Reactive Oxygen Species and Cancer Cell Invasion

描述（申请人提供）：癌细胞在宿主体内采用多种策略来维持其生长和存活。其中一些策略允许原发肿瘤持续生长；其他的则促进了转移的过程。转移的肿瘤细胞需要具有运动性，并且能够穿过包围血管和器官的基底膜。一般认为基底膜是由肿瘤和宿主细胞提供的被肿瘤激活的胞外蛋白酶降解的。近年来，越来越多的人关注被称为足质体或侵过体的特殊膜突起在控制基底膜蛋白水解中的作用。足小体的外表面富含蛋白酶，包括金属蛋白酶、丝氨酸蛋白酶和半胱氨酸蛋白酶家族，它们可以降解细胞外基质（ECM）。我们最近发现了一种名为Tks5的Src底物和接头蛋白，它定位于正常细胞和癌细胞的足小体/侵过足。用siRNA减少Tks5的表达可以抑制足质体/侵过体的形成和侵袭，即使蛋白酶仍在分泌，这与该结构在侵袭表型中的重要性保持一致。控制足体/侵入足形成的机制尚不清楚。我们发现癌细胞在1%的氧气中孵育增加了足质体/侵入性足质体的形成。此外，用抗氧化剂或黄素蛋白抑制剂DPI处理细胞可以阻止podosome的形成，这表明活性氧（ROS）参与其中。在Src转化、缺氧和经历上皮间质转化的癌细胞中，Tks5 mRNA水平上调。我们的假设是，侵袭前信号转导通路上调Tks5的水平，而这反过来又促进了足质体的形成和ROS的产生。Tks5和ROS共同增加足质体/侵过体的形成，从而增加入侵。为了验证这一假设，我们提出以下具体目标：1。以评估Tks5的转录控制。2. 阐明NADPH氧化酶的组成及其在ROS形成中的作用。3. 表征ROS促进入侵的机制。这项研究的意义在于，它将增加我们对癌细胞侵袭受到调节的机制的理解。该研究的影响在于有机会阐明转移控制的新分子靶点。

DESCRIPTION (provided by applicant): Cancer cells adopt many strategies to maintain their growth and survival in their host. Some of these strategies allow the sustained growth of a primary tumor; others promote the process of metastasis. To metastasize tumor cells need to be motile, and to be able to cross the basement membrane which encloses blood vessels and organs. It is generally thought that basement membrane is degraded by extracellular proteases, provided by both tumor and host cells, that are activated by the tumor. In recent years, growing attention has focused on the role of specialized membrane protrusions called podosomes or invadopodia in control of basement membrane proteolysis. The outer surface of the podosome is rich in proteases, including those of the metallo-, serine- and cysteine protease families, which act to degrade the extracellular matrix (ECM). We recently identified a Src substrate and adaptor protein called Tks5 which is localized to podosomes/ invadopodia in both normal and cancer cells. Reducing Tks5 expression with siRNA inhibits the formation of podosomes/invadopodia and invasion, even though proteases are still secreted, in keeping with the importance of this structure in the invasive phenotype. The mechanisms which control podosome/invadopodia formation are not yet well understood. We have found that incubation of cancer cells in 1% oxygen increased the formation of podosomes/invadopodia. Furthermore, podosome formation was prevented by treating the cells with anti-oxidants or the flavoprotein inhibitor DPI, suggesting that reactive oxygen species (ROS) were involved. The level of Tks5 mRNA was upregulated by Src transformation, by hypoxia, and in cancer cells that had undergone an epithelial mesenchymal transition. Our hypothesis is that pro-invasive signal transduction pathways upregulate the level of Tks5, and that this in turn promotes podosome formation the production of ROS. Together, Tks5 and ROS increase the formation of podosomes/invadopodia and thus invasion. To test this hypothesis, we propose the following specific aims: 1. to evaluate the transcriptional control of Tks5. 2. To elucidate the composition and role of NADPH oxidases in the formation of ROS. 3. To characterize the mechanisms by which ROS promotes invasion. The significance of this research is that it will increase our understanding of the mechanisms by which cancer cell invasion is regulated. The impact of the research lies in the opportunity to elucidate new molecular targets for metastasis control.