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Reprogramming somatic cells to a pluripotent state drastically reconfigures the cellular anabolic requirements, thus potentially inducing cancer-like metabolic transformation. Accordingly, we and others previously showed that somatic mitochondria and bioenergetics are extensively remodeled upon derivation of induced pluripotent stem cells (iPSCs), as the cells transit from oxidative to glycolytic metabolism. In the attempt to identify possible regulatory mechanisms underlying this metabolic restructuring, we investigated the contributing role of hypoxia-inducible factor one alpha (HIF1a), a master regulator of energy metabolism, in the induction and maintenance of pluripotency. We discovered that the ablation of HIF1a function in dermal fibroblasts dramatically hampers reprogramming efficiency, while small molecule-based activation of HIF1a significantly improves cell fate conversion. Transcriptional and bioenergetic analysis during reprogramming initiation indicated that the transduction of the four factors is sufficient to upregulate the HIF1a target pyruvate dehydrogenase kinase (PDK) one and set in motion the glycolytic shift. However, additional HIF1a activation appears critical in the early upregulation of other HIF1a-associated metabolic regulators, including PDK3 and pyruvate kinase (PK) isoform M2 (PKM2), resulting in increased glycolysis and enhanced reprogramming. Accordingly, elevated levels of PDK1, PDK3, and PKM2 and reduced PK activity could be observed in iPSCs and human embryonic stem cells in the undifferentiated state. Overall, the findings suggest that the early induction of HIF1a targets may be instrumental in iPSC derivation via the activation of a glycolytic program. These findings implicate the HIF1a pathway as an enabling regulator of cellular reprogramming. STEM CELLS 2014;32:364-376

BackgroundReduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1α has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1α's role in determining chemosensitivity focussing on responsible molecular pathways.Methodology and Principal FindingsRNA interference was applied to inactivate HIF-1α or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-κB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1α in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1α-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1α markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1α-competent cells was identified as the molecular mechanism of HIF-1α-mediated inhibition of p53. Furthermore, loss of HIF-1α abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-κB and expression of anti-apoptotic NF-κB target genes. Accordingly, reconstitution of the NF-κB subunit p65 reversed the increased chemosensitivity of HIF-1α-deficient cells.Conclusion and SignificanceIn summary, we identified HIF-1α as a potent regulator of p53 and NF-κB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy.

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