Adenylate Kinase 4 Modulates the Resistance of Breast Cancer Cells to Tamoxifen through an N6-Methyladenosine-based Epitranscriptomic Mechanism

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and this methylation constitutes an important regulatory mechanism for the stability and translational efficiency of mRNA. In this study, we found that the protein levels of adenylate kinase 4 (AK4) and m6A writer METTL3 are significantly higher in tamoxifen-resistant (TamR) MCF-7 cells than in parental cells. The TamR MCF-7 cells also exhibit increased methylation at multiple m6A consensus motif sites in the 5' untranslated region (5' UTR) of AK4 mRNA, and genetic depletion of METTL3 in TamR MCF-7 cells led to a diminished AK4 protein level and attenuated resistance to tamoxifen. In addition, we observed augmented levels of reactive oxygen species (ROS) and p38 activity in TamR MCF-7 cells, and both are diminished upon genetic depletion of AK4. Reciprocally, overexpression of AK4 in MCF-7 cells stimulates ROS and p38 phosphorylation levels, and it suppresses mitochondrial apoptosis. Moreover, scavenging of intracellular ROS leads to reduced p38 activity and re-sensitizes TamR MCF-7 cells to tamoxifen. Thus, our results uncover a novel m6A-mediated epitranscriptomic mechanism for the regulation of AK4, illustrate the cellular pathways through which increased AK4 expression contributes to tamoxifen resistance, and reveal AK4 as a potential therapeutic target for overcoming tamoxifen resistance.