1. Metabolism and Cancer Bob Harris D-3034 Roudebush VA Medical Center 988-4544 raharris@iupui.edu Fall 2010

2. Metabolism and Cancer Objectives: 1.Define and explain the Pasteur effect. 2.Define and explain the Warburg effect. 3.Define aerobic glycolysis. 4.Give a biochemical explanation for why cancer cells accumulate greater amounts 18 F- fluorodeoxyglucose than normal cells. 5.Explain why the Warburg hypothesis for cancer is not considered correct.

3. Metabolism and Cancer Objectives 6. Explain what causes cancer. 7. Describe how the metabolism of glucose in cancer cells differs from normal cells. 8. Describe how the metabolism of glutamine in cancer cells differs from normal cells. 9. What purpose does altered metabolism serve in cancer cells? 10. List things that promote ROS production. 11. List things that decrease ROS.

4. Metabolism and Cancer Objectives 12. Illustrate how mitochondria are involved in ROS production. 13. Explain why uncouplers of oxidative phosphorylation decrease ROS production. 14. Discuss whether the difference in metabolism is important for growth and survival of cancer cells. 15. Explain whether you think the metabolic difference between normal cells and cancer cells has therapeutic potential.

5. Normal cell metabolism Tight coupling of glucose to pyruvate and pyruvate to CO 2 and H 2 O Glucose 2 Pyruvate 6 CO 2 O2O2 2 Lactate

6. Normal cell metabolism Tight coupling of glucose to pyruvate and of pyruvate to CO 2 and H 2 O Glucose 2 Pyruvate 6 CO 2 2 Lactate Effect of Lack of O 2

7. Normal cell metabolism Louis Pasteur: 1822-1895 –Rates of fermentation are high anaerobically but low aerobically Pasteur effect: inhibition of fermentation by oxygen Glucose 2 pyruvate 2 lactate

8. Normal cell metabolism Louis Pasteur: 1822-1895 –Rates of fermentation are high anaerobically but low aerobically Pasteur effect: inhibition of fermentation by oxygen Glucose 2 pyruvate 2 lactate O2O2 6 CO 2

9. Cancer Cell Metabolism Defective coupling of glucose to pyruvate and pyruvate to CO 2 and H 2 O Cancer cells produce large amounts of lactate in the presence of oxygen Pasteur effect is defective in cancer cells

10. Metabolism and Cancer Otto Warburg: 1883-1970 –Warburg effect: “Aerobic glycolysis”: generation of lactate in the presence of oxygen Glucose 2 pyruvate 6 CO 2 Effect of lack of O 2 “Dysfunctional mitochondria likely responsible” 2 lactate O2O2

11. Is aerobic glycolysis an in vitro artifact? 18 F-fluorodeoxyglucose Positron emission tomography Vander Heiden et al. Science 2009; 329: 1029

12. Metabolism and Cancer Otto Warburg:1930. –recognized Pasteur effect is defective in cancer –discovered cancer cells carry out aerobic glycolysis (Warburg effect) –suggested “deficiencies in mitochondrial oxidative metabolism is responsible”. –proposed “replacement of respiration by fermentation is the primary cause of malignant cell transformation” (Warburg hypothesis) –suggested “altered metabolism of cancer cells might provide a means to treat cancer”

13. Metabolism and Cancer The Warburg hypothesis (replacement of respiration by fermentation is the primary cause of cancer) is not correct Cancer is caused by mutations that: –inactivate tumor suppressor genes –activate proto-oncogenes

14. Why altered metabolism in cancer? Warburg was wrong about what causes cancer – but he discovered an intrinsic difference in metabolism between cancer cells and normal cells Are there other metabolic differences between normal and cancer cells? Is the difference important for growth and survival of cancer cells? Does the difference in metabolism between normal and cancer cells have therapeutic potential?

15. Glutamine metabolism also differs between normal and cancer cells

16. Metabolism and Cancer The metabolism of glucose and glutamine by normal cells is very efficient. Primary end products are CO 2, H 2 O, and ammonia (or urea). Maximum ATP yield per mole of glucose and glutamine is achieved. The metabolism of glucose and glutamine by cancer cells is very wasteful. Primary end products are CO2, H2O, lactate, pyruvate, alanine, and aspartate. Maximum ATP yield per mole of glucose and glutamine is not achieved.

17. What purpose does altered metabolism serve in cancer cells? Assures ATP synthesis when tumor outgrows its oxygen supply Assures supply of building blocks for proliferation and growth Creates space by starving neighboring cells for nutrients Release of acid lowers extracellular which favors tumor invasion and suppresses immune effectors Increases resistance to oxidative stress by promoting NADPH production and reduction of glutathione Reduces production of reactive oxygen species (ROS) by mitochondria

18. Can normal cell metabolism cause cancer? Normal metabolism produces reactive oxygen species (ROS) ROS can induce cancer

19. Reactive oxygen species and cancer The good things about ROS –Second messenger in signal transduction –Kills bacteria that invade cells –Induces senescence and apoptosis The bad thing about ROS –High concentrations react with DNA

20. What increases ROS production and oxidative damage? Smoking Chemicals (carcinogens) UV radiation Over eating

21. Production of ROS by mitochondria Brownlee Diabetes 2005; 54: 1615

22. What decreases ROS? Uncoupling of oxidative phosphorylation Enzymes that destroy superoxide radicals and hydrogen peroxide Caloric restriction Warburg effect

23. Mechanisms responsible for Warburg effect Induction of glycolytic enzymes Induction of pryuvate dehydrogenase kinases Down regulation of mitochondrial enzymes and decrease in the number of mitochondria

24. Multiple changes in gene expression are responsible for aerobic glycolysis in cancer cells Inactivation of p53 Activation of HIF-1

25. How “aerobic” glycolysis is increased in cancer cells Hypoxia induces HIF-1 which induces expression of glycolytic enzymes. Tumor suppressor p53, which normally maintains low concentration of F2,6P 2 (activator of PFK1), is down regulated in cancer cells. HIF-1 induces expression of PDK1 which inhibits the pyruvate dehydrogenase complex, which inhibits pyruvate oxidation.

26. p53 reduces “aerobic” glycolysis p53 promotes transcription of TIGAR, a phosphatase, that hydrolyzes F2,6P 2, a positive effector of PFK1. p53 promotes expression of the thiamine transporter. Greater uptake of thiamine increases cellular [TPP], which increases activities of the pyruvate dehydrogenase and  -ketoglutarate dehydrogenase complexes.

27. P53 reduces “aerobic” glycolysis p53 increases activity of the cytochrome oxidase. Greater cytochrome oxidase promotes ATP production. Greater cellular ATP suppresses glycolysis at PFK1. p53 down regulates expression of PDK2. This increases pyruvate dehydrogenase complex which decreases the need to generate ATP by glycolysis and therefore decreases aerobic glycolysis.

28. p53 and Cancer Mutations in the p53 gene is the most common cause of cancer. Loss of p53 function in cancer cells causes greater glycolytic flux, reduced pyruvate oxidation, and reduced production of ATP by oxidative phosphorylation.

29. HIF-1 increases “aerobic” glycolysis HIF-1 increases glucose uptake by up regulating GLUT1 expression. HIF-1 increases glucose phosphorylation by up regulating hexokinase 2 expression. HIF-1 increases flux through PFK-1 by up regulating expression of the “hypoxia-inducible 6-PF-2-K/F-2,6- P 2 ase”, a form of this bifunctional enzyme in which the kinase moiety is activated by AMPK.

30. HIF-1 increases “aerobic” glycolysis HIF-1 up regulates expression of pyruvate kinase M2, aldolase A, enolase 1, and carbonic anhydrase IX. HIF-1 up regulates LDHA and the lactate transporter MCT4. HIF-1 up regulates expression of PDK1 and PDK3.

31. HIF-1, p53 and Cancer HIF-1 is activated in many cancers. Increase in HIF-1 induces the same effects as loss of p53 function, i.e. causes greater glycolytic flux, reduced pyruvate oxidation, and reduced production of ATP by oxidative phosphorylation. Therefore, because of increase in HIF-1 and decrease in p53, many tumors use “aerobic” glycolysis as their major energy pathway.

32. Activation of glycolysis and inhibition of the mitochondria in cancer Glucose Pyruvate PDCAcetyl-CoA CAC CO 2 ATP Mitochondrion Lactate ― + H + Tumor

33. Is the metabolic difference important for growth and survival of cancer cells? Inhibitors of: Hexokinase Pyruvate kinase LDH-A Monocarboxylate translocase (MCT) Pyruvate dehydrogenase kinase Glutaminase

34. Does the metabolic difference have therapeutic potential? Vander Heiden et al. Science 2009; 329: 1029