Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth

Abstract

Recent studies of tissue culture cells have defined a widespread system of oxygen-regulated gene expression based on the activation of a heterodimeric transcription factor termed hypoxia-inducible factor-1 (HIF-1). To determine whether the HIF-1 transcriptional response is activated within solid tumors and to define the consequences, we have studied tumor xenografts of a set of hepatoma (Hepa-1) cells that are wild type (wt), deficient (c4), and revertant (Rc4) for an obligatory component of the HIF-1 heterodimer, HIF-1beta. Because HIF-1beta is also essential for the xenobiotic response (in which it is termed the aryl hydrocarbon receptor nuclear translocator), we also studied c31 cells, which have a different defect in the xenobiotic response and form the HIF-1 complex normally. Two genes that show different degrees of HIF-1-dependent hypoxia-inducible expression in cell culture were selected for analysis-the glucose transporter, GLUT3, and vascular endothelial growth factor (VEGF). In situ hybridization showed intense focal induction of gene expression in tumors derived from wt, Rc4, and c31 cells, which was reduced (VEGF) or not seen (GLUT3) in those derived from c4 cells. In association with these changes, tumors of c4 cells had reduced vascularity and grew more slowly. These findings show that HIF-1 activation occurs in hypoxic regions of tumors and demonstrate a major influence on gene expression, tumor angiogenesis, and growth.

Figures

Figure 1

In situ hybridization for GLUT3 mRNA. Bright-field (Upper) and dark-field (Lower) views of sections of wt Hepa-1, c4, Rc4, and c31 xenografts. Regions of high-intensity signal are seen in the dark-field views of wt Hepa-1, Rc4, and c31 tumors, but not in the c4 tumors. Each of the four views includes areas of necrosis. Arrows within necrotic areas in the bright-field views point toward the boundary with viable tumor cells. In the wt, Rc4, and c31 tumors regions of high-intensity signal border on these areas of necrosis. Final magnification, ×80.

Figure 2

In situ hybridization for VEGF and GLUT3 mRNA in wt Hepa-1 (Left) and c4 (Right) tumors. Bright-field (Top) and dark-field (Middle) views of sections hybridized to the antisense VEGF probe. In the wt Hepa-1 tumor high-level VEGF expression borders on an area of necrosis (arrows). Semi-serial sections hybridized for GLUT3 mRNA are also shown (Bottom). In the wt Hepa-1 tumor the same regions express high levels of GLUT3 and VEGF mRNA. Final magnification, ×80.

Figure 3

Immunoperoxidase labeling for the vascular endothelial marker CD31/PECAM. (A) Representative sections through wt Hepa-1 (Upper) and c4 (Lower) xenotransplants viewed with phase contrast. Vascular density is greater in the wt Hepa-1 xenograft. Final magnification, ×80. (B) Histogram showing maximum microvessel density assessed by Chalkley counting. The mean Chalkley score for tumors of each cell type is given (±SEM). n, number of tumors assessed. ∗, significant difference from c4; •, significant difference from wt (P < 0.05). (C) Histograms showing the median and 90th centile distances to the nearest labeling for CD31/PECAM from an array of points within each tumor. Mean values for the tumors of each cell type are given (±SEM). ∗, significant difference from c4; •, significant difference from wt (P < 0.05).

Figure 4

Growth of cells in tissue culture monolayers and as xenografts. (A) Growth curves for wt and c4 xenografts from the first series of tumors. Growth of the c4 tumors is seen to be generally slower than the wt tumors. One of the wt Hepa-1 implants did not form a detectable tumor. (B) Histogram showing the time taken for growth from 50 to 200 mm3 for tumors of each cell type from all three series of experiments (mean ± SEM). ∗, significant difference from c4; •, significant difference from wt (P < 0.05). (C) Histograms showing the doubling time and maximum number of cells per 60-mm dish for each of the four cell types in normoxic culture (mean of 3–6 observations, ±SEM). The differences between cell types were not statistically significant. (D) Histograms showing doubling times for cells cultured in parallel in normoxia (solid bars) and 1% oxygen (open bars). The mean from two experiments is shown.