ls Feedback loops to strengthen the metabolic reprogramming Although over-active PDH can promote reactive oxygen species production during high mitochondrial flux, dysfunction of PDH within the mitochondrion 
is invariably associated with increased production of ROS. Thus, Pvract cells exhibit high ROS levels detected by direct staining with the DHE dye or as monitored by gstD-GFP expression frequently used as a surrogate for ROS, marking cells under oxidative stress. This ROS induction is suppressed when PDHKRNAi is co-expressed. Co-expression of hRafact and PI3Kact does not cause ROS production unlike that seen for Pvract. Instead, Pvract induced ROS is effectively suppressed in a BskDN genetic background. Similar effects are seen upon knockdown of hep or Src42A. Thus Src-JNK signaling plays an important role in the inhibition of mitochondrial activity and in raising ROS levels in Pvract tumors. Two R-roscovitine notable facts about ROS have a bearing on the data presented here. The first is that ROS can activate the JNK pathway through interaction with the upstream component ASK . The second is that high ROS conditions result in potent stabilization of the Hif protein even under normoxic conditions. This provides the opportunity for ROS generated downstream of both Hif and JNK, to feedback and reinforce these two pathways. To test this hypothesis, several antioxidant proteins were expressed to reduce the level of ROS. Of these, the strongest ROS scavenging activity was seen upon Peroxidasin expression. In addition to a decrease in ROS, we found that JNK is no longer activated when Pxn is co-expressed in Pvract discs. Similarly, LDH-GFP expression and the accumulation of Sima protein are strongly suppressed by co-expression of Pxn. These results establish ROS as the central player in enforcing the metabolic reprogramming. Initially established through the activation of three oncogenic pathways by Pvr, the shift to glycolysis is then reinforced when ROS is generated as a subsequent step. This establishes one means to maintain a stable Warburg effect by oncogene activation. Discussion A model can be constructed that is consistent with all our observations on the Pvract induced interacting network that leads to aerobic glycolysis and potentially also allows this new metabolic state to be maintained through later stages of tumor growth. This is a genetic model derived from analysis that allows placement of gene function according to their hierarchy along a pathway. Many genetic backgrounds that achieve full or partial Warburg effect have been described in the literature. Although the details may seem to vary, the three critical components involved in this process are the two pyruvate-metabolism enzymes, LDH and PDH and the free radical metabolite class designated as ROS. During the acquisition of the aerobic glycolytic activity in a tumor environment, LDH actively converts pyruvate to lactate, and high PDHK inactivates PDH resulting in low pyruvate to acetyl-CoA conversion, low TCA flux and electron transport activity. Our results suggest that these events can be sustained over long periods of tumor growth only when coupled with a feed back signal from accumulating ROS in a mechanism that reinforces and enhances the high-LDH/low-PDH activity state. This genetic study was achieved in an in vivo context of an animal that is PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826115 only mutant for the one specific activated oncogene that we introduce and is otherwise normal. Additional pathways are activated with