SnoN and TGFbeta signaling

描述（由申请人提供）：转化生长因子-？（TGF?）调节多种正常细胞过程，包括增殖、存活、细胞-基质相互作用、分化，在哺乳动物肿瘤发生过程中发挥复杂作用。SnoN是TGF？通过结合和拮抗Smad蛋白的活性来传递信号。它是经典定义的原癌基因Ski家族的一员，当过度表达时，诱导鸡和鹌鹑胚胎成纤维细胞的转化。它在所有成人细胞和组织中都有低水平表达，但在许多人类癌细胞中其表达被改变（上调或下调）。以往有关SnoN功能的研究主要集中在其促进鸡胚细胞癌性转化的能力上。它在正常哺乳动物上皮细胞中的功能几乎一无所知，它在哺乳动物肿瘤发生中的作用也没有很好地定义。本研究的长期目标是了解SnoN和SnoN/Smad相互作用在调节细胞增殖、存活和衰老以及哺乳动物肿瘤发生中的功能，并确定这些过程的分子机制。我们将利用小鼠胚胎成纤维细胞（MEF）和正常人乳腺上皮细胞系MCF10A来研究SnoN在正常哺乳动物细胞中的功能。为了确定SnoN/Smad相互作用的生理意义，我们从一株敲入小鼠中分离出MEF，该敲入小鼠表达与Smad蛋白结合缺失的SnoN突变体。这些MEF细胞对凋亡刺激表现出增强的敏感性，更有趣的是，提早衰老，表明SnoN/Smad相互作用可能调节凋亡和衰老反应。我们还在MCF10A细胞中使用了小干扰RNA方法，并发现SnoN以基底膜依赖的方式促进上皮细胞的存活。在本提案中，我们想通过smad依赖和smad独立的方式调节细胞的衰老、存活和增殖，来验证SnoN同时具有抗肿瘤和促癌活性的假设。具体目的是：1)确定SnoN调控细胞衰老的分子机制；2)研究SnoN能否通过诱导早衰而发挥抑癌作用；3)确定SnoN在正常人上皮细胞中的功能。这些研究将使我们了解SnoN在正常哺乳动物细胞中的功能，以及这些活动的解除如何促进肿瘤发生。公共卫生相关性：SnoN是转化生长因子-？（TGF-?）信号，在哺乳动物肿瘤发生过程中发挥着重要而复杂的作用。它调节许多方面的细胞功能，包括增殖、分化、衰老和运动，并含有致癌和抗致癌活性。识别介导这些活性的信号通路并破译这些通路的特异性不仅对于理解控制恶性进展的机制至关重要，而且对于开发新的癌症治疗方法至关重要，这些治疗方法可以特异性地靶向SnoN的致癌活性，同时保持其抗致癌活性。

DESCRIPTION (provided by applicant): Transforming growth factor-? (TGF?) regulates a wide variety of normal cellular processes including proliferation, survival, cell-matrix interaction, differentiation and plays a complex role during mammalian tumorigenesis. SnoN is a potent negative regulator of TGF? signaling through binding to and antagonizing the activity of the Smad proteins. It is a member of the Ski family of classically defined proto-oncogenes that when overexpressed, induces transformation of chicken and quail embryo fibroblasts. It is expressed in all adult cells and tissues at a low level but its expression is altered (up- or down-regulated) in many human cancer cells. Previous studies related to SnoN function mostly focused on its ability to promote oncogenic transformation in chicken embryo cells. Virtually nothing is known about its function in normal mammalian epithelial cells, and its role in mammalian tumorigenesis has not been well defined. The long-term goal of this proposal is to understand the function of SnoN and the SnoN/Smad interaction in regulation of cell proliferation, survival and senescence as well as mammalian tumorigenesis and to determine the molecular mechanisms underlying these processes. We will employ the mouse embryo fibroblasts (MEF) and MCF10A normal human mammary epithelial cell line to investigate the function of SnoN in normal mammalian cells. In an effort to determine the physiological significance of the SnoN/Smad interaction, we have isolated MEF from a strain of knock-in mice that express a mutant SnoN deficient in binding to the Smad proteins. These MEF cells display enhanced sensitivity to apoptotic stimuli and more interestingly, premature senescence, indicating that the SnoN/Smad interaction may regulate the apoptosis and senescence responses. We have also employed small-interference RNA approach in MCF10A cells and showed that SnoN promotes epithelial survival in a basement membrane-dependent manner. In this proposal, we would like to test the hypothesis that SnoN possess both anti-oncogenic and pro-oncogenic activities through regulation of cell senescence, survival and proliferation in both Smad-dependent and Smad-independent manner. The specific aims are: 1) To determine the molecular mechanism by which SnoN regulates cell senescence; 2) To determine whether SnoN can function as a tumor suppressor through its ability to induce premature senescence; 3) To determine the function of SnoN in normal human epithelial cells. These studies will allow us to understand the function of SnoN in normal mammalian cells and how deregulation of these activities facilitates tumorigenesis. PUBLIC HEALTH RELEVANCE: SnoN is a potent negative regulator of transforming growth factor-? (TGF-?) signaling, which play important but complex roles during mammalian tumorigenesis. It regulates many aspects of cellular functions including proliferation, differentiation, senescence and motility and contains both oncogenic and anti-oncogenic activities. Identification of the signaling pathways mediating each of these activities and deciphering the specificity of these pathways are crucial not only for understanding the mechanisms controlling malignant progression but also for development of novel cancer therapy that specifically targets the oncogenic activity of SnoN while preserving its anti-oncogenic activity.