Epithelial mesenchymal transition in gastrointestinal homeostasis and disease

描述（申请人提供）：上皮-间充质转化（EMT）是一个高度保守的细胞程序，它允许极化、不动的上皮细胞转化为可动的间充质细胞。EMT是发育过程中组织重塑事件的基础，但这一过程在成人的伤口愈合、组织再生、器官纤维化、肿瘤侵袭和转移过程中也会重新参与。虽然EMT的生理和临床意义是压倒性的，但EMT的精确分子和功能特征仍然缺乏表征。绒毛蛋白是一种上皮细胞特异性肌动蛋白结合蛋白，在胃肠道、泌尿生殖道和呼吸道中表达量最大。绒毛蛋白是一种调节上皮细胞可塑性和EMT等功能的多功能蛋白。用绒毛蛋白敲除小鼠进行的研究清楚地表明，缺乏绒毛蛋白会损害上皮细胞对调节EMT的信号的反应能力，导致伤口修复和细胞迁移的缺陷。尽管有这些发现，绒毛蛋白如何驱动EMT的变化仍有待确定。在我们的初步研究中，我们首次证明了绒毛蛋白定位于细胞核，并直接与转录共调节因子ZBRK1相互作用。此外，我们报道了核绒毛蛋白激活转录因子Slug的表达来调节上皮可塑性和EMT。最重要的是，我们的研究表明，远离刷状缘膜的绒毛蛋白错误定位是结直肠癌患者死亡率增加的预后因素。本应用程序设计的研究将验证我们的新假设，即绒毛蛋白的细胞质-核运输调节转录共调节剂ZBRK1的稳定性和/或周转，从而调节关键转录因子Slug的表达以诱导EMT。更多的研究旨在将绒毛蛋白的异常核定位与结直肠癌患者的转移联系起来。具体来说，拟议研究的目标是：(i)验证调节绒毛蛋白的细胞质-核运输和核保留的分子机制；（ii）表征villin-ZBRK1复合物及其在Slug表达中的作用，Slug是EMT的关键转录激活因子；（iii）阐明核绒毛蛋白在EMT及转移调控中的作用。我们提出的实验方法结合了机制和功能生化，细胞和分子生物学研究以及绒毛蛋白-/-，绒毛蛋白+/+，ApcMin/绒毛蛋白-/-的体内研究；ApcMin/villin+/+和严重联合免疫缺陷小鼠（SCID）使我们能够解开如何调节EMT的复杂问题。另外的研究建议使用两种独特的人类结肠癌组织微阵列资源，即334个临床试验样本的队列，来自III期MAX试验的患者，以及29个匹配的原发性和转移性结肠肿瘤样本的组织微阵列（TMA）。我们研究的长期目标是将我们的发现转化为临床结果，以诊断、预防和/或治疗纤维化和转移。

DESCRIPTION (provided by applicant): Epithelial-mesenchymal transition (EMT) is a highly conserved cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells. EMT is fundamental for tissue remodeling events during development, but this process is re-engaged in adults during wound healing, tissue regeneration, organ fibrosis, tumor invasion and metastasis. While the physiological and clinical significance of EMT is overwhelming, the precise molecular and functional features of EMT remain poorly characterized. Villin is an epithelial cell specific actin-binding protein that is expressed in mos significant amounts in the gastrointestinal, urogenital and respiratory tracts. Villin is a multifunctional protein that regulates epithelial cell plasticity and EMT amongst other functions. Studies done with the villin knockout mice have clearly demonstrated that the absence of villin impairs the ability of epithelial cells to respond to signals that regulate EMT, resulting in a deficiency in wound repair and cell migration. Despite these findings, how villin actuates changes in EMT remains to be determined. In our preliminary studies, we demonstrate for the first time that villin localizes to the nucleus and interacts directly with a transcriptional co-regulator, ZBRK1. Furthermore, we report that nuclear villin activates the expression of the transcriptional factor Slug to modulate epithelial plasticity and EMT. Most importantly, our studies suggest that mislocalization of villin away from the brush border membrane is prognostic of increased mortality in patients with colorectal cancer. Studies designed in this application wil test our novel hypothesis that cytoplasmic-nuclear trafficking of villin regulates the stability and/or turnover of the transcriptional co-regulator ZBRK1, thereby modulating the expression of the key transcriptional factor Slug to induce EMT. Additional studies are designed to link the abnormal nuclear localization of villin with metastasis in patients with colorectal cancer. Specifically, the goal of the proposed studies are: (i) to validate the molecular mechanism(s) that regulates the cytoplasmic-nuclear trafficking and nuclear retention of villin; (ii) to characterize the villin-ZBRK1 complex and its role in the expression of Slug, a key transcriptional activator of EMT and; (iii) to elucidate the function of nuclear villin in the regulation of EMT and metastasis. The experimental approach we have proposed combines mechanistic and functional biochemical, cell and molecular biological studies together with in vivo studies in the villin-/-, villin+/+, ApcMin/villin-/-; ApcMin/villin+/+, and severe combined immunodeficiency mice (SCID) mice to allow us to unravel the complex question of how EMT is regulated. Additional studies are proposed using two unique human colon cancer tissue microarray resources namely, a cohort of 334 clinical trial specimens from patients enrolled in phase III MAX trial and a tissue microarray (TMA) of 29 matched primary and metastatic colon tumor specimens. The long-term goal of our studies is to translate our findings into clinical outcomes to diagnose, prevent and/or treat fibrosis and metastasis.