Reduced glucose signaling and downregulation of survivin as potential anticancer therapeutic strategies
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The avid acquisition of glucose is a basic characteristic of cancer cells. This property is exploited to detect cancer and to monitor therapeutic response by employing glucose tracers in positron emission tomography (PET) scans. The higher the accumulation of glucose in tumors the more likely the malignancy will be clinically aggressive. Over the years many genes have been shown to play a role in cancer. One such gene is survivin, an antiapoptotic protein in the Inhibitor of Apoptosis (IAP) gene family that is invariably upregulated in common cancers. Survivin is a cornerstone member of the Chromosome Passenger Complex (CPC). The survivin protein is indispensable for replicating chromosomes during cell division. Survivin has also been previously implicated in our lab and others in anti-cancer drug resistance and radiation resistance mechanisms. Survivin expression in cancer cells circumvents physiological apoptosis mechanisms and promotes cell proliferation by disrupting cell division checkpoints. Survivin is also a biomarker associated with shortened duration of patient survival. The first part of the research described in this dissertation was to search for environmental factors that would reduce survivin expression without the use of drugs, in part because of the inherent challenges associated with drug delivery to cancer cells. We hypothesized that high glucose induces survivin expression, and that glucose deprivation therefore might be a suitable approach to achieve this goal. To test our hypothesis, we searched for direct evidence that glucose regulates survivin expression. Our results show that in breast cancer cells, survivin expression is inhibited by low glucose. Using a luciferase reporter gene assay we sought to identify sequences in the survivin promoter that are regulated by glucose. Analysis of various promoter deletions revealed the presence of a putative glucose response element in the survivin core promoter. In silico analyses of the putative glucose response element led to identification of a candidate transcription factor binding motif within the glucose response element that potentially regulates survivin expression by glucose. Sequence analysis of this putative transcription factor binding site indicates it corresponds to a degenerate consensus sequence that in other promoters is recognized by the Carbohydrate Response Element Binding Protein (ChREBP), and its co-factors. ChREBP is a transcription factor that upregulates genes involved in lipogenesis when glucose levels are high, such as postprandially. Using Electrophoretic Mobility Shift Assays (EMSA), we demonstrated that glucose stimulates the binding of nuclear proteins to radiolabeled versions of this motif significantly more when the cells from which the nuclear extracts were derived had been cultured in the presence of glucose compared to its absence. DNA protein complexes were formed at specific sequences, because their formation was blocked by competition with both non-radiolabeled survivin promoter sequences and a consensus ChoRE oligonucleotide, but not random sequences. Enforced expression of ChREBP upregulated survivin promoter activity and attenuated the ability of low glucose to down-regulate survivin. We also found that the longevity-associated Silent Information Regulator (SIRT1, sirtuin), expression was inversely related to expression of survivin; whereas survivin expression decreased in low glucose, SIRT1 expression increased. This is consistent with the demonstration by others that the mouse survivin promoter is bound by SIRT1 and that in mice survivin transcription is inhibited by SIRT1 deacetylase activity. The second part of the research described in this dissertation was to explore the potential of low glucose and/or drugs that reduce glucose signaling (metformin and rapamycin) to reduce toxic effects of chemotherapy. We hypothesized that reducing glucose signaling would protect normal cells expressing wild type p53 from mitotic poisons by arresting them in G1 and/or G2 through a p53-dependent mechanism. In contrast, reduced glucose signaling would not protect cancer cells harboring mutant p53 genes. Chemotherapeutic strategies that take advantage of differences between normal and cancer cells to preferentially kill cancer cells are called cyclotherapy. Cyclotherapy exploits the absence of checkpoint regulation by p53 and other proteins in cancer cells, but not in normal cells. Our results demonstrate that rapamycin, metformin or low glucose protect normal epithelial and fibroblast cells, but not cancer cells from the toxic effects of the mitotic inhibitors nocodazole and paclitaxel. Treatment of cells with a combination of both rapamycin and metformin in the presence of low glucose enhanced the toxic effects of nocodazole and paclitaxel in breast cancer cells, but protected normal cells from these drugs. This suggests that lowering glucose signaling by fasting in combination with metformin and rapamycin might substantially increase the therapeutic window of mitotic inhibitors employed to treat cancer.