Metabolic targeting of hypoxia and HIF1 in solid tumors can enhance cytotoxic chemotherapy

Abstract

Solid tumors frequently contain large regions with low oxygen concentrations (hypoxia). The hypoxic microenvironment induces adaptive changes to tumor cell metabolism, and this alteration can further distort the local microenvironment. The net result of these tumor-specific changes is a microenvironment that inhibits many standard cytotoxic anticancer therapies and predicts for a poor clinical outcome. Pharmacologic targeting of the unique metabolism of solid tumors could alter the tumor microenvironment to provide more favorable conditions for anti-tumor therapy. Here, we describe a strategy in which the mitochondrial metabolism of tumor cells is increased by pharmacologic inhibition of hypoxia-inducible factor 1 (HIF1) or its target gene pyruvate dehydrogenase kinase 1 (PDK1). This acute increase in oxygen consumption leads to a corresponding decrease in tumor oxygenation. Whereas decreased oxygenation could reduce the effectiveness of some traditional therapies, we show that it dramatically increases the effectiveness of a hypoxia-specific cytotoxin. This treatment strategy should provide a high degree of tumor specificity for increasing the effectiveness of hypoxic cytotoxins, as it depends on the activation of HIF1 and the presence of hypoxia, conditions that are present only in the tumor, and not the normal tissue.

Fig. 1.

Inhibition of HIF1 and PDK1 increases oxygen consumption in vitro and in vivo. (a) Western blots of extracts from RKO and Su.86 cells exposed to normoxia (N) or hypoxia (H) (0.5% O₂) for 24 h in the presence or absence of 2 ng/ml echinomycin probed for the indicated proteins. (b) Oxygen consumption rates of RKO and RKOShHIF1α cells (Left) and Su.86 cells (Right) after 24 h treatment with normoxia or hypoxia (0.5% O₂) with or without 2 ng/ml echinomycin. Data are normalized to normoxic samples. (c) Relative oxygen consumption of RKO cells treated with hypoxia (0.5% O₂) for 24 h in the presence of increasing concentrations of echinomycin. (d) Oxygen consumption rates of freshly explanted tumor tissue from RKO (n = 16) and RKOShHIF1α (n = 8) xenografts. (e) Oxygen consumption rates of RKO and RKOShHIF1α tumors from mice treated with 0.12 mg/kg echinomycin i.p. 24 h prior (n = 8), or 50 mg/kg DCA i.p. 4 h prior (n = 8). Data are normalized to PBS treated controls (n = 8–16). ∗, Significant difference relative to control (P < 0.05).

Fig. 2.

Increasing oxygen consumption by inhibition of PDK activity increases tumor hypoxia. (a) Luciferase activity of WT and HIF1α knockdown RKO cells stably transfected with a HIF1 responsive luciferase reporter gene. Cells were exposed to 0.5% O₂ for 24 h in triplicate. Luminescence is normalized to normoxic HIF WT cells. (b) Luciferase activity of WT RKO HIF1 reporter cells exposed to 0.5% O₂ for 24 h in the presence of increasing concentrations of echinomycin. Data are normalized to the increase in signal observed in the absence of drug. (c) Bioluminescent imaging in vivo. Images show a representative animal bearing an RKO reporter tumor on the left flank and an RKOShHIF1α reporter tumor on the right flank as a function of time after i.p. injection of 50 mg/kg DCA. The pseudocolor overlay shows the intensity of bioluminescence. (d) Quantification of in vivo bioluminescence. The graph shows the change in signal intensity after DCA treatment for RKO parent and RKOShHIF1α tumors. The data represent the mean of three independent experiments, each comprising five RKO and five RKOShHIF1α reporter tumors.

Fig. 3.

Increasing oxygen consumption by inhibition of HIF increases tumor hypoxia. (a–d) Pimonidazole staining of tumor sections from RKO (a and b) and RKOShHIF1α (c and d) tumors 24 h after treatment with PBS (a and c) or echinomycin (b and d). The tumor section is outlined in white, pimonidazole staining is shown in green, and the necrotic areas and cutting artifacts are masked in gray. (e) The mean hypoxic fraction of RKO and RKOShHIF1α tumors 24 h after treatment with PBS or echinomycin (n = 4–5 tumors per group). Error bars represent the standard error of the mean. ∗, Significant difference (P < 0.05).

Fig. 4.

Pharmacologic inhibition of HIF1 or PDK1 enhances the response of tumor xenografts to the hypoxic cytotoxin TPZ. (a) RKO tumor bearing mice were treated with 0.12 mg/kg echinomycin i.p. followed by 30 mg/kg TPZ i.p. at 24 h, followed by a rest day for 3 cycles. (b) RKO tumor-bearing mice were treated with 50 mg/kg DCA i.p. followed by 20 mg/kg TPZ i.p. at 4 h daily for 14 days. (c) RKO tumor-bearing mice were treated with 0.12 mg/kg echinomycin i.p. followed by 10 mg/kg etoposide i.p. at 24 h, followed by a rest day for 3 cycles. For all experiments, single agents were given at the same dose, on the same schedule. Arrows indicate the end of treatment.

Fig. 5.

Enhancement of TPZ efficacy is HIF-dependent and schedule-dependent. RKO (a), RKOShHIF1α (b), or Su.86 (c) tumor-bearing mice were treated with echinomycin followed by TPZ for six cycles (Ech+TPZ), or TPZ followed by echinomycin for 6 cycles (TPZ+Ech) as described above. Arrows indicate the end of treatment.