Role of p53-mediated unconventional functions in tumor suppression

描述(由申请人提供)：越来越多的证据表明，在正常生理条件下，P53在糖酵解、活性氧自由基(ROS)的产生和氧化应激反应的调节中起关键作用。许多研究表明，p53的丢失会导致线粒体呼吸减少和糖酵解增强，从而导致Warburg效应。P53还诱导几个抗氧化基因，包括TIGAR和GLS2，以降低活性氧物种(ROS)的水平。值得注意的是，ROS的产生通过增加8-羟基-22-脱氧鸟苷(8-OH-DG)水平来氧化核苷酸碱基，从而导致DNA损伤。因此，P53的抗氧化活性参与了通过降低ROS水平来限制DNA损伤。然而，目前尚不清楚P53的这一部分功能是否直接参与了P53作为肿瘤抑制因子的作用。我们最近已经证明，P53的乙酰化是激活细胞周期停滞和细胞凋亡所必需的。在我们的初步研究中，我们已经产生了在关键的p53乙酰化位点中的一个(P53K117R)或三个(p533KR；K117R+K161R+K162R)具有赖氨酸到精氨酸突变的小鼠。P53K117R/K117R细胞具有P53介导的细胞周期停滞和衰老功能，但不能诱导细胞凋亡，而p533KR/3KR细胞的上述三个过程均被阻断。令人惊讶的是，与迅速死于自发性胸腺淋巴瘤的p53缺失小鼠不同，p53K117R/K117R或p533KR/3KR动物都不会出现早发性肿瘤形成。由于p533KR/3KR小鼠缺乏 P53介导的细胞周期停滞、细胞凋亡和衰老，这一观察表明，P53功能的其他方面足以抑制早发性肿瘤的发生。值得注意的是，p533KR保留了反式激活代谢靶基因的能力，如TIGAR和GLS2，并随后抑制糖酵解和活性氧(ROS)的产生。这里要检验的中心假设是，在没有细胞周期停滞、细胞凋亡和衰老的情况下，P53介导的糖酵解、ROS产生和氧化应激反应是否作为一种独立的机制在肿瘤抑制中发挥作用。拟议的研究包括以下两个具体目标。在目标1中，我们将剖析在没有细胞周期停滞、细胞凋亡和衰老的情况下，p53在调节活性氧簇(ROS)产生、氧化应激反应和糖酵解中的作用。在目标2中，我们将研究P53对活性氧(ROS)产生、DNA氧化损伤和糖酵解的影响是否在抑制癌基因介导的肿瘤发生中起关键作用。

DESCRIPTION (provided by applicant): There is accumulating evidence indicating that p53 is critical in regulation of glycolysis, reactive oxygen species (ROS) production and oxidative stress responses under normal physiological conditions. A number of studies showed that loss of p53 results in decreased mitochondrial respiration and enhanced glycolysis, leading to the Warburg effect. p53 also induces several antioxidant genes, including TIGAR and GLS2, to reduce the levels of reactive oxygen species (ROS). Notably, ROS production causes DNA damage through oxidation of nucleotide bases by increasing 8-hydroxy-22-deoxyguanosine (8-OH-dG) levels. Thus, antioxidant activities of p53 are involved in limiting DNA damage through reducing ROS levels. Nevertheless, it remains unclear whether this part of p53 functions directly contributes to the role of p53 as a tumor suppressor. We have recently demonstrated that acetylation of p53 is required for its activation of cell cycle arrest and apoptosis. In our preliminary studies, we have generated mice bearing lysine to arginine mutations at one (p53K117R) or three (p533KR; K117R+K161R+K162R) of the critical p53 acetylation sites. While p53K117R/K117R cells are competent for p53-mediated cell-cycle arrest and senescence, but not apoptosis, all three of these processes are ablated in p533KR/3KR cells. Surprisingly, unlike p53-null mice, which rapidly succumb to spontaneous thymic lymphomas, early-onset tumor formation does not occur in either p53K117R/K117R or p533KR/3KR animals. Since p533KR/3KR mice lack p53-mediated cell cycle arrest, apoptosis, and senescence, this observation suggests that other aspects of p53 function are sufficient for suppression of early-onset tumorigenesis. Notably, p533KR retains the ability to transactivate metabolic target genes, such as TIGAR and GLS2, and subsequently suppresses glycolysis and reactive oxygen species (ROS) production. The central hypothesis to be tested here is whether p53-mediated effects in glycolysis, ROS production and oxidative stress responses act as an independent mechanism in tumor suppression in the absence of cell-cycle arrest, apoptosis and senescence. The proposed studies include the following two specific aims. In Aim 1, we will dissect the role of p53 in regulating reactive oxyge species (ROS) production, oxidative stress responses and glycolysis, in the absence of cell cycle arrest, apoptosis and senescence. In Aim 2, we will examine whether p53 mediated effects on reactive oxygen species (ROS) production, DNA oxidation damage and glycolysis are critical in suppressing oncogene-mediated tumorigenesis.