15-Lipoxygenase 2 is a Prostate Senescence Gene

描述(申请人提供)：前列腺癌(PCa)是一种与年龄相关的疾病。癌基因的激活和抑癌基因的缺失都与前列腺癌的发生有关。我们最近的工作表明，代谢膜磷脂花生四烯酸(AA)的15-1环氧合酶2(15-LOX-2)是一种功能性的前列腺癌抑制因子。该分子在成年前列腺癌中大量表达，但在前列腺癌中缺失。15-LOX-2在PCa细胞中的重新表达在体外抑制其增殖，在体内抑制肿瘤的发展。15-LOX-2如何抑制前列腺癌的发生尚不完全清楚。我们实验室新出现的数据表明，15-LOX-2是一个前列腺衰老基因。在培养的正常人前列腺(NHP)上皮细胞中，15-LOX-2及其剪接变异体的mRNA和蛋白水平在复制细胞衰老之前积累。体内NHP细胞中15-LOX-2的表达也与年龄相关。功能获得实验表明，恢复15-LOX-2或其剪接变异体在PCa细胞中的表达可诱导衰老样表型。在转基因动物中靶向表达15-LOX-2或15-LOX-2Sv-b，这是一种缺乏AA代谢活性的剪接变体，会导致与氧化应激和衰老相关的上皮改变。使用小干扰RNA(SiRNA)瞬时敲除15-LOX-2表达的功能丧失实验促进NHP细胞的细胞周期进展和年轻特征。 机制研究表明，15-LOX-2诱导的NHP细胞衰老与其AA代谢酶活性无关，而与氧化应激增加引起的p53和pl6tpRb相关的细胞周期停滞有关。基于这些发现，我们推测15-LOX-2是一个具有肿瘤抑制功能的前列腺癌衰老基因，15-LOX-2表达的缺失可能使细胞逃避复制性衰老，从而促进前列腺癌的发展。我们进一步假设，15-LOX-2介导的NHP细胞衰老可能与氧化应激增加引起的细胞周期停滞有关。我们提出以下三个具体目标来验证我们的假设：1)研究15-LOX-2剪接变体在NItP细胞衰老中的作用：它们的表达与体内前列腺年龄的相关性；2)建立因果关系：利用转基因动物模型和稳定的RNAi敲除研究；3)剖析15-LOX-2及其剪接变体介导的NHP细胞衰老的分子机制。 这些目标将通过结合细胞生物学、生化和分子方法以及利用转基因动物来实现。我们的长期目标是利用从这个项目中获得的数据来开发15-LOX-2靶向的抗前列腺癌治疗药物，从而触发细胞周期停滞和衰老。

DESCRIPTION (provided by applicant): Prostate cancer (PCa) is an age-related disease. Both activation of oncogenes and loss of tumor suppressor genes have been implicated in PCa development. Our recent work suggests that 15-1ipoxygenase 2 (15-LOX-2), an enzyme that metabolizes membrane phospholipid arachidonic acid (AA), is a functional prostate tumor suppressor. The molecule is abundantly expressed in the adult prostate gland but lost in PCa. 15-LOX-2 re-expression in PCa cells inhibits their proliferation in vitro and tumor development in vivo. How 15-LOX-2 suppresses PCa development is incompletely understood. Newly emerged data from our lab suggests that 15-LOX-2 is a prostate senescence gene. In cultured normal human prostate (NHP) epithelial cells, the mRNA and protein levels of 15-LOX-2 and its splice variants accumulate, preceding the replicative cell senescence. 15-LOX-2 expression in NHP cells in vivo is also correlated with age. Gain-of-function experiments reveal that restoration of 15-LOX-2 or its splice variant expression in PCa cells induces a senescence-like phenotype. Targeted expression of 15-LOX-2 or 15-LOX-2sv-b, a splice variant that lacks AA-metabolizing activity, in transgenic animals causes epithelial alterations associated with oxidative stress and senescence. Loss-of-function experiments using small interfering RNA (siRNA) to transiently knock down 15-LOX-2 expression promotes cell-cycle progression and youthful characteristics in NHP cells. Mechanistic studies suggest that the 15-LOX-2-induced NHP cell senescence is not related to its AA-metabolizing enzymatic activity, but rather to p53- and pl6tpRb-related cell-cycle arrest caused by increased oxidative stress. Based on these findings, we hypothesize that 15-LOX-2 is a prostate senescence gene with tumor suppressive functions and loss of 15-LOX-2 expression may allow cells to escape replicative senescence and thus contributes to PCa development. We further hypothesize that 15-LOX-2-mediated NHP cell senescence may involve cell-cycle arrest caused by increased oxidative stress. We propose the following three Specific Aims to test our hypotheses: 1) To study the role of 15-LOX-2 splice variants in NItP cell senescence: Correlation of their expression with prostate age in vivo; 2) To establish the cause and effect relationship: Studies using transgenic animal models and stable RNAi knockdown; and 3) To dissect the molecular mechanisms of NHP cell senescence mediated by 15-LOX-2 and its splice variants. These aims will be accomplished by a combination of cell biological, biochemical, and molecular methods, as well as utilizing transgenic animals. Our long-term goal is to utilize the data obtained from this project to develop 15-LOX-2-targeted anti-PCa therapeutics that trigger cell-cycle arrest and senescence.