High Throughput Microscopy Assays to Identify Inhibitors of Metastasis

描述(由申请人提供)：当癌症被及早发现时，单靠手术通常是可以治愈的。然而，在确诊时，癌症已经经常通过转移过程扩散到身体的其他部位。与癌症相关的许多疼痛、痛苦和死亡都是转移性疾病的结果。我们需要更好的方法来早期诊断转移疾病，需要更好的方法来治疗转移疾病。最终目标是防止转移的发生，特别是那些已被诊断为浸润性疾病的患者，如乳腺导管原位癌和前列腺上皮内瘤变。当我们能够定义和开发针对转移细胞中存在的靶点的治疗方法时，我们将实现这一目标，而这些靶点并不是大多数正常生理所必需的，从而允许安全有效的长期治疗。转移性肿瘤和良性肿瘤的增殖都与通常控制细胞分裂和细胞死亡的细胞信号无关。但只有转移细胞才有能力从它们产生的器官中挣脱出来，进入血管系统，并进入其他器官并在其中生长。这些步骤统称为入侵。人们普遍认为，在转移过程中，最常见的侵袭方式是通过降解细胞外基质(ECM)蛋白的蛋白酶的作用。由于ECM的持续降解也被认为是次级器官部位播散性肿瘤生长所必需的，抑制这一过程可能提供一种控制转移的手段。癌细胞经常过度表达一些能够降解细胞外基质的蛋白酶。其中一些是分泌的，而另一些则与质膜有关。近年来，人们越来越清楚地看到，癌细胞腹膜的特殊突起称为足体(也称为内陷)，是这些蛋白水解酶的集中作用部位。例如，使用一个侵袭的模型系统，由激活版本的Src癌基因转化的小鼠成纤维细胞，我们已经证明了在体外和体内，典型的Src转化的侵袭性都需要足体。缺乏足体的细胞仍能分泌蛋白水解酶，但不能有效地降解细胞外基质。此外，人类癌细胞也需要足体/侵袭体才能侵袭。这些结果与一个模型是一致的，在该模型中，需要足体/内囊体来协调蛋白酶的活性。此外，这些数据支持我们的假设，即防止足小体形成的小分子将是限制肿瘤转移生长的有效药物。在这里，我们建议开发和利用基于细胞的高通量筛选系统来识别抑制足体形成和入侵的化学实体。转移是这个国家癌症死亡的主要原因。我们需要更好的方法来早期诊断转移疾病，需要更好的方法来治疗转移疾病。这项研究旨在建立高通量筛选方法来识别转移抑制物，因此是开发具有新作用机制的抗转移药物的第一步。

DESCRIPTION (provided by applicant): When cancer is detected early enough, surgery alone can often be curative. However, by the time of diagnosis, the cancer has frequently spread to other sites in the body, by the process of metastasis. Much of the pain, suffering, and mortality associated with cancer are the result of metastatic disease. We need better ways to diagnose metastatic disease early, and better ways to treat metastatic disease therapeutically. The ultimate goal is to prevent metastasis from occurring, particularly in those who have been diagnosed with pre-invasive conditions such as ductal carcinoma in situ of the breast, and prostate intra-epithelial neoplasia. We will achieve this goal when we are able to define and develop therapeutics against targets present in metastatic cells which are not essentially required for most normal physiology, thus allowing for safe and effective long term treatment. Both metastatic and benign tumors proliferate without regard to the cellular cues that normally control cell division and cell death. But only the metastatic cells have the ability to break free from the organ in which they arise, enter the vasculature, and move into and grow in other organs. These steps are collectively called invasion. It is widely believed that the most common way in which invasion occurs during metastasis is through the action of proteases, which degrade extracellular matrix (ECM) proteins. Since continued ECM degradation is also thought to be required for disseminated tumor growth in secondary organ sites, inhibiting this process may provide a means to control metastasis. Cancer cells frequently over-express a number of proteases that are capable of degrading ECM. Some of these proteases are secreted, while others remain associated with the plasma membrane. In recent years, it has become increasingly clear that specialized protrusions of the ventral membrane of cancer cells called podosomes (also known as invadopodia) are the concentrated sites of action of these proteases. For example, using a model system of invasion, mouse fibroblasts transformed by an activated version of the Src oncogene, we have shown that podosomes are required for the invasiveness that typifies Src transformation, both in vitro and in vivo. Cells lacking podosomes still secrete proteases, but they are unable to degrade ECM efficiently. Furthermore, human cancer cells also require podosomes/invadopodia for invasion. These results are consistent with a model in which podosomes/invadopodia are required for the coordinated activity of proteases. Further, these data support our hypothesis that small molecules that prevent the formation of podosomes would be effective agents to limit the growth of tumor metastases. Here, we propose to develop and utilize cell-based, high throughput screening systems to identify chemical entities that inhibit podosome formation and invasion. Metastasis is the leading cause of cancer mortality in this country. We need better ways to diagnose metastatic disease early, and better ways to treat metastatic disease therapeutically. The proposed research aims to set develop high throughput screening assays to identify inhibitors of metastasis, and therefore represents the first step to developing anti-metastatic drugs with a new mechanism of action.