Transformed cells are commonly thought to be associated with increased glycolytic flux and tend to direct the majority of glucose towards lactate fermentation, thus covering part or most of the cellular energy (ATP) requirement. This apparently inefficient way of energy production, normally activated under hypoxia, occurs even under normoxic conditions (Warburg effect)6
. The molecular switches involved in these metabolic changes and the significance of these events have just begun to be elucidated. One surprising observation of our cell line data is the complete dependency on glucose for cell growth and viability, hinting at glycolysis as a crucial pathway in transformation of cells with an active JAK2/STAT5 pathway. Our results indicate that metabolic changes are not simply intrinsic activities of cancer cells but coordinated events that can be specifically induced by the JAK2V617F oncogenic tyrosine kinase. Signaling mechanisms that regulate changes in the expression of glycolytic enzymes have been mainly associated with transcription factors that have a rather global effect on expression, including Myc and Hif1α/Hif2α16
. In our model the STAT5 transcription factor was found to be a strong inducer of PFKFB3. It is not known whether STAT5 can directly enhance expression of PFKFB3, or if this occurs through other mechanisms, such as upregulation of MYC or HIF2α by active STAT512,19
. Both, MYC and HIF proteins are thought to be key regulators of metabolic reprogramming in transformed cells16,17
. An additional possibility could be the involvement of downstream effectors of STAT5 that regulate PFKFB3 expression. It should be emphasized that PFKFB3 can not only be regulated at the transcriptional level but it is also a target of ubiquitination and proteasomal degradation20
, alternative splicing21, 22
as well as activation by serine phosphorylation21
, which all may have additional effects on the functional expression of this enzyme.
STAT5 is frequently but not exclusively activated in hematologic malignancies with tyrosine kinase oncogenes and can also be found, for example in lung, prostate and breast cancer23–25
. Consistent with this observation, high levels of PFKFB3 have also been frequently observed in a variety of solid tumors, including brain, breast, colon, kidney, lung, ovary, prostate, stomach and thyroid cancer26, 27
. It is therefore possible that STAT5 participates in the regulation of PFKFB3 in these diseases. Even though our studies focused on cells with active JAK2/STAT5, it cannot be excluded that mechanisms independent of this pathway may be involved in the regulation of PFKFB3 as well. As pointed out before, a major function of PFKFB3 is to allow high glycolytic flux through PFK1, even in the presence of elevated levels of ATP. Transformed cells already have an intrinsically high demand for energy, thus limiting excess ATP levels. This may be in particular true for cells transformed by tyrosine kinase oncogenes, which lead to abundant phosphorylation of cellular proteins. Recently, Fang et al.28
described an ATP-regulating mechanism in cancer cells that is associated with a deregulated PI3K pathway. Overexpression of the UTPase ENTPD5 (ectonucleoside triphosphate diphosphohydrolase 5), localized to the endoplasmic reticulum, was associated with ATP consumption and increased glycolysis. ENTPD5 promotes protein N-glycosylation and folding, and acts in concert with cytidine monophosphate kinase1 and adenylate kinase 1 to hydrolyze ATP to AMP. Thus, this pathway may constitute an alternative mechanism to the regulation of PFK-1 by PFKFB3 in the absence of sufficient JAK2/STAT5 activation or it may complement the regulation of PFK-1 by PFKFB3 through lowering of cellular ATP levels.
High glycolytic flux and the Warburg effect do not preclude the possibility that mitochondrial activity can be increased in cancer cells. For example, we have previously demonstrated that elevated glucose metabolism in BCR-ABL transformed cells is associated with elevated levels of intracellular ROS29, 30
. The majority of these ROS are a byproduct of reactions from the mitochondrial electron transport chain13
. This would support the notion that increased glycolytic flux and pyruvate production do not only elevate lactate levels but fuel mitochondrial electron flux as well. Nevertheless, mitochondria may display reduced functionality in transformed cells and may not be a significant factor in providing cellular energy7
, but a causal relationship between increased glucose metabolism and change in these activities has not been established. Our results, demonstrating reduced ROS in cells with PFKFB3 knockdown, would indicate that this enzyme may also be a key molecule in regulating mitochondrial ROS. This pathway is of particular relevance to the disease process since ROS may not simply be a by-product. ROS are involved in regulating cellular signaling events, can be a major cause for DNA damage and have the potential to cause genomic instability31
. In chronic myelogenous leukemia, ROS have been implicated in DNA damage and drug resistance, caused by point mutations in the BCR-ABL kinase domain32
. It would be expected that this basic mechanism of drug resistance has broad implications to all cancers with high glycolytic activity and elevated intracellular ROS. Limiting PFKFB3 function may therefore help to maintain genomic stability and would be predicted to delay the onset of drug resistance and disease progression.
Our data using a lentiviral shRNA approach suggest a crucial role of PFKFB3 for in vivo
tumor growth and expand previous data33
by linking its regulation, at least in part, to activation of the JAK2/STAT5 pathway. The role of JAK/STAT signaling for dysregulated glucose metabolism has not been sufficiently appreciated and targeting mechanism involved in its regulation may open a venue for novel therapeutic approaches. We have used the small molecule drug 3PO, a compound previously identified as a potent inhibitor of PFKFB318
, to efficiently inhibit lactate production and longer exposure of cells to this drug led to a drastic loss in cell growth. It is not known whether 3PO has additional off target effects, but these results are consistent with our genetic approaches. Targeting glycolytic mechanisms, as a central pathway in carbon metabolism, is of obvious concern. PFKFB3 knockout mice are known to be embryonically lethal34
, suggesting a crucial role for this enzyme not only in transformed cells but also in proliferating or differentiating tissue. The functional role of PFKFB3 in adult mice has not yet been studied and may be quite different. Nevertheless, the PFKFB3 inhibitor 3PO showed efficacy in a mouse model, without causing apparent side effects18
, suggesting the feasibility of this approach. In humans, there are three isoforms of the PFKFB3 target PFK-1. Defects in the muscle specific isoform PFKM causes glycogen storage disease type 7 (GSD7 or Tarui disease), characterized by different degrees, of exercise intolerance, myopathy, loss of muscle and red cell PFK activity or congenital nonspherocytic hemolytic anemia35
. One would expect that targeting PFKFB3 leads to a somewhat increased risk of side effects, similar to the symptoms observed in this disease. It would now be important to see whether different tumors are similarly dependent on PFKFB3 and whether targeting its expression has similar effects on tumor growth as in our model.