Arginine methylation of ALKBH5 by PRMT6 promotes breast tumorigenesis via LDHA-mediated glycolysis

Abstract

ALKBH5 is a master regulator of N6-methyladenosine (m⁶A) modification, which plays a crucial role in many biological processes. Here, we show that ALKBH5 is required for breast tumor growth. Interestingly, PRMT6 directly methylates ALKBH5 at R283, which subsequently promotes breast tumor growth. Furthermore, arginine methylation of ALKBH5 by PRMT6 increases LDHA RNA stability via m⁶A demethylation, leading to increased aerobic glycolysis. Moreover, PRMT6-mediated ALKBH5 arginine methylation is confirmed in PRMT6-knockout mice. Collectively, these findings identify a PRMT6-ALKBH5-LDHA signaling axis as a novel target for the treatment of breast cancer.

Keywords

PRMT6 / ALKBH5 / N6-methyladenosine / glycolysis / tumor growth

1 Introduction

N⁶-methyladenosine (m⁶A) is one of the most mRNA modifications in the more than 100 types of RNA modifications, which mediate gene expression by changing RNA splicing, editing, stability, and degradation [1]. At least one-fourth of all RNAs are modified by m⁶A, which is a reversible modification tested in at least 25% of human and mouse mRNAs [2]. The m⁶A modification depends on the RNA methyltransferases, the demethylases, and m⁶A binding proteins, which are alternatively expressed in multiple cancers and might play an essential role in the cancer progress [3]. As a demethylase, alkylation repair homolog protein 5 (ALKBH5) could reverse m⁶A modification, which is abnormally expressed in multiple human cancers [4]. ALKBH5 is highly expressed in breast cancer [5], and ALKBH5 could promote hypoxia induced by the stem cell phenotype by mediating the m⁶A demethylation of NANOG mRNA [6]. However, the mechanism underlying the post-transcriptional modification of ALKBH5 remains elusive in breast cancer.

Protein arginine methyltransferase 6 (PRMT6) belongs to the protein arginine methyltransferase family, including PRMT1-9 [7]. The PRMT family could be divided into three types according to the property of the methylarginine. Type I (PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8) generates asymmetric dimethylarginine, Type II (PRMT5 and PRMT9) forms symmetric dimethylarginine, and Type III (PRMT7) only generates monomethylarginines [8]. PRMT6 has many cellular substrates, which regulate transcription, DNA damage responses, splicing, translation, and phase separation [9]. PRMT6 is highly expressed in multiple human cancers, which could promote tumor malignancy [9]. However, the roles of PRMT6 in breast cancer progress remain largely unclear.

Here, we report that ALKBH5 is essential for breast tumorigenesis, especially about demethylation of LDHA mRNA, which enhances LDHA protein expression that induces breast cancer cell high glycolysis. As a ubiquitous protein arginine methyltransferase, PRMT6 is continuously active in breast cancer, which could methylate and activate ALKBH5. We demonstrate that a PRMT6-ALKBH5-LDHA signaling axis, which might become a novel potential therapeutic target for breast cancer, is important for breast tumor growth in vivo and in vitro.

2 Materials and methods

2.1 Cell lines and cell culture

The cell lines used in this study (HEK293T, MCF-7, and MB231) were purchased from Cell Bank of the Chinese Academy of Sciences and have been described previously [10]. These cell lines were cultured in DMEM supplemented with 10% fetal bovine serum and 100 μg/mL streptomycin and 100 U/mL penicillin at 37 °C and 5% CO₂.

2.2 Western blot analysis

The indicated cells were lysed with Triton X-100 lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5) and 1% Triton X-100) with multiple protease inhibitors (Sigma-Aldrich). Using the BCA Protein Assay Kit (Beyotime) quantified the concentration of protein samples according to the manufacturer instructions. The indicated samples were analyzed by SDS-PAGE and transferred to methanol-activated PVDF membranes, and then indicated antibodies were added overnight at 4 °C. The blot was further incubated with IRDye secondary antibodies and developed with Odyssey® Fc Imaging System to obtain Western blots [11,12].

2.3 Immunoprecipitation and glutathione S-transferase pull-down assay

The indicated cells were extracted in a buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, and 0.5% IGEPAL CA-630) with multiple protease inhibitors (Sigma-Aldrich) and then incubated on ice [13]. The cell lysate was centrifuged at 13 400× g at 4 °C for 10 min. The cell lysate was used for immunoprecipitation (IP) with the indicated antibodies and beads overnight at 4 °C. The indicated beads were further cleaned five times using the IP buffer. The samples were detected by immunoblot analysis with indicated antibodies. The indicated purified proteins were extracted from Escherichia coli BL21 (DE3), and glutathione S-transferase (GST) pull-down assays were described previously [14,15].

2.4 Plasmid construction and mutagenesis

The lentiviral shRNAs for PRMT6 and ALKBH5 were constructed using pLVX-shRNA1. The CRISPR-cas9 sgPRMT6 plasmid was obtained from Genepharma. PCR-amplified human indicated genes were cloned into pcDNA3.0/HA, pFlag-CMV4, pEGFP-C1, PET28a, or pGEX-4T-1. The mutants used in the study were generated by using overlap PCR. The plasmid information is shown Tables S1 and S2.

2.5 Real-time PCR

The indicated cells were treated with Trizol Reagent to isolate total RNA, and RNAs were reverse transcribed using a cDNA synthesis kit (Takara). The primers used in this study are shown in Table S3.

2.6 Arginine methylation assay

His-tagged PRMT6 and His-tagged ALKBH5 proteins were incubated in a methylation reaction buffer (50 mmol/L Tris-HCl, pH8.0, 20 mmol/L KCl, 5 mmol/L DTT, 4 mmol/L EDTA) containing 200 µmol/L S-adenosyl-L-methionine (Sigma) and then incubated at 37 °C for 1 h in a final volume of 50 µL. Reactions were resolved by adding 5× SDS-PAGE loading buffer, followed by immunoblotting analysis.

2.7 Measurement of m⁶A levels

The total RNA was extracted from indicated cells. The m⁶A levels were determined using an m⁶A RNA methylation quantification kit (Epigentek) according to the manufacturer’s instruction. To detect m⁶A levels of LDHA mRNA, m⁶A immunoprecipitation was determined as described previously. Briefly, a protein A/G agarose-conjugated m⁶A antibody was added to the total RNA in the IP buffer (50 mmol/L Tris-HCl, pH 8.0, 750 mmol/L NaCl, and 0.5% IGEPAL CA-630) at 4 °C. The m⁶A RNA was eluted using an elution buffer (5 mmol/L Tris-HCl, pH 8.0, 1 mmol/L EDTA, and 0.05% SDS) with proteinase K at 50 °C. The RNA was purified by phenol/chloroform extraction followed by RT-PCR.

2.8 Xenograft tumor studies

Four-week-old female BALB/c nude mice were injected subcutaneously with indicated MCF-7 cells (5 × 10⁶ in 100 μL of PBS). After three weeks, the mice were sacrificed, and the tumors were harvested. The tumor volume was calculated as follows: volume = (length × width²)/2. The in vivo experiments were ethically authorized by the Ethics Committee of Weifang Medical University. The animals were administered treatment in accordance with relevant institutional and national guidelines and regulations.

2.9 Statistical analysis

GraphPad Prism 7.0 software was used for statistical analysis. Student’s t-test (unpaired, two-tailed) was used for two group comparisons. Data were presented as mean ± s.e.m. P values < 0.05 were considered statistically significant. (*P < 0.05, n.s. = not significant).

3 Results

3.1 ALKBH5 is required for breast tumor growth

ALKBH5 is a crucial m⁶A demethylase, which is highly expressed in many human cancers. Thus, we used the TIMER database to assess ALKBH5 expression in multiple human cancers. Data showed that high expression of ALKBH5 had a bad survival rate in breast cancer (Fig. S1A). To further examine the functions of ALKBH5 in breast tumorigenesis, we knocked down ALKBH5 (Fig. S1B) and then rescued ALKBH5 expression in breast cancer cells (Fig.1). As expected, the knockdown of ALKBH5 in breast cancer cells demonstrated declined proliferation rate and decreased colonies, suggesting its promotion effect on cell proliferation (Fig.1 and 1C). Moreover, the knockdown of ALKBH5 greatly reduced cell migration and invasion (Fig.1 and 1E). To explore the oncogenic roles of ALKBH5 in vivo, we performed a xenograft mice model. Consistently, data showed that the knockdown of ALKBH5 substantially decreased tumor growth in vivo (Fig.1–1I). Thus, ALKBH5 plays an important role in breast tumor growth.

3.2 PRMT6 interacts with ALKBH5

To identify ALKBH5 partners that mediated ALKBH5 functions in breast cancer, we conducted mass spectrometry analysis of proteins that are bound to endogenous ALKBH5 in MCF-7 cells. Among the candidates, we found arginine methyltransferase-PRMT6 association with ALKBH5 (Table S4). To confirm their interaction further, we performed Co-IP assays using ectopic or endogenous expression. The results showed that protein–protein interactions occurred between PRMT6 and ALKBH5 (Fig.2–2D). To explore the direct interaction of PRMT6 with ALKBH5, we performed GST pull-down assay, which suggested that PRMT6 directly interacted with ALKBH5 in vitro (Fig.2). Furthermore, PRMT6 could colocalize with ALKBH5 in the nucleus (Fig.2). To confirm the structural fragments for the association between PRMT6 and ALKBH5, IP assays were performed, and results showed that the P3 (185–375 aa) of PRMT6 was necessary for their interaction (Fig.2 and 2H). These data suggest that PRMT6 is a new binding partner of ALKBH5.

3.3 PRMT6 methylates ALKBH5 at R283

Given that PRMT6 was an arginine methyltransferase, we explored whether PRMT6 could directly methylate ALKBH5. Data showed that PRMT6 induced arginine methylation of ALKBH5, but the inactivated enzyme could not (Fig.3). Interestingly, PRMT1, PRMT2, PRMT3, PRMT4, or PRMT8 as another member of Type I arginine methyltransferase family could not regulate arginine methylation of ALKBH5 (Fig.3). To identify the arginine residues on ALKBH5 protein methylated by PRMT6, we mutated all arginine residues into lysine residues on the ALKBH5 protein. Interestingly, PRMT6 did not affect the arginine methylation level of the ALKBH5-R283K mutant (Fig.3 and 3D). Furthermore, we produced an antibody that specifically recognizes ALKBH5 R283 methylation, and its validity was verified (Fig. S1C–S1E). We then used this ALKBH5-methylation antibody and determined that PRMT6 has no effect on the ALKBH5 R283K mutant (Fig.3). Moreover, an in vitro methylation assay demonstrated that PRMT6 directly methylated ALKBH5 at R283 (Fig.3). Consistently, mass spectrometry demonstrated that the R283 site of ALKBH5 was directly methylated by PRMT6 (Fig.3). However, ALKBH5 R283 methylation did not affect its interaction with PRMT6 (Fig. S1F). Furthermore, we studied whether PRMT6 could regulate ALKBH5 protein stability. Interestingly, PRMT6 did not mediate ALKBH5 protein expression and ubiquitination (Fig. S2). Together, these results suggest that PRMT6 directly methylates ALKBH5 at R283.

3.4 ALKBH5 R283 methylation promotes breast cancer cell glycolysis by elevating LDHA expression

To explore whether ALKBH5 R283 methylation mediates glycolysis, we rescued ALKBH5 expression in ALKBH5-depleted breast cancer cells (Fig. S3A). Next, we performed mass spectrometry to explore cellular metabolites, which were extracted from cultured ALKBH5-depleted MCF-7 cells. The knockdown of ALKBH5 decreased most cellular metabolites in MCF-7 cells, consistent with previous data (Fig.4 and Fig. S3B–S3D). Furthermore, the mutant R283K ALKBH5 decreased more extracellular glucose consumption and lactate production than the WT or R283F ALKBH5 (Fig.4 and 4C). Consistently, ALKBH5 R283 methylation accelerated extracellular acidification rate in breast cancer cells, which could reflect high glycolytic rate (Fig.4 and 4E). The cell ability to use glucose as an energy source was strictly mediated by key glucose transporters and a series of enzymes that determined the fate of glucose in cells. To study why ALKBH5 R283 methylation promotes glycolysis in breast cancer cells, we measured the expressions of 28 glycolysis-related genes in rescued ALKBH5 (WT, R283K, or R283F) cells (Fig.4). Several of the enzymes involved in glucose metabolism in the mutant cells were less expressive than the wild type cells, in which LDHA was the most different. LDHA promoted glycolysis by converting pyruvate to lactic acid. Many studies have shown that LDHA was abnormally overexpressed in various cancers, which was related to malignant progression [16]. Furthermore, we tested LDHA protein expression in rescued ALKBH5 cells and showed that ALKBH5 R283 methylation promoted LDHA protein expression (Fig.4 and 4H). However, ALKBH5 did not change LDHA protein ubiquitination and half-life, which suggested that ALKBH5 might regulate the RNA level of LDHA (Fig. S4A–S4C). Thus, ALKBH5 R283 methylation promotes breast cancer cell glycolysis via elevating LDHA expression.

3.5 ALKBH5 R283 methylation promotes LDHA RNA stability via m⁶A demethylation

Our previous study showed that ALKBH5 R283 methylation promoted LDHA expression; hence, we explored whether ALKBH5 directly regulated LDHA mRNA stability through m⁶A modification. The overexpression of ALKBH5 increased LDHA protein expression but not the enzyme inactivity mutant, suggesting that this regulation relied on its m⁶A demethylation (Fig.5 and 5B). Consistently, the knockdown of ALKBH5 blocked LDHA expression in breast cancer cells (Fig.5 and 5D). Furthermore, the knockdown of ALKBH5 or ALKBH5 R283K mutant increased the global m⁶A levels (Fig. S4D and S4E) and m⁶A methylation levels of LDHA mRNA (Fig. S4F), whereas ALKBH5 R283 methylation inhibited levels of m⁶A methylation LDHA mRNA (Fig.5 and 5F). Interestingly, we identified several motifs on the LDHA mRNA, which contained a match to the m⁶A consensus sequence, 5′-RRACU-3′ (Fig. S4G). We found that P1 mutant expression could not rise again, which was a potential site of m⁶A demethylation (Fig.5). Luciferase assays demonstrated that the knockdown of ALKBH5 resulted in unchanged luciferase activity of P1 mutation, suggesting that the mutation increased the stability of the luciferase–LDHA fusion mRNA (Fig.5). Moreover, ALKBH5 R283 methylation enhanced the luciferase activity of the P1 wild type, indicating that ALKBH5 R283 methylation increased the m⁶A demethylation function (Fig.5). METTL3 and METTL14 are the main m⁶A methylation enzymes [17]. Interestingly, METTL14 could decrease LDHA protein expression, whereas METTL3 could not. Thus, METTL14 might be the upstream enzyme for its m⁶A methylation (Fig. S4H). These results indicate that ALKBH5 R283 methylation promotes LDHA RNA stability via m⁶A demethylation in breast cancer.

3.6 ALKBH5 R283 methylation contributes to breast tumor growth

To explore the role of ALKBH5 R283 methylation in breast tumor growth, we tested the breast cancer cell proliferation with rescued ALKBH5 expression. As expected, the ALKBH5 R283K mutant decreased breast cancer cell proliferation (Fig.6 and 6B). Moreover, ALKBH5 R283 methylation promoted cell invasion and migration, which was studied by transwell migration and cell-scratch assays (Fig.6 and 6D). To determine the role of ALKBH5 R283 methylation in breast tumor growth in vivo, we subcutaneously injected manipulated ALKBH5-rescued MCF-7 cells into athymic nude mice. Consistently, ALKBH5 R283K mutant markedly reduced tumor growth compared with ALKBH5 (WT or R283F) (Fig.6–6G). Furthermore, IHC analysis showed that the ALKBH5 R283K mutant led to a significant decrease in cell proliferation (Fig.6). Collectively, these data indicate that ALKBH5 R283 methylation promotes breast tumor growth.

3.7 Protein expressions of PRMT6, meR283-ALKBH5, and LDHA positively correlate with each other in human breast cancer tissues

To explore the clinical significance of the PRMT6-mediated ALKBH5 R283 methylation, we performed IHC analyses. Data showed that the expression levels of PRMT6, meR283-ALKBH5, and LDHA were high in breast tumor tissues from 80 patients (Fig.7). Moreover, we demonstrated that meR283-ALKBH5 expression level had a positive correlation with PRMT6 and LDHA expression levels in the breast cancer samples (Fig.7). In addition, we manipulated PRMT6 expression in breast cancer cells (Fig. S5). Consistently, PRMT6 depletion led to the decreases in meR283-ALKBH5 and LDHA (Fig.7 and 7D). Furthermore, the knockout of PRMT6 decreased the expression meR283-ALKBH5 and LDHA in PRMT6 knockout mouse embryonic fibroblast (MEF) cells (Fig.7). Collectively, these data reveal a close relationship between ALKBH5 meR283 and clinical aggressiveness of human breast tumor.

4 Discussion

ALKBH5, which plays an important role as m⁶A demethylase, is usually dysregulated in various malignant tumors. Based on different cancer types, changing ALKBH5 could promote and inhibit the carcinogenesis. ALKBH5 could induce the formation and development of various malignant tumors by removing m⁶A methylation on crucial RNA. The overexpression of ALKBH5 in glioma, as a central nervous system tumor, could promote metabolism. Especially, ALKBH5 demethylates the rate limiting enzyme of the pentose phosphate pathway-G6PD mRNA and increases its mRNA stability, thus enhancing pentose phosphate pathway flux and enhancing the malignance of glioma [18]. For lung cancer, ALKBH5 increases cell proliferation and inhibits apoptosis in nonsmall-cell lung cancer cells by repressing TIMP3 mRNA stability and translation [19]. In ovarian cancer, ALKBH5 regulates autophagy and increases malignancy by stabilizing BCL-2 mRNA and enhancing the interaction between BCL-2 and BECN1 [20]. In gastric and colon cancers, ALKBH5 promotes NEAT1 gene expression via m⁶A modification, thus increasing cancer cell invasion and metastasis [21,22]. In breast cancer, we found that ALKBH5 was an oncogene, which could promote breast tumorigenesis. Further study showed that ALKBH5 contributed to aerobic glycolysis via demethylation of LDHA mRNA. Thus, our study revealed a new mechanism of ALKBH5 promoting breast tumor growth, consistent with a previous study [6].

PRMT6 plays a crucial role in many cancers, including endometrial cancer [23], colorectal cancer [24], gastric cancer [25], prostate cancer [26], breast cancer [27], bladder cancer [28], and lung cancer [29], in which PRMT6 is highly expressed. As an arginine methylase, PRMT6 plays its main role in its enzyme activity, which could methylate a series of substrates. PRMT6 could methylate histone H3 at R2 and inhibit H3 K4 trimethylation to recruit WDR5 to the MLL complex, leading to a decrease in the transcription of Hox genes [30,31]. Moreover, asymmetric dimethylation of histone H3 at R2 by PRMT6 recruits the chromosome passenger complex to the chromosome arm and promotes H3 S10 phosphorylation through AURKB for chromosome concentration [32]. PRMT6 methylation of RCC1 at R214 is required for RCC1 to interact with chromatin, which mediates a biological phenotype of glioblastoma stem cells [33]. PRMT6 directly binds to and methylates SIRT7 at R388 and suppresses the H3K18 deacetylase activity of SIRT7 without modulating its subcellular localization, which promotes mitochondria biogenesis and maintains mitochondria respiration [34]. PRMT6 could methylate CRAF at R100 and change extracellular signal-regulated kinase (ERK)-mediated PKM2 translocation into the nucleus, which promotes aerobic glycolysis and hepatocarcinogenesis [35]. However, no study has reported that arginine dimethylation could regulate m⁶A modification. In this study, we found that PRMT6 methylated ALKBH5 at R283, enhancing its enzyme activity. However, how R283 methylation could enhance ALKBH5 activity remains unsolved, and we speculated that R283 methylation might change the spatial structure of the ALKBH5 protein. ALKBH5 R283 methylation could increase LDHA RNA m⁶A demethylation, which promotes its RNA stability. Elevated expression of LDHA contributes to glycolysis and breast tumorigenesis.

As a double-edged sword, m⁶A methylation plays different functions on promoting or inhibiting the occurrence and development of different cancer types. However, the mechanisms of the duality of m⁶A in breast cancer are still not fully understood. METTL3 as a main m⁶A methylation enzyme could promote the breast cancer progression via methylation of Bcl-2 and promote its translation [36]. Furthermore, METTL3 promotes the progression of breast cancer via blocking tumor suppressor let-7g [14]. PIN1 binds to METTL3 and decreases its ubiquitination and lysosomal degradation to promote breast tumorigenesis [37]. However, METTL3 as a tumor suppressor inhibits tumor progression and drug resistance via an N6 methyladenosine-dependent mechanism in breast cancer [38]. Thus, controversy over the study of METTL3 remains. ALKBH5 is reported as an oncogene to promote breast tumor progression. Hypoxia-induced ALKBH5 expression promotes m⁶A-demethylation of NANOG mRNA to induce the breast cancer stem cell phenotype [6]. Moreover, ALKBH5 expression is correlated with HIF-1α expression in human breast cancer [39]. Recently, ALKBH5 has been found to enhance m⁶A demethylation of GLUT4 mRNA and promote its mRNA stability in a YTHDF2-dependent manner in breast cancer cells [40]. Here, we find that ALKBH5 enhances glycolysis via demethylation of LDHA mRNA, which is consistent with a previous study [40]. Moreover, METTL14 as a tumor suppressor in breast cancer [41,42] could decrease LDHA expression, which suggests that METTL14 might act as a LDHA m⁶A modification enzyme.

In summary, we find a new substrate of PRMT6, i.e., the demethylation enzyme ALKBH5, and build the relationship between PRMT6 and m⁶A modification. We also illuminate the molecular mechanism of PRMT6-mediated ALKBH5 R283 methylation and demonstrate the functions of ALKBH5 R283 methylation in promoting breast tumorigenesis in vitro and in vivo (Fig.7). Thus, PRMT6-ALKBH5-LDHA signaling axis serves as a potential therapeutic target in breast cancer.

References

[1] Wiener D, Schwartz S. The epitranscriptome beyond m⁶A. Nat Rev Genet 2021; 22(2): 119–131

[2] Oerum S, Meynier V, Catala M, Tisne C. A comprehensive review of m⁶A/m⁶Am RNA methyltransferase structures. Nucleic Acids Res 2021; 49(13): 7239–7255

[3] Huang H, Weng H, Chen J. m⁶A modification in coding and non-coding RNAs: roles and therapeutic implications in cancer. Cancer Cell 2020; 37(3): 270–288

[4] Qu J, Yan H, Hou Y, Cao W, Liu Y, Zhang E, He J, Cai Z. RNA demethylase ALKBH5 in cancer: from mechanisms to therapeutic potential. J Hematol Oncol 2022; 15(1): 8

[5] Wu L, Wu D, Ning J, Liu W, Zhang D. Changes of N⁶-methyladenosine modulators promote breast cancer progression. BMC Cancer 2019; 19(1): 326

[6] Zhang C, Samanta D, Lu H, Bullen JW, Zhang H, Chen I, He X, Semenza GL. Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m⁶A-demethylation of NANOG mRNA. Proc Natl Acad Sci USA 2016; 113(14): E2047–E2056

[7] Gupta S, Kadumuri RV, Singh AK, Chavali S, Dhayalan A. Structure, activity and function of the protein arginine methyltransferase 6. Life (Basel) 2021; 11(9): 951

[8] Blanc RS, Richard S. Arginine methylation: the coming of age. Mol Cell 2017; 65(1): 8–24

[9] Chen Z, Gan J, Wei Z, Zhang M, Du Y, Xu C, Zhao H. The emerging role of PRMT6 in cancer. Front Oncol 2022; 12: 841381

[10] Han X, Ren C, Lu C, Qiao P, Yang T, Yu Z. Deubiquitination of MYC by OTUB1 contributes to HK2 mediated glycolysis and breast tumorigenesis. Cell Death Differ 2022; 29(9): 1864–1873

[11] Lu C, Ren C, Yang T, Sun Y, Qiao P, Han X, Yu Z. Fructose-1, 6-bisphosphatase 1 interacts with NF-κB p65 to regulate breast tumorigenesis via PIM2 induced phosphorylation. Theranostics 2020; 10(19): 8606–8618

[12] Lu C, Qiao P, Sun Y, Ren C, Yu Z. Positive regulation of PFKFB3 by PIM2 promotes glycolysis and paclitaxel resistance in breast cancer. Clin Transl Med 2021; 11(4): e400

[13] Ren C, Han X, Lu C, Yang T, Qiao P, Sun Y, Yu Z. Ubiquitination of NF-κB p65 by FBXW2 suppresses breast cancer stemness, tumorigenesis, and paclitaxel resistance. Cell Death Differ 2022; 29(2): 381–392

[14] Cai X, Wang X, Cao C, Gao Y, Zhang S, Yang Z, Liu Y, Zhang X, Zhang W, Ye L. HBXIP-elevated methyltransferase METTL3 promotes the progression of breast cancer via inhibiting tumor suppressor let-7g. Cancer Lett 2018; 415: 11–19

[15] Han X, Ren C, Yang T, Qiao P, Wang L, Jiang A, Meng Y, Liu Z, Du Y, Yu Z. Negative regulation of AMPKα1 by PIM2 promotes aerobic glycolysis and tumorigenesis in endometrial cancer. Oncogene 2019; 38(38): 6537–6549

[16] Feng Y, Xiong Y, Qiao T, Li X, Jia L, Han Y. Lactate dehydrogenase A: a key player in carcinogenesis and potential target in cancer therapy. Cancer Med 2018; 7(12): 6124–6136

[17] Sendinc E, Shi Y. RNA m⁶A methylation across the transcriptome. Mol Cell 2023; 83(3): 428–441

[18] Liu Z, Chen Y, Wang L, Ji S. ALKBH5 promotes the proliferation of glioma cells via enhancing the mRNA stability of G6PD. Neurochem Res 2021; 46(11): 3003–3011

[19] Zhu Z, Qian Q, Zhao X, Ma L, Chen P. N⁶-methyladenosine ALKBH5 promotes non-small cell lung cancer progress by regulating TIMP3 stability. Gene 2020; 731: 144348

[20] Zhu H, Gan X, Jiang X, Diao S, Wu H, Hu J. ALKBH5 inhibited autophagy of epithelial ovarian cancer through miR-7 and BCL-2. J Exp Clin Cancer Res 2019; 38(1): 163

[21] Guo T, Liu DF, Peng SH, Xu AM. ALKBH5 promotes colon cancer progression by decreasing methylation of the lncRNA NEAT1. Am J Transl Res 2020; 12(8): 4542–4549

[22] Zhang J, Guo S, Piao HY, Wang Y, Wu Y, Meng XY, Yang D, Zheng ZC, Zhao Y. ALKBH5 promotes invasion and metastasis of gastric cancer by decreasing methylation of the lncRNA NEAT1. J Physiol Biochem 2019; 75(3): 379–389

[23] Jiang N, Li QL, Pan W, Li J, Zhang MF, Cao T, Su SG, Shen H. PRMT6 promotes endometrial cancer via AKT/mTOR signaling and indicates poor prognosis. Int J Biochem Cell Biol 2020; 120: 105681

[24] Pan R, Yu H, Dai J, Zhou C, Ying X, Zhong J, Zhao J, Zhang Y, Wu B, Mao Y, Wu D, Ying J, Duan S. Significant association of PRMT6 hypomethylation with colorectal cancer. J Clin Lab Anal 2018; 32(9): e22590

[25] Okuno K, Akiyama Y, Shimada S, Nakagawa M, Tanioka T, Inokuchi M, Yamaoka S, Kojima K, Tanaka S. Asymmetric dimethylation at histone H3 arginine 2 by PRMT6 in gastric cancer progression. Carcinogenesis 2019; 40(1): 15–26

[26] Almeida-Rios D, Graca I, Vieira FQ, Ramalho-Carvalho J, Pereira-Silva E, Martins AT, Oliveira J, Goncalves CS, Costa BM, Henrique R, Jerónimo C. Histone methyltransferase PRMT6 plays an oncogenic role of in prostate cancer. Oncotarget 2016; 7(33): 53018–53028

[27] Dowhan DH, Harrison MJ, Eriksson NA, Bailey P, Pearen MA, Fuller PJ, Funder JW, Simpson ER, Leedman PJ, Tilley WD, Brown MA, Clarke CL, Muscat GEO. Protein arginine methyltransferase 6-dependent gene expression and splicing: association with breast cancer outcomes. Endocr Relat Cancer 2012; 19(4): 509–526

[28] Yoshimatsu M, Toyokawa G, Hayami S, Unoki M, Tsunoda T, Field HI, Kelly JD, Neal DE, Maehara Y, Ponder BA, Nakamura Y, Hamamoto R. Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types of human cancers. Int J Cancer 2011; 128(3): 562–573

[29] Avasarala S, Wu PY, Khan SQ, Yanlin S, Van Scoyk M, Bao J, Di Lorenzo A, David O, Bedford MT, Gupta V, Winn RA, Bikkavilli RK. PRMT6 promotes lung tumor progression via the alternate activation of tumor-associated macrophages. Mol Cancer Res 2020; 18(1): 166–178

[30] Hyllus D, Stein C, Schnabel K, Schiltz E, Imhof A, Dou Y, Hsieh J, Bauer UM. PRMT6-mediated methylation of R2 in histone H3 antagonizes H3 K4 trimethylation. Genes Dev 2007; 21(24): 3369–3380

[31] Guccione E, Bassi C, Casadio F, Martinato F, Cesaroni M, Schuchlautz H, Luscher B, Amati B. Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature 2007; 449(7164): 933–937

[32] Kim S, Kim NH, Park JE, Hwang JW, Myung N, Hwang KT, Kim YA, Jang CY, Kim YK. PRMT6-mediated H3R2me2a guides Aurora B to chromosome arms for proper chromosome segregation. Nat Commun 2020; 11(1): 612

[33] Liu L, Zhang X, Ding H, Liu X, Cao D, Liu Y, Liu J, Lin C, Zhang N, Wang G, Hou J, Huang B, Zhang Y, Lu J. Arginine and lysine methylation of MRPS23 promotes breast cancer metastasis through regulating OXPHOS. Oncogene 2021; 40(20): 3548–3563

[34] Yan WW, Liang YL, Zhang QX, Wang D, Lei MZ, Qu J, He XH, Lei QY, Wang YP. Arginine methylation of SIRT7 couples glucose sensing with mitochondria biogenesis. EMBO Rep 2018; 19(12): e46377

[35] Wong TL, Ng KY, Tan KV, Chan LH, Zhou L, Che N, Hoo RLC, Lee TK, Richard S, Lo CM, Man K, Khong PL, Ma S. CRAF methylation by PRMT6 regulates aerobic glycolysis-driven hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation. Hepatology 2020; 71(4): 1279–1296

[36] Wang H, Xu B, Shi J. N6-methyladenosine METTL3 promotes the breast cancer progression via targeting Bcl-2. Gene 2020; 722: 144076

[37] Bhattarai PY, Kim G, Lim SC, Mariappan R, Ohn T, Choi HS. METTL3 stabilization by PIN1 promotes breast tumorigenesis via enhanced m⁶A-dependent translation. Oncogene 2023; 42(13): 1010–1023

[38] Ouyang D, Hong T, Fu M, Li Y, Zeng L, Chen Q, He H, Wen Y, Cheng Y, Zhou M, Zou Q, Yi W. METTL3 depletion contributes to tumour progression and drug resistance via N6 methyladenosine-dependent mechanism in HR+HER2-breast cancer. Breast Cancer Res 2023; 25(1): 19

[39] Zhang C, Zhi WI, Lu H, Samanta D, Chen I, Gabrielson E, Semenza GL. Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells. Oncotarget 2016; 7(40): 64527–64542

[40] Liu H, Lyu H, Jiang G, Chen D, Ruan S, Liu S, Zhou L, Yang M, Zeng S, He Z, Wang H, Li H, Zheng G, Liu B. ALKBH5-mediated m⁶A demethylation of GLUT4 mRNA promotes glycolysis and resistance to HER2-targeted therapy in breast cancer. Cancer Res 2022; 82(21): 3974–3986

[41] Gong PJ, Shao YC, Yang Y, Song WJ, He X, Zeng YF, Huang SR, Wei L, Zhang JW. Analysis of N6-Methyladenosine methyltransferase reveals METTL14 and ZC3H13 as tumor suppressor genes in breast cancer. Front Oncol 2020; 10: 578963

[42] Dong XF, Wang Y, Huang BF, Hu GN, Shao JK, Wang Q, Tang CH, Wang CQ. Downregulated METTL14 expression correlates with breast cancer tumor grade and molecular classification. BioMed Res Int 2020; 2020: 8823270

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