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着关键作用，但其在肿瘤抑制中发挥作用的转录网络尚不完全清楚。这一知识对于开发更好的诊断和治疗癌症至关重要，其中p53是失活的，包括胰腺癌。在这里，我们建议使用小鼠胰腺癌模型来确定p53转录程序对抑制胰腺癌进展至关重要，目的是确定新的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）突变敲入小鼠菌株来定义下游p53转录靶点，包括mrna和非编码rna，这对抑制胰腺癌进展至关重要。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，并且仍然保留肿瘤抑制活性，这使我们能够确定与肿瘤抑制最相关的p53靶点。利用krasg12d表达的癌前胰腺导管上皮细胞（PDECs）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，这些靶基因与抑制癌前病变向胰腺导管腺癌（PDA）的进展有关。我们将在软琼脂测定和原位胰腺癌模型中使用RNA干扰方法识别具有肿瘤抑制活性的细胞。这些实验提供了一种创新的方法来定义p53靶基因，这些基因在抑制胰腺癌进展中对p53活性最关键，并且将提出潜在的新治疗靶点。此外，我们建议确定直接p53抑制的靶基因，这些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PDECs中p53存在时被抑制的基因进行分析，并结合p53 chip测序数据，将确定p53被直接抑制的基因，这些基因可能是p53活性的读数。特别是，我们将专注于鉴定胰腺癌患者血液中p53抑制的mirna，其水平可能会升高。在PDECs中鉴定出p53抑制的mirna后，我们将评估是否可以在表达krasg12d的血液中检测到同源mirna水平升高；p53-/-小鼠与KRasG12D；p53 + / +老鼠。然后，我们将评估我们是否可以在KRasG12D中p53 LOH（杂合性缺失）发生PDA时检测到这种p53抑制miRNAs水平的增强；p53 + / -老鼠。识别随着进展而增加的生物标志物将有助于pda的早期发现，并且可以通过手术根除。总之，利用小鼠模型的拟议研究为理解p53缺失如何促进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强有力的策略。

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.