Reactive Oxygen Species and Cancer Cell Invasion

描述(由申请者提供)：癌细胞采用多种策略来维持它们在宿主中的生长和存活。其中一些策略允许原发肿瘤的持续生长，另一些策略则促进了转移过程。要转移肿瘤细胞，需要能够移动，并能够穿过包裹血管和器官的基底膜。通常认为，基底膜是由肿瘤和宿主细胞提供的细胞外蛋白水解酶降解的，这些酶被肿瘤激活。近年来，越来越多的人关注被称为足体或内足突的特殊膜突起在控制基底膜蛋白分解中的作用。足体外表面含有丰富的蛋白酶，包括金属、丝氨酸和半胱氨酸蛋白酶家族，它们能降解细胞外基质(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中的足体/内陷。用siRNA降低Tks5的表达可以抑制足体/侵袭体的形成和侵袭，即使蛋白酶仍然被分泌，这与这种结构在侵袭表型中的重要性是一致的。控制足体/内侧体形成的机制还不是很清楚。我们发现，癌细胞在1%的氧气中孵育会增加足体/内足的形成。此外，用抗氧化剂或黄素蛋白抑制剂DPI处理细胞可以防止足体的形成，这表明活性氧物种(ROS)参与其中。在经历了上皮间充质转化的癌细胞中，Src转化、低氧和癌细胞中Tks5的mRNA水平被上调。我们的假设是，前侵袭性信号转导途径上调了Tks5的水平，进而促进了足体的形成和ROS的产生。Tks5和ROS共同促进了足体/侵袭体的形成，从而促进了侵袭。为了验证这一假说，我们提出了以下具体目标：1.评估Tks 5的转录调控。2.阐明NADPH氧化酶的组成及其在ROS形成中的作用。3.研究ROS促进侵袭的机制。这项研究的意义在于，它将增加我们对癌细胞侵袭调控机制的理解。这项研究的影响在于有机会阐明控制转移的新分子靶点。

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.