ABSTRACT
BACKGROUND: Hypoxia-inducible factors (HIF)1 and 2 are transcription factors that regulate the homeostatic response to low oxygen conditions. Since data related to the importance of HIF1 and 2 in hematopoietic stem and progenitors is conflicting, we investigated the chromatin binding profiles of HIF1 and HIF2 and linked that to transcriptional networks and the cellular metabolic state. METHODS: Genome-wide ChIPseq and ChIP-PCR experiments were performed to identify HIF1 and HIF2 binding sites in human acute myeloid leukemia (AML) cells and healthy CD34+ hematopoietic stem/progenitor cells. Transcriptome studies were performed to identify gene expression changes induced by hypoxia or by overexpression of oxygen-insensitive HIF1 and HIF2 mutants. Metabolism studies were performed by 1D-NMR, and glucose consumption and lactate production levels were determined by spectrophotometric enzyme assays. CRISPR-CAS9-mediated HIF1, HIF2, and ARNT-/- lines were generated to study the functional consequences upon loss of HIF signaling, in vitro and in vivo upon transplantation of knockout lines in xenograft mice. RESULTS: Genome-wide ChIP-seq and transcriptome studies revealed that overlapping HIF1- and HIF2-controlled loci were highly enriched for various processes including metabolism, particularly glucose metabolism, but also for chromatin organization, cellular response to stress and G protein-coupled receptor signaling. ChIP-qPCR validation studies confirmed that glycolysis-related genes but not genes related to the TCA cycle or glutaminolysis were controlled by both HIF1 and HIF2 in leukemic cell lines and primary AMLs, while in healthy human CD34+ cells these loci were predominantly controlled by HIF1 and not HIF2. However, and in contrast to our initial hypotheses, CRISPR/Cas9-mediated knockout of HIF signaling did not affect growth, internal metabolite concentrations, glucose consumption or lactate production under hypoxia, not even in vivo upon transplantation of knockout cells into xenograft mice. CONCLUSION: These data indicate that, while HIFs exert control over glycolysis but not OxPHOS gene expression in human leukemic cells, this is not critically important for their metabolic state. In contrast, inhibition of BCR-ABL did impact on glucose consumption and lactate production regardless of the presence of HIFs. These data indicate that oncogene-mediated control over glycolysis can occur independently of hypoxic signaling modules.
ABSTRACT
Hypoxia and hypoxia-induced factors (HIFs) are main regulators for tumor stem cells, metastasis-initiating cells and their differentiated progenies to adapt to the environment which lacks oxygen and nutrient in the process of cancer development. HIFs are up-regulated in many tumors, including leukemia, glioblastoma, melanoma, prostate cancer, breast cancer and pancreatic cancer, in where they are especially highly expressed in hypoxic regions. HIFs activation can induce expression of numerous stem cell related genes and multidrug resistance genes, which may play important roles in tumour and stem cell-mediated self-renewing, energy metabolism alternation, invasion, metastasis, angiogenesis and treatment resis?tance of neoplastic cells. Consequently, it will provide new clues for cancer therapy after investigating the role of HIFs in tar?geted regulation and metabolic pathway modulation in various stem cell-mediated tumor cells.
