Biological and cancer-associated role of epitranscriptomic gene expression regulation

项目概述我的实验室发现NNMT是TNBC中一个直接的GR转录靶基因。然后，我观察到在几个侵袭性患者来源的TNBC细胞系中，NNMT的表达相对较高。NNMT消耗通用的甲基供体S-腺苷蛋氨酸(SAM)进行烟酰胺的甲基化。高NNMT活性会耗尽SAM；因此，在NNMT高表达的细胞中，甲基转移酶靶标的甲基化程度较低。NNMT诱导的DNA和组蛋白低甲基化导致癌基因在癌细胞中的表达，但NNMT在TNBC生物学中的作用机制尚不清楚。NNMT的表达和mRNA低甲基化之间的联系以前还没有被确定为促进癌症进展的机制。N-6-甲基腺苷(M6A)是真核生物中含量丰富且可逆的RNA修饰产物。我们的合作者川和博士发现，m6A结合蛋白通过改变m6A修饰的mRNAs的稳定性和翻译效率来介导翻译调控。重要的是，m6A基因甲基化改变通过引起癌症途径转录后基因表达的变化而与几种人类癌症的进展有关。据我们所知，我是第一个描述患者来源的TNBC细胞系模型(MDA-MB-231)的m6A甲基组的人：~7000m6A修饰的转录本显著丰富了参与细胞应激反应、细胞死亡和细胞生存的途径。此外，我有数据表明，在MDA-MB-231 TNBC细胞系中，NNMT的活性导致1)调节关键癌症途径的mRNAs的m6A修饰减少，2)体内肿瘤生长增加。在我的论文研究中，我验证了这样一个假设，即TNBC细胞中NNMT的活性导致1)m6A mRNA修饰减少，与介导细胞应激反应的通路的蛋白表达改变有关；2)癌症干细胞样特征与生存、转移潜能和体内成瘤能力增加有关。在我的博士后研究期间，我的目标是测试外转录基因表达是否调节动态的细胞表型，包括对不断变化的微环境的适应。我将首先用聚合酶ChIPseq鉴定活跃转录的基因，并用质谱仪对暴露于不同微环境应激源(如营养剥夺、低氧)的细胞进行整体蛋白质组定量。然后，我将确定基因的差异转录是否与不同细胞状态下的蛋白质表达相关。如果没有很强的相关性，我将对所有已知的m6A-调控基因进行单独的siRNA敲除，并确定对蛋白质表达的影响。然后，我将利用患者来源的异种移植小鼠模型和自发性乳腺癌的SpragueDowley大鼠模型，通过单细胞RNA测序来确定m6A调节蛋白是否在不同的肿瘤区域有差异表达。

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. N 6 - 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 m 6A-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 m 6A-regulatory proteins are differentially expressed in distinct tumor regions with single-cell RNA sequencing.