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

RhoGEF Net1 在乳腺癌细胞运动和转移中的作用

• 批准号: 8697496
• 负责人: Jeffrey A. Frost
• 金额: $ 31.54万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8697496
• 关键词: Acetylation; Actomyosin; Address; Affect; Automobile Driving; Biological Assay; Breast Cancer Cell; Breast Cancer Treatment; Cell Nucleus; Cell Proliferation; Cell membrane; Cells; Cytoskeletal Gene; Cytoskeletal Modeling; Data; Deacetylase; Defect; Development; Disease; EGF gene; ERBB2 gene; Epidermal Growth Factor Receptor; Extracellular Matrix; Focal Adhesions; GTP-Binding Proteins; Gene Expression; Genes; Genetic; Growth Factor Receptors; Guanine Nucleotide Exchange Factors; HDAC6 gene; Histone Deacetylase; Human; In Vitro; Integrins; Intervention; Knockout Mice; Ligands; MMP14 gene; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Mediator of activation protein; Mesenchymal; Metastatic to; Mitotic; Molecular; Movement; Mus; Mutation; Neoplasm Metastasis; Neuroepithelial; Nodal; Oncogenes; Oncogenic; PTK2 gene; Phosphorylation; Process; Protein Family; Protein Isoforms; Proteins; Receptor Activation; Role; Signal Transduction; Site; Tubulin; United States; Woman; Work; ameboid movement; base; cancer cell; cell motility; cell transformation; in vivo; killings; malignant breast neoplasm; mammary gland development; new therapeutic target; novel; novel therapeutics; overexpression; prevent; public health relevance; rho; rho GTP-Binding Proteins; tumorigenesis

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.

描述（由申请人提供）：控制乳腺癌转移的分子机制尚不清楚。在细胞外基质（ECM）的侵袭期间，癌细胞在快速、变形虫和缓慢间充质类型的运动之间交替。这种运动的可塑性对于有效的入侵是必不可少的，但人们还不清楚它是如何发生的。 控制。我们发现RhoGEF Net 1A是乳腺癌细胞侵袭可塑性的重要调节因子，刺激变形虫并抑制间充质运动。Net 1A通过控制RhoA和去乙酰化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。Net 1A在RhoGEFs中是例外的，因为它被隔离在细胞核中以防止异常的RhoA激活，并且很明显，亚细胞定位是Net 1亚型致癌潜力的关键决定因素。最近，我们已经表明，Rac 1激活下游的整合素或生长因子受体激活刺激Net 1A重新定位到质膜。我们还发现，Net 1A的重新定位是通过位点特异性乙酰化来维持的，并且特异性脱乙酰酶的作用对于抑制Net 1A对RhoA的活性至关重要。此外，我们已经观察到，Net 1A激酶控制特定脱乙酰酶的活性，以调节细胞骨架组织和与细胞侵袭相关的关键基因（如MT 1-MMP）的表达。这些发现支持我们的假设，即Net 1A作为节点的功能，在时间和空间上调节RhoA激活和脱乙酰酶活性，这是乳腺癌细胞运动，侵袭和转移的基础。在这个建议中，我们将使用基于细胞和体内试验的组合，以了解Net 1A定位是如何调节，以驱动RhoA激活和ECM入侵过程中控制脱乙酰酶活性。我们还将确定Net 1亚型如何在体内促进乳腺肿瘤发生和转移。在目标1中，我们将展示EGFR和HER 2激活如何刺激Net 1A从细胞核输出，以刺激细胞运动和ECM侵袭。我们将展示Net 1A乙酰化如何影响其功能，并确定调节Net 1A的脱乙酰酶。在目标2中，我们将确定Net 1A如何控制HDAC和Sirtuin功能，以调节细胞骨架组织蛋白和基因表达的乙酰化。在目标3中，我们将确定Net 1基因缺失如何影响小鼠乳腺发育的分子基础，并确定Net 1是否是HER 2/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一个机制的理解如何Net 1亚型有助于乳腺癌细胞的运动性和侵袭性的潜力，并将提供新的目标，在转移性疾病的干预。