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. 2014 Jan;12(1):48-57.
doi: 10.1158/1541-7786.MCR-13-0258-T. Epub 2013 Dec 2.

Autophagy-dependent metabolic reprogramming sensitizes TSC2-deficient cells to the antimetabolite 6-aminonicotinamide

Andrey A Parkhitko  1 , Carmen Priolo, Jonathan L Coloff, Jihye Yun, Julia J Wu, Kenji Mizumura, Wenping Xu, Izabela A Malinowska, Jane Yu, David J Kwiatkowski, Jason W Locasale, John M Asara, Augustine M K Choi, Toren Finkel, Elizabeth P Henske
Affiliations

Autophagy-dependent metabolic reprogramming sensitizes TSC2-deficient cells to the antimetabolite 6-aminonicotinamide

Andrey A Parkhitko et al. Mol Cancer Res. 2014 Jan.

Abstract

The mammalian target of rapamycin complex 1 (mTORC1) is hyperactive in many human cancers and in tuberous sclerosis complex (TSC). Autophagy, a key mTORC1-targeted process, is a critical determinant of metabolic homeostasis. Metabolomic profiling was performed to elucidate the cellular consequences of autophagy dysregulation under conditions of hyperactive mTORC1. It was discovered that TSC2-null cells have distinctive autophagy-dependent pentose phosphate pathway (PPP) alterations. This was accompanied by enhanced glucose uptake and utilization, decreased mitochondrial oxygen consumption, and increased mitochondrial reactive oxygen species (ROS) production. Importantly, these findings revealed that the PPP is a key autophagy-dependent compensatory metabolic mechanism. Furthermore, PPP inhibition with 6-aminonicotinamide (6-AN) in combination with autophagy inhibition suppressed proliferation and prompted the activation of NF-κB and CASP1 in TSC2-deficient, but not TSC2-proficient cells. These data demonstrate that TSC2-deficient cells can be therapeutically targeted, without mTORC1 inhibitors, by focusing on their metabolic vulnerabilities.

Implications: This study provides proof-of-concept that therapeutic targeting of diseases with hyperactive mTORC1 can be achieved without the application of mTORC1 inhibitors.

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Conflict of interest statement

The authors disclose no potential conflicts of interest

Figures

Figure 1
Figure 1. Chloroquine inhibits the proliferation and tumorigenicity of Tsc2-deficient cells and reprograms their metabolism
(A) Proliferation of Tsc2−/− and Tsc2+/+ MEFs treated with chloroquine (CQ, 5 μM) for 4 days (crystal violet staining). Time points represent average of 3 independent experiments ± SD. Comparison of Tsc2−/− treated with CQ vs. control: *p<0.05. B) Macroscopic (upper panel) and microscopic (lower panel) score of spontaneous renal tumors in Tsc2+/− mice (n=15 for macroscopic and n=8 for microscopic score) following 4 month of i.p. treatment with chloroquine (CQ, 50 mg/kg/day, 5 days/week) or placebo (n=14 for macroscopic and n=14 for microscopic score). Photographs show representative kidneys of a control mouse and a CQ-treated mouse. Arrows indicate macroscopic lesions. **p<0.01 (C) Heat map showing the top 40 metabolites significantly changed in Tsc2−/− MEFs treated with Chloroquine (CQ, 5 μM) for 24 hours (n= 4 samples) versus control (n= 4 samples; left panel). Immunoblot analysis of phospho-S6 Ser 235/236 in Tsc2−/− MEFs treated with chloroquine (CQ, 5 μM) for 24 hours. Metabolic Set Enrichment Analysis (MSEA) of the Tsc2−/− MEFs treated with CQ vs. control (right panel). (D) Box plots of individual pentose phosphate pathway metabolites that were significantly changed in Tsc2−/− MEFs treated with chloroquine (CQ, 5 μM) for 24 hours.
Figure 2
Figure 2. Autophagy inhibition enhances glucose uptake and suppresses oxygen consumption
(A) Glucose uptake and lactate secretion measured by YSI in Tsc2−/− and Tsc2+/+ MEFs treated with chloroquine (CQ, 5 μM) or control for 24 hours (left panel) or infected with shRNA against the autophagy genes Atg5 (Atg5-1 and Atg5-2) or Beclin1 (right panel). Bars represent average of 4 independent samples ± SD. *p<0.05 **p<0.01 (B) Intact cellular respiration measured using the Seahorse Bioscience XF24 analyzer, under basal conditions or in the presence of FCCP in Tsc2−/− MEFs treated with chloroquine (CQ, 5 μM) for 24 hr. Levels of oxygen consumption were normalized to cell number. Bars represent average of 3 independent experiments ± SD. *p<0.05
Figure 3
Figure 3. Autophagy inhibition enhances glucose oxidative metabolism in mTORC1-dependent manner
(A) Glucose oxidation measured by 14C–CO2 production in Tsc2−/− MEFs following 24-hour treatment with chloroquine (CQ, 5 μM) and 3-hour labeling with D[U-14C]glucose (left panel). Bars represent average of 5 independent experiments ± SD. **p<0.01 (B) Fatty acid oxidation measured by 14C–CO2 production in Tsc2−/− MEFs following 24-hour treatment with chloroquine (CQ, 5 μM) and 3-hour labeling with [U-14C]palmitate (right panel). Bars represent average of 4 independent experiments ± SD. *p<0.05 (C) Glucose oxidation measured by 14C–CO2 production in Tsc2−/− MEFs following 24-hour treatment with chloroquine (CQ, 5 μM), rapamycin (20 nM), or both and 3-hour labeling with D[U-14C]glucose. Bars represent average of 3 independent experiments ± SD. *p<0.05, **p<0.01 (D) Glucose and fatty acid oxidation measured by 14C–CO2 production in Tsc2+/+ MEFs following 24-hour treatment with chloroquine (CQ, 5 μM) and 3-hour labeling with D[U-14C]glucose (left panel) or [U-14C]palmitate (right panel). Bars represent average of 3 independent experiments ± SD.
Figure 4
Figure 4. Autophagy inhibition and 6-aminonicotinamide act synergistically to suppress the growth of Tsc2-null cells
(A, B) Proliferation of Tsc2−/− and Tsc2+/+ MEFs treated with chloroquine (CQ, 5 μM), 6-aminonicotinamide (6-AN, 10 μM) or both for 4 days, measured by crystal violet staining. Right panel: crystal violet staining. **p<0.01 (C) Proliferation of Tsc2−/− and Tsc2+/+ MEFs treated with the autophagy inhibitor spautin-1 (5 μM), 6-AN (10 μM) or both for 4 days (crystal violet staining). **p<0.01 (D) Proliferation of Tsc2−/− MEFs treated with chloroquine (CQ, 5 μM) plus 6-aminonicotinamide (6-AN, 10 μM), NADPH (0.1 mM) alone, or all three for 4 days (crystal violet staining).
Figure 5
Figure 5. Genetic inhibition of macro- or chaperone mediated- autophagy sensitize Tsc2-null cells to 6-aminonicotinamide
(A) Proliferation of Tsc2−/− MEFs infected with control shRNA or shRNA against Atg5 (Atg5-1 and Atg5-2) or Lamp2A and treated with 6-aminonicotinamide (6-AN, 10 μM) for 4 days (crystal violet staining). Time points represent average of 3 independent experiments ± SD. *p<0.05, **p<0.01 (B) Immunoblot analysis of Atg5 (left panel) and Lamp2A (right panel) in shRNA-infected Tsc2−/− MEFs. (C) Autophagy flux analysis in in Tsc2−/− MEFs infected with control shRNA or shRNA against Atg5 (Atg5-1 and Atg5-2) or Lamp2A. Immunoblot analysis of LC3 following 6 hr- treatment with BafilomycinA 20 nM.
Figure 6
Figure 6. Combined autophagy and pentose phosphate pathway inhibition lead to activation of NF-kB and the inflammasome
(A) Immunoblot analysis of phospho-p65 (RelA, ser 536), total RelA, phospho-S6 (ser 235/236) and total S6 in Tsc2−/−and Tsc2+/+ MEFs treated with chloroquine (CQ, 5 μM), 6-AN (10 μM) or both for 4 days. (B) Immunoblot analysis of caspase-1 (pro- and cleaved-form) in Tsc2−/− and Tsc2+/+ MEFs treated with chloroquine (CQ, 5 μM) and 6-AN (10 μM) for 4 days. (C) Immunoblot analysis of caspase-1 (pro- and cleaved-form) in Tsc2−/− MEFs treated with chloroquine (CQ, 5 μM) and 6-AN (10 μM), NADPH (0.1 mM) alone, or all three for 4 days. (D) Working model: autophagy inhibition enhances ROS production, leading to PPP dependency and inflammasome activation. G6P (glucose-6-phosphate); G6PD (glucose-6-phosphate dehydrogenase); 6P-G (6-phosphogluconate); 6PGD (6-phosphogluconate dehydrogenase); R-5P (ribose-5-phosphate); ROS (reactive oxygen species); CQ (chloroquine); 6-AN (6-aminonicotinamide).
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