Ets-1 Regulates Energy Metabolism in Cancer Cells

Cancer cells predominantly utilize glycolysis for ATP production even in the presence of abundant oxygen, an environment that would normally result in energy production through oxidative phosphorylation. Although the molecular mechanism for this metabolic switch to aerobic glycolysis has not been fully elucidated, it is likely that mitochondrial damage to the electron transport chain and the resulting increased production of reactive oxygen species are significant driving forces. In this study, we have investigated the role of the transcription factor Ets-1 in the regulation of mitochondrial function and metabolism. Ets-1 was over-expressed using a stably-incorporated tetracycline-inducible expression vector in the ovarian cancer cell line 2008, which does not express detectable basal levels of Ets-1 protein. Microarray analysis of the effects of Ets-1 over-expression in these ovarian cancer cells shows that Ets-1 up-regulates key enzymes involved in glycolysis and associated feeder pathways, fatty acid metabolism, and antioxidant defense. In contrast, Ets-1 down-regulates genes involved in the citric acid cycle, electron transport chain, and mitochondrial proteins. At the functional level, we have found that Ets-1 expression is directly correlated with cellular oxygen consumption whereby increased expression causes decreased oxygen consumption. Ets-1 over-expression also caused increased sensitivity to glycolytic inhibitors, as well as growth inhibition in a glucose-depleted culture environment. Collectively our findings demonstrate that Ets-1 is involved in the regulation of cellular metabolism and response to oxidative stress in ovarian cancer cells

Ets-1 global gene expression profile reveals associations with metabolism and oxidative stress in ovarian and breast cancers

BACKGROUND: The Ets-1 proto-oncogene is frequently upregulated in cancer cells, with known involvement in cancer angiogenesis, metastasis, and more recently energy metabolism. In this study we have performed various bioinformatic analyses on existing microarray data to further clarify the role of Ets-1 in ovarian cancer, and validated these results with functional assays. METHODS: Functional pathway analyses were conducted on existing microarray data comparing 2008 and 2008-Ets1 ovarian cancer cells. Methods included over-representation analysis, functional class scoring and pathway topology, and network representations were visualized in Cytoscape. Oxidative stress regulation was examined in ovarian cancer cells by measuring protein expression and enzyme activity of glutathione peroxidases, as well as intracellular reactive oxygen species using dichlorofluorescin fluorescence. A stable Ets-1 knockdown MDA-MB-231 cell line was created using short hairpin RNA, and glycolytic dependence of these cells was measured following treatment with 2-deoxy-D-glucose and Hoechst nuclear staining to determine cell number. High-resolution respirometry was performed to measure changes in basal oxygen flux between MDA-MB-231 cells and MDA-Ets1KD variants. RESULTS: Enrichments in oxidoreductase activity and various metabolic pathways were observed upon integration of the different analyses, suggesting that Ets-1 is important in their regulation. As oxidative stress is closely associated with these pathways, we functionally validated our observations by showing that Ets-1 overexpression resulted in decreased reactive oxygen species with increased glutathione peroxidase expression and activity, thereby regulating cellular oxidative stress. To extend our findings to another cancer type, we developed an Ets-1 knockdown breast cancer cell model, which displayed decreased glycolytic dependence and increased oxygen consumption following Ets-1 knockdown confirming our earlier findings. CONCLUSIONS: Collectively, this study confirms the important role of Ets-1 in the regulation of cancer energy metabolism in ovarian and breast cancers. Furthermore, Ets-1 is a key regulator of oxidative stress in ovarian cancer cells by mediating alterations in glutathione antioxidant capacity

Primer sequences used for real time qRT-PCR.

<p>Primer sequences used for real time qRT-PCR.</p

Effect of Ets-1 over-expression in oxidative stress.

<p>p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***.</p

Ets-1 expression did not affect the expression of similar ETS family members.

<p>The protein expression of ETS family transcription factors (<b>A</b>) Ets-2 and (<b>B</b>) PEA3 were compared in 2008 and 2008-Ets1 ovarian cancer cells to determine the specificity of our Ets-1 expression model. Western blot analysis showed that neither of these transcription factors was affected by Ets-1 expression in 2008 cells.</p

Real time qRT-PCR validation of microarray findings.

<p>Correlation Coefficient = 0.99; p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***.</p

Effect of Ets-1 over-expression on the ETC.

<p>p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***.</p

Effect of Ets-1 over-expression on glycolysis, glycolytic feeder pathways, and lipid metabolism.

<p>p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***.</p

Glycolytic dependence of Ets-1-expressing ovarian cancer cells.

<p>(<b>A</b>) 2008, C13*, and 2008-Ets1 cells were treated with the glycolytic inhibitor 2-DG, and cellular growth was measured following 96 hours of incubation. 2008-Ets1 and C13* cells which express Ets-1 showed significantly decreased growth following glycolytic inhibition in 2008-Ets1 compared to parental 2008 cells (n = 4). (<b>B</b>) 2008 and 2008-Ets1 cells were grown in the absence of glucose, and proliferation assays were conducted at 24 hour intervals. 2008-Ets1 ovarian cancer cells expressing high levels of Ets-1 showed a decreased ability for growth in glucose-free culture medium, suggesting an increased reliance on glycolysis for energy (n = 3).</p