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，p53在正常生理条件下对糖酵解、活性氧（ROS）产生和氧化应激反应的调节至关重要。许多研究表明，p53的缺失导致线粒体呼吸降低和糖酵解增强，从而导致瓦尔堡效应。p53还诱导几种抗氧化剂基因，包括TIGAR和GLS 2，以降低活性氧（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，通过核苷酸碱基的氧化引起DNA损伤。因此，p53的抗氧化活性通过降低ROS水平参与限制DNA损伤。然而，目前还不清楚p53的这部分功能是否直接有助于p53作为肿瘤抑制因子的作用。我们最近已经证明，乙酰化的p53是需要其激活细胞周期阻滞和凋亡。在我们的初步研究中，我们已经产生了在一个（p53 K117 R）或三个（p533 KR; K117 R + K161 R + K162 R）的关键p53乙酰化位点的赖氨酸精氨酸突变的小鼠。虽然p53 K117 R/K117 R细胞能够进行p53介导的细胞周期停滞和衰老，但不能进行凋亡，但所有这三个过程在p533 KR/3 KR细胞中都被消除。令人惊讶的是，不像p53-null小鼠，迅速死于自发性胸腺淋巴瘤，早发性肿瘤形成不发生在p53 K117 R/K117 R或p533 KR/3 KR动物。由于p533 KR/3 KR小鼠缺乏 p53介导的细胞周期停滞、凋亡和衰老，这一观察结果表明p53功能的其他方面足以抑制早发性肿瘤发生。值得注意的是，p533 KR保留了反式激活代谢靶基因（如TIGAR和GLS 2）的能力，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、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的作用是否作为肿瘤抑制的独立机制。拟议的研究包括以下两个具体目标。在目的1中，我们将剖析p53在调节活性氧（ROS）的产生，氧化应激反应和糖酵解，在细胞周期停滞，凋亡和衰老的情况下的作用。在目标2中，我们将研究p53介导的对活性氧（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至关重要。

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.