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

doi:10.1007/s11684-023-1028-4

Front. Med.

2024, Vol. 18

Issue (2) : 344-356 https://doi.org/10.1007/s11684-023-1028-4

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

Xue Han¹, Chune Ren¹, Aifang Jiang¹, Yonghong Sun², Jiayi Lu¹, Xi Ling¹, Chao Lu¹, Zhenhai Yu¹(

)

¹. Department of Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang 261053, China
². Department of Pathology, Affiliated Hospital of Weifang Medical University, Weifang 261053, China

Download: PDF(7410 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

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

Corresponding Author(s): Zhenhai Yu

Just Accepted Date: 12 January 2024 Online First Date: 12 March 2024 Issue Date: 27 May 2024

Cite this article:

Xue Han,Chune Ren,Aifang Jiang, et al. Arginine methylation of ALKBH5 by PRMT6 promotes breast tumorigenesis via LDHA-mediated glycolysis[J]. Front. Med., 2024, 18(2): 344-356.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-023-1028-4
https://academic.hep.com.cn/fmd/EN/Y2024/V18/I2/344

Fig.1 ALKBH5 is required for breast tumor growth. (A) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. Immunoblotting analysis was used to determine ALKBH5 protein expression. (B) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. Cell proliferation was evaluated by CCK-8 assay. (C) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. Cell proliferation was evaluated by plate colony formation assay. (D) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. Cell migration was evaluated by wound healing assay. (E) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. Cell invasion was evaluated by transwell assay. (F) Representative images of tumors. (G, H) ALKBH5-depleted MCF-7 cells with reconstituted expression of rALKBH5 were subcutaneously injected into nude mice. After three weeks, the mice were sacrificed. Tumor weight and growth were measured. (I) Representative images of H&E and Ki67 staining of tumor tissues (scale bar, 20 μm). (All data represent mean ± SEM, n≥ 3), *, P < 0.05.

Fig.2 PRMT6 interacts with ALKBH5. (A, B) HEK293T cells were cotransfected with HA-tagged ALKBH5 and Flag-tagged PRMT6 plasmids. IP assays were performed with anti-HA (A) or anti-Flag (B) antibodies, followed by immunoblot analysis. (C, D) IP assays were performed with indicated antibodies, followed by immunoblot analysis using MCF-7 cell lysates. (E) GST pull-down assays were performed, followed by Western blot analysis with indicated antibodies using purified proteins. (F) Immunofluorescence was performed to determine the colocalization of endogenous PRMT6 and ALKBH5 in MCF-7 cells. (G, H) Crucial domains required for the interaction between PRMT6 and ALKBH5 were identified.

Fig.3 PRMT6 methylates ALKBH5 at R283. (A) HEK293T cells were cotransfected with HA-tagged ALKBH5 and Flag-tagged PRMT6 (WT or KD) proteins. IP assay with an anti-HA antibody was performed, followed by Western blot analysis with indicated antibodies. (B) HEK293T cells were cotransfected with HA-tagged ALKBH5 and Flag-tagged PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, or PRMT8 proteins. IP assay with an anti-HA antibody was performed, followed by immunoblot analyses with indicated antibodies. (C, D) HEK293T cells were cotransfected with HA-tagged ALKBH5 (mutants) and Flag-tagged PRMT6 proteins. IP assay with an anti-HA antibody was performed, followed by immunoblot analyses with indicated antibodies. (E) HEK293T cells were cotransfected with HA-tagged ALKBH5 (WT or R283K) and Flag-tagged PRMT6 plasmids. IP assay was performed, followed by immunoblot analysis with indicated antibodies. (F) Purified His-tagged ALKBH5 was incubated with the purified His-tagged PRMT6 proteins. An in vitro arginine methylation assay was performed, followed by immunoblot analysis. (G) Mass spectrometry analysis of in vitro methylated His-ALKBH5 arginine demethylation was performed.

Fig.4 ALKBH5 R283 methylation promotes breast cancer cell glycolysis by elevating LDHA expression. (A) Heatmap showed the level of 27 metabolites related to energy metabolism in MCF-7 cells stably knocked down ALKBH5. (B) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5 (WT, R283K, or R283F). Glucose consumption was measured using a glucose uptake assay kit. (C) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5 (WT, R283K, or R283F). Lactate production was measured using a lactate assay kit. (D, E) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5 (WT, R283K, or R283F). The glycolysis rate was measured using seahorse analysis. (F) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5 (WT, R283K, or R283F). The mRNA levels of metabolic genes were quantitated by RT-PCR. (G, H) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5 (WT, R283K, or R283F). LDHA protein expression was determined by immunoblot analysis.

Fig.5 ALKBH5 R283 methylation promotes LDHA RNA stability via m⁶A demethylation. Immunoblot analyses were performed with the indicated antibodies. (A) HEK293T cells were overexpressed in HA-tagged LDHA and Flag-tagged ALKBH5 (WT or mutants) proteins, followed by immunoblot analysis. (B) HEK293T cells were overexpressed in HA-tagged LDHA and Flag-tagged ALKBH5 (0, 0.5 or 1 μg) proteins, followed by immunoblot analysis. (C, D) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5, followed by immunoblot analysis. (E) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. m⁶A IP assay and RT-PCR were performed to determine the percentage of LDHA mRNA with methylation. (F) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5 (WT, R283K, or R283F). m⁶A IP assay and RT-PCR were performed to determine the percentage of LDHA mRNA with methylation. (G) Sequence motif identified from sequencing profile. HEK293T cells were overexpressed in HA-tagged LDHA (WT or mutants) and Flag-tagged ALKBH5 proteins, followed by immunoblot analysis. (H) MCF-7 cells were stably knocked down ALKBH5 and dual LDHA reporter plasmids, followed by luciferase reporter assay. (I) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5 (WT, R283K or R283F) with dual LDHA reporter plasmids, determined by luciferase reporter assay. (All data represent mean ± SEM, n = 3), *, P < 0.05.

Fig.6 ALKBH5 R283 methylation contributes to breast tumor growth. (A) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5 (WT, R283K, or R283F). Cell proliferation was evaluated by CCK-8 assay. (B) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. Cell proliferation was evaluated by plate colony formation assay. (C) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. Cell migration was evaluated by wound healing assay. (D) ALKBH5-depleted breast cancer cells were a reconstituted expression of rALKBH5. Cell invasion was evaluated by transwell assay. (E) Representative images of tumors were shown. (F, G) ALKBH5-depleted MCF-7 cells with reconstituted expression of rALKBH5 were subcutaneously injected into nude mice. After three weeks, the mice were sacrificed. Tumor weight and growth were examined. (H) Representative images of H&E and Ki67 staining of tumor samples (scale bar, 20 μm). (All data represent mean ± SEM, n≥ 3), *, P < 0.05.

Fig.7 Protein expressions of PRMT6, meR283-ALKBH5 and LDHA positively correlate with each other in human breast cancer tissues. (A) A total of 80 breast cancer samples were immunohistochemically stained with the indicated antibodies. Representative photos of these tumors were shown (scale bar, 20 μm). (B) The Pearson correlation test was used to determine the correlation between PRMT6, meR283-ALKBH5, and LDHA in breast cancer tissues (n = 80, *P < 0.05). (C, D) PRMT6 knockdown causes decrease in meR283-ALKBH5 and LDHA protein levels in breast cancer cells, followed by immunoblot analysis. (E) PRMT6 depletion causes decrease in meR283-ALKBH5 and LDHA protein levels in MEF cells, followed by immunoblot analysis. (F) Mechanism of ALKBH5 R283 methylation-promoted breast tumor growth.

1	D Wiener, S Schwartz. The epitranscriptome beyond m6A. Nat Rev Genet 2021; 22(2): 119–131 https://doi.org/10.1038/s41576-020-00295-8
2	S Oerum, V Meynier, M Catala, C Tisne. A comprehensive review of m6A/m6Am RNA methyltransferase structures. Nucleic Acids Res 2021; 49(13): 7239–7255 https://doi.org/10.1093/nar/gkab378
3	H Huang, H Weng, J Chen. m6A modification in coding and non-coding RNAs: roles and therapeutic implications in cancer. Cancer Cell 2020; 37(3): 270–288 https://doi.org/10.1016/j.ccell.2020.02.004
4	J Qu, H Yan, Y Hou, W Cao, Y Liu, E Zhang, J He, Z Cai. RNA demethylase ALKBH5 in cancer: from mechanisms to therapeutic potential. J Hematol Oncol 2022; 15(1): 8 https://doi.org/10.1186/s13045-022-01224-4
5	L Wu, D Wu, J Ning, W Liu, D Zhang. Changes of N6-methyladenosine modulators promote breast cancer progression. BMC Cancer 2019; 19(1): 326 https://doi.org/10.1186/s12885-019-5538-z
6	C Zhang, D Samanta, H Lu, JW Bullen, H Zhang, I Chen, X He, GL Semenza. 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 https://doi.org/10.1073/pnas.1602883113
7	S Gupta, RV Kadumuri, AK Singh, S Chavali, A Dhayalan. Structure, activity and function of the protein arginine methyltransferase 6. Life (Basel) 2021; 11(9): 951 https://doi.org/10.3390/life11090951
8	RS Blanc, S Richard. Arginine methylation: the coming of age. Mol Cell 2017; 65(1): 8–24 https://doi.org/10.1016/j.molcel.2016.11.003
9	Z Chen, J Gan, Z Wei, M Zhang, Y Du, C Xu, H Zhao. The emerging role of PRMT6 in cancer. Front Oncol 2022; 12: 841381 https://doi.org/10.3389/fonc.2022.841381
10	X Han, C Ren, C Lu, P Qiao, T Yang, Z Yu. Deubiquitination of MYC by OTUB1 contributes to HK2 mediated glycolysis and breast tumorigenesis. Cell Death Differ 2022; 29(9): 1864–1873 https://doi.org/10.1038/s41418-022-00971-8
11	C Lu, C Ren, T Yang, Y Sun, P Qiao, X Han, Z Yu. Fructose-1, 6-bisphosphatase 1 interacts with NF-κB p65 to regulate breast tumorigenesis via PIM2 induced phosphorylation. Theranostics 2020; 10(19): 8606–8618 https://doi.org/10.7150/thno.46861
12	C Lu, P Qiao, Y Sun, C Ren, Z Yu. Positive regulation of PFKFB3 by PIM2 promotes glycolysis and paclitaxel resistance in breast cancer. Clin Transl Med 2021; 11(4): e400 https://doi.org/10.1002/ctm2.400
13	C Ren, X Han, C Lu, T Yang, P Qiao, Y Sun, Z Yu. Ubiquitination of NF-κB p65 by FBXW2 suppresses breast cancer stemness, tumorigenesis, and paclitaxel resistance. Cell Death Differ 2022; 29(2): 381–392 https://doi.org/10.1038/s41418-021-00862-4
14	X Cai, X Wang, C Cao, Y Gao, S Zhang, Z Yang, Y Liu, X Zhang, W Zhang, L Ye. HBXIP-elevated methyltransferase METTL3 promotes the progression of breast cancer via inhibiting tumor suppressor let-7g. Cancer Lett 2018; 415: 11–19 https://doi.org/10.1016/j.canlet.2017.11.018
15	X Han, C Ren, T Yang, P Qiao, L Wang, A Jiang, Y Meng, Z Liu, Y Du, Z Yu. Negative regulation of AMPKα1 by PIM2 promotes aerobic glycolysis and tumorigenesis in endometrial cancer. Oncogene 2019; 38(38): 6537–6549 https://doi.org/10.1038/s41388-019-0898-z
16	Y Feng, Y Xiong, T Qiao, X Li, L Jia, Y Han. Lactate dehydrogenase A: a key player in carcinogenesis and potential target in cancer therapy. Cancer Med 2018; 7(12): 6124–6136 https://doi.org/10.1002/cam4.1820
17	E Sendinc, Y Shi. RNA m6A methylation across the transcriptome. Mol Cell 2023; 83(3): 428–441 https://doi.org/10.1016/j.molcel.2023.01.006
18	Z Liu, Y Chen, L Wang, S Ji. ALKBH5 promotes the proliferation of glioma cells via enhancing the mRNA stability of G6PD. Neurochem Res 2021; 46(11): 3003–3011 https://doi.org/10.1007/s11064-021-03408-9
19	Z Zhu, Q Qian, X Zhao, L Ma, P Chen. N6-methyladenosine ALKBH5 promotes non-small cell lung cancer progress by regulating TIMP3 stability. Gene 2020; 731: 144348 https://doi.org/10.1016/j.gene.2020.144348
20	H Zhu, X Gan, X Jiang, S Diao, H Wu, J Hu. ALKBH5 inhibited autophagy of epithelial ovarian cancer through miR-7 and BCL-2. J Exp Clin Cancer Res 2019; 38(1): 163 https://doi.org/10.1186/s13046-019-1159-2
21	T Guo, DF Liu, SH Peng, AM Xu. ALKBH5 promotes colon cancer progression by decreasing methylation of the lncRNA NEAT1. Am J Transl Res 2020; 12(8): 4542–4549
22	J Zhang, S Guo, HY Piao, Y Wang, Y Wu, XY Meng, D Yang, ZC Zheng, Y Zhao. ALKBH5 promotes invasion and metastasis of gastric cancer by decreasing methylation of the lncRNA NEAT1. J Physiol Biochem 2019; 75(3): 379–389 https://doi.org/10.1007/s13105-019-00690-8
23	N Jiang, QL Li, W Pan, J Li, MF Zhang, T Cao, SG Su, H Shen. PRMT6 promotes endometrial cancer via AKT/mTOR signaling and indicates poor prognosis. Int J Biochem Cell Biol 2020; 120: 105681 https://doi.org/10.1016/j.biocel.2019.105681
24	R Pan, H Yu, J Dai, C Zhou, X Ying, J Zhong, J Zhao, Y Zhang, B Wu, Y Mao, D Wu, J Ying, S Duan. Significant association of PRMT6 hypomethylation with colorectal cancer. J Clin Lab Anal 2018; 32(9): e22590 https://doi.org/10.1002/jcla.22590
25	K Okuno, Y Akiyama, S Shimada, M Nakagawa, T Tanioka, M Inokuchi, S Yamaoka, K Kojima, S Tanaka. Asymmetric dimethylation at histone H3 arginine 2 by PRMT6 in gastric cancer progression. Carcinogenesis 2019; 40(1): 15–26 https://doi.org/10.1093/carcin/bgy147
26	D Almeida-Rios, I Graca, FQ Vieira, J Ramalho-Carvalho, E Pereira-Silva, AT Martins, J Oliveira, CS Goncalves, BM Costa, R Henrique, C Jerónimo. Histone methyltransferase PRMT6 plays an oncogenic role of in prostate cancer. Oncotarget 2016; 7(33): 53018–53028 https://doi.org/10.18632/oncotarget.10061
27	DH Dowhan, MJ Harrison, NA Eriksson, P Bailey, MA Pearen, PJ Fuller, JW Funder, ER Simpson, PJ Leedman, WD Tilley, MA Brown, CL Clarke, GEO Muscat. Protein arginine methyltransferase 6-dependent gene expression and splicing: association with breast cancer outcomes. Endocr Relat Cancer 2012; 19(4): 509–526 https://doi.org/10.1530/ERC-12-0100
28	M Yoshimatsu, G Toyokawa, S Hayami, M Unoki, T Tsunoda, HI Field, JD Kelly, DE Neal, Y Maehara, BA Ponder, Y Nakamura, R Hamamoto. 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 https://doi.org/10.1002/ijc.25366
29	S Avasarala, PY Wu, SQ Khan, S Yanlin, M Van Scoyk, J Bao, A Di Lorenzo, O David, MT Bedford, V Gupta, RA Winn, RK Bikkavilli. PRMT6 promotes lung tumor progression via the alternate activation of tumor-associated macrophages. Mol Cancer Res 2020; 18(1): 166–178 https://doi.org/10.1158/1541-7786.MCR-19-0204
30	D Hyllus, C Stein, K Schnabel, E Schiltz, A Imhof, Y Dou, J Hsieh, UM Bauer. PRMT6-mediated methylation of R2 in histone H3 antagonizes H3 K4 trimethylation. Genes Dev 2007; 21(24): 3369–3380 https://doi.org/10.1101/gad.447007
31	E Guccione, C Bassi, F Casadio, F Martinato, M Cesaroni, H Schuchlautz, B Luscher, B Amati. Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature 2007; 449(7164): 933–937 https://doi.org/10.1038/nature06166
32	S Kim, NH Kim, JE Park, JW Hwang, N Myung, KT Hwang, YA Kim, CY Jang, YK Kim. PRMT6-mediated H3R2me2a guides Aurora B to chromosome arms for proper chromosome segregation. Nat Commun 2020; 11(1): 612 https://doi.org/10.1038/s41467-020-14511-w
33	L Liu, X Zhang, H Ding, X Liu, D Cao, Y Liu, J Liu, C Lin, N Zhang, G Wang, J Hou, B Huang, Y Zhang, J Lu. Arginine and lysine methylation of MRPS23 promotes breast cancer metastasis through regulating OXPHOS. Oncogene 2021; 40(20): 3548–3563 https://doi.org/10.1038/s41388-021-01785-7
34	WW Yan, YL Liang, QX Zhang, D Wang, MZ Lei, J Qu, XH He, QY Lei, YP Wang. Arginine methylation of SIRT7 couples glucose sensing with mitochondria biogenesis. EMBO Rep 2018; 19(12): e46377 https://doi.org/10.15252/embr.201846377
35	TL Wong, KY Ng, KV Tan, LH Chan, L Zhou, N Che, RLC Hoo, TK Lee, S Richard, CM Lo, K Man, PL Khong, S Ma. CRAF methylation by PRMT6 regulates aerobic glycolysis-driven hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation. Hepatology 2020; 71(4): 1279–1296 https://doi.org/10.1002/hep.30923
36	H Wang, B Xu, J Shi. N6-methyladenosine METTL3 promotes the breast cancer progression via targeting Bcl-2. Gene 2020; 722: 144076 https://doi.org/10.1016/j.gene.2019.144076
37	PY Bhattarai, G Kim, SC Lim, R Mariappan, T Ohn, HS Choi. METTL3 stabilization by PIN1 promotes breast tumorigenesis via enhanced m6A-dependent translation. Oncogene 2023; 42(13): 1010–1023 https://doi.org/10.1038/s41388-023-02617-6
38	D Ouyang, T Hong, M Fu, Y Li, L Zeng, Q Chen, H He, Y Wen, Y Cheng, M Zhou, Q Zou, W Yi. 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 https://doi.org/10.1186/s13058-022-01598-w
39	C Zhang, WI Zhi, H Lu, D Samanta, I Chen, E Gabrielson, GL Semenza. 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 https://doi.org/10.18632/oncotarget.11743
40	H Liu, H Lyu, G Jiang, D Chen, S Ruan, S Liu, L Zhou, M Yang, S Zeng, Z He, H Wang, H Li, G Zheng, B Liu. 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 https://doi.org/10.1158/0008-5472.CAN-22-0800
41	PJ Gong, YC Shao, Y Yang, WJ Song, X He, YF Zeng, SR Huang, L Wei, JW Zhang. Analysis of N6-Methyladenosine methyltransferase reveals METTL14 and ZC3H13 as tumor suppressor genes in breast cancer. Front Oncol 2020; 10: 578963 https://doi.org/10.3389/fonc.2020.578963
42	XF Dong, Y Wang, BF Huang, GN Hu, JK Shao, Q Wang, CH Tang, CQ Wang. Downregulated METTL14 expression correlates with breast cancer tumor grade and molecular classification. BioMed Res Int 2020; 2020: 8823270 https://doi.org/10.1155/2020/8823270

[1]

FMD-23047-OF-YZH_suppl_1

Download

[1]	Ronghui Yang, Guoguang Ying, Binghui Li. Potential of electron transfer and its application in dictating routes of biochemical processes associated with metabolic reprogramming[J]. Front. Med., 2021, 15(5): 679-692.
[2]	Jianye WU, Chuanyong GUO, Jun LIU, Xuanfu XUAN. Expression of integrin in hepatic fibrosis and intervention of resveratrol[J]. Front Med Chin, 2009, 3(1): 100-107.

Viewed

Full text

Abstract

Cited

Shared

Discussed