Targeting histone deacetylase suppresses tumor growth through eliciting METTL14-modified m6 A RNA methylation in ocular melanoma

Abstract

Background: Diversified histone deacetylation inhibitors (HDACis) have demonstrated encouraging outcomes in multiple malignancies. N6-methyladenine (m⁶ A) is the most prevalent messenger RNA modification that plays an essential role in the regulation of tumorigenesis. Howbeit, an in-depth understanding of the crosstalk between histone acetylation and m⁶ A RNA modifications remains enigmatic. This study aimed to explore the role of histone acetylation and m⁶ A modifications in the regulation of tumorigenesis of ocular melanoma.

Methods: Histone modification inhibitor screening was used to explore the effects of HDACis on ocular melanoma cells. Dot blot assay was used to detect the global m⁶ A RNA modification level. Multi-omics assays, including RNA-sequencing, cleavage under targets and tagmentation, single-cell sequencing, methylated RNA immunoprecipitation-sequencing (meRIP-seq), and m⁶ A individual nucleotide resolution cross-linking and immunoprecipitation-sequencing (miCLIP-seq), were performed to reveal the mechanisms of HDACis on methyltransferase-like 14 (METTL14) and FAT tumor suppressor homolog 4 (FAT4) in ocular melanoma. Quantitative real-time polymerase chain reaction (qPCR), western blotting, and immunofluorescent staining were applied to detect the expression of METTL14 and FAT4 in ocular melanoma cells and tissues. Cell models and orthotopic xenograft models were established to determine the roles of METTL14 and FAT4 in the growth of ocular melanoma. RNA-binding protein immunoprecipitation-qPCR, meRIP-seq, miCLIP-seq, and RNA stability assay were adopted to investigate the mechanism by which m⁶ A levels of FAT4 were affected.

Results: First, we found that ocular melanoma cells presented vulnerability towards HDACis. HDACis triggered the elevation of m⁶ A RNA modification in ocular melanoma. Further studies revealed that METTL14 served as a downstream candidate for HDACis. METTL14 was silenced by the hypo-histone acetylation status, whereas HDACi restored the normal histone acetylation level of METTL14, thereby inducing its expression. Subsequently, METTL14 served as a tumor suppressor by promoting the expression of FAT4, a tumor suppressor, in a m⁶ A-YTH N6-methyladenosine RNA-binding protein 1-dependent manner. Taken together, we found that HDACi restored the histone acetylation level of METTL14 and subsequently elicited METTL14-mediated m⁶ A modification in tumorigenesis.

Conclusions: These results demonstrate that HDACis exert anti-cancer effects by orchestrating m⁶ A modification, which unveiling a "histone-RNA crosstalk" of the HDAC/METTL14/FAT4 epigenetic cascade in ocular melanoma.

Keywords: N6-methyladenine; epigenetics; histone deacetylation inhibitors; histone-RNA crosstalk; melanoma.

FIGURE 1

LBH589 selectively attenuates the oncogenesis of ocular melanoma. (A) Schematic diagram of high‐throughput histone modification drug screening procedures. Selective index = IC₅₀ (average in tumor cells) / IC₅₀ (normal melanocytes). (B) Heatmap of histone modification drug screening results in ocular melanoma cell lines (92.1 and CRMM1) upon treatment with DMSO or inhibitors (100 nmol/L) for 24 h. Data represent three biological replicates. (C) Selective indices of the six histone modification inhibitor candidates (LBH589, I‐BET726, RG2833, LMK‐235, EED226, and Chidamide) for ocular melanoma cells. Each of these six inhibitors exhibited an inhibitory rate greater than 60% in ocular melanoma cells and less than 30% in PIG1 cells. Inhibitory rate (%) = 1 ‐ cell viability (%). (D) Western blotting of H3K9Ac and H3K27Ac relative to histone H3 in ocular melanoma cells (92.1, OMM2.3, and CRMM1) after treatment with different concentrations of LBH589 for 24 h. Data are representative of triplicate experiments. (E) Densitometric analysis of the expression levels of H3K9Ac and H3K27Ac relative to histone H3 in ocular melanoma cells (92.1, OMM2.3, and CRMM1) upon treatment with different concentrations of LBH589. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (F‐G) A Colony formation assay was performed to assess the growth of normal melanocytes (PIG1) and ocular melanoma cells (92.1, OMM2.3, and CRMM1) upon treatment with LBH589 (100 nmol/L). Representative images (F) of three experimental replicates are shown. Data (G) are presented as the mean ± standard deviation of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (H) Images acquired with an in vivo small‐animal imaging system showing the suppression of bioluminescent signals in orthotopic xenografts derived from cells pretreated with DMSO or LBH589 (100 nmol/L, 24 h) before intraocular injection. Representative images of five biological replicates are shown. The data are presented as mean ± standard error of mean. Overall and eyeball appearances showing the suppressive effects of LBH589 on tumor volumes in orthotopic xenografts. Representative images of five biological replicates are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: HDAC, Histone deacetylase; IC₅₀, half maximal inhibitory concentration; BET family, bromodomain and extra‐terminal family; BETi, BET inhibitor; PRC2i, polycomb repressive complex 2 inhibitor; rep, replicate; H3, histone 3; K9: lysine 9; K27: lysine 27; Ac, acetylation; DMSO, dimethylsulfoxide; ns, no significance.

FIGURE 2

LBH589 fuels global m⁶A modification through activating METTL14. (A) Volcano plot showing 2,086 downregulated and 2,841 upregulated genes in LBH589‐treated ocular melanoma cells (92.1) (|log₂Fold Change| > 1, P < 0.05) compared to the control group. (B) GO analysis showing the functions of the downregulated genes in ocular melanoma cells (92.1). Three biological replicates were analyzed. (C) Venn diagram showing the overlap between 2,841 genes upregulated after LBH589 treatment and 2,357 inequable m⁶A methylated genes in ocular melanoma (92.1). (D) m⁶A dot blot of global m⁶A levels in normal control cells (ARPE‐19 and PIG1), ocular melanoma cells (OMM1, OMM2.3, MUM2B, OCM1, 92.1, MEL290, CM2005.1, CRMM2, and CRMM1), and cutaneous melanoma cells (A375 and SK28). (E) m⁶A dot blot of m⁶A levels in ocular melanoma cells (92.1, OMM2.3, and CRMM1) after treatment with different concentrations of LBH589 for 24 h. The images are representative of experimental triplicates. Data are presented as the mean ± standard deviation of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (F) Heatmap of the relative expression levels of m⁶A‐related genes in DMSO‐ and LBH589‐treated groups. (G) RNA‐seq results showing the fold change in expression levels of m⁶A‐related genes in ocular melanoma cells (92.1) after LBH589 treatment. (H) IGV tracks for METTL14 from RNA‐seq data in DMSO‐ and LBH589‐treated ocular melanoma cells (92.1). Three biological replicates were analyzed. (I)Western blotting of m⁶A‐related proteins relative to GAPDH in ocular melanoma cells (92.1 and CRMM1) upon treatment with LBH589 at different concentrations. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: RNA‐seq, RNA sequencing; GO, Gene Ontology; m⁶A, N6‐methyladenine; IGV, Integrative Genomics Viewer; No., number; TSS, transcriptional start site. METTL14, methyltransferase‐like 14; METTL3, methyltransferase‐like 3; WTAP, Wilms tumor 1‐associating protein; ALKBH5, AlkB homolog 5 RNA demethylase; FTO, fat mass and obesity‐associated protein; YTHDF1, YTH N6‐methyladenosine RNA binding protein 1; YTHDF2, YTH N6‐methyladenosine RNA binding protein 2; YTHDF3, YTH N6‐methyladenosine RNA binding protein 3; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; DMSO, dimethylsulfoxide; ns, no significance.

FIGURE 3

METTL14 is downregulated and serves as a tumor suppressor in ocular melanomas. (A) IGV tracks from CUT&Tag analysis showing H3K27Ac enrichment at the promoter region of METTL14 in ocular melanoma cells and normal melanocytes. Three biological replicates were analyzed. (B) ChIP‐qPCR assay of H3K27Ac status at the METTL14 TSS region in ocular melanoma cells (92.1, OMM2.3, and CRMM1) and normal control cells (PIG1 and ARPE‐19) compared with IgG. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (C) ChIP‐qPCR assay of H3K27Ac status at the METTL14 TSS region in ocular melanoma cells (92.1 and CRMM1) after LBH589 exposure compared to the DMSO‐treated group. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (D) The IHC assay showed that METTL14 in ocular melanoma and normal melanocyte samples. Representative images were shown. IHC scores of METTL14 in ocular melanoma tissues (n = 56) and normal melanocyte tissues (n = 27) are presented as the median with interquartile range. Significance was determined using unpaired Mann‐Whitney nonparametric test. (E) Correlation analysis of METTL14 expression and functional states in single‐cell datasets from CancerSEA (GSE139829). Significance was determined using Pearson correlation analysis (filtered by correlation strength < ‐0.1, P < 0.001). (F) Kaplan–Meier analysis of the correlation between IHC scores of METTL14 and recurrence‐free rate in the internal cohort (n = 56). Significance was determined by a two‐sided log‐rank test. (G) qPCR data showing METTL14 expression in ocular melanoma cells (92.1 and CRMM1) after DMSO or LBH589 exposure in METTL14 knockdown or control groups, respectively. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (H) Western blotting of METTL14 relative to GAPDH in ocular melanoma cells (92.1 and CRMM1) after DMSO or LBH589 exposure in METTL14 knockdown or control groups, respectively. The images are representative of experimental triplicates. (I) A colony formation assay was performed to assess the growth of ocular melanoma cells (92.1 and CRMM1) upon DMSO or LBH589 treatment in METTL14 knockdown or control groups, respectively. The colony formation assay data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (J) A transwell assay was performed to assess the cell migration ability of ocular melanoma cells (92.1 and CRMM1) upon DMSO or LBH589 treatment in METTL14 knockdown or control groups, respectively. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (K) Images acquired with an in vitro small animal imaging system showing bioluminescent signals in orthotopic xenografts derived from 92.1 cells upon DMSO or LBH589 (100 nmol/L, 24 h) treatment in METTL14 knockdown or control groups, respectively. Representative images of five biological replicates are shown. Data are presented as the mean ± SEM. The overall and eyeball appearances exhibited tumor volumes in the orthotopic xenografts. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: UM, uveal melanoma; CUT&Tag, Cleavage Under Targets and Tagmentation; No., number; rep, replicate; METTL14, methyltransferase‐like 14; IGV, Integrative Genomics Viewer; NC, normal control; H3K27Ac, acetylation of histone 3 at lysine 27; ChIP, Chromatin immunoprecipitation; qPCR, quantitative real‐time polymerase chain reaction; TSS, transcriptional start site; EMT, epithelial‐mesenchymal transition; SD, standard deviation; SEM, standard error of mean; DMSO, dimethylsulfoxide; IHC, immunohistochemistry; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

FIGURE 4

FAT4 serves as the downstream candidate of METTL14. (A) Venn diagram identifying CRYBG3, FAT4, and SEMA3D as downstream target candidates of METTL14. (B) Kaplan‐Meier analysis showing the correlation between METTL14 expression and overall survival in TCGA‐uveal melanoma patients stratified by METTL14 expression levels. Statistical significance was determined using a two‐sided log‐rank test. (C) Correlation analysis of METTL14 and FAT4 expression in the TCGA‐uveal melanoma cohort. Significance was determined using Pearson’s correlation analysis (R = 0.471, P < 0.001). (D) IGV tracks for FAT4 from RNA‐seq data in the control and METTL14‐overexpressed ocular melanoma cell lines. Biological triplicates were analyzed. (E) IGV tracks for FAT4 from RNA‐seq data in control and LBH589‐treated ocular melanoma cell lines. Biological triplicates were analyzed. (F) Real‐time PCR (top) data showing FAT4 expression in ocular melanoma cells (92.1 and CRMM1) after DMSO or LBH589 treatment in METTL14 knockdown group and control group, respectively. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. Western blotting (bottom) of FAT4 relative to GAPDH in ocular melanoma cells (92.1 and CRMM1) after DMSO or LBH589 treatment in METTL14 knockdown group and control group, respectively. Representative images from three experimental replicates are shown. (G) Real‐time PCR (top) data showing FAT4 expression in control and METTL14‐overexpressed ocular melanoma cells (92.1 and CRMM1). Western blotting (bottom) of FAT4 relative to GAPDH in control and METTL14‐overexpressed ocular melanoma cells (92.1 and CRMM1). Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: HDACis, histone deacetylation inhibitors; m⁶A, N6‐methyladenine; CRYBG3, crystallin beta‐gamma domain containing 3; SEMA3D, Semaphorin 3D; FAT4, FAT Tumor Suppressor Homolog 4; METTL14, methyltransferase‐like 14; TCGA, The Cancer Genome Atlas; IGV, Integrative Genomics Viewer; RNA‐seq, RNA sequencing; rep, replicate; TSS, transcriptional start site; No., number; DMSO, dimethylsulfoxide; SD, standard deviation; NC, normal control; oe, overexpression; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

FIGURE 5

FAT4 acts as a tumor suppressor. (A) The IHC assay showed that FAT4 in ocular melanoma and normal melanocyte samples. Representative images were shown. IHC scores of FAT4 in ocular melanoma tissues (n = 56) and normal melanocyte tissues (n = 27) are presented as the median with interquartile range. Significance was determined using unpaired Mann‐Whitney nonparametric test. (B) Kaplan–Meier analysis of the correlation between IHC scores of FAT4 and recurrence‐free rate in the internal cohort (n = 56). Significance was determined by a two‐sided log‐rank test. (C) Real‐time PCR data showing the expression levels of FAT4 in ocular melanoma cell lines and retinal pigment epithelial cells, relative to normal melanocytes. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (D) Western blotting of FAT4 relative to GAPDH in ocular melanoma cells, retinal pigment epithelial cells, and normal melanocytes. The images are representative of experimental triplicates. (E) Integrative Genomics Viewer tracks showing the expression levels of FAT4 from RNA‐seq data in ocular melanoma cells and normal melanocytes. The biological triplicates were analyzed. (F) Western blotting of FAT4 relative to GAPDH in control or METTL14‐overexpressed ocular melanoma cells (92.1 and CRMM1) upon FAT4 knockdown or not. The images are representative of experimental triplicates. (G) A colony formation assay was performed to assess the growth of control or METTL14‐overexpressed ocular melanoma cells (92.1 and CRMM1) upon FAT4 knockdown. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (H) A transwell assay was performed to assess the cell migration ability of control or METTL14‐overexpressed ocular melanoma cells (92.1 and CRMM1) upon FAT4 knockdown. Data are presented as the mean ± SD of triplicate experiments. Significance was determined using unpaired two‐tailed Student's t‐tests. (I) Representative images from five biological replicates showing the bioluminescent signals (top) and eyeball appearances (bottom) of orthotopic xenografts derived from control or METTL14‐overexpressed ocular melanoma cells (92.1 and CRMM1) upon FAT4 knockdown. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: FAT4, FAT Tumor Suppressor Homolog 4; IHC, immunohistochemistry; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; SD, standard deviation; RNA‐seq, RNA sequencing; rep, replicate; TSS, transcriptional start site; No., number; METTL14, methyltransferase‐like 14; NC, normal control; oe, overexpression; SEM, standard error of mean.

FIGURE 6

The YTHDF1 recognition of FAT4 m⁶A modification enhances RNA stability. (A‐B) Integrative Genomics Viewer tracks showing m⁶A status at FAT4 from miCLIP‐seq (A) and meRIP‐seq (B) data in ocular melanoma cells and normal melanocytes. (C) RIP‐qPCR assay of YTHDF1, YTHDF2, and YTHDF3 status in the transcripts of METTL14 in ocular melanoma cells (92.1) and normal melanocytes (PIG1). Data are presented as the mean ± SD of triplicate experiments. (D) Western blotting of FAT4, YTHDF1, YTHDF2, and YTHDF3 relative to GAPDH in control or YTHDF1/2/3 knockdown ocular melanoma cells (92.1) and normal melanocytes (PIG1). (E) Lifetime FAT4 mRNA levels in control or METTL14‐overexpressed ocular melanoma cells upon YTHDF1 silencing. (F) Correlation analysis of FAT4 expression and YTHDF1 expression in TCGA‐uveal melanoma cohort. Statistical significance was determined using Pearson’s correlation analysis (R = 0.359, P < 0.01). (G) Schematic diagram of the regulatory mechanism by which METTL14 functions as a tumor suppressor in ocular melanoma. METTL14 deposits m⁶A modifications in FAT4 transcripts YTHDF1 recognizes m⁶A modification sites in FAT4 mRNA and enhances its stability, which facilitates the expression of FAT4 protein. *P < 0.05, **P < 0.01. Abbreviations: FAT4, FAT Tumor Suppressor Homolog 4; rep, replicate; m⁶A, N6‐methyladenine; meRIP‐seq, methylated RNA immunoprecipitation sequencing; miCLIP‐seq, m⁶A individual‐nucleotide‐resolution cross‐linking and immunoprecipitation; No. number; RIP, RNA‐binding protein immunoprecipitation; qPCR, quantitative real‐time polymerase chain reaction; YTHDF1, YTH N6‐methyladenosine RNA binding protein 1; YTHDF2, YTH N6‐methyladenosine RNA binding protein 2; YTHDF3, YTH N6‐methyladenosine RNA binding protein 3; METTL14, methyltransferase‐like 14; NC, normal control; oe, overexpression; SD, standard deviation; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; TCGA, The Cancer Genome Atlas.

FIGURE 7

Schematic diagram depicting that HDAC inhibitors exert anti‐cancer effects by HDAC/METTL14/FAT4 axis in ocular melanoma. In normal melanocytes, histone acetylation (H3K9Ac and H3K27Ac) at the transcription start site maintains the transcription of METTL14. Aberrant hypoacetylation “locks” the expression of METTL14, which contributes to the tumorigenesis of uveal melanoma. Target correction by the pan‐HDAC inhibitor, LBH589, which acts as an “unlocking key” to restore the acetylation levels of histone H3 at the transcription start site of METTL14, exerts a potent anti‐tumor effect. Abbreviations: H3, histone 3; H3K9Ac, acetylation of histone 3 at lysine 9; H3K27Ac, acetylation of histone 3 at lysine 27; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; METTL14, methyltransferase‐like 14; FAT4, FAT Tumor Suppressor Homolog 4.