Molecular Mechanisms of Cell Invasion

描述(由申请人提供)：这是一份研究侵袭性肿瘤细胞中微泡生物发生机制的申请。它建立在先前NIH对一种独特的囊泡群体进行资助时产生的令人兴奋的发现，这种囊泡被称为微泡，含有功能活跃的蛋白水解酶，并在肿瘤细胞获得侵袭潜力时释放出来。携带蛋白水解酶的微囊的释放可能是一种导致基质降解的机制，甚至可能在远端位置沉积旁分泌信息，从而在肿瘤细胞通过周围组织侵袭和迁移时创造出“最小抵抗”的路径。这不同于内侧足底的细胞周围蛋白分解，后者使局部的基质降解并列在前沿。发现可能存在一种以上的蛋白水解性侵袭模式，限制了任何不包括局灶性和远端蛋白水解性侵袭的侵袭靶向治疗策略的有效性。虽然已经有大量的研究针对了解侵袭细胞部位的侵袭足的形成和功能的机制，但微囊的生物发生和功能仍然是肿瘤生物学中相对较少研究的领域。然而，最近越来越多的证据表明，体液(血液、尿液和腹水)中确实存在微泡，并有可能作为疾病的指示物，这引起了人们对微泡生物学和功能的兴趣，并加强了研究工作。这项应用的首要目标是确定微泡形成的分子机制。该项目的核心假设是，特定的ARF和Rab蛋白将膜型蛋白酶和其他蛋白质定向到微囊生物发生的位置，而RhoA和rac1信号之间的紧密交换控制着在微泡和内毒素介导的蛋白水解性入侵之间切换所需的可塑性。我们将解决两个具体目标。在第一个目标中，我们将定义将货物引导到微囊泡生物发生部位的内吞循环途径，并研究特定Rab效应子的招募如何调节微囊泡生物发生所需的基于肌球蛋白的收缩。在第二个目标中，我们将检测RhoA和rac1在侵袭性肿瘤细胞中的空间激活。我们还将研究在微泡形成过程中调节rac1下调的潜在机制，以及Rho信号如何促进这一过程。鉴于最近对微囊的生物学和临床前景的高度兴趣，这些研究是非常新的。它们将促进目前对微囊生物发生的理解，并有可能为诊断和治疗应用提供靶点。

DESCRIPTION (provided by applicant): This is an application to investigate the mechanisms of microvesicle biogenesis in invasive tumor cells. It builds on exciting findings generated with previous NIH funding on a unique population of vesicles, called microvesicles that contain functionally active proteases and are released by tumor cells as they acquire invasive potential. The release of protease-loaded microvesicles may serve as a mechanism to bring about matrix degradation and perhaps even deposit paracrine information at distal locations, thus creating paths of "least resistance" as tumor cells invade and migrate through surrounding tissue. This is distinct from pericellular proteolysis at invadopodia, which enables localized matrix degradation juxtaposed to the leading edge. Discovering that there may exist more than one mode of proteolytic invasion, limits the effectiveness of any invasion-targeted therapeutic strategy that does not include both focal and distal proteolysis. While a significant amount of research has been directed to the understanding mechanisms of invadopodia formation and function at sites of cell invasion, microvesicles biogenesis and function remains a relatively understudied area of tumor biology. However, recent accruing evidence demonstrating the bona fide presence of microvesicles in body fluids (blood, urine and ascites), and their potential to serve as indicators of disease, has extended interest and intensified research efforts in microvesicle biology and function. The overarching objective of this application is to define molecular mechanisms of microvesicle formation. The project focuses on the central hypothesis that specific ARF and Rab proteins direct membrane type proteases and other proteins to sites of microvesicle biogenesis and that tight interchanges between RhoA and Rac1 signaling governs the plasticity required for switching between microvesicle and invadopodia-mediated proteolytic invasion. We will address two specific aims. In the first aim, we will define endocytic recycling pathways that direct cargo to sites of microvesicle biogenesis as well as examine how recruitment of specific Rab effectors regulate actomyosin-based contraction required for microvesicle biogenesis. In the second aim, we will examine the spatial activation of RhoA and Rac1 in invasive tumor cells. We will also investigate potential mechanisms that regulate Rac1 down regulation during microvesicle formation and how Rho signaling facilitates the process. Given recent heightened interest in the biology and clinical promise of microvesicles, these investigations are highly current. They will advance present understanding of microvesicle biogenesis and have potential to provide targets for diagnostic as well as therapeutic application.