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. 2013 Sep 1;73(17):5459-72.
doi: 10.1158/0008-5472.CAN-13-1429. Epub 2013 Jun 24.

Inhibition of AMPK and Krebs cycle gene expression drives metabolic remodeling of Pten-deficient preneoplastic thyroid cells

Valeria G Antico Arciuch  1 , Marika A Russo, Kristy S Kang, Antonio Di Cristofano
Affiliations

Inhibition of AMPK and Krebs cycle gene expression drives metabolic remodeling of Pten-deficient preneoplastic thyroid cells

Valeria G Antico Arciuch et al. Cancer Res. .

Abstract

Rapidly proliferating and neoplastically transformed cells generate the energy required to support rapid cell division by increasing glycolysis and decreasing flux through the oxidative phosphorylation (OXPHOS) pathway, usually without alterations in mitochondrial function. In contrast, little is known of the metabolic alterations, if any, which occur in cells harboring mutations that prime their neoplastic transformation. To address this question, we used a Pten-deficient mouse model to examine thyroid cells where a mild hyperplasia progresses slowly to follicular thyroid carcinoma. Using this model, we report that constitutive phosphoinositide 3-kinase (PI3K) activation caused by PTEN deficiency in nontransformed thyrocytes results in a global downregulation of Krebs cycle and OXPHOS gene expression, defective mitochondria, reduced respiration, and an enhancement in compensatory glycolysis. We found that this process does not involve any of the pathways classically associated with the Warburg effect. Moreover, this process was independent of proliferation but contributed directly to thyroid hyperplasia. Our findings define a novel metabolic switch to glycolysis driven by PI3K-dependent AMPK inactivation with a consequent repression in the expression of key metabolic transcription regulators.

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Figures

Figure 1
Figure 1. Metabolic reprogramming in Ptenthyr−/− mice
(A) Relative expression of the indicated genes in wild type and mutant thyroids. Bars represent mean ± SD of triplicate measurements. Asterisks indicate significant (P<0.05) differences. (B) Western blot showing down-regulation of representative TCA cycle enzymes in mutant glands. (C) TCA cycle diagram showing the genes down-regulated in Ptenthyr−/− thyroids. The color scale reflects changes calculated from the Affymetrix data. Genes circled in red have been validated by qPCR. (D) Heat map showing the repression of TCA cycle genes in Ptenthyr−/− thyroids and in follicular carcinomas (FTC) arising in older mice. (E) DNA content-based assessment of mitochondria number in 3 month-old wild type and mutant mice (top panel). Expression levels of selected mitochondrial genes in the thyroids of wild type and mutant mice (bottom panel). Bars represent mean ± SD of triplicate measurements. Asterisks indicate significant (P<0.05) differences. (F) Mitochondrial damage (swelling, clarification, cristae disruption) in Pten−/− glands detected by transmission electron microscopy. (G) Oxygen consumption rates (OCR) in control and mutant primary thyrocytes cells in response to 1 μg/ml Oligomycin, 1 μM Fluoro-carbonyl cyanide phenylhydrazone (FCCP) or 2 μM Antimycin A + 2 μM Rotenone. (H) Extracellular acidification rate (ECAR) in control and mutant primary thyrocytes cells. P= 0.004). (I) Mitochondrial membrane polarization was measured in primary thyrocytes by flow cytometry using TMRE. FCCP pretreatment of wt cells was used to determine the baseline.
Figure 2
Figure 2. Enhanced glycolysis in Pten−/− thyroids
(A) Lactate levels in the thyroids of 3 month-old wild type and mutant mice. Bars represent mean ± SD (n=4 per pool). (B) Lactate production rate in primary cultures of wild type and mutant thyrocytes. (C) 18FDG microPET analysis showing increased glucose uptake in 3 month-old mutant mice compared to wild type controls. The inset shows a transverse section centered on the thyroid. (D) Expression levels of the indicated genes in wild type and mutant thyroids. Bars represent mean ± SD of triplicate experiments. (E) Western blot showing no deregulation of Hif1α and PKM2 in mutant glands. Asterisks indicate significant (P<0.05) differences.
Figure 3
Figure 3. TCA cycle/OXPHOS gene repression depends on Pdk1 but not mTOR
(A, B) Loss of Pdk1 restores normal levels of TCA cycle (A) and mitochondrial (B) gene expression in Ptenthyr−/− mice. Bars represent mean ± SD of triplicate assays. (C) Loss of Pdk1 restores normal levels of lactate in Ptenthyr−/− thyroid glands. Bars represent mean ± SD (n=4 per genotype). (D) Scheme of RAD001 administration to 3-month old mice and western blot verification of effective inhibition of mTOR activity. (E) mTOR inhibition suppresses thyrocyte proliferation. Bars represent mean ± SD (n=5 per genotype). (F, G) mTOR inhibition fails to restore normal TCA cycle (F) and mitochondrial (G) gene expression. Bars represent mean ± SD of triplicate assays. (H) mTOR inhibition fails to rescue lactate increase in mutant mice. Bars represent mean ± SD (n=4 per genotype). Asterisks indicate significant (P<0.05) differences.
Figure 4
Figure 4. AMPK is repressed in Ptenthyr−/− thyroids
(A) qPCR profiling of metabolic transcriptional regulators in the thyroids of control and mutant mice. Bars represent mean ± SD of triplicate assays. Asterisks indicate significant (P<0.05) differences. (B) Western blotting analysis of AMPK activation in wild type and mutant glands. (C) AMP, ADP, and ATP levels in thyroids from wild type and Ptenthyr−/− mice. Bars represent mean ± SD (n=3 pools of 10 thyroids per genotype).
Figure 5
Figure 5. AICAR-mediated AMPK activation reverts the metabolic switch in Ptenthyr−/− thyroids
(A) Top panel: Scheme of AICAR administration: 3 month-old mice were injected IP with AICAR (400 mg/kg/day) for 4 weeks. Bottom panel: Western blotting analysis of the effect of in vivo AICAR treatment on the phosphorylation of AMPK. (B, C) Expression levels of selected metabolic transcriptional regulators (B) and TCA cycle genes (C) upon in vivo AICAR treatment. Bars represent mean ± SD of triplicate assays. (D) Western blotting showing that in vivo AICAR treatment rescues TCA cycle enzymes expression. (E) Expression levels of mitochondrial genes in the thyroids of control and AICAR-treated mice. Bars represent mean ± SD of triplicate assays. (F) Transmission electron microscopy showing mitochondria structure in the thyroids of wild type, Pten−/− and AICAR-treated Pten−/− mice. (G) Effect of the in vivo AICAR treatment on lactate production. Bars represent mean ± SD (n=4 per genotype). (H) Thyroid weight in wild type, mutant, and AICAR treated mutant mice. (I) Proliferation index of thyroids from wild type, mutant, and AICAR treated mutant mice as measured by BrdU incorporation. Asterisks indicate significant (P<0.05) differences.
Figure 6
Figure 6. The AKT/AMPK/PGC1α/TCA cycle axis is conserved in human thyroid cancer cells
(A) Western blotting analysis of the effect of AICAR treatment on AMPK phosphorylation status in FTC-133 cells. (B) Expression analysis of representative TCA cycle genes in FTC-133 cells upon AICAR treatment. Bars represent mean ± SD of triplicate assays. (C) AICAR-induced increase in the expression levels of representative TCA cycle genes in FTC-133 cells transfected with wild type or mutant (AA) PGC1α. On the top, western blotting analysis showing comparable expression levels of transfected wild type and mutant (AA) PGC1α (specific band marked with an asterisk). (D) Western blotting analysis of the effect of H89, BKM120, and their combination on AMPK phosphorylation in FTC-133 cells. (E) Expression levels of representative metabolic transcriptional regulators and TCA cycle genes upon H89, BKM120, and their combination treatment in FTC-133 cells. Bars represent mean ± SD of triplicate assays. (F) Luciferase assay showing PGC1α promoter activity in FTC-133 cells after treatment with the indicated inhibitors. Bars represent mean ± SD (n=3 per treatment). (G) Western blotting analysis of the effect of AICAR treatment of THJ16T and 8505c cells on AMPK phosphorylation status. (H) Western blotting analysis of the effect of H89, BKM120 and their combination on AMPK phosphorylation status in THJ16T and 8505c cells. (I) Expression levels of the ESRRG and PPARGC1B metabolic transcriptional regulators upon H89, BKM120, and their combination treatment in THJ16T and 8505c cells. Bars represent mean ± SD of triplicate assays. Asterisks indicate significant (P<0.05) differences. (J) Proliferation of mouse and human thyroid cancer cells treated with 1mM AICAR and counted after 72h. Bars represent mean ± SD of triplicate assays.
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