m⁶A methyltransferase complex, which is named 'writers' as well, is composed of core proteins methyltransferase-like 3 (METTL3), METTL14, Wilms tumor 1-associated protein (WTAP) and other proteins. METTL3 and METTL14 form stable heterodimers, which are responsible for the majority of m⁶A sites in mRNA (34). Among them, METTL3 functions as the fundamental catalytic enzyme by binding to S-adenosylmethionine as a subunit, and is capable of enhancing the translation of most oncogenic mRNAs (31,36). METTL14 acts as an allosteric activator of METTL3, thereby stabilizing its structure and recognizing target RNAs (37). WTAP is essential for the proper localization of the METTL3-METTL14 complex to nuclear speckles and for the enhancement of its catalytic activity (38). Furthermore, m⁶A methylation is regulated by several other proteins. METTL16 has been demonstrated to function independently and regulate m⁶A modification in several RNAs. Specifically, in the nucleus, it deposits N⁶ into specific mRNA targets, while separately stimulating translation in the cytoplasm (39). KIAA1429 [also known as vir-like m⁶A methyltransferase-associated protein (VIRMA)], a newly confirmed ingredient of the m⁶A methyltransferase complex, is crucial in guiding the process of regioselective m⁶A deposition. It means that KIAA1429 can mediate preferential m⁶A deposition in the 3'UTR and around the termination codon (40). RNA binding motif protein 15/15B (RBM15/15B) can attract and bind cone proteins complexes, directing them to specific RNA positions (41). Zinc finger CCCH domain-containing protein 13 (ZC3H13) serves as an anchor for the complex (WTAP, VIRMA and Cbl proto-oncogene like 1) within the nucleus so as to promote m⁶A methylation and mouse embryonic stem cell self-renewal (42). The summarized functions of m⁶A writers are presented in Table I.

The eraser enzyme is capable of facilitating m⁶A demethylation through the involvement of two proteins, namely Fat mass and obesity-associated protein (FTO) and AlkB homolog 3/5 RNA demethylase (ALKBH3/5) (33). FTO and ALKBH5, which belong to the α-ketoglutarate-dependent dioxygenase family, only work in the presence of oxygen, ferrous ions and α-ketoglutarate (43). They initiate the conversion of m⁶A into N⁶ hydroxymethyl adenosine and subsequently into N⁶ formyl adenosine (f⁶A). Ultimately, f⁶A is hydrolyzed to adenosine to complete the demethylation process (43). FTO is the first m⁶A demethylase and it exhibits a strong correlation with weight gain, obesity and other metabolic diseases in humans (44). FTO is situated in both the nucleus and cytoplasm, and directly participates in the regulation of multiple pre-nuclear mRNA processing pathways, as well as other processes such as mRNA splicing (45). The second eraser, ALKBH5, is located in the nucleus and regulates gene expression mainly by mediating the transportation, metabolism and assembly of mRNA (43). The expression level of FTO and ALKBH5 affects the level of m⁶A in mRNA. Numerous studies have reported that the overexpression of m⁶A erasers is closely related to the occurrence and development of cancer (43,44,46). The functions of m⁶A erasers are summarized in Table II.

The reader is another participant in the dynamic and reversible regulation of m⁶A methylation, which can recognize and bind to m⁶A targets. The readers comprise the YTH domain family of proteins (YTHDC1/2, YTHDF1/2/3), insulin-like growth factor 2 mRNA binding protein (IGF2BP1/2/3), the heterokaryotic nuclear RNA protein family [heterogeneous nuclear ribonucleoprotein C (HNRNPC), HNRNPG] and eukaryotic initiation factor 3 (eIF3) (47). Diverse species rely on different m⁶A readers to perform specific biological functions.

The YTH family members are the most vital readers with a conservative m⁶A binding domain. YTHDF2 recruits RNA decay mechanism factor (C-C motif chemokine receptor 4-NOT deaminase complex) directly, which has an important role in accelerating the degradation of m⁶A-modified RNA (48). Unlike YTHDF2, YTHDF1 may neither take part in mRNA decay directly nor alter the total methylation mRNA level, but it facilitates translation by interacting with translation initiation factors and ribosomes (49,50). YTHDF3 is considered an assistant to boost the translation or degradation of target RNA with two other YTHDF members (51). YTHDC1 facilitates exon inclusion in the nucleus by recruiting splicing factor 3 (SRSF3) and preventing SRSF10 from binding to mRNA (52). It can also promote the nuclear export of m⁶A-labeled mRNA by interacting with nuclear transport receptors, and it is involved in promoting the stability of mRNA transcripts (47,53). YTHDC2 has 3′→5′ RNA helicase activity and improves the translation efficiency of target mRNA (54).

In the HNRNP family, HNRNPA2/B1 contains two RNA-specific recognition motifs and governs the directional sorting of miRNAs, thereby promoting primary miRNA processing (55). HNRNPC and HNRNPG can modulate mRNA abundance and splicing (43). IGF2BPs have been proven to be a unique and conservative family of m⁶A readers, which can enhance translation efficiency in an m⁶A-dependent way by regulating alternative splicing and improving stability (56). In addition, eIF3 promotes cap (m⁷GPPPN)-independent and YTHDF1-dependent mRNA translation (57). The functions of m⁶A readers are summarized in Table III. The functions of m⁶A regulators are illustrated in Fig. 1.

As an m⁶A methyltransferase, METTL3 has a crucial role in cancer. Mostly, METTL3 stimulates the occurrence and progression of diverse cancers as an oncogene, through depositing m⁶A modification on key transcripts (37). Numerous studies have validated that METTL3 expression is elevated in diverse cancerous tissues. However, the mechanism by which METTL3 promotes carcinogenesis may differ across various cancer types. The reported mechanisms mainly entail activating multiple m⁶A-dependent signaling pathways, increasing m⁶A modification of carcinogenic primary miR-25 and mediating the binding of m⁶A-modified target transcripts with specific cytokines, so as to promote mRNA translation or degradation, and ultimately facilitate tumor-cell proliferation and migration (61-64).

However, in certain cases, contrary results have been reported for similar tumors, implying that METTL3 may at times function as a tumor suppressor (65). For instance, certain researchers have detected that METTL3 methylation of basic leucine zipper ATF-like transcription factor (BATF) mRNA inhibits its expression in gastric cancer (GC), and low expression of BATF mRNA is significantly associated with postoperative recurrence of GC (66). In addition, there have been reports indicating that the knockdown of METTL3 significantly hastened tumor progression and reduced the lifespan of animals implanted with glioblastoma stem cells (67). Other studies have demonstrated that METTL3 expression is decreased in certain cases of renal cell carcinoma and bladder cancer (68,69). Shi et al (70) found that a low level of METTL3 in TNBC is indicative of a poor prognosis, suggesting that the reduced presence of m⁶A markers contributes to the progression of TNBC.

To date, certain studies on the mechanism underlying the role of METTL3 in BC have been published. The present study only provides a summary of recent findings. Wan et al (71) discovered that METTL3 enhances the m⁶A modification of programmed cell death ligand 1 (PD-L1) mRNA in BC cells, thereby improving the stability and expression of PD-L1 mRNA. Knocking down METTL3 can boost anti-tumor immunity and reduce PD-L1 expression, thus alleviating the progression of BC. Cai et al (72) have shown that the expression levels of METTL3 and hepatitis B x-interacting protein (HBXIP) are very high in BC tissues. HBXIP increases the expression of METTL3 through restraining the expression of tumor suppressor let-7g, and METTL3 in turn upregulates HBXIP via m⁶A modification, thus forming a positive feedback regulatory loop of HBXIP/let-7g/METTL3/HBXIP, and ultimately causing the malignant growth of BC cells (72).

It has also been observed that the METTL3 level in BC is significantly higher than that in surrounding normal tissues, particularly in patients with T3-T4 BC or lymph node metastasis (73). Studies revealed that METTL3 overexpression can upregulate enhancer of zeste homolog 2 through m⁶A modification. This process results in the suppression of tumor suppressor genes and promotion of epithelial-mesenchymal transformation (EMT), which triggers the occurrence, migration and invasion of BC cells (74,75). In addition, another study indicated that METTL3 can accelerate the proliferation of BC by regulating the methylation of BCL-2 or the metastasis associated lung adenocarcinoma transcript 1 (MALAT1)/miR-26b/high mobility group AT-hook 2 axis (76).

To sum up, METTL3 has been observed to be overexpressed in most BC samples, and its expression level appears to be positively correlated with the malignancy and metastasis of BC. The specific mechanism of the connection between METTL3 and BC-cell proliferation may involve multiple signaling pathways, but the exact mechanism requires to be further studied and clarified. The functions of METTL3 in BC are shown in Fig. 2.

KIAA1429 acts as a scaffold for bridging the core protein of methyltransferase and it is also involved in the positive regulation of diverse tumorigenesis. Certain studies have indicated that KIAA1429 promotes the proliferation and growth of BC in a way independent of m⁶A, and the overall survival period of patients with BC is positively associated with KIAA1429 (77,78).

Zhang et al (78) found that KIAA1429 can improve the stability of structural maintenance of chromosomes 1A (SMC1A) mRNA via binding to the motif of SMC1A mRNA. Subsequently, SMC1A further increases snail family transcriptional repressor 1 (SNAIL) expression via binding to the promoter region of the SNAIL gene, which promotes the migration and invasion of BC. Another study illustrated that KIAA1429 targets to regulate cyclin-dependent kinase 1 (CDK1) (77), which is an oncogene related to the proliferation and metastasis of BC. The functions of KIAA1429 in BC are illustrated in Fig. 3.

It is known that FTO, as an obesity-related protein, can catalyze the demethylation of m⁶A. Numerous studies have indicated that FTO is significantly upregulated in various cancerous tissues, including but not limited to cervical squamous cell carcinoma (79), lung squamous cell carcinoma (80), gastric cancer (81) and pancreatic cancer (82). FTO is involved in the regulation of tumor progression by decreasing the abundance of m⁶A and activating specific signaling pathways, reducing the overall survival rate of patients afflicted with malignant tumors (83). In a significant proportion of BC specimens, an elevated expression of FTO was observed compared to the adjacent normal breast tissue. Furthermore, it has been strongly associated with tumor proliferation, invasion and metastasis (83-85).

Niu et al (84) reported that, in MCF7 and MDA-MB231 cells, the expression of FTO was negatively correlated with BCL2 interacting protein 3 (BNIP3) in BC. Due to the overexpression of FTO in BC, the level of BNIP3 is downregulated, which is necessary for cell apoptosis (84). This change inhibits the cleavage of apoptosis factor caspase-3 and promotes the expression of anti-apoptotic protein Bcl-2 (86,87), thus reducing cell apoptosis and promoting the proliferation and colony formation of BC cells.

Xu et al (85) demonstrated that in SKBR3 and MDA-MB453 cells, FTO overexpression decreased the expression of miR-181b-3p, increasing the expression of ADP ribosylation factor like GTPase 5B (ARL5B) directly and indirectly. ARL5B subsequently drives the migration and invasion of HER2+ BC tissue.

Liu et al (88) indicated that FTO overexpression promotes aerobic glycolysis and increases ATP production via improving the activity of pyruvate kinase and hexokinase. Subsequently, the PI3K/AKT signaling pathway is abnormally activated, thus accelerating the progression of BC.

In conclusion, deregulation of FTO is a tumorigenic factor that cannot be ignored. The FTO-m⁶A axis can be considered a potential new target for the treatment and diagnosis of BC. The functions of FTO in BC are presented in Fig. 4.

A growing body of evidence indicates that ALKBH5 is commonly dysregulated in malignant tumors, which regulates the expression of multiple oncogenes and contributes to tumor immune evasion through post-transcriptional mechanisms (89). However, studies indicated that ALKBH5 has a dual role in cancer, as its expression is not consistently upregulated or downregulated across all cancer types. Certain studies have shown a positive association between ALKBH5 levels and BC (90-92).

Under anoxic conditions, ALKBH5 mediates the pluripotency factor Nanog homeobox (NANOG) to regulate the BC stem cell characteristic specification in a hypoxia-inducible factor-dependent manner. In other words, ALKBH5 enhances the demethylation of NANOG mRNA and upregulates NANOG, while knocking down ALKBH5 inhibits this pluripotency factor (93,94). Therefore, ALKBH5 disorder is considered to be an important link in the proliferation, metastasis and enhancement of the stem cell phenotype of BC.

In addition, ALKBH5 upregulates the expression of ubiquitin conjugating enzyme E2 C (UBE2C) and reduces that of p53 by modifying the m⁶A of the downstream target gene UBE2C (91). Among them, UBE2C has been proven to exert a carcinogenic effect (95). The upregulated p53 is conducive to decreasing cancer cells and preventing the occurrence of cancer (96). Therefore, the ALKBH5/UBE2C/p53 axis is regarded as a potential mechanism for promoting the tumorigenesis and metastasis of TNBC cells (91).

In general, before ALKBH5 can be utilized as a therapeutic target for BC, its expression and specific regulatory mechanism should be further clarified. The functions of ALKBH5 in BC are presented in Fig. 5.

As a m⁶A binding protein, YTHDF1 amplification is universal in cancer tissues. The level of YTHDF1 is negatively associated with survival and positively correlated with the degree of malignancy and metastasis (97-99). In the experimental report by Sun et al (98), YTHDF1 and its downstream target transcription factor 8 (E2F8) were indicated to promote the transition to S-phase by regulating cell cycle-related factors, and to be involved in DNA replication and DNA damage repair (DDR). Furthermore, YTHDF1 blocked the cleavage of E2F8 mRNA, which is dependent on METTL14. All of these findings indicate that YTHDF1 functions as a promoter of tumor growth. As reported by Chen et al (97), low YTHDF1 restrained the proliferation, invasion and EMT of BC cells, and blocked cell-cycle progression. YTHDF1 also accelerated the translation of forkhead box (FOX)M1 by combining with m⁶A-modified FOXM1 mRNA, thus promoting its carcinogenic effect.

YTHDF2 can selectively bind m⁶A-modified sites and promote mRNA decay, but its function in solid tumors is still controversial. Recent reports have mentioned that YTHDF2 can degrade tumor promoter and tumor suppressor gene mRNA and have a dual role in tumor progression (100,101). For instance, YTHDF2 acts as a cancer-promoting regulator in certain tumors, such as glioblastoma, acute myeloid leukemia and prostate cancer (102-104). However, it has a tumor suppressor function in other tumor types, such as melanoma and liver cancer (100). As Einstein et al (105) suggested, moderate expression of YTHDF2 is essential to maintain the survival of cells driven by MYC proto-oncogene, bHLH transcription factor (MYC). Depletion of YTHDF2 activates the EMT-specific pathway in BC cells, particularly in TNBC, leading to further activation of cancer-related translation initiation factors. However, in MYC-addicted cells, over-translation of these target mRNAs eventually activates programmed cell death, leading to TNBC tumor-cell apoptosis. This result proves the importance of YTHDF2 for the survival of TNBC cells and the feasibility of knocking down YTHDF2 as a potential therapeutic method.

YTHDF3 may boost translation by interacting with ribosomal protein and significantly raise the translation efficiency of YTHDF1/3 common target (106). YTHDF3 can enhance the stability of its target factor zinc finger E-box binding homeobox 1 (ZEB1) mRNA, which is an EMT transcription factor (107). Chang et al (108)'s study on brain metastasis of BC indicated that YTHDF3 regulates its own mRNA translation by binding to m⁶A residues in its 5'UTR. YTHDF3 also combines with m⁶A-modified mRNA to promote the expression of brain metastasis genes, such as ST6 N-acetylgalactosaminide α-2,6-sialyltransferase 5, gap junction protein α1 and EGFR. It is noteworthy that in comparison to primary BC, YTHDF3 expression was significantly increased in its brain metastases, but not in other organs such as lung, bone, liver, spleen, lymph nodes and adrenal glands.

In conclusion, YTHDF disorder is a prevalent occurrence in cancer tissues. YTHDF1 and YTHDF3 are responsible for improving the translation efficiency of m⁶A-modified mRNA, and they are frequently amplified in BC cells. Their high levels are closely related to poor prognosis and low survival rates. Conversely, YTHDF2 promotes mRNA degradation and also acts as a carcinogen most of the time. It is plausible that the YTH family proteins work collaboratively to execute their regulatory role in translation, but their respective roles in cancer cannot be replaced, providing potential targets for BC treatment. The functions of YTHDFs in BC are displayed in Fig. 6.

IGF2BP protein is a newly discovered m⁶A binding protein, which selectively binds to mRNA transcripts (109). The carcinogenic effect of IGF2BPs depends on its function of improving the stability and translation efficiency of certain oncogene mRNAs, such as MYC (109,110). The translocation of IGF2BPs may lead to the anomalous accumulation of carcinogenic products, thus stimulating the malignant development of cancer tissue (109).

Qiao et al (111) found that long intergenic ncRNA 483 (LINC00483) can promote the proliferation of BC cells and is negatively associated with the survival rate of patients with BC. A high level of IGF2BP1 significantly increased the expression of LINC00483, thus inducing carcinogenesis. Shi et al (112) reported that proto-oncogene MYCN activates IGF2BP1, and subsequently, IGF2BP1 enhances the stability of the carcinogen miR210HG and mediates its carcinogenic function in BC. According to the latest research, the ubiquitin specific peptidase 10 (USP10)/IGF2BP1/carnitine palmitoyl transfer 1A (CPT1A) axis plays an important role in BC metastasis (113). They found that the de-ubiquitination enzyme USP10 reduces its cleavage by de-ubiquitination of IGF2BP1. Subsequently, IGF2BP1 binds to the m⁶A site on CPT1A mRNA and makes it more stable, thus promoting the growth and metastasis of BC (114,115).

In BC, the level of PD-L1 increases with the increase of IGF2BP3. Knocking down IGF2BP3 significantly inhibited the expression of PD-L1, which cooperates with tumor cells to escape immune surveillance (70). In addition, the IGF2BP3/tripartite motif containing 25 (TRIM25)/miR-3614 axis represents a new way to regulate tumor cell proliferation. TRIM25 is mainly expressed in estrogen target tissues, which can improve cell viability and promote cell proliferation. MiR-3614-3p can be used as a tumor suppressor to inhibit the growth of BC cells. IGF2BP3 can induce the expression of TRIM25 and inhibit the maturation of miR-3614, which conversely protects TRIM25 mRNA from miR-3614-mediated degradation (116).

In short, the IGF2BP gene and its downstream targets are generally amplified in BC, thereby resulting in enhanced proliferation, metastasis and poor prognosis. These results provide a foundation for evaluating IGF2BP as a potential target for BC treatment, while the specific mechanism of IGF2BP should be further studied. The functions of IGF2BPs in BC are shown in Fig. 7.

The dysfunction of METTL14, WTAP, RBM15/15B and ZC3H13 in methyltransferase are also commonly recorded in cancer databases. METTL14 has been reported as an oncogene in most studies and its expression is usually positively correlated with the expression of METTL3 and WTAP. It can improve the stability of target mRNA through HuR (RNA-binding protein) mediation, involving in the regulation of cell cycle, EMT and other tumor growth processes (90). The expression level of WTAP in BC is higher than that in normal breast tissue, and it is positively correlated with tumor size and grade (117). Certain scholars have reported that the complement C5a receptor 1 (C5AR1)+/WTAP/enolase 1 (ENO1) axis regulates the glycolytic activity of BC cells and the lncRNA DLG-associated protein 1-antisense 1/miR-299-3p/WTAP axis promotes the proliferation of drug-resistant BC cells, which is worthy of further exploration (118,119). However, the function of WTAP in tumors cannot be separated from the expression of METTL3 (120).

HNRNPs, another family of m⁶A readers, are also involved in regulating various types of RNA processing, including translation and splicing (121). The tumor suppressor HNRNP E1 regulates the expression of EMT-related genes. Silencing HNRNP E1 increased BC-cell migration and endowed cells with stem cell characteristics, which promoted abnormal proliferation and metastatic growth of cancer cells (122). Knocking down HNRNP A1 accelerated cell death and reduced cell invasion (123). HNRNPC is upregulated in diverse cancers and HNRNPC silencing significantly suppressed BC-cell proliferation and tumor growth (124). The functions of these regulators in BC are illustrated in Fig. 8.

METTL3 can be specifically recruited to gene fragments damaged by ultraviolet radiation and rapidly methylated RNA; subsequently, m⁶A-modified RNA starts the DDR pathway to improve the cell survival rate (126,127). METTL3-mediated m⁶A methylation also regulates homologous recombination (HR)-mediated double-stranded DNA break (DSB) repair (128). Phosphorylated METTL3 can be localized in the DSB region so that the damaged chromatin region of the RNA is modified by m⁶A. The m⁶A-modified RNA is then recognized by YTHDC1 and forms a DNA-RNA hybrid with DSBs, which recruits repair-related proteins and promotes HR-mediated repair (129). It has been reported that a low level of METTL3 increases the sensitivity of cancer cells to the treatment of DNA damage, while upregulated METTL3 reduces the survival rate of patients with head and neck squamous cell carcinoma who have received cisplatin or radiation treatment of DNA damage (126). Knocking down METTL3-mediated and YTHDC2-mediated m⁶A modification led to the accumulation of DNA-RNA hybridization (R loop) and γH2AX (a DSB marker), which plays a key role in inhibiting cell growth and regulating genome stability (130).

A study revealed that METTL3-mediated m⁶A modification improves the stability of transcription factor activated enhancer binding protein 2C mRNA, thus increasing the abundance of DNA repair genes, which endows spermatogonioma cells with resistance to DNA damage induced by cisplatin treatment, promoting tumor cell survival (131). METTL14 arginine methylation is positively correlated with enhanced translation of DNA repair genes (132). VIRMA was also demonstrated to enhance the invasion and cisplatin resistance of teratoma cells by regulating DNA damage (133). On the contrary, FTO participates in the upregulation of repair gene resection repair cross complementation group 1 through β-catenin mRNA demethylation, equipping cervical squamous cell carcinoma with radiochemotherapy resistance (79,134). ALKBH5 can also be inhibited by small ubiquitin-like modifier, which upregulates DNA repair genes and protects cells from reactive oxygen species (ROS)-induced DNA damage (126). This indicates that m⁶A plays a dual role in anti-cancer therapy based on DNA damage through DDR.

M⁶A modification is also involved in the regulation of telomere length and genomic integrity in human cancer (135). Telomere shortening is closely associated with cancer-related genomic changes (136). Homebox-containing protein 1 (HMBOX1) is a telomere-binding protein. HMBOX1 mRNA has been identified as the real target of m⁶A modification in cancer cells. HMBOX1 degradation caused by upregulation of METTL3 and YTHDF2 in cancer cells leads to telomere shortening and dysfunction of p53-dependent DNA damage response pathway inactivation. This change is likely to lead to various types of telomere-related chromosome aberrations, thus enhancing the tumorigenicity and invasiveness of cancer cells. Conversely, the malignant progression of cancer cells caused by METTL3-induced genomic instability can be alleviated or even reversed by introducing HMBOX1 (135).

The pathogenesis of BC primarily entails the hyperactivation and overexpression of oncogenes, coupled with deficiencies in DDR gene defects, DDR gene transcription defects and mitotic defects, among others. The defective repair of damaged DNA leads to genomic instability, which is closely related to the malignant progress and poor prognosis of BC.

It has been found that tumor genome subtypes of BC are related to tumor gene expression, which involves the methylation gain and loss processes of a large number of loci (137). The researchers suggested that extensive aberrations in methylation induce epigenomic instability, rendering tumors more prone to regulatory mutation and deterioration. They even linked different methylation scores with higher epigenetic instability and higher chromosomal instability in BC, predicting the disease stage and progress (137).

Based on the above principles and experimental evidence, it may be reasoned that METTL3-mediated modification of m⁶DSB repair may serve as a promising target for cancer treatment, including BC. Whether targeted inhibition of METTL3 can reduce the proliferation activity and invasiveness of BC cells by inhibiting DNA repair or improve the sensitivity of BC cells to DNA damage therapies (such as chemotherapy or radiotherapy) is also likely to become a new topic. It may also be true for other m⁶A methylases and demethylases.