Elucidating the molecular determinants of p53-mediated pleiotropic effects

项目摘要：P53是一种转录因子，通过诱导基因表达而发挥多效性作用 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，细胞环境如何影响P53的多效性还不是很清楚。 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 可以有针对性地促进细胞毒计划的机制，这些计划在 癌症治疗比不上持久的细胞抑制计划。我们的实验室已经开发出基因工具来可逆地 灭活小鼠和肿瘤细胞系中不同肺癌亚型中的P53。这些系统允许 美国将识别具有细胞背景可变性影响p53基因的不同肺癌亚型 功能。使用我们独特的工具箱，我们确定重新激活来自Kras的肿瘤来源细胞系中的P53- 驱动肺腺癌(LUAC)在小细胞肺癌中诱导衰老，而重新激活P53 (SCLC)诱导一种非凋亡性的细胞死亡。我们进行了差异基因表达分析和 发现在LUAC细胞中，当P53重新激活时，自噬特征变得丰富。在小细胞肺癌细胞中，我们 研究发现，P53的重新激活丰富了一个受调控的坏死基因表达程序。这些发现表明， 不同的肺癌亚型包含影响基因表达程序和抗肺癌基因表达程序的上下文变异性 P53重新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性细胞死亡反应的关键 在LUAC和SCLC中重新激活。在目标1中，我将研究诱导细胞死亡的非凋亡性形式 P53在小细胞肺癌中的重新激活。我将验证小细胞肺癌细胞在P53重新激活后发生程序性坏死 使用细胞和分子生物学分析，并通过遗传和药理学方法抑制 调节坏死，以确定细胞是否存活。在目标2中，我将确立自噬的重要性。 在p53介导的LUAC衰老中起重要作用。我将通过测量p53重新激活后的自噬通量来实现这一点， 通过抑制自噬来确定它影响细胞存活、衰老诱导的程度， 和细胞周期进程。这项提案的目标是揭示p53多效性的关键决定因素 以及最终在治疗方面具有靶向性的分子，这些分子在癌症治疗过程中实施细胞毒性结果。

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.