Elucidating the molecular determinants of p53-mediated pleiotropic effects

项目概述：p53是一种转录因子，通过诱导基因表达以多效性方式发挥作用 在某些情况下协调细胞毒性反应，但在其他情况下仅协调细胞抑制反应的程序。 然而，目前还不清楚细胞环境如何影响p53的多效性行为。 了解细胞环境如何影响p53介导的抗肿瘤反应将突出关键控制 可以靶向促进细胞毒性程序的机制， 癌症治疗比不太持久的细胞抑制方案。我们的实验室已经开发出基因工具， 小鼠和肿瘤衍生细胞系中不同肺癌亚型中的p53。这些系统允许 我们确定遗传上不同的肺癌亚型，这些亚型具有影响p53的细胞环境变异性 功能使用我们独特的工具集，我们确定在来自Kras的肿瘤衍生细胞系中重新激活p53， 驱动型肺腺癌（LUAC）诱导衰老，而在小细胞肺癌中重新激活p53 （SCLC）诱导非凋亡形式的细胞死亡。我们进行了差异基因表达分析， 鉴定了自噬特征在LUAC细胞中p53再活化后变得富集。在SCLC细胞中，我们 发现p53再活化丰富了调节性坏死基因表达程序。这些发现表明 不同的肺癌亚型具有影响基因表达程序和抗肿瘤活性的环境变异性。 p53再激活诱导的肿瘤反应。我的中心假设是自噬和程序化 坏死分别是p53诱导的衰老和非凋亡细胞死亡反应的关键 在LUAC和SCLC中重新激活。在目标1中，我将研究诱导的非凋亡形式的细胞死亡， p53在小细胞肺癌中的再激活。我将证实在p53重新激活后，SCLC细胞中发生程序性坏死 使用细胞和分子生物学测定，并通过使用遗传和药理学方法来抑制 调节坏死以确定细胞存活是否被拯救。在目标2中，我将阐述自噬的重要性， p53介导的LUAC衰老。我将通过测量p53重新激活后的自噬通量来实现这一点， 并通过抑制自噬来确定其影响细胞存活，衰老诱导， 和细胞周期进程。这项提议的目的是揭示p53多效性行为的关键决定因素 以及最终在癌症治疗过程中增强细胞毒性结果的治疗靶向分子。

Project Summary: p53 is transcription factor that acts in a pleiotropic manner by inducing gene expression programs that orchestrate cytotoxic responses in certain contexts, but only cytostatic responses in others. However, it is not well-understood how cellular context influences the pleiotropic behavior of p53. Understanding how cellular context influences p53-mediated anti-tumor responses will highlight critical control mechanisms that could be targeted to promote cytotoxic programs that would be more efficacious during cancer therapy than less durable cytostatic programs. Our laboratory has developed genetic tools to reversibly inactivate p53 in distinct lung cancer sub-types in the mouse and tumor-derived cell lines. These systems allow us to identify genetically distinct lung cancer sub-types that harbor cellular context variability to influence p53 function. Using our unique toolset, we determined that reactivating p53 in tumor-derived cell lines from Kras- driven lung adenocarcinoma (LUAC) induces senescence, whereas reactivating p53 in small cell lung cancer (SCLC) induces a non-apoptotic form of cell death. We conducted differential gene expression analysis and identified that autophagy signatures become enriched upon p53 reactivation in LUAC cells. In SCLC cells, we found that p53 reactivation enriches a Regulated Necrosis gene expression program. These findings show that distinct lung cancer sub-types harbor context variability that influences the gene expression programs and anti- tumor responses induced by p53 reactivation. My central hypothesis is that autophagy and programmed necrosis are respectively critical for the senescence and non-apoptotic cell death responses induced by p53 reactivation in LUAC and SCLC. In Aim 1, I will investigate the non-apoptotic form of cell death that is induced by p53 reactivation in SCLC. I will verify that programmed necrosis occurs in SCLC cells after p53 reactivation using cell and molecular biological assays, and by using genetic and pharmacological approaches to inhibit regulated necrosis to determine if cell survival is rescued. In Aim 2, I will establish the importance of autophagy in p53-mediated senescence in LUAC. I will achieve this by measuring autophagic flux after p53 reactivation, and by inhibiting autophagy to determine the extent to which it influences cell survival, senescence induction, and cell cycle progression. The goal of this proposal is expose key determinants of p53 pleiotropic behavior and ultimately therapeutically targetable molecules that enforce cytotoxic outcomes during cancer treatment.