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. 2016 Aug 8;6:31146.
doi: 10.1038/srep31146.

Hypoxic Repression of Pyruvate Dehydrogenase Activity Is Necessary for Metabolic Reprogramming and Growth of Model Tumours

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Free PMC article

Hypoxic Repression of Pyruvate Dehydrogenase Activity Is Necessary for Metabolic Reprogramming and Growth of Model Tumours

Tereza Golias et al. Sci Rep. .
Free PMC article

Abstract

Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs.

Figures

Figure 1
Figure 1. Hypoxia inhibits mitochondrial OCR and PDH activity and induces PDHK1 protein and activity.
(a) Ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) measured by Seahorse XF in MIA PaCa-2, PANC-1, and SU.86.86 cell lines in high (25 mM) or low (0.5 mM) glucose incubated overnight with or without 1 mM DMOG. (mean ± SEM, two-tailed Student’s t-test, **p < 0.01, ***p < 0.001) (b) Cell-based PDH activity assay in cells incubated 16 h in normoxia, hypoxia (0.5% O2) or 1 mM DMOG. (mean ± SD, one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001) (c) Western blots of HIFα isoforms, pyruvate dehydrogenase kinase isoforms (PDHKs), phosphatase (PDP1 – lower band *), target phosphorylated serine residues on E1α and total E1α after overnight incubation in normoxia or hypoxia (0.5% O2) at 25 or 5 mM glucose as indicated.
Figure 2
Figure 2. PDHK1 kinase activity can be induced by hypoxia, and it is essential for serine 232 phosphorylation.
(a) Western blots of MIA PaCa-2 control (Hg) and shHIF1α cells treated for 16 h with decreasing oxygen levels showing effect on HIF-regulated PDHK1 and 3 expression and phosphorylation of PDH E1α. (b) WT and HIF1α KO MEFs were treated with hypoxia (0.5% O2) or 1 mM DMOG and levels of PDHK1 and pSer232-E1α detected by Western blotting. (c) PANC-1 control (Hg (pX335)) and PDHK1 null cells (PDHK1 KO, two clones) treated with hypoxia (0.5% O2) overnight showing no kinase activity on Ser232 of E1α. Note that phosphorylation of Ser293 and 300 remain unaffected, reflecting activity of the remaining PDHKs. (d) PDH activity measured in extracts of PANC-1 control and PDHK1 null cells described in (c), after treatment with hypoxia (0.5%) or 1 mM DMOG overnight. (mean ± SD, one-way ANOVA, *p < 0.05, **p < 0.01).
Figure 3
Figure 3. Genetic manipulations can alter PDH E1α phosphorylation in response to hypoxia.
(a) MIA PaCa-2 control (Hg), silenced HIF1α (shHIF1α) or silenced PDHK1 (shPDHK1) and PDP1 overexpressing cells were incubated for 16 h in normoxia or hypoxia (0.5% O2) and phospho-serine E1α detected by Western blot showing that all three modifications can have an inhibitory effect on hypoxic Ser232 phosphorylation. (b) Increased pSer232-E1α detection after E1α immunoprecipitation from lysates of cells treated as in (a). (c) The same analysis as in (a) after treatment with 16 h 1 mM DMOG.
Figure 4
Figure 4. Genetic manipulations can alter hypoxic reduction in PDH activity and OCR.
(a) PDH activity assay performed on immuno-captured PDH from MIA PaCa-2 Hg, shHIF1α, shPDHK1, and PDP1 cells treated with 16 h normoxia or hypoxia (0.5% O2) showing a blunted response to hypoxia. (mean ± SD, two-tailed Student’s t-test, **p < 0.01) (b) Western blot of captured PDH from samples in (a) showing a commensurate reduction in phosphorylation of E1α. (c) Quantization of the phospho signal in panel (b) normalized to the signal for total E1α in (b) by ImageJ software. (d) OCR to ECAR ratios of modified MIA PaCa-2 cells incubated with or without 1 mM DMOG overnight showing modification of hypoxic OCR/ECAR. (mean ± SEM, two-tailed Student’s t-test, *p < 0.05, ***p < 0.001).
Figure 5
Figure 5. Decreased hypoxic response of PDH activity can slow the growth of xenografted tumours.
(a) Tumour growth curves of MIA PaCa-2 genetically modified cells (HIF1α and PDHK1 knockdown, PDP1 overexpression) described in Fig. 4 grown in nude mice (n = 8 per group in replicate experiments). Statistically significant differences exist between control (Hg) and shPDHK1 and PDP1 tumours as indicated. (mean ± SEM, two-way ANOVA) (b) Western blots from three random tumours of each group in (a) showing decreased PDHK1 expression and activity in vivo. (c) Tumour volumes of SU.86.86 control (LUC) and PDHK1 knockdown cells grown in nude mice (n = 8 per group). The growth rate differences were statistically significant. (mean ± SEM, two-way ANOVA) (d) Western blots from three random tumours in (c) showing decreased PDHK1 expression and activity in vivo. (e) Tumour volumes of PANC-1 control (Hg (pX335)) and 2 clones of PDHK1 null cells (PDHK1 KO) described in Fig. 2 grown in nude mice (n = 4 per group). The growth rate differences were statistically significant. (mean ± SEM, two-way ANOVA) (f) Western blots from three random tumours in (e) showing no PDHK1 expression and activity in vivo.
Figure 6
Figure 6. PDHK1 and pSer232-E1α are prognostic markers in oropharyngeal cancers.
(a) Examples of low (left) and high (right) magnifications images of staining patterns for negative and positive cores detecting either PDHK1 (top) or PDH pSer232-E1α (bottom). (b) Kaplan Meier (KM) survival curves showing univariate analysis of overall survival based on PDHK1 stain intensity. (c) KM curves showing univariate analysis of survival based on pSer232-E1α stain intensity. (d) Dual marker analysis combining both markers.

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