Elucidating p53 transcriptional networks involved in pancreatic cancer suppressio

描述（由申请人提供）：尽管p53转录调节因子在肿瘤抑制中起关键作用，但其在肿瘤抑制中发挥作用的转录网络尚未完全了解。这些知识对于开发更好的诊断和治疗干预措施对于p53失活的癌症（包括胰腺癌）至关重要。在这里，我们建议使用小鼠胰腺癌模型来定义p53转录程序的胰腺癌进展的抑制至关重要，其目的是确定新的诊断标志物和治疗策略，这种疾病。我们建议利用一组独特的p53转录激活结构域（p53）突变敲入小鼠品系，我们已经产生了定义下游p53转录靶点，包括mRNA和非编码RNA，最重要的抑制胰腺癌的进展。一个特别强大的突变体是p5325，它不能诱导大多数已知的p53靶基因，但仍然激活一小部分新的p53诱导基因，并且仍然保留肿瘤抑制活性，使我们能够确定与肿瘤抑制最相关的p53靶点。利用KRasG 12 D表达的癌前胰腺导管上皮细胞（PDEC）中的基因表达谱和ChIP测序，我们将确定一组有限的直接p53和p5325，26激活的靶基因，这些基因与抑制癌前病变向胰腺导管腺癌（PDA）的进展相关。我们将在软琼脂试验和原位胰腺癌模型中使用RNA干扰方法鉴定具有肿瘤抑制活性的那些。这些实验提供了一种创新的方法来定义那些p53靶基因最关键的p53活性在抑制胰腺癌的进展，此外，将建议潜在的新的治疗靶点。此外，我们建议确定直接p53抑制的靶基因，可以作为胰腺癌进展过程中p53失活的生物标志物。我们对PDEC中存在p53时抑制的基因的分析，结合p53 ChIP测序数据，将鉴定出可以读出p53活性的直接p53抑制基因。特别是，我们将专注于识别p53抑制的miRNA，其水平可能在胰腺癌患者的血液中增加。在鉴定PDEC中p53抑制的miRNA后，我们将评估与KRasG 12 D; p53+/+小鼠相比，在表达KRasG 12 D;p53-/-小鼠的血液中是否可以检测到增加水平的同源miRNA。然后，我们将评估在KRasG 12 D;p53+/-小鼠中p53洛缺失（LosofHeterozygosity）后出现PDA期间，我们是否可以检测到这种p53抑制的miRNA水平的提高。识别疾病进展时增加的生物标志物将有助于早期发现PDA，因为它们可以通过手术根除。总之，利用小鼠模型的拟议研究提供了一个强大的策略，以了解p53的丢失如何有助于胰腺癌的进展，并开发新的临床方法来诊断和治疗这种致命的疾病。 公共卫生相关性：胰腺癌是美国癌症死亡的第四大原因，几乎总是致命的。因此，了解胰腺癌的发病机制，以便更有效地诊断和治疗胰腺癌是非常重要的。本研究旨在阐明一种称为p53的基因如何防止胰腺癌的发展，最终目的是为这种癌症设计更好的诊断和治疗策略。

DESCRIPTION (provided by applicant): Although the p53 transcriptional regulator plays a critical role in tumor suppression, the transcriptional networks through which it functions in tumo suppression are incompletely understood. This knowledge is critical for developing better diagnostics and therapeutic interventions for cancers in which p53 is inactivated, including pancreatic cancer. Here we propose to use mouse pancreatic cancer models to define p53 transcriptional programs critical for the suppression of pancreatic cancer progression, with the aim of identifying novel diagnostic markers and therapeutic strategies for this disease. We propose to take advantage of a set of unique p53 transcriptional activation domain (TAD) mutant knock-in mouse strains we have generated to define the downstream p53 transcriptional targets, including both mRNAs and non-coding RNAs, most essential for suppressing pancreatic cancer progression. A particularly powerful mutant is one known as p5325,26, which fails to induce the majority of known p53 target genes but still activates a small subset of novel p53-inducible genes and still retains tumor suppressor activity, allowing us to pinpoint the p53 targets most relevant for tumor suppression. Using gene expression profiling and ChIP-sequencing in KRasG12D-expressing, premalignant pancreatic ductal epithelial cells (PDECs), we will identify a limited set of direct p53 and p5325,26- activated target genes associated with suppressing the progression of premalignant lesions to Pancreatic Ductal Adenocarcinoma (PDA). We will identify those with tumor suppressor activity using RNA interference approaches in soft agar assays and orthotopic pancreatic cancer models. These experiments provide an innovative approach to defining those p53 target genes most critical for p53 activity in suppressing pancreatic cancer progression, and furthermore, will suggest potential novel therapeutic targets. In addition, we propose to identify direct p53-repressed target genes that could serve as biomarkers of p53 inactivation during pancreatic cancer progression. Our analysis of genes repressed in the presence of p53 in PDECs, combined with p53 ChIP-sequencing data, will identify direct p53-repressed genes that could be readouts of p53 activity. In particular, we will focus on identifying p53-repressed miRNAs whose levels may be increased in the blood of patients with pancreatic cancer. Upon identifying p53-repressed miRNAs in PDECs, we will assess whether increased levels of cognate miRNAs can be detected in the blood of KRasG12D-expressing;p53-/- mice compared to KRasG12D;p53+/+ mice. We will then assess whether we can detect enhanced levels of such p53- repressed miRNAs during the emergence of PDA occurring upon p53 LOH (Los of Heterozygosity) in KRasG12D;p53+/- mice. Identifying biomarkers increased upon progression will facilitate early detection of PDAs when they can be eradicated through surgery. Together, the proposed studies leveraging mouse models provide a powerful strategy for understanding how loss of p53 contributes to pancreatic cancer progression and for developing novel clinical approaches to diagnose and treat this deadly disease. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is the 4th leading cause of cancer deaths in the US and is nearly always fatal. It is therefore very important to understand the basis of thi disease in order to be able to more effectively diagnose and treat it. This research aims to elucidate how a gene known as p53 acts to prevent the development of pancreatic cancer, with the ultimate goal of devising better diagnostic and treatment strategies for this cancer.