Research Advances in the Roles of N6-Methyladenosine Modification in Ovarian Cancer

Ovarian cancer and METTL3

METTL3, the most critical component of the m6A methyltransferase complex, is a highly conserved S-adenosylmethionine (SAM)-binding protein. ²⁰ Several studies have underscored the significance of METTL3 in the development and progression of various cancers. ²¹ In studies related to hepatocellular carcinoma and lung cancer, METTL3 has been identified as an oncogene that promotes tumor proliferation, migration, and colony-forming ability, thereby correlating with poor prognosis in cancer patients.^22,23

The expression of METTL3 in OC tissues has been reported to be significantly higher than that in normal ovarian tissues. ²⁴ Additionally, METTL3 has been identified as a regulator of m6A methylation in epithelial ovarian cancer (EOC), independent of METTL14 and WTAP. ²⁵ Regarding the specific mechanism of METTL3 in OC development, various studies have proposed different explanations. For instance, Bi et al demonstrated that METTL3 could recognize the m6A modification site of pri-miR-1246, leading to the upregulation of miR-1246 expression and the inhibition of cyclin G2 (CCNG2), which accelerated the proliferation, migration, and invasion of OC cells while inhibiting their apoptosis, ultimately promoting OC progression. ²⁶ Cui et al demonstrated that in cisplatin-resistant OC cells, METTL3 could enhance the stability of RHPN1-AS1 (lncRNA RHPN1 antisense RNA 1) through m6A modification, ²⁷ which in turn promoted the expression of RHPN1-AS1, activated the PI3K/AKT signaling pathway, and subsequently enhanced cisplatin resistance in OC cells. Additionally, Wenfeng Hua et al reported that METTL3 could indirectly regulate AXL receptor tyrosine kinase (AXL), which plays a pro-oncogenic role in various cancers, including OC. ²⁴ Likewise, Liang et al proposed that METTL3 promotes the malignant progression of OC by activating the AKT-related signaling pathway and thereby stimulating a series of downstream effectors, including p70S6K and Cyclin D1. ²⁸ Moreover, Zhang et al experimentally validated that the low-level expression of oncogene intermediate filament family orphan 1 (IFFO1) in OC was modulated by METTL3/YTHDF2 participation. METTL3, when highly expressed, reduced the post-transcriptional stability of IFFO1, which in turn was degraded by YTHDF2. Consequently, the mechanisms of anti-tumor metastasis and IFFO1-mediated drug resistance reversal were inhibited. ²⁹ Bi et al, conversely, inhibited the development of OC by knocking down METTL3 to suppress the upregulation of phosphatase and tensin homolog (PTEN) mediated by miR-126-5p (a key biomarker for several cancers), thereby preventing the activation of the PI3K/Akt/mTOR pathway. ³⁰ Additionally, Fan et al reported an upregulation of baculoviral IAP repeat containing 5 (BIRC5), an inhibitor of the apoptosis (IAP) family protein, in cisplatin-resistant OC cell lines. They further showed that BIRC5 was upregulated and stabilized by METTL3-mediated m6A modification and IGF2BP1 binding, which in turn promoted platinum resistance in OC. ³¹ Furthermore, Yin et al demonstrated that the upregulation of receptor-interacting protein kinase 4 (RIPK4), which acts as an oncogene in OC, was facilitated by METTL3-mediated m6A modification. This process was accompanied by concurrent enhancement of RIPK4 mRNA stability mediated by YTHDF1, leading to NF-κB activation and subsequent tumor growth and cisplatin resistance. ³² Moreover, CircPLPP4 was found to be remarkably overexpressed in chemoresistant OC tissues and cells, with its increased levels regulated by METTL3/IGF2BP1 through modulation of its stability. ³³ Similarly, vestigial-like 1 (VGLL1) was also found to be upregulated by the combined action of METTL3 and IGF2BP2 in an m6A-dependent manner, which in turn promoted OC proliferation and metastasis. ³⁴ Additionally, a mechanistic study revealed that METTL3/YTHDF2-mediated methylation induces the degradation of the tumor suppressor lncRNA MEG3, which in turn releases the tumor suppression effect and subsequently promotes OC proliferation and metastasis through the miR-885-5p/VASH1 axis. ³⁵ These findings collectively affirm that METTL3 is dysregulated in OC and affects multiple signaling pathways in an m6A-dependent manner, thus playing a pro-cancer role in OC.

Ovarian cancer and METTL14

METTL14 acts as a key component of the m6A methyltransferase complex and influences oncogene expression in tumors by modulating RNA stability and degradation.^36-38 Interestingly, in breast cancer cells, METTL14 has been implicated in a pro-tumorigenic role, where it regulates the m6A methylation level of key mRNAs involved in the epithelial-to-mesenchymal transition (EMT). However, its expression is downregulated in various tumors, including hepatocellular carcinoma, bladder cancer, endometrial carcinoma, and glioblastoma, where it functions as a tumor suppressor.^39,40 Moreover, in digestive malignant tumors, METTL14 has been observed to play a contradictory role in the regulation of digestive system tumors, ⁴¹ with similar findings reported in the studies related to OC. Trophinin-associated protein (TROAP, also known as TASTIN) has been implicated in the proliferation, invasion, and migration processes of various cancers. ⁴² Li et al demonstrated that METTL14 negatively regulates the expression of TROAP in an m6A-dependent manner, thereby inhibiting OC cell proliferation. ⁴³ However, in another retrospective study, Wei et al proposed a different hypothesis suggesting that high METTL14 expression elevates the level of m6A modification in OC cells, thereby promoting their proliferation, invasion, and migration. ⁴⁴ Presently, there are limited studies on the association of METTL14 with OC; therefore, further exploration is needed to elucidate the multiple molecular mechanisms underlying the effect of METTL14 on the malignant biological behaviors of OC and the potential theoretical rationale for targeted therapies.

Ovarian cancer and WTAP

WTAP plays a crucial role in localizing the methyltransferases METTL3–METTL14 to nuclear speckles and engages in post-translational regulation, including binding to the 3′-untranslated region (UTR) to enhance mRNA stability and acting as a spliceosome for selective splicing of precursor mRNAs.^45,46 Similar to METTL3, WTAP is highly expressed in high-grade serous OC tissues, plays pro-proliferative, migratory, and anti-apoptotic roles, and is associated with patient prognosis and survival. ⁴⁷ Wang et al confirmed a significant positive correlation between WTAP expression and the expression of Family with sequence similarity 76 member A (FAM76A) and HBS1-like translational GTPase (HBS1L). They hypothesized that WTAP mediates the m6A modification of FAM76A and HBS1L, thereby synergizing with positive transcripts to promote their stability, ⁴⁸ which in turn promotes OC progression. Additionally, Lyu et al reported that WTAP drives the malignant progression of OC by promoting the maturation of miR-200, which subsequently affects hexokinase 2 (HK2), a key gene in the glycolytic pathway. ⁴⁹ Furthermore, Fu et al proposed that highly expressed WTAP promotes aberrant mRNA methylation, inhibiting the transcription and translation of the target gene early growth response 3 (EGR3), and should be considered a potential marker for OC subtype diagnosis. ⁵⁰

Ovarian cancer and VIRMA

VIRMA, also known as KIAA1429, is a recently identified m6A methyltransferase that is highly amplified or mutated in various cancer types, indicating its potential involvement in cancer development. Studies have shown that VIRMA is transcriptionally activated by SPI1, highly expressed in OC, and enhances the stability of enolase1 (ENO1) mRNA in an m6A-dependent manner, thereby promoting aerobic glycolysis and contributing to OC development. ⁵¹

Ovarian cancer and YTH domain family

YTHDF1–3 are highly homologous proteins that recognize and bind to m6A modification sites through the C-terminal YTH domain. However, they diverge in their main functions: YTHDF1 interacts with translation initiation factors, inducing target mRNA translation; YTHDF2 affects target mRNA stability; and YTHDF3 modulates translation and decay of m6A-modified mRNAs by interacting with YTHDF1 and YTHDF2. ⁵⁴ Liu’s team showed that YTHDF1 promoted ovarian carcinogenesis and metastasis by binding to the m6A-modified protein translation initiation factor EIF3C mRNA, thereby enhancing EIF3C translation in an m6A-dependent manner, ultimately affecting total protein translation. ⁵⁵ Hao et al demonstrated that YTHDF1 is recruited by m6A-modified tripartite motif protein 29 (TRIM29) and promotes TRIM29 translation in cisplatin-resistant OC cells. Knockdown of YTHDF1 in these cells suppresses their cancer stem cell (CSC)-like features, which can be reversed by TRIM29 overexpression, suggesting a dependency on m6A-YTHDF1 for its oncogenic role. ⁵⁶ Furthermore, Xu et al revealed the clinical significance of a novel regulatory axis involving the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7), which mediates the ubiquitination and subsequent proteasomal degradation of multiple oncogenic proteins, in OC. This regulatory axis is known as the FBW7-YTHDF2-BMF (Bcl-2 modifying factor) axis. FBW7 antagonizes YTHDF2, leading to the inhibition of its destabilizing effect on the m6A-modified pro-apoptotic BMF mRNA, which ensures the stable expression of BMF mRNA, consequently hindering OC growth and progression. ⁵⁷ Li et al found that YTHDF2 expression was significantly up-regulated in OC, where it disrupted miR-145 via a bidirectional negative feedback loop, promoting the proliferation and migration of OC cells. ⁵⁸ These findings underscore the multifaceted role of the YTH domain family in driving the malignant progression of OC through multiple mechanisms. However, significant uncharted territory remains in the exploration of the role of YTH domain family in OC.

Ovarian cancer and IGF2BPs

The IGF2BP family exhibits high structural similarity and plays crucial roles in regulating mRNA stability, translation, and localization. ⁵⁹ Upregulation of IGF2BP proteins has been associated with poor prognosis in malignant tumors such as breast, colorectal, glioma, hepatocellular, and pancreatic cancers. Specifically, IGF2BP1 recognizes and binds to m6A-modified serum response factor (SRF) mRNA, thereby enhancing its transcriptional activation and maintaining the expression of various IGF2BP1-SRF target genes, which in turn contributes to tumor growth and invasion, while also promoting CSC-like tumor cell characteristics. Müller et al observed the role of this mechanism in ovarian, hepatocellular, and lung cancers, correlating with poor prognosis and low overall survival. ⁶⁰ Wang et al found that an OC-inhibiting lncRNA called ubiquitin-like modifier-activating enzyme 6 antisense RNA 1 (UBA6-AS1) increased the m6A level of its target UBA6 mRNA by recruiting RBM15. They further noted an increase in the stability of UBA6-AS1 upon IGF2BP1 binding, which in turn suppressed the malignant phenotype of OC. ⁶¹ Additionally, Sun et al demonstrated that IGF2BP2 recognized and bound to m6A modification sites, promoting mRNA stability, and that FTO inhibited SNAI1 mRNA expression in an m6A-IGF2BP2-dependent manner, thereby attenuating OC growth and metastasis. ⁶² Furthermore, M6A-circNFIX, which is highly expressed in OC under the coregulation of IGF2BP1/2/3, up-regulates the expression of PD-L1 and activates the JAK1/STAT3 pathway via circNFIX/miR-647/IL-6R, thereby promoting OC proliferation, metastasis, and immune escape. ⁶³ The aforementioned studies suggest that IGF2BPs contribute to OC progression primarily through their roles in promoting the transcriptional activity of target mRNAs and enhancing their stability.

Ovarian cancer and HNRNPs

The HNRNP family differs from the aforementioned “readers” in that these proteins do not exclusively recognize m6A-modified sites through direct binding. For example, HNRNPC and HNRNPG preferentially bind to m6A-modified RNAs through an “m6A-switch” mechanism. ⁶⁴ HNRNPA2B1 is highly expressed in OC tissues, garnering attention for its role as an oncogene in OC development. Chen et al identified HNRNPA2B1 as a potential target of miR-30c-5p in OC. They found that miR-30c-5p inhibition led to decreased CDK19 mRNA stability and reduced m6A levels, thereby inhibiting OC progression. ⁶⁵

Ovarian cancer and FTO

Initially identified for its involvement in adipogenesis and obesity, ⁶⁷ FTO was later found to have demethylase activity. As the first m6A demethylase discovered, FTO is highly expressed and functions as an oncogene in various malignant tumors such as acute myeloid leukemia, pancreatic cancer, hepatocellular carcinoma, and non-small-cell lung cancer (NSCLC).^68-70 However, this was not exactly the case in OC. Huang et al demonstrated that FTO acts as an OC suppressor and inhibits the CSC-like features of OC. They further identified 2 phosphodiesterase genes, PDE4B and PDE1C, as downstream FTO targets that regulate the cAMP signaling pathway and play a key role in maintaining ovarian CSC-like features. ⁷¹ Based on previous studies showing the involvement of m6A in autophagy, Zhang et al found that CircRAB11FIP1 binds to and upregulates FTO mRNA expression, thereby modulating the m6A methylation levels of ATG5 and ATG7 mRNA through FTO. This mechanism ultimately enhances autophagy and malignant behavior in OC. ⁷² Additionally, Sun et al demonstrated that decreased FTO expression elevates m6A levels in SNAI1 mRNA, upregulating SNAI1 expression and consequently promoting OC proliferation, invasion, and migration. ⁶² Furthermore, Huang et al revealed that FTO overexpression enhances OC sensitivity to platinum and induces DNA double-strand breaks, with nicotinamide N-methyltransferase (NNMT) emerging as a novel FTO target regulating OC response to platinum in an m6A-dependent manner. ⁷³ Tumor-associated long non-coding RNAs (lncRNAs) modulated by epigenetic modification switches play pivotal roles in immune evasion and chemoresistance in OC. Chen et al developed a prognostic model integration methylation-associated and immune-associated lncRNAs, revealing that FTO inhibits OC proliferation and drug resistance by regulating the RP5-991G20.1/hsa-miR-1976/MEIS1 signaling pathway. ⁷⁴ Several studies have confirmed FTO’s role as a suppressor influencing various malignant behaviors in OC, including proliferation, metastasis, autophagy, and drug resistance, through diverse ontogenetic mechanisms.

Ovarian cancer and ALKBH5

ALKBH5, another significant m6A demethylase, modulates various biological processes, such as nuclear RNA export, metabolism, and gene expression, with its involvement in different RNA interactions playing distinct roles across various cancer types. ⁷⁵ In ovarian cancer, Zhu et al identified ALKBH5 overexpression in tumor tissues, correlating with cancer progression through the inhibition of autophagy in OC cells. ⁷⁶ In contrast, ALKBH5, in conjunction with FTO, contributes to PARP inhibitor resistance in BRCA1/2 mutant OC cells by regulating frizzled class receptor 10 (FZD10) mRNA expression and mediating the Wnt signaling pathway. ⁷⁷ Liu et al observed upregulated ALKBH5 expression in cisplatin-resistant OC, leading to enhanced OC cell proliferation and cisplatin resistance both in vivo and in vitro. They identified homeobox A10 (HOXA10) as a transcription factor promoting ALKBH5 transcription, reciprocally regulated by ALKBH5. Further analysis revealed that the sustained upregulation of ALKBH5-HOXA10 promoted OC tumor growth and cisplatin resistance via m6A-dependent activation of the JAK2/STAT3 signaling pathway. ⁷⁸ Sun et al found that hypoxia could stimulate the expression of hypoxia inducible factor 1 subunit alpha (HIF-1α) and ALKBH5. They observed that ALKBH5 regulated the expression of integrin subunit beta 1 (ITGB1) in an m6A-YTHDF2-dependent manner, which triggered the phosphorylation of focal adhesion kinase (FAK), thus promoting OC lymphangiogenesis and metastasis. ⁷⁹ In the field of immunology, Jiang et al established an in vitro model by co-culturing M1 and M2 macrophages with OC cells and found that this co-culture significantly increased tumor cell invasiveness. Additionally, they observed that Toll-like receptor 4 (TLR4) upregulated the expression of ALKBH5 in M2 co-cultured group through activation of the NF-κB signaling pathway. ALKBH5, in turn, promoted OC development by demethylating Nanog homeobox (NANOG). ⁸⁰