Abstract 1290: Role of NNMT-regulated m6A mRNA modification in triple-negative breast cancer oncogenic gene expression

Abstract

Abstract Background: Post-transcriptional RNA methylation is implicated as a mechanism regulating gene expression associated with stem cell differentiation and malignant transformation. N6-methyladenosine (m6A) is the most abundant eukaryotic mRNA methylation modification. Proteins that selectively bind m6A alter m6A-modifed mRNA stability and translational efficiency. One mechanism regulating nucleic acid methylation is methyl donor availability and nicotinamide N-methyltransferase (NNMT) removes the methyl moiety from S-adenosyl methionine (SAM) and uses it to methylate nicotinamide, reducing methyl donor availability. Therefore, high NNMT activity depletes SAM; as a result, SAM-dependent methyltransferase targets such as mRNA become hypomethylated. NNMT-induced DNA and histone hypomethylation have been shown to result in oncogenic gene expression in cancer cells, although m6A hypomethylation of mRNA is less well understood. We found consistently high NNMT protein expression in triple-negative breast cancer (TNBC) cell lines relative to estrogen receptor (ER)+ lines. We also discovered the glucocorticoid receptor (GR) activation, which is associated with aggressive TNBC, further increased NNMT expression in MDA-MB-231 and HCC1937 cells. We therefore hypothesized that NNMT overexpression/activity might increase oncogenic gene expression(by reducing m6A modification)of mRNA transcripts involved in TNBC biology. Methods and Results: We found that TNBC cells upregulate NNMT expression following GR activation and therefore asked whether GR activation (and concomitant NNMT induction) significantly decrease global m6A mRNA modification as measured by mass spectrometry. Furthermore, either GR or NNMT antagonism restored m6A mRNA modification, suggesting that high NNMT activity may decrease m6A mRNA methylation. We then performed m6A immunoprecipitation-coupled RNAseq to determine m6A-modification levels on transcripts of individual genes before and after GR activation. We identified ~7000 m6A-modified transcripts among ~9000 expressed transcripts. Importantly, GR activation and concomitant NNMT induction significantly reduced the m6A modification of a subset of ~3000 transcripts that were found to be relatively enriched with cancer-promoting pathways (e.g. RAF signaling). Conclusions: NNMT overexpression is implicated in the biology of several epithelial cancers; however, its mechanism of action is not fully understood. Our ongoing work will determine whether reduced m6A modification of selective TNBC mRNAs contribute to the oncogenic phenotype via increased half-life and protein production. We are using inducible NNMT-knockdown to determine whether reduced NNMT expression results in increased m6A modification of pro-oncogenic mRNAs and thereby inhibits oncogenic phenotypes such as invasion and cell survival. Citation Format: Deniz N. Dolcen, Bryan Harada, Chuan He, Suzanne D. Conzen. Role of NNMT-regulated m6A mRNA modification in triple-negative breast cancer oncogenic gene expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1290.