Hypoxia is a mediator of malignant progression, with mechanisms including resistance to apoptosis and expression of angiogenic factors. The adaptive response to hypoxia is mediated through the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Solid tumors contain hypoxic cells that are resistant to many forms of anti-cancer agents. The aim of this study was to analyse the effect of HIF-1 inhibition on the drug responsiveness of hypoxic tumor cells. For this purpose a dominant negative form of HIF-1α (HIF-1α no TAD/EGFP) that lacks the transactivation and oxygen dependent degradation (ODD) domains has been developed to target HIF-1. Transient co-transfection experiments in HT1080 fibrosarcoma cells with the HIF-1α no TAD/EGFP and hypoxia-responsive (PGK HRE) luciferase reporter expression plasmids confirmed that HIF-1α no TAD/EGFP blocks normal HIF-1 function. Subsequently, the HIF-1α no TAD/EGFP cDNA was inserted into the pEF IRES-P expression vector for stable cell line generation. Two clones of HIF-1α no TAD/EGFP-transfected HT1080 cells, DN4 and DN5, were tested in all following experiments. Initial analysis of these cell lines showed that HIF-1α expression is hypoxia-dependent and remains unaltered when compared to wild-type and transfection control (pEF IRES-P or EV) cells. Experiments were then completed to look at the effect of HIF-1 inhibition on downstream targets of HIF-1. Immunohistochemical analysis of wild-type and EV cells revealed that the expression of the glucose transporter-1 (Glut-1) gene is hypoxia-dependent and that this upregulation is ablated in hypoxic DN4 and DN5 cells. However, analysis of wild-type and EV cells by ELISA revealed that expression of VEGF was high in both normoxic and hypoxic conditions yet could be down-regulated by HIF-1α no TAD/EGFP expression in DN4 and DN5 cells in both conditions. To assess the effect of HIF-1 inhibition on drug responsiveness, normoxic and hypoxic DN4 and DN5 cells were treated with the anti-cancer drug etoposide for 16 h before evaluation by clonogenic assay. It was found that DN4 and DN5 cells were more sensitive to killing by etoposide than wild-type or EV cells. Interestingly, this trend was not hypoxia-specific suggesting again that HIF-1α no TAD/EGFP can influence the phenotype of aerobic cells. A potential mechanism by which this could occur is through functional counteraction of Myc. Recent studies have shown that a constitutively expressed form of HIF-1α lacking the ODD can override Myc target gene expression (Koshiji et al. (2004), EMBO 23: 1949-55). The differential impact of HIF-1α no TAD/EGFP on VEGF versus Glut-1 expression in the DN4 and DN5 cells may be supportive of a Myc interaction as VEGF is a Myc target gene. The precise mechanism by which HIF-1α no TAD/EGFP affords chemosensitisation in both aerobic and hypoxic conditions is the focus of ongoing studies.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]