Malignant cells produce energy by preferentially utilizing glycolytic pathways instead of oxidation of pyruvate in mitochondria under normal oxygen states, a metabolic alteration known as the Warburg effect. Hypoxia-inducible transcription factor 1 (HIF-1) plays a crucial role in this major shift. Some Warburg effect mediators are SLC2A1 (glucose transporter1), PDK1 (pyruvate dehydrogenase kinase, isozyme 1) and VEGF-A (vascular endothelial growth factor A). Whether the Warburg effect is exhibited by pre-malignant, clonal disorders such as Ph-negative myeloproliferative neoplasms (MPN) is not known. We investigated the presence of the Warburg effect and levels of other selected HIF-1 regulated genes in clonal granulocytes and platelets of patients with MPN, i.e. polycythemia vera (PV), essential thrombocythemia (ET), and primary or secondary myelofibrosis (MF).
We collected blood samples from 21 PV, 9 ET, 9 MF patients and 14 healthy controls. Total RNA from granulocytes and platelets was isolated by Tri-Reagent and reverse transcribed using SuperScript III First-Strand Synthesis kit. Expression of HIF-1 target genes SLC2A1, PDK1, VEGF A, NFE-2 and RUNX2 was determined by qPCR using hydrolysis probes. Relative gene expression was calculated using Rest 2009 Qiagen software.
We report upregulation of SLC2A1 in granulocytes and PDK1 transcripts in platelets and down regulation of PDK1 transcripts in granulocytes. HIF-1 regulated NFE-2 and RUNX1 genes transcripts were also upregulated in PV patients (see K. Kapralova abstract at this ASH meeting). The VEGF-A transcript was increased in granulocytes and platelets, and SLC2A1was increased in platelets of MPN patients, compared to controls, but this did not reach statistical significance.
We then directly evaluated glucose transport in red blood cells (another feature of the Warburg effect) in some of the PV, ET and MF studied patients using Raman scattering (RS). RS is a process by which the energy of an impinging photon is shifted due to an interaction with a molecule. The energy shift of the scattered photon usually corresponds to a vibrational mode of the involved molecule. The RS spectrum is highly molecule specific, as each molecule has a distinctive set of vibrational modes. However, the RS cross-section is very small, usually requiring an enhancement mechanism. RS, as well as other optical processes like fluorescence, can be enhanced in the vicinity of metallic nanoparticles, giving rise to surface-enhanced Raman scattering (SERS) and metal-enhanced fluorescence (MEF).
SERS and MEF spectroscopy measurements done on granulocytes isolated from MPN patients show that fluorescence contribution to the spectra is higher and shifted when compared to spectra from control subjects. When healthy granulocytes were incubated with various concentrations of deoxyglucose (the glucose analog not metabolized but actively transported), there was an increase of the fluorescence contribution with higher concentrations, as well as a shift in the spectrum. Together, these spectroscopic results confirm a higher glucose uptake by granulocytes from MPN patients.
The increased expression of SLC2A1 and PDK1 genes and enhanced uptake of glucose in MPN patients suggest the presence of Warburg effect. The difference in regulation of transcripts of PDK1 in granulocytes and platelets may be due to tissue-specific differences of gene transcription as the HIF-1 activity is often content-specific. To validate the likely presence of the Warburg effect in MPN patients, we are now analyzing the expression of HIF-1-regulated genes in hematopoietic stem cells, as well as in reticulocytes and in the BFU-E colonies in JAK2V617Fpositive and negative MPN.
Note: First and second authors contributed equally.
Swierczek:University of Utah : No financial compensation, No financial compensation Patents & Royalties.
Asterisk with author names denotes non-ASH members.