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.