Role of cyclic AMP response element-binding protein in cancer cells in response to glucose stress

Abstract

Background: cyclic AMP response element-binding protein (CREB) is a leucine zipper transcription factor that regulates expression of target genes involved in metabolism, transcription, cell cycle, survival, DNA repair, growth control, immune regulation, signaling, transport, and reproduction. However, there are very limited data available regarding the role of CREB in human cancer. CREB mediates energy homeostasis and promotes stress resistance in non-cancerous tissues, which is similar to active reprogramming systems of cancer that enable adaptation and survival under environmental metabolic stress. Thus, it is plausible that CREB plays a role in stress resistance or energy compensation in cancer. This study was conducted to characterize the role of CREB in cancer cells in response to glucose starvation and investigate differentially expressed genes under glucose deprivation conditions.Methods: Several human cancer cell lines (A549, H1299, MDA-MB-231, MDA-MB-468, BT474, and SKOV-3) were used in this study. Deprivation of glucose or fetal bovine serum from culture media was used to mimic metabolic stress. Transient transfection with siRNA was performed to test CREB-specific roles. Proliferation under normal culture conditions and cell viability assays under extremely low glucose and FBS-free conditions were conducted to explore the metabolic phenotype of CREB. To investigate cell death mode, assessment of poly(ADP-ribose) polymerase (PARP) cleavage by Western blot and an apoptosis assay using a flow cytometer were performed. And the CREB-specific gene expression signature under glucose deprivation conditions was investigated by microarray experiments and gene set enrichment analysis. Finally, microarray results were validated by quantitative real-time-PCR.Results: Expression of phosphorylated CREB was increased under glucose-free media conditions. Proliferation of CREB-knockdown cells was significantly suppressed compared with that of control cells under normal media conditions (p < 0.001). The viability of CREB-knockdown cells was significantly higher than that of control ones under metabolic stress conditions (p < 0.001). Western blot analysis revealed that PARP cleavage induced by glucose deprivation was attenuated by knockdown of CREB. Additionally, apoptotic assay revealed that the number of cells undergoing apoptotic death decreased in response to knockdown of CREB under glucose deprivation conditions. CREB-specific gene expression signatures under glucose deprivation conditions revealed that energy metabolism-related gene sets such as glycolysis/gluconeogenesis, oxidative phosphorylation, and ATP synthesis were highly enriched (p < 0.00001). Expression of selected putative CREB target genes such as NDUFV1, ATP5G1, and MVD genes was significantly lower in CREB-knockdown cells than in control cells under glucose deprivation conditions (p < 0.001).Conclusion: CREB is essential for cellular proliferation under normal culture media conditions. Under glucose starvation conditions, CREB is activated and mediates apoptotic cell death in correlation with expression of energy metabolism-related gene sets. These findings provide dynamic role of CREB in cancer cells according to the presence or absence of glucose in culture medium.