Role of the RhoGEF Net1 in breast cancer cell motility and metastasis

描述(申请人提供)：控制乳腺癌转移的分子机制还不是很清楚。在细胞外基质(ECM)侵袭期间，癌细胞在快型、阿米巴型和慢型间充质运动之间交替。这种运动的可塑性对于有效入侵是必不可少的，但它是如何形成的还不是很清楚。 控制住了。我们发现，Rhogef Net1a是乳腺癌细胞侵袭可塑性的重要调节因子，刺激变形虫和抑制间充质运动。Net1A通过控制RhoA和脱乙酰酶功能来调节这一过程，从而影响细胞骨架组织和基因表达。Net1A在RhoGEF中是例外的，因为它隔离在细胞核中以防止异常的RhoA激活，显然，亚细胞定位是Net1亚型致癌潜力的关键决定因素。最近我们发现，整合素下游的rac1激活或生长因子受体的激活可以刺激Net1a重新定位到质膜上。我们还发现Net1A的重新定位是通过位点特异性的乙酰化来维持的，并且特定的脱乙酰酶的作用对于抑制Net1A对RhoA的活性是至关重要的。此外，我们还观察到，Net1A相互控制特定脱乙酰酶的活性，以调节细胞骨架的组织和与细胞侵袭相关的关键基因的表达，如MT1-MMP.这些发现支持我们的假设，即Net1A作为一个结点在时间和空间上调节RhoA的激活和脱乙酰酶的活性，这是乳腺癌细胞运动、侵袭和转移的基础。在这项提案中，我们将使用基于细胞的分析和体内分析相结合的方法来了解Net1A的定位是如何调节的，以推动RhoA的激活，并在细胞外基质侵袭过程中相互控制脱乙酰酶活性。我们还将确定Net1亚型如何在体内促进乳腺肿瘤的发生和转移。在目标1中，我们将演示EGFR和HER2的激活如何刺激Net1A从细胞核输出，从而刺激细胞运动和ECM侵袭。我们将展示Net1A乙酰化如何影响其功能，并确定调节Net1A的脱乙酰酶。在目标2中，我们将确定Net1A如何控制HDAC和Sirtuin的功能，以调节细胞骨架组织蛋白的乙酰化和基因表达。在目标3中，我们将确定Net1基因缺失如何影响小鼠乳腺发育的分子基础，并确定Net1是否是HER2/Neu驱动的乳腺肿瘤发生和转移所必需的。这些目标的完成将提供对Net1亚型如何促进乳腺癌细胞运动和侵袭潜力的机械理解，并将为转移性疾病的干预提供新的靶点。

DESCRIPTION (provided by applicant): Molecular mechanisms controlling metastasis in breast cancer are not well understood. During invasion of the extracellular matrix (ECM) cancer cells alternate between fast, amoeboid, and slow mesenchymal types of movement. This plasticity in movement is essential for efficient invasion, but it is not well understood how it is controlled. We have found that the RhoGEF Net1A is an important regulator of invasion plasticity in breast cancer cells, stimulating amoeboid and suppressing mesenchymal movement. Net1A regulates this process by controlling RhoA and deacetylase function to impact cytoskeletal organization and gene expression. Net1A is exceptional among RhoGEFs in that it is sequestered in the nucleus to prevent aberrant RhoA activation, and it is clear that subcellular localization is a key determinant of the oncogenic potential of Net1 isoforms. Recently we have shown that Rac1 activation downstream of integrin or growth factor receptor activation stimulates Net1A relocalization to the plasma membrane. We have also found that Net1A relocalization is maintained by site-specific acetylation, and that the actions of specific deacetylases are critical to suppressing Net1A activity towards RhoA. Moreover, we have observed that Net1A reciprocally controls the activity of specific deacetylases to regulate cytoskeletal organization and expression of key genes associated with cell invasion, such as MT1-MMP. These findings support our hypothesis that that Net1A functions as a nodal point to temporally and spatially regulate RhoA activation and deacetylase activity which underlie breast cancer cell motility, invasion and metastasis. In this proposal we will use a combination of cell-based and in vivo assays to understand how Net1A localization is regulated to drive RhoA activation and reciprocally control deacetylase activity during ECM invasion. We will also determine how Net1 isoforms contribute to mammary gland tumorigenesis and metastasis in vivo. In Aim 1 we will demonstrate how EGFR and HER2 activation stimulates Net1A export from the nucleus to stimulate cell motility and ECM invasion. We will demonstrate how Net1A acetylation affects its function and identify the deacetylases that regulate Net1A. In Aim 2 we will determine how Net1A controls HDAC and Sirtuin function to regulate acetylation of cytoskeletal organizing proteins and gene expression. In Aim 3 we will identify the molecular basis for how genetic deletion of Net1 affects mammary gland development in mice, and determine whether Net1 is required for HER2/Neu driven mammary gland tumorigenesis and metastasis. Completion of these aims will provide a mechanistic understanding of how Net1 isoforms contribute to breast cancer cell motility and invasive potential, and will provide novel targets for intervention in metastatic disease.