A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation

Abstract

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development and progression by regulating genes that are vital for proliferation, glycolysis, angiogenesis, and metastasis. To identify strategies of targeting the HIF-1 pathway, we screened a siRNA library against the entire druggable genome and a small-molecule library consisting of 691,200 compounds using a HIF-1 reporter cell line. Although the siRNA library screen failed to reveal any druggable targets, the small-molecule library screen identified a class of alkyliminophenylacetate compounds that inhibit hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations. These compounds were found to inhibit hypoxia but not deferoxamine-induced HIF-1alpha protein stabilization. Further analysis indicated that the alkyliminophenylacetate compounds likely inhibit the HIF-1 pathway through blocking the hypoxia-induced mitochondrial reactive oxygen species (ROS) production. Strikingly, all of the nonalkyliminophenylacetate HIF-1 inhibitors identified from the small-molecule library screen were also found to target mitochondria like the alkyliminophenylacetate compounds. The exclusive enrichment of mitochondria inhibitors from a library of >600,000 diverse compounds by using the HIF-1 reporter assay highlights the essential role of mitochondria in HIF-1 regulation. These results also suggest that targeting mitochondrial ROS production might be a highly effective way of blocking HIF-1 activity in tumors.

Fig. 1.

siRNA library screen using the HIF-1 reporter assay. (A) The H1299_HRE cells transfected with a control siRNA (Con) or HIF-1α siRNA (Hif-1α) were subjected to hypoxia treatment and assayed for luciferase activity afterward. (B) The H1299_HRE cells were plated in 96-well plates. For each plate, half of the plate was transfected with a scramble siRNA and the other half of the plate was transfected with a HIF-1α siRNA. Forty-eight hours after transfection, the siRNA-transfected cells were subjected to hypoxia treatment for 16 h. The cells were then collected to determine the firefly luciferase activity. Z′ was calculated as Z′ = 1 − (3 σ_H + 3 σ_N)/μ_H − μ_N. In both A and B, siRNA was transfected at a final concentration of 20 nM.

Fig. 2.

Small-molecule library screen using the HIF-1 reporter assay. The structures and EC₅₀s of representative alkyliminophenylacetate compounds.

Fig. 3.

The alkyliminophenylacetate compounds are specific HIF-1 inhibitors. (A) The H1299_HRE cells, HeLa_HSP70P cells, and A549_NF-κB cells were treated with C2 by using indicated concentrations for 4 h. The cells were then treated with hypoxia, heat shock, or IL-1 to activate each reporter, respectively. At the end of each treatment, the cells were collected and assayed for luciferase activity. (B) The HCT116 and DLD-1 cells were transiently transfected with the HIF-1 reporter, pHRE, and the constitutive renilla luciferase reporter, pRL-SV40. The reporter-transfected cells were then incubated with each compound at indicated concentrations, treated with hypoxia, and assayed for luciferase activity. (C) The H1299 cells cultured at the normoxic conditions (Con-N) or the H1299 cells cultured in the presence of DMSO (Con) or 1 μM compounds (samples 1–4 represent compounds C1, C2, C3, and C4) were treated with hypoxia or DFO. The mRNA levels of PGK1 and Enolase 1 in cells were then determined by qPCR.

Fig. 4.

The alkyliminophenylacetate compounds inhibit hypoxia-induced but not DFO-induced HIF-1α accumulation. The H1299 cells cultured without the presence of compounds at the normoxic conditions (Normoxia, Con) and the H1299 cells cultured in the presence of DMSO (Con) or 1 μM compounds C1, C2, C3, and C4 (1, 2, 3, and 4, respectively) were treated with hypoxia or 100 μM DFO for 4 h. The cells were then lysed and analyzed by Western blotting.

Fig. 5.

The alkyliminophenylacetate compounds likely inhibit HIF-1 by blocking mitochondrial ROS production. (A) The EC₅₀s of the alkyliminophenylacetate compounds for the HIF-1 reporter inhibition and the cytotoxicity in the galactose and glucose media. (B) The log(EC₅₀) of each compound for the HIF-1 reporter inhibition (x axis) and the cytotoxicity in the galactose media (y axis) was plotted. (C) The H1299 cells cultured under nomoxic or hypoxic conditions were either untreated or treated with various alkyliminophenylacetate compounds. The ROS levels in cells were determined by quantifying the DCF fluorescence.

Fig. 6.

Inhibiting mitochondrial ROS generation is a common mechanism used by the HIF-1 inhibitors identified from our chemical genomic screening. (A) The H1299_HRE cells were cultured in the glucose or galactose media in the presence of 1 μM (Left), 0.3 μM (Center), or 0.1 μM (Right) concentrations of different HIF-1 inhibitors. The cell viability was determined 72 h after compound treatment, and the percent cell survival was calculated as the percentage of viable cells in the compound-treated samples versus the DMSO-treated control sample. Results from a subset (19 compounds) of the 200 HIF-1 inhibitors tested were shown here. (B) The H1299_HRE cells cultured in the galactose media were transfected with different siRNAs or treated with 1 μM C3, and cell death was determined 72 h after siRNA transfection or compound treatment by using the Toxilight assay. (C) The H1299 cells cultured under nomoxic or hypoxic conditions were either untreated or treated with various compounds. The ROS levels in cells were determined by quantifying the DCF fluorescence.