Regulation of the protumorigenic senescence-associated factor OPN

描述(由申请人提供)：年龄是癌症发展的主要危险因素(1)。除了上皮细胞突变的积累外，衰老基质细胞的年龄相关性积累在肿瘤的促进中起着重要作用(1-5)。虽然衰老细胞不再复制，但它们仍保持代谢活性，并分泌多种促肿瘤因子，称为衰老相关分泌表型(2，6-8)(SASP)。通过SASP，衰老细胞在体外促进癌前细胞的转化，并在异种移植模型中促进肿瘤生长(2，6-8)。骨桥蛋白(OPN)是一种促肿瘤蛋白，随着衰老在mRNA水平上调。重要的是，OPN缺失的衰老细胞缺乏正常衰老成纤维细胞的促肿瘤能力。此外，在癌前上皮细胞中加入重组OPN足以诱导细胞增殖。了解SASP因子是如何调节的，是设计间质靶向癌症疗法的重要一步。许多SASP因子的调节已被很好地研究，并且依赖于ATM和NF-βB。然而，OPN的诱导不依赖于这两个因子。为了研究OPN对衰老的调控，我们使用OPN启动子报告结构来确定OPN转录在衰老过程中被激活所需的启动子区域。我们发现，转录起始点上游190个碱基对的区域包含激活OPN响应衰老所需的关键序列。该衰老反应元件(OPN-SRE)含有c-Myb和C/EBP？转录因子结合位点，提示这些因子对于OPN的转录在衰老反应中的激活是重要的。我假设c-Myb和C/EBP？协同作用直接激活OPN转录以应对衰老。为了验证这一假说，本研究的目的有三：1)确定c-Myb在衰老过程中骨桥蛋白表达中的作用；2)确定C/EBP在骨桥蛋白表达中的作用；3)确定c-Myb和C/EBP在骨桥蛋白表达中的作用。协调激活骨桥蛋白以应对衰老。为了达到这些目标，我将阐明一个独立的SASP调控臂和OPN的转录调控机制。另外，我会决定C/EBP是否？作为SASP的重要中央调节因子，提供了对SASP激活的整体机制的洞察。由于衰老细胞通过SASP促进肿瘤形成过程的每个阶段，了解这些调控途径将提供潜在的癌症治疗机会。

DESCRIPTION (provided by applicant): Age is a major risk factor in the development of cancer(1). In addition to the accumulation of epithelial cell mutations, the age-dependent accumulation of senescent stromal cells plays an important role in tumor promotion(1-5). Although senescent cells no longer replicate, they remain metabolically active and secrete a wide variety of protumorigenic factors known as the senescence-associated secretory phenotype(2,6-8) (SASP). Through the SASP, senescent cells promote the transformation of preneoplastic cells in vitro and promote tumor growth in xenograft models(2,6-8). Osteopontin (OPN) is a protumorigenic protein that is upregulated at the mRNA level in response to senescence. Importantly, senescent cells depleted of OPN lack the tumor-promoting ability of normal senescent fibroblasts. Furthermore, the addition of recombinant OPN to preneoplastic epithelial cells is sufficient to induce cell proliferation. Understanding how SASP factors are regulated is an important step in designing stroma-targeted cancer therapies. The regulation of many SASP factors is well studied and dependent on ATM and NF-?B. However, the induction of OPN is independent of both of these factors. To study the regulation of OPN in response to senescence, we used OPN promoter reporter constructs to identify regions of the promoter required for robust activation of OPN transcription during senescence. We found that a region 190 base pairs upstream of the transcription start site housed critical sequences required for activation of OPN in response to senescence. This senescence response element (OPN-SRE) contains c-Myb and C/EBP? transcription factor binding sites, suggesting that these factors are important for the activation of OPN transcription in response to senescence. I hypothesize that c-Myb and C/EBP? cooperate to directly activate OPN transcription in response to senescence. To test this hypothesis, this proposal will pursue three aims: 1) determine c-Myb's role in OPN expression during senescence; 2) determine C/EBP?'s role in OPN expression in during senescence 3) determine whether c-Myb and C/EBP? coordinately activate OPN in response to senescence. In addressing these aims, I will elucidate an independent SASP regulatory arm and the mechanism of transcriptional regulation of OPN. Further, I will determine whether C/EBP? acts as an essential central regulator of SASP, providing insight into the mechanism of SASP activation as a whole. Because senescent cells promote every stage of the tumorigenic process through the SASP, understanding these regulatory pathways will provide potential cancer therapeutic opportunities.