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. ? ?
| Kim, William Y; Perera, Samanthi; Zhou, Bing et al. (2009) HIF2alpha cooperates with RAS to promote lung tumorigenesis in mice. J Clin Invest 119:2160-70 |
