Pancreatic cancer stem cells: PD2-mediated novel mechanistic link and metabolomic alterations

项目摘要/摘要 这项研究建议旨在提供博士后和博士后培训，以发展必要的 作为一名癌症生物学独立调查员的职业技能。长期的研究重点是发展 通过1)确定肿瘤干细胞对胰腺癌(PC)有效治疗的作用 (2)了解CSC维持和CSC-2的机制。 介导的耐药性，3)确定CSC的遗传、表观遗传和代谢因素 维护，目的是确定新的表观遗传和代谢靶点，可用于 针对PC的综合治疗。我的论文研究(F99阶段)的目的是确定 胰腺CSCs中胰腺分化2(PD2)和CSC介导的PC进展的研究 破译依赖PD2的CSC维持机制。PD2是一种普遍存在的多功能蛋白质，是一种 人RNA聚合酶II相关因子1复合体(PAF1C)的核心成分，在 转录延伸和信使核糖核酸加工。我们发现PD2是一种新的胰腺CSC标志物， 介导CSCs的耐药。对PD2依赖的CSC分子机制的认识 维持和抗药性至关重要。我们最近有了几项与这一概念相关的发现。 首先，PD2基因敲除显著降低了已建立的CSC和自我更新标志物的水平。 重要的是，PD2耗尽显著降低了体内的肿瘤负担。此外，RNA测序和 转录因子聚合酶链式反应阵列分析显示，几个茎和转移基因显著 在PD2耗尽后下调。根据上述信息和其他数据，我们 假设PD2作为干细胞维持的主调节器发挥作用，从而介导 个人电脑的进步。我们的研究将利用高通量基因组技术，如染色质 存在和不存在PD2的免疫沉淀测序和RNA测序以确定下游 PD2的靶点，并确定依赖PD2的胰腺干细胞维持的途径。我们还将 用新的基于CRISPR的PD2基因敲除模型确定PD2在CSC介导的PC进展中的作用 与PC进度的KPC模型交叉。拓展未来研究方向，构筑 为了独立，我将继续进行癌症新陈代谢方面的博士后培训。建议的目标是 K00博士后培训的目的是获得以下方面的专业知识：1)当前的代谢策略；2)了解 促进肿瘤干细胞和分化肿瘤细胞获得不同代谢程序的机制；3) 代谢在CSC介导的耐药性中的作用；4)新的组合药物的开发 基于新陈代谢靶向的治疗癌症。最终，拟议的F99/K00培训将提供 R01资金的强大智力基础将建立我的独立性，并将提供专业的 培训成为一名有效的PI和导师所需的技能。

PROJECT SUMMARY / ABSTRACT This research proposal is intended to provide predoctoral and postdoctoral training to develop the necessary skills for a career as an independent investigator in cancer biology. The long-term research focus is development of effective therapy for pancreatic cancer (PC) through 1) determining the contribution of cancer stem cells (CSCs) to PC progression and metastasis, 2) understanding the mechanism of CSC maintenance and CSC- mediated drug resistance, 3) identifying genetic, epigenetic, and metabolic factors essential for CSC maintenance with the aim of identifying novel epigenetic and metabolic targets that can be exploited for combinatorial therapy against PC. The objective of my dissertation research (F99 phase) is to define the role of Pancreatic Differentiation 2 (PD2) in pancreatic CSCs and CSC-mediated PC progression, with the goal of deciphering the mechanism of PD2-dependent CSC maintenance. PD2 is a ubiquitous multifunctional protein, a core component of human RNA Polymerase II-Associated Factor 1 complex (PAF1C) that functions in transcription elongation and mRNA processing. We discovered that PD2 is a novel pancreatic CSC marker and mediates drug resistance of CSCs. Knowledge of the molecular mechanism of PD2-dependent CSC maintenance and drug resistance is critical. We have recently made several discoveries relevant to this concept. First, knockdown of PD2 significantly reduces the levels of established CSC and self-renewal markers. Importantly, PD2 depletion significantly reduces tumor burden in vivo. Moreover, RNA-sequencing and transcription factor PCR array analyses revealed that several stemness and metastasis genes were significantly downregulated following PD2 depletion. Based on aforementioned information and additional data, we hypothesize that PD2 functions as a master-regulator of stem cell maintenance and thereby mediates PC progression. Our research will utilize high throughput genomic techniques such as chromatin immunoprecipitation sequencing and RNA-sequencing in presence and absence of PD2 to define downstream targets of PD2, and identify the pathway for PD2-dependent maintenance of pancreatic CSCs. We will also determine the role of PD2 in CSC-mediated PC progression using a novel CRISPR- based PD2 knockout model crossed with KPC model of PC progression. To expand upon the future research direction and to build the foundation for independence, I will pursue postdoctoral training in cancer metabolism. The goal of the proposed K00 postdoctoral training is to gain expertise on :1) the current metabolomic strategies; 2) understand the mechanisms that promote acquisition of different metabolic programs by CSCs and differentiated tumor cells; 3) contribution of metabolism to CSC-mediated drug resistance; and 4) development of novel combinatorial therapeutics based on metabolic targeting to treat cancer. Ultimately, the proposed F99/K00 training will provide a strong intellectual foundation for R01 funding that will establish my independence and will provide professional training in the skills required to be an effective PI and mentor.