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p53的失活，通常通过错义突变发生，明确地促进癌症，但突变的p53蛋白也显示出促进癌症的功能获得（GOF）特性。在本提案中，我们努力解构野生型p53抑制癌症和错义突变型p53发挥GOF效应促进癌症的转录程序。我们建议使用综合遗传学、基因组学、细胞生物学和生化方法来定义p53转录程序，这些转录程序对p53介导的胰腺癌的抑制至关重要。胰腺癌是一种致命的癌症，5年生存率仅为6%，通常与p53突变相关。我们提出的工作建立在对一组独特的p53转录激活域（TAD）突变敲入小鼠的初步研究之上，我们生成了这些小鼠来定义抑制癌症发展最重要的下游p53转录靶点。一个特别强大的突变体是TAD1突变体，称为p5325,26，它对大多数已知p53靶基因的激活严重受损，但仍然有效地激活成纤维细胞中一小部分新的p53依赖基因，并且仍然保持抑制多种癌症的充分活性。此外，TD2突变体p5353,54可以超激活p53靶基因的一个亚群，并表现为超级肿瘤抑制因子。p5325,26和p53533,54突变体激活的p53“肿瘤抑制相关靶基因”（tumor suppression associated target genes, TSAGs）在很大程度上代表了新的p53靶点，我们假设这些靶点对肿瘤抑制至关重要，因此有很大的潜力扩展我们目前对p53肿瘤抑制机制的认识。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，以鉴定与胰腺癌抑制相关的TSAGs。然后，我们提出通过在小鼠胰腺癌模型中进行CRISPR筛选，鉴定p53抑制肿瘤功能的关键介质，以鉴定p53 tags的缺失促进癌症的组合。我们将通过过表达和敲低的方法，确定p53 tags编码的蛋白在p53调控的不同细胞过程中的细胞功能，包括细胞周期进程、细胞凋亡、代谢和侵袭/转移。详细分析p53 TSAG功能将有助于阐明肿瘤抑制中最关键的细胞功能。接下来，我们将使用质谱法和siRNA筛选来确定TADs诱导p53 TSAGs并抑制胰腺癌的辅助因子，从而为肿瘤抑制提供另一个层面的理解。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活性的重要性。我们将采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突变体如何发挥其作用。总的来说，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.