m5C RNA methylation in cancer: from biological mechanism to clinical perspectives.

Introduction

Cancer, a leading cause of death globally, is characterized by high incidence and mortality rates, presenting a significant challenge to public health [ 1 ]. While traditional therapies such as surgery and chemoradiotherapy, combined with emerging treatments like targeted therapy and immune checkpoint inhibitors (ICIs), have led to improved clinical outcomes for some patients [ 2 – 4 ], challenges such as treatment resistance, recurrence, metastasis, and immune microenvironment suppression—driven by tumor heterogeneity—persist [ 5 , 6 ]. Consequently, the urgent discovery of new therapeutic targets and intervention strategies remains critical. The advent of precision medicine further emphasizes the need to unravel the epigenetic mechanisms underpinning tumorigenesis and progression, alongside the identification of novel biomarkers and therapeutic targets [ 7 , 8 ], which are essential for overcoming existing diagnostic and therapeutic limitations.

The growing field of epigenetics has illuminated the significant role of regulatory mechanisms such as DNA methylation, histone modifications, and non-coding RNAs in the onset and progression of various diseases [ 9 – 11 ]. Among these, RNA post-transcriptional modifications have emerged as a pivotal mechanism in cancer research [ 12 ]. RNA modification involves chemical alterations to nucleotide structures, influencing gene function without altering the underlying DNA sequence [ 13 , 14 ]. Various RNA modifications, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanine (m7G), and pseudouracil (Ψ), have been identified [ 15 – 17 ]. Research indicates that aberrant RNA modification reprogramming is closely associated with the malignant phenotype of tumor cells and is a critical mechanism in the pathogenesis and progression of cancers, including liver, colorectal, lung, and bladder cancers [ 18 – 21 ].

Among the various RNA modifications, m5C has garnered significant attention due to its widespread distribution and dynamic regulation [ 22 ]. RNA m5C methylation involves the addition of a methyl group to the fifth carbon atom of the cytosine base in RNA, resulting in the formation of m5C [ 23 , 24 ]. This modification influences RNA metabolism and function through the dynamic regulation by methyltransferases, demethylases, and recognition proteins, thereby participating in gene expression regulation [ 25 ]. It is closely associated with the onset and progression of numerous diseases. Notably, dysregulated RNA m5C methylation is widely implicated in modulating oncogenic signaling pathways, metabolic reprogramming, and phenotypic transformation, playing a pivotal role in tumor microenvironment remodeling [ 26 – 29 ]. Therefore, an in-depth exploration of the m5C methylation mechanism and its clinical translational potential holds promise for advancing cancer research.

Building on recent breakthroughs, RNA m5C methylation has demonstrated considerable potential for clinical applications, particularly in the precise diagnosis, targeted therapy, and prognostic evaluation of cancer [ 30 , 31 ]. It is poised to become a novel diagnostic and prognostic biomarker as well as a therapeutic target. This review provides a comprehensive analysis of the multifaceted regulatory mechanisms of RNA m5C modification in cancer, highlighting its clinical translational value. Emphasis is placed on the development of diagnostic and prognostic markers and the application of combination therapy strategies. Through a systematic analysis of m5C modification, this review aims to advance epigenetic research to a new phase of clinical translation, paving the way for precision cancer diagnosis and treatment.

Writers, erasers, and readers: dynamic regulatory proteins for RNA m5C methylation

The dynamic regulation of RNA m5C methylation is orchestrated by three key groups of regulatory proteins: methyltransferases (writers), demethylases (erasers), and recognition proteins (readers) [ 32 ]. These proteins play critical roles in various biological processes and disease mechanisms, particularly in cancer, where they influence RNA splicing, nuclear export, stability, and translation during RNA metabolism [ 22 ] (Fig. 1 , Table 1 ). A deeper understanding of these regulatory proteins not only helps elucidate the molecular mechanisms behind cancer initiation and progression but also provides a strong foundation for translating m5C modification into potential cancer biomarkers and therapeutic targets. Fig. 1 The dynamic regulatory network of m5C RNA methylation. RNA m5C methylation is co-regulated by methyltransferases (Writers), demethylases (Erasers), and recognition proteins (Readers), playing an important role by modifying different types of RNA. Writers (NSUN1-7, and DNMT2) catalyze m5C modifications, promoting the nuclear export, stability, and translation of mRNA, and enhancing the proliferation and invasion of cancer cells; they also regulate the stability and translation of tRNA, and influence mitochondrial protein synthesis; they can also affect ribosome biogenesis and protein synthesis by modifying rRNA; in addition, m5C modifications can upregulate the expression of lncRNA, promote the nuclear export of circRNA (circERI3), and inhibit the expression and function of microRNA (miR-125b). Erasers (TET1-3 and ALKBH1) inhibit the stability of mRNA and enhance the codon decoding efficiency of tRNA under stress through demethylation. Readers (ALYREF and YBX1) recognize and bind to m5C modified RNA, mediating the nuclear export, stability, and expression of mRNA, and can also regulate glucose metabolism reprogramming. Through different mechanisms and interactions, these regulatory proteins together constitute a complex dynamic regulatory network of RNA m5C modification, and are widely involved in a variety of biological processes Table 1 Regulators of m5C Category Regulator Target RNA Function Reference(s) Writers NSUN1/NOP2 28S rRNA (C4447) Regulates ribosomal biogenesis [ 36 ] RAPGEF4 mRNA Promotes the proliferation, migration, and invasion of high-grade serous ovarian cancer cells [ 37 ] NSUN2 mRNA Promotes mRNA nuclear export [ 44 ] HGH1 mRNA Enhances mRNA stability and protein synthesis to promote breast cancer progression [ 33 ] FABP5 mRNA Enhances mRNA stability to promote osteosarcoma progression [ 47 ] SKIL mRNA Enhances mRNA stability to promote colorectal cancer progression [ 46 ] tRNA Affects synaptic signaling and behavior [ 38 ] tRNA Enhances tRNA stability and promotes translation efficiency [ 45 ] mt-tRNA Affects mitochondrial protein synthesis [ 40 ] H19 lncRNA Associated with poor differentiation of hepatocellular carcinoma [ 41 ] MALAT1 LncRNA Promotes the expression of MALAT1 LncRNA in multiple myeloma cells [ 42 ] microRNA 125b Inhibits the level and function of microRNA 125b [ 43 ] NSUN3 mt-tRNA (C34) Affects the efficiency of mitochondrial protein synthesis [ 49 ] NSUN4 12S rRNA (C911) Associated with the assembly of ribosomes [ 50 ] CDC42 mRNA Enhances CDC42 mRNA stability to promote glioma progression [ 51 ] circERI3 Promotes circERI3 nuclear export and alter mitochondrial energy metabolism to promote lung cancer development [ 52 ] NSUN5 28S rRNA (C3782) Maintains the stability of ribosomal structure and protein synthesis [ 53 ] NSUN6 mRNA In connection with the termination of translation [ 54 ] tRNA-Cys/Thr (C72) / [ 55 ] NSUN7 eRNA Enhances the coactivator function of PGC-1α [ 56 ] DNMT2 tRNA-Asp (C38) Maintains tRNA stability and regulate protein translation [ 57 , 58 ] mRNA Promotes the migration and invasion of HEK293 cells [ 59 ] Erasers TET1 mRNA Associated with transcription-coupled homologous recombination [ 68 ] TET2 mRNA Reduces mRNA stability and expression to inhibit leukemia progression [ 64 – 66 ] tRNA Promotes translation efficiency [ 67 ] TET3 miR-30d precursor gene promoter Blocking epithelial–mesenchymal transition to inhibit ovarian cancer [ 70 ] ALKBH1 tRNALeu (C34) Promotes decoding of the Leu codon under stress [ 71 , 72 ] tRNAMet (C34) Affects mitochondrial translation ability [ 72 ] Readers ALYREF mRNA Promotes mRNA nuclear export [ 44 ] RPS6KB2 and RPTOR mRNA Promotes mRNA nuclear export to promote colorectal cancer progression [ 75 ] NOTCH1 mRNA Enhances mRNA stability to promote nasopharyngeal carcinoma progression [ 76 ] PKM2 mRNA Enhances mRNA stability to promote bladder cancer progression [ 77 ] ENO1 mRNA Enhances mRNA stability to promote non-small cell lung cancer progression [ 78 ] YBX1 YAP2 mRNA Leads to reprogramming of glucose metabolism to promote colorectal cancer progression [ 27 ] PFKFB4 mRNA Enhances mRNA stability and transcription to promote lung squamous cell carcinoma progression [ 79 ] E2F1 mRNA Enhances mRNA stability and expression to promote ovarian cancer progression [ 80 ]

Writers

Methyltransferases, the"writers"of m5C modifications, catalyze the addition of methyl groups to cytosine residues in RNA, marking the presence of m5C modifications [ 33 ]. The identified"writers"of RNA m5C methylation include the NOL1/NOP2/SUN (NSUN) family and DNA methyltransferase 2 (DNMT2), each performing specific functions depending on the RNA type and modification site [ 34 ]. The NSUN family consists of seven members (NSUN1-7) and is primarily responsible for RNA methylation modifications in the nucleus and mitochondria [ 35 ]. NSUN1, also known as nucleolar protein 2 (NOP2), catalyzes the methylation of cytosine at position C4447 in 28S rRNA, playing a key role in ribosomal biogenesis [ 36 ]. NSUN1 also forms non-catalytic complexes with box C/D snoRNAs to regulate pre-rRNA processing [ 36 ]. Additionally, NOP2 is overexpressed in high-grade serous ovarian cancer (HGSOC), promoting cancer cell proliferation, migration, and invasion via regulation of RAPGEF4 mRNA [ 37 ]. NSUN2, a key methyltransferase, modifies mRNA and non-coding RNAs such as tRNA [ 38 – 40 ], lncRNA [ 41 , 42 ], and miRNA [ 43 ], influencing RNA nucleoplasmic transport [ 44 ], stability, and translation efficiency [ 45 ]. Dysregulation of NSUN2 expression is closely linked to the development of various cancers, including breast, colorectal, and osteosarcoma [ 33 , 46 , 47 ]. In the 3'UTR region of p21 mRNA, NSUN2-catalyzed m5C modification and METTL3/METTL14-catalyzed m6A modification mutually reinforced [ 48 ]. NSUN3 specifically mediates m5C methylation at position C34 in mitochondrial tRNA (mt-tRNA), which is subsequently oxidized to 5-formylcytidine (f5C); impaired NSUN3 expression disrupts mitochondrial protein synthesis efficiency [ 49 ]. NSUN4 catalyzes m5C methylation at C911 of mitochondrial 12S rRNA and is associated with ribosome assembly [ 50 ]. In addition, NSUN4 is involved in the regulation of tumor progression in glioma and lung cancer by affecting mRNA stability and circERI3 nuclear export, respectively [ 51 , 52 ]. NSUN5 modifies the C3782 site of 28S rRNA, maintaining ribosomal structure stability and protein synthesis [ 53 ]. Notably, NSUN6 regulates translational termination by specifically targeting the CTCCA motif in the 3'-UTR of mRNA [ 54 ] and can also catalyze m5C methylation of tRNA at specific sites [ 55 ]. NSUN7 modifies enhancer RNA (eRNA) by m5C methylation, enhancing the coactivator function of PGC-1α [ 56 ]. DNMT2, also referred to as aspartate tRNA methyltransferase 1 (TRDMT1), is traditionally recognized as a DNA methyltransferase. However, it also plays a significant role in the m5C modification of RNA. DNMT2 catalyzes m5C methylation at the C38 position of tRNA-Asp, crucial for tRNA stability and protein translation [ 57 , 58 ]. Additionally, DNMT2 affects HEK293 cell migration and invasion by regulating m5C methylation on mRNA [ 59 ]. It has also been shown that a heart necroptosis-associated piRNA regulates cardiomyocyte necroptosis by inhibiting DNMT1-mediated m5C methylation of Atf7 mRNA [ 60 ].

Erasers

Demethylases, known as the"erasers"of m5C modifications, regulate RNA methylation dynamically and reversibly by removing m5C modifications [ 61 ]. Key demethylases include AlkB homolog 1 (ALKBH1) and members of the 10–11 translocation (TET) family, which consists of TET1, TET2, and TET3 [ 62 ]. Among these, TET2 is the most extensively studied m5C demethylase. Its catalytic domain (CD) oxidizes m5C to an intermediate product, such as 5-hydroxymethylcytosine (hm5C), before completing the demethylation process through the base excision repair pathway [ 63 ]. In leukemia, TET2 inhibits cancer progression by removing methylation modifications from oncogene mRNA and reducing its stability and expression [ 64 – 66 ]. Additionally, TET2 can oxidize m5C modifications on tRNA, reducing methylation levels and promoting translation efficiency [ 67 ]. TET1 facilitates the demethylation of mRNA by catalyzing the oxidation of m5C [ 68 , 69 ] and TET3’s demethylation activity inhibits cancer progression by blocking the epithelial–mesenchymal transition (EMT) in ovarian cancer cells [ 70 ]. ALKBH1, a dioxygenase, plays a pivotal role in demethylating the C34 position of cytoplasmic tRNALeu and mitochondrial tRNAMet, with loss of function resulting in impaired mitochondrial translation capacity [ 71 , 72 ].

Readers

Recognition proteins, the"readers"of m5C modifications, specifically recognize and bind to m5C-modified RNA methylation sites, thereby regulating downstream functions [ 73 ]. Notable readers of m5C modifications include Aly/REF export factor (ALYREF) and Y-box binding protein 1 (YBX1) [ 74 ]. ALYREF is a critical component of the transcription-export complex (TREX) in the nucleus, binding specifically to m5C-modified mRNA and promoting its transport from the nucleus to the cytoplasm. It also enhances mRNA stability and translation efficiency [ 44 , 75 ]. In colorectal tumors, ALYREF forms a complex with ELAV like RNA binding protein 1 (ELAVL1) that synergistically promotes m5C recognition and nuclear export of mRNA to exert procancer effects [ 75 ]. ALYREF is implicated in the development and progression of various cancers, including nasopharyngeal cancer [ 76 ], bladder cancer [ 77 ], and non-small cell lung cancer [ 78 ]. YBX1, one of the most studied readers of m5C modifications, binds specifically to RNA containing m5C modifications. In cancers such as colorectal cancer (CRC) [ 27 ], lung squamous cell carcinoma [ 79 ], and ovarian cancer [ 80 ], YBX1 enhances RNA stability or promotes translation, thereby contributing to cancer progression.

Mechanism insights: m5C-driven regulations in cancers

The regulation of m5C RNA modification in carcinoma cells

As a key component of epigenetic regulation, m5C RNA modification plays a multifaceted role in tumorigenesis and the progression of malignant tumors [ 81 ]. It has been shown to modulate tumor-associated signaling pathways through precise regulation of various aspects of RNA metabolism, exerting both pro- and anti-tumorigenic effects across multiple cancer types [ 76 , 82 ]. Notably, m5C modification exhibits a dual regulatory impact on the metabolic reprogramming of cancer cells [ 28 ]. It also influences tumor cell proliferation, invasion, metastasis potential, and autophagy by dynamically regulating RNA stability and protein translation efficiency [ 83 , 84 ]. Consequently, m5C modification presents itself as a promising molecular target for the development of precision therapeutic strategies (Table 2 ). Table 2 Regulation of m5C RNA modification in carcinoma and immune cells Regulator Category Target Function Tumor Role Mechanism References NSUN2 Writer SARS2 mRNA / Hepatocellular carcinoma Promoting tumor progression Acting Wnt signaling pathway [ 85 ] ALYREF Reader NOTCH1 mRNA Enhancing stability Nasopharyngeal cancer Promoting tumor metastasis Acting Notch signaling pathway [ 76 ] YBX1 Reader SMOX mRNA Enhancing stability Esophageal squamous cell carcinoma Promoting tumor progression Acting mTORC1 signaling pathway [ 87 ] NSUN2 Writer GRB2 mRNA Inducing m5C modification Esophageal squamous cell carcinoma Promoting tumor progression Acting PI3K/AKT and ERK/MAPK signaling pathway [ 88 ] NSUN2 Writer PKM2 mRNA Inducing m5C modification Hepatocellular carcinoma Promoting tumor progression Regulating glycolysis metabolism [ 92 ] ALYREF Reader PKM2 mRNA Enhancing stability Bladder cancer Promoting tumor progression Regulating glycolysis metabolism [ 77 ] NSUN2 Writer SREBP2 mRNA Enhancing stability Hepatocellular carcinoma Promoting tumor metastasis Regulating cholesterol metabolism [ 93 ] NSUN2 Writer GLS mRNA Inducing m5C modification Gastric cancer Promoting tumor progression Regulating glutamine metabolism [ 94 ] NSUN4 Writer circERI3 Increasing nuclear export Lung cancer Promoting tumor progression Regulating mitochondrial energy metabolism [ 52 ] NSUN2 Writer p57 Kip2 mRNA Decreasing stability Gastric cancer Promoting tumor cell proliferation Regulating cell cycle [ 96 ] NSUN5 Writer / / Colorectal cancer Promoting tumor cell proliferation Regulating cell cycle [ 97 ] YBX1 Reader mTOR mRNA Enhancing stability Triple-negative breast cancer Promoting tumor progression Inhibiting autophagy [ 98 ] NSUN2 Writer CircFam190b Enhancing stability Lung cancer Promoting tumor progression Inhibiting autophagy [ 99 ] NOP2/AlYREF Writer/Reader EZH2 mRNA Enhancing stability Lung cancer Promoting tumor progression Inducing epithelial–mesenchymal transition [ 101 ] ALYREF Reader EGFR mRNA Enhancing stability Hepatocellular carcinoma Promoting tumor progression Inducing epithelial–mesenchymal transition [ 102 ] NSUN3 Writer / / Head and neck squamous cell carcinoma Promoting tumor progression Increasing M1, decreasing M2 [ 111 ] NSUN6 Writer / / Bladder cancer / Reducing M2 recruitment [ 114 ] YBX1 Reader / / Prostate Cancer Inhibiting tumor progression Enhancing anti-tumor response [ 115 ] NSUN2 Writer / / Nasopharyngeal carcinoma Promoting tumor progression Inhibiting immune infiltration [ 116 ] ALYREF Reader JunD mRNA Enhancing stability Pancreatic ductal adenocarcinoma Promoting tumor progression Suppressing CD8 + T cells [ 122 ] NSUN2 Writer SOAT2 mRNA Enhancing stability Hepatocellular carcinoma Promoting tumor progression Suppressing CD8 + T cells [ 123 ]

m5C RNA modifications involved in signaling pathways

m5C RNA modification significantly influences tumor progression by impacting the behavior and fate of carcinoma cells through the regulation of signaling pathways. For example, NSUN2 has been shown to enhance the activity of the Wnt/β-catenin signaling pathway in hepatocellular carcinoma (HCC), promoting the nuclear translocation of β-catenin and the transcription of its target genes [ 85 ]. Its stabilization and nuclear translocation are regulated by small ubiquitin-like modifier (SUMO)−2/3 modification [ 86 ], which increases its oncogenic activity and involvement in various cancer-related signaling pathways. In nasopharyngeal cancer, ALYREF drives tumor metastasis by recognizing the m5C-modified NOTCH1 mRNA site, prolonging its half-life and activating NOTCH signaling [ 76 ]. Additionally, YBX1, promotes the progression of esophageal squamous cell carcinoma (ESCC) by stabilizing spermine oxidase (SMOX) mRNA to activate mTORC1 pathway [ 87 ]. Furthermore, Su and colleagues demonstrated that the development of ESCC is associated with the activation of PI3K/AKT and ERK/MAPK signaling pathways by growth factor receptor binding protein 2 (GRB2) [ 88 ]. They found that NSUN2 induced m5C modification of GRB2, stabilizing its mRNA and thereby increasing GRB2 levels. These findings underscore the importance of m5C modification in the tumor signaling network through complex mechanisms, offering a novel molecular perspective for understanding tumorigenesis and development.

m5C RNA modifications involved in metabolic reprogramming

In tumor biology, m5C RNA methylation is recognized as a key epitranscriptomic modification that regulates metabolic reprogramming. Numerous studies have demonstrated that m5C modification plays a pivotal role in the dynamic reprogramming of glucose, amino acid, and metabolic pathways [ 89 – 91 ]. In HCC, NSUN2 mediates the hypermethylation of pyruvate kinase muscle isozyme M2 (PKM2) mRNA at the m5C site (C773) within the 3'-untranslated region (UTR) [ 92 ]. Similarly, in bladder cancer, the stability of PKM2 mRNA is enhanced via the HIF-1α/ALYREF axis, facilitating the Warburg effect [ 77 ]. These mechanisms promote glycolysis and tumor progression. NSUN2 also binds lncRNA LINC00618 to inhibit degradation and increase SREBP2 m5C modification, promoting cholesterol biosynthesis [ 93 ]. Furthermore, NSUN2-mediated lncRNA methylation stabilizes the mRNA of glutaminase (GLS), a key enzyme in glutamine metabolism, which drives the production of α-ketoglutarate and forms a positive feedback loop to accelerate tumor growth [ 94 ]. Notably, Wu et al. discovered that NSUN4-mediated m5C modification of circERI3 enhances its nuclear export efficiency [ 52 ]. They suggested that circERI3 interacts with DNA damage binding protein 1 (DDB1) to activate the transcription of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), ultimately regulating mitochondrial function and energy metabolism [ 52 ]. These findings underscore the profound impact of m5C modification on metabolic reprogramming in carcinoma cells, supporting their malignant progression through diverse molecular mechanisms.

m5C RNA modifications involved in cell phenotype

m5C methylation plays a pivotal role in driving phenotypic changes in tumor cells, significantly influencing key biological behaviors such as proliferation, autophagy, and invasion [ 95 ]. Additionally, NSUN2 regulates the cell cycle of gastric cancer cells in an m5C-dependent manner [ 96 ]. Notably, the inhibitory effect of NSUN2 on cell cycle protein-dependent kinase inhibitor 1 C (CDKN1C) highlights its critical role in regulating tumor cell cycles. A similar regulatory mechanism was observed in CRC, where NSUN5 not only induced cell cycle arrest in vitro but also promoted tumor growth in vivo by modulating the oncogene Rb and cell cycle protein-dependent kinases [ 97 ]. In various murine acute myeloid leukemia models, the loss of TET2 increased the self-renewal of leukemia stem cells and suppressed myeloid differentiation [ 64 ]. Moreover, m5C methylation’s involvement in regulating autophagy in tumor cells is particularly significant. Tian et al. demonstrated that SAT1 inhibited autophagy in breast cancer cells by accumulating stable mTOR mRNA through YBX1-mediated m5C modification [ 98 ]. Additionally, the current study indicated that NSUN2-mediated m5C modification of circFAM190B suppressed autophagy in lung cancer cells via the mTOR/ULK1 pathway, underscoring the critical role of m5C modification in autophagy regulation within tumor cells [ 99 ]. In the EMT—a key process driving tumor invasion and metastasis—m5C modification also plays a pivotal role [ 100 ]. Yang et al. identified a co-regulatory relationship between NOP2 and ALYREF in stabilizing EZH2 mRNA [ 101 ]. They found that the NOP2/EZH2 axis promoted the malignant phenotype of lung cancer cells through EMT, both in vitro and in vivo. Similarly, ALYREF facilitated the EMT of HCC cells by stabilizing epidermal growth factor receptor (EGFR) mRNA and activating the signal transducer and activator of transcription 3 (STAT3) signaling pathway [ 102 ].

In summary, m5C methylation significantly influences signaling pathways, metabolic reprogramming, and cell phenotypes in tumor cells. The critical role of this modification in tumor cell remodeling cannot be overlooked.

The regulation of m5C RNA modification in immune cells

The dynamic interaction between tumors and the immune system unfolds in three phases: elimination, equilibrium, and escape [ 103 , 104 ]. During the elimination phase, the immune system effectively eradicates tumor cells. However, in the escape phase, residual tumor cells survive and progress through genetic mutations or immunosuppressive mechanisms [ 105 , 106 ]. RNA modifications, including m5C, influence immune cell infiltration and function [ 107 , 108 ], although the precise role of m5C RNA modification in tumor immunity remains inadequately understood (Table 2 ).

m5C RNA modifications involved in immune cell infiltration

Immune cell infiltration refers to the process by which immune cells enter the tumor microenvironment from the bloodstream, with the extent and pattern of infiltration directly impacting the efficacy of tumor immunotherapy and patient prognosis [ 109 , 110 ]. A study demonstrated that NSUN3 knockdown increased the infiltration of M1-type macrophages and reduced the percentage of M2-type macrophages in head and neck squamous cell carcinoma [ 111 ]. This shift in macrophage polarization affected the immune properties of the tumor microenvironment [ 112 , 113 ]. Yan et al. found a strong correlation between NSUN6 and macrophage recruitment, revealing that m5C methylation promoted HDAC10 expression [ 114 ], which inhibited the transcription of macrophage-associated chemokines and consequently reduced M2-type macrophage recruitment. Furthermore, YBX1 knockdown led to decreased infiltration of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg), while enhancing CD8 + T cell infiltration, significantly strengthening the anti-tumor immune response and highlighting a potential target for immunotherapy intervention [ 115 ]. In the tumor microenvironment of nasopharyngeal carcinoma, NSUN2 expression was negatively correlated with immune cell infiltration, suggesting its potential influence on the sensitivity to immunotherapy and chemotherapy [ 116 ]. Additionally, NSUN3 and NSUN4 were linked to the infiltration of various major immune cells, with NSUN3 associated with CD8 + T cells and NSUN4 with neutrophils, emphasizing their role in regulating immune cell infiltration and function [ 117 ].

m5C RNA modifications involved in immune cell function

The influence of RNA modifications on the enhancement or suppression of immune cell function in tumors is a growing area of research, with multiple factors being explored [ 118 – 120 ]. m5C RNA modification plays a key role in regulating immune cell functions. For example, YBX1 has been shown to upregulate PD-L1 expression, which significantly inhibits the proliferation and function of cytotoxic CD8 + T cells [ 121 ]. In contrast, ALYREF stabilizes JunD mRNA through selective recognition at the m5C site, thereby limiting the activity and function of CD8 + T cells [ 122 ]. Furthermore, Jiang et al. revealed that NSUN2 suppresses the activity and toxicity of CD8 + T cells by promoting metabolic reprogramming [ 123 ] (Fig. 2 ). Fig. 2 Multifaceted roles of m5C RNA methylation in cancer and immune cells. m5C RNA methylation dynamically regulates the following: (i) oncogenic signaling (including Wnt/β-catenin, NOTCH1, mTORC1, and PI3K/AKT/ERK/MAPK signaling pathways), (ii) Cell phenotypes (comprising cell cycle arrest, autophagy inhibition, and EMT promotion), (iii) Metabolic reprogramming (such as glycolysis and mitochondrial function), and (iv) immune cell infiltration and function. These mechanisms collectively influence the progression and immune response of tumors

These findings highlight the critical role of m5C RNA modification in modulating the tumor immune microenvironment, suggesting it as an important molecular target for the development of novel immunotherapeutic strategies.

The potential clinical application of m5C in tumors

Previous research has established m5C as a significant epigenetic modification in RNA [ 124 ], with its methylation playing a critical role in tumor onset and progression. This modification presents potential targets for innovative therapeutic approaches. However, investigations into m5C methylation are still in the early stages, with a lack of targeted intervention strategies and clinical applications. Therefore, further research is needed to advance this field (Fig. 3 ). Fig. 3 Potential clinical roles of m5C RNA methylation in cancer. The potential of m5C functions as the biomarkers for diagnosis and prognosis and therapeutic targets enhancing chemosensitivity and immunotherapy

Diagnostic and prognostic value of m5C

m5C modifications have emerged as potential biomarkers for the diagnosis and prognosis of various cancers. Dysregulated expression of m5C methyltransferases and m5C"readers"is strongly linked to tumor progression, poor prognosis, and treatment resistance across multiple cancer types. These regulatory factors present considerable potential as biomarkers for cancer prediction and disease monitoring. The modification of RNA by m5C, particularly through the NSUN gene family (NSUN1-NSUN7) and DNMT2, plays a significant role in altering cellular biology and influencing tumorigenesis [ 84 ]. DNMT2 is upregulated in individuals with HCC, and its increased expression correlates with adverse prognostic outcomes [ 125 ]. Similarly, NSUN2 is upregulated in various cancer types and correlates with poor prognosis [ 126 ]. NSUN2-mediated m5C modification enhances glycolysis and promotes HCC progression by stabilizing the mRNA of PKM2, suggesting its potential as both a prognostic indicator and therapeutic target in HCC [ 92 ]. In HCC, NSUN4 and ALYREF serve as independent prognostic markers with notable diagnostic precision [ 127 ]. NSUN3 and NSUN4 have been identified as potential prognostic indicators for lung squamous cell carcinoma and are involved in modulating the immune microenvironment [ 117 , 128 ]. Furthermore, NSUN5 knockout in HCC cells reduces their invasion and migration abilities, both in vitro and in vivo, while overexpression of NSUN5 increases these abilities, offering a basis for potential therapeutic strategies in HCC [ 129 ]. Increased expression of NSUN6 in colorectal adenocarcinoma (COAD) tissues and cells correlates with poor prognosis, and NSUN6 expression is negatively associated with infiltrating immune cells in COAD [ 130 ]. Elevated levels of YBX1 correlate with unfavorable outcomes in patients with advanced gastric cancer undergoing 5-fluorouracil (5-FU) chemotherapy, suggesting that YBX1 may play a pivotal role in mediating resistance to 5-FU and could serve as a novel therapeutic target for overcoming this resistance [ 131 ]. Additionally, YBX1 contributes to the progression of ESCC by stabilizing spermidine oxidase mRNA through an m5C-dependent mechanism, further underscoring its potential as a therapeutic target for ESCC [ 87 ]. Moreover, alterations in the stability of m5C-modified RNA molecules, including lncRNAs and messenger RNAs (mRNAs), in tumor cells have led to the development of diagnostic markers [ 41 ]. For example, LINC02167 enhances the ERK/MAPK signaling pathway and promotes CRC metastasis through m5C modification, positioning it as a valuable therapeutic target in the treatment of metastatic CRC [ 132 ]. These findings collectively suggest that m5C modification plays a significant role in the early detection and ongoing monitoring of tumor progression, with substantial implications for the development of novel diagnostic and therapeutic strategies.

The targeted therapeutic value of m5C modification inhibitors

The inhibition of RNA methyltransferases using small molecules is an emerging area within epitranscriptomics and medicinal chemistry [ 133 ]. Research has demonstrated that inhibiting NSUN2 holds significant therapeutic potential across various diseases [ 134 , 135 ]. Additionally, NSUN2 inhibitors substantially enhance the anti-tumor efficacy of doxorubicin, cisplatin, and lenvatinib [ 136 ], suggesting that NSUN2 may be a promising target for combating multidrug resistance, particularly in anaplastic thyroid carcinoma. In pancreatic ductal adenocarcinoma, suppressing NSUN2 could reduce matrix fibrosis and promote the differentiation of ductal epithelial cells, potentially slowing tumor progression. This highlights the viability of NSUN2 as a therapeutic target [ 137 ]. However, the development of specific inhibitors must account for the spatiotemporal specificity of m5C modification. Future research should focus on creating inhibitors that selectively target m5C methyltransferases to minimize adverse effects on normal cells. Network pharmacology-based drug development of m5C inhibitors can coordinate multiple omics techniques to shorten drug development time and greatly improve drug research efficiency [ 138 ]. Additionally, the majority of current research primarily utilizes preclinical models, and ClinicalTrials.gov indicates that only a limited number of m5C inhibitors, such as STC-15, have entered early-stage clinical trials ( NCT05584111 ). Ongoing studies ( NCT03525002 and NCT04205318 ) highlight the complex role of FTO in obesity and cancer, demonstrating the importance of stratifying patients based on genotype. The inclusion of FTO genotyping in trials like NCT03719118 (focused on inflammatory bowel disease) and NCT02886013 (focused on diabetes risk) indicates that personalized treatment approaches may help reduce unintended effects. Nevertheless, there remains a lack of clinical evidence specifically related to oncology applications.

The combined therapeutic approach aimed at m5C modification and pharmacological agents

The m5C methyltransferase NSUN2 is upregulated in HCC tissues and modulates the sensitivity of HCC cells to sorafenib by affecting the activity of the Ras signaling pathway [ 82 ]. Additionally, high levels of YBX1 are associated with decreased resistance to platinum-based chemotherapy and improved overall survival rates [ 139 ]. Inhibiting YBX1 could increase ovarian cancer sensitivity to platinum treatments, positioning it as a potential therapeutic target for overcoming platinum resistance in ovarian cancer [ 140 ]. YAP, a key transcriptional coactivator, plays an essential role in tumor development [ 141 ]. In lung adenocarcinoma (LUAD), m5C modification of YAP reduces its interaction with miR-582-3p and enhances exosome secretion, promoting aggressive tumor behavior and resistance to AZD9291, a third-generation EGFR tyrosine kinase inhibitor [ 142 ]. The m5C modification of YAP facilitates tumor progression, suggesting that targeting this modification could offer a potential therapeutic strategy for LUAD. These findings propose innovative approaches for overcoming resistance to targeted therapies in cancer treatment.

m5C regulation and immunotherapy

Recent years have witnessed significant advancements in the application of m5C methylation in tumor immunotherapy [ 143 ]. Exploring the mechanisms by which m5C modification influences cancer progression and immune responses could provide innovative strategies for enhancing the efficacy of immunotherapeutic interventions. Research has shown that the combined use of m5C inhibitors and ICIs effectively induces apoptosis in cancer cells and promotes immunogenic cell death [ 144 ]. m5C-modified mRNA has the ability to reprogram tumor-associated macrophages and anti-cancer T cells, facilitating the activation of anti-tumor immune responses and contributing to tumor regression [ 145 ]. YTHDF2, which is overexpressed in B-cell malignancies, plays a key role in promoting cancer cell survival and immune evasion [ 146 ]. Additionally, studies have shown remarkable progress in modulating YTHDF2 to enhance the proliferation and cytotoxicity of NK cells in vitro, which may inspire future NK cell-based immunotherapy regimens [ 147 ]. Modifications of N6-methyladenosine by RBM15B and IGFBP2, along with m5C modification by NSUN5, significantly enhance anti-cancer immunity through the activation of the cyclic GMP-AMP synthase–stimulator of interferon signaling (cGAS–STING) pathway, which is maintained by redox homeostasis in COAD [ 148 ]. Genetic deletion of the glucose/NSUN2/TREX2 pathway inhibits tumor development and improves the efficacy of anti-PD-L1 immunotherapy in cold tumors by activating the cGAS/STING pathway, promoting apoptosis, and increasing CD8 + T cell infiltration [ 149 ]. Significant advancements have been achieved in regulating YTHDF2 to improve the growth and killing ability of NK cells in laboratory settings. These findings may guide the development of future methods for NK cell-based immunotherapy [ 150 ]. These findings suggest that targeting m5C modification may offer novel approaches for improving immunotherapeutic efficacy. Although m5C inhibitors demonstrate encouraging potential for therapy, their application in clinical settings may be constrained by unintended effects on non-target molecules and toxicity in healthy cells. Future research should focus on creating targeted delivery methods or combined treatment approaches to reduce these risks. Moreover, comprehensive toxicity assessments using preclinical models will be crucial to ensure safety.

Future research should integrate clinical data to enhance the application of m5C methylation markers in clinical settings. It is critical to correlate m5C methylation markers with clinical medical data and validate their diagnostic and prognostic potential through comprehensive analyses of large-scale clinical samples. The development of specific inhibitors targeting m5C methyltransferases and demethylases is essential, as is the investigation of their potential for clinical therapeutic applications. Additionally, incorporating multi-omics approaches, including transcriptomics, proteomics, and metabolomics, could provide a deeper understanding of the role of m5C methylation in tumorigenesis and progression. Further translational research should assess the role of m5C methylation in personalized medicine, offering a theoretical foundation for precision treatment strategies.

Conclusion

In conclusion, m5C modification dynamically alters RNA molecules through the action of three key regulatory proteins—methyltransferases, demethylases, and recognition proteins—thereby modulating oncogene signaling, metabolic reprogramming, and tumor microenvironment remodeling. Notably, m5C modification holds significant clinical potential for cancer diagnosis, treatment, and prognostic prediction, serving as a diagnostic marker, prognostic predictor, and targeted therapeutic agent. This review provides a comprehensive exploration of the molecular mechanisms and clinical conversion pathways of m5C methylation, offering new perspectives for precision medicine research in cancer, enhancing cancer diagnosis and treatment, and improving patient prognosis.

However, the clinical translation of m5C still faces several challenges. First, m5C detection technologies, including bisulfite sequencing and mass spectrometry, face technical bottlenecks such as DNA and RNA degradation and insufficient single-base resolution. Secondly, the development of targeted inhibitors for RNA m5C methylation is still in its infancy, and clinical trials are lacking. Therefore, in future, it is necessary to combine experimental methods and computational tools to optimize the detection method of m5C and enhance its clinical application value. For example, enzymatic methyl-seq was used to replace sulfite treatment to reduce DNA damage [ 151 ], and computational tools were developed to optimize the efficiency and accuracy of sulfite sequencing data [ 152 ]. In addition, small molecule inhibitors or protein degraders of m5C methylation regulatory proteins should be actively developed for targeted therapy of RNA m5C methylation. At the same time, drawing on the experience of clinical trial design of m6A inhibitors, preclinical research and clinical trial research on solid tumors should be carried out. We can also construct single-cell resolution m5C profiles to further elucidate the dynamic modification of m5C in tumor heterogeneity for better clinical application. In conclusion, future researches must focus on deeper exploration and study of the clinical application and translation of m5C in cancer, ultimately providing new hope for improving the prognosis and quality of life for cancer individuals.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Acknowledgements

None.

Author contributions

Fang Yuan, Yuxuan Xia, and Xiangqin Zhou contributed to the concept the work and wrote the manuscript. Fang Yuan participated in original draft preparation. Zhen Huang and Huihui Gao designed the figures. Yuqi Bian completed the tables. All the authors contributed to the manuscript and reviewed and approved the published version of the manuscript.

Funding

This work was supported by the Zhejiang Provincial Medical and Health Science and Technology Program: Research and Application of a Tumor Staging NLP Model for Improving Cancer Care Quality (2023KY555).

Availability of data and materials

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.

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