Molecular Mechanisms of Cell Invasion

这是一项研究肿瘤细胞侵袭的分子机制的建议。获得…… 侵袭潜能赋予肿瘤细胞从原发肿瘤分离并向远端扩散的能力 网站。以侵袭性黑色素瘤细胞系为模型系统，我们发现小分子GTP结合 ARF6蛋白调节细胞侵袭。近年来，ARF6已经成为一个重要的信号 分子，并已被证明调节内体循环，以及多种细胞类型的肌动蛋白重塑， 这两者都会影响细胞迁移/侵袭潜能的获得。我们的研究表明 ARF6的激活与ERK的激活有关，该信号通路似乎是必不可少的 用于细胞入侵。通过siRNA沉默ARF6或表达ARF6显性负性突变体 取消基于细胞的体外检测中的侵袭性。我们还表明，内源性ARF6的激活 对刺激肿瘤细胞侵袭的生理刺激的反应增加。在本应用程序中，我们的目标是 探讨ARF6调节细胞侵袭的机制。根据初步调查结果，我们 假设ARF6激活至少部分地通过调节(1) 金属蛋白水解酶的运输和(2)细胞骨架重塑是内侧足细胞形成所必需的。通过使用 在细胞和动物模型系统中的细胞生物学、分子和生化方法，我们将 探讨ARF6在肿瘤细胞侵袭中的作用。我们提出了三个具体目标。在目标1中，我们将 通过检测ARF6对MT1-MMPs的调节来研究ARF6在调节蛋白水解酶运输中的作用 交通和细胞表面的蛋白水解酶。在目标2中，我们将阐明信号转导 在细胞侵袭过程中将ARF6与rac1激活联系起来的途径，并询问内向突起的形成是否重要 用来分泌蛋白酶。在目标3中，我们将研究ARF6 GTP酶周期在黑色素瘤细胞中的作用 用小鼠模型系统进行体内侵袭。我们相信，从调查中获得的信息 将为蛋白酶运输的监管提供新的见解，这是一个研究相对较少的癌症领域 生物学，并将拓宽关于肿瘤进展过程中发生的分子变化的现有知识。 希望这些调查结果最终将有助于设计新的诊断和/或战略 癌症进展的治疗干预。

This is a proposal to investigate the molecular mechanisms underlying tumor cell invasion. Acquistion of invasive potential confers on tumor cells the ability to detach from the primary tumor and spread to distal sites. Using an invasive melanoma cell line as a model system, we have found that the small GTP-binding protein, ARF6, regulates cell invasion. In recent years, ARF6 has emerged as an important signaling molecule and has been shown to regulate endosome cycling, and actin remodeling in a variety of cell types, both of which can impinge on the acquisition of cell migratory/invasive potential. Our studies demonstrate that ARF6 activation is linked to the activation of ERK via a signaling pathway that appears to be essential for cell invasion. Silencing of ARF6 by siRNAs or expression of an ARF6 dominant negative mutant abolishes invasion in cell-based in vitro assays. We have also shown that activation of endogenous ARF6 increases in response to physiological stimuli that stimulate tumor cell invasion. In this application, we aim to investigate the mechanisms by which ARF6 regulates cell invasion. Based on preliminary findings, we hypothesize that ARF6 activation promotes tumor cell invasion at least in part, by regulating (1) the trafficking of metalloproteases and (2) cytoskeletal remodeling required for invadopodia formation. By using cell biological, molecular and biochemical approaches in both, cell and animal model systems, we will investigate the role of ARF6 in tumor cell invasion. We propose three specific aims. In aim 1, we will investigate the role of ARF6 in the regulation of protease traffic by examining its regulation of MT1-MMP traffic and the release of proteases from the cell surface. In aim 2, we will elucidate the signal transduction pathway linking ARF6 to Rac1 activation during cell invasion and ask if invadopodia formation is important for protease secretion. In aim 3, we will investigate the role of the ARF6 GTPase cycle in melanoma cell invasion in vivo using a mouse model system. We believe that the information gained from investigations will provide new insights into the regulation of protease trafficking, a relatively understudied area of cancer biology, and will broaden existing knowledge on the molecular changes that occur during tumor progression. It is hoped that these findings will ultimately aid in the design of new strategies for diagnostic and/or therapeutic intervention of cancer progression.