Disruption of the circadian clock by a cancer/testis antigen

描述(申请人提供)：在人类中，倒班工作时夜间暴露在光线下会扰乱正常的昼夜节律，导致癌症发病率增加；然而，支持昼夜节律中断的分子机制在肿瘤发生中的作用还不是很清楚。人体内几乎每个细胞都有一个内源性的分子时钟，它控制着大约24小时的综合生化过程。这个分子时钟的核心是昼夜节律的基本螺旋-环-螺旋PER：ARNT：SIM转录因子复合时钟：BMal1，它控制着近15%的基因组的有节奏的转录，包括新陈代谢、激素分泌和细胞周期中的必要基因。在健康的体细胞组织中，生物钟和细胞周期是联系在一起的，以至于时钟将细胞分裂控制在一次 日，从而防止了不受控制的扩散。细胞周期/昼夜节律的联系已被确认多年，然而，在癌症中使这两个节律系统分离的细胞扰动仍未阐明。这项建议的目的是了解一种在健康的体细胞组织中不表达但在癌症中上调的蛋白质如何抑制生物钟以及内源性时钟抑制对细胞增殖的影响。我们的初步数据显示，这种癌症特异性蛋白PASD1抑制时钟：BMal1的活性。基于初步研究，我们的中心假设是，当PASD1在癌症中表达时，会沉默时钟功能，消除时钟控制的动态平衡和细胞周期门控，引导细胞走上致癌途径。我们将研究PASD1在时钟调节中的作用，具体目的如下：1)确定PASD1抑制时钟的机制：BMal1活性。初步数据表明，PASD1中与染色质靶向结构域一起保守的区域是抑制CLOCK：BMal1活性所必需的。我们的工作假设是，PASD1使用分子模仿来干扰CLOCK：BMal1在一天中的正确时间访问目标基因的能力。为了验证这一假设，我们将进行芯片序列实验，在PASD1存在和不存在的情况下检查CLOCK：BMal1基因组靶向。2)确定PASD1的表达如何调节人癌细胞的昼夜周期和增殖。我们假设，在癌细胞系中敲除PASD1将改善CLOCK：BMAL1靶基因的昼夜循环，提供更强劲的节律和降低的增殖率。研究在癌症中上调的蛋白质直接作用于核心昼夜节律转录反馈环的机制，将建立昼夜节律中断与癌症之间的分子联系，并以识别癌症治疗为长期目标。

DESCRIPTION (provided by applicant): In humans, exposure to light at night during shift work disrupts normal circadian rhythms, leading to an increased incidence of cancer; however, the molecular mechanisms underpinning circadian disruption in oncogenesis are not well understood. Nearly every cell in the human body has an endogenous molecular clock that controls integrated biochemical processes on a ~24-hour period. At the core of this molecular clock is the circadian basic helix-loop-helix Per:Arnt:Sim transcription factor complex CLOCK:BMAL1 that controls the rhythmic transcription of nearly 15% of the genome including essential genes in metabolism, hormone secretion and the cell cycle. In healthy somatic tissues the circadian clock and the cell cycle are linked such that the clock gates cell division to once a day thus preventing uncontrolled proliferation. The cell cycle/circadian link has been acknowledged for many years, however, the cellular perturbations that uncouple these two rhythmic systems in cancer remain to be elucidated. The objective of this proposal is to understand how a protein that is not expressed in healthy somatic tissues, yet is upregulated in cancer, represses the circadian clock and the effect of endogenous clock repression on cell proliferation. Our preliminary data show that this cancer-specific protein, PASD1, represses the activity of CLOCK:BMAL1. Based upon preliminary studies, our central hypothesis is that PASD1 silences clock function when expressed in cancer, removing clock-controlled homeostasis and cell cycle gating, leading cells down an oncogenic path. We will examine the role of PASD1 in clock regulation with the following specific aims: 1) Determine the mechanism by which PASD1 represses CLOCK:BMAL1 activity. Preliminary data demonstrate that a region of PASD1 that is conserved with a chromatin targeting domain in CLOCK is required for inhibition of CLOCK:BMAL1 activity. Our working hypothesis is that PASD1 uses molecular mimicry to interfere with the ability of CLOCK:BMAL1 to access target genes at the right time of day. To test this hypothesis we will perform ChIP-seq experiments to examine CLOCK:BMAL1 genomic targeting in the presence and absence of PASD1. 2) Determine how expression of PASD1 modulates circadian cycling and proliferation in human cancer cells. We hypothesize that knockdown of PASD1 in cancer cell lines will improve circadian cycling of CLOCK:BMAL1 target genes, providing more robust rhythms and decreased rates of proliferation. Investigating the mechanism by which a protein that is upregulated in cancer acts directly on the core circadian transcriptional feedback loop will establish a molecular link between circadian disruption and cancer with the long-term goal of identifying cancer therapeutics.