SnoN and TGFbeta signaling

说明(申请人提供)：转化生长因子-？(转化生长因子？)它调控多种正常的细胞过程，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扮演着复杂的角色。SnoN是一种强有力的转化生长因子？负调控因子。通过与Smad蛋白的结合和拮抗Smad蛋白的活性来传递信号。它是经典定义的原癌基因Ski家族的成员之一，当过表达时，可以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低水平表达，但在许多人类癌细胞中表达改变(上调或下调)。以往关于SnoN功能的研究大多集中在其促进鸡胚胎细胞致癌转化的能力上。它在正常哺乳动物上皮细胞中的功能几乎一无所知，在哺乳动物肿瘤发生中的作用也没有很好的确定。这一建议的长期目标是了解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。我们将使用小鼠胚胎成纤维细胞(MEF)和MCF10A正常人乳腺上皮细胞系来研究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，我们从一种表达突变的SnoN与Smad蛋白结合不足的敲入小鼠中分离出MEF。这些MEF细胞对凋亡刺激的敏感性增强，更有趣的是，它们提前衰老，表明SnoN/Smad相互作用可能调节细胞的凋亡和衰老反应。我们还在MCF10A细胞中使用了小干扰RNA方法，并表明SnoN以基底膜依赖的方式促进上皮细胞存活。在这项研究中，我们希望通过Smad依赖和非Smad依赖的方式来调节细胞的衰老、存活和增殖，从而验证SnoN同时具有抗肿瘤和促肿瘤活性的假说。其具体目的是：1)确定SnoN调控细胞衰老的分子机制；2)确定SnoN是否可以通过诱导早衰发挥肿瘤抑制作用；3)确定SnoN在正常人类上皮细胞中的功能。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调控如何促进肿瘤的发生。

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.