Integrative approaches to elucidate p53 transcriptional networks during carcinogenesis

描述(申请人提供)：P53转录因子在肿瘤发生中起关键作用。肿瘤中的P53失活，通常通过错义突变发生，明确地促进癌症，但突变的P53蛋白也表现出促进癌症的功能获得(GOF)特性。在这个提案中，我们努力解构野生型p53抑制癌症和错义突变体p53发挥GOF效应促进癌症的转录程序。我们建议使用综合的遗传、基因组、细胞生物学和生化方法来确定P53转录程序，该程序对于P53介导的抑制胰腺癌至关重要。胰腺癌是一种致命的癌症，5年生存率仅为6%，通常与P53突变有关。我们拟议的工作建立在对一组独特的P53转录激活结构域(TAD)突变敲入小鼠的初步研究基础上，我们建立了这些小鼠来定义下游的P53转录靶标，这些靶标对于抑制癌症的发展最为关键。一个特别强大的突变体是TAD1突变体，被称为p5325，26，它在激活大多数已知的p53靶基因时受到严重损害，但仍然有效地反式激活成纤维细胞中一小部分新的p53依赖基因，并在抑制各种癌症方面仍然保持全部活性。此外，TD2突变体p5353，54可以过度激活P53靶基因的一个子集，并发挥超级肿瘤抑制因子的作用。由突变体p5325、26和p5353、54激活的P53“肿瘤抑制相关靶基因”(TSAGs)主要代表新的P53靶点，我们推测这些靶点对肿瘤抑制至关重要，因此有很大的潜力扩大我们目前对P53肿瘤抑制机制的了解。在这里，我们建议对表达这些突变的癌前胰腺导管上皮细胞进行CHIP-SEQ和RNA-SEQ，以确定与胰腺癌抑制相关的TSAG。然后，我们建议通过在体内小鼠胰腺癌模型中进行CRISPR筛查来确定p53 TSAG缺失促进癌症的组合，以确定P53在肿瘤抑制中发挥作用的关键介质。我们将使用过表达和基因敲除的方法，确定P53 TSAGs编码的蛋白在P53调控的不同细胞过程中的细胞功能，包括细胞周期进展、细胞凋亡、代谢和侵袭/转移。对P53 TSAG功能的详细分析将有助于阐明对肿瘤抑制至关重要的细胞功能。接下来，我们将使用质谱学和siRNA筛选来确定TADS作用于诱导P53 TSAG和抑制胰腺癌的辅助因素，从而提供对肿瘤抑制的另一个层次的理解。最后，我们将利用我们在研究P53 TADS方面的专业知识来确定TADS对P53 GOF活动的重要性。我们将使用系统的方法在遗传、基因组和生化水平上分析TADS，以了解肿瘤来源的P53 GOF突变体是如何发挥其作用的。总而言之，这些研究将为如何在癌症治疗策略中调节P53通路提供重要的见解。

DESCRIPTION (provided by applicant): The p53 transcription factor plays a pivotal role in tumorigenesis. p53 inactivation in tumors, which occurs typically through missense mutation, unequivocally promotes cancer, but the mutated p53 protein also displays gain-of-function (GOF) properties that promote cancer. In this proposal, we strive to deconstruct the transcriptional programs through which wild-type p53 suppresses cancer and through which missense mutant p53 exerts GOF effects to promote cancer. We propose to use integrated genetic, genomic, cell biological and biochemical approaches to define the p53 transcriptional programs critical for p53-mediated suppression of pancreatic cancer, a deadly cancer with a mere 6% 5-year survival rate and which is typically associated with p53 mutation. Our proposed work builds on initial studies of a unique panel of p53 transcriptional activation domain (TAD) mutant knock-in mice that we generated to define the downstream p53 transcriptional targets most essential for suppressing cancer development. A particularly powerful mutant is the TAD1 mutant, known as p5325,26, which is severely compromised for activation of most known p53 target genes yet still efficiently transactivates a small set of novel p53-dependent genes in fibroblasts and still retains full activity in suppressing a variety of cancers. In addition, the TD2 mutant, p5353,54 can hyperactivate a subset of p53 target genes and behaves as a super-tumor suppressor. The p53 "tumor suppression associated target genes" (TSAGs) activated by the p5325,26 and p5353,54 mutants largely represent novel p53 targets, which we hypothesize are critical for tumor suppression and therefore have great potential to expand our current knowledge of p53 tumor suppression mechanisms. Here, we propose to perform ChIP-seq and RNA-seq in premalignant pancreatic ductal epithelial cells expressing these mutants to identify TSAGs associated with pancreatic cancer suppression. We then propose to identify key mediators of p53 function in tumor suppression by performing CRISPR screening in mouse pancreas cancer models in vivo to identify combinations of p53 TSAGs whose loss promotes cancer. We will define the cellular functions of the proteins encoded by p53 TSAGs in different cellular processes regulated by p53, including cell-cycle progression, apoptosis, metabolism, and invasion/metastasis, using overexpression and knockdown approaches. Detailed analyses of p53 TSAG function will help elucidate cellular functions most critical for tumor suppression. We will next use mass spectrometry and siRNA screening to identify the co-factors through which the TADs act to induce p53 TSAGs and to suppress pancreatic cancer, providing another level of understanding of tumor suppression. Finally, we will leverage our expertise in studying p53 TADs to define the importance of TADs for p53 GOF activity. We will use a systematic approach to analyze the TADs at the genetic, genomic and biochemical levels to understand how tumor-derived p53 GOF mutants exert their effects. Collectively, these studies will provide crucial insight into how to modulate p53 pathways during therapeutic strategies for cancer.