Roles of N6-methyladenosine (m6A) modifications in gynecologic cancers: mechanisms and therapeutic targeting.

Numerous studies have explored the participation of m 6 A regulators in many functions in the EC, such as cell cycle regulation and self-renewal of CSCs. The role and mechanism of m 6 A regulators in EC are summarized in Fig.  3 and Table 3 . Fig. 3 In EC, m 6 A regulatory proteins contribute to tumorigenesis and metastasis by interacting with various RNAs. METTL14 mutation or reduced expression of METTL3 increases the proliferation and tumorigenicity of EC by activating the AKT pathway. WTAP downregulates CAV‐1 expression to activate the NF‐κB signaling pathway in EC, promoting EC progression. HIF-1α and HIF-2α activate the expression of ALKBH5 under hypoxic conditions, facilitating the SOX2 expression by demethylating the SOX2 mRNA, leading to the tumorigenesis of EC. ALKBH5 demethylates the target transcript IGF1R and enhances its mRNA stability to promote tumorigenesis and metastasis of EC. FTO promotes HOXB13 protein expression, activates the WNT signaling pathway, and promotes EC invasion and metastasis. PADI2 activates the IGF2BP1 expression and helps in maintaining the mRNA stability and expression of SOX2, thereby supporting the malignancy state of EC. IGF2BP1 recruits PABPC1 to promote PEG10 protein expression, contributing to the tumorigenesis of EC. YTHDF2 inhibits the expression of IRS1 and inhibits IRS1/AKT signaling pathway, consequently inhibiting the tumorigenicity of EC. YTHDF2-mediated LncRNA FENDRR degradation promotes cellular proliferation by elevating the SOX4 expression in EC Table 3 The role and mechanism of m 6 A regulators in EC m 6 A regulators Roles Genes/RNAs Mechanisms Model Refs. Writers METTL3/METTL14 Tumor suppressor PHLPP2; mTORC2 METTL14 mutation or reduced METTL3 reduces the level of m 6 A methylation, leading to decrease expression of the negative AKT regulator PHLPP2 and increase expression of the positive AKT regulator mTORC2 In vitro; in vivo [ 168 ] WTAP Oncogene CAV-1 WTAP methylates 3′‐UTR of CAV‐1 and downregulates CAV‐1 expression to activate NF‐κB signaling pathway In vitro; in vivo [ 159 ] Readers YTHDF2 Oncogene FENDRR YTHDF2-mediated LncRNA FENDRR degradation promotes cell proliferation by elevating SOX4 expression In vitro; in vivo [ 170 ] YTHDF2 Tumor suppressor IRS1 YTHDF2 binds the methylation sites of target transcripts IRS1 and promotes IRS1 mRNA degradation, consequently inhibiting the expression of IRS1 and inhibiting IRS1/AKT signaling pathway In vitro [ 169 ] IGF2BP1 Oncogene PEG10 IGF2BP1 can recognize m 6 A sites in the 3′UTR of PEG10 mRNA and recruit PABPC1 to enhance PEG10 mRNA stability, which consequently promotes PEG10 protein expression In vitro; in vivo [ 176 ] IGF2BP1 Oncogene SOX2 Dysregulation of IGF2BP1 by PADI2/MEK1/ERK signaling results in abnormal accumulation of oncogenic SOX2 expression In vitro; in vivo [ 183 ] Erasers FTO Oncogene HOXB13 FTO demethylates m 6 A modifications in HOXB13 mRNA and promotes EC metastasis by activating the WNT signaling pathway In vitro; in vivo [ 146 ] ALKBH5 Oncogene IGF1R ALKBH5 demethylates target transcripts IGF1R and enhances IGF1R mRNA stability, consequently promoting IGF1R translation and activating IGF1R signaling pathway In vitro [ 189 ] ALKBH5 Oncogene SOX2 ALKBH5 in promoting SOX2 transcription via mRNA demethylation, thereby maintaining the stem-like state and tumorigenicity potential of ECSCs In vitro; in vivo [ 197 ] Immunoregulators ZCH3H13, METTL14, and YTHDC1 NA NA the expression, mutation, and SCNAs of these genes are associated with the immune cell infiltration NA [ 198 ] NA not available In EC, m 6 A regulatory proteins contribute to tumorigenesis and metastasis by interacting with various RNAs. METTL14 mutation or reduced expression of METTL3 increases the proliferation and tumorigenicity of EC by activating the AKT pathway. WTAP downregulates CAV‐1 expression to activate the NF‐κB signaling pathway in EC, promoting EC progression. HIF-1α and HIF-2α activate the expression of ALKBH5 under hypoxic conditions, facilitating the SOX2 expression by demethylating the SOX2 mRNA, leading to the tumorigenesis of EC. ALKBH5 demethylates the target transcript IGF1R and enhances its mRNA stability to promote tumorigenesis and metastasis of EC. FTO promotes HOXB13 protein expression, activates the WNT signaling pathway, and promotes EC invasion and metastasis. PADI2 activates the IGF2BP1 expression and helps in maintaining the mRNA stability and expression of SOX2, thereby supporting the malignancy state of EC. IGF2BP1 recruits PABPC1 to promote PEG10 protein expression, contributing to the tumorigenesis of EC. YTHDF2 inhibits the expression of IRS1 and inhibits IRS1/AKT signaling pathway, consequently inhibiting the tumorigenicity of EC. YTHDF2-mediated LncRNA FENDRR degradation promotes cellular proliferation by elevating the SOX4 expression in EC The role and mechanism of m 6 A regulators in EC NA not available The signal transduction network is a communication line among cells, and is used for perceiving signals, including those from the extracellular environment, and transmitting them to the downstream targets for the proper functioning and maintenance of cells. The abnormal changes in the signal transduction pathways are the important biological characteristics of the tumor cells. Current studies have shown that the alternation in the signal transduction pathways affects the cellular metabolism and immune response in the tumor cells [ 157 , 158 ]. In EC, the mechanism of m 6 A methylation effects on tumor growth through signaling pathways has been elucidated. Liu et al. reported higher expression levels of WTAP in the EC tissues as compared to the adjacent normal tissues [ 159 ]. Their further investigations confirmed that the expression of CAV-1 was regulated by WTAP in an m 6 A-dependent manner [ 159 ]. They also showed that, after being regulated by WTAP, the CAV-1 could activate the downstream NF-κB pathway [ 159 ]. PI3K/AKT pathway also plays an important role in various biological processes. The dysregulation of the AKT signaling pathway has shown crucial roles in the proliferation and apoptosis of numerous tumor cells [ 160 – 162 ]. Some studies showed that the stem cells and cancer cells proliferated with the reduction in the m 6 A methylation [ 36 , 163 , 164 ]. However, other studies reported that some cancers were related to the high expression of METTL3 and increased m 6 A methylation, which might involve different mechanisms and require more in-depth and detailed studies [ 35 , 165 , 166 ]. Researchers have shown that the dynamic m 6 A-modification in mRNAs, particularly in the key transcripts, might alter the physiology of cells [ 167 ]. For instance, the decreased m 6 A mRNA methylation could stimulate cellular proliferation by modulating the expression of the critical enzymes, which were involved in the AKT signaling pathway [ 168 ]. Hong et al. showed that the overexpressed YTHDF2 could bind to the m 6 A-modified insulin receptor substrate 1 (IRS1), which reduced its translation, thereby blocking the IRS1/AKT pathway [ 169 ]. In the same study, the results indicated that several vital proteins could regulate the cellular activities of EC cells through the AKT pathway; these proteins were also responsible for regulating the dynamic equilibrium of EC cells [ 169 ]. On the other hand, studies showed that YTHDF2 could also regulate the proliferation of EC cells by affecting the metabolism of lncRNA. According to Shen et al., the expression levels of lncRNA FENDRR in the EC tissues were reduced; however, its m 6 A methylation levels showed a negative effect trend [ 170 ]. The subsequent in-vitro experiments in the same study demonstrated that YTHDF2 could recognize the abundance of m 6 A-modified lncRNA FENDRR in the EC cells and degrade it [ 170 ]. After expressing the YTHDF2 gene, the expression levels of lncRNA FENDRR were restored, thereby inhibiting proliferation and stimulating the apoptosis of EC cells [ 170 ]. Furthermore, they reported that overexpressing the lncRNA FENDRR could reduce SOX4 translation and result in inhibiting EC cell proliferation and promoting cellular apoptosis [ 170 ]. These results were consistent with those of the previous study by Liu et al., which reported an adverse effect of the lncRNA FENDRR on the SOX4 expression in CRC [ 171 ]. Among the molecular mechanisms of cellular proliferation, cell cycle acceleration is of great importance, which is regulated by the CDK-cyclin complexes and cyclin-dependent kinase inhibitors [ 172 ]. A recent study reported that paternally expressed gene 10 (PEG10), a critical factor, which directly modulates the key proteins, was involved in cell cycle proteins [ 173 ]. Numerous studies have demonstrated the contributions of PEG10 to cellular proliferation; however, its mechanism has rarely been studied [ 174 , 175 ]. The knockdown of the PEG10 increased the p21 and p27 expression levels [ 173 ]. Zhang et al. reported that IGF2BP1 could recognize the m 6 A site in the 3′-UTR of the PEG10 mRNA in EC and recruit the polyadenylate binding protein 1 (PABPC1) to stabilize PEG10 mRNA, thereby increasing its protein expression levels and accelerating the cell cycle [ 176 ]. The peptide arginine deaminases (PADIs) family contains five members, including PADI1-4 and PADI6. Except for the PADI6 which has no enzymatic activity and is expressed only in the ovary [ 177 ], other PADIs can deaminate positively charged arginine residues in substrate proteins into the neutral non-coding residues called citrulline [ 178 , 179 ]. The expression of PADIs was higher in various malignant tumor tissues as compared to healthy tissues [ 180 , 181 ]. Numerous studies showed that the PADIs-catalyzed protein citrullination could alter signal transduction, cell differentiation, and EMT in a variety of human cancer cells [ 180 , 182 ]. Xue et al., for the first time, reported that m 6 A reader IGF2BP1 could be used as a downstream factor of PADI2 and could regulate it to promote tumor progression in EC [ 183 ]. They further showed that PADI2 could interact with MEK1 kinase in the MAPK pathway and catalyze the citrullination, which was beneficial for the phosphorylation of ERK1/2 by MEK1, thereby activating the expression of IGF2BP1. In addition, IGF2BP1 could also bind to the m 6 A site in the 3′-UTR of SOX mRNA to prevent its degradation [ 183 ]. This study revealed that the PADI2/MEK1/ERK/IGF2BP1 pathway could promote the characteristics of carcinogenic tumor cells in EC, and the combination of specific PADI2 and MEK1 inhibitors might provide a novel therapeutic target site for the treatment of the MEK inhibitor-resistant EC patients [ 183 ]. The insulin-like growth factor (IGF) is involved in many functions in most organs [ 184 – 186 ]. IGF1 and IGF2 can affect EC, as observed in both clinical and experimental data [ 187 ]. Both the IGF1 and IGF2 ligands can activate insulin-like growth factor 1 receptor (IGF1R), a tyrosine kinase receptor present on the cell surface, which is coupled with several intracellular secondary messenger pathways, including Ras/Raf/MAPK and PI3K/AKT signaling pathways, especially in regulating the normal uterine physiology [ 188 ]. Pu et al. reported that ALKBH5 enhanced the stability and translation of IGF1R mRNA by the demethylation of m 6 A and increased the protein levels of COL1A1 and MMP9, thereby promoting the tumorigenesis of EC cells [ 189 ]. However, the possibility of involving other signaling pathways cannot be excluded. Other signaling pathways may alter either directly or indirectly due to the changes in ALKBH5 and require further investigation. The role of m 6 A methylation, participating in the Wnt signaling pathway by regulating the related RNAs or proteins, has been elucidated in the CC [ 56 , 135 , 136 ]. FTO could remove the m 6 A modification of HOXB13 mRNA, abolish the degradation of HOXB13 mRNA mediated by YTHDF2, promote the expression of HOXB13 protein, activate the Wnt signaling pathway, and promote the invasion and metastasis of EC [ 146 ]. Hypoxia is an important niche feature of the CSCs, positively affecting the growth of stem cells and tumor progression [ 190 , 191 ]. Hypoxia-inducible factors (HIFs), including HIF-1α and HIF-2α, are the main media of hypoxia and indispensable for the activation and self-renewal of CSCs; they are strongly associated with tumors [ 192 ]. The ability of CSCs to tolerate hypoxia can be attributed to the rearrangement of genes involved in cellular multipotency and differentiation [ 193 ]. All these studies further deepen the understanding of the correlations among hypoxia, HIFs, and SOX2 [ 194 – 196 ]. Chen et al. demonstrated that the hypoxia and high levels of ALKBH5 could restore the stemness of differentiated endometrial CSCs (ECSCs) and increase the ECSC-like phenotype [ 197 ]. In addition, a recent study showed that the changes in mRNA stability were negatively correlated with the expression of these multipotency factors [ 65 ]. The m 6 A reader IGF2BP1 could stabilize the degradation of SOX2 mRNA in EC, thereby promoting tumor progression [ 183 ]. Similarly, Chen et al. verified that ALKBH5 could stimulate SOX2 mRNA expression by reducing its m 6 A methylation level, thereby increasing the stemness and carcinogenicity of ECSCs [ 197 ]. These studies revealed that the decrease in the m 6 A mRNA methylation in the key mRNAs might be a potential mechanism of most EC. These studies also confirmed that m 6 A methylation was a regulatory factor for cell growth. In EC, the correlation between m 6 A methylation modification and TME has rarely been reported. Recently, Ma et al. analyzed the EC patients’ data from TCGA and identified the genetic changes in the m 6 A regulatory genes. The results showed a significant correlation between the negative changes in the m 6 A levels and adverse prognostic outcomes [ 198 ]. The study identified ZC3H13, METTL14, and YTHDC1 as independent prognostic factors for EC patients [ 198 ]. Noteworthy, the expression, mutation, and somatic copy number alterations (SCNAs) of these genes were associated with immune cell infiltration [ 198 ]. The incidence of EC has increased over the past few decades, making it one of the most prevalent gynecologic cancers [ 199 ]. In 2020, 417,367 new cases of EC were diagnosed, causing 97,370 deaths [ 1 ]. The global incidence rate of EC is continuously increasing, while those of several other types of cancer have decreased over the past two decades [ 200 – 204 ]. Despite the better prognosis of EC than that of CC and OC, screening for the high-risk parts of EC patients is imperative due to more likeliness of developing advanced cancer and early death. With the advancements in the studies on m 6 A methylation modification in EC, numerous research groups have reported the prognostic potential of m 6 A regulatory factors and m 6 A-related genes in EC (Table 4 ). In a recent study, Zhai et al. analyzed the TCGA dataset of EC patients and established a risk model based on the m 6 A regulators, particularly FTO, RBM15, and YTHDF1, and revealed their crucial roles in the development and prognosis of EC [ 205 ]. Further analysis of the datasets showed that FTO and RBM15 could affect the survival of EC patients by regulating the m 6 A-associated genes involved in the development of connective tissue, catabolism, RNA stability, oxidative demethylation, temperature balance, and energetic metabolism [ 205 ]. Zhang et al. established a reliable protective model based on seven significant CpG sites located in the m 6 A regulators. The model could effectively predict the prognosis of EC, indicating that the CpG sites might be advantageous in predicting the EC [ 206 ]. Three m 6 A-associated lncRNAs revealed by Shi et al. that, in contrast to the TRAF3IP2-AS1, AL133243.2 , the patients with the high SCARNA9 expression tended to have a worse prognosis [ 207 ]. These lncRNAs were verified to participate in the development of endometriosis by modulating m 6 A-related enzymes, suggesting that these RNAs might be associated with the diagnosis and treatment of EC [ 207 ]. Table 4 The prognostic potential of m 6 A regulatory genes and m 6 A associated genes Prognostic genes/model Univariate Cox Analyses Multivariate Cox Analyses Sample size Refs. HR (95 CI%) P HR (95 CI%) P OC WTAP NA P  = 0.021 1.191 (1.023–1.385) P  = 0.024 231 [ 84 ] YTHDC2 and KIAA1429 3.143 (1.516–6.517) P  = 0.002 2.330 (1.116–4.865) P  = 0.024 379 [ 88 ] IGF2BP1, VIRMA and ZC3H13 1.25 (1.10–1.43) P  < 0.001 1.24 (1.08–1.41) P  = 0.002 379 [ 86 ] VIRMA, IGF2BP1, and HNRNPA2B1 1.62 (1.20–2.20) P  = 0.002 1.60 (1.18–2.17) P  = 0.003 374 [ 87 ] WTAP, LGR6, ZC2HC1A, SLC4A8, AP2A1, NRAS, CUX1, HDAC1, CD79A, ACE2, FLG2, and LRFN1 1.664 (1.481–1.868) P  = 8.5E-18 1.699 (1.508–1.913) P  = 2.29E-18 373 [ 89 ] CC HNRNPC, KIAA1429, ZC3H13 2.268 (1.161–4.431) P  = 0.017 2.583 (1.289–5.174) P  = 0.007 306 [ 155 ] ZC3H13, YTHDC1, and YTHDF1 NA NA 4.592 (2.788–7.562) P  < 0.001 304 [ 156 ] EC FTO, RBM15, and YTHDF1 1.129 (1.065–1.197) P  < 0.001 1.090 (1.023–1.161) P  = 0.008 406 [ 205 ] 7 CpG sites (cg13823621, cg08881614, cg07867023, cg22247039, cg00624976 cg06778680 and, cg13204529) NA NA 4.3 (2.4–7.6) P  < 0.001 312 [ 206 ] NA not available The prognostic potential of m 6 A regulatory genes and m 6 A associated genes NA not available

The main types of gynecologic cancers, which seriously damage the female reproductive organs, include vulvar cancer, vaginal cancer, cervical cancer (CC), endometrial cancer (EC), uterine cancer, and ovarian cancer (OC). CC and OC are the most frequent types of gynecologic cancer, accounting for 6.5% and 3.4%, respectively, of all the new cancers in women [ 1 ]. A population-based study conducted on the epidemiological trends of gynecologic cancer from 1990 to 2019 indicated that the incidence and mortality of gynecologic cancers might have geographical variations and changes along with sociodemographic index value [ 2 ]. Most gynecologic cancer patients have no distinct symptoms or physical signs in the early stages. In addition, the specific biomarkers for the early diagnosis of gynecologic cancer are also lacking. Moreover, most of the cases are in advanced stages at the time of diagnosis. Therefore, understanding the pathogenesis of gynecologic cancer is particularly important. This might identify specific markers for early diagnosis and therapeutic targets for the related therapeutic drugs, thereby ultimately improving the prognosis and quality of patients [ 3 – 5 ]. N 6 -methyladenosine (m 6 A) was first discovered in 1974 as an internal chemical modification, which was widely observed in the messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) [ 6 ]. The m 6 A plays important role in numerous aspects of RNA metabolism, such as pre-mRNA splicing, processing of 3′-untranslated region (UTR), export, translation, and degradation of mRNA, and processing of non-coding RNA [ 7 – 10 ]. Recent studies have shown that the m 6 A regulatory proteins act as writers, erasers, and readers, thereby modulating the dynamic deposition of mRNAs and other nuclear RNAs [ 11 , 12 ]. These findings strongly suggest the dynamic regulatory role of m 6 A modification is similar to the other well-known chromosomal reversible epigenetic modifications. This reversible RNA methylation provides a new dimension in the post-transcriptional regulation of gene expression [ 11 ]. In addition to mRNAs, m 6 A is also reported in a variety of ncRNAs, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and ribosomal RNAs (rRNAs) and has been indicated to be crucial for their metabolism and function [ 13 – 15 ]. In addition, abnormal m 6 A modifications in ncRNA by some m 6 A regulatory proteins participate in the proliferation, invasion, and drug resistance of cancer cells, thereby indicating their potential association with cancer [ 16 , 17 ]. Therefore, this new field in cancer pathogenesis might provide new opportunities for the diagnosis and treatment of cancer. This review summarizes the recent studies on m 6 A modifications in OC, CC, and EC, particularly focusing on the regulatory mechanism of m 6 A regulatory proteins in promoting the proliferation, invasion, and metastasis of these three gynecologic cancers. Finally, the current knowledge and prospects of m 6 A modifications in the tumor immune microenvironment, diagnosis, and prognosis of gynecologic cancer are also discussed.

In this review, the studies on the m 6 A methylation modification in OC, CC, and EC were summarized from the aspects of tumor development, immune microenvironment, and prognosis. From the existing studies, it was concluded that the abnormal expression of the m 6 A regulator in gynecological tumors might lead to an increase or decrease in the m 6 A modification level of RNA. The m 6 A modifications of RNA might affect the fate of various RNAs and lead to the proliferation, invasion, and metastasis of tumors as well as also alter the tumor immune microenvironment of the patients, thereby participating in the occurrence and development of tumors. The advancements in medical technology have greatly improved the survival rate of gynecologic cancer patients than before. However, due to the lack of specific biomarkers, early diagnosis is still challenging. At the same time, the resistance to the anti-tumor drugs in some patients also urges researchers to deepen the understanding of tumorigenesis and identify new immune targets for the development of anti-tumor drugs. This review summarized the results of numerous studies, which showed that the m 6 A regulator and related genes could be used as potential biomarkers or prognostic indicators for the early diagnosis of gynecologic cancers. Studying the mechanism of the m 6 A regulator in tumor development also supported this view. The role of m 6 A modification in the drug resistance mechanism has also been reported, which showed that the inhibitors of m 6 A regulators might have the potential of being used as anti-tumor drugs in drug-resistant patients. At present, numerous studies have reported the mechanism of m 6 A-promoting effects on the development of gynecologic cancer; however, the knowledge is still insufficient for a deeper understanding of tumorigenesis. The mechanisms, explaining the upregulation of the m 6 A regulator in gynecologic cancer and their relationship with oncogenes, are still unclear. Therefore, further studies are needed in the future to explain these mechanisms in order to develop effective therapeutic strategies.