Objective
Up-regulation of glycolysis has been demonstrated in multiple tumor types and is believed to originate as an adaptive response to the selective pressure of the tumor microenvironment. We hypothesized that ovarian cancer cells are dependent on the glycolytic pathway for adenosine triphosphate generation and that this phenotype could be exploited for therapeutic intervention.
Study Design
Expression of glucose transporter 1 (Glut1), phosphorylated protein kinase B (pPKB/pAkt), and phosphorylated mammalian target of rapamycin (pmTOR) was assessed in ovarian carcinoma tumors and cell lines. Cells were incubated with 2-deoxyglucose and rapamycin; growth inhibition, viability, and mechanism of cell death were determined.
Results
Ovarian carcinoma cells overexpress Glut1, pAkt, and pmTOR compared with benign ovarian epithelial cells. 2-deoxyglucose and rapamycin markedly enhance apoptotic and nonapoptotic cell death in ovarian cancer cells.
Conclusion
The glycolytic phenotype of ovarian cancer cells can be targeted for therapeutic intervention. Combined treatment modalities that target multiple cellular pathways hold promise for the treatment of chemoresistant ovarian cancer cells.
Throughout their lifetime, mammalian cells are exposed to a wide range of oxygen concentrations and have developed the ability to maintain adequate energy production in a myriad of environmental states. In untransformed cells, the preferred method of adenosine triphosphate (ATP) production is through oxidative phosphorylation, which is an efficient process that produces a net gain of 36 ATPs per molecule of glucose. In contrast, cancer cells rely on aerobic glycolysis for energy production, which is an inefficient series of events in which 1 molecule of glucose is broken down into 2 molecules of pyruvate, which produces a net gain of 2 molecules of ATP. The survival advantage of using aerobic glycolysis, otherwise known as the Warburg effect , is unclear. Regardless of the cause, the dependence of malignant cells on glycolysis has been observed in many tumor types and lends itself as an attractive target for cancer therapeutics.
2-Deoxyglucose (2-DG) is a glucose analog currently under intense investigation as a cancer therapeutic. 2-DG competes with native glucose for entry into the glycolytic pathway; however, once inside the cell, it is phosphorylated by hexokinase to 2-deoxyglucose-phosphate. However, unlike native glucose, 2-DG cannot be further metabolized and begins to accumulate inside the cell. This accumulation effectively inhibits the glycolytic machinery from further metabolizing its natural substrate and depresses energy production. The subsequent fall in cellular ATP levels leads to the inhibition of cell cycle progression and eventual cell death. The cytotoxic properties of 2-DG have been demonstrated in in vitro experiments and in xenograft models with the use of osteosarcoma and nonsmall lung cancer cells.
Another crucial factor in cellular energy signaling is the mammalian target of rapamycin (mTOR). MTOR is conserved evolutionarily and integrates nutrient- and growth factor–derived signals to control the cell growth machinery. In normal cells, mTOR signaling is up-regulated when the cellular environment is favorable (replete with growth factors and nutrients) and is diminished in periods of stress. In transformed cells, stress-induced inhibition of mTOR signaling is lost, and deregulated mTOR signaling is thought to drive tumorigenesis. Rapamycin, a macrolide ester produced by Streptomyces hygroscopicus , inhibits mTOR and arrests cells in the G 1 phase of the cycle. Today, a myriad of mTOR inhibitors are being investigated as anticancer agents; preliminary results are mixed, which suggests that the ideal, molecularly selected patient population has not yet been identified.
Our goal in undertaking this study was to determine whether targeting metabolic signaling pathways in ovarian carcinoma cells would have antineoplastic activity. Aberrant expression of the facilitative glucose transporter 1 (Glut1) and phosphorylation of protein kinase B (PKB/Akt) and mTOR have been described in epithelial ovarian cancer, yet it is not clear whether ovarian cancer cells are susceptible to the inhibition of these factors. We hypothesized that ovarian carcinoma cells would display increased glucose uptake and that this phenotype could be exploited for therapeutic intervention by using the glucose analog 2-DG. Furthermore, we reasoned that the inhibition of mTOR dependent signaling with rapamycin should mimic nutrient and growth factor deprivation and result in growth inhibition and cell death. Finally, because prolonged nutrient deprivation is thought to result in autophagic cell death, we hypothesized that the induction of nonapoptotic cell death could enhance the effect of chemotherapy and/or circumvent chemoresistance because of alterations in apoptosis.
Materials and Methods
Reagents and cell lines
Rapamycin was purchased from LA Laboratories (Los Angeles, CA); 2-DG was purchased from Sigma-Aldrich (St. Louis, MO), and cisplatin was obtained from Bedford Laboratories (Bedford, OH). Ovarian carcinoma cell lines TOV21G and SKOV3 cells were provided by Dr K. Cho (University of Michigan, Ann Arbor, MI).
Growth inhibition, subG 0 , and viability assays
The sulforhodamine B assay was used to assess in vitro growth inhibition, as previously described. The percentage of apoptotic cells was determined at the indicated time points in triplicate cultures by nuclear propidium iodide staining. In this assay, the nuclei of apoptotic cells exhibit a subG 0 profile characteristic of DNA fragmentation. Analysis was performed with Lysis II software (Becton Dickinson, Franklin Lakes, NJ) on a FACScan flow cytometer (Becton Dickinson).
Immunoblotting
For caspase-9 immunoblotting, S-100 cytosolic lysates were prepared as previously described. For all other immunoblots, cells were lysed in radioimmunoprecipitation buffer. Antibodies for caspase-9 (Stressgen, Ann Arbor, MI), total and phosphorylated Akt (pAkt) and mTOR (pmTOR) (Cell Signaling Technology Inc, Danvers, MA) and for GAPDH (Chemicon International, Temecula, CA) were used for immunoblotting and were detected with enhanced chemiluminescence Western blot detection kit (Amersham Biosciences, Danvers, MA).
ATP assay
SKOV3 and TOV21G cells were plated and treated with indicated agents. ATP levels were assessed with ENLITEN ATP Assay System Bioluminescence Detection Kit (Promega Corp, Madison, WI) and analyzed with the LMax II384/LMax II Microplate reader (Molecular Devices, Sunnyvale, CA) at 560 nm.
Tissue microarray analysis
Tissue microarrays were constructed with 302 cores from 86 patients with epithelial ovarian cancer and 25 cores from benign ovarian samples. Tumors were classified histopathologically according to the International Federation of Gynecology and Obstetrics criteria. The histologic evidence of tumors in the ovarian carcinoma microarray included papillary serous (52%), endometrioid (9%), clear cell (9%), undifferentiated (3%), and mixed histologic findings (27%). Clinicopathologic and demographic data were collected from medical records under an institutional review board-approved protocol (IRBMED # 2004-0814).
All hematoxylin and eosin-stained slides of the tumors were reviewed, and areas of tumor were identified. Two to 3 tissue cores per tumor were taken from spatially separate areas in a single donor block from each case with the use of a tissue microarrayer (Chemicon Advanced Tissue Arrayer; Chemicon International). Cores were arrayed into a recipient block at predetermined coordinates. The hematoxylin and eosin-stained sections from donor and recipient paraffin blocks were used to confirm the area of tumor from which cores were retrieved.
For immunohistochemistry, 5 μmol/L–thick sections were cut from the respective arrays, deparaffinized, and dehydrated. Immunohistochemical staining for pAkt, pmTOR, and Glut1 was performed by a streptavidin peroxidase procedure. The antibodies were obtained from Cell Signaling Technology Inc and Millipore (Billerica, MA), respectively. Antigen-bound primary antibody was detected with a standard avidin-biotin immunoperoxidase complex (DAKO Corp, Carpinteria, CA).
Expression scores for membrane staining of Glut1 and cytoplasmic expression of pAkt and pmTOR were based on the percentage of cells that were reactive with antibodies to Glut-1 (1:800), pAkt (1:100), or pmTOR (1:50) in the following manner: negative (score = 1; no staining); weak (score = 2; <25% of cells staining, any intensity); moderate (score = 3; 25-75% of cells staining, any intensity); and strong (score = 4; >75% of cells staining, any intensity). All scoring was performed by a single author (M.K.).
Statistical analysis
Expression scores (1-4) per patient for primary tumor were calculated with the median of the core-specific scores. In cases in which the median was the mid point between score values, the score was rounded. The potential association between tumor expression of Glut1, pAkt, and pmTOR was explored with contingency tables; significant associations were determined with the Fisher’s exact test statistic. For in vitro experiments, differences were expressed as percentage change from control. The arcsine transformation of the proportion was used to normalize the distribution for statistical testing. To detect significant differences between groups, Tukey’s HSD multiple comparison correct was used to control the overall experimental type-1 error at 5%. For all statistical tests, probability values ≤5% were considered to indicate meaningful associations.
Results
Glut1, pAkt, and activated mTOR are expressed in ovarian cancer
Membranous Glut1 was strongly overexpressed in most epithelial ovarian carcinomas (93.9% of tumors). In contrast, only 12.5% of benign ovarian samples overexpressed Glut1 ( Figure 1 , A-D, and data not shown [refers to normal ovarian tissue stained with Glut1, pAkt, and pmTOR]; P < .0001). Because there is some evidence that activated Akt may increase Glut1 protein synthesis and promote the accumulation of Glut1 on the surface of tumor cells, we next analyzed expression of phosphorylated Akt in ovarian tumors using an antibody against phosphorylated Ser473 of Akt. Immunostaining of our ovarian carcinoma tissue microarrays revealed that 69 of 82 ovarian tumors (84.1%) stained positively for pAkt. In benign ovarian tissue, a smaller proportion of samples (13/25; 52.0%) expressed pAkt ( Figure 1 , E-H, and data not shown; P = .007). The downstream effector of Akt (mTOR) was of particular interest, because mTOR can regulate transcription and translation in response to the nutrient milieu. Immunohistochemical analysis of mTOR activation was performed with an antibody that was directed at phosphorylated mTOR Ser2448, which recognizes the active form of the mTOR kinase. In total, 63 of 82 ovarian tumors (76.8%) that were analyzed demonstrated high expression of phospho-mTOR; only 6 of 21 benign ovarian samples (28.6%) demonstrated the same high expression ( Figure 1 , I-L, and data not shown; P < .0001). Glut 1 expression was associated significantly with expression of pAkt. Sixty-four of 82 tumors (78.0%) expressed both Glut1 and pAkt (expression scores, >1; P = .0088). pAkt was also associated significantly with pmTOR expression. Fifty of 82 tumors (61.0%) expressed both pAkt and pmTOR (expression scores, >1; P = .0264).
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