Imaging Rho-C Oncogene Function in Human Mammary Cells

描述(申请人提供)：快速表征活细胞中癌基因的分子功能和/或激活的能力将是癌症诊断、基于分子的治疗计划和癌症预防的根本进步。炎症性乳腺癌(IBC)是一种侵袭性的局部晚期乳腺癌，具有高度血管生成、侵袭性和转移性。RHOC-GTP酶已被Merajver实验室确定为侵袭性乳腺癌表型的特异性标记，尤其是IBC。RhoC是一种人类乳腺上皮细胞的转化癌基因，其过度表达可导致高度侵袭、血管生成和转移表型，与IBC极其相似。该癌基因的激活和抑制的生物物理机制(包括详细的分子结合和细胞定位)尚不完全清楚，但这些特征影响肿瘤的功能和生物活性。在这项R21应用中，我们建议使用Mycek实验室开发的用于细胞和分子成像的强度和寿命荧光共振能量转移(FRET)技术和方法，在活体人类IBC细胞模型中研究RHOC-GTP酶的定位、激活和抑制。FRET测量检测具有纳米尺度空间灵敏度的分子定位和结合。荧光寿命传感的使用为在活细胞中执行定量FRET测量提供了额外的对比源，同时通常独立于影响荧光强度的伪影(包括荧光团浓度、光漂白和生物系统中的光损失(吸收和散射)源)，这些伪影可能使强度测量的解释复杂化。具体目的1：研究RhoC-GTPase癌基因在活体正常乳腺上皮细胞和RhoC诱导的恶性肿瘤细胞中的定位。具体目标2：确定RhoC在与关键效应因子Rhotekin结合和不结合的情况下的激活状态。具体目标3：确定已知的RHOC-GTP酶激活抑制剂：C3外转移酶和法尼基转移酶抑制剂(FTI 832)引起的FRET和RHOC定位的变化。R21提案中开发的分子成像方法本质上是交叉的，因为它们广泛适用于从患者那里获得的活细胞中细胞过程和结构的基础生物学研究，并将很快应用于临床。

DESCRIPTION (provided by applicant): The ability to rapidly characterize molecular function and/or activation of oncogenes in living cells would be a fundamental advance in the diagnosis of cancer, the planning of molecular-based therapies, and in cancer prevention. Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer that is highly angiogenic, invasive, and metastatic. RhoC-GTPase has been identified in the Merajver lab as a specific marker of aggressive breast cancer phenotypes and of IBC in particular. RhoC behaves as a transforming oncogene of human mammary epithelial cells whose overexpression can lead to a highly invasive, angiogenic, and metastatic phenotype, extremely akin to IBC. The biophysical mechanisms for activation and inhibition of this oncogene (including detailed molecular associations and cellular localization) are not completely understood; however, these features impact on function and biological activity in tumors. We propose in this R21 application to investigate RhoC-GTPase localization, activation, and inhibition in living human cellular models of IBC using intensity and lifetime fluorescence resonance energy transfer (FRET) technology and methods developed in the Mycek lab for cellular and molecular imaging. FRET measurements detect molecular localization and binding with nanoscale spatial sensitivity. The use of fluorescence lifetime sensing offers an additional source of contrast for performing quantitative FRET measurements in living cells, while being generally independent of artifacts influencing fluorescence intensity (including fluorophore concentration, photobleaching, and sources of optical loss (absorption and scattering) in biological systems) that may complicate the interpretation of intensity measurements. Specific Aim 1: Characterize RhoC-GTPase oncogene localization in living normal mammary epithelial cells and in RhoC-driven malignant cells. Specific Aim 2: Determine RhoC activation states with and without binding to the key effector, rhotekin. Specific Aim 3: Define the changes in FRET and RhoC localization caused by known inhibitors of RhoC-GTPase activation: C3 exotransferase and a farnesyl transferase inhibitor (FTI 832). The molecular imaging methods developed in this R21 proposal are cross-cutting in nature in that they are broadly applicable to basic biological studies of cellular processes and structures in living cells obtained from patients, and would find rapid translation to the clinic.