The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation

Abstract

Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway.

Figure 1. PI3K/mTOR inhibition reduces O₂ consumption of SQ20B cells in vitro

(A) After 16 hrs. of treatment with BEZ235 (50 nM), SQ20B cells were harvested as single cells and placed in Clark electrode chambers for O₂ measurement. Slopes of lines represent rate of O₂ consumption. Bar graph to right of line graph shows O₂ consumption rate (OCR) determined from these slopes (*: p = 0.014). (B) Cells were seeded into flux analyzer plates and allowed to attach before BEZ235 or BGT226 was added for 16 hours prior to measurement of OCR. Vertical lines labeled A, B, and C indicate respectively times when ATPase inhibitor oligomycin, mitochondrial uncoupler FCCP, or complex I inhibitor rotenone was added. Bar graph to right of flux analyzer tracing shows baseline OCR as determined from flux analyzer tracing at start of measurements (average of readings at 0, 8, and 16 minutes). (*: p = 0.001; **: p = 0.006). (C) SQ20B cells were transfected with scrambled siRNA or siRNA directed against Akt1. 48, 72 or 96 hours later cells were trypsinized and Western blotting was performed. Numbers below P-Akt lane represent fold-increase in intensity relative to lane 1. (D) cells were plated into flux analyzer plates and allowed to attach prior to OCR measurement. This panel shows bar graph using T=0 measurements from flux analyzer tracing (*: p = 0.006). (E) Cells were treated with KU-0063794 for indicated lengths of time prior to harvesting. Lysates were collected, and immunoblotting was performed. (F) Cells were seeded into flux analyzer plates and allowed to attach before KU-0063794 was added for 16 hours prior to measurement of OCR. Bar graph shows baseline OCR as determined from flux analyzer tracing (at start of measurements).

Figure 2. PI3K/Akt/mTOR inhibition increases E1α phosphorylation in SQ20B cells

Panels (A) – (C) are immunoblots using antibodies as indicated. Fold increase refers to increase in P-PDH-E1α (Ser293) relative to baseline lane, which is given the value 1.0. (A) SQ20B cells were treated with BEZ235 or BGT226 for indicated lengths of time prior to harvesting. Numbers below P-PDH (S293) lane represent fold-increase in intensity relative to lane 2. (B) Cells were harvested 48 hours after transfection with Akt1 siRNA or 16 hours after BEZ235 or BGT226 (50 nM) treatment. Numbers below P-PDH (S293) lane represent fold-increase in intensity relative to lane 1. (C) SQ20B cells were treated with GDC-0068 (5 μM) or GDC-0980 (1 μM) for indicated lengths of time before harvest. Numbers below P-PDH (S293) lane represent fold-increase in intensity relative to lane 1. (D) SQ20B cells were seeded into flux analyzer plates and allowed to attach before GDC-0068 or GDC-0980 was added for 16 hours prior to measurement of OCR. Bar graph (2D) shows baseline OCR as determined from flux analyzer tracing (Suppl. Fig. 6A, B) at start of measurements (average of readings at 0, 11, and 21 minutes). (*: p = 0.004). (E) Same as in (D) except MK-2206 was added for 16 hours prior to measurement of OCR. Lysates were collected, and immunoblotting was performed. (F) Cells were seeded into flux analyzer plates and allowed to attach before MK-2206 was added for 16 hours prior to measurement of OCR. Bar graph shows baseline OCR as determined from flux analyzer tracing (at start of measurements).

Figure 3. PI3K/mTOR inhibition decreases O₂ consumption and increases PDH-E1α phosphorylation in other cell lines

(A) FaDu cells were treated with BEZ235 or BGT226 for indicated lengths of time prior to harvesting. Lysates were collected, and immunoblotting was performed. Fold increase refers to increase in P-PDH-E1α (Ser293) relative to baseline lane, which is given the value 1.0. (B) FaDu cells were seeded into flux analyzer plates and allowed to attach before BEZ235 or BGT226 was added for 16 hours prior to measurement of OCR. Bar graph shows baseline OCR as determined from flux analyzer tracing (Suppl. Fig. 6) at start of measurements (average of readings at 0, 11, and 21 minutes). (*: p = 0.004; **: p = 0.008). (C) U251MG cells engineered to be inducible for either wild-type PTEN or phosphatase-dead PTEN (C124S) were exposed to doxycycline (1μg/ml). After 24 hours, cells from each group were harvested and the resultant lysates immunoblotted. (D) U251-PTEN cells were treated with doxycycline as described in panel C, then placed into flux analyzer plates for OCR measurement. Flux analyzer tracing is shown in Suppl. Fig. 7A. Lines A, B, C represent times when oligomycin, FCCP, and rotenone were added respectively. Bar graph (Fig. 3D) representing baseline OCR taken from flux analyzer tracing. (*: p = 0.0001). (E) U251-wtPTEN cells were treated with BEZ235 (50 nM) or BGT226 (50 nM) for indicated lengths of time prior to harvesting. Lysates were collected, and immunoblotting was performed. Fold increase refers to increase in P-PDH-E1α (Ser293) relative to baseline lane, which is given the value 1.0.

Figure 4. Downregulating PDH decreases O₂ consumption and reversing drug-induced PDH phosphorylation abrogates effect of PI3K inhibitors on O₂ consumption

(A, B) SQ20B cells were seeded and allowed to attach prior to transfection with siRNA PDH-E1α (50 nM). Parallel dishes of cells were treated with BEZ235 (50 nM) or BGT226 (50 nM). Either 48 hours after siRNA transfection or 16 hours after drug treatment, (A) cells were harvested and immunoblotting was performed or (B) OCR measurements were made on the flux analyzer. Bar graph shows baseline OCR as determined from flux analyzer tracing (Suppl. Fig. 8A) at start of measurements (average of readings at 0, 11, and 21 minutes). The difference between the scrambled siRNA and any of the other 3 groups, PDH siRNA, BEZ235 or BGT266 was statistically significant (*: p = 0.00001). (C, D) Cells were treated with 20 mM DCA (dicholoroacetate) for one hour prior to being treated for 16 hours with either BEZ235 or BGT226. Either (C) immunoblotting was performed and numbers below P-PDH (S293) lane represent fold-increase in intensity relative to lane 1. (D) OCR measurements were made on the flux analyzer. Bar graph shows baseline OCR as determined from flux analyzer tracing (Suppl. Fig. 8B) at start of measurements (average of readings at 0, 11, and 21 minutes). (*: p = 0.0002, **: p = 0.00003).

Figure 5. Ectopic expression of PDH-E1α mutants that cannot be phosphorylated blunts BEZ235-mediated decrease in OCR

(A) Schematic representation of plasmids encoding C terminal FLAG-tagged mouse PDHA1 (WT E1α) and single point mutation of serine232 -> alanine at position (1S-1A) or triple mutation of serine232, serine 292, and serine 300 -> alanine (3S-3A). (B) SQ20B cells were infected with retrovirus expressing plasmids encoding WT E1α) or 1S-1A mutant or 3S-3A mutant. 72 hours later cells were trypsinized and Western blot analysis was performed for indicated proteins. (C) Alternatively similarly infected cells were plated into flux analyzer plates and allowed to attach prior to OCR measurement. The bar graph shows the OCR values for control and infected cells +/− BEZ235 treatment. (D) The same OCR data from (C) are presented as % change (decrease in OCR following BE235 treatment divided by OCR without BEZ2345 treatment). (*: p = 0.004; **: p <0.001).

Figure 6. BEZ235 decreases tumor hypoxia in human xenografts

(A, B) When the mice were 5 to 7 weeks of age, tumors were initiated in the flank by subcutaneous injection of 1 × 10⁶ SQ20B cells. Starting 10–14 days later, when tumors were 50–100 mm³ in size, mice in the treatment group were gavaged daily with BEZ235 (50 mg/kg) suspended in 0.1% Tween 80 and 0.3% carboxymethylcellulose in sterile physiological saline. After 5 days of drug treatment, the mice were injected with EF3. 1.5 hours later they were sacrificed, and the tumors were removed and stained using an antibody to EF3. Panel A shows representative samples with staining. Panel B shows the quantitation of EF3 binding as bar graph (*: p = 0.05). (C) SQ20B xenografts were grown subcutaneously in nude mice. Tumors were measured every two days through the experiment and plotted in panel. (D) When tumors reached a size of at least 400 mm³, mice were assigned to either control (4 tumors) or BEZ235 treatment (6 tumors). The OxyLab pO₂^™ probe was used to measure pO₂ levels (see Materials and Methods) for the control and BEZ-treated tumors on day 0 (just prior to drug treatment) and then on day 1 and day 3 following the start of drug treatment. Each animal served as its own control, and the fold change was calculated relative to the day 0 measurement for each individual mouse and plotted in whisker graph. (E) Following pO₂ measurement on day 3, mice used in (D) were sacrificed and tumors were removed to measure the in vivo level of PDH 293 phosphorylation by immunoblot analysis. Numbers below P-PDH (S293) lane represent fold-increase in intensity relative to lane 1.