Dynamics of Gene and Isoform Regulation during EMT and tumor progression

描述(申请人提供)：上皮-间充质转化(EMT)是一个复杂的细胞生物学程序，在癌细胞向高度恶性发展的过程中运行，赋予这些细胞许多与侵袭性肿瘤相关的属性，包括扩散到远处和种植转移克隆的能力。这 该程序由一系列多向性作用的主要转录因子(EMT-TF)协调，这些转录因子组织基因表达的复杂变化，导致大量上皮细胞蛋白被与间充质细胞状态相关的蛋白取代。然而，对侵袭性间充质状态表达所需的一个主要的、关键的控制水平却知之甚少：许多在EMT期间表达变化的mRNAs的前体也经历了选择性剪接(AS)，从而导致成熟的、加工的mRNAs的性质改变，包括稳定性、蛋白质编码信息和对microRNA介导的抑制的反应性的变化。目前对AS对EMT程序执行的影响的零星见解使其不可能形成对这一关键的细胞生物学程序如何影响的合理完整的理解。这项拟议的研究将通过列举在培养细胞和活组织中诱导EMT程序的几种替代机制而发生的数百种AS事件开始。在这样做之后，将使用生物信息学算法来确定与所涉及的剪接点相邻的序列。此后，利用大量已经表征的RNA结合、剪接调节蛋白的已知核苷酸识别特性，这些算法将对剪接调节因子的身份做出预测，这些剪接调节因子可能对观察到的在通过EMT过程中发生的大规模移位负责。这一实验策略应该产生AS的关键调节者的身份，这些AS在执行EMT计划时在功能上可能与EMT-TF一样重要。将进行旨在从功能上测试这些因素的候选资格的实验测试。这些测试将衡量这些因子的强制或阻断表达是否影响EMT计划的关键组成部分的执行，以及如预测的那样，AS因子表达的这种强加变化是否影响关键EMT相关蛋白的产生，即在恶性进展过程中观察到的上皮细胞与间质细胞表型的表达中发挥关键作用的蛋白质。这项工作还有可能确定EMT计划的新生物标记物，例如，可用于检测各种上皮组织中的干细胞。

DESCRIPTION (provided by applicant): The epithelial-mesenchymal transition (EMT) is a complex cell-biological program that operates during the progression of carcinoma cells to high-grade malignancy, conferring on these cells many of the attributes associated with aggressive tumors, including the ability to disseminate to distant sites and to seed metastatic colonies. This program is orchestrated by a series of pleiotropically acting master transcription factors (EMT- TFs) that organize the complex changes in gene expression causing the replacement of a large cohort of epithelial cell proteins with those associated with the mesenchymal cell state. A major, critical level of control required for expression of the aggressive mesenchymal state is poorly understood however: the precursors of many of the mRNAs whose expression changes during the EMT also undergo alternative splicing (AS) that confer on resulting mature, processed mRNAs altered properties, including changes in stability, protein-coding information, and responsiveness to microRNA-mediated inhibition. The current fragmentary insights into the effects of AS on the execution of the EMT program make it impossible to form a reasonably complete understanding of how this critical cell-biological program is effected. The proposed research will begin by enumerating the hundreds of AS events that occur in response to several alternative mechanisms of inducing an EMT program both in cultured cells and in a living tissue. Having done so, bioinformatics algorithms will be employed to determine the sequences adjacent to involved splice sites. Thereafter, using the known nucleotide-recognizing properties of the large array of already-characterized RNA- binding, splice-regulating proteins, predictions will be made by these algorithms about the identities of the splice-regulators that are likely to b responsible for the observed large-scale shifts in AS occurring during passage through an EMT. This experimental strategy should yield the identities of key regulators of AS that are likely to b as important functionally as the EMT-TFs in executing the EMT program. Experimental tests designed to functionally test the candidacies of these AS factors will be performed. These tests will gauge whether the forced or blocked expression of these factors affect execution of critical components of the EMT program, and whether, as predicted, such imposed changes in AS factor expression affect the production of key EMT-associated proteins, i.e., proteins that play key roles in the expression of the epithelial versus mesenchymal cell phenotypes observed during malignant progression. This work also has the potential to identify novel biomarkers of the EMT program that are applicable, for example, for the detections of stem cells in a variety of epithelial tissues.