Dysregulations and functions of RNA modifying enzymes METTL3 and TRMT6 in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma is the most common type of primary liver cancer, ranking the third leading cause of cancer-related death worldwide. However, only limited therapeutic strategies are available for HCC patients. Thus, understanding the detailed mechanism of HCC progression might shed light on the development of new therapeutic treatments. Epigenetic alterations immensely contribute to human carcinogenesis. Traditional epigenetic studies focus on DNA methylation, histone modifications, and chromatin remodeling. Recently, diverse and reversible chemical modifications on RNAs emerge as a new layer of epigenetic regulation.

N-6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA, which regulates mRNA stability, alternative splicing, and translation efficiency. However, the roles of m6A deregulation in human carcinogenesis remain elusive. Firstly, through whole-transcriptome sequencing of 16 paired primary HCC and their corresponding non-tumor liver tissues, we identified that METTL3, the major m6A methyltransferase, was significantly up-regulated in human HCC. Up-regulation of METTL3 associated with poor overall and disease-free survivals of HCC patients. Knockdown of METTL3 dramatically inhibited HCC proliferation, migration and colony formation abilities in vitro and significantly suppressed HCC tumorigenicity and metastasis in vivo. Mechanistically, through RNA-Seq, m6A-qPCR and m6A-Seq, we identified SOCS2, a prominent tumor suppressor, as a direct down-stream target of METTL3. A m6A peak was identified in SOCS2 transcript by m6A-seq which diminished upon METTL3 knockdown. Inactivation of METTL3 by RNAi or methylation inhibitor (DAA) treatment impaired m6A mediated mRNA degradation, thereby stabilized SOCS2 mRNA. In conclusion, our findings suggested that deregulation of METTL3 and its associated m6A modification could contribute to human carcinogenesis by imposing an epigenetic control on the stability and expressions of critical tumor suppressor gene.

tRNAs are subject to extensive chemical modifications, among which N1-methyladenosine (m1A) has been suggested as a common reversible modification to dynamically regulate tRNA stability and implicated in protein translation. However, little is known about the functions of the tRNA modifying proteins in human carcinogenesis. We proved that TRMT6, which catalyzes the installation of m1A at position 58 of tRNA, is an oncogene in HCC. Genetic ablation of TRMT6 altered tRNA m1A level, inhibited HCC proliferation and migration in vitro and suppressed HCC tumorigenicity and lung metastasis in vivo. tRNA sequencing and proteomics analysis uncovered that knockout of TRMT6 resulted in a distinct tRNA repertoire and suppressed stemness genes Nestin and CD44 at protein level in a codon usage dependent manner. TRMT6 knockout HCC cells displayed compromised stemness properties, as reflected by impaired sphere formation and tumor initiating ability, and increased sensitivity to molecular target drug sorafenib. Our findings, for the first time, uncovered that dysregulation of tRNA modifying enzyme TRMT6 could promote liver carcinogenesis through codon dependent translational regulations of stemness factors.

In conclusion, we proved that METTL3 and TRMT6 are novel oncogenes in human HCCs and comprehensively illustrated their underlying mechanisms in promoting hepatocarcinogenesis. Our findings added a new dimension to epigenetic alterations in human malignancy and suggest RNA modifying proteins are vulnerability of HCC which could be applied to the inventions of novel diagnostic and therapeutic strategies for clinical cancer treatments.