Epigenetic and epitranscriptomic determinants of treatment resistance in cancer

生物学教科书上的核心原理是DNA被转录成mRNA， mRNA被翻译成蛋白质。这种简化忽略了使选择性外显子转录和翻译成为可能的复杂过程，这是正常细胞功能所必需的，而现在已知的对外显子转录和翻译的放松会导致癌症和其他疾病。因此，本项目将研究正常和恶性细胞中选择性转录和翻译调控的基本机制。我们最近的工作已经确定了涉及替代外显子利用的新机制。我们发现，涉及组蛋白赖氨酸甲基化的表观遗传机制和涉及RNA甲基化的表观转录组学机制似乎趋同于(i)选择DNA的哪些区域被积极转录和（ii） mrna内的哪些外显子被翻译。最重要的是，我们发现与非恶性细胞相比，这些机制在癌细胞中被破坏，使这些癌细胞能够选择性地改变异构体表达，以逃避目前的癌症治疗。这导致了一种假设，即异常RNA甲基化有助于在癌症中发现的促癌选择性剪接转录组。由于转移性癌症是无法治愈的，迫切需要新的治疗方法。因此，我们的研究将建立基础科学，即靶向rna甲基化的新药是否可以逆转对现有癌症药物的耐药性，从而原则上延长转移性疾病患者的生命。本项目的具体目的是破译YTHDC1 m6A读取器在确定外显子利用方面的功能。为此，我们将在m6A修饰子（METTL3, METTL14, CBLL1， FTO和ALKBH5）和调节选择性剪接的组蛋白赖氨酸去甲基化酶（KDM1A, KDM5B, KDM7A）的现有数据背景下比较YTHDC1的功能。要做到这一点，DTP学生将1。评估YTHDC1 m6A读取器在我们的人类前列腺癌和乳腺癌标本中的表达（所有其他相关标记的表达已经确定）。2. 利用CRISPR-Cas9和/或siRNA选择性靶向乳腺癌和前列腺癌细胞系中的YTHDC1，确定qRTPCR3对雄激素和雌激素调控基因表达的影响。使用RNAseq和western blotting来确定YTHDC1、m6A和组蛋白赖氨酸去甲基化酶在AR蛋白的选择性剪接和外显子利用中的相对作用4。将比较YTHDC1和m6A调节复合物的其他成分的功能缺失与RNA甲基转移酶的新型药物抑制剂对癌细胞转录组、增殖和体外侵袭的影响。总的来说，这些目标将促进对YTHDC1在基因调控中的作用的基本理解，并将指导m6a靶向癌症治疗的未来发展。DTP学员将接受以下技术方面的培训。细胞培养，CRISPR-cas9，体外药理学，报告基因分析基础分子生物学，克隆，qRTPCR, western blotting生物信息学：我们的团队已经优化了管道和现有的数据集，可供比较。临床基因组学和数据解释：学生将学习如何完成免疫组织化学和临床相关性

Biology textbooks teach that the central dogma is that DNA is transcribed to mRNA and mRNA is translated to protein. This simplification overlooks the complex processes that enable selective exon transcription and translation which are essential for normal cellular function and the deregulation of which are now known to contribute to cancer and other diseases. Therefore this project will investigate the fundamental mechanisms that enables the regulation of selective transcription and translation in normal and malignant cells. Our recent work has identified novel mechanisms involved in alternative exon utilisation. We have found that epigenetic mechanisms involving histone lysine methylation and epitranscriptomic mechanisms involving RNA methylation appear to converge to (i) select which regions of DNA are actively transcribed and (ii) which exons within mRNAs are translated. Most importantly we have found that these mechanisms are disrupted in cancer cells as compared to non-malignant cells, enabling these cancer cells to selectively change isoform expression to evade current cancer treatments. This leads to the hypothesis that aberrant RNA methylation contributes to the pro-oncogenic alternatively spliced transcriptome found in cancer. As metastatic cancer is incurable, new treatments are urgently required. Our research will therefore establish the fundamental science as to whether new drugs which target RNA-methylation can reverse resistance to existing cancer drugs and thereby in principle extend the life of patients with metastatic disease. The specific aim of this project is to decipher the function of the YTHDC1 m6A reader in determining exon utilization. To do this we will compare YTHDC1 function in the context of our existing data on m6A modifiers (METTL3, METTL14, CBLL1, FTO and ALKBH5) and histone lysine demethylases (KDM1A, KDM5B, KDM7A) which regulate alternative splicing. To do this the DTP student will 1. assess expression of the YTHDC1 m6A reader in our human prostate and breast cancer specimens (expression of all other relevant markers have already been determined). 2. use CRISPR-Cas9 and/ or siRNA to selectively target YTHDC1 in breast and prostate cancer cell lines and determine the effect on androgen and estrogen regulated gene expression by qRTPCR3. use RNAseq and western blotting to determine the relative role of YTHDC1 and the m6A and histone lysine demethylases in alternative splicing and exon utilization of the AR protein4. will compare the effects of functional depletion of YTHDC1, and other components of the m6A regulating complex, with new pharmaco-inhibitors of RNA methyltransferases on the transcriptome, proliferation and in vitro invasion of cancer cells. Collectively these aims will advance fundamental understanding of the role of YTHDC1 in gene regulation and will guide the future development of m6A-targeted therapies in cancer. The DTP student will receive training in the following techniques1. Cell culture, CRISPR-cas9, in vitro pharmacology, reporter assays2. Basic molecular biology, cloning, qRTPCR, western blotting3. Bioinformatics: our group has optimized pipelines and existing datasets already available for comparison 4. Clinical genomics and data interpretation: the student will learn how to complete immunohistochemistry and clinical correlations