Pi3k/akt inhibition decreases oxygen consumption in tumor cells by phosphorylating and inactivating pyruvate dehydrogenase (PDH) e1a subunit

Purpose/Objective(s): Hypoxia is associated with resistance to radiation. We have found that the dual PI3K/mTOR inhibitor NVP-BEZ235 reduces tumor hypoxia, which may contribute to its ability to increase radiation response. In vitro exposure of cells to the drug results in decreased O2 consumption rate (OCR). Our objective in this study was to determine the mechanism underlying this phenomenon. Materials/Methods: We measured OCR using the Clark electrode and the XF Extracellular Flux Analyzer. Tissue culture and immunoblotting were performed using standard laboratory protocols. Results: Using the XF Extracellular Flux Analyzer we found that treatment of SQ20B head and neck squamous cell carcinoma cells for 16 hours with another dual PI3K/mTOR inhibitor (NVP-BGT226) or a selective PI3K inhibitor (NVP-BKM120), decreased OCR by 30-40%, similar to the effect seen with NVP-BEZ235. Likewise, siRNA directed against either Akt1 or the p110alpha subunit of PI3K led to a similar decrease in OCR. A key regulator of mitochondrial respiration is the PDH complex, which decarboxylates pyruvate to acetyl CoA, which can then be metabolized via the Krebs cycle. Hence, we examined PDH and found that treatment with any of the 3 drugs led to increased Ser293 phosphorylation of its E1alpha subunit. This phosphorylation inactivates the complex, preventing the conversion of pyruvate to acetyl CoA, which should lead to decreased OCR. We made similar observations with FaDu, another head and neck cancer line, and A549, a lung adenocarcinoma line. To make a stronger link between PDH E1alpha phosphorylation and OCR, we used siRNA against PDH E1alpha and found that it decreased OCR. There was no additional effect when combined with NVP-BEZ235, suggesting an epistatic effect and that the two may lie in a parallel pathway. Lastly, we used dichloroacetate (DCA), which is known to block PDH E1alpha phosphorylation. DCA pre-treatment prevented the upregulation in PDH E1alpha phosphorylation in response to NVP-BEZ235 and also reversed the decrease in OCR the drug causes by itself. Conclusions: Inhibition of the PI3K pathway by several drugs leads to decreased O2 consumption. This effect appears to be mediated, at least in part, by increased phosphorylation of the E1alpha subunit of the PDH complex. Our findings offer mechanistic insight as to how PI3K inhibitors currently being tested in clinical trials alter in vitro O2 metabolism and may play a role in their effects on tumor hypoxia in vivo.