Tumor cells upregulate glucose metabolism and oxidative phosphorylation (OXPHOS) to support their enhanced anabolic demands [1
]. Apart from glycolysis, glucose is also utilized by the pentose phosphate pathway, the hexosamine pathway, and the one-carbon metabolism pathway to generate building blocks and reducing power for the cell [1
]. Indeed, glucose uptake is upregulated by various oncogenes, such as MYC [3
] and RAS [4
], with such tumor-driven high glucose uptake being exploited for diagnosis using radiolabelled glucose analogues such as 18
F-FDG and positron emission tomography. However, a number of tumors develop within a glucose-depleted tumor microenvironment [4
], as measurements of glucose levels in tumor tissues indicate a 2–45-fold reduction in glucose in such tumors compared to normal surrounding tissues [9
]. Hence, these tumor cells must evolve proper cellular mechanisms to adapt to low-glucose conditions.
In an unbiased shRNA-based (short hairpin RNA) screen targeting metabolic proteins specifically aimed at identifying proteins that support the survival of tumor cells in low-glucose-containing medium, Birsoy et al. identified mitochondrial OXPHOS proteins, and in particular, complex I proteins, as being central for the metabolic adaptation of tumor cells to low-glucose conditions [9
]. In addition, other studies have identified several protective cellular pathways supporting the survival of tumor cells growing in glucose deprivation, including the unfolded protein response (UPR) pathway [11
], the mTOR pathway [12
], and the NF-ĸB pathway [17
These requirements support the premise that proteins and cellular pathways which support cell survival under glucose starvation represent potential drug targets, as their inhibition is expected to selectively kill glucose-depleted tumor cells, but not normal tissues that do not experience glucose deprivation [9
Based on the observations described above, we performed a phenotypic synthetic lethality high-throughput (HTP) drug screen in the hope of finding new potential anticancer drugs. In doing so, we identified compounds which selectively kill tumor cells growing in glucose-free media. Strikingly, we found significant enrichment for mitochondrial poisons. We validated the activity of two such compounds, QNZ (EVP4593) and papaverine, in culture and demonstrated that a combination of the antiangiogenic agent bevacizumab with QNZ or papaverine had superior antitumor activity in vivo than shown by either agent alone. These findings support the concept that pharmacological targeting of glucose-deprived tumor cells in vitro has the power to uncover compounds that can act synergistically with antiangiogenic agents to inhibit tumor growth in vivo.
2. Materials and Methods
2.1. Cell Cultures
DLD, MCF7, PANC1, H1299, and U87 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Biological Industries, Kibbutz Beit-Haemek, Israel) containing antibiotic–antimycotic (Tivan Biotech, Kfar-Saba, Israel), 1 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (Biological Industries). All cell cultures were incubated in 5% CO2 at 37 °C. For glucose starvation, the medium was replaced with glucose-free DMEM containing antibiotic–antimycotic and 10% FBS.
2.2. Cell Viability Assays
For compound screening, cells were plated on 384-well plates containing one compound/vehicle per well with or without glucose in the medium. The plates were incubated in 5% CO2 at 37 °C for 48 h. Cell viability was measured using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA). For validation of compound screening and determination of the IC50 values, cells were plated in 24-well plates with glucose-depleted medium and the compound of interest. The plates were incubated in 5% CO2 at 37 °C for 48 h, followed by aspirating the medium and washing with PBS. Two hundred and fifty microliters of crystal violet staining solution (0.5% (w/v) crystal violet powder, 20% methanol) was added to the plates. After incubation for 10 min at room temperature, the staining solution was discarded, the plates were washed 5 times with distilled water and dried, and 250 µL of 10% acetic acid was added to each well. Absorbance was measured at 570 nm.
2.3. Cell Death Assays
Cell death was determined using flow cytometry. Cells were harvested at 48 h post-treatments and stained with annexin V/PI using an apoptosis detection kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were analyzed using a flow cytometer (Sysmex, Kobe, Japan) equipped with FCS Express software according to the manufacturer’s instructions.
2.4. Cell Lysis and Sample Preparation
Cell lysis was performed on ice. Cells were washed one time with PBS and scraped with RIPA lysis buffer (150 mM NaCl; 50 mM Tris pH = 8.0; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS). Samples were sonicated and centrifuged at 4 °C for 20 min. The supernatant was collected and stored at −20 °C. Before proceeding, protein amounts were quantified using a PierceTM BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).
2.5. Gel Electrophoresis and Immunoblotting
Protein lysates were mixed with 5× sample loading buffer (250 mM Tris–HCl pH 6.8; 10% SDS; 30% glycerol; 10 mM DTT; 0.05% (w/v) bromophenol blue), followed by 10 min at 96 °C and spin-down. Samples were then loaded onto SDS-PAGE gels and gels were transferred on a nitrocellulose membrane. Membranes were blocked with blocking solution (5% skim milk; Tris buffer saline (TBS)) for 1 h. Membranes were incubated with primary antibody solution for 1 h at room temperature or overnight at 4 °C, followed by washing with TBS. Membranes were incubated with secondary antibodies for 1 h at room temperature. Images were developed using the Western blotting Serius HRP substrate kit and an electrochemiluminescence imager (Thermo Scientific, Waltham, MA, USA) and processed using GIMP 2.8 software.
HSC70 antibody (sc-7298) was obtained from Santa Cruz. Antibodies for phospho-p70S6K (Thr389) (9206s), total p70S6K (2708s), total 4EBP1 (C-9644S), phospho-S6RP (Ser240/244) (2215S), phospho-4EBP1 (Ser65) (9451S), and anti-rabbit (C-7074S) and anti-mouse (C-7076S) phosphor-AKT (Ser473) (9271S) were obtained from Cell Signaling Technology (Danvers, MA, USA).
2.7. Tumor Xenografts and Drug Treatments in Vivo
For the xenograft experiments, 6-week-old NOD–SCID (NOD.CB17-Prkdcscid/NCrCrl) mice were injected subcutaneously in the flank with DLD1 cells (5 × 106 cells per injection in PBS). Two independent experiments were performed and each experiment consisted of 20 mice. All mice developed tumors. When the tumor volume reached 150–200 mm3, they were randomized into four groups of five mice in each experiment. Animals were treated with vehicle (PBS/saline), papaverine (10 mg/kg), or QNZ (EVP4593) (1 mg/kg) daily via intraperitoneal injection, or bevacizumab (5 mg/kg) twice weekly in combination with papaverine (10 mg/kg) or QNZ (1 mg/kg). Tumors were measured with digital caliper twice a week, and tumor volumes were determined using the formula: length × width2 × π/6. At the end of the experiment, animals were sacrificed using CO2 inhalation and the tumors were harvested for investigation. Tumor volumes were normalized to initial volumes and presented as an averaged percentage of the initial volumes ±standard error of the mean (SEM).
Mice were maintained and treated according to the institutional guidelines of Ben-Gurion University of the Negev. Mice were housed in air-filtered laminar flow cabinets with a 12-h light/dark cycle and food and water ad libitum. Animal experiments were approved by the Ben Gurion University of the Negev animal care and use committee (license number: IL.80-12-2015).
2.8. Immunohistochemistry (IHC) and Analysis
Following mice sacrifice, tumors were fixed in 4% paraformaldehyde. Tissues were dehydrated using alcohol gradient and embedded in paraffin. Five-micrometer sections were taken, deparaffinized, and dehydrated using xylene and alcohol gradients, respectively. The slides were incubated in 10 mM citric acid buffer, pH 6.0 at 100 °C for 20 min for antigen retrieval. The endogenous peroxidase activity was blocked with H2O2 (0.3%). Sections were then blocked for 1 h at room temperature with blocking solution (PBS, 0.1% TWEEN, 5% BSA), followed by incubation with primary antibodies. Ki67 (275R-1, Sigma, St. Louis, MO, USA), pS6RP (4857S, Cell Signaling, Danvers, MA, USA), and CD31 (ab28364, Abcam, Cambridge, UK) antibodies were diluted in blocking solution and incubated overnight at 4 °C. The ABC kit (VECTASTAIN Cat. VE-PK-6200) was used for detection according to the manufacturer’s protocol. Sections were counterstained with hematoxylin, dehydrated, and mounted with mounting media (Micromount, Leica, Cat. 380-1730, Wetzlar, Germany). IHC slides were digitalized using the Pannoramic Scanner (3DHISTECH, Budapest, Hungary) and analyzed using QuantCenter (3DHISTECH) using a single threshold parameter for all images of a specific staining sample in each experiment.
Statistical analyses were done using GraphPad Prism 7.03 software. All cellular experiments were repeated at least three times. A two-tailed Student’s unpaired t test was performed to compare control versus treated group. p values of 0.05 (*), 0.01 (**), and 0.001 (***) were considered statistically significant. For experiments with more than two groups, a one-way ANOVA was calculated using Turkey’s multiple comparison test. In vivo experiments were performed with indicated n values, and a one-way ANOVA test was performed to compare between groups.
Tumor cells often grow within a metabolically deprived tumor microenvironment. This implies that tumor cells need to adapt to such conditions in order to develop into macroscopic tumors. In many cases, tumor cells exploit endogenous protective pathways for their own advantage, including those supporting survival under metabolic stress [14
]. Tumor cells, however, become addicted to such survival mechanisms [6
], thereby offering potential weaknesses that can be targeted. To uncover compounds targeting these adaptive mechanisms, we carried out a phenotypic drug screen in cells growing in glucose-deprivation versus normal conditions. This led to the identification of 67 compounds presenting selective toxicity towards glucose-starved tumor cells. We validated two of these drugs, papaverine and QNZ, which are both mitochondrial complex I inhibitors. Our data demonstrate that both papaverine and QNZ exhibited selective toxicity under glucose starvation, and more importantly, enhanced the efficacy of the antiangiogenic bevacizumab against tumor xenografts in vivo. Bevacizumab is an anti-VEGF inhibitor which inhibits the growth of blood vessels in colon cancer tumors [44
] and is used clinically to treat colon cancer [44
]. Our results thus highlight how compounds that sensitize tumors cells to glucose starvation can enhance the antitumorigenic activity of bevacizumab in vivo. This can lay the groundwork for further developing drug synergies between glucose deprivation-selective compounds and metabolic stress-inducing agents (such as antiangiogenics and 2-deoxyglucose) to improve tumor targeting.
The ability of tumor cells to survive glucose deprivation relies on profound metabolic reprogramming. This includes the blocking of anabolic processes, such as protein and fatty acid synthesis, and the activation of catabolic processes, such as fatty acid oxidation, together allowing cells to maintain redox balance and prevent energy depletion [1
]. However, how mitochondrial activity responds to glucose-deprived conditions is still poorly understood. The induction of mitochondrial activity (i.e., oxygen consumption) in response to low-glucose conditions has been reported, but only in cells resistant to such growth conditions [9
]. This suggests that an increase in mitochondrial activity may be linked to improved survival under glucose-deprived conditions. While glycolysis is less active under glucose starvation, it is possible that mitochondrial activity is enhanced to compensate and generate sufficient ATP and cofactors involved in redox reactions. Strikingly, a high proportion of the glucose-starved cell-selective compounds we identified are mitochondrial toxins (approximately 50% of the compounds). This supports the notion that tumor cells rely heavily on mitochondrial activity to survive and adapt to glucose deprivation. Therefore, employing mitochondrial toxins to selectively target tumor cells experiencing metabolic stress may be a promising therapeutic approach. Notably, a number of mitochondrial toxins have been successfully used to restrict tumor growth in vivo, such as doxorubicin [9
] and metformin [46
], although not in combination with metabolic stress-inducing agents.
Papaverine is a non-narcotic opiate alkaloid commonly used in the clinic for treatment of spasms [47
] and erectile dysfunction [48
]. Recently, papaverine was found to sensitize tumors to radiation therapy by inhibiting mitochondrial complex I, thus reducing tumor cell respiration and promoting tumor oxygenation [49
]. We identified papaverine in our HTP screen for compounds sensitizing cells to glucose starvation in culture. In vivo, papaverine significantly enhanced the activity of bevacizumab in reducing tumor growth, thus providing support to our model. Interestingly, since papaverine is regularly used in the clinic and as bevacizumab is used as a first-line treatment in colon cancer, it is conceivable that the combination of the two would be beneficial and quickly available in patients. In addition, QNZ was not only shown to act as a mitochondrial complex I inhibitor in vitro [21
], but also to inhibit NF-ĸB in a pathway known to promote survival under glucose starvation [17
]. It is, therefore, possible that NF-ĸB inhibition may contribute to the selective toxicity of QNZ under glucose starvation.
Finally, we found that both papaverine and QNZ selectively inhibited the mTOR pathway in glucose-starved cells, which was confirmed in vivo in combined treatment with bevacizumab. Since mTOR inhibition is required to prevent cell death in response to glucose deprivation [12
], these results may be indicative of the energy-depleted status of the cell. Indeed, by inhibiting mitochondrial activity, papaverine and QNZ may deplete mitochondrial ATP levels under glucose deprivation. Given that mTOR is a hub whose activity depends on ATP levels, this scenario would be expected to lead to an inhibition of the mTOR pathway, in accordance with our observations.
Limitations of Our Study
We identified compounds exhibiting selective toxicity upon glucose starvation in vitro and found that two of the identified compounds synergize with avastin in inhibiting tumor growth in vivo. The synergic effect we observed in vivo may be due to the induction of glucose starvation by avastin treatment, but it may also be caused by hypoxia triggered by this compound or by a combination of both. It also remains to be determined whether such synergic action is specific to avastin or will occur with other antiangiogenic compounds and calorie-restriction mimetics. In addition, given that glucose-starved cells are dependent on mitochondria to survive under glucose starvation and since the compounds we identified are known inhibitors of mitochondrial complex 1, it is likely that QNZ and papaverine enhance the antitumoral activity of avastin by targeting the mitochondria in vivo. However, potential other mechanisms cannot be excluded until this hypothesis is formally tested.