structure and function of the modified RNA.⁸⁵ During translation, m¹A modifications affect the initiation and elongation processes by regulating tRNA, mRNA, and rRNA.⁸⁵ Furthermore, these modifications increase the thermal stability of tRNA structures and contribute to the processing of nascent polycistronic mitochondrial RNA.^{86, 87}

Although m¹A is one of the most common RNA modifications in humans, its mechanisms and biological functions remain poorly understood. As m¹A shares some regulators, such as YTHDF1–3, with m⁶A, studying m⁶A may provide insights into m¹A. Due to its effect on RNA base pairing, m¹A is expected to influence RNA interactions, including those involving mRNA, microRNA (miRNA), long ncRNA (lncRNA), and circular RNA (circRNA).

2.4 N⁷-methylguanosine

m⁷G, the methylation of guanine at the N⁷ position in RNA, occurs in roughly 0.4% of all guanosines.⁸⁸ These m⁷G modifications are commonly located at the 5′ cap and internal sites of mRNA, as well as within rRNA, tRNA, and miRNA.^{89, 90} They actively affect biological and pathological processes via the metabolism of various RNA molecules.⁹¹

The primary enzyme responsible for this modification is METTL1, which works with the WD repeat domain 4 (WDR4) complex to enact m⁷G modifications in tRNA, miRNA, and mRNA, thereby influencing miRNA structure and biogenesis.⁹² The expression of WDR4 is closely correlated with METTL1 levels, underscoring WDR4's role as a crucial cofactor for METTL1.⁹³ Reduced levels of METTL1 and WDR4 are strongly linked to neurological disorders, including brain ischemia⁹⁴ and Alzheimer's disease.⁹⁵ The METTL1/WDR4 complex modifies a specific subset of tRNAs with m⁷G, stabilizing these mRNAs against decay, increasing translation efficiency, reducing ribosome stalling, and increasing the expression of growth-promoting proteins, which facilitates cancer development.^{91, 96, 97}

RNA guanine-7 methyltransferase (RNMT) and RNMT-activating mini-protein (RAM) are essential for effective mRNA cap methylation via m⁷G modifications.⁹² During T cell activation, stimulation of the T cell receptor induces RNMT, which coordinates the production of the mRNA, small nucleolar RNA (snoRNA), and rRNA necessary for ribosome biogenesis, improving translation capacity.⁹⁸ Williams-Beuren syndrome chromosome region 22 (WBSCR22) and tRNA methyltransferase 112 (TRMT112) mediate m⁷G methylation in rRNA.⁹⁹ WBSCR22, which was originally identified as one of the 26 genes associated with Williams syndrome, contains a nuclear localization signal and a highly conserved S-adenosyl-l-methionine binding motif.¹⁰⁰ As a ribosome biogenesis factor, WBSCR22 has been reported in various human cancers,¹⁰¹ significantly contributing to tumor proliferation, migration, and development.^{102, 103}

The m⁷G cap is recognized by eukaryotic translation initiation factor 4E (eIF4E) and the cap-binding complex (CBC), influencing mRNA maturation, nuclear export, and translation.¹⁰⁴

m⁷G is commonly found in mRNA, where it plays a crucial role in regulating the translation process. Its role varies across different RNA types and diseases. m⁷G promotes some cancers, such as BCa,¹⁰⁵ lung cancer,⁹³ liver cancer,¹⁰⁶ and gliomas,¹⁰³ but has opposite effects in teratomas¹⁰⁷ and PC.¹⁰⁷ Our knowledge of m⁷G regulators remains quite limited. To date, no specific demethylase has been discovered that can control m⁷G levels. The interactions between m⁷G and other posttranscriptional modifications are gaining greater attention and their underlying mechanisms invite further exploration.

2.5 Pseudouridine

Discovered 70 years ago, Ψ is the C5-glycoside isomer of uridine, as well as the earliest and most abundantly modified nucleoside in RNA.¹⁰⁸ Normal pyrimidine nucleosides form glycosidic bonds between the N-1 atom of the heterocycle and the C-1′ atom of the pentose. However, Ψ nucleosides bond the C-5 atom of the heterocycle to the C-1′ atom of the pentose.¹⁰⁹ Ψ is found in nearly all types of RNA, both coding and noncoding, and is highly conserved across species.^110-112

In humans, 14 Ψ writers have been identified that are responsible for catalyzing Ψ formation through either RNA-dependent or RNA-independent mechanisms. The RNA-dependent process involves dyskerin Ψ synthase 1 (DKC1), which is the catalytic subunit of the H/ACA snoRNA complex that catalyzes rRNA pseudouridylation.¹¹³ The remaining 12 writers are the RNA-independent single Ψ synthases (PUSs) PUS1/2/3/4/6/7/7L/9/10 and the RNA Ψ synthase domain-containing genes 1–4 (RPUSD1–4), each with specific cellular localization and RNA targets.^114-118

Currently, no specific “readers” or “erasers” for Ψ have been identified. The lack of erasers might reflect the inertness of the C−C bond between the base and ribose in Ψ compared with the C−N bond, making pseudouridylation irreversible.

When Ψ is incorporated into RNA, it increases the thermodynamic stability and spatial conformation of the RNA by increasing base stacking, improving base pairing, and rigidifying the sugar–phosphate backbone.^{119, 120} tRNA contains numerous pseudouridylation sites, including in the anticodon stem and loop, TΨC loop, and the D stem. These modifications contribute to tRNA's structural stability, codon–anticodon recognition, and translation efficiency and accuracy.^{121, 122} In rRNA, Ψ is found in critical regions such as the decoding site, mRNA channel, peptidyl transferase center, tRNA binding sites, and ribosomal subunit interface. These sites play crucial roles in ribosome assembly, function, and protein synthesis.^{123, 124} In mRNA, Ψ can improve precursor mRNA splicing, facilitate the conversion of nonsense codons to sense codons and improve base pairing at the ribosome decoding center, contributing to protein diversity.^{111, 117, 125} Other in vitro studies have reported that mRNA with Ψ translates more slowly and affects mRNA decoding more than unmodified mRNA.¹²⁶

Nucleoside modifications can effectively increase mRNA stability and translation efficiency while reducing immunogenicity in vivo. The benefits conferred by Ψ make mRNA a promising tool for gene replacement and vaccination.¹²⁷ Ψ deposition can endow modified RNA with distinct molecular properties, altering its fate or activity. In addition, as a prevalent RNA modification, Ψ plays a crucial role in various cancers.^128-130

2.6 Adenosine-to-inosine editing

Discovered over 30 years ago, adenosine-to-inosine (A-to-I) editing was initially identified for its role in introducing a premature stop codon in mRNA, resulting in the production of apolipoprotein B.^{131, 132} Recently, A-to-I editing has been linked to tumorigenesis and cancer progression. This process is facilitated by the adenosine deaminase acting on the RNA (ADAR) family of proteins, which deaminate adenosine to produce inosine in double-stranded RNA (dsRNA).^{133, 134}

In humans, two catalytic forms, ADAR1 and ADAR2, mediate dsRNA-specific A-to-I editing. These enzymes comprise deaminase domains and dsRNA-binding domains (dsRBDs), functioning as both writers and editors of A-to-I modifications.¹³⁵ In contrast, ADAR3 lacks editing capacity and functions as a negative regulator of ADAR1-mediated editing. It competitively binds to dsRNA, reducing the efficiency of ADAR1 and ADAR2.¹³⁶

In 1995, researchers reported that ADAR1 comprises two main isoforms: the interferon-inducible ADAR1 p150 and the constitutively expressed ADAR1 p110. ADAR1 p150 is located in both the cytoplasm and the nucleus, while ADAR1 p110 primarily exists in the nucleus.¹³⁷ In addition, ADAR1 contains a z-DNA binding domain.¹³⁸ Both ADAR1 and ADAR2 are ubiquitously expressed, whereas ADAR3 is predominantly found in the brain. In UTRs, A-to-I editing can regulate various RNA processes, including transport, translation, and degradation.^139-142

A-to-I editing facilitates the recruitment of the RNA-binding protein human antigen R to the 3ʹ UTR of cathepsin S (CTSS) mRNA, improving the stability and translation of CTSS mRNA.¹⁴³ In miRNAs, A-to-I editing can affect miRNA biogenesis and function.^{144, 145} Furthermore, A-to-I editing inhibits the formation of dsRNA structures in Alu elements, promoting canonical linear mRNA splicing and suppressing circRNA formation.^{146, 147} A-to-I editing influences lncRNAs’ secondary structure, stability, and interactions with other molecules.¹⁴⁸ Adenosine deaminase acting on tRNA 2 (ADAT2) and adenosine deaminase acting on tRNA 3 (ADAT3) are closely associated with tRNA decoding capabilities.¹⁴⁵

3.1 Metabolic reprogramming

During the metabolic reprogramming of tumor cells, the glycolytic pathway is particularly crucial (Figure 2A). Due to the Warburg effect, tumor cells preferentially use glycolysis even under aerobic conditions.¹⁴⁹ This metabolic strategy not only provides the energy required for rapid proliferation but also generates precursors for biosynthesis.¹⁵⁰ Glycolysis metabolites are essential for tumor cell growth and survival.¹⁵¹

Mitochondrial dysfunction and the activation of glycolysis are key characteristics of HCC. Nucleolar protein 2 (NOP2) promotes HCC progression by regulating Myc proto-oncogene protein (c-Myc) expression through m⁵C modification, increasing glycolysis.¹⁵² FTO suppresses apolipoprotein E (APOE) expression by reducing its m⁶A modification. This regulates the interleukin 6 (IL-6)/Janus kinase (JAK2)/STAT3 signaling pathway, inhibiting glycolysis and tumor growth in papillary thyroid carcinoma (PTC) cells.¹⁵³

In CRC, METTL3 directly induces the m⁶A–glucose transporter type 1 (GLUT1)–mechanistic target of the rapamycin complex 1 (mTORC1) axis, promoting glucose uptake, lactate production, and CRC progression. Inhibiting mTORC1 increases the anticancer effects of METTL3 silencing in CRC patients’ tissues and METTL3 transgenic mouse models.¹⁵⁴ In PC, YTHDF3 interacts with lncRNA DICER1-AS1, promoting its degradation under glucose depletion. This degradation reduces the inhibitory effect lncRNA DICER1-AS1 imposes on glycolysis, resulting in more available energy and metabolic intermediates for rapid cancer cell proliferation.¹⁵⁵ In lung adenocarcinoma (LUAD), m⁶A modification via METTL3 upregulation and ALKBH5 downregulation increases ENO1 translation, promoting glycolysis and providing energy for cancer cell proliferation. Increased ENO1 expression directly fosters tumor cell proliferation.¹⁵⁶ LHPP, a histidine phosphatase, inhibits glycolysis and proliferation in gastric cancer cells by suppressing glycogen synthase kinase 3 beta (GSK3b) phosphorylation and mediating hypoxia-inducible factor 1 alpha (HIF1A). METTL14 adds m⁶A modifications to LHPP mRNA, inhibiting its expression, which may increase GSK3b activity and reduce HIF1A activity to promote cancer cell proliferation.¹⁵⁷

Glucose not only promotes tumor progression through glycolysis but also acts as a signaling molecule to regulate tumor proteins. In prostate, liver, colon, and breast cancer (BC) cells, as well as melanoma, glucose binds to the N-terminal region of methyltransferase NSUN2, promoting its oligomerization and activation. Activated NSUN2 maintains overall m⁵C RNA methylation levels, stabilizing TREX2 and limiting dsDNA accumulation at the cell membrane and cGAS/STING activation, which promotes tumorigenesis.⁶⁵

In addition to glycolysis, glutaminolysis is a crucial component of the metabolic reprogramming of tumor cells. Through glutaminolysis, tumor cells obtain energy and carbon sources that are essential for biosynthesis and contribute to antioxidation. Glutaminolysis is vital for tumor cell growth and survival.^{158, 159}

NSUN2 methylates lncRNA NR-033928, stabilizing glutaminase (GLS) mRNA and promoting glutamine metabolism reprogramming, which increases gastric cancer proliferation.¹⁶⁰ Methyltransferase-like 16 (METTL16) influences gene expression by regulating m⁶A modification, promoting genes associated with cell proliferation and supporting leukemia cell survival and proliferation.¹⁶¹ IGF2BP2 binds to m⁶A-modified mRNA, increasing its stability and translation and thus regulating key genes involved in the glutamine metabolic pathway, such as MYC, GPT2, and solute carrier family 1 member 5 (SLC1A5), promoting acute myeloid leukemia (AML) cell proliferation and leukemia stemness/self-renewal.¹⁶²

The regulation of lipid metabolism also plays a vital role in the metabolic reprogramming of tumor cells. Tumor cells increase fatty acid synthesis and β-oxidation to meet the demands of rapid proliferation. Lipid metabolism is essential not only in the construction of tumor cell membranes but also in signal transduction.¹⁶³

The TRMT6/TRMT61A complex boosts the m¹A methylation of specific tRNAs, increasing peroxisome proliferator-activated receptor (PPAR) translation. This process stimulates cholesterol synthesis, activates Hedgehog signaling, and, ultimately, promotes the self-renewal and tumorigenesis of liver cancer stem cells (CSCs).¹⁶⁴ Cancer cells persistently engage in elevated glycolysis and glutaminolysis by reprogramming their internal metabolism, which drives the progression of HCC. Understanding these mechanisms can provide new insights for cancer therapies. Hepatitis B virus X-interacting protein (HBXIP) is upregulated in liver cancer tissues and mediates the METTL3-induced metabolic reprogramming and malignant behavior of HCC cells, accelerating tumor progression.¹⁶⁵ The elevated expression of FTO correlates with poor prognoses in patients with esophageal cancer (EC). FTO promotes lipid droplet (LD) formation by regulating hydroxysteroid 17-beta dehydrogenase 11 (HSD17B11) expression, affecting cell proliferation.¹⁶⁶ In advanced BCa, m⁶A modification regulated by methyltransferase METTL14 increases the expression of lncDBET, which activates the PPAR signaling pathway and leads to increased lipid metabolism and cancer proliferation.¹⁶⁷

3.2 Biosynthetic pathways

In tumor cell biosynthetic pathways, nucleic acid synthesis and ribosome synthesis are critical processes (Figure 2B). Tumor cells increase purine and pyrimidine synthesis to support rapid DNA and RNA replication and boost ribosome synthesis to meet high protein production demands.^{168, 169} These biosynthetic activities provide the necessary genetic material and proteins for rapid cell proliferation, promoting the cancer's growth and spread. This surge in nucleic acid and ribosome synthesis is closely tied to the swift proliferation of tumor cells, playing a pivotal role in their growth mechanisms.

The m⁷G methyltransferase RNMT regulates ribosome synthesis, making it essential for T cell activation. m⁷G in tRNAs maintains tRNA structural integrity by promoting stability and translation efficiency and reducing ribosome stalling, thus influencing the mRNA translatome.⁹⁸ WBSCR22 is a ribosome biogenesis factor. The protein TRMT112 interacts with and promotes the overproduction of WBSCR22, which reduces ISG15 levels. This decrease in ISG15 expression reduces PC cells’ ability to migrate to and invade surrounding tissues.¹⁰²

m⁵C RNA modification regulators, such as TRDMT1, NSUN1, and NSUN4, are differentially expressed in LUAD, potentially affecting RNA translation and stability and, thus, regulating tumor cell proliferation. Different m⁵C modification clusters are associated with differences in patient survival, supporting this interpretation.¹⁷⁰ NOP2/NSUN1, an oncogene, is overexpressed in various cancers. By regulating rRNA processing and function, NOP2/NSUN1 may influence ribosome synthesis and promote cancer cell proliferation.¹⁷¹

In the nucleus, METTL1 regulates gene expression by modulating m⁶A modifications to mRNA. METTL16 operates in the cytoplasm, where its methyltransferase domain facilitates interactions with the eukaryotic initiation factors eIF3a and eIF3b, along with rRNA. These interactions aid in the formation of the translation initiation complex and promote mRNA translation, and they collectively promote HCC proliferation.¹⁷² The depletion of methyltransferase-like 5 (METTL5)-mediated m⁶A modification on 18S rRNA impairs 80S ribosome assembly, affecting mRNA translation and, consequently, protein synthesis related to cell proliferation.¹⁷³ In gastric cancer, METTL3 associates with PABPC1, stabilizing the attachment of the eIF4F complex and preferentially boosting the translation of specific epigenetic factors. This mechanism enables cancer cells to more efficiently synthesize proteins related to proliferation, thereby promoting tumor growth.¹⁷⁴ In glioblastoma, upregulated METTL3 methylates ADAR1 mRNA, increasing the resulting protein levels and establishing a protumor mechanism that involves METTL3, YTHDF1, and ADAR1. ADAR1 promotes cancer by binding to CDK2 mRNA independently of deaminase activity, and ADAR1 knockdown significantly inhibits glioblastoma growth in vivo. The METTL3/ADAR1 axis establishes a connection between m⁶A modification and A-to-I RNA editing during posttranscriptional regulation, which reveals a novel pathway involved in cancer progression.¹⁷⁵

ADAR is markedly upregulated in BC tissues and may drive the progression of the disease by interacting with OASL, STAT2, and IFIT3. In vitro experiments show that ADAR knockdown hinders BC cell proliferation, invasion, and migration, while ADAR overexpression promotes these activities.¹⁷⁶ The ADAR1 enzyme performs A-to-I edits, preventing the sensing of endogenous dsRNA. In triple-negative BC (TNBC) cells, ADAR1 knockdown inhibits cell proliferation and tumorigenesis, causing strong translational suppression.

TNBC cell lines that rely on ADAR1 also demonstrate increased levels of genes activated by interferon.^{177, 178}

3.3 Regulation of signaling pathways

The signaling pathway comprising phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) is essential for tumor cell communication, influencing cell growth, division, and maintenance (Figure 2C). The dysregulation of this pathway is strongly linked to the augmentation and persistence of numerous tumor types. Modulating this pathway can effectively influence tumor cell behavior.¹⁷⁹

FTO facilitates pancreatic cancer cells’ growth and resistance to gemcitabine by modulating phosphatase and tensin homolog (PTEN) expression and affecting the PI3K/AKT signaling cascade.¹⁸⁰ Meanwhile, YTHDF1 stabilizes m⁶A-modified mRNA, boosting the expression of its downstream target, RPN2, which then activates the PI3K/AKT/mTOR pathway, thereby promoting BCa cells’ growth and resistance to cisplatin. Suppressing METTL3 and YTHDF1 expression decreases RPN2 levels, which inhibits this pathway and affects both cancer cell proliferation and chemotherapy efficacy.¹⁸¹ High YTHDF2 expression promotes diffuse large B-cell lymphoma (DLBCL) cell proliferation and supports tumor growth by inhibiting apoptosis. YTHDF2 also influences ceramide metabolism and related pathways (e.g., extracellular signal-regulated kinase [ERK] and PI3K/AKT), further driving tumor progression.¹⁸² IGF2BP2 augments cellular survival and proliferation by upregulating the activity of p-AKT and c-Myc. The activation of the p-AKT pathway is intimately connected with cellular growth, metabolic processes, and survival.¹⁸³

3.4 Cell cycle regulation

Cell cycle regulation is fundamental to tumor cell proliferation (Figure 2D). Cell cycle advancement is driven by cyclins and cyclin-dependent kinases (CDKs), which together facilitate tumor cell growth. Abnormal cell cycle regulation drives the uncontrolled proliferation of tumor cells.¹⁸⁴ Understanding this regulatory process is essential for elucidating tumor cell growth mechanisms.¹⁸⁵

Serine hydroxymethyltransferase 2 (SHMT2) influences c-Myc mRNA stability and expression by regulating its m⁶A modification, inhibiting c-Myc expression through METTL3/FTO/ALKBH5/IGF2BP2 and, thereby, blocking EC cell proliferation.¹⁸⁶ The oncogene c-Myc is instrumental in advancing the cell cycle from the G1 to the S phase by upregulating genes such as Cyclin D and CDK4/6, which are essential for cellular proliferation.¹⁸⁷ The tRNA m⁷G modification that is mediated by METTL1 and WDR4 is linked to unfavorable prognoses in HCC. Silencing METTL1 or WDR4 curtails HCC cell proliferation, migration, and invasion; conversely, elevated METTL1 expression facilitates HCC progression.⁹⁷ METTL1 regulates epidermal growth factor receptor (EGFR)/EFEMP1 translation by modifying certain tRNAs, leading to BCa proliferation, migration, and invasion.¹⁰⁵ FTO, an m⁶A demethylase, regulates RNA methylation status, affecting the cell cycle and proliferation-related gene expression. The upregulation of FTO may promote uterine leiomyosarcoma cell proliferation.¹⁸⁸ ALKBH5 demethylates m⁶A modifications, influencing gene expression and cell processes, including proliferation. Inhibiting ALKBH5 increases m⁶A levels, which generally decreases mRNA stability and translation efficiency, thereby affecting cell cycle regulation.¹⁸⁹ This inhibition can lead to reduced cancer cell proliferation by altering mRNA dynamics.¹⁹⁰ IGF2BP1 promotes HCC proliferation by regulating c-Myc expression.¹⁹¹

In summary, RNA modifications play pivotal roles in metabolic reprogramming, biosynthetic pathways, signaling pathway regulation, and cell cycle control in tumor cells. These mechanisms collectively affect tumor cells’ rapid proliferation, driving cancer progression. A thorough investigation of RNA modification mechanisms and functions deepens our comprehension of tumor biology and offers potential targets for new cancer treatment strategies.

4.1 Epithelial–mesenchymal transition

EMT plays a critical role in normal embryonic development and tissue repair (Figure 3A). However, its abnormal reactivation is linked to tumor cells’ malignant traits during cancer advancement and metastasis. EMT equips tumor cells with increased migratory and invasive properties by downregulating epithelial markers, such as E-cadherin, and upregulating mesenchymal markers, including N-cadherin and vimentin.¹⁹²

YTHDF2 overexpression triggers EMT in lung squamous cell carcinoma (LUSC), promoting greater cell migration and invasion. This phenomenon has been substantiated by Transwell invasion assays and wound healing assays.¹⁹³ In pancreatic ductal adenocarcinoma (PDAC), circNEIL3 enhances proliferation and metastasis through the circNEIL3/miR-432-5p/ADAR1/GLI1 axis, affecting both the cell cycle and EMT processes. Its expression is modulated by ADAR1 through a negative feedback mechanism.¹⁹⁴ In nasopharyngeal carcinoma (NPC), METTL1 facilitates proliferation and EMT by activating the WNT/β-catenin signaling pathway.¹⁹⁵ In HCC, m⁷G modification increases EMT by promoting the translation of SLUG and SNAIL, key EMT regulators, increasing cell invasion and metastasis, especially after radiofrequency ablation (RFA).¹⁹⁶

FTO knockdown significantly inhibits the invasion and stemness of EC cells, while FTO overexpression promotes these traits. Invasion and stemness are crucial for cancer cell migration and metastasis, and FTO enhances EC cell migration and invasion.¹⁶⁶ circGPR137B inhibits distant metastasis in HCC by upregulating FTO.¹⁹⁷ MIR100HG is a positive regulator of EMT; m⁶A modification strengthens the association between MIR100HG and hnRNPA2B1, stabilizing TCF7L2 mRNA. This activation of the Wnt/β-catenin signaling pathway promotes CRC cell invasion and metastasis.¹⁹⁸ METTL14 regulates m⁶A modification of lncRNA–NEAT1_1, affecting renal cell carcinoma (RCC) cell migration. YTHDF2 selectively recognizes m⁶A modifications on NEAT1_1 and accelerates its degradation, inhibiting cell metastasis and demonstrating the critical role m⁶A modification plays in cancer metastasis.¹⁹⁹

4.2 Regulation of the immune microenvironment

Another crucial factor in cancer metastasis is the regulation of the tumor immune microenvironment (Figure 3B). RNA modifications significantly affect tumor immune evasion and metastatic potential by altering immune cell function and infiltration. These modifications crucially mediate interactions between tumor cells and immune cells.^200-202

The loss of METTL14 exacerbates the macrophage response to acute bacterial infections and drives CD8+ T cells toward dysfunction, impairing their ability to eliminate CRC tumors.²⁰³ FTO-mediated m⁶A demethylation increases the levels of the transcription factors c-Jun, JunB, and C/EBPβ in tumor cells, which alters glycolytic metabolism, impairs CD8+ T cell function, and inhibits tumor progression. Furthermore, m⁶A modifications alter the tumor immune microenvironment by modulating immune cell infiltration, contributing to cancer metastasis.²⁰⁴ The accumulation of MDSCs is intricately linked to tumor metastasis. METTL3 facilitates MDSC migration through the basic helix-loop-helix family member e41 (BHLHE41)–CXC motif chemokine ligand 1 (CXCL1)/CXCR2 axis, which may influence tumor cells’ metastatic potential.²⁰⁵ The subcellular localization of METTL3 plays a critical role in cancer metastasis. An IL-6-dependent positive feedback loop amplifies the function of nuclear METTL3, initiating BC metastasis. In addition, METTL3 deacetylation and nuclear translocation are associated with increased m⁶A modification levels, which may influence the expression of genes related to metastasis and, ultimately, promote it.²⁰⁶ ALKBH5 promotes cancer cell migration and invasion by modulating m⁶A modifications, a phenomenon confirmed by studies on glioma cells in vitro and in vivo. It also recruits M2 macrophages into the TME, potentially promoting tumor metastasis.²⁰⁷

m⁷G modification may increase cancer cells’ invasiveness and promote metastasis by altering the composition of immune cells within the TME. An immunosuppressive microenvironment can diminish the immune system's ability to monitor tumor cells, elevating the risk of cancer cell metastasis.²⁰⁸ Core m⁷G-modified genes FN1 and ITGB1 regulate immune cell infiltration (e.g., macrophage and neutrophil upregulation) by fostering interactions between the stroma and cancer cells, driving invasion and metastasis.²⁰⁹ Although glioma metastasis is less pronounced than that of other cancers, m⁷G can still influence tumor cell invasion via the SPP1 and PTN signaling pathways, facilitating its expansion into surrounding tissues.²¹⁰

A-to-I editing of AZIN1 promotes tumor angiogenesis and increases tumor cell invasion and metastasis by upregulating IL-8.²¹¹ Having completed our discussion of the effects of RNA modifications on the immune microenvironment, we will next analyze their role in signaling pathway regulation.

4.3 Regulation of signaling pathways

The regulation of signaling pathways is essential for tumor cell migration and invasion (Figure 3C). RNA modifications affect tumor cell behavior through multiple signaling pathways, such as the PI3K/AKT pathway, the MAPK/ERK pathway, and others.

The PI3K/AKT pathway is fundamental for cell growth, proliferation, and survival. The upregulation of fibroblast growth factor 19 (FGF19) and its receptor, fibroblast growth factor receptor 4 (FGFR4), is associated with HCC cells’ invasiveness. By driving IGF2BP1 expression, FGF19/FGFR4 may foster metastasis. These proteins can promote programmed death ligand 1 (PD-L1) upregulation via the PI3K/AKT pathway, boosting tumor cells’ immune evasion and metastatic potential.²¹² METTL14 upregulation also inhibits RCC cell migration. The PI3K/AKT pathway is integral not only to cell proliferation but also to cell migration and invasion. By inhibiting this pathway via m⁶A modification, METTL14 indirectly suppresses cancer cell migration and invasion.²¹³ FTO inhibitors may also alter tumor cell invasiveness by affecting cell proliferation and survival, influencing their metastatic capacity. Furthermore, FTO inhibitors can regulate the Wnt/PI3K–AKT signaling pathway, affecting cancer metastasis.²¹⁴ The expression of EphA2 and vascular endothelial growth factor A (VEGFA) are closely linked to tumor metastasis. Research indicates that METTL3, through an IGF2BP2/3-dependent mechanism, inhibits the degradation of EphA2 and VEGFA mRNA by modulating the PI3K/AKT and ERK1/2 pathways, facilitating metastasis.²¹⁵

The MAPK/ERK pathway facilitates tumor cell growth and migration by controlling genes related to the cell cycle and cell proliferation. RNA modifications are also crucial in this pathway. ALKBH5 boosts both the proliferation and metastasis of HCC cells by upregulating MAP3K8, which, in turn, activates the JNK and ERK signaling pathways that are essential for cell migration and metastasis.²¹⁶ Deficiencies of the m⁶A methyltransferase METTL3 hinder YTHDF1-mediated translation of Sprouty-related EVH1 domain-containing protein 2 (SPRED2), augmenting the activation of nuclear factor kappa B (NF-κB) and STAT3. This augmentation promotes tumor growth and metastasis through the ERK pathway.²¹⁷ METTL3 also plays a pivotal role in cancer metastasis through its regulation of the MAPK signaling pathway and related immune responses.²¹⁸

In addition to these pathways, RNA modifications influence tumor cell migration and invasion through other signaling pathways.

METTL3 governs the m⁶A modification of Frizzled-10, increasing its expression in liver cancer stem cells and activating the β-catenin and yes-associated protein 1 (YAP1) signaling pathways. This increases tumor cell self-renewal and tumorigenicity. The upregulation of Frizzled-10 forms a positive feedback loop, further activating METTL3 and driving HCC metastasis and drug resistance.²¹⁹

FTO not only promotes cell proliferation but also supports tumor metastasis by affecting cell migration and invasion. It regulates heat shock factor 1(HSF1)Heat Shock ProteinsHeat Shock Proteins expression through demethylation, altering HSP levels, which promotes the proliferation, survival, migration, and invasion of multiple myeloma cells.²²⁰ Carbon ion radiotherapy elevates METTL3 levels and its associated m⁶A modifications in NSCLC cells, influencing cancer cell migration and invasion. By regulating critical gene expression, m⁶A modifications can either promote or suppress cancer metastasis.²²¹ The overexpression of WTAP markedly increases ovarian cancer cells’ invasive potential by modulating miR-200 expression and affecting genes involved in cell migration and invasion.²²² The upregulation of METTL16 in HCC induces m⁶A modifications of lncRNA–RAB11B-AS1, decreasing its transcript stability and resulting in its downregulation. This process promotes the proliferation, migration, and invasion of HCC cells, inhibits apoptosis, and facilitates tumor growth in vivo.²²³

4.4 Metabolic reprogramming in cancer metastasis

Metabolic reprogramming is essential for tumor cells to adapt to their rapid proliferation and challenging environmental conditions. Because RNA modifications regulate metabolic pathways, affecting energy metabolism and biosynthesis, they can enhance migration and invasion.

YTHDF1 increases BC cell tumorigenicity and metastasis by upregulating PKM2, promoting glycolysis. Inhibiting YTHDF1 eliminates YTHDF1-dependent tumor growth and metastasis.²²⁴ Targeting METTL3/14 increases ACLY and SCD1 protein levels, along with the production of triglycerides and cholesterol and the accumulation of LDs. This change in membrane lipid composition fosters tumor cell migration and invasion.²²⁵ Mitochondrial m⁵C modification is essential for cancer cell metastasis. Tumor cells that are dependent on CD36 rely on m⁵C modifications to initiate invasion and dissemination. Cells deficient in m⁵C exhibit impaired metastasis, underscoring the significance of RNA modifications in modulating mitochondrial oxidative phosphorylation and metabolic pathways.²²⁶

In conclusion, RNA modifications drive numerous mechanisms underlying cancer metastasis. A comprehensive investigation of the specific functions and mechanisms of these modifications will improve our understanding of the intricate processes driving tumor metastasis and offer novel insights for future therapeutic strategies.

5.1 Types of programmed cell death

Programmed cell death is an active cellular process that occurs under particular conditions, encompassing apoptosis, autophagy, and ferroptosis. Apoptosis is triggered by internal and external signals involving enzymes, such as caspases, and various signaling pathways (Figure 4A). Many anticancer drugs kill tumor cells by inducing apoptosis.

The m¹A modification influences the stability of E2 promoter binding factor 1 (E2F1) mRNA, a key regulator of the cell cycle and apoptosis. By regulating E2F1 expression, m¹A can indirectly affect programmed cell death in cancer cells.²²⁷ The m⁵C reader ALYREF is notably upregulated in HCC tissues and cell lines. ALYREF knockdown markedly suppresses the proliferation of Huh7 and HepG2 cells and elevates apoptosis rates, demonstrating tumor-suppressive effects in vivo.²²⁸

YTHDF1 upregulation may inhibit antitumor immune responses, affecting apoptosis and programmed cell death. FTO inhibitors, including Compound 18097, augment the m⁶A modification in suppressor of cytokine signaling 1 (SOCS1) mRNA, which increases its stability and activates the P53 pathway, facilitating apoptosis.²²⁹ FTO inhibitor 44/ZLD115 upregulates RARA and downregulates MyC in leukemia cells, inducing apoptosis.²³⁰ IGF2BP2 regulates m⁶A modifications, affecting mitochondrial activity and gene expression in hematopoietic stem cells HSCs and, thereby, influencing cell survival and programmed death. Mitochondrial activity is closely linked to both energy metabolism and apoptosis.²³¹ IGF2BP1 knockdown induces cancer cell apoptosis; this effect highlights its role in programmed death. Cucurbitacin B (CuB), a chemogenetic small molecule, promotes apoptosis by blocking IGF2BP1's recognition of m⁶A-modified mRNA, which affirms m⁶A's critical role in cancer cell survival and death regulation.¹⁹¹ High FTO expression inhibits apoptosis and promotes leukemia cell survival by affecting the PDGFRB/ERK signaling axis.^{232, 233} IGF2BP2 knockdown reduces T-ALL cell proliferation and increases apoptosis by modulating m⁶A modifications and interacting with NOTCH1.²³⁴

ADAR1 prevents the accumulation of Z-RNA, inhibiting ZBP1 activation and RIPK3-mediated necroptosis and helping cancer cells evade programmed cell death.²³⁵

Autophagy is a cellular process that involves the degradation and recycling of intracellular components to maintain homeostasis (Figure 4B). Under specific conditions, autophagy can result in cell death. m⁶A modifications can affect cell viability by altering the expression of genes associated with autophagy.²³⁶ The m⁶A methyltransferase METTL3 targets DCP2, influencing the regulation of the Pink1-Parkin pathway, which, in turn, affects mitochondrial autophagy and damage in small-cell lung cancer (SCLC) cells. This interaction contributes to chemotherapy resistance in SCLC patients.²³⁷ METTL3 and YTHDF1 facilitate the m⁶A modification of Rubicon mRNA, increasing transcript stability. Rubicon, in turn, inhibits the fusion process between autophagosomes and lysosomes, obstructing the clearance of LDs.²³⁸

Ferroptosis is an emerging form of programmed cell death characterized by the accumulation of intracellular iron and subsequent peroxidation of lipids²³⁹ (Figure 4C). FTO promotes OTU deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1) expression by demethylating its m⁶A, inhibiting ferroptosis and increasing radioresistance.²⁴⁰ FTO acts as a tumor suppressor in PTC by downregulating the cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) via the ferroptosis pathway.²⁴¹ METTL3 engages the m⁶A reader YTHDF1 to boost m⁶A modification and the translation of SLC7A11, facilitating LUAD cell proliferation and suppressing ferroptosis.²⁴² The METTL3/IGF2BP1/m⁶A axis stabilizes SLC7A11 mRNA, upregulating its expression by preventing deadenylation. This upregulation facilitates hepatoblastoma (HB) cell proliferation and mitigates ferroptosis in vitro and in vivo.²⁴³ The upregulation of m⁶A–SLC7A11 in glioblastoma confers resistance to ferroptosis, suggesting that targeting the m⁶A–SLC7A11 axis could, instead, promote cancer cell ferroptosis.²⁴⁴

Hypoxia-induced lncRNA–CBSLR interacts with YTHDF2, forming a signaling axis that reduces cystathionine beta-synthase (CBS) mRNA stability. This affects the methylation and degradation of ACSL4 protein, lowering proferroptotic phosphatidylethanolamine levels and leading to ferroptosis resistance.²⁴⁵ CD8 T cells stimulate ACSL4 expression via IFNγ, altering tumor cell lipid profiles and promoting the incorporation of arachidonic acid (AA) into phospholipids, inducing ferroptosis. Low-dose AA further promotes this mechanism and boosts immune checkpoint blockade (ICB)-induced antitumor immunity. Targeting the ACSL4 pathway could be an anticancer strategy.²⁴⁶ The inhibition of protein arginine methyltransferase 3 (PRMT3) leads to the upregulation of METTL14, which increases m⁶A–YTHDF2-dependent methylation. This process diminishes the stability of glutathione peroxidase 4 (GPX4) mRNA, heightens lipid peroxidation, and accelerates ferroptosis in endometrial cancer.²⁴⁷ YTHDF1 suppresses CD8+ T cell-induced ferroptosis in PCa, affecting programmed cell death. YTHDF1 upregulation increases PD-L1 expression, diminishing T cell cytotoxicity and ferroptosis; this effect affirms YTHDF1’s critical role in tumor immune evasion.²⁴⁸

5.2 Tumor immune microenvironment

The regulation of the tumor immune microenvironment is another key factor in cancer programmed cell death. CD8+ T cells serve as key effector cells that can directly eliminate tumor cells, and their infiltration levels within the TME are strongly linked to prognoses (Figure 5A). Boosting the infiltration and activity of CD8+ T cells is a primary objective of numerous immunotherapy approaches.

After RFA in HCC, METTL1 upregulation induces TGF-β2 translation, creating an immunosuppressive environment and inhibiting CD8+ T cell activity. Fewer CD8+ T cells decrease tumor cells’ sensitivity to programmed cell death. METTL1 influences tumor cell survival and programmed cell death by regulating the immune microenvironment.²⁴⁹ Within the TME, a deficiency in YTHDF2 augments the antigen-presenting capacity of tumor-associated macrophages (TAMs), facilitating the activation and proliferation of CD8+ T cells. Activated CD8+ T cells effectively recognize and kill tumor cells, promoting programmed cell death. Therefore, YTHDF2 affects tumor cell survival and death by regulating TAM function.²⁵⁰ Elevated YTHDF2 expression in SCLC is associated with decreased immune infiltration, manifesting as reductions in cluster of differentiation 4 (CD4)+ and cluster of differentiation 8 (CD8)+ T cells, affecting immune-related treatments and resulting in poor prognoses.²⁵¹ ALKBH5 promotes CD8+ T cell infiltration in the CRC microenvironment by inhibiting the NF-κB pathway and reducing C-C motif chemokine ligand 5 (CCL5) expression. Increased CD8+ T cell infiltration may promote tumor cell programmed cell death.²⁵²

circCCAR1 induces CD8+ T cell dysfunction by stabilizing programmed cell death protein 1 (PD-1), potentially reducing tumor cells’ sensitivity to programmed cell death. IGF2BP3 increases circCCAR1 stability, indirectly affecting tumor cell survival and death.²⁵³ YTHDF1 inhibits CD8+ T cell cytotoxicity by inducing IL-6 secretion, allowing tumor cells to evade immune surveillance and inhibiting programmed cell death. This immunosuppressive environment facilitates tumor cells’ survival and proliferation.²⁵⁴ The reduction of YTHDF1 impedes tumor growth by reinstating CD8+ T cell infiltration, suggesting its significant role in programmed cell death.²⁵⁵ IGF2BP1 affects programmed cell death by reducing CD8+ T cell-mediated tumor cytotoxicity. The upregulation of IGF2BP1 increases PD-L1 stability, inhibiting CD8+ T cell function and reducing apoptosis in CRC.²⁵⁶ Retinoic acid-inducible gene I (RIG-I) (encoded by DDX58 mRNA) influences immune cell activity through the interferon alpha (IFNα) signaling pathway. The upregulation of ALKBH5 inhibits this mechanism, possibly enabling tumor cells to evade programmed cell death and, thereby, fostering tumor growth.²⁵⁷

Conversely, regulatory T (Treg) cells help tumor cells evade immune attack by inhibiting effector T cell activity (Figure 5B). Targeting Treg cells can improve antitumor immune responses.²⁵⁸ In tumors with high glycolytic activity, Treg cells actively uptake lactate through monocarboxylate transporter 1 (MCT1), which facilitates the translocation of nuclear factor of activated T-cells 1 (NFAT1) to the nucleus and increases PD-1 expression. Effector T cells, however, have suppressed PD-1 expression. Lactate in the high-glycolytic TME significantly affects cancer programmed cell death and immunotherapy efficacy by affecting Treg cells’ PD-1 expression.²⁵⁹ YTHDF2 deficiency increases apoptosis and impairs the suppressive function of Treg cells in the TME. Reduced Treg cells may increase tumor cells’ sensitivity to programmed cell death as Treg cells typically protect tumor cells by inhibiting effector T cell activity.²⁶⁰

Beyond CD8+ T cells and Treg cells, numerous other critical immune cells and factors populate the tumor immune microenvironment (Figure 5C). Specifically, in head and neck squamous cell carcinoma (HNSCC), the overexpression of ALKBH5 suppresses RIG-I-mediated IFNα secretion via the IκB kinase epsilon/TBK1/interferon regulatory factor 3 (IRF3) signaling pathway, which is closely related to tumor cell programmed cell death. In addition, the overexpression of ALKBH5 diminishes the population of tumor-infiltrating lymphocytes, which could reduce immune surveillance and programmed cell death.²⁵⁷ METTL14-mediated m⁶A modification may influence tumor cell programmed cell death by regulating the mRNA decay of negative immune regulators. By promoting positive selection, METTL14 may increase B cells’ resistance to immune attacks.²⁶¹

The immunosuppressive function of tumor-infiltrating myeloid cells (TIMs) influences both tumor cell survival and the process of programmed cell death, primarily through METTL3 regulation. The presence of TIMs may inhibit T cell activity, affecting tumor cell programmed cell death.²⁶² YTHDF3 may influence programmed cell death mechanisms by promoting M1 macrophage polarization, increasing the immune response against tumor cells, and increasing apoptosis rates. Conversely, M2 macrophage polarization may lead to immunosuppression and reduced tumor cell apoptosis.²⁶³ IGF2BP1 is linked to immune checkpoint expression and tumor mutational burden (TMB), potentially affecting programmed cell death mechanisms through immune pathways and altering cancer cell survival and death rates.²⁶⁴

m⁶A/m⁵C modifications can influence tumor cell sensitivity to programmed cell death by affecting the immune microenvironment. Early-stage LUAD with high m⁶A/m⁵C expression presents with elevated immune checkpoint gene and immune cell expression, potentially enabling tumor cells to evade immune surveillance and reducing their sensitivity to programmed cell death.²⁶⁵ m⁵C modification regulates the immune microenvironment by modulating immune cell infiltration and function, which can alter cancer cells’ sensitivity to programmed cell death (e.g., immune-mediated apoptosis), further aiding their survival.^{266, 267} m⁷G modification can affect tumor cells’ response to programmed death signals. Cancer patients with a high m⁷G risk also exhibit higher TMB and immunosuppressive states.^268-270 ADAR1's two isoforms (p150 and p110) have dual roles in the immune response, indicating their potential regulation of cancer cell programmed death. The p150 isoform may increase immune surveillance in the TME by promoting T cell infiltration, facilitating tumor cell clearance. Conversely, silencing the constitutive p110 isoform reduces T cell chemotaxis, potentially allowing cancer cells to evade immune-mediated programmed death and fostering their survival.²⁷¹

5.3 Signaling pathway regulation

The regulation of signaling pathways is essential to orchestrate programmed cell death. RNA modifications affect tumor cell survival and death through various pathways, including the PD-L1/PD-1 and mTOR/AKT pathways. The PD-L1/PD-1 pathway is significant in immune evasion and tumor survival²⁷²; meanwhile, the mTOR/AKT pathway contributes crucially to governing cell growth and metabolic regulation.²⁷³

Seven in absentia homolog 2 (Siah2), a RING E3 ubiquitin ligase, plays a vital role in tumorigenesis and cancer progression. In cholangiocarcinoma (CCA), METTL14 increases m⁶A modifications in the 3′UTR region of Siah2 mRNA, leading to its degradation via a YTHDF2-dependent mechanism. This degradation of Siah2 is crucial to sustaining PD-L1 expression on tumor cells, which, in turn, inhibits T cell proliferation and cytotoxicity, modulating programmed cell death in CCA.²⁷⁴

METTL3 posttranscriptionally augments PD-L1 expression in an IGF2BP3-dependent fashion, stabilizing PD-L1 mRNA. By inhibiting either METTL3 or IGF2BP3, antitumor immunity can be improved as this modification affects PD-L1-mediated T cell activation, exhaustion, and infiltration in vitro and in vivo.^{20, 275-277} METTL3 is essential for BCa cells to evade CD8+ T cell cytotoxicity as it controls PD-L1 expression. In addition, JNK signaling facilitates tumor immune evasion through a METTL3-dependent pathway.²⁷⁸ In HCC, LPS induces the upregulation of METTL14, which, in turn, increases the m⁶A methylation of MIR155HG. MIR155HG acts as a competing endogenous RNA, regulating PD-L1 expression via the miR-223/signal transducer and activator of transcription 1 (STAT1) axis, promoting immune evasion in HCC.²⁷⁹ In HCC, ALKBH5 increases the expression of MAP3K8, which then activates the JNK and ERK signaling pathways, regulating IL-8 expression and promoting PD-L1+ macrophage recruitment.²¹⁶ METTL1 promotes PMN–MDSC accumulation via m⁷G, inhibiting tumor-specific T cell activity and reducing ICC tumor cell sensitivity to PD-1 therapy. PMN–MDSCs secrete inhibitory cytokines and express suppressive molecules, decreasing T cell function and affecting tumor cell survival and death.²⁸⁰ ALKBH5 affects T cell activity by regulating PD-L1 expression. PD-L1 upregulation typically inhibits T cell cytotoxicity, increasing tumor cell survival.²⁸¹

Hypoxic conditions significantly elevate YTHDF2 expression, which activates the mTOR/AKT signaling pathway in LUSC, increasing its antiapoptotic capabilities and reducing programmed cell death. The mTOR/AKT pathway facilitates cell proliferation and suppresses apoptosis via multiple mechanisms.¹⁹³ mTORC1, a crucial element of the mTOR signaling pathway, governs cellular growth and metabolism, especially in response to nutrient availability and growth factor cues. Postchemotherapy, the upregulation of L-amino acid transporter 2 increases amino acid uptake, activating mTORC1. This activation is associated with c-Myc-mediated cluster of differentiation 47 (CD47) transcription, where CD47 upregulation may inhibit macrophage phagocytosis of tumor cells, inhibiting programmed cell death.²⁸² The inhibition of YTHDF1 results in the overexpression of the interferon-gamma (IFN-γ) receptor and activation of the JAK/STAT1 signaling pathways, which increase tumor cells’ sensitivity to the immune response.²⁸³ In HCC, m⁶A-modified circMDK is upregulated, increasing cell proliferation, migration, and invasion. This is achieved by sponging miR-346 and miR-874-3p and upregulating autophagy-related 16 like 1 (ATG16L1), which activates the PI3K/AKT/mTOR signaling pathway.²⁸⁴

In conclusion, RNA modifications regulate various mechanisms of programmed cell death in cancer. A comprehensive exploration of the specific roles and mechanisms of RNA modifications can improve our understanding of tumor cell survival and apoptosis, providing new perspectives for developing therapeutic strategies.

6.1 Conventional therapies

Traditional cancer treatments include chemotherapy and radiotherapy. However, tumor cells often develop resistance to these therapies through various mechanisms. RNA modifications are essential to modulate tumors’ resistance to both chemotherapy and radiotherapy. By affecting drug metabolism, DNA repair, and apoptosis, RNA modifications can significantly alter tumor cells’ treatment sensitivity.

Chemoresistance presents a significant obstacle in cancer treatment (Figure 6A). RNA modifications affect tumor cells’ tolerance of chemotherapeutic agents by modulating the expression of drug-metabolizing enzymes and drug efflux transporters.^{285, 286}

Chemoresistance to 5-fluorouracil (5-FU) significantly contributes to poor prognoses in CRC patients. Resistant cells bolster 5-FU resistance through the METTL3/lactate dehydrogenase A (LDHA) pathway. Inhibiting METTL3 can restore chemosensitivity in CRC cells.²⁸⁷ In CRC, METTL3-dependent m⁶A methylation upregulates miR-181b-5p in Cancer-Associated Fibroblasts (CAFs), reducing 5-FU sensitivity.²⁸⁸ METTL3-mediated m⁶A modification also upregulates Sec62, promoting CRC stemness and chemoresistance through β-catenin and Wnt signaling.²⁸⁹ Complement C1q subcomponent subunit A (C1qA) modulates rituximab resistance in DLBCL cells. The methylation of C1qA is regulated by METTL3 and YTHDF2, and knocking out these factors decreases rituximab resistance.²⁹⁰ METTL3 also increases lenvatinib resistance in liver cancer by regulating frizzled class receptor 10 (FZD10) m⁶A modification, activating the β-catenin and YAP1 pathways.²¹⁹ TNF receptor-associated factor 1 (TRAF1) expression is linked to sunitinib resistance, and METTL14-dependent m⁶A modification stabilizes TRAF1. Targeting METTL14 and TRAF1 may present new intervention strategies.²⁹¹ METTL16 expression correlates with PDAC cell sensitivity to Poly(ADP-ribose) Polymerase (PARP) inhibitors, especially when combined with gemcitabine.²⁹² NSUN2 overexpression correlates with a poor prognosis in prostate cancer and decreased sensitivity to chemotherapeutic agents, such as docetaxel and cisplatin, underscoring the significance of the m⁵C modification to the chemotherapeutic response.²²⁶ RNA modification might alter treatment outcomes by regulating drug metabolism-related genes or affecting immune infiltration in the TME.²⁹³

Elevated METTL1 expression in tumor cells increases their sensitivity to specific chemotherapeutic drugs that target chromatin histone methylation, as well as the ERK–MAPK and WNT signaling pathways, suggesting METTL1 as a potential biomarker for drug sensitivity. Elevated METTL1 expression is linked to effective responses to anti-PD-L1 therapy, highlighting its potential to guide immunotherapy.²⁰⁹ Beyond common RNA modifications, such as m⁶A and m⁵C, ncRNA itself is a crucial regulatory mechanism, playing key roles in various diseases, including cancer.²⁹⁴ Research on ncRNA combined with drug therapy represents a development of traditional cancer treatments.^{295, 296}

As a novel approach to combat chemotherapy-resistant tumors, an increasing number of clinical studies are being conducted utilizing RNA modification and RNA sequencing, offering promising prospects for cancer treatment (Table 1). Current RNA-focused clinical research primarily targets two areas: Due to the minimally invasive nature and lower risk of RNA detection, which only requires blood or bodily fluids for extraction, RNA is predominantly studied as a biomarker for precancerous conditions and postoperative monitoring. Given the complexity of RNA regulatory mechanisms, inherent medical risks, and ethical considerations, direct RNA-based therapeutic approaches remain limited. At present, RNA therapies are primarily employed in combination with traditional chemotherapeutic agents or monoclonal antibodies as RNA vaccines. In addition, most research is concentrated in Phase I/II clinical trials to assess the safety of RNA therapies. Much research remains before RNA therapies become a standard clinical treatment modality.

Radiotherapy resistance affects cancer treatment outcomes (Figure 6B). RNA modifications regulate DNA re2pair and antioxidant genes, influencing the tumor response. MDSCs expand after radiotherapy, causing radioresistance and immunosuppression. YTHDF2 inhibition, via the ionizing radiation (IR)-YTHDF2-NF-κB circuit, enhances antitumor immunity and overcomes radioresistance, making it a promising target for RT and RT/immunotherapy combinations.²⁹⁷ Carbon ion radiotherapy increases the METTL3 and m⁶A levels in NSCLC cells. Targeting m⁶A and METTL3 may offer new strategies by affecting cancer cell proliferation, migration, and survival.²²¹ Bone-specific m⁶A-modified eRNA is essential for mediating radiotherapy resistance in metastatic prostate cancer (mPCa) by averting XRN2 degradation through KHSRP. Disrupting the MLXIPe/KHSRP/PSMD9 complex suppresses tumor growth and heightens radiotherapy sensitivity.²⁹⁸ METTL1 increases radiotherapy resistance in HCC by mediating m⁷G tRNA modifications and promoting Double-Strand Break repair. High METTL1 expression correlates with poor radiotherapy outcomes in HCC patients.²⁹⁹

6.2 Immunotherapy

Immunotherapy represents a major advancement in cancer treatment. RNA modifications modulate the efficacy of checkpoint inhibitors, facilitating tumor immune evasion, and transforming cold tumors into hot tumors. In addition, these modifications affect the immune response within the TME by regulating inflammatory mediators and the infiltration of immune cells.

Immune checkpoint inhibitors (ICIs) boost antitumor immunity by relieving the immune system's suppression of tumor cells (Figure 7A). RNA modifications control the expression levels of checkpoint molecules, influencing the effectiveness of these inhibitors.

Many CRC patients respond poorly to ICI therapy. Reduced METTL16 and elevated PD-L1 were observed in CRC tissues and cell lines. The overexpression of METTL16 in CRC cells reduces the presence of PD-1-positive T cells. The METTL16/PD-L1/PD-1 axis is a promising therapeutic target.³⁰⁰ ALKBH5 demethylates axis inhibition protein 2 (AXIN2) mRNA, leading to its degradation and Wnt/β-catenin overactivation, inducing Dickkopf-related protein 1 (DKK1) and recruiting MDSCs and, therefore, driving CRC immunosuppression and tumorigenesis. Targeting ALKBH5 could sensitize CRC to immunotherapy.³⁰¹ The depletion of YTHDF1 suppresses tumor growth by promoting CD8+ T cell infiltration, and this effect increases when combined with PD-1 blockade. Clinically, high YTHDF1 expression is linked to poor prognoses and suboptimal immunotherapy outcomes.²⁵⁵ IGF2BP1 influences antitumor immunity by modulating PD-L1 expression in colon and liver cancers. Targeting IGF2BP1 could improve the effectiveness of PD-1/PD-L1 blockade therapies.^{212, 256} The reduced expression of METTL3 is associated with resistance to anti-PD-1 therapy. METTL3 downregulation stabilizes CD70 mRNA, promoting an immunosuppressive microenvironment. Inhibiting CD70 using cusatuzumab can counteract M2 macrophage-induced resistance to anti-PD-1 therapy, highlighting METTL3's role in regulating the immunotherapy response.³⁰²

In EC cells and CD8+ T cell coculture, elevated METTL3 expression suppresses the proliferation and migration of endometrial carcinoma (EC) cells while encouraging the proliferation of CD8+ T cells. Conversely, the depletion of METTL3 yields the opposite effect. METTL3 bolsters immune surveillance by hindering the YTHDF2-mediated degradation of NLRC5 mRNA, indicating the METTL3/YTHDF2/NLRC5 axis as a potential target for EC immunotherapy.³⁰³ The arginine methylation of METTL14 by PRMT3 facilitates the progression of EC and contributes to treatment resistance. Blocking PRMT3 sensitizes EC to immunotherapy and increases anti-PD-1 efficacy by accelerating ferritin deposition.²⁴⁷ The m⁷G risk score (MRS) can be used to identify CRC patients who may benefit from ICIs. To optimize treatment strategies, m⁷G-related gene expression can be assessed.³⁰⁴

Tumor immune evasion refers to the strategies employed by tumor cells to escape attacks from the immune system (Figure 7B). RNA modifications influence immune evasion by regulating relevant gene expression.²⁰⁰ m⁶A modifications affect cancer immunotherapy by regulating immune cell functions. The aberrant expression of m⁶A methyltransferases or demethylases is linked to CD8+ T cell infiltration, as well as tumor immune evasion.^{204, 305} METTL3 mediates m⁶A modifications on Jak1 mRNA in TIMs, increasing JAK1 translation and STAT3 phosphorylation. Elevated METTL3 expression in TIMs is associated with a poor prognosis in colon cancer patients. Conversely, a deficiency of METTL3 in myeloid cells reduces tumor growth in mouse models.²⁶² Targeting METTL3 could present new avenues for cancer immunotherapies. METTL3-induced circMYO1C promotes PDAC proliferation and migration in vitro and increases immune evasion by stabilizing PD-L1. Regulating m⁶A modifications may offer new strategies for PDAC immunotherapies as well.³⁰⁶ METTL3 knockdown in CRC cells diminishes the accumulation of MDSCs and sustains the activation and proliferation of CD4+ and CD8+ T cells. Inhibiting METTL3 could counteract immunosuppression and bolster antitumor immunity.²⁰⁵ Keratin 17 KRT1 increases T cell infiltration in CRC by promoting YTHDF2 degradation via the ubiquitin–proteasome system. This increases CXC motif chemokine ligand 10 (CXCL10) expression, improving tumors’ response to immunotherapy and highlighting RNA modifications’ role in immune evasion.³⁰⁷ NSUN5 increases TAM phagocytosis in glioma by regulating m⁵C-modified RNA and inhibiting β-catenin. It interacts with and degrades CTNNB1 caRNA, linking its function to DNA methylation and emphasizing RNA modifications’ importance in the tumor immune microenvironment.³⁰⁸

Cold tumors lack immune cell infiltration and are often unresponsive to immunotherapy (Figure 7C). RNA modifications can convert cold tumors to hot tumors by upregulating immune cell recruitment and activation, increasing immunotherapy efficacy.^{309, 310}

In HCC, m⁶A methylation and ferroptosis-related lncRNA (mfrlncRNA) signatures have significant prognostic value, predicting both survival and risk stratification. High-risk patients show increased immunosuppressive cell infiltration and reduced cytotoxic immune cell infiltration, suggesting mfrlncRNA's role in converting cold tumors to hot tumors.³⁰⁵ In PCa, m⁶A-identified immune microenvironment subtype 2 (IMS2) shows a cold phenotype and a poor prognosis. HNRNPC serves as a biomarker for IMS2 and is also a promising therapeutic target to increase the efficacy of immunotherapy.³¹¹ NF-κB, a key transcription factor, regulates apoptosis and survival signals. Regulating the ARID1A/NF-κB pathway can improve the immunotherapy response.³¹²

YTHDF1 deficiency inhibits lysosomal gene translation, limiting major histocompatibility complex class I (MHC-I) and antigen lysosomal proteolysis and, thereby, restoring tumor immune surveillance. It also converts cold tumors to hot ones, increasing ICI efficacy.³¹³ YTHDF2 targets Treg cells in the TME, boosting antitumor immunity without affecting peripheral immune homeostasis, reducing unnecessary inflammation, and improving immunotherapy outcomes.²⁶⁰ NSUN2, a glucose sensor, maintains TREX2 expression upon glucose activation, promoting tumor cell proliferation by inactivating cGAS/STING. Deleting the glucose/NSUN2/TREX2 axis inhibits tumorigenesis and increases apoptosis and CD8+ T cell infiltration, overcoming resistance to anti-PD-L1 therapy in cold tumors.⁶⁵ The thermal ablation (TA) of HCC has high recurrence and metastasis rates. Xiao et al.³¹⁴ created a nanoparticle platform that releases tumor-associated antigens and FTO inhibitors to dendritic cells (DCs). In vivo, postintratumoral administration of these nanoparticles, they facilitate DC maturation, boost T cell infiltration, and establish immune memory. This synergizes with ICB therapy to suppress the growth of distant tumors and prevent metastasis.³¹⁴

Inflammatory factors and the infiltration of immune cells are essential components of the TME (Figure 7D). RNA modifications modulate the expression of genes associated with these factors, affecting the immune response within the TME.³¹⁵ The METTL1-mediated m⁷G modification of tRNA protects them from stress-induced cleavage. The loss of tRNA m⁷G methylation activates the interferon pathway, making tumor cells stress-sensitive.³¹⁶ Reducing METTL1 expression in prostate cancer models results in increased proinflammatory immune cell infiltration and improves the response to immunotherapy, suggesting METTL1 as a potential target to improve chemotherapy sensitivity.³¹⁷ IGF2BP3, an m⁶A reader, facilitates macrophage infiltration and M2 polarization by upregulating the expression of CCL5 and transforming growth factor beta 1 (TGF-β1), which, in turn, inhibits CD8+ T cell activation and contributes to the progression of liver cancer. The combination of IGF2BP3 inhibition with anti-CD47 therapy markedly suppresses liver cancer growth.³¹⁸ In the BCa TME, NSUN6 mediates m⁵C methylation to promote histone deacetylase 10 (HDAC10) expression, inhibiting macrophage-related chemokine transcription and M2 macrophage recruitment. This provides insights into epigenetic regulation in TME and potential immunotherapy strategies.³¹⁹

6.3 Molecular targeted therapy

Molecular targeted therapy offers effective treatment with fewer side effects by targeting key molecules in tumor cells (Figure 6C). RNA modifications affect these therapies by regulating key molecules’ expression and function.

In DLBCL, Hippo–YAP pathway inactivation via carbohydrate sulfotransferase 11 (CHST11) and KIAA1429/YTHDF2 repression inhibits cell proliferation and tumor growth and induces cell cycle arrest and apoptosis.³²⁰ METTL3 inhibitors are potential treatments for myeloid leukemia, according to Yankova et al.³²¹ Increased METTL7B in drug-resistant LUAD cells induces EGFR-TKI resistance, but METTL7B knockdown resensitizes cells to gefitinib and osimertinib.³²² A photosensitive, dual-targeting nanoparticle system (M.RGD@Cr-CTS)–small interfering RNA-targeting YTHDF1 (siYTHDF1) nanoparticles target TAMs to deliver the drug, altering the STAT3/STAT1 balance, reducing IL-10, increasing IL-12 and IFN-γ, and promoting CD8+ T cell infiltration, highlighting the potential of RNA interference in cancer immunotherapy.³²³

6.4 Metabolism-related therapies

Metabolic therapies target tumor cell metabolic pathways, offering a novel treatment approach (Figure 6D). RNA modifications are essential in the regulation of tumor metabolic reprogramming, as they affect the expression of metabolic enzymes and pathways. This, in turn, alters both energy metabolism and biosynthetic processes within the tumor.

m¹A regulates cancer cell glycolysis.²²⁷ Xie et al.³²⁴ developed an abscisic acid-induced reversible m¹A demethylation tool (AI-dm¹A) to facilitate photoinduced m¹A methylation or demethylation. AI-dm¹A specifically reduces m¹A levels in ATP synthase subunit delta (ATP5D), promoting ATP5D expression and increasing glycolysis. This m¹A editing tool offers a flexible approach to studying m¹A's effects on tumors and holds therapeutic potential.³²⁴ circRHBDD1 increases glycolysis and recruits the m⁶A reader YTHDF1 to PI3K regulatory subunit 1 (PIK3R1) mRNA, limiting the efficacy of anti-PD-1 therapy. Targeting circRHBDD1 may improve anti-PD-1 therapy's effects in HCC patients.³²⁵ NOP2 promotes HCC progression via MAZ/NOP2/c-Myc-mediated aerobic glycolysis. NOP2 knockdown combined with sorafenib increases sorafenib sensitivity, significantly inhibiting tumor growth.¹⁵²

In summary, RNA modifications play a critical role in various cancer therapy mechanisms. By thoroughly investigating the specific functions and mechanisms of these modifications, we can better understand tumor cells’ responses to different treatments and develop novel therapeutic strategies.