Reactive Oxygen Species and Cancer Cell Invasion

描述（由申请人提供）：癌细胞采取许多策略来维持其在宿主中的生长和存活。这些策略中的一些允许原发性肿瘤的持续生长;另一些促进转移的过程。要转移肿瘤细胞需要是能动的，并且能够穿过包围血管和器官的基底膜。一般认为，基底膜被肿瘤和宿主细胞提供的细胞外蛋白酶降解，这些蛋白酶被肿瘤激活。近年来，越来越多的注意力集中在专门的膜突起称为podosomes或invadopodia在控制基底膜蛋白水解的作用。podosome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的蛋白酶，其作用是降解细胞外基质（ECM）。我们最近发现了一种Src底物和衔接蛋白Tks 5，它定位于正常细胞和癌细胞中的podosomes/ invadopodia。用siRNA减少Tks 5表达抑制了足体/侵袭足的形成和侵袭，即使蛋白酶仍然被分泌，这与该结构在侵袭表型中的重要性一致。控制podosome/invadopodia形成的机制还没有很好的理解。我们已经发现，在1%的氧气中孵育癌细胞增加了足体/侵入伪足的形成。此外，通过用抗氧化剂或黄素蛋白抑制剂DPI处理细胞来防止足状体形成，这表明涉及活性氧（ROS）。Src转化、缺氧和经历上皮间质转化的癌细胞中Tks 5 mRNA水平上调。我们的假设是，促侵袭信号转导途径上调Tks 5的水平，这反过来又促进了podosome的形成和ROS的产生。Tks 5和ROS共同增加了足体/侵入伪足的形成，从而增加了侵袭。为了验证这一假设，我们提出了以下具体目标：1。以评估Tks 5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.描述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节机制的理解。这项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.