Role, mechanism, and application of N6-methyladenosine in hepatobiliary carcinoma

Abstract

Hepatobiliary carcinoma is a frequently occurring and highly invasive cancer within the digestive tract, known for its rapid progression. Due to its difficult diagnosis and treatment in clinical practice, hepatobiliary carcinoma is a serious threat to human life and health. In recent years, the incidence of hepatobiliary carcinoma has gradually increased. N6-methyladenosine (m6A) modification, as a reversible post-transcriptional modification of the adenosine N6 site, is one of the most important RNA modifications in eukaryotes. Emerging research indicates that m6A affects the biological process of cells through the regulation of gene expression. m6A modification also plays a key role in the occurrence and development of various cancers. This review summarizes the role and mechanism of m6A modification in hepatobiliary carcinoma, and discussed its potential clinical application, so as to provide a theoretical reference for the individualized treatment of hepatobiliary carcinoma.

Key Words: Hepatobiliary carcinoma; RNA modification; N6-methyladenosine; Therapeutic targets; Treatment

Core Tip: N6-methyladenine (m6A) modifications is one of the most common RNA modifications in eukaryotes, and it has been reported to affect biological process of cells via regulation of gene expression, and play a crucial role in occurrence and development in various cancers by regulating RNA stability, decay, spicing and transport. This article summarized the role and mechanism of m6A modification in hepatobiliary carcinoma, and discussed its potential clinical application in an attempt to provide theoretical evidences for the individualized treatment of hepatobiliary carcinoma.

INTRODUCTION

Hepatobiliary carcinoma is a common malignant disease of the digestive system, and mainly includes cholangiocarcinoma (CCA) and gallbladder cancer. Its onset is hidden and progress is rapid, which is a serious threat to human life and health[1]. Since early hepatobiliary carcinoma has no significant clinical symptoms, most patients are diagnosed at an advanced stage and have a high recurrence rate after radical surgery. The 5-year overall survival rate is < 10%[1-5].

RNA modification is important in epigenetics and is closely related to the occurrence and development of cancer. As a reversible post-transcriptional modification, N6-methyladenosine (m6A) is the most common and important modification of eukaryotic cells[6,7]. m6A modification plays a significant role in many biological processes such as cell proliferation, differentiation and apoptosis[8,9]. Emerging research indicates that m6A modification plays a crucial role in the initiation and development of tumors by regulating RNA stability, decay, splicing and transport[7-12].

COMPOSITION OF M6A MODIFICATION

As a reversible post-transcriptional modification, the level of m6A modification depends on the activity of m6A methyltransferase ("writer") and demethylase ("eraser")[13-15]. m6A writers include but are not limited to methyltransferase-like (METTL)3, METTL14, WT1-associated protein (WTAP) and RNA-binding motif protein (RBM)15, and erasers include fat mass and obesity-associated protein (FTO) and AlkB homolog (ALKBH)5[6]. RNA-binding proteins ("readers") specifically recognize the m6A site to regulate RNA export, translocation, translation and decay (Figure 1)[14,16,17].

Figure 1 Schematic depiction of N6-methyladenosine modification in RNA metabolism. The installation of N6-methyladenosine (m6A) is facilitated by writers such as METTL3, METTL14, WTAP, RBM15, VIRMA, and METTL16. Conversely, erasers like FTO and ALKBH5 are responsible for removing these m6A modifications. Essential to this process are readers, which recognize m6A and implement post-transcriptional regulation. Collectively, writers, erasers, and readers intricately orchestrate RNA’s splicing, export, translation, decay, and overall stability.

Writers

Emerging research indicates that the ability of writers to regulate m6A modification is achieved through a multicomponent methyltransferase complex[6,18]. As the first identified m6A methyltransferase, METTL3 was able to catalyze the m6A modification process by forming a stable methyltransferase complex with METTL14[19-22]. WTAP, as an adaptor protein, can interact with the above dimer complex to determine its location in the nuclear spots and play a catalytic role[20,23,24]. Subsequently, members of m6A methyltransferase such as RBM15 and METTL16 were successively found, which played an irreplaceable role in the process of m6A modification[25-27].

Erasers

Erasers, namely m6A demethylase, can reverse m6A methylation modification, and co-regulate m6A RNA modification with writers[28]. These demethylases promote the conversion of m6A to N6-hydroxymethyl adenosine, thereby hydrolyzing it to adenosine[7,28]. Currently, few m6A demethylases are known, only ALKBH5/3 and FTO have been identified. Among them, FTO was the first m6A demethylase to be discovered[6,29,30]. Unlike FTO and ALKBH5, which mainly regulate mRNA, ALKBH3 was able to catalyze the demethylation of m6A to affect tRNA[31,32]. According to the latest reports, KIAA1429 may also function as an "eraser" to reverse m6A methylation modifications, but this requires further research for verification[33].

Readers

Although the level of m6A modification depends on the activities of writers and erasers, its biological function also requires different readers to induce a variety of biological phenotypes by recognizing and combining m6A methylation targets[34]. The readers mainly include the YTH domain family (including YTHDC1-2 and YTHDF1-3)[35-37], insulin-like growth factor 2 mRNA binding protein (IGF2BP)1-3[38,39], heterogeneous nuclear RNA protein family (HNRNP, including HNRNPC and HNRNPG)[40-42], and eukaryotic initiation factor (eIF)3[43]. YTHDF1-3 and YTHDC1-2 can directly recognize the conserved m6A binding domain, thereby directly reading and binding to m6A modified RNA[6]. IGF2BPs possess the ability to recognize m6A modifications and thus modulate the abundance and function of m6A-modified RNA[38]. HNRNP, also known as the m6A switch, can directly bind to mRNA possessing m6A recognition sites, indirectly regulating its abundance, translation, and stability by affecting the secondary structure of RNA[44,45].

Since the biological function of m6A modification depends on readers, different reader groups can regulate mRNA transcription by influencing the biological function of m6A modification, thus affecting cell function and regulating physiological status. Once there is an error in regulation, it may lead to the initiation and development of various diseases, including carcinomas. Therefore, blocking the binding sites of certain specific readers may become a new method for cancer treatment.

FUNCTION OF M6A

As a reversible post-transcriptional modification, m6A methylation is modulated by writers, erasers and readers, which is a dynamic balancing process. Through m6A methylation modification, various RNA splicing, translation, transport and stability can be regulated in an orderly manner[46-48].

Splicing

Splicing mRNA precursors into mature mRNA can affect the biological functions of eukaryotes, which is a complex and dynamically balanced process. The m6A site overlaps spatially with the splicing enhancer binding region of the serine/arginine-rich (SR) protein exon, and FTO can preferentially bind the alternative splicing exons and polyA sites, thereby modulating the RNA binding ability of SR splicing factor (SRSF)2 and further controlling mRNA splicing[7,29,49]. ALKBH5 colocalizes with ASF/SF2; it is able to alter the phosphorylation status of ASF/SF2, and the hyperphosphorylated ASF/SF2 is involved in mRNA splicing[50]. Previous studies have confirmed that loss of the m6A reader protein HNRNPG regulates alternative splicing in an m6A-dependent way[7,40]. Also, downregulation of m6A writers has been found to interfere with the processes of splicing and gene expression[51,52].

Nuclear export

It has been reported that ALKBH5 can regulate the nuclear export process, thereby influencing the subcellular distribution of mRNAs[7]. Mechanistically, ALKBH5 colocalizes with ASF/SF2; it is able to alter the phosphorylation status of ASF/SF2, thereby facilitating TAP/p15-complex-mediated mRNA export[50]. YTHDC1 is able to promote RNA binding to both nuclear RNA export factor 1 and export adaptor protein SRSF3, thus facilitating nuclear export[53]. Previous studies have confirmed that fragile X mental retardation protein, as an m6A reader, is able to facilitate nuclear export mediated by nuclear export protein chromosome region maintenance 1[54].

Translation

The function of METTL3 in regulating translation is enabled by the specific binding of different readers to the m6A sites. METTL3 can interact with eIF3h to modulate and facilitate translation, which is independent of methyltransferase activity[55,56]. Previous studies have confirmed that METTL16 and YTHDF proteins play pivotal roles in regulating translation[57-60]. YTHDF1, which is spatially adjacent to the translation start site bridged by eIF4G, plays a pivotal role in constructing loop structures with eIF4G and eIF3, as well as in the recruitment of ribosomes, thereby facilitating cap-dependent translation initiation[60]. YTHDC2 can interact with the 5'→3' exoribonuclease XRN1 and enhance helicase activity to promote translation, which is independent of m6A modification[61]. YTHDF3, in cooperation with YTHDF1, can interact with the 40S and 60S ribosome subunits to promote translation[59]. Besides, YTHDF3 facilitate the malignant development of bladder carcinoma through improving the translation of ITGA6[62].

Stability

As a reversible post-transcriptional modification, m6A modification bidirectionally regulates mRNA stability. For instance, YTHDF1 destabilizes MAT2A mRNA through binding to the m6A site 3’-untranslated region[63]. YTHDF2 is able to directly recruit the CCR4-NOT deadenylase complex to accelerate the process of deadenylation, thus promoting RNA degradation[64]. In synergism with YTHDF2, YTHDF3 promotes decay of m6A-modified RNA[65]. YTHDF2 can directly identify the methylation of Arrestin domain containing 4 and suppressor of cytokine signaling (SOCS)2 and destabilize their mRNAs, thereby facilitating the malignant progression of tumors[66,67]. It has been reported that the IGF2BPs family of proteins can significantly bolster the stability of mRNAs through the binding of their KH domains to specific m6A sites[38].

FUNCTIONAL ROLE OF m6A IN HEPATOBILIARY CARCINOMA

Modification of m6A plays a significant role in various biological processes, such as cell proliferation, differentiation and apoptosis, and it is crucial in the initiation and progression of tumors. The function of m6A modification depends on its readers, which means that the same m6A modification can have completely opposite effects when interacting with different readers. In this context, we have comprehensively summarized the research progress on the role of m6A in hepatobiliary carcinoma (Table 1).

Table 1 The roles and mechanism of N6-methyladenosine modification in hepatobiliary carcinoma.

Tumor typed role	Regulators	m6A-relate	Functions	Mechanisms	Ref.
Hepatocellular carcinoma	METTL3	m6A writers	Promotes HCC cell proliferation, migration, and tumorigenicity	Accelerates the degradation of SOCS2	[66]
			Promotes HCC progression	Modulates MAPK cascade	[78]
			Promotes the formation of vasculogenic mimicry	Enhances the translation efficiency of YAP1 mRNA	[133]
	METTL14	m6A writers	Inhibits tumor invasion and metastasis	Regulates the pri-microRNA 126 processing	[69]
			Inhibits HCC progression	Inhibits the EGFR/PI3K/AKT signaling pathway	[74]
			Inhibits HCC tumor growth	Regulates degradation of SLC7A11 mRNA	[75]
			Boosts the differentiation of liver CSCs and repress HCC growth	Maintains the stability of HNF3γ mRNA	[76]
			Impairs the metabolic reprogramming of HCC cells	Stabilizes USP48 mRNA	[77]
Pancreatic cancer	METTL3	m6A writers	Promotes tumorigenesis and chemoradiation tolerance	Modulates MAPK cascade	[52]
	METTL14	m6A writers	Promotes PC proliferation and migration	Induces PERP mRNA degradation	[81,82]
			Inhibits PC cells apoptosis and autophagy, and enhances chemotherapy tolerance	Facilitates AMPKα, ERK1/2, and mTOR pathways	[83]
			Enhances PC cells chemotherapy tolerance	Maintains CDA mRNA stability	[84]
			Inhibits PC progression	Induces PIK3CB mRNA degradation	[85]
	ALKBH5	m6A erasers	Inhibits the initiation and progression of PC	Reduce WIF-1 mRNA methylation and down-regulate the Wnt pathway	[86]
			Prevents PC tumorigenesis	Activates PER1 via YTHDF2-dependent manner	[87]
	FTO	m6A erasers	Promotes PC progression	Stabilizes proto-oncogene MYC mRNA	[88]
	YTHDF2	m6A readers	Inhibits PC progression	Curtails cell invasion and migration via the YAP pathway	[89]
			Accelerating the growth of PC cells	Activates Akt/GSK3b/CyclinD1 pathway	[89]
Gallbladder carcinoma	IGF2BP3	m6A readers	Promotes the aggressive progression of GBC	Stabilizes CLDN4 mRNA	[93]
			Promotes GBC progression	Stabilizes KLK5 mRNA	[94]
	YTHDF2	m6A readers	Promotes GBC progression and chemoresistance	Decreases DAPK3 mRNA stability	[95]
	METTL3	m6A writers	Promotes GBC cell proliferation, migration, and tumorigenicity	Destabilizes DUSP5 mRNA	[96]
			Promotes GBC progression	Regulates miR-92b-3p expression	[97]
Cholangiocarcinoma	METTL3	m6A writers	Facilitates glycolysis and CCA progression	Increase AKR1B10 expression via m6A-dependent manner	[101]
			Promotes CCA progression	Mediates IFIT2 mRNA degradation	[102]
			Facilitates iCCA proliferation and metastasis	Stabilizes NFAT5 mRNA	[103]
	METTL5	m6A writers	Promotes CCA progression	Mediating 18S rRNA m6A methylation	[104]
	METTL14	m6A writers	Promotes CCA progression	Disrupts Siah2 mRNA stability	[105]
	METTL16	m6A writers	Promotes ICC proliferation and metastasis	Up-regulates PRDM15-mediated FGFR4 expression	[106]
	VIRMA	m6A writers	Facilitates the malignant development of iCCA	Enhances the abundance of TMED2	[107]
			Facilitates the malignant development of iCCA	Enhances the abundance of PARD3B	[107]
			Promotes CCA progression	Activates the downstream target SIRT1	[108]
	IGF2BP1	m6A readers	Promotes iCCA progression	Activates the c-Myc/p16 and ZIC2/PAK4/AKT/MMP2 pathways	[109]
	YTHDF1	m6A readers	Induces CCA growth and metastasis	Regulates EGFR mRNA translation	[110]
			Induces CCA growth and metastasis	Mediating the m6A methylation of lncRNA CTBP1-AS2	[111]
	YTHDF2	m6A readers	Facilitates iCCA progression and chemoresistance	Destabilizes CDKN1B mRNA	[112]
	ALKBH5	m6A erasers	Promotes the malignant development of CCA	Increases the expression of PD-L1 to facilitate immune evasion	[113]

m6A: N6-methyladenosine; GBC: Gallbladder carcinoma; HCC: Hepatocellular carcinoma; PC: Pancreatic cancer; CCA: Cholangiocarcinoma.

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC), as the main type of primary liver cancer, is the sixth most common malignancy worldwide and the third most common cause of cancer-related deaths[68]. Previous studies have confirmed that abnormal m6A methylation plays a vital role in the progression of liver cancer (Figure 2). METTL14 expression is decreased in HCC tissues, which usually represents poor prognosis[69-73]. Knockdown-level METTL14 promotes malignant progression of HCC through activating the EGFR/PI3K/AKT signaling pathway[74]. METTL14 can interact with DiGeorge Critical Region 8 to modulate m6A-dependent pri-miR126 processing, thereby suppressing the metastatic potential of HCC[69]. METTL14 decreases expression of solute carrier family 7 (SLC7)A11 and facilitates m6A methylation of SLC7A11 mRNA, thereby promoting hypoxia-blocked ferroptosis in HCC cells[75]. Similarly, the expression level of Hepatocyte nuclear factor 3γ is modulated by METTL14, which can affect the differentiation of HCC cells and the resistance of sorafenib[76]. In addition, ubiquitin-specific peptidase 48, which is upregulated by METTL14-triggered m6A methylation, attenuates HCC tumorigenesis by deubiquitinating and stabilizing sirtuin (SIRT)6[77]. The above studies show that decreased m6A modification is crucial in the initiation and malignant progression of HCC.

Figure 2 Concise overview of N6-methyladenosine modifiers’ functions in hepatobiliary carcinoma. Diverse N6-methyladenosine (m6A)-related regulators, through aberrant m6A modifications, specifically target various genes or pathways, influencing the onset and progression of the tumor.

Likewise, up-regulated the expression of m6A is also closely related to HCC. For instance, the expression and prognosis of METTL3 are absolutely inverse to METTL14[70]. As a writer of m6A, excessive METTL3 accelerates the degradation of tumor suppressor SOCS2 through a YTHDF2-dependent mechanism, and ultimately promotes tumorigenesis[66]. METTL3 affects extracellular signaling pathways and the cell cycle through regulating the mitogen-activated protein kinase (MAPK) cascade, thus facilitating HCC malignant progression[78]. Recent studies have demonstrated that METTL3 enhanced beaded filament structural protein 1 stability by upregulating m6A modification, ultimately promoting tumorigenesis[79]. Therefore, upregulated m6A modification also plays a vital role in the initiation and malignant progression of HCC.

Pancreatic cancer

Pancreatic cancer (PC) could become the world’s third-most lethal cancer, according to projections, and the 5-year survival rate remains < 10% due to its late diagnosis, chemotherapy resistance and metastasis[80]. Recent studies have confirmed that abnormal m6A modification plays a pivotal role in the progression of PC (Figure 2). The MAPK cascade, as a ubiquitin-dependent modification in PC, can be modulated by m6A writer METTL3, thereby facilitating tumorigenesis and chemoradiation tolerance[52]. In addition, METTL14 is highly expressed in PC tissues, which can induce p53 effector related to PMP-22 mRNA degradation in an m6A-dependent manner, significantly promoting PC proliferation and migration[81,82]. Another study showed that downregulation of METTL14 facilitates apoptosis and autophagy in PC cells, while also enhancing sensitivity to chemotherapy. This effect is achieved through the inhibition of key signaling pathways, including AMP-activated protein kinase α, extracellular signal-regulated kinase (ERK)1/2 and mammalian target of rapamycin (mTOR)[83]. Similarly, downregulated METTL14 reduces cytidine deaminase mRNA stability through m6A-dependent manner, thereby enhancing the sensitivity of PC cells to chemotherapy[84]. Emerging research indicates that the m6A modification, catalyzed by METTL14 and recognized by YTHDF2, can enhance the rs142933486[G] allele variation of oncogene PIK3CB, thereby promoting the degradation of mRNA and decreasing PIK3CB expression, which is significant in PC, especially pancreatic ductal adenocarcinoma[85]. The eraser ALKBH5 can reduce WIF-1 mRNA methylation and downregulate the Wnt pathway, further inhibiting the initiation and progression of PC[86]. ALKBH5 also activates period circadian regulator 1 via YTHDF2-dependent manner, which activates the ATM/CHK2/P53/CDC25C pathway and ultimately prevents PC tumorigenesis[87]. Another type of eraser, FTO, is highly expressed in PC. Previous studies have confirmed that FTO reduces the methylation level of the proto-oncogene MYC mRNA and improves its stability, which ultimately promotes PC progression[88]. Moreover, YTHDF2 functions as an inhibitor, curtailing cell invasion and migration via the YAP signaling pathway. Conversely, it also acts as a promoter, accelerating the growth of PC cells through the Akt/GSK3b/CyclinD1 pathway[89]. Recent studies have confirmed that Proteasome 26S subunit non-ATPase 14 enhanced spondin 2 mRNA stability through RBM15B-mediated m6A modification, thereby facilitating PC proliferation, migration, and invasion[90]. Overall, all aspects of m6A modification, including writers, readers and erasers, can affect the initiation and development of PC, and it may be of importance to target one of these aspects as a therapeutic target for PC.

Gallbladder carcinoma

Gallbladder carcinoma (GBC) is the most common and deadly type of hepatobiliary carcinoma, with 5-year survival < 5% in the advanced stages, and most patients already present with metastases at initial diagnosis, frequently to the liver[91,92]. At present, research on the role of m6A in GBC is still in the initial stage, and the m6A aspects that are involved in the malignant progression of GBC include IGF2BP3, YTHDF2 and METTL3 (Figure 2). IGF2BP3 is expressed at elevated levels in GBC, which implies poor prognosis. IGF2BP3 stabilizes claudin 4 mRNA via an m6A-dependent manner, thereby activating the nuclear factor-kB pathway and ultimately promoting the aggressive progression of GBC[93]. Another study showed that IGF2BP3 stabilizes KLK5 mRNA via an m6A-dependent manner to activate the PAR2/AKT pathway, ultimately promoting the progression of GBC. It also confirmed that let-7 g-5p negatively regulates IGF2BP3 and the let-7 g-5p/IGF2BP3/KLK5/PAR2/AKT pathway is a potential treatment strategy for GBC[94]. Another type of reader, YTHDF2, decreases tumor suppressor death-associated protein kinase 3 mRNA stability and abundance via an m6A-dependent manner, ultimately promoting GBC malignant progression and chemoresistance[95]. METTL3 destabilizes dual specificity phosphatase 5 mRNA in an m6A/YTHDF2-dependent manner, thereby promoting malignant prognosis of GBC[96]. Emerging research indicates that deoxycholic acid (DCA) regulates GBC progression by inhibiting miRNA maturation via an m6A-dependent pathway. Mechanistically, DCA interacts with METTL3, disrupting the METTL3/METTL14/WTAP complex, which leads to a decrease in miR-92b-3p expression, and subsequently inactivates the PI3K/AKT pathway, which inhibits initiation and progression of GBC[97]. m6A methylation is important in GBC, and it may be important to target part of this modification process as a therapeutic strategy.

CCA

CCA is a primary liver cancer with an incidence second only to that of HCC, and is anatomically divided into intrahepatic CCA (iCCA) and extrahepatic CCA (eCCA). The early symptoms are not obvious; therefore, most patients are diagnosed at an advanced stage, and the 5-year survival rate is only 5%-15%[98-100]. Many studies have indicated that m6A methylation modification is critical in CCA (Figure 2). As a writer, METTL3 expression levels are elevated in CCA cells, which means poor prognosis. Highly expressed METTL3 bind to aldo-keto reductase family 1 (AKR1)B10 at m6A modification sites, and increase the expression level of AKR1B10 in an m6A-dependent manner, thereby facilitating glycolysis and CCA progression[101]. METTL3 also mediates interferon-induced protein with tetratricopeptide repeats (IFIT)2 mRNA degradation via a YTHDF2-dependent manner, thereby downregulating IFIT2 expression levels and ultimately promoting malignant prognosis of iCCA[102]. Importantly, METTL3-mediated nuclear factor of activated T-cells (NFAT)5 m6A modification stabilizes NFAT5 mRNA in the m6A-IGF2BP1-dependent pathway, thereby increasing the abundance of GLUT1 and PGK1, resulting in iCCA proliferation and metastasis[103]. The above views suggest that down-regulation of METTL3 may be a potential therapeutic target for CCA. Similarly, as methyltransferases, METTL5, METTL14, and METTL16 also play significant roles in CCA. Mechanistically, METTL5 facilitates ribosome synthesis and oncogenic mRNA translation by mediating 18S rRNA m6A methylation, and ultimately promoting the malignant prognosis of CCA[104]. METTL14 inhibits Siah2-mediated antitumor T-cell activity by disrupting Siah2 mRNA stability in an m6A-YTHDF2-dependent way, ultimately leading to CCA progression[105]. METTL16 upregulates members of the PRDI-BF1 and RIZ homology domain 15-mediated fibroblast growth factor receptor 4 expression in a YTHDF1-dependent manner to promote ICC proliferation and metastasis[106]. As another type of writer, vir-Like m6A methyltransferase associated (VIRMA) enhances the abundance of transmembrane effector 2 and par-3 homolog B through an m6A-HuR-mediated manner, activates the Akt/GSK/β-catenin and MEK/ERK/Slug pathways, thus facilitating malignant development of iCCA[107]. The pro-oncogenic effect of VIRMA-mediated m6A modification is also achieved by activating the downstream target SIRT1[108]. Readers and erasers also participate in the progression of CCA. For instance, IGF2BP1-mediated m6A modification activates the c-Myc/p16 and ZIC2/PAK4/AKT/MMP2 pathways, promoting iCCA progression[109]. YTHDF1 induces CCA growth and metastasis through several pathways, such as mediating the m6A methylation of lncRNA CTBP1-AS2 and regulation of EGFR mRNA translation[110,111]. YTHDF2 facilitates iCCA progression and chemoresistance by destabilizing cyclin-dependent kinase inhibitor 1B mRNA in an m6A-dependent manner[112]. As an important m6A demethylase, ALKBH5 increases the expression of programmed death protein ligand (PD-L)1 through Ya THDF2-dependent manner to facilitate immune evasion and ultimately promote malignant development of CCA[113]. To summarize, the role of m6A methylation modifications is pivotal and irreplaceable in the development and evolution of CCA. Exploring elements of this mechanism as potential therapeutic targets presents a highly promising avenue for research.

M6A MODIFICATION AND IMMUNE MICROENVIROMENT

Tumor immune microenvironment (TIME) is crucial in the progression of hepatobiliary carcinoma. In recent years, the important role of epigenetic regulation in TIME has gradually emerged, among which m6A has received special attention as the most abundant post-transcriptional modification[114]. As an important writer protein, METTL3-mediated m6A modification increases SHP-2 expression to activate interleukin (IL)-15-dependent pathways AKT/mTOR and MAPK/ERK, ultimately promoting the antitumor immunity of natural killer (NK) cells[115]. METTL3 also facilitates immunosuppression of tumor-infiltrating myeloid cells by elevating Janus kinase 1 expression in a YTHDF1-dependent manner[116]. Emerging research indicates that METTL3 increases the expression of protumor chemokines such as CXCL1, CXCL5 and 20, and promotes the degradation of PD-L1 mRNA through m6A methylation, resulting in the formation of non-inflamed tumor microenvironment[117]. Targeting METTL3 is a potential method to improve immunotherapy in hepatobiliary carcinoma patients. m6A modification mediated by YTHDF1 plays a pivotal role in facilitating hypoxia-induced autophagy and the advancement of HCC, primarily by enhancing the translation of autophagy-related genes (ATG)2A and ATG14[118]. Additionally, YTHDF2 can positively regulate the proliferation and survival of NK cells, and improve their antitumor and antiviral immune effects[119]. The RNA m6A eraser ALKBH5 regulates MAP3K8 m6A methylation, and recruits PD-L1⁺ tumor-associated macrophages in an IL-8-dependent manner, ultimately promoting HCC advancement[120]. Previous studies have suggested that dysregulated m6A modification implies a greater tumor mutational burden, thereby upregulating expression of pivotal factors such as PD-1, cytotoxic T-lymphocyte-associated antigen 4, T-cell immunoglobulin and mucin domain-containing protein (TIM)3, and lymphocyte activation gene 3, inducing the TIME, and leading to poor prognosis[121]. Currently, accumulating evidence indicates that m6A modification plays a prominent role in the advancement of hepatobiliary carcinoma, so that selecting rational therapeutic strategies against m6A is valuable.

ANTICANCER SMALL-MOLECULE TARGETING M6A REGULATORS

Presently, an accumulating body of evidence suggests that abnormal m6A modification is critically involved in the progression of various carcinomas. Consequently, targeting m6A regulators with precision might offer a promising therapeutic approach for treating hepatobiliary carcinoma.

As FTO inhibitors, natural product rhein, meclofenamic acid and fluorescein derivatives were among the first small-molecule identified to target m6A regulators, but their activity and specificity are relatively limited[122-124]. In recent years, a new generation of more potent and selective FTO inhibitors, including FTO inhibitor (FB)23, FB23-2, compounds from NCI DTP library NSC337766 (CS1) and NSC368390 (CS2), has been discovered[125-127]. FB23 and FB23-2, in particular, are known to positively regulate m6A modification, playing a pivotal role in leukemia treatment[125]. CS1 and CS2 impede tumor progression via the FTO/m6A/LILRB4 pathway[126]. Emerging research highlights the significant impact of FTO-04 in glioblastoma treatment through its inhibition of FTO[127]. At present, there are few studies on small-molecule inhibitors of demethylase ALKBH5, and two compounds have been discovered including 2-[(1-hydroxy-2-oxo-2-phenylethyl)sulfanyl]acetic acid(3) and 4-{[(furan-2-yl)methyl]amino}-1,2-diazinane-3,6-dione(6), which can inhibit the proliferation of cancer cells and play a considerable role in the progression of leukemia[128]. Although these small-molecule inhibitors have been reported to have significant effects in hematological tumors, these inhibitors may also become potential therapeutic modalities in hepatobiliary carcinoma overexpressing FTO or ALKBH5. The current small-molecule modulators of m6A writers are poorly studied, and STM2457, as the earliest bioavailable METTL3 small-molecule inhibitor, which has significant antitumor effects[102,129,130]. UZH1a, an emerging small-molecule METTL3 inhibitor, also plays a significant role in anticancer treatment[131,132].

Overall, RNA epigenetics-based tumor-targeted treatment is still in its infancy, and research on small-molecule drugs targeting m6A regulators is insufficient. It is necessary to further explore the mechanism of m6A modification, which is the basis for the development of new antitumor drugs.

CONCLUSION

Hepatobiliary carcinoma is a prevalent malignancy in the digestive system, and presents with a stealthy onset and swift progression, posing a significant threat to human life and health. m6A methylation, the most copious post-transcriptional modification, is currently the focus of extensive research. It is a dynamic and balanced process, intricately regulated by a trio of elements known as writers, erasers and readers. Accumulating evidence indicates that aberrations in m6A modification play a pivotal role in the pathophysiological development of the hepatobiliary system, substantially influencing the advancement of hepatobiliary carcinoma. This review systematically elucidates the dual roles of m6A modification in hepatobiliary carcinoma, revealing its molecular mechanisms in either promoting or suppressing tumor progression through dynamic regulation of oncogene expression, cancer stem cell properties, and tumor microenvironment interactions. Notably, the spatiotemporal specificity of m6A regulation leads to opposing effects in similar tumors, a phenomenon that may stem from: (1) Site-specific methylation differences (e.g., distinct functions of m6A in coding sequences vs 3'UTRs); (2) Variable expression profiles of reader proteins; and (3) Context-dependent integration of downstream signaling pathways. Although targeting m6A regulators (such as METTL3 inhibitors or FTO activators) has shown promise in preclinical models, current research faces three major challenges: First, at the technical level, existing m6A detection methods are challenging to resolve methylation dynamics at single-cell resolution, limiting our understanding of tumor heterogeneity. Second, at the biological level, the cooperative mechanisms between m6A regulatory networks and other epigenetic modifications (such as m5C or pseudouridylation) remain unclear. Finally, at the translational medicine level, developing highly specific small-molecule compounds still requires solutions to key issues: How to overcome off-target effects caused by structural similarities among reader proteins, how to achieve tissue-specific delivery, and how to establish reliable efficacy-predictive biomarkers. Future research should focus on: (1) Developing in situ m6A imaging technologies; (2) Constructing hepatobiliary carcinoma-specific m6A regulatory maps; and (3) Optimizing organoid models for drug screening. These breakthroughs will advance m6A-targeted therapies from bench to bedside, providing new paradigms for precision medicine in hepatobiliary carcinoma.