Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality

Abstract

Identifying new targeted therapies that kill tumor cells while sparing normal tissue is a major challenge of cancer research. Using a high-throughput chemical synthetic lethal screen, we sought to identify compounds that exploit the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in about 80% of renal cell carcinomas (RCCs). RCCs, like many other cancers, are dependent on aerobic glycolysis for ATP production, a phenomenon known as the Warburg effect. The dependence of RCCs on glycolysis is in part a result of induction of glucose transporter 1 (GLUT1). Here, we report the identification of a class of compounds, the 3-series, exemplified by STF-31, which selectively kills RCCs by specifically targeting glucose uptake through GLUT1 and exploiting the unique dependence of these cells on GLUT1 for survival. Treatment with these agents inhibits the growth of RCCs by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue. Activity of STF-31 in these experimental renal tumors can be monitored by [(18)F]fluorodeoxyglucose uptake by micro-positron emission tomography imaging, and therefore, these agents may be readily tested clinically in human tumors. Our results show that the Warburg effect confers distinct characteristics on tumor cells that can be selectively targeted for therapy.

Fig. 1

Chemical synthetic lethal screen identifies compounds that specifically target loss of VHL in renal carcinoma. (A) Effect of STF-31 on clonogenic survival of RCC4 with and without VHL (10 days) (*P < 0.00005). (B) Representative plates of clonogenic survival in RCC4 and RCC4/VHL cells (5 μM STF-31; 10 days). (C) Time course of STF-31 (5 μM) effect on cells (*P < 0.0005). (D) Effect of STF-31 on clonogenic survival of ACHN cells with and without shRNA to VHL (*P < 0.0001). (E) Effect of STF-31 (5 μM) on cell death in RCC4 and RCC4/VHL cells treated for 3 days. Cell death was measured with trypan blue staining (*P < 0.01). (F) Effect of STF-31 on RCC4, RCC4/VHL, or RCC4/VHL cell clones overexpressing HIF-2α (*P < 0.005). All error bars represent the SEM (n = 3).

Fig. 2

STF-31 inhibits glucose metabolism in VHL-deficient cells. (A) Lactate (μmol/cell), which is converted from pyruvate, the end product of glycolysis, in RCC4 and RCC4/VHL cells treated with either vehicle or STF-31 (5 μM) (*P < 0.01). (B) Relative extracellular acidification rate (ECAR) (mpH/min) of VHL-deficient cells and cells with wild-type VHL in response to STF-31 (5 μM) for 48 hours. Cells were stained with crystal violet and absorbance was measured for normalization (*P < 0.0005). (C) Relative glucose uptake after treatment with STF-31 (5 μM). Counts are normalized to cell number (*P < 0.000005). (D) Effect of STF-31 concentration on glucose uptake (*P < 0.00005). (E) Effect of STF-31 on hexokinase activity in whole-cell lysates after STF-31 treatment (5 μM). (F) Effect of STF-31 (5 μM) on glucose uptake in RCC4 cells transfected with small interfering RNA (siRNA) to HIF-1β (*P < 0.05). (G) Effect of STF-31 (5 μM) on oxygen consumption (nmol/min/10⁶ cells). (H) Effect of STF-31 (5 μM) on relative ATP levels in cells with and without VHL (*P < 0.005). (I) Effect of STF-31 on ATP levels in cells with and without VHL (*P < 0.01). (J) Effect of STF-31 (5 μM) on glucose uptake and clonogenic cell survival in cells without VHL up to 72 hours. (K) Effect of STF-31 (5 μM) on glucose uptake and clonogenic cell survival in cells with VHL up to 72 hours. All error bars represent the SEM (n = 3).

Fig. 3

VHL-deficient renal carcinomas are more sensitive to glucose deprivation and have higher GLUT1 levels than RCCs with wild-type VHL. (A) Effect of lack of glucose and/or pyruvate for 6 days on the number of RCC4 and RCC4/VHL cells (*P < 0.005). (B) Effect of lack of glucose and/or pyruvate for 6 days on the number of 786-O and 786/VHL cells (*P < 0.005). (C) Expression of GLUT1, GLUT2, GLUT3, and GLUT4 in a renal cancer data set containing normal renal tissue (Normal) and renal clear cell carcinomas (RCC) (17). (D) Relative mRNA expression of GLUT1 and GLUT2 in RCC4 and RCC4/VHL as determined by quantitative real-time PCR [normalized to TATA box binding protein (TBP)]. (E) Correlation of GLUT1 and GLUT2 expression in a renal cancer data set (17). All error bars represent the SEM (n = 3). (F) VHL status, HIF-1, HIF-2, GLUT1, and GLUT2 expression, and sensitivity to STF-31 in a panel of RCC cell lines.

Fig. 4

Inhibition of GLUT1 leads to cell death in VHL-deficient cells. (A) Schematic representation of STF-31 docking within the solute channel of GLUT1. (B) Cell lysates of RCC4 and RCC4/VHL were incubated with Affi-Gel–immobilized STF-31 derivatized with a linker, and then eluted with urea buffer. Elutions were probed with antibodies to GLUT1, GLUT2, or GLUT3. (C) Relative mRNA levels of GLUT1 from RCC4 and RCC4/VHL cells carrying a stable, inducible shRNAmir to GLUT1. Cells were treated with doxycycline (DOX) (500 ng/ml) for 5 days. (D) Cell viability of RCC4 and RCC4/VHL after induction of the shRNAmir to GLUT1. All error bars represent the SEM (n = 3). (E) Fluorescence-activated cell sorting analysis of RCC4 and RCC4/VHL cells expressing an inducible shRNAmir to GLUT1. Cells were treated with doxycycline (500 ng/ml) to induce the shRNAmir, which also turns on the expression of TurboRFP. After 4 days, cells were collected, stained with a marker of cell necrosis, and subjected to flow cytometry.

Fig. 5

Efficacy of 3-series drugs can be monitored in vivo. (A) Glucose uptake measured in human erythrocytes after treatment with a soluble STF-31 analog (5 μM) (*P < 0.001) (n = 3). (B) Representative photos of human red blood cells treated with either red blood cell (RBC) lysis buffer or an STF-31 analog (5 μM). (C) FDG-PET imaging of 786-O cells, an RCC with a naturally occurring VHL mutation, implanted subcutaneously into the flanks of CD-1 nude mice. Representative axial cross section of a mouse before treatment (left) and after three daily intraperitoneal injections of a soluble analog of STF-31 (11.6 mg/kg) (right), overlaid with CT scan. (D) Quantification of FDG-PET inhibition as determined by the 90th percentile region of interest for percent injected dose per gram (ID/g) (*P < 0.01). (E) (a) Kidney from vehicle-treated mouse. (b) Kidney from STF-31 analog–treated mouse. (c) Spleen from vehicle-treated mouse. (d) Spleen from STF-31 analog–treated mouse. (e) Liver from vehicle-treated mouse. (f) Liver from STF-31 analog–treated mouse. (g) Heart from vehicle-treated mouse. (h) Heart from STF-31 analog–treated mouse. (i) Salivary gland from vehicle-treated mouse. (j) Salivary gland from STF-31 analog–treated mouse. (k) Brain from vehicle-treated mouse. (l) Brain from STF-31 analog–treated mouse. Animals were treated for 10 days with vehicle or drug (11.6 mg/kg for the first 3 days, followed by 7.8 mg/kg for the next week). Scale bar, 100 μm. (F) 786-O tumor-bearing mice were treated once or twice a day with vehicle (11 animals) or a soluble STF-31 analog (12 animals) (11.6 mg/kg for the first 3 days, followed by 7.8 mg/kg for the next week) (*P < 0.005). (G) ACHN cells expressing a short hairpin RNA to VHL were implanted subcutaneously into the flanks of immunocompromised mice (five mice per group). Once tumors reached an average of >60 mm³, mice were treated daily with STF-31 analog or vehicle (*P < 0.01). All error bars represent the SEM.

Fig. 6

STF-31 is synthetically lethal to cells dependent on GLUT1 for aerobic glycolysis. VHL regulates both HIF-1 and HIF-2, which can increase GLUT1 expression. HIF-1 also inhibits the mitochondria, causing cells to switch to aerobic glycolysis. STF-31 compounds inhibit GLUT1 in RCCs with high levels of GLUT1. These RCCs are dependent on GLUT1 for glycolysis and cell viability.