Imaging VHL-Associated Tumors with Labile O2 Biosensors

描述(申请人提供)：缺氧诱导因子(HIF)在多种肿瘤中过度产生，是涉及血管生成和肿瘤细胞代谢的基因的关键调节因子。Von HippeI-Lindau肿瘤抑制蛋白(PVHL)是泛素连接酶的一种成分，它针对HIF进行多泛素化，从而在有氧的情况下降解蛋白酶体。最近的研究表明，pVHL与HIF的相互作用是由HIF中存在的一种保守肽(以下称为奇肽)的酶促脯氨酸羟化来调节的。我的初步研究表明，奇数肽与荧光素酶(‘ODDLuc’)的融合产生了一种嵌合蛋白，这种嵌合蛋白在氧气存在的情况下被pVHL泛素化并被破坏。由于荧光素酶活性可以非侵入性地测量，我假设ODDLuc嵌合体将允许我实时测量完整组织和动物中氧气的可用性和pVHL途径的完整性。在特定的目标1：我将验证ODDLuc荧光素酶在肿瘤移植瘤中的表达反映了氧气的可获得性和VHL泛素连接酶的活性。在特定的目标2：我将通过建立ODDLuc敲入小鼠(rosa26-ODDLuc)并确定该小鼠中的荧光素酶信号是否可用于监测缺氧来进行体内研究。虽然显然，rosa26-ODDLuc小鼠在研究缺乏氧气(缺血)的疾病方面有很大的实用价值，但我的目标是建立一个小鼠模型，用于对VHL相关肿瘤进行成像。因此，在特定的目标3：rosa26-ODDLuc小鼠将被杂交到已经被设计成发展VHL(-/-)肿瘤的小鼠。这些小鼠将通过荧光素酶成像来验证检测VHL丢失的能力。我为这些小鼠设想的未来可能性之一是将它们用于小分子的药效学研究，这些小分子可能被预测为预防或治疗VHL相关肿瘤。

DESCRIPTION (provided by applicant): Hypoxia-inducible factor (HIF) is overproduced in a wide variety of tumors and is a key regulator of genes involved in angiogenesis and tumor cell metabolism. The von HippeI-Lindau tumor suppressor protein (pVHL) is a component of a ubiquitin ligase that targets HIF for polyubiquitination, and hence proteasomal degradation, in the presence of oxygen. Recent studies have shown that the interaction of pVHL with HIF is regulated by enzymatic prolyl hydroxylation of a conserved peptide (hereafter called the 'ODD' peptide) present in HIF. My preliminary studies suggest that fusion of the ODD peptide to luciferase ('ODDLuc') gives rise to a chimeric protein that is ubiquitinated and destroyed by pVHL in the presence of oxygen. Since luciferase activity can be measured non-invasively, I hypothesize that the ODDLuc chimera will allow me to measure, in real-time, the availability of oxygen and the integrity of the pVHL pathway in intact tissues and animals. In Specific Aim 1: I will validate that ODDLuc luciferase expression in tumor xenografts reflects the availability of oxygen and the activity of the VHL ubiquitin ligase. In Specific Aim 2: I will proceed to in vivo studies by generating an ODDLuc knock-in mouse (ROSA26-ODDLuc) and determining whether the luciferase signal in this mouse can be used to monitor hypoxia. Although clearly the ROSA26-ODDLuc mouse would have great utility in studying diseases in which there is a lack of oxygenation (ischemia) my goal is to develop a mouse model for imaging VHL-associated neoplasms. Therefore, in Specific Aim 3: the ROSA26-ODDLuc mouse will be crossed to mice that have been engineered to develop VHL (-/-) tumors. These mice will be validated for the ability to detect VHL loss by luciferase imaging. One of the future possibilities I envision for these mice is their use in pharmacodynamic studies of small molecules that might be predicted to prevent or treat VHL-associated neoplasms.