Biological and cancer-associated role of epitranscriptomic gene expression regulation

项目摘要 本实验室发现NNMT是TNBC中GR直接转录的靶基因。然后我观察到， NNMT在几种侵袭性患者来源的TNBC细胞系中的高表达。NNMT消耗通用 甲基供体S-腺苷甲硫氨酸（SAM）用于烟酰胺的甲基化。高NNMT活性耗尽SAM; 结果，甲基转移酶靶在NNMT高表达的细胞中被低甲基化。NNMT诱导 DNA和组蛋白低甲基化已显示导致癌细胞中致癌基因表达， NNMT在TNBC生物学中的作用机制仍不清楚。NNMT表达与mRNA之间的联系 低甲基化以前没有被确定为促进癌症进展的机制。N6- 甲基腺苷（m6 A）是真核生物中大量存在的可逆RNA修饰。我们的合作者博士。 Chuan He发现m6 A结合蛋白通过改变稳定性介导翻译调节， m6 A修饰的mRNA的翻译效率。重要的是，m6 A mRNA甲基化的改变与肿瘤的发生有关。 通过引起癌症转录后基因表达的变化导致几种人类癌症的进展 途径。据我们所知，我是第一个表征患者来源的TNBC细胞的m6 A甲基化组的人。 线模型（MDA-MB-231）：约7000 m6 A修饰的转录物显著富集参与的途径， 细胞应激反应、细胞死亡和细胞存活。此外，我有数据表明，NNMT活性在 MDA-MB-231 TNBC细胞系导致1）调节关键癌症的mRNA的m6 A修饰减少 途径和2）增加体内肿瘤生长。在我的论文研究中，我正在验证一个假设， TNBC细胞中的NNMT活性导致1）与改变的蛋白质相关的m6 A mRNA修饰减少 介导细胞应激反应的途径的表达和2）与之相关的癌症干细胞样特征 存活率、转移潜能和增加的体内肿瘤形成能力。 在我的博士后研究期间，我的目标是测试表观转录组基因表达是否调节 动态细胞表型，包括对变化的微环境的适应。首先，我将描述 使用聚合酶ChIPseq主动转录基因，并使用质谱进行全蛋白质组定量 在暴露于不同微环境应激源（例如营养剥夺、缺氧）的细胞中，光谱分析是一个重要的研究领域。我会 然后确定基因的差异转录是否与不同细胞中的蛋白质表达相关， states.如果没有很强的相关性，我将对所有已知的m6 A进行单独的siRNA敲除。 调节基因并决定对蛋白质表达的影响。然后我会用病人的异种移植物 小鼠模型和Sprague Dowley大鼠模型的自发性乳腺癌，以确定是否 m6 A调节蛋白在不同的肿瘤区域用单细胞RNA测序差异表达。

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. 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.