METTL1-mediated tRNA m7G methylation and translational dysfunction restricts breast cancer tumorigenesis by fueling cell cycle blockade

Abstract

Background: RNA modifications of transfer RNAs (tRNAs) are critical for tRNA function. Growing evidence has revealed that tRNA modifications are related to various disease processes, including malignant tumors. However, the biological functions of methyltransferase-like 1 (METTL1)-regulated m⁷G tRNA modifications in breast cancer (BC) remain largely obscure.

Methods: The biological role of METTL1 in BC progression were examined by cellular loss- and gain-of-function tests and xenograft models both in vitro and in vivo. To investigate the change of m⁷G tRNA modification and mRNA translation efficiency in BC, m⁷G-methylated tRNA immunoprecipitation sequencing (m⁷G tRNA MeRIP-seq), Ribosome profiling sequencing (Ribo-seq), and polysome-associated mRNA sequencing were performed. Rescue assays were conducted to decipher the underlying molecular mechanisms.

Results: The tRNA m⁷G methyltransferase complex components METTL1 and WD repeat domain 4 (WDR4) were down-regulated in BC tissues at both the mRNA and protein levels. Functionally, METTL1 inhibited BC cell proliferation, and cell cycle progression, relying on its enzymatic activity. Mechanistically, METTL1 increased m⁷G levels of 19 tRNAs to modulate the translation of growth arrest and DNA damage 45 alpha (GADD45A) and retinoblastoma protein 1 (RB1) in a codon-dependent manner associated with m⁷G. Furthermore, in vivo experiments showed that overexpression of METTL1 enhanced the anti-tumor effectiveness of abemaciclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor.

Conclusion: Our study uncovered the crucial tumor-suppressive role of METTL1-mediated tRNA m⁷G modification in BC by promoting the translation of GADD45A and RB1 mRNAs, selectively blocking the G2/M phase of the cell cycle. These findings also provided a promising strategy for improving the therapeutic benefits of CDK4/6 inhibitors in the treatment of BC patients.

Keywords: Breast cancer; CDK4/6; Cell cycle; GADD45A; METTL1.

Fig. 1

Downregulation of METTL1 and WDR4 in BC patients. (A-B) qRT-PCR analysis revealed a significant decrease in the expression of METTL1 (A) and WDR4 (B) in BC tissues compared to adjacent non-tumor tissues. (C) Western blotting of METTL1 and WDR4 protein levels in four pairs of human BC specimens. N: Normal, T: Tumor. (D-G) Stratification of METTL1 and WDR4 mRNA expression levels in BC tissues based on different subtypes (D-E) and tumor stages (F-G). (H-J) qRT-PCR (H-I) and western blotting (J) of METTL1 and WDR4 mRNA and protein levels in BC cell lines. The normal human mammary epithelial cells, MCF10A, were used as a normal control. (K-L) Representative images (K) and quantification (L) of METTL1 and WDR4 immunostaining in BC specimens and adjacent normal tissues. Scale bar = 100 μm. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Inhibition of METTL1 promotes BC progression in vitro. (A-B) qRT-PCR analysis of relative METTL1 expression after METTL1 knockdown in MDA-MB-231 and MCF-7 cells. (C) Verification of METTL1 knockdown by western blotting in MDA-MB-231 and MCF-7 cells. (D) Colony formation assays assessing the clonogenicity of MDA-MB-231 and MCF-7 cells upon METTL1 silencing. (E) Flow cytometric analysis of cell cycle distribution. (F-G) EdU assays to evaluate cell proliferation. (H-I) Transwell assays to analyze cell migration and invasion ability. Scale bar = 200 μm. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Overexpression of METTL1 impedes BC progression in vitro. (A-B) qRT-PCR analysis of relative METTL1 levels after forced expression of wild-type and catalytically inactive METTL1 in MDA-MB-231 and MCF-7 cells. (C) Western blotting confirmation of METTL1 in indicated MDA-MB-231 and MCF-7 cells. (D) Colony formation assays to assess the effects of METTL1 overexpression on the clonogenicity of MDA-MB-231 and MCF-7 cells. (E) Flow cytometric analysis of cell cycle distribution. (F-G) EdU assays to evaluate changes in BC cell proliferation. (H-I) Transwell assays to profile cell migration and invasion capacity. Scale bar = 200 μm. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ns., not significant

Fig. 4

METTL1 enhances m⁷G tRNA methylation levels and global mRNA translation. (A) List of m⁷G-modified tRNAs identified by m⁷G tRNA MeRIP-seq in MCF-7 cells. (B) Quantitative comparison of fold change in expression between m⁷G and non-m⁷G tRNAs. (C) Representative images from Integrative genome viewer (IGV) displaying different IP/Input at the motif sequence of indicated tRNA. (D) Relative expression profile of m⁷G-modified tRNAs based on m⁷G tRNA MeRIP-seq. The relative expression of each tRNA type was calculated by combining the expression of all tRNA genes belonging to the same tRNA type. (E) Polysome profiling of MCF-7 cells with or without METTL1 overexpression. (F) Distribution of ribosome-protected fragments (Rfs). (G) Measurement of puromycin intake in MCF-7 cells overexpressing wild-type or mutant METTL1 compared to control, and in METTL1-depleted MCF-7 cells compared to control. Coomassie brilliant blue staining of the gel was used as a control. The data are presented as mean ± SD. *P < 0.05

Fig. 5

METTL1-mediated m⁷G tRNA modification selectively modulates the translational efficiency of specific transcripts. (A) Scatterplot of TE in MCF-7 cells with or without METTL1 overexpression. TE was calculated as the ratio of polyribosome signals to input signals. (B) Frequencies of m⁷G-related codons in TE-increased genes (TE-up), TE-decreased genes (TE-down), and other genes (others) in METTL1-overexpressing cells. (C) Pathway analysis (upper panel) and Gene Ontology analysis (lower panel) of TE-up genes upon METTL1 overexpression. (D) Frequency analysis of the 19 m⁷G-modified tRNAs decoded codons in 15 mRNAs associated with negative regulation of the cell cycle. (E-F) Western blotting analysis of GADD45A protein expression in 293T control and METTL1-knowdown cells overexpressing GADD45A WT-Flag, GADD45A mut-Flag (mutant 1 codons, CTA), or GADD45A mut-13-Flag (mutant 13 codons). (G) Relative expression and translation efficiency of RB1, GADD45A, CDK1, and CCNB1 mRNA in METTL1-overexpressed and control MCF-7 cells. Tubulin was used as an internal control. TE was calculated as the ratio of polyribosome signals to input signals. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ns., not significant

Fig. 6

METTL1 controls BC malignant phenotypes through GADD45A. (A) qRT-PCR analysis of relative METTL1 expression after forced expression of wild-type METTL1 (oeM1) and catalytically inactive METTL1 (oeMut), followed by knockdown of METTL1 (oeM1 + si-M1, oeMut + si-M1) in MDA-MB-231 cells. (B) Western blotting analysis of GADD45A, RB1, and their downstream signaling molecules in different treatment groups of MDA-MB-231. (C) qRT-PCR analysis of relative METTL1 expression in oeM1, oeMut, oeM1 + si-M1, and oeMut + si-M1 in MCF-7 cells. (D) Western blotting analysis of GADD45A, RB1, and their downstream signaling molecules in different treatment groups of MCF-7. (E-F) Flow cytometry analysis of cell cycle phase distribution in MDA-MB-231 and MCF-7 treatment groups (oeM1, oeMut, oeM1 + si-45 A, oeMut + si-45 A) and control group (NC). (G-H) Assessment of proliferation ability using CCK-8 assays in MDA-MB-231 and MCF-7 cells. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ns., not significant

Fig. 7

METTL1 enhances the anti-oncogenic effects of abemaciclib in vivo. (A) Schematic diagram to illustrate the treatment model protocol. (B) Representative images of tumors in xenograft mice. (C-E) Statistical analysis of body weight, tumor volume, and tumor weight in different groups of nude mice (n = 6). Data were measured every three days. (F) Representative IHC images of METTL1, GADD45A, RB1, and Ki67 expression in serial segments of tumor tissue separated from the subcutaneous model. (G) Quantification of METTL1, GADD45A, RB1, and Ki67 expression in xenograft tumor tissues. Scale bar = 100 μm. The data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ns., not significant

Fig. 8

METTL1 positively correlates with GADD45A and RB1 expression in BC samples. (A-B) Representative images of IHC staining for GADD45A and RB1 expression in 50 BC samples with different levels of METTL1, Scale bar = 100 μm. (C) Schematic model to illustrate the role and potential mechanisms of m⁷G tRNA modification in regulating BC tumorigenesis and development. **P < 0.01, ***P < 0.001