The importance of RNA methylation in biological processes is an emerging focus of investigation. University of Texas Health Science Center at San Antonio researchers report that altering m6A levels by silencing either N6-adenosine methyltransferase METTL14 (methyltransferase-like 14) or demethylase ALKBH5 (ALKB homolog 5) inhibits cancer growth and invasion. METTL14/ALKBH5 mediate their protumorigenic function by regulating m6A levels of key epithelial-mesenchymal transition and angiogenesis-associated transcripts, including transforming growth factor–β signaling pathway genes.
Using MeRIP-seq (methylated RNA immunoprecipitation sequencing) analysis and functional studies, the researchers found that these target genes are particularly sensitive to changes in m6A modifications, as altered m6A status leads to aberrant expression of these genes, resulting in inappropriate cell cycle progression and evasion of apoptosis. Their results reveal that METTL14 and ALKBH5 determine the m6A status of target genes by controlling each other’s expression and by inhibiting m6A reader YTHDF3 (YTH N6-methyladenosine RNA binding protein 3), which blocks RNA demethylase activity. Furthermore, they showed that ALKBH5/METTL14 constitute a positive feedback loop with RNA stability factor HuR to regulate the stability of target transcripts. The researchers discovered that hypoxia alters the level/activity of writers, erasers, and readers, leading to decreased m6A and consequently increased expression of target transcripts in cancer cells.
(A and B) MeRIP-seq analysis showing the number of peaks (A) and m6A peak–containing transcripts (B) identified in scrambled-siRNA (siCntrl)– and METTL14-siRNA (METTL14 KD)–transfected breast cancer cells. Common m6A-containing genes share at least one common peak, while unique m6A-containing genes share no peak between scrambled-siRNA and METTL14 KD breast cancer cells. (C) TGFβ1, CCNE1, and SMAD3 showing significantly enriched m6A peaks in METTL14 KD MDA-MB-231 cells compared to scrambled-siRNA. Top two tracks represent MeRIP and input for METTL14-siRNA–transfected MDA-MB-231 cells, while bottom two tracks represent MeRIP and input for scrambled-siRNA–transfected MDA-MB-231 cells. bp, base pairs; RefSeq, reference sequence. (D) Pie chart of m6A peak distribution showing proportion of total (top) and unique (bottom) peaks in different regions of genes in scrambled-siRNA and METTL14 KD cells. (E) Ingenuity Pathway Analysis (IPA) using m6A peak–containing genes shows TGFβ as one of the top upstream regulators. (F) Bar graphs show enriched canonical pathways derived from IPA using m6A-containing genes. (G) qRT-PCR showing m6A abundance (normalized to input) of target genes in MeRIP samples from MDA-MB-231 and MCF-7 cells transfected with scrambled-siRNA or METTL14-siRNA (METTL14 KD). The data shown are means ± SEM of three independent experiments (n = 3 biological replicates per experiment).
This study unveils a previously undefined role for m6A in cancer and shows that the collaboration among writers-erasers-readers sets up the m6A threshold to ensure the stability of progrowth/proliferation-specific genes, and protumorigenic stimulus, such as hypoxia, perturbs that m6A threshold, leading to uncontrolled expression/activity of those genes, resulting in tumor growth, angiogenesis, and progression.