Biological and cancer-associated role of epitranscriptomic gene expression regulation

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PROJECT SUMMARY My lab discovered that NNMT is a direct GR transcriptional target gene in TNBC. I then observed relatively high NNMT expression in several aggressive patient-derived TNBC cell lines. NNMT consumes the universal methyl donor S-adenosyl methionine (SAM) for methylation of nicotinamide. High NNMT activity depletes SAM; as a result, methyltransferase targets are hypomethylated in cells with high NNMT expression. NNMT-induced DNA and histone hypomethylation have been shown to result in oncogenic gene expression in cancer cells but NNMT mechanism of action in TNBC biology remains unclear. A link between NNMT expression and mRNA hypomethylation has not previously been established as a mechanism contributing to cancer progression. N6- methyladenosine (m6A) is an abundant and reversible RNA modification in eukaryotes. Our collaborator Dr. Chuan He discovered that m6A-binding proteins mediate translational regulation by altering stability and translational efficiency of m6A-modifed mRNAs. Importantly, altered m6A mRNA methylation is implicated in the progression of several human cancers via causing changes in post-transcriptional gene expression of cancer pathways. To our knowledge, I am the first to characterize the m6A methylome of a patient-derived TNBC cell line model (MDA-MB-231): ~ 7000 m6A-modified transcripts are significantly enriched for pathways involved in cellular stress response, cell death and cell survival. In addition, I have data suggesting that NNMT activity in the MDA-MB-231 TNBC cell line results in 1) reduced m6A modification of mRNAs regulating key cancer pathways and 2) increased in vivo tumor-growth. In my dissertation research, I am testing the hypothesis that NNMT activity in TNBC cells results in 1) reduced m6A mRNA modification associated with altered protein expression of pathways mediating cellular stress response and 2) cancer stem cell-like traits associated with survival, metastatic potential and increased in vivo tumor-forming capacity. During my postdoctoral research, I aim to test whether epitranscriptomic gene expression regulates dynamic cellular phenotypes including adaptation to the changing microenvironment. I will first characterize the actively transcribed genes with polymerase ChIPseq and perform whole proteome quantification with mass spectrometry in cells exposed to distinct microenvironmental stressors (e.g. nutrient deprivation, hypoxia). I will then determine whether differential transcription of genes correlate with protein expression in different cellular states. If there is not a strong correlation, I will perform individual siRNA knockdown of all known m6A- regulatory genes and determine the effect on protein expression. I will then utilize patient-derived xenograft mouse models and the Sprague Dowley rat model of spontaneous breast cancer to determine whether the m6A-regulatory proteins are differentially expressed in distinct tumor regions with single-cell RNA sequencing.