Identifying the Mechanisms Governing Mena-Induced Tumor Cell Dissemination

描述(申请人提供)：超过三分之一的人会在他们的一生中患上癌症，在美国，每天大约有1500人死于癌症。转移是癌症相关死亡的主要原因，需要侵袭和血管内注射。关键的细胞骨架、增殖和凋亡蛋白在细胞侵袭和血管内渗入过程中会动态改变其基因表达模式，提示它们参与调控这些细胞迁移表型所涉及的部分或全部过程。这些蛋白质被统称为“入侵标志”。“侵袭标志”的组成部分之一是MENA，它是Enable(Ena)/血管扩张剂刺激的磷酸蛋白(VASP)家族的成员。Ena/Vasp蛋白是肌动蛋白动态的高度保守的调节因子，已知在细胞迁移中发挥关键作用。EnA/Vasp蛋白含有几个保守的结构域，被认为通过选择性剪接来诱导MENA功能的特异性。MENA异构体MenaINV的表达使癌细胞对表皮生长因子(EGF)诱导的癌细胞侵袭和转移变得敏感。作为对低水平EGF的反应，MenaINV的表达导致体内转移的增强。此外，MenaINV的表达导致癌细胞向血管定向移动，增加了内毒素的稳定性，并使跨内皮细胞迁移(TEM)增加了200倍。另一种MENA亚型Mena11a的表达也导致肿瘤细胞迁移增加。因此，MENA在侵袭和血管内侵袭中起着关键作用。虽然已经发现了MENA在肿瘤细胞转移中的许多作用，但表达MENA的癌细胞执行这些功能的机制仍不清楚。因此，我们将利用简化的体外系统来具体控制参数，以及我们实验室开发的体内系统来考虑肿瘤微环境。有了这些方法，具体地说，我将测试在Mena诱导的趋化、侵袭和血管内凝血过程中对内向足类的需求，以及调节这些过程的上游信号事件。具体地说，我提出了以下目标：1)确定MENA异构体表达细胞是否能够自主地化学感知一种趋化物质，以及在化学感觉中什么步骤(S)是关键的；2)研究MENA诱导的趋化作用与体内INVE形成的关系；以及3)确定在表达MenaINV的细胞中透射电子显微镜活性增加200倍的机制。最终，这些实验将提供对体内观察到的膜诱导转移的调控机制的洞察，这可能对开发新的抗转移疗法至关重要。 公共卫生相关性：超过三分之一的人会在一生中患上癌症，在美国，每天约有1500人死于癌症。及早识别癌症，并了解肿瘤的转移潜力，可能有助于治疗，从而提高存活率。通过这项工作，我们希望了解肿瘤细胞扩散的分子基础，目标是发现新的标记物来识别高危患者，并最终开发新的抗转移疗法。

DESCRIPTION (provided by applicant): More than 1 in 3 people will develop cancer in their lifetime, and approximately 1,500 people die from cancer each day in the United States. Metastases, which require invasion and intravasation, are the major causes of cancer-related deaths. Key cytoskeletal, proliferative and apoptotic proteins have been shown to dynamically alter their gene expression patterns during cell invasion and intravasation, suggesting their involvement in regulating some or all of the processes involved in these cell migration phenotypes. These proteins have been termed collectively as the "Invasion Signature". One of the components of the "Invasion Signature" is Mena, a member of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family. Ena/VASP proteins are highly conserved regulators of actin dynamics, known to play critical roles in cell migration. Ena/VASP proteins contain several conserved domains that are thought to elicit specificity of Mena function through alternative splicing. Expression of a Mena isoform, MenaINV, sensitizes carcinoma cells to epidermal growth factor (EGF)- induced carcinoma cell invasion and metastasis. In response to lower levels of EGF, expression of MenaINV leads to enhanced metastasis in vivo. Furthermore, expression of MenaINV results in directional movement of carcinoma cells toward blood vessels, increased invadopodium stability, and a 200-fold increase in transendothelial migration (TEM). Expression of another Mena isoform, Mena11a, also results in increased tumor cell migration. Therefore, Mena plays critical roles in invasion and intravasation. Although many roles for Mena in tumor cell metastasis have been discovered, the mechanisms by which Mena-expressing carcinoma cells use to carry out these functions are still unknown. Thus, we will take advantage of a simplified in vitro system for specific control of parameters, as well as an in vivo system developed in our lab to take into account the tumor microenvironment. With these approaches in hand, specifically, I will test the requirement of invadopodia during Mena-induced chemotaxis, invasion and intravasation, and the upstream signaling events that regulate these processes. In particular, I propose the following aims: 1) Determining whether Mena isoform-expressing cells can autonomously chemosense a chemoattractant, and what step(s) of invadopodium formation is critical for chemosensing; 2) Investigating the relationship between Mena-induced chemotaxis and invadopodium formation in vivo; and 3) Identifying the mechanism underlying the 200 fold increase in TEM activity in MenaINV-expressing cells. Ultimately, these experiments will provide insight into the mechanisms governing the observed Mena-induced metastasis in vivo, which may be critical to the development of new anti-metastatic therapies. PUBLIC HEALTH RELEVANCE: More than 1 in 3 people will develop cancer in their lifetime, and approximately 1,500 people die from cancer each day in the United States. Early identification of cancer, and knowledge about the metastatic potential of a tumor may facilitate treatment, and thus increase survival. With this work, we hope to understand the molecular basis of tumor cell spread, with the goal of uncovering new markers to identify high-risk patients and ultimately develop new anti-metastatic therapies.