VHL-deficient renal cancer cells gain resistance to mitochondria-activating apoptosis inducers by activating AKT through the IGF1R-PI3K pathway

Abstract

We previously developed (2-deoxyglucose)-(ABT-263) combination therapy (2DG-ABT), which induces apoptosis by activating Bak in the mitochondria of highly glycolytic cells with varied genetic backgrounds. However, the rates of apoptosis induced by 2DG-ABT were lower in von Hippel-Lindau (VHL)-deficient cancer cells. The re-expression of VHL protein in these cells lowered IGF1R expression in a manner independent of oxygen concentration. Lowering IGF1R expression via small interfering RNA (siRNA) sensitized the cells to 2DG-ABT, suggesting that IGF1R interfered with the activation of apoptosis by the mitochondria. To determine which of the two pathways activated by IGF1R, the Ras-ERK pathway or the PI3K-AKT pathway, was involved in the impairment of mitochondria activation, the cells were treated with a specific inhibitor of either PI3K or ERK, and 2DG-ABT was added to activate the mitochondria. The apoptotic rates resulting from 2DG-ABT treatment were higher in the cells treated with the PI3K inhibitor, while the rates remained approximately the same in the cells treated with the ERK inhibitor. In 2DG-ABT-sensitive cells, a 4-h 2DG treatment caused the dissociation of Mcl-1 from Bak, while ABT treatment alone caused the dissociation of Bcl-xL from Bak without substantially reducing Mcl-1 levels. In 2DG-ABT-resistant cells, Mcl-1 dissociated from Bak only when AKT activity was inhibited during the 4-h 2DG treatment. Thus, in VHL-deficient cells, IGF1R activated AKT and stabilized the Bak-Mcl-1 complex, thereby conferring cell resistance to apoptosis.

Keywords: 2-deoxyglucose; ABT-263; AKT; Apoptosis; ERK1/2; PI3K; Ras.

Fig. 1

The rates of apoptosis induced by 2DG-ABT were lower in VHL-deficient cancer cells under both normoxic and hypoxic conditions. a The renal cancer cell lines, RCC4, RCC4 + VHL, UOK121, UOL121 + VHL, and UOK121 were treated with 5-Aza-dC (UOK121 + 5-Aza-dC) to restore VHL expression. Subsequently, the cells were tested for sensitivity to 2DG-ABT combination therapy. First, the cells were treated with 10 mM 2DG and, 2 h later, with 10 mM ABT. At 4 h from the start of the combination treatment, the cells were washed and re-incubated in fresh media. Cells were assayed under either normoxic (21 % oxygen) or hypoxic (1 % oxygen) conditions and analyzed for propidium iodide incorporation by FACS. Graphs of the FACS data are shown. Inserted Panel. Western blots of UOK121 cells treated with or without 5-Aza-dCyd. b For ease of comparison, the rates of apoptosis of cells with or without VHL were placed next to each other in this panel

Fig. 2

The absence of VHL stabilized IGF1R expression independent of oxygen concentrations and interfered with mitochondria-dependent apoptosis. a Western blots of RCC4, RCC4 + VHL, UOK121, and UOK121 + VHL cells cultured under normoxic (N) or hypoxic (H) conditions and probed with antibodies against IGF1R, Hif1a, VHL, and beta-tubulin. b Western blots of either RCC4- or IGF1R-depleted RCC4 (RCC4ΔIGF1R) cells treated with 2DG, the IGF1R specific inhibitor, picropodophyllin (PPP), both, or left untreated. The blots were probed with antibodies against phosphor-tyrosine-IGF1R, phosphor-serine473-AKT, pan-IGF1R, pan-AKT ,and β-tubulin. c FACS analysis of RCC4- and IGF1R-depleted RCC4 cells treated with 0–3 mM ABT, with or without 2DG. d Comparison of apoptotic rates using 10 mM 2DG and 1 mM ABT-263 in RCC4 (28+/−3 %), RCC4-VHL (46.206 %+/−3.91) and RCC4 cells in which IGF1R had been depleted, designated RCC4 delIGF1R (77+/−2). The unpaired t test results were *1p = 0.0052, *2p = 0.0024, and *3p = 0.0003. We noted that RCC4 delIGF1R was even more sensitive than RCC4 + VHL, in which there is some IGF1R expression

Fig. 3

IGF1R activated both the Ras-ERK and PI3K-AKT pathways, but only the latter pathway interfered with mitochondria-dependent apoptosis. a Serum-starved UOK121 cells were stimulated with or without 20 ng/ml of IGF1 for 10 min in the presence or absence of either 50 μM PD98509 (ERK inhibitor) or 50 μM LY29402 (PI3K inhibitor). Cells were harvested and analyzed by western blotting using anti-phospho-IGF1R, anti-pS473-AKT, anti-P(T202/Y204)-ERK, and anti-IGF1R, PI3K, AKT, and ERK antibodies, as indicated. b UOK121 cells were first treated with or without 10 mM 2DG for 2 h, after which, either PD98509, LY294002, or DMSO was added for 5 min, prior to the addition of either 2 μM ABT-263 or DMSO for another 2 h. Then, the cells were washed, re-seeded in fresh media, and incubated overnight. The experiment was performed in triplicate, and live cells were counted the next day. The error bars indicate the standard deviation

Fig. 4

Inhibiting AKT sensitized UOK121 cells to 2DG-induced dissociation of Mcl-1 from Bak. Cells were treated with 10 mM 2DG, 50 μM LY294002, both, or left untreated for 4 h. The cells were harvested, and whole cell extracts were analyzed by western blotting (a). Some cells were also resuspended in immunoprecipitation buffer (see Materials and Methods section) and immunoprecipitated with anti-Bak antibody (b; upper panel ), anti-Bak antibody in the presence of 2 μM ABT-263 (b; middle panel ), or with anti-Mcl-1 antibody (b; lower panel ). The immunoprecipitates were analyzed by western blotting. For a preliminary experiment showing that ABT-263 could disrupt binding between Bak and Bcl-xL in vitro, see Sup Fig. 2

Fig. 5

Partial depletion of AKT sensitizes RCC4 cells to 2DG-induced dissociation of Mcl-1 from Bak, thereby sensitizing cells to 2DG-ABT induced apoptosis. (a; left panel ) AKT isoforms 1 and 2 were depleted from RCC4 with siRNA for 36 h, and the resulting cells are indicated as ΔAKT1/2. These cells and untreated cells were analyzed by western blotting. (a; right panel ) These cells were assayed for 2DG-ABT-induced apoptosis, as in Fig. 3b. Live cells were counted, and the results are shown in the graphs. (b; WCL) RCC4 and ΔAKT1/2 were also incubated with 10 mM 2DG for 4 h, and the whole cell lysates analyzed by western blotting. (b; Mcl-1 IP and Bak IP panels). 2DG-treated and untreated cells were also re-suspended in immunoprecipitation buffer and incubated with anti-Mcl-1 or anti-Bak antibodies. The immunoprecipitates were analyzed by western blotting

Fig. 6

The model of signaling pathways in RTK-expressing cells. (1) In RTK-activated cells, AKT is activated by PI3K, stabilizing the Bak-Mcl-1 association in the mitochondria. (2) In the absence of activated AKT, 2DG can induce the dissociation of Mcl-1 from Bak. (3) ABT-263 binds to Bcl-xL with high affinity, dissociating it from Bak. (4) When freed from all its inhibitory associations, Bak is activated, forming a pore on the mitochondria. (5) Cytochrome c is released into cytosol. (6) The release of cytochrome c triggers the assembly of the death inducing apoptosome in the cytosol