83: Molecular Imaging of HIF-1α and pVHL Interaction in Living Subjects Using Firefly Luciferase Complementation - Improvement Over the Renilla Luciferase System

Abstract

Tumor hypoxia induces genomic and proteomic changes which enhance tumor cell survival and dissemination. Clinically, hypoxic tumors are more resistant to therapy and portends for a worse outcome. Under hypoxia, HIF-1α (Hypoxia-inducible factor) regulates the activation of genes promoting malignant progression. Under normoxia, HIF-1α is hydroxylated by the prolyl hydroxylases (PHDs) and is targeted for ubiquitin mediated degradation by interacting with the von Hippel Lindau (VHL) protein complex. The study of HIF-1α and pVHL interaction has been challenging due to the transient nature of the interaction between HIF-1α and pVHL and the rapid rate of HIF-1α degradation. Here, we present a novel method of studying the interaction between HIF-1α and pVHL using the split firefly luciferase complementation based bioluminescence system and compare this to the split Renilla luciferase system. HIF-1α and pVHL split Renilla and firefly luciferase chimeras were generated by fusing the amino- and carboxy-terminal regions of the luciferase to HIF-1α and pVHL, respectively. In vitro split luciferase activity was assayed by co-transfecting the split luciferase expression plasmids into mammalian cells and measuring the complementation based luciferase activity using a luminometer. Imaging studies were performed in nude mice implanted with HIF-1α and pVHL split firefly luciferase expressing mammalian cells using an optical cooled charge-coupled devise camera. The split firefly luciferase fragments, in the absence of HIF-1α and pVHL, produce minimal complementation. Upon HIF-1α proline hydroxylation, pVHL interacts with hydroxylated-HIF-1α, leading to complementation of the split luciferase fragments and generation of an active enzyme. HIF-1α proline hydroxylation by the PHDs is a requisite step, and interfering with proline hydroxylation by using 1) HIF-1α mutants (P402A and P564G), 2) small molecule inhibitors of proline hydroxylation, or 3) genetic silencing of PHD2 using the shRNA approach leads to inhibition of HIF-1α and pVHL mediated split luciferase activity. The interaction between HIF-1α and pVHL was also imaged in living subjects. Confirming our cell culture results, the HIF-1α and pVHL split luciferase interaction in live mice also requires HIF-1α hydroxylation. In comparison to the HIF-1α and pVHL split Renilla luciferase system, the split firefly luciferase system has several advantages, including a lower background, more stable bioluminescence signal, easier administration of the luciferase substrate, and less dependence on the size of the HIF-1a construct used. The split firefly luciferase complementation based bioluminescence system represents a novel approach for studying the interaction between HIF-1α and pVHL in vivo and has several advantages over the split Renilla system. We have established a new technique for visualizing the interaction between an E3 ubiquitin ligase and its substrate and investigating the importance of post-translational modifications in protein-protein interaction. This system will allow high-throughput screening to identify compounds that modulate the interaction between HIF-1α and pVHL, and imaging studies will allow for intra-tumor target validation assessment in live animals.