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. 2014 Jun;124(6):2640-50.
doi: 10.1172/JCI71749. Epub 2014 Apr 24.

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation

Ming Yan, Marie-Claude Gingras, Elaine A Dunlop, Yann Nouët, Fanny Dupuy, Zahra Jalali, Elite Possik, Barry J Coull, Dmitri Kharitidi, Anders Bondo Dydensborg, Brandon Faubert, Miriam Kamps, Sylvie Sabourin, Rachael S Preston, David Mark Davies, Taren Roughead, Laëtitia Chotard, Maurice A M van Steensel, Russell Jones, Andrew R Tee, Arnim Pause

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation

Ming Yan et al. J Clin Invest. 2014 Jun.

Abstract

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

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Figures

Figure 1
Figure 1. Loss of FLCN stimulates HIF-dependent glycolysis and ATP production.
(A, D, and E) Western blot analysis of FLCN, HIF-1α, and HIF-2α expression levels in the indicated MEF cell lines. Actin was used as loading control. Results are representative of 3 independent experiments. (B) Fold HIF activity assessed under hypoxic conditions using HIF reporter assay. (C and F) Relative mRNA expression of HIF target genes determined by qRT-PCR (C) in the indicated MEFs or (F) in KO MEFs downregulated (shHIF-1α cell lines) or not (shNT) for HIF-1α. (G) Fold change in glucose uptake (Glu up), lactate production (Lact), and extracellular acidification rate (ECAR) in the indicated MEFs. (H) Glucose uptake and ATP levels measured in the indicated MEF cell lines. Data in B, C, and F–H represent the mean ± SD of 4 independent experiments performed in triplicate. *P < 0.05 **P < 0.01, ***P < 0.001.
Figure 2
Figure 2. Enhancement of mitochondrial biogenesis upon loss of FLCN activates HIF in MEFs.
(A) Fold changes in total mitochondrial respiration, divided in ATP turnover and proton leak, determined following inhibition of ATP synthase by oligomycin treatment. (B) Fold changes in mitochondrial load and potential determined using MitoTracker Green FM and MitoTracker Red CMXROS, respectively. Pot, mitochondrial membrane potential; Load, mitochondrial mass. (C and F) Fold ROS levels assessed using the general oxidative stress indicator CM-H2DCFDA incubated with cells for (C) 30 minutes or (F) the indicated time points in a time course experiment. Data are expressed as fold ROS levels normalized to (C) WT or as (F) the KO/WT ROS ratio. (D) Extent of ROS-dependent protein and (E) DNA damage quantified using the OxyBlot Protein Oxidative Detection Kit and the OxiSelect Oxidative DNA Damage Kit, respectively. Data are representative of 3 independent experiments. (G) Relative mRNA expression of HIF target genes in MEFs treated with 10 mM of the antioxidant NAC for 24 hours. Data in A–C and E–G represent the mean ± SD of 4 independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3. ROS-mediated HIF activation depends on PGC-1α upregulation in FLCN-null cells.
(A) PPARGC1A and PPARGC1B relative mRNA expression measured by qRT-PCR in the indicated MEFs. (B) Western blot analysis of the PGC-1α and FLCN expression levels. Actin was blotted as loading control. Results are representative of 3 independent experiments. (C) Relative mRNA expression of PGC-1α target genes and coactivators determined by qRT-PCR. (D) PGC-1α protein and relative mRNA expression levels in KO MEFs downregulated using shRNA for PGC-1α (shPGC-1α-A and -B) or control (shNT), as measured by Western blot and qRT-PCR. (E) Relative ROS levels and (F) HIF target gene expression determined in the indicated cell lines. Data represent the (A and D–F) mean ± SD or (C) mean ± SEM of 4 independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4. AMPK activation upon loss of FLCN binding drives the PGC-1α-ROS–mediated HIF induction.
(A) Western blot analysis of AMPK expression (AMPKα) and activation (pT172 AMPKα [pAMPKα]) levels and acetyl-CoA carboxylase (ACC) expression and activation (pS79 ACC [pACC]) levels in the indicated MEFs. Actin was used as loading control. (B) Western blot analysis of Ampk–/– or Ampk+/+ MEFs downregulated (shFlcn) for FLCN or not (shEV). Actin was used as loading control. (C) Relative PPARGC1A mRNA expression and (D) ROS production levels in the indicated MEFs. (E–G) Flcn KO MEFs were rescued with FLCN WT, FLCN S62A mutant (S62A), or EV constructs, and (E) the extent of FLCN binding to AMPKα and FNIP1 was determined by coimmunoprecipitation. The effect of the S62A mutation on (F and G) PGC-1α, (G) HIF target gene expression, and (F) AMPK activation (pT172 AMPKα) was assayed by (F) Western blot and (G) qRT-PCR. Results in A, B, E, and F are representative of 3 independent experiments and data in C, D, and G represent the mean ± SD of 4 independent experiments performed in triplicate. **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. FLCN-null cell survival depends on glucose-derived biosynthetic precursor production.
(A) Metabolic signature of Flcn KO cells determined by GC/MS and LC/MS. Data are expressed as fold of the WT MEF metabolite quantification and are indicated in the schematic representation of the metabolic pathways. (B and C) Mass isotopomer labeling of MEFs pulsed with (B) 13C glucose or (C) 13C glutamine. Relative incorporation of (B) 13C glucose (dark gray) and (C) 13C glutamine (light gray) to total metabolite pool are indicated, and the metabolite abundance relative to WT condition was measured. (D and E) Percentage cell survival under (D) glucose and (E) glutamine starvation relative to cell number at day 0. Data represent the mean ± SD of (A) 8 or (B–E) 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6
Figure 6. HIF-dependent metabolic advantage drives tumorigenesis in FLCN-deficient cancer cells.
(A) Western blot analysis of the AMPK expression (AMPKα) and activation (pT172 AMPKα) and PGC1-α and HIF-1α expression levels in FTC-133 cells deficient (EV) or rescued (Resc) for FLCN expression. (B–D) Fold (B) mitochondrial respiration, (C) load and potential, and (D) ROS production in FTC-133 cells. (E) Fold mRNA levels of the indicated genes relative to FTC-133 cells rescued for FLCN expression. (F) Glycolysis (glucose uptake) and ATP levels quantified in the FLCN-null (EV) cells downregulated (shHIF-1α) or not (shScram) for HIF-1α expression and compared with FTC-133 cells rescued for FLCN expression (Resc). (G) Fold change in soft agar colony number and (H) xenograft tumor volume 42 days after subcutaneous tumor cell injection in nude mice using the indicated FTC-133 cell lines. Data represent the mean ± SD of (A–G) 4 independent experiments or (H) 5 tumors per group. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7
Figure 7. Increased mitochondrial content and HIF target gene expression in a BHD tumor.
(A and B) Representative images of immunohistochemistry staining performed on a BHD kidney chromophobe tumor and (A) normal kidney or (B) adjacent unaffected tissues. Scale bar: 50 μm (A); 100 μm (B).
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