1. The SIRT1/HIF2α Axis Drives Reductive Glutamine Metabolism under Chronic Acidosis and Alters Tumor Response to Therapy 석사 1 학기 이완주

2. Introduction

3. When body fluids contain too much acid, this is known as acidosis. Acidosis occurs when kidneys and lungs can’t keep your body’s pH in balance. The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. Acidosis in the tumor microenvironment may have many effects on the malignancy and development of a tumor. Acidosis

5. Under conditions of hypoxia or mitochondrial dysfunction, isocitrate dehydrogenase (IDH1 in cytosol, IDH2 in mitochondria) uses CO 2 and NADPH to convert α-KG into isocitrate. Citrate produced downstream of this reaction is converted into cytosolic acetyl-CoA without passing through the conventional clockwise steps of the TCA cycle. Acetyl-CoA generated by this pathway can function as a precursor for fatty acid synthesis. Glutamine metabolism

6. Sirtuin Silent information regulator 2 (Sir2) mediates gene silencing and lifespan extension in yeast and Drosophila. Seven Sir2 homologs (sirtuins, SIRT1–7) have been identified in mammals, and all catalyze protein deacetylation or adenosine diphosphate (ADP) ribosylation.

7. Characteristically, sirtuins require oxidized nicotinamide adenine dinucleotide (NAD+ ) for enzymatic reaction and generate nicotinamide (NAM), which then acts as a negative feedback inhibitor. Because of this NAD+ dependency, sirtuins are categorized as class III histone deacetylases (HDACs). In particular, SIRT1 has protein deacetylase activity, but not ADP-ribosyl transferase activity. Furthermore, SIRT1 targets many transcription factors, such as, p53, FOXO, E2F1, NF-kB, PGC-1a, LXR, and MyoD.

8. Hypoxia-inducible factors (HIF) Hypoxia-inducible factors (HIFs) are transcriptional regulators that control genes induced during hypoxia and other stresses. HIF-1α, but not HIF-2α, is transcriptionally regulated during hypoxia, and the activity of HIF-1α is proportional to its abundance. Amounts of HIF-2α protein increase modestly during hypoxia, but HIF-2α–dependent transactivation increases markedly, which suggests that additional posttranslational mechanisms besides oxygen-dependent hydroxylation regulate HIF-2α activity. pVHL(Von Hippel-Lindau) : Tumor suppressor protein FIH : Factor inhibiting HIF-1 PHD : Prolyl hydroxylase

9. In this study We postulated that long-term selection of tumor cells able to survive and proliferate under acidic conditions could offer more relevant models to get insights on the influence of acidosis on tumor metabolism. The capacity of different tumor cells to develop resistance to the intracellular acidification was associated with an increase in SIRT1- driven protein deacetylation leading to a reduction in HIF1a activity and abundance concomitantly to a net increase in HIF2a activity and expression

10. Results

11. Fig 1. Chronic acidosis results in a metabolic switch from glucose to glutamine utilization. S1AS1B S1C

12. S1DS1E GPNA : Glutamine metabolism inhibitor BPTES : Glutaminase(GLS)-selective inhibitor Chronic acidosis causes a shift from glucose to glutamine metabolism in three different tumor cell lines

13. Fig 2. Oxidative and reductive glutamine metabolism pathways are both increased under chronic. GLS1 : Kidney-type glutaminase GLS2 : Liver-type glutaminase GS : Glutamine Synthetase Rotenone, antimycin A : Mitochondria respiratory inhibitor

14. Chronic acidosis enhances both glutamine-fueled respiration and reductive glutamine metabolism

15. Fig 3. Glycolytic pathway is inhibited under chronic low pH conditions. S3A S3B

16. S3C MCT4 : Monocarboxylate transporter Glycolysis inhibition only partly mimics acidosis-triggered increase in glutamine metabolism

17. Fig 4. MCT1 and SIRT1 contribute to the maintenance of the intracellular pH in tumor cells exposed to chronic acidosis. MCT1 and Sirt1 contribute to the maintenance of the intracellular pH in tumor cells chronically exposed to acidosis

18. Fig 5. Changes in both acetylation and abundance of HIF1α and HIF2α support the metabolic adaptation of tumor cells to chronic acidosis. S4C EX-527 : Sirt1 selective inhibitor

19. Ac-Lys HIF-1α Ac-Lys HIF-2α SIRT1 Flag IP: HIF-1α HIF-2α inputs Flag-SIRT1 wild-type Flag-SIRT1 H363Y SiHa/7.4SiHa/6.5 - - + +- -- - + +- - actin ctrl EX-527 ctrl EX-527 SiHa/7.4SiHa/6.5 SIRT1 actin Ac-Lys HIF-1α Ac-Lys HIF-2α IP: HIF-1α HIF-2α inputs I S4D S4E Sirt1 supports the adaptation of tumor cells to chronic acidosis through changes in HIF1a and HIF2a acetylation and abundance

20. Fig 6. Acidosis-triggered metabolic shift is reversible and parallels the opposite alterations in the abundance of HIF1α and HIF2α. Acidosis-triggered metabolic shift toward glutamine metabolism is reversible and parallels the opposite alterations in HIF1a and HIF2a abundance

21. Fig 7. Inhibition of SIRT1-driven glutamine metabolism delays the growth of tumor cells preadapted to acidic pH. Inhibition of Sirt1-driven glutamine metabolism delays the growth of tumors arising from acidic pH-adapted cancer cells

22. Conclusion

24. Discussion

25. The chronic adaptation to acidic pH led to a cell phenotype with the exact same proliferation rate as that of parental cells maintained at pH 7.4 but exhibiting dramatic alterations in the cellular metabolic preferences. The acidification-triggered alterations in the metabolic profile include a dramatic reduction in the use of glucose and thus in the glycolytic flux, an increase in the reductive metabolism of glutamine together with an increase in glutamine-fueled OXPHOS. We report here that in addition to a global unspecific deacetylation process, the NAD+ -dependent histone deacetylase SIRT1 drives this phenomenon in an unexpected specific manner. SIRT1-mediated deacetylation of both HIF1a and HIF2a is actually observed in cells chronically exposed to acidic pH and accounts for a reduction in HIF1a activity and a net increase in HIF2a activity. Altogether, these alterations in the expression of key metabolic actors resulting from deacetylation of the HIF protein family support the preferential use of glutamine and the reduced glucose metabolism in tumor cells adapted to the acidic pH environment.

26. In conclusion, we report here how acidosis occurring in the tumor microenvironment may dramatically influence the tumor metabolic preferences and thereby directly modulate sensitivity (and resistance) to therapeutic modalities.