Introduction
Gynaecologic cancers encompass life-threatening malignancies that can affect the vulva, vagina, cervix and uterus, with the potential to spread to other organs associated with the functioning of the female reproductive system (Refs Reference Wang1, Reference Lõhmussaar, Boretto and Clevers2, Reference Huang3). In particular, carcinomas of the cervix, ovaries and endometrium are the most prevalent gynaecologic cancers, collectively representing 95% of all diagnosed cases (Ref. Reference Sung4). Cervical cancer (CC), comprising highly prevalent squamous cell carcinoma and rare adenocarcinomas, is the leading cause of gynaecologic cancer-related deaths among women, primarily affecting those between the ages of 35 and 44 (Refs Reference Sung4, Reference Arbyn5). CC currently presents several challenges, including elevated incidence and recurrence rates, resistance to current treatments, poor prognosis at advanced stages (i.e., 5-year survival rate consistently lower than 50%) and high mortality rates (Refs Reference Salani6, Reference Condic7, Reference Geng8). Therefore, there is an urgent need to deepen our understanding of the pathogenesis and progression of CC to overcome those limitations, which would unravel novel diagnostic markers and anti-cancer therapeutics.
Cervical cancer cells undergo both epigenetic and genetic changes that play a significant role in the disease progression, including the dysregulation of tumour-suppressing agents and oncogenes, from low-grade squamous intra-epithelial lesions to metastatic cancer (Ref. Reference Wang9). The research literature encompasses a variety of approaches aimed at understanding the mechanisms and components of these changes. Researchers have explored the role of DNA methylation, non-coding RNA and histone modifications in understanding CC initiation, as well as the potential impact on tumour immunity within the complex microbial landscape (Refs Reference Huang3, Reference Guo10). Technological advancements in genomic sequencing, particularly in studying epigenetic modifications, are continuously revolutionizing our understanding of the human genome and its health implications (Refs Reference Jonkhout11, Reference Lucas and Novoa12). Modern analysis techniques involve mapping the location and abundance of epigenetic modifications by combining antibody immunoprecipitation and chemical administration with next-generation sequencing (Refs Reference Jonkhout11, Reference Lucas and Novoa12).
Epitranscriptomic modifications, reversible epigenetic modifications of RNA, enable the regulation of various biological processes by RNA metabolism, localization, degradation, splicing, translation, stability, turnover and their intricate interactions. Groups of endogenous RNA-editing proteins have been identified to regulate epitranscriptomic modifications, including the ‘writers’ that facilitate the deposition of specific modifications, ‘erasers’ that remove particular modifications, and ‘readers’ that interpret the modifications and trigger downstream effects (Fig. 1) (Ref. Reference Jonkhout11). These modifications are observed across diverse RNA types, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), enhancer RNA (eRNA), viral transfer RNA (vtRNA), small nuclear RNA (snRNA), non-coding small RNA (sncRNA), long non-coding RNA (lncRNA), microRNAs (miRNAs) and circular RNAs (circRNAs) (Refs Reference Ma13, Reference Yu14, Reference Zhang15). Sequencing technologies have revealed over 145 post-transcriptional RNA modifications, with RNA methylation comprising a significant portion, around 60%, of all RNA modifications (Refs Reference Jonkhout11, Reference Lucas and Novoa12). The dynamic nature of RNA modifications enables swift cellular responses to environmental stimuli. The crucial role of RNA modifications in the fate of cancer tumour cells becomes apparent in their adaptation to rapidly changing and harsh conditions, such as those induced by drugs or stress. Epitranscriptomic modifications play a crucial role in the spatial and temporal expression of genes, and there is compelling evidence suggesting their involvement in tumour development, regulation and progression (Refs Reference Wang9, Reference Yu14). Epitranscriptomic modifications are linked to various hallmarks of cancer including survival, growth, restoration, differentiation, stress adaptation, invasion and drug resistance (Refs Reference Cui16, Reference Su17, Reference Janin18). Therefore, gaining a comprehensive understanding of molecular mechanisms, including the dysregulated endogenous RNA-editing proteins and epitranscriptomic modifications, that underlie the development and metastasis of CC is crucial for discovering diagnostic biomarkers, advancing therapeutic strategies and drug development. Notably, N6-methyladenosine (m6A), 5-methylcytosine (m5C) and N1-methyladenosine (m1A) are among the only epitranscriptomic modifications currently being researched in the context of CC.
Figure 1. Epitranscriptomic mechanism in cervical cancer cells. DNA is transcribed into RNA, which undergoes various modifications including N6-methyladenosine (m6A), 5-methylcytosine (m5C) and N1-methyladenosine (m1A). These RNA modifications are regulated by specific endogenous RNA-editing proteins, categorized as: (1) ʻwriters', facilitating modification deposition; (2) ʻerasers', removing modifications; and (3) ʻreaders', interacting with modified RNA. This epitranscriptomic mechanism intricately governs RNA fate, influencing processes such as export, localization, stabilization, translation and degradation. Consequently, this modulation of genetic expression profoundly impacts cellular functions in cervical cancer. Created with BioRender.com.
In this comprehensive review, we have elucidated the intricate cellular and molecular mechanisms governed by endogenous RNA-editing proteins and their associated epitranscriptomic modifications, with a particular focus on especially m6A, m5C and m1A, in modulating expression of both coding and non-coding RNAs (i.e., oncogenes and oncosuppressor genes) within CC cells. This review delves into the multifaceted roles of epitranscriptome in regulating key features of CC. We highlight the pivotal implications of altered epitranscriptome in conferring resistance to conventional therapies and recurrence in CC. Furthermore, we provide a concise overview of the therapeutic avenues that emerge from targeting the altered expression of endogenous RNA-editing proteins and aberrant deposition of RNA modifications, underscoring the potential for precision medicine strategies in combating CC. Table 1 summarizes the role of epitranscriptomics in cervical cancer as discussed in this review.
Table 1. Epitranscriptomic Regulation of Gene Expression by Endogenous RNA-Editing Proteins Impacting Key Hallmarks of Cervical Cancer.
Role of m6A modification in cervical cancer
N6-methyladenosine (m6A) RNA modification entails the methylation of the sixth nitrogen on the adenosine base and is currently the most researched chemical modification (Ref. Reference Zhang and Liu19). m6A modifications modulate RNA transcription, processing, splicing and translation to regulate oncogenic and tumour-suppressing gene activity (Ref. Reference Zhang and Liu19). CC cells have shown altered global m6A levels (Refs Reference Wang20, Reference Wang21), suggesting differential activity of writers and erasers promotes CC development and metastasis. The endogenous RNA-editing proteins for m6A modifications include (1) the writers METTL3/4/14/16, CBLL1, KIAA1429, ZC3H13, RBM15 and WTAP, (2) the erasers ALKBH3/5 and FTO, and (3) the readers YTHDC1/2, YTHDF1/2/3, HNRNPC, HNRNPA2B1, ELAVL1, ABCF1, FXR1, FMR1, LRPPRC and IGF2BP1/2/3. m6A regulators act as independent prognostic biomarkers, tumour microenvironment modulators and therapeutic targets for CC patients (Refs Reference Zhu22, Reference Lu23, Reference Zou24). Throughout the literature, various m6A-related independent prognostic signatures have been identified to predict CC patient survival including (1) ZC3H13, YTHDC1 and YTHDF1 (Ref. Reference Pan, Xu and Pan25), (2) ZC3H13, RBMX, ALKBH5, YTHDC1/2 and YTHDF1 (Ref. Reference Wu26), (3) METTL16, ZC3H13 and YTHDF1 (Ref. Reference Ji27), (4) ZC3H13 and G3BP1 (Ref. Reference Wang28), (4) ZC3H13, KIAA1429, HNRNPC and YTHDF1 (Ref. Reference Ma13), and (5) IGF2BP1, IGF2BP2, HNRNPA2B1, YTHDF1, and RBM15 (Ref. Reference Chen29). Moreover, ZC3H13 has shown the highest genetic alteration (especially deep deletion) frequency of 6% (Refs Reference Pan, Xu and Pan25, Reference Ji27), followed by 4% in LRPPRC and 3% in YTHDC2 (Refs Reference Guo10, Reference Ji27). ELAVL1, IGF2BP2, RBM15, WTAP, YTHDF2 and ZC3H13 show high frequencies of CNV deletions, while ABCF1, ALKBH3, FMR1, FXR1, IGF2BP2 and RBMX show high probabilities of CNV amplification (Refs Reference Guo10, Reference Wang28, Reference Zhang30). Among 297 cervical cancer patients, genetic alterations in endogenous RNA-editing proteins responsible for m6A modification were observed in 275 patients (93%) (Fig. 2), emphasizing the promising translational potential of these alterations as therapeutic targets and diagnostic markers warranting further investigation.
Figure 2. OncoPrint depicting the landscape of endogenous RNA-editing proteins responsible for N6-methyladenosine (m6A) modification in cervical cancer patients. Writers including CBLL1, METTL3/4/14/16, RBM15, VIRMA, WTAP and ZC3H13, as well as erasers ALKBH3/5 and FTO, are shown alongside readers such as ABCF1, ELAVL1, FMR1, FXR1, HNRNPA2B1, HNRNPC, IGF2BP1/2/3, LRPPRC, YTHDC1/2 and YTHDF1/2/3. Each column represents an individual patient sample and displays a comprehensive overview of the mutation spectrum, diagnosis age (years), overall survival (months), radiation therapy and genetic alterations, along with mRNA expression levels of m6A-associated endogenous RNA-editing proteins. mRNA expression is represented by z-scores relative to diploid samples (RNA Seq V2 RSEM). The Cancer Genome Atlas Program (TCGA) data of 297 cervical cancer patients were analysed and visualized using cBioPortal for Cancer Genomics (Refs Reference Cerami115, Reference Gao116, Reference de Bruijn117).
m6A writers METTL3/4/14, ZC3H13, RBM15, WTAP and CBLL1
Methyltransferase 3 (METTL3), an oncogenic m6A writer, is highly expressed in CC and associated with poor prognosis in patients (Refs Reference Wang20, Reference Chen31, Reference Liu32, Reference Du33). While METTL3 is highly expressed in tumours of all CC patients, human papillomavirus (HPV)-positive patients exhibit even higher METTL3 expression compared to HPV-negative patients (Ref. Reference Nie34). Mechanistically, ETS proto-oncogene 1 (ETS1) activates the transcription of METTL3 mRNA by mediating H3K4me3 and H3K27ac histone modifications through WDR5 and EP300, respectively, at the METTL3 promoter region in CC cells (Ref. Reference Du33). Also, the binding of TATA-binding protein to the METTL3 promoter region enhances METTL3 expression in CC cells (Refs Reference Huang3, Reference Li35). High levels of METTL3 lead to alteration in mRNA stability, degradation and translation of several genes. This cascade of changes contributes to cell proliferation, migration, chemotaxis, lymph node metastasis, immunosuppressive tumour microenvironment (i.e., reduced immune cell infiltration) and survival of CC cells (Refs Reference Huang3, Reference Wang20, Reference Liu32, Reference Li36, Reference Zhang and Zhang37, Reference Yu38). The involvement of METTL3 in cell cycle checkpoints and progression is critical for development and continuous growth of CC. The initiation of the G2/M phase is controlled by cell division cycle 25B (CDC25B), which stimulates the activation of CDK1/cyclin B and is considered an oncogene that is frequently altered in tumours (Ref. Reference Li36). High levels of METTL3 in the M phase upregulates CDC25B expression to promote cell cycle progression and tumorigenesis by inducing m6A modifications on CDC25B mRNA that are stabilized by m6A reader YTHDF1 (Ref. Reference Li36). Unique to METTL3, other key m6A writers do not exhibit remarkable expression during cell cycle progression (Ref. Reference Li36). Further interactions involve METTL3-induced m6A modifications on nuclear receptor NR4A1 mRNA, interacting with m6A reader YTHDF2 and DDX6, promoting NR4A1 mRNA degradation and facilitating malignancy in CC (Ref. Reference Yu39). Overexpressing NR4A1 impairs CC progression by recruiting transcription repressing LSD1/HDAC1/CoREST complex that inhibits AKT1 expression and consequent activation of the Akt signalling pathway (Ref. Reference Yu39). Moreover, the global increase in the transcription and translation rates within CC cells to support their malignant behaviour induces endoplasmic reticulum (ER) stress and demands proper protein folding to inhibit the activation of apoptosis pathways. TXNDC5, an ER protein that aids in correct protein folding, is highly expressed in CC patients (Ref. Reference Du33). METTL3 promotes CC cell proliferation and metastasis by inducing m6A modifications on the TXNDC5 mRNA, which are stabilized and signalled for translation by m6A readers IGF2BP2/3 and YTHDF1, respectively (Ref. Reference Du33). Also, METTL3 downregulates YTHDF2 expression and prevents consequent YTHDF2-mediated degradation of TXNDC5 mRNA (Ref. Reference Du33). METTL3 and TXNDC5 overexpression in CC reduces sensitivity to tunicamycin (i.e., glycosylation inhibitor) treatment, autophagy and apoptosis (i.e., low levels of Bax, active caspase 3 and LC3B-I/II) (Ref. Reference Du33). Furthermore, aggressiveness and metastasis of CC cells is mediated by METTL3-induced m6A modifications on apoptotic chromatin condensation inducer 1 (ACIN1) and cathepsin L (CTSL) mRNA, which are stabilized by IGF2BP3 and IGF2BP2, respectively, to upregulate their expression (Refs Reference Liu32, Reference Su40). Overexpressing IGF2BP3 in the METTL3 knockdown cells can rescue the decreased ACIN1 levels by prolonging the half-life of its mRNA (Ref. Reference Su40). Overall, METTL3 knockdown inhibits cell proliferation (i.e., by arresting cancer cells at the G0/G1 phase of the cell cycle, increasing apoptosis, lowering ACIN1 and TXNDC5 mRNA stability), migration (i.e., by lowering ACIN1 and TXNDC5 mRNA stability) and invasion (Refs Reference Wang20, Reference Liu32, Reference Su40). The involvement of CD33+ myeloid-derived suppressor cells (MDSCs) add another layer to the complexity, with METTL3 expression positively associated with CD33+ MDSC density (Ref. Reference Ni41). Given the role of MDSCs in enhancing tumour growth, establishing a pre-metastatic and immunosuppressive niche, and strengthening resistance to currently available immunotherapies for CC, high levels of both are correlated with shorter disease-free and overall survival of CC patients (Ref. Reference Ni41). Hence, combining multiple therapies such as immune-checkpoint inhibition (i.e., anti-PD-1) and MDSC-targeted therapy with METTL3 inhibitor presents a promising therapeutic approach for CC patients. METTL3 plays a pivotal role in regulating the expression of factors packaged within tumour-derived exosomes crucial for intercellular communication within the tumour microenvironment (Ref. Reference Ao42). It facilitates m6A modification on heat shock protein HSPA9 mRNA, thereby enhancing both their stability and translation in CC (Ref. Reference Ao42). This elevates the levels of exosomal mortalin HSPA9 protein, which correlates with tumour formation and progression (Ref. Reference Ao42).
Methyltransferase 14 (METTL14), an oncogenic m6A writer, is highly expressed in both HPV-positive and HPV-negative patients and is associated with reduced overall survival (Refs Reference Geng8, Reference Wang43). Upregulated METTL14 promotes the proliferation, migration, invasion and survival of CC cells (Refs Reference Geng8, Reference Wang43). Conversely, METTL14 knockdown impairs the malignant properties, induces cell cycle arrest, inactivates the PI3 K/AKT/mTOR signalling pathway (i.e., reduces AKT and mTOR phosphorylation), upregulates pro-apoptotic protein expression (i.e., active Caspase 9, BAX and BIM) and downregulates anti-apoptotic protein expression (i.e., BCL-2) in CC cells (Ref. Reference Geng8). METTL14 induces m6A modifications on tripartite motif-containing 11 (TRIM11) mRNA, a member of the E3 ubiquitin ligase family (Ref. Reference Zhang44). This modification enhances the stability of TRIM11 mRNA through an interaction with IGF2BP1 (Ref. Reference Zhang44). Elevated levels of TRIM11 contribute to increased ubiquitination of PHLPP1, consequently activating the AKT signalling pathway, thereby promoting tumorigenesis, proliferation, migration and invasion of CC cells (Ref. Reference Zhang44). Suppressing TRIM11 expression to enhance PHLPP1 levels represents a promising therapeutic avenue for inhibiting tumour growth in CC (Ref. Reference Zhang44).
Zinc finger CCCH-type containing 13 (ZC3H13) is a highly expressed oncogenic m6A writer that modulates centromere protein K (CENPK) and cytoskeleton-associated protein 2 (CKAP2) expression to promote malignant properties, tumour stemness and chemoresistance in CC patients (Refs Reference Lin45, Reference Zhang46). CENPK is a crucial protein in mitosis (especially chromosome segregation) while CKAP2, an intrinsically disordered protein, plays a key role in mitotic progression and exhibits cell-cycle-dependent expression (i.e., highest in the G2/M phase with localization in mitotic spindle and centrosome) (Refs Reference Lin45, Reference Zhang46). ZC3H13-induced m6A modifications on CENPK and CKAP2 mRNA upregulate their expression in CC, which is associated with cancer recurrence and shorter overall survival of patients (Refs Reference Lin45, Reference Zhang46). The binding of CENPK and SOX6 disrupts the potential interaction of CENPK and β-catenin resulting in nuclear translocation and enhanced expression of β-catenin, p53 ubiquitination, activated Wnt/β-catenin signalling pathway and inactivated p53 pathway (Ref. Reference Lin45). This alteration in cell activity results in proliferation (i.e., enhanced DNA replication), stemness (i.e., correlated with CD133, EPCAM, OCT4 and SOX2 expression), metastasis (i.e., enhanced epithelial-mesenchymal transition) and chemoresistance (i.e., enhanced DNA repair mechanism against cisplatin/carboplatin drugs) in CC (Ref. Reference Lin45). CENPK knockdown impairs those malignant properties of CC (Ref. Reference Lin45). While ZC3H13 inhibition reduces proliferation, migration and invasion of CC cells, overexpression of CKAP2 following ZC3H13 inhibition leads to partial restoration of those malignant properties (Refs Reference Lin45, Reference Zhang46). This suggests that either inhibiting ZC3H13 or synergistically inhibiting both CENPK and CKAP2 presents a promising therapeutic approach for CC patients. Contradictory to the studies by Lin et al. (Ref. Reference Lin45) and Zhang et al. (Ref. Reference Zhang46), Lu et al. (Ref. Reference Lu23) showed down-regulation of ZC3H13 in CC cells and knockdown of ZC3H13 enhanced the proliferation, migration and invasion of CC cells; hence, requiring further investigation to fully elucidate the complexity of ZC3H13's role in CC.
RNA binding motif protein 15 (RBM15), an oncogenic m6A writer, is highly expressed in HPV-positive as compared to HPV-negative CC patients (Refs Reference Nie34, Reference Zhang47, Reference Song and Wu48). The presence of HPV-E6 further exacerbates CC cell proliferation by enhancing intracellular RBM15 mRNA accumulation (i.e., inhibits its degradation), RBM15-induced m6A modifications-mediated c-MYC upregulation and inhibition of autophagy (Ref. Reference Nie34). Notably, HPV-E6 siRNA inhibits CC cell proliferation by promoting autophagy (Ref. Reference Nie34). RBM15 promotes proliferation, metastasis and stemness of CC cells (Refs Reference Quan49, Reference Wang50). Moreover, RBM15-induced m6A modification on deubiquitinase otubain 2 (OTUB2) mRNA upregulates its expression, correlating with stage progression of CC and predicting poor prognosis (Ref. Reference Song and Wu48). Also, RBM15 downregulates decorin (DCN) expression by inducing m6A modification on DCN mRNA, thereby enhancing the progression of CC (Ref. Reference Wang50). Conversely, RBM15 knockdown (i.e., upregulates DCN expression) supresses tumorigenesis, proliferation, migration and invasion of CC cells (Ref. Reference Wang50). Silencing RBM15 has been shown to suppress the malignant properties of CC cells by inhibiting the JAK-STAT signalling pathway and reducing OTUB2 expression (Refs Reference Zhang47, Reference Song and Wu48, Reference Wang50). Inhibition of OTUB2 promotes apoptosis and attenuates proliferation and metastasis of CC cells by downregulating the AKT/mTOR signalling pathway (Ref. Reference Song and Wu48). While a study conducted by Yuan et al. (2024) suggests that RBM15 might not a play role in apoptosis of CC cells (Ref. Reference Quan49), indicating a need for further investigation into this aspect.
Methyltransferase 4 (METTL4) and WT1-associated protein (WTAP) are highly expressed m6A writers in CC cells, especially in HPV-positive patients (Refs Reference Chen31, Reference Nie34). The expression of WTAP in CC cells appears enigmatic, with conflicting reports indicating both upregulation (Ref. Reference Nie34) and downregulation (Ref. Reference Zhang47), highlighting the complexity of its role and urging further investigation to reconcile these divergent observations. This difference in WTAP expression could be attributed to variations in CC cells samples, especially HPV status, but requires validation in future studies. Notably, the m6A writer Cbl proto-oncogene like 1 (CBLL1) exhibits significant downregulation in CC (Ref. Reference Zhang47), contrary to its overexpression observed in various other cancers. However, the oncogenic role of METTL4, WTAP and CBLL1 and underlying mechanisms remain largely unexplored.
m6A erasers FTO and ALKBH5
Fat mass and obesity-associated protein (FTO), a prominent m6A eraser/demethylase, was initially identified for regulating body mass and obesity. However, emerging research has demonstrated its involvement in the proliferation of various cancers, including acute myeloid leukaemia, melanoma, and breast, lung, endometrial and pancreatic cancers (Refs Reference Zou51, Reference Li52, Reference Yang53, Reference Niu54, Reference Zhou55, Reference Zhu56). In CC, FTO overexpression is associated with poor prognosis and regulates tumour cell proliferation, migration and invasion by upregulating the expression of cancer-promoting genes such as E2F1, ZEB1 and MYC (Refs Reference Zhang and Liu19, Reference Zou51, Reference Wang57). FTO achieves this modulation by reducing the deposition of m6A modifications on their mRNA, thereby enhancing their translation efficiency (Refs Reference Zou51, Reference Wang57). Knocking down FTO impairs the expression of genes E2F1 and MYC, leading to a reduction in cell proliferation, migration and invasion (Refs Reference Zou51, Reference Wang57). Notably, the ectopic expression of E2F1/ZEB1/MYC can restore the lost aggressiveness of CC (Refs Reference Zou51, Reference Wang57). Furthermore, FTO knockdown downregulates E2F1 downstream targets, impacts epithelial-mesenchymal transition and glycolysis while simultaneously activating the p53 pathway and DNA damage repair mechanisms (Ref. Reference Zou51). FTO also modulates the m6A-deposition on genes involved with the BMP4/Hippo/YAP1/TAZ pathway, influencing CC proliferation, migration and invasion (Ref. Reference Huang58). Importantly, BMP4 overexpression can restore the lost malignant behaviour in an FTO knockdown model (Ref. Reference Huang58). Along with a crucial role in CC pathogenesis, FTO also interferes with the currently available treatments by enhancing the chemoradiotherapy resistance of CC cells (Ref. Reference Zhou55). FTO reduces the presence of m6A modifications on β-catenin mRNA, enhancing its translation efficiency, which upregulates the expression of the downstream DNA excision repair protein ERCC1 (Refs Reference Huang3, Reference Zhou55, Reference Qin59). Cells overexpressing FTO exhibit higher survival rates following cisplatin and irradiation treatment, while FTO inhibition increases the chemoradiotherapy sensitivity (Ref. Reference Zhou55). Inhibition of β-catenin counteracts FTO-induced chemoradiotherapy resistance in CC (Ref. Reference Zhou55). Elevated levels of FTO and β-catenin are associated with poorer prognosis of patients and reduced success rate of currently available cancer therapies (Ref. Reference Zhou55). Developing clinically safe drugs to inhibit oncogenic regulator FTO presents a promising therapeutic strategy for CC patients.
In contrast, m6A demethylase alkB homolog 5 (ALKBH5) acts as an oncosuppressor, limiting CC proliferation, migration, invasion and epithelial-mesenchymal transition (Ref. Reference Zhen and Pan60). Inhibition of ALKBH5 promotes the malignant behaviour of CC, downregulating E-cadherin expression and upregulating N-cadherin and vimentin expression (Ref. Reference Zhen and Pan60). Lower ALKBH5 levels are associated with a poorer prognosis in CC patients (Ref. Reference Zhen and Pan60). However, contradictory findings by Huo et al. (Ref. Reference Huo61) suggest an oncogenic role of ALKBH5 in CC progression. The HPV E7 oncoprotein activates histone modifications (i.e., H3K4Me3 and H3K27Ac) via E2F1 and modulates post-translation modifications via DDX3, which promotes the expression of ALKBH5 in CC cells (Ref. Reference Huo61). ALKBH5-mediated m6A demethylation on p21 activated kinase 5 (PAK5) mRNA stabilizes and enhances PAK5 expression in a YTHDF2-dependent manner, contributing to CC progression (Ref. Reference Huo61). Also, METTL3, METTL14, FTO and ALKBH5 have been identified as regulators of the expression of the tumour suppressor DIRAS family GTPase1 (DIRAS1) (Ref. Reference Wang62). While FTO and ALKBH5 play crucial roles in regulating malignant properties, non-coding RNAs and metabolism in CC (discussed in the below sections), the role and underlying mechanism of another m6A eraser, ALKBH3, remain unexplored.
m6A readers YTHDC1/2, YTHDF1/2/3 and IGF2BP1/2/3
YTH N6-methyladenosine RNA binding protein C1 (YTHDC1), identified as a tumour-suppressing m6A reader, interacts with m6A modifications on the suppressor of cytokine signalling 4 (SOCS4) mRNA (Ref. Reference Chen63). This interaction enhances SOCS4 expression, leading to the inhibition of angiogenesis and proliferation of CC cells (Ref. Reference Chen63). Notably, CC patients exhibit low levels of YTHDC1 (Ref. Reference Chen63). Overexpressing YTHDC1 counteracts CC progression by inhibiting proliferation, migration, invasion, impairing angiogenesis through reduced vascular endothelial growth factor A (VEGF) expression and facilitating CC cell apoptosis (Ref. Reference Chen63). Conversely, YTH N6-methyladenosine RNA binding protein C2 (YTHDC2) is highly expressed in CC, yet its role and underlying mechanisms in pathogenesis remain elusive (Ref. Reference Chen31).
YTH N6-methyladenosine RNA binding protein F1 (YTHDF1), an oncogenic m6A reader, exhibits high expression in CC, correlating with poor recurrence-free survival (Refs Reference Wang20, Reference Wang64). YTHDF1 upregulates RAN binding protein 2 (RANBP2) expression by interacting with m6A modifications on its mRNA (Ref. Reference Wang64). This interaction promotes proliferation, migration and invasion while inhibiting apoptosis of CC cells (Ref. Reference Wang64). YTHDF1 knockdown suppresses tumorigenesis and metastasis of CC cells and induces their apoptosis through downregulating RANBP2 expression (Ref. Reference Wang64). While the RANBP2 knockdown impairs the migrative and invasive properties of YTHDF1-overexpressing cells (Ref. Reference Wang64). Hence, targeting the YTHDF1-m6A-RANBP2 axis offers potential therapeutic avenues. YTH N6-methyladenosine RNA binding protein F2 (YTHDF2), an oncogenic m6A reader, interacts with the m6A modifications on the 5-hydroxytryptamine receptor 7 (HTR7) mRNA, contributing to tumorigenesis and dysregulated cell cycle in CC (Ref. Reference Chen31). Elevated expression of YTHDF2 and its target, receptor HTR7, is associated with poor prognosis in CC patients (Refs Reference Huang3, Reference Chen31, Reference Wu65). Inhibiting YTHDF2 emerges as a potential strategy to enhance the survival rate of CC patients. Additionally, YTHDF2 interacts with m6A modifications on the AXIN1 mRNA, stabilizing its expression (Ref. Reference Wu65). This interaction promotes CC progression and chemotherapy resistance of CC (Ref. Reference Wu65). YTHDF2 inhibition reduces migration, invasion and epithelial-mesenchymal transition, and enhances cisplatin chemosensitivity through regulating AXIN1 expression and inhibiting the Wnt/β-catenin signalling pathway (Ref. Reference Wu65). YTHDF2 knockdown arrests tumour cells in the S phase, impairing the growth of CC (Ref. Reference Huang3). YTH N6-methyladenosine RNA binding protein F3 (YTHDF3), an oncogenic m6A reader, is upregulated in CC through transcriptional activation by the transcription factor SREBF1 (Ref. Reference Zhong66). YTHDF3 promotes the proliferation, migration and invasion of CC cells, thereby regulating tumorigenesis and lymph node metastasis (Ref. Reference Zhong66). Radiotherapy-resistant CC cells exhibit elevated expression of hepatocyte nuclear factor 1-alpha (HNF1α) (Ref. Reference Du67). Highly expressed HNF1α upregulates the expression of YTHDF3, which interacts with m6A modifications on DNA repair protein RAD51 homologue 4 (RAD51D) mRNA (Ref. Reference Du67). This interaction accelerates RAD51D mRNA translation, preventing and repairing radiation-induced DNA damage (i.e., breakage) to enhance cancer cell viability (Ref. Reference Du67). The HNF1α/YTHDF3/RAD51D axis is a critical regulatory mechanism in patients resistant to currently available radiotherapy. Targeting this pathway in conjunction with radiotherapy could promote the survival of advanced-stage CC patients.
Insulin-like growth factor 2 mRNA binding proteins 1/2/3 (IGF2BP1/2/3) are highly expressed oncogenic m6A readers that enhance the stability and translation efficiency of proto-oncogene MYC (Refs Reference Huang68, Reference Sun69, Reference Han70). PARKIN (i.e., E3 ubiquitin ligase) ubiquitinates IGF2BP3, promoting its degradation and loss of oncogenic function in normal cervical tissue (Ref. Reference Sun69). However, low levels of PARKIN in CC cells result in IGF2BP3 overexpression, activating PI3 K and MAPK signalling pathways to promote tumorigenesis (Refs Reference Sun69, Reference Zhou71). IGF2BPs knockdown reduces MYC expression, inhibiting proliferation, migration and invasion of CC cells (Refs Reference Huang68, Reference Han70). HPV-induced carcinogenesis relies on the translation of viral early protein 7 (E7) in CC cells (Ref. Reference Wang72). IGF2BP1 interacts with m6A modifications on the E7 mRNA, stabilizing and promoting its translation (Ref. Reference Wang72). Mild daily heat stress treatment destabilizes the oncotranscript complex, including IGF2BP1 and results in the formation of E7-IGF2BP1 aggregates (Ref. Reference Wang72). These aggregates are targeted by the ubiquitin-proteasome system, downregulating E7 expression and reversing HPV-induced carcinogenesis (Ref. Reference Wang72). This suggests an epitranscriptomic-associated heat-based treatment strategy for patients with HPV-positive CC.
Table 2 illustrates the synergistic and sequential interaction of writers/erasers and readers with RNA, elucidating their role in regulating the expression of over 50 oncogenes and oncosuppressors in cervical cancer. Consequently, the identification of therapeutic targets becomes imperative for disrupting this intricate network of endogenous RNA-editing proteins and advancing the development of effective therapies.
Table 2. Synergistic interaction of endogenous RNA-editing proteins to modulate epitranscriptomic modifications and expression of specific genes in cervical cancer
m6A-associated long non-coding, micro, circular and PIWI-interacting RNAs
Long non-coding RNAs (lncRNAs), the largest group of non-coding RNA in mammals, manage around 70% of gene expression through DNA/RNA/protein interactions and have a potential role in cancer development (Refs Reference Jia73, Reference Bhan, Soleimani and Mandal74, Reference Cáceres-Durán, Ribeiro-dos-Santos and Vidal75). In CC, the oncogenic lncRNA DARS-AS1 regulates cytoprotective autophagy in the hypoxic tumour microenvironment (Ref. Reference Zhu76). Hypoxia-inducible factor 1-alpha (HIF1α) transcriptionally upregulates the expression of DARS-AS1 in CC cells (Ref. Reference Zhu76). DARS-AS1 binds to the DARS mRNA to enhance its stability and recruits METLL3 and METTL14 to promote the translation of the DARS mRNA in CC cells (Ref. Reference Zhu76). Upregulated DARS modulates the expression of downstream targets, ATG3 and ATG5, to promote cryoprotective autophagy in CC (Ref. Reference Zhu76). This unveils the HIF1α/DARS-AS1/DARS/ATG5/ATG3 axis as a promising therapeutic target for CC patients. Another CC-associated lncRNA, FOXD2-AS1, is associated with poor prognosis in patients and promotes cell proliferation and migration in CC (Ref. Reference Ji77). The expression of FOXD2-AS1 is maintained by METTL3, which enhances its transcript stability through inducing m6A modifications (Ref. Reference Ji77). FOXD2-AS1 can lower p21 mRNA expression by recruiting and supporting lysine-specific demethylase 1 (LSD1) (Ref. Reference Ji77). FOXD2-AS1 knockdown inhibits proliferative and migrative abilities, while promoting apoptosis in CC cells (Ref. Reference Ji77). METTL3 also regulates lncRNA METTL4-2, promoting its expression through YTHDF1-mediated mechanisms, ultimately enhancing epithelial-mesenchymal transformation in CC (Ref. Reference Shen78). METTL3 knockdown results in the upregulation of E-cadherin and downregulation of FN1, N-cadherin and vimentin (Ref. Reference Shen78). The expression of lncRNA HOXC13-AS is upregulated and stabilized by the demethylase activity of FTO in CC cells (Ref. Reference Wang79). HOXC13-AS upregulates frizzled class receptor 6 (FZD6) expression through H3K27ac modification induced by cAMP-response element binding protein (CBP) (Ref. Reference Wang79). The FZD6-mediated activation of Wnt/β-catenin signalling pathway promotes cell proliferation and invasion and epithelial-mesenchymal transformation in CC (Ref. Reference Wang79). Another m6A-regulaed lncRNA LINC00426 plays a crucial role in promoting epithelial-mesenchymal transition in CC cells via LINC00426/miR-200a-3p/ZEB1 axis (Ref. Reference Shen80). METTL3-induced m6A modification on LINC00426 promotes its expression in CC cells, which makes those cells resistant to bleomycin and cisplatin and sensitive to imatinib (Ref. Reference Shen80). LncRNA can also modulate the activity of RNA-editing proteins to promote epithelial-mesenchymal transition in CC (Ref. Reference Sui81). LncRNA LRRC75A-AS1 competitively binds with the IGF2BP1 protein, hindering its interaction with m6A modifications present on SYVN1 mRNA (Ref. Reference Sui81). This interference reduces the stability and translation of SYVN1 mRNA, which inhibits the degradation of NLRP3 through SYVN1-mediated ubiquitination and activates IL-1β/Smad2/3 signalling pathways to facilitate the progression of epithelial-mesenchymal transition in CC (Ref. Reference Sui81). Tumour-suppressing lncRNA GAS5-AS1 is significantly downregulated in CC, leading to cell proliferation, migration and invasion, while its overexpression suppresses the development and metastasis of CC (Ref. Reference Wang, Zhang and Wang82). Reduced GAS5-AS1 levels minimize the interactions between GAS5 mRNA and ALKBH5 (i.e., regulates m6A modifications) (Ref. Reference Wang, Zhang and Wang82). YTHDF2 interacts with m6A modifications on GAS5 mRNA, which destabilizes them and lowers the expression of GAS5 in CC (Ref. Reference Wang, Zhang and Wang82). While overexpression of GAS5-AS1 upregulates tumour-suppressing GAS5 expression in the ALKBH5-m6A-YTHDF2-dependent pathway to inhibit CC tumorigenesis and metastasis (Ref. Reference Wang, Zhang and Wang82). m6A modification-associated regulation of lncRNA MALAT1 expression has a critical role in CC (Ref. Reference Peng83), however, its underlying upstream mechanism remains elusive. HPV-positive CC cells show high expression of MALAT1 while silencing MALAT1 attenuates the proliferative, migrative and invasive properties of those cells (Ref. Reference Wu84). Also, silencing MALAT1 modulates miR-141-3p expression, resulting in reduced ALKBH5 expression and consequent downregulation of MMP2 and MMP9, which suppresses migration and invasion of CC cells (Ref. Reference Wu84). Moreover, the necroptosis-related lncRNA prognostic signature can predict the expression of m6A-associated writers, erasers and readers (Ref. Reference Lin85). m6A-related lncRNAs can act as accurate biomarkers for predicting prognosis, tumour microenvironment, immune cell infiltration, response to immunotherapies and patient survival (Refs Reference Jia73, Reference Zhang86, Reference Jia87, Reference Liu88, Reference Pan89). Downregulated lncRNAs AL109811.2, AC024270.4 and AC008124.1 and upregulated lncRNAs AC025176.1 and RPP38-DT are positively associated with the overall survival of CC patients, while the downregulated lncRNA AC015922.2 and upregulated lncRNA AC099850.4 are negatively associated with the overall survival of CC patients (Refs Reference Jia73, Reference Jia87).
Micro RNAs (miRNAs), a class of small non-coding RNAs, perform negative modulation of gene expression post-transcription and are widely known for their adamant roles in carcinogenesis (Ref. Reference Huang90). Highly expressed lncRNA ZNFX1 antisense RNA 1 (ZF-AS1) in CC indicates poor survival of patients, higher metastatic potential and advanced FIGO stage (Ref. Reference Yang91). Oncogenic ZF-AS1 suppresses miR-647 in a METTL3-mediated manner to promote CC development and metastasis while ZF-AS1 knockdown inhibits cell proliferation, migration and invasion (Ref. Reference Yang91). Overexpressing miR-647 partially inhibits the malignant properties of CC (Ref. Reference Yang91); hence, there would be missing parts to the METTL3-ZF-AS1-miR-647 axis that needs to be explored. Highly expressed lncRNA KCNMB2-AS1 in CC is associated with poor prognosis of patients while inhibiting KCNMB2-AS1 suppresses proliferation and induces apoptosis of CC cells (Ref. Reference Zhang92). KCNMB2-AS1 silences the expression of miR-130b-5p and miR-4294 resulting in the upregulation of oncogenic IGF2BP3 (Ref. Reference Zhang92). IGF2BP3 interacts with the m6A modifications on KCNMB2-AS1 to enhance its stability and expression (i.e., positive feedback loop), which results in pronounced tumorigenicity (Ref. Reference Zhang92). YTHDF2 interacts with METTL3/METTL14-induced m6A modification on tumour-suppressing lncRNA CARMN, promoting the degradation of CARMN (Ref. Reference Yu93). miR-21-5p is a downstream target gene of CARMN that can bind to CARMN and negatively regulate expression (i.e., causes degradation) of CARMN (Ref. Reference Yu93). Hence, targeting the interplay of m6A modification and miR-21-5p could reduce the occurrence and development of CC. RBM15 induces m6A modification to promote the stability and expression of lncRNA HEIH, which in turn promotes tumour cell proliferation, migration and stemness through the miR-802/EGFR axis (Ref. Reference Quan49). METTL3-induced m6A modifications on tumour-suppressing miR-193b downregulate its expression in CC cells (Ref. Reference Huang90). Low levels of miR-193b enable the overexpression of CCND1, which promotes deeper stromal invasion and tumorigenesis (Ref. Reference Huang90). Overexpression of miR-30c-5p emerges as a promising therapeutic strategy to inhibit tumour growth and metastasis in CC (Ref. Reference Gong94). miR-30c-5p exerts its effects by suppressing METTL3 expression, consequently reducing METTL3-induced m6A modifications on proto-oncogene KRAS mRNA. This leads to decreased expression of KRAS and promotes ferroptosis of CC cells (i.e., increases accumulation of Fe2+) (Ref. Reference Gong94).
Circular RNAs (circRNAs) play a critical role in cancer progression by regulating gene expression, sequestering miRNA and RNA-binding proteins, and interfering with transcription and splicing mechanisms (Ref. Reference Chen95). METTL3-induced m6A modifications increase the stability and expression of circ0000069, which suppresses miR-4426 expression to promote CC proliferation and migration (Ref. Reference Chen95). However, the downstream mechanism of miR-4426 remains elusive. hsa_circRNA_101996 acts as a miR-8075 sponge and modulates the expression of microtubule nucleation factor TPX2 to inhibit cell proliferation, migration and invasion in CC (Ref. Reference Qin59). Low levels of ALKBH5 in CC enable the presence of m6A modifications on circCCDC134, which significantly enhances its stability and expression in a YTHDF2-dependent manner (Ref. Reference Liang96). circCCDC134 regulates proto-oncogene MYB expression by recruiting p65 and functioning as a miR-503-5p sponge, which enhances HIF1α transcription and consequent CC development and metastasis (Ref. Reference Liang96). Overexpression of ALKBH5 or HIF1α in CC cells prolongs or shortens the overall survival, respectively (Ref. Reference Liang96). m6A-dependent upregulation of circARHGAP12 in CC promotes tumorigenesis (Ref. Reference Ji97). Moreover, circARHGAP12 combines with FOXM1 mRNA by interacting with IGF2BP2, which enhances FOXM1 translation and consequent malignant behaviour of CC cells (Ref. Reference Ji97). High expression levels of circRNF13 promote the stability and expression of CXC motif chemokine ligand 1 (CXCL1), which results in enhanced radiotherapy resistance of CC cells (Ref. Reference Shi98). Overexpressing METTL3 induces m6A modifications on circRNF13 and promotes its YTHDF2-mediated degradation, which results in reduced expression of circRNF13 and improved radiosensitivity in CC cells (i.e., similar to in CC cells with circRNF13 inhibition) (Ref. Reference Shi98).
Piwi-interacting RNAs (piRNAs) are widely expressed PIWI proteins-interacting small non-coding RNAs with dual roles in cancer, exhibiting both cancer-promoting and inhibiting properties (Refs Reference Xie99, Reference Vinasco-Sandoval100, Reference Zhong101). The highly expressed piRNA-14633 in CC enhances the stability and expression of METTL14 in a concentration-dependent manner, leading to increased cytochrome CYP1B1 expression and promoting cell proliferation, migration and invasion (Ref. Reference Xie99). Knockdown of piRNA-14633 or METTL14 impairs the malignant properties of CC cells (Ref. Reference Xie99). Additionally, the oncogenic role of highly expressed piRNA-17458 CC involves the promotion of cell proliferation (i.e., S/G2 arrest), migration and invasion without influencing apoptosis (Ref. Reference Liu102). piRNA-17458 enhances the stability of WTAP mRNA (i.e., no effect on METTL3/14, ALKBH5 and FTO mRNA stability), increasing m6A levels in CC cells and promote tumorigenesis (Ref. Reference Liu102). Knockdown of piRNA-17458 or WTAP abolishes the malignant properties of CC cells (Ref. Reference Liu102).
m6A-regulated metabolism
Understanding the impact of m6A modification on metabolism-related genes is crucial for unravelling the intricate mechanisms of cancer development and identifying potential therapeutic targets (Refs Reference Wang20, Reference Li35). In CC, METTL3-induced m6A modifications on pyruvate dehydrogenase kinase 4 (PDK4) mRNA play a pivotal role in enhancing its stability (facilitated by IGF2BP3) and translation (facilitated by YTHDF1/eEF-2 complex) (Ref. Reference Li35). This cascade of events leads to the activation of glycolysis, characterized by increased glucose and oxygen consumption rates, and ATP generation pathways, ultimately promoting CC tumour growth (Ref. Reference Li35). ALKBH5 overexpression or METTL3 knockdown in CC cells demonstrates a decrease in glucose consumption, ATP levels, extracellular acidification rate and lactate production rate, while increasing the oxygen consumption rate (Refs Reference Wang20, Reference Li35). Promoting the expression of glucose transporters and aerobic glycolysis enzymes becomes a strategy to increase glucose supply in the tumour microenvironment, heightening cell proliferation and inhibiting apoptosis (Refs Reference Hu103, Reference Reinfeld104, Reference Boese and Kang105). METTL3-induced m6A modifications on the growth factor HDGF mRNA enhance its stability and translation in an IGF2BP3-dependent manner (Ref. Reference Wang20). This, in turn, promotes glycolysis by activating ENO2 and GLUT4 in CC cells (Ref. Reference Wang20). METTL3-induced m6A modifications on hexokinase 2 (HK2) mRNA contribute to the enhancement of its stability and translation (mediated by YTHDF1) (Ref. Reference Wang20). This process improves glycolytic capacity, highlighting the significance of METTL3 in driving the Warburg effect and aerobic glycolysis, ultimately promoting the proliferation of CC cells (Ref. Reference Wang20). Exogenous expression of HPV oncogenes E6/E7 enhances intracellular HK2 and GSK3β expression, contributing to CC tumorigenesis and metastasis (Ref. Reference Liu106). Overexpressing FTO downregulates HK2 expression by inhibiting the nuclear export of HK2 pre-mRNA, while GSK3β overexpression promotes ubiquitin-proteasomal FTO degradation (Ref. Reference Liu106). E6/E7 proteins further regulate IGF2BP2 to interact with METTL14-induced m6A modifications on MYC mRNA, enhancing its translation to promote aerobic glycolysis, cancer development and metastasis (Ref. Reference Hu103). The knockout of IGF2BP2 and E6/E7 demonstrates inhibitory effects on CC progression and glycolytic capacity (Ref. Reference Hu103). METTL14 can boost glycolysis by activating the AMPK signalling pathway, leading to the production of lactic acid (Ref. Reference Wang43). Elevated levels of lactic acid in the tumour microenvironment foster the M2 phenotype of macrophages, characterized by heightened expression of PD-1 (Ref. Reference Wang43). This shift to the M2 phenotype correlates with reduced phagocytic activity, ultimately contributing to enhanced tumour growth (Ref. Reference Wang43). The intricate involvement of ALKBH5 in lipid metabolism adds another layer to the intricate landscape of m6A-regulated metabolic pathways in CC. Low levels of tumour-suppressing ALKBH5 in CC are associated with enhanced fatty acid metabolism and poor patient prognosis (Ref. Reference Zhen and Pan60). Low levels of ALKBH5 enhance the presence of m6A modifications on SIRT3 mRNA, which improves their stability and translation in an IGF2BP1-dependent manner (Ref. Reference Zhen and Pan60). Elevated expression of SIRT3 causes an increase in ACC1 expression resulting in enhanced lipid metabolism in CC cells (Ref. Reference Zhen and Pan60). Overexpressing ALKBH5 in CC cell lines results in removal of m6A modifications on SIRT3 mRNA (i.e., lowers SIRT3 expression) and consequent reduction in ACC1 expression, which suppresses lipid metabolism and malignant behaviour of CC cells (Ref. Reference Zhen and Pan60). IGF2BP3 interacts with METTL14-induced m6A modifications on stearoyl-CoA desaturase (SCD) mRNA, leading to upregulated SCD expression in CC cells (Ref. Reference Han70). Elevation in SCD levels accelerates lipid metabolism, ultimately promoting the proliferation and metastasis of CC cells (Ref. Reference Han70). YTHDF3 interacts with m6A modification on LRP6 mRNA, boosting its translation efficiency in CC cells (Ref. Reference Zhong66). LRP6's pivotal role lies in activating the Wnt/ß-catenin signalling pathway, which in turn reprograms fatty acid metabolism to promote lymph node metastasis via the LRP6-YAP-VEGF-C axis in CC (Ref. Reference Zhong66). IGF2BP3 plays a critical role in enhancing glutamate and glutamine metabolism by stabilizing and upregulating the expression of GLS and GLUD1 mRNA through an m6A-mediated mechanism (Ref. Reference Zhou71). This regulatory process leads to heightened lactate production and secretion, thereby facilitating Treg cell-mediated immune evasion (Ref. Reference Zhou71). The complex regulatory network involving m6A modifications, metabolic enzymes and oncogenic factors sheds light on the multifaceted nature of metabolic reprogramming in CC.
Role of m5C modification in cervical cancer
Recent research findings have shed light on the multifaceted role of 5-methylcytosine (m5C), a post-transcriptional modification characterized by cytosine methylation at the 5th position, in various molecular processes. These encompass RNA export, fragmentation, translation, transcription, ribosome composition, tRNA homeostasis maintenance, stress regulation, codon-anticodon pairing, translation control, rRNA glioma sensitivity to stress-related enzyme NQO1 substrates, structural preservation of the tertiary rRNA–tRNA–mRNA complex, mRNA nuclear cytoplasmic-shuttling, splicing, DNA damage repair, migration, proliferation, development, differentiation, stability and stem cell augmentation (Ref. Reference Zhang15). Despite the well-established associations of m5C modifications with the development and aetiology of various cancers, autoimmune diseases and cardiovascular conditions, there exists a notable lack of research on their role and mechanisms in CC initiation and progression (Refs Reference Yu14, Reference Guo107, Reference Chen108). This highlights the critical necessity to unravel the mechanisms and functionalities of m5C modifications in the specific context of CC. A comprehensive exploration of the functions of the writers, readers and erasers involved in the formation and removal of m5C modifications holds the promise of providing valuable insights into the intricate landscape of CC (Refs Reference Yu14, Reference Chen108). The writers or methyltransferases responsible for catalysing m5C modification include NSUN1/2/3/4/5/6/7, DNMT1, DNMT3A/B and TRDMT1 (Refs Reference Wang1, Reference Yu14, Reference Yang109). On the other hand, TET2 acts as an eraser or demethylase, while ALYREF and YBX1 serve as readers or distinct effector proteins in the complex regulatory network of m5C modification (Refs Reference Wang1, Reference Yu14, Reference Yang109). Among 297 cervical cancer patients, genetic alterations in endogenous RNA-editing proteins responsible for m5C modification were observed in 236 patients (79%) (Fig. 3), emphasizing the promising translational potential of these alterations as therapeutic targets and diagnostic markers warranting further investigation.
Figure 3. OncoPrint depicting the landscape of endogenous RNA-editing proteins responsible for 5-methylcytosine (m5C) modification in cervical cancer patients. Writers including DNMT1, DNMT3A/B, NOP2, NSUN2/3/4/5/6/7 and TRDMT1, as well as eraser TET2, are shown alongside readers such as ALYREF and YBX1. Each column represents an individual patient sample and displays a comprehensive overview of the mutation spectrum, diagnosis age (years), overall survival (months), radiation therapy and genetic alterations, along with mRNA expression levels of m6A-associated endogenous RNA-editing proteins. mRNA expression is represented by z-scores relative to diploid samples (RNA Seq V2 RSEM). The Cancer Genome Atlas Program (TCGA) data of 297 cervical cancer patients was analysed and visualized using cBioPortal for Cancer Genomics (Refs Reference Cerami115, Reference Gao116, Reference de Bruijn117).
m5C-associated prognostic gene signature
Genes intricately linked with m5C modification emerge as potent prognostic indicators in CC, offering accurate predictions of 1-, 3- and 5-year survival rates for patients (Ref. Reference Yu14). Notably, a 4-gene signature comprising CPE, FNDC3A, OPN3 and VEGFA has demonstrated remarkable prognostic capabilities (Ref. Reference Yu14). Elevations in this gene signature within CC patients correlate with adverse prognoses, while therapeutic interventions targeting oncogenes CPE, FNDC3A or VEGFA exhibit promising outcomes by restraining cancer cell proliferation, migration and invasion (Ref. Reference Yu14). The modulation of key m5C writers and erasers is intricately linked with the survival rates of CC patients. Downregulation of writers NSUN2/3/6, DNMT1 and DNMT3B and eraser TET2, coupled with the upregulation of writer NSUN5 and reader ALYREF, is associated with improved survival outcomes of CC patients (Ref. Reference Yu14). However, the expression of writers NSUN1/4/7, TRDMT1 and DNMT3A appears to have no influence on patient survival rates (Ref. Reference Yu14). Intriguingly, immune cell infiltration emerges as a pivotal factor influencing CC patient survival. Robust infiltration of activated CD8T cells, natural killer cells, macrophages and myeloid-derived suppressor cells is correlated with enhanced survival rates of CC patients (Ref. Reference Yu14). Conversely, CC patients exhibiting central memory CD4 T cells and neutrophil infiltration tend to face a less favourable prognosis (Ref. Reference Yu14). This nuanced understanding of gene signatures and m5C regulators opens avenues for a novel molecular diagnostic clinical test, facilitating prognostic risk assessment and identifying potential therapeutic targets for CC patients.
m5C writer NSUN2 and reader YBX1
NSUN family of proteins emerges as pivotal players in tumour development and maintenance, offering potential m5C modified-oncogene biomarkers across various cancer types (Ref. Reference Yu14). NSUN2, in particular, not only catalyses mRNA methylation but also contributes to critical cellular functions such as promoting cell proliferation, maintaining mitotic spindle stability, and responding to diverse cellular stressors (Refs Reference Wang1, Reference Sajini110). In CC, the upregulation of NSUN2 takes centre stage, fostering the migration and invasion of cancer cells through m5C methylation on keratin 13 (KRT13) mRNA and consequent interaction/stabilization of those mRNA with highly expressed oncogenic reader YBX1 that promotes KRT13 expression (Refs Reference Wang1, Reference Zheng111). KRT13, a 54-kDa type 1 acidic intermediate filament protein, is recognized as both tumour suppressor and tumour promoter depending on the type of cancer (Ref. Reference Wang1). Potential therapeutic strategies involve inhibiting NSUN2 or introducing catalytically inactive mutations in NSUN2, disrupting the m5C-dependent NSUN2-YBX1-KRT13 axis to impede tumorigenesis in CC and improve patient survival. Inducing KRT13 overexpression can counteract the beneficial effects of inhibiting NSUN2 in CC, while overexpression of NSUN2 in KRT13 knockdown cells is unable to rescue the migration and invasion of CC (Ref. Reference Wang1). While the impact of NSUN2 depletion on CC cell proliferation remains debatable, it consistently hampers their migration and invasion (Refs Reference Wang1, Reference Chen112). Notably, the impact of inhibiting YBX1 in CC pathogenesis remains unexplored, warranting further exploration.
LRRC8A (leucine-rich repeat-containing 8 volume-regulated anion channel subunit A), a regulator of cellular homeostasis and osmoregulation, assumes a dual role by promoting cell survival under physiological stresses and facilitating tumorigenesis in in vitro and in vivo models by suppressing apoptosis (Ref. Reference Chen112). In CC, NSUN2 upregulation triggers m5C modification on LRRC8A mRNA, subsequently binding to the reader YBX1 and elevating mRNA stability, leading to enhanced LRRC8A expression (Ref. Reference Chen112). This overexpression is associated with increased cell survival, growth, migration and invasion, thereby shortening recurrence-free survival for CC patients (Ref. Reference Chen112). Knockdown of LRRC8A, conversely, inhibits CC cell proliferation, migration and invasion, accompanied by promoting the swelling and breaking of the cancer cells (Ref. Reference Chen112). Additionally, LRRC8A knockdown reduces reactive oxygen species production and inactivates the PI3K/AKT signalling pathway, while inducing AKT activation in LRRC8A knockdown rescues the cell migration and inhibits Caspase-3 expression in CC (Ref. Reference Chen112). Moreover, the LRRC8A knockdown cells are highly sensitive to cisplatin, suggesting its potential role in chemotherapy resistance in CC patients (Ref. Reference Chen112). Consequently, targeting the NSUN2-mediated m5C-LRRC8A-YBX1 axis emerges as a promising therapeutic strategy to prevent the malignant properties of CC.
Role of m1A modification in cervical cancer
Existing literature highlights the significance of N1-methyladenosine (m1A), a post-transcriptional modification involving adenosine methylation at the N1 position, in influencing RNA structure and protein interactions, with potential implications for gynaecological cancer cell proliferation (Refs Reference Wang9, Reference Xiong, Li and Yi113). Despite this, the specific role of m1A in CC remains largely underexplored. TRMT10C, an m1A writer, has garnered attention due to its distinct expression and functional consequences in these malignancies (Ref. Reference Wang9). Elevated TRMT10C expression in CC has been associated with poor patient survival, and its silencing has demonstrated suppressive effects on cancer cell proliferation, migration and colony formation (Ref. Reference Wang9). TRMT10C could potentially be associated with diverse cellular processes, including rRNA and tRNA metabolism, protein localization to the endoplasmic reticulum and chromosomes, nucleotide excision repair, endothelium and endoderm growth, integrin-mediated signalling and amoeboid-type cell migration (Ref. Reference Wang9). Furthermore, advanced stages of CC are associated with a decreased expression of the m1A eraser ALKBH3 and m1A writer TRMT6 (Ref. Reference Wang9). Conversely, high expressions of m1A writers TRMT6 and TRMT61A, along with m1A readers YTHDC1 and YTHDF2, have been correlated with better survival outcomes in CC patients, positioning them as promising prognostic biomarkers (Ref. Reference Wang9). Notably, a significant correlation exists between the expression of m1A regulators and the expression of m6A and m5C regulators during oncogenesis (Refs Reference Wang9, Reference Wang114). Low-risk m6A/m5C/m1A-regulated genes (CHAF1A, DUOX1, IGBP1 and STAC3) are associated with the infiltration of dendritic cells, macrophages, natural killer cells and T cells (Ref. Reference Wang114). Conversely, high-risk m6A/m5C/m1A-regulated genes (CA2, CUX1, IQGAP3, PTBP1, SLC2A1 and STAC3) are associated with infiltration of mast cells and poor survival duration of CC patients (Ref. Reference Wang114). This intricate interplay between m6A/m5C/m1A regulatory genes showcases their association with the immune microenvironment and immunotherapy, suggesting that anti-CTLA-4 therapeutics and pazopanib might be most suitable for the high-risk group (Ref. Reference Wang114). Among 297 cervical cancer patients, genetic alterations in endogenous RNA-editing proteins responsible for m1A modification were observed in 140 patients (47%) (Fig. 4), emphasizing the promising translational potential of these alterations as therapeutic targets and diagnostic markers warranting further investigation.
Figure 4. OncoPrint depicting the landscape of endogenous RNA-editing proteins responsible for N1-methyladenosine (m1A) modification in cervical cancer patients. Writers including TRMT10C, TRMT6 and TRMT61A, as well as eraser ALKBH3, are shown alongside readers such as YTHDC1 and YTHDF2. Each column represents an individual patient sample and displays a comprehensive overview of the mutation spectrum, diagnosis age (years), overall survival (months), radiation therapy and genetic alterations, along with mRNA expression levels of m6A-associated endogenous RNA-editing proteins. mRNA expression is represented by z-scores relative to diploid samples (RNA Seq V2 RSEM). The Cancer Genome Atlas Program (TCGA) data of 297 cervical cancer patients was analysed and visualized using cBioPortal for Cancer Genomics (Refs Reference Cerami115, Reference Gao116, Reference de Bruijn117).
Expert and topical summary
Epitranscriptomic modifications, reversible epigenetic RNA modifications, have emerged as a crucial factor in the development and progression of various cancers. This review explores the impact of epitranscriptomic modifications on CC, shedding light on endogenous RNA-editing proteins involved in this intricate process. Dysregulation of RNA modifications, specifically m6A, m5C and m1A, along with their associated writers, erasers and readers, significantly influences critical aspects of CC such as cell proliferation, migration, invasion, tumorigenicity and resistance to chemoradiotherapy. The review emphasizes the potential of targeting aberrant deposition of epitranscriptomic modifications by correcting the altered expression of associated RNA-editing proteins as a novel and promising therapeutic strategy for CC. The field of epitranscriptomics in CC is still in its infancy. With over 145 epitranscriptomic modifications and 20 of them being detectable with the currently available technologies, it presents a vast opportunity to explore the functional roles of unexplored RNA modifications in CC and opens avenues for developing drugs targeting epitranscriptomic modifications and RNA-editing proteins. In conclusion, epitranscriptomics stands out as a promising field in understanding the molecular mechanisms underlying CC. Further research should incorporate the use of single-cell RNA sequencing technology and multi-omics approach to elucidate the cell-specific functions of epitranscriptomic players and their cell-specific therapeutic potential in CC. The ongoing exploration and translation of those findings into clinically relevant diagnostic kits and treatment strategies holds a great promise that can potentially save lives and contribute to the well-being of women globally.
Acknowledgements
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Funding statement
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
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