The goal of this proposal is to apply molecular imaging and reporter gene techniques to create in vivo sensors of HIF-1 and XBP1 activity, and to use them to study hypoxia-inducible cell signaling in animal models of cancer therapy. Identification of the hypoxia-induced transcription factors HIF-1 and XBP1 has given insight into how changes in oxygenation activate gene expression, but has also demonstrated that these factors may cause increased tumor malignancy independent of oxygen. The importance of these pathways as markers as well as effectors of the hypoxic tumor phenotype has been demonstrated, however a complete understanding of their behavior and dynamics in the in vivo tumor microenvironment has been lacking. To accomplish this task, we propose a two-year project balanced between technique development and application. We will first develop and rigorously validate a set of HIF-1- and XBP1-responsive reporter constructs in cell cultures, focusing on methods for distinguishing reporter gene expression from hypoxia-mediated loss of reporter protein activity. After evaluation of their ability to independently measure HIF-1a stabilization and HIF-1- and XBP1-induced transcriptional activity in cell culture models of oxidative stress, we will generate murine tumor models expressing each reporter gene and demonstrate the robustness of each construct to report on its target under in vivo conditions. These models will then be treated with novel anti-HIF-1 chemotherapeutics, over the course of which HIF-1 and XBP1 response will be monitored by imaging the reporters. We will therefore generate molecular biology tools to study hypoxia-inducible physiology in living subjects, and apply these tools towards the validation and optimization of novel targeted chemotherapies, accelerating their progress towards clinically useful cancer treatments. This research will develop reporter gene techniques for detecting and imaging the activity of two transcription factors, HIF-1 and XBP1, within intact tumors. By developing novel methods to quantify these factors and applying them towards the study of these pathways during cancer therapy, this project will further our understanding of tumor biology and will accelerate the development and evaluation of targeted therapies for cancer. ? ? ?
