Elucidating p53 transcriptional networks involved in pancreatic cancer suppressio

描述(申请人提供)：尽管P53转录调节因子在肿瘤抑制中发挥关键作用，但其在肿瘤抑制中发挥作用的转录网络尚不完全清楚。这一知识对于开发对P53失活的癌症(包括胰腺癌)的更好诊断和治疗干预措施至关重要。在这里，我们建议使用小鼠胰腺癌模型来定义对抑制胰腺癌进展至关重要的P53转录程序，目的是识别这种疾病的新诊断标记和治疗策略。我们建议利用我们产生的一组独特的P53转录激活域(TAD)突变敲入小鼠品系来定义下游的P53转录靶点，包括mRNAs和非编码RNAs，这对抑制胰腺癌的进展是最重要的。一个特别强大的突变体是p5325，26，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，并且仍然保持肿瘤抑制活性，使我们能够精确定位与肿瘤抑制最相关的P53靶基因。利用KrasG12D表达的癌前胰腺导管上皮细胞(PDECs)的基因表达谱和芯片测序，我们将确定一组有限的直接激活的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。我们将在软琼脂实验和原位胰腺癌模型中使用RNA干扰方法识别那些具有肿瘤抑制活性的基因。这些实验提供了一种创新的方法来确定那些对抑制胰腺癌进展中的P53活性最关键的P53靶基因，并将进一步提出潜在的新的治疗靶点。此外，我们建议识别直接抑制p53的靶基因，这些基因可以在胰腺癌进展过程中作为p53失活的生物标志物。我们对PDECs中存在P53时被抑制的基因的分析，结合P53芯片测序数据，将识别直接被P53抑制的基因，这些基因可能是P53活性的读数。特别是，我们将专注于识别胰腺癌患者血液中可能升高的P53抑制的miRNAs。一旦在PDECs中发现了p53抑制的miRNAs，我们将评估与KrasG12D；p53+/+小鼠相比，是否可以在KrasG12D表达的p53-/-小鼠的血液中检测到同源miRNAs水平的增加。然后，我们将评估是否可以在KrasG12D；p53+/-小鼠的P53杂合性缺失(Los Of Heterozygity)出现PDA期间检测到这种P53抑制的miRNAs水平升高。当PDA可以通过手术根除时，识别进展增加的生物标记物将有助于及早发现PDA。总之，拟议的利用小鼠模型的研究为了解p53缺失如何导致胰腺癌进展以及开发诊断和治疗这种致命疾病的新临床方法提供了一个强大的策略。 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，了解这种疾病的基础是非常重要的，以便能够更有效地诊断和治疗它。这项研究旨在阐明一种名为p53的基因如何作用于预防胰腺癌的发展，最终目标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.