Combining Optical Reporter Proteins with Different Half-lives Driven by Hypoxia to Monitor Hypoxia-inducible Factor-1 (HIF-1) Activity in Prostate Cancer Cells

Metastasis is a leading cause of death linked to prostate cancer and the altered tumor microenvironment. Hypoxia, specifically, plays a pivotal role in metastatic dissemination. Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor that mediates the deleterious effects of tumor hypoxia on cancer progression. HIF-1 is composed of 2 subunits: HIF-1β, which is constitutively nuclear and HIF-1α, which is constitutively transcribed but degraded under normoxia due to the presence of an oxygen–dependent degradation domain (ODD). At low pO2, HIF-1α protein is stabilized, undergoes nuclear–translocation, and binds to the hypoxia-response element (HRE) sequences of many genes involved in the metastatic cascade. Previous studies have shown that tumor hypoxia promotes spontaneous metastasis to the lymph nodes [1]. Using a combination of optical reporters with different half-lives that are induced under hypoxia, we aim to determine whether metastatic prostate cancer cells in the lymph nodes become hypoxic at the metastatic site or were already hypoxic in the primary tumor. This information is important to determine the role of hypoxic environments in the metastatic process. For this purpose, human prostate cancer PC3 cells were transfected either with the luciferase gene fused with ODD (ODD-luc, kindly provided by H. Harada)[2] or a variant of the enhanced green fluorescent protein (pd2EGFP, Clontech). Both ODD-luciferase and pd2EGFP are under the promotion of a poly-HRE sequence (5xHRE). PC3-5xHRE-ODD-luc or PC3-5xHRE-GFP cells were incubated under hypoxia (1%O2) for 48 hours and then reoxygenated. Our data indicate that the luciferase activity (luciferase assay kit, Promega) of PC3-5xHRE-ODD-luc cells rapidly decreased after reoxygenation (Fig 1A). On the other hand, EGFP levels in PC3-5xHRE-GFP cells remained stable for several hours after reoxygenation (Fig 1B&1C). To characterize the half-life of the optical reporters, cells were treated with cycloheximide (CHX, 100 µg/ml) just after reoxygenation to inhibit protein synthesis. Using CHX, the half-life of the fusion protein ODD-luc was approximately 15 minutes (Fig 1A), whereas the pd2EGFP half-life was approximately 2 hours (Fig 1B&1C). The combination of these two optical reporter genes with different half-lives will be useful for imaging temporal changes in oxygen in vivo in tumors derived from prostate cancer cells carrying both constructs (Fig 1D). This novel imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP will advance our understanding of the temporal changes in hypoxia during the metastatic journey. 1. Cairns RA, et al, Cancer Res 2004;64: 2054-2061.2. Harada H, et al, Biochem Biophys Res Commun 2007;360: 791-796