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. 2008 Nov 15;68(22):9323-30.
doi: 10.1158/0008-5472.CAN-08-2873.

Preferential cytotoxicity of bortezomib toward hypoxic tumor cells via overactivation of endoplasmic reticulum stress pathways

Diane R Fels  1 , Jiangbin Ye, Andrew T Segan, Steven J Kridel, Michael Spiotto, Michael Olson, Albert C Koong, Constantinos Koumenis
Affiliations

Preferential cytotoxicity of bortezomib toward hypoxic tumor cells via overactivation of endoplasmic reticulum stress pathways

Diane R Fels et al. Cancer Res. .

Abstract

Hypoxia is a dynamic feature of the tumor microenvironment that contributes to drug resistance and cancer progression. We previously showed that components of the unfolded protein response (UPR), elicited by endoplasmic reticulum (ER) stress, are also activated by hypoxia in vitro and in vivo animal and human patient tumors. Here, we report that ER stressors, such as thapsigargin or the clinically used proteasome inhibitor bortezomib, exhibit significantly higher cytotoxicity toward hypoxic compared with normoxic tumor cells, which is accompanied by enhanced activation of UPR effectors in vitro and UPR reporter activity in vivo. Treatment of cells with the translation inhibitor cycloheximide, which relieves ER load, ameliorated this enhanced cytotoxicity, indicating that the increased cytotoxicity is ER stress-dependent. The mode of cell death was cell type-dependent, because DLD1 colorectal carcinoma cells exhibited enhanced apoptosis, whereas HeLa cervical carcinoma cells activated autophagy, blocked apoptosis, and eventually led to necrosis. Pharmacologic or genetic ablation of autophagy increased the levels of apoptosis. These results show that hypoxic tumor cells, which are generally more resistant to genotoxic agents, are hypersensitive to proteasome inhibitors and suggest that combining bortezomib with therapies that _target the normoxic fraction of human tumors can lead to more effective tumor control.

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Figures

Figure 1
Figure 1. Hypoxic tumor cells are sensitive to ER stressors
Tumor cells were exposed to normoxic or hypoxic conditions for 12h before treatment with the indicated agents for 12h (DLD1) or 24h (HeLa). Error bars represent s.e. values. (A) Clonogenic survival of HeLa cells treated with thapsigargin. ** p = 0.001; *** p = 0.0002. (B) Hypoxic HeLa cells are more resistant to etoposide. *** p < 0.001. (C) Clonogenic survival of HeLa cells treated with bortezomib. * p ≤ 0.03; *** p < 0.001. (D) Hypoxic DLD1 cells are also more sensitive to bortezomib than normoxic cells. *** p < 0.001.
Figure 2
Figure 2. Bortezomib enhances UPR signaling under normoxia and hypoxia
(A) ATF4 levels in HeLa cells exposed to normoxic or hypoxic conditions for 2h before treatment with DMSO or bortezomib for 6h. Thapsigargin (TH; 1μM, 4h) was used as a positivecontrol. CHOP levels in HeLa cells exposed to hypoxia 6h before treatment with DMSO or bortezomib for 12h. Thapsigargin (TH; 500nM, 12h) was used as a positive control. Nuclear protein was isolated and immunoblotted for ATF4 and CHOP; lamin A/C (loading control). (B) Active XBP-1 protein (XBP-1376aa) was induced in HeLa cells exposed to normoxia or hypoxia for 6h before treatment with DMSO or bortezomib for 18h. Thapsigargin (1μM, 12h) was used as a control. Nuclear protein was isolated and immunoblotted for XBP-1; lamin A/C (loading control). (C) HT1080-ATF4-Luc cells were treated with DMSO or bortezomib and luminescence was measured 8h later. (D) Left Panel: mice with HT1080-CMV-Luc tumors on the left flank and HT1080-ATF4-Luc tumors on the right flank were given IP injections of the vehicle or bortezomib (1mg/kg) and imaged for optical bioluminescence 8h later. Right panel: Quantitation of in vivo bioluminescence of mice shown on the left panel. Results are normalized to CMV-luc activity in untreated (vehicle-injected) animals.
Figure 3
Figure 3. Ameliorating ER stress with CHX reverses the enhanced cytotoxic activity of bortezomib towards hypoxic tumor cells
(A) Unspliced XBP-1U (473bp) and spliced XBP-1S (447bp) mRNA detected by RT-PCR on RNA from HeLa cells exposed to normoxia or hypoxia for 6h before treatment with DMSO, bortezomib and cycloheximide (1μg/ml) for 18h. Thapsigargin (250nM, 8h) was used as a control; GAPDH (loading control). (B) Clonogenic survival of HeLa cells exposed to normoxic or hypoxic conditions for 12h before treatment with DMSO, bortezomib and cycloheximide (1μg/ml) for 24h. Experiments were performed in triplicate and error bars represent S.E. values.
Figure 4
Figure 4. Bortezomib induces apoptotic cell death in hypoxic DLD1 but not in HeLa cells
(A) DLD1 cells were exposed to normoxic or hypoxic conditions for 12h before treatment with DMSO or bortezomib for 24h. Immunoblotting was performed using antibodies specific for cleaved PARP and β-actin (loading control). (B) HeLa cells were exposed to normoxic or hypoxic conditions for 12h before treatment with DMSO or bortezomib for 24h. Staurosporine (STS; 1μM, 4hr) was used as a control. Immunoblotting was performed using antibodies specific for cleaved caspase-3, cleaved PARP and β-actin (loading control).
Figure 5
Figure 5. Bortezomib induces increased autophagy in hypoxic HeLa cells
(A) HeLa cells 24h-post transfection with GFP-LC3 were exposed to normoxic or hypoxic conditions with bortezomib for 4h. Chloroquine (CQ; 10μM, 4h) was used as a control. Two different cells are shown for each treatment. (B) HeLa cells were exposed to normoxic or hypoxic conditions for 6h before treatment with DMSO or bortezomib for 18h. EBSS was used as a control. Cells were stained with acridine orange and red fluorescence was detected by flow analysis. (C) HeLa cells were exposed to normoxic or hypoxic conditions for 12h before treatment with DMSO or bortezomib for 24h. Chloroquine (10μM) was present during the last 4h of treatment. TRAIL (TR; 50ng/ml, 4h) was used as a control. Immunoblotting was performed using antibodies specific for cleaved PARP and β-actin (loading control). (D) HeLa cells, transfected with si RNA against Beclin1 or a non-_targeting control siRNA, were treated 30h-post transfection as in B. Staurosporine (STS; 1μM, 4hr) was used as a control. Immunoblotting was performed using antibodies specific for Beclin1, cleaved PARP and β-actin (loading control).
Figure 6
Figure 6. Hypoxic HeLa cells treated with bortezomib undergo necrotic death
(A) HeLa cells were exposed to normoxic or hypoxic conditions for 12h before treatment with DMSO or bortezomib for 24h. LDH activity was assayed in media off each sample at the indicated doses. Error bars represent s.e. values. (B) Protein was isolated from the media off HeLa cells treated as in A. Freeze/thawed HeLa cells (FT) were used as a control. Immunoblotting was performed using an antibody specific for HMGB1; the Ponceau-stained membrane is shown as a loading control. (C). Model describing the lethal effects of the combination of hypoxia and the proteasome inhibitor bortezomib in DLD1 cells (top) or HeLa cells (bottom). See Discussion for more details.
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