Abstract
BackgroundThe 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.MethodsFunctional 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.ResultsEnrichments 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.ConclusionsCollectively, 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.
Highlights
The Ets-1 proto-oncogene is frequently upregulated in cancer cells, with known involvement in cancer angiogenesis, metastasis, and more recently energy metabolism
The functional interaction analyses in this study further strengthen the importance of Ets-1 in regulating cancer metastasis due to the pathway associations we have observed in WNT, vascular endothelial growth factor, and matrix metalloproteinase (MMP) signaling, as well as extracellular-signalregulated kinase 5 (ERK5), mitogen-activated protein kinase (MAPK), epidermal growth factor (EGF), plateletderived growth factor (PDGF), MET and G protein-coupled receptor (GPCR) pathways
We have found that several enriched metabolic and oxidative stress pathways are differentially expressed in an ovarian cancer cell model of Ets-1 overexpression
Summary
The Ets-1 proto-oncogene is frequently upregulated in cancer cells, with known involvement in cancer angiogenesis, metastasis, and more recently energy metabolism. Ets-1 is a member of the E-26 (Ets) family of transcription factors, and was first characterized as a protooncogene of the retroviral v-ets oncogene in avian leukemia retrovirus E26 [1] This family of transcription factors currently comprises 28 members, many of which are known to be elevated in various cancers [2,3] including Ets-2 [4,5,6,7,8,9], Friend leukemia integration 1 [10], Etsrelated gene [4], Polyomavirus enhancer activator 3 homolog [11,12], Ets-related molecule [11], Prostate epithelium-specific Ets transcription factor [13] and E74-like factor-3 [11]. Comparing the transcriptional programs of cancer cells that express low levels of Ets-1 protein to those that express Ets-1 protein in abundance will create a gene expression profile illustrating some of the key differences between invasive and non-invasive ovarian cancer cells
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