Cancer as a metabolic disease 1 Yoav Shaul Department of Biochemistry and Molecular Biology The Institute for Medical Research Israel-Canada The Hebrew University Medical School Jerusalem Israel https://amit1b.wordpress.com/the-molecules-of-life/10-the-living-cell-gallery/ Mammalian cell 2 Fatty acids EnergyNucleotides Amino acids Glycans Mammalian cell as a factory 3 Hallmark of cancer Hanahan, D., and Weinberg, R.A. (2000). The Hallmarks of Cancer. Cell 100, 57–70. The most fundamental trait of cancer cells involves their ability to sustain chronic proliferation. 4 Emerging hallmark of cancer Cancer metabolism Hanahan, D., and Weinberg, R. A. (2011) Hallmarks of Cancer: The Next Generation. Cell. 144, 646–674 5 The metabolism of cell proliferation Resting cells Maintain homeostatic processes EnergyATP Proliferating cells Nucleotides Amino Acids Fatty Acids Redox Building blocks Support rapid growth EnergyATP 6 Proliferating cells Building blocks Support rapid growth Cancer metabolism: current challenges Which metabolic pathways are essential for cancer cells? Enzyme Signaling pathways Metabolic processes that are essential for cancer cells 7 Course leading Questions 8 Will it inhibit or accelerate the cancer cells' growth? Warburg Effect What is the molecular mechanism that regulates the WA? Metabolism and cancer Can metabolism be the driver of cancers? Amino acid metabolism in cancer What is the role of AA metabolism in cancer Oncogenic signaling and metabolic pathways in cancer mTOR pathway as an example How can we exploit metabolic dependency for tumor inhibition? Antimetabolites -1 ATP -1 ATP +2 ATP +2 ATP 2x Pyruvate 6 carbon molecule 3 carbon molecule The glycolysis pathways yields 2mol ATP/mol of glucose Glycolysis 9 Catabolic fate of pyruvate Fermentation TCA cycle Figure 14-4 Lehninger Lactate 10 Lactic acid is produced under aerobic conditions 11 Otto Warburg First indication of cancer dependent metabolic remodelling Pyruvate +O2 Anaerobic glycolysis -O2 Aerobic glycolysis 31 mol ATP/mol of glucose2 mol ATP/mol of glucose X The Warburg effect 12 Vander Heiden, M. G., Cantley, L. C., and Thompson, C. B. (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 324, 1029–1033 Aerobic glycolysis Cell produce large amount of lactate regardless of the availability of oxygen 13 Vander Heiden, M. G., Cantley, L. C., and Thompson, C. B. (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 324, 1029–1033 Decreased metabolism of glucose by tumors, visualised by PET with the glucose analog FDG Detection of tumors by glucose analogs 14 Course leading Questions Will it inhibit or accelerate the cancer cells’ growth? Warburg Effect What is the molecular mechanism that regulates the WA? LDH LDHA (LDH5) functions as a prognosis marker in many tumors including: lymphoma, prostate cancer, renal cell carcinoma (RCC), and melanoma -1 ATP -1 ATP +2 ATP +2 ATP Lactate 16 Suggested molecular mechanics for Warburg effect Lactate Dehydrogenase as a tumor marker (A) Normal kidney proximal tubular epithelium, (B) weak (C) moderate (D) strong staining Clear Cell Renal Cell Carcinoma LDHA protein expression by immunohistochemistry Box plot representing LDHA expression levels in relation to tumor size Girgis, H., Masui, O., White, N. M., Scorilas, A., Rotondo, F., Seivwright, A., Gabril, M., Filter, E. R., Girgis, A. H., Bjarnason, G. A., Jewett, M. A., Evans, A., Al-Haddad, S., Siu, K. M., and Yousef, G. M. (2014) Lactate Dehydrogenase A is a potential prognostic marker in clear cell renal cell carcinoma. Molecular Cancer. 13, 101 17 LDH-A suppression interferes with tumorigenicity of malignant cell L2-5/10 LDH-A-KD L2-5.c 15 LDH-A rescue Ki67- mitotic marker Lactate Dehydrogenase KD 18 -1 ATP -1 ATP +2 ATP +2 ATP Lactate Pyruvate kinase -1 ATP -1 ATP +2 ATP +2 ATP Pyruvate kinase 19 There are four isoforms of Pyruvate kinase Encoded by two genes: Different isoforms of PK 1. Chaneton, B., and Gottlieb, E. (2012) Rocking cell metabolism: revised functions of the key glycolytic regulator PKM2 in cancer. Trends Biochem Sci. 37, 309–316 PKR: two isoforms PKM: two isoform PKL: Liver PKR: red blood cells PKM2: Embryo PKM1: Adult 20 PKM2 activity is tightly regulated High activity Ability to inhabit PKM2 provides advantage to prolifertating cells Low activity PKM2 enzymatic activity can be inhibited by a variety of mechanisms Dayton, T. L., Jacks, T., and Vander Heiden, M. G. (2016) PKM2, cancer metabolism, and the road ahead. EMBO Rep. 17, e201643300–1730 SAICAR, an intermediate of de novo purine biosynthesis 21 There are two isoforms of PKM2 PKM PKM1 expressed in normal tissues Christofk, H. R., Vander Heiden, M. G., Harris, M. H., Ramanathan, A., Gerszten, R. E., Wei, R., Fleming, M. D., Schreiber, S. L., and Cantley, L. C. (2008) The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature. 452, 230–233 PKM2 expressed in cancer PKM1 is a more active enzyme than PKM2 -1 ATP -1 ATP +2 ATP +2 ATP Lactate 22 Activating PKM2 suppress tumorgenesis TEPP-46 activates PKM2 Anastasiou, D., Yu, Y., Israelsen, W. J., Jiang, J.-K., Boxer, M. B., Hong, B. S., Tempel, W., Dimov, S., Shen, M., Jha, A., Yang, H., Mattaini, K. R., Metallo, C. M., Fiske, B. P., Courtney, K. D., Malstrom, S., Khan, T. M., Kung, C., Skoumbourdis, A. P., Veith, H., Southall, N., Walsh, M. J., Brimacombe, K. R., Leister, W., Lunt, S. Y., Johnson, Z. R., Yen, K. E., Kunii, K., Davidson, S. M., Christofk, H. R., Austin, C. P., Inglese, J., Harris, M. H., Asara, J. M., Stephanopoulos, G., Salituro, F. G., Jin, S., Dang, L., Auld, D. S., Park, H.-W., Cantley, L. C., Thomas, C. J., and Vander Heiden, M. G. (2012) Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat Chem Biol. 8, 839–847 TEPP-46 decrease tumor growth TEPP-46 affects glycolysis intermediates 23 Mitochondrial Pyruvate Carrier (MPC) Bender, T., and Martinou, J.-C. (2016) The mitochondrial pyruvate carrier in health and disease: To carry or not to carry? Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1863, 2436–2442 -1 ATP -1 ATP +2 ATP +2 ATP Lactate Gatekeeper of pyruvate entry to the mitochondria Two inform MPC1 and 2 24 MPC1/2 re-expression decreases tumor size MPC1/2 slowed tumor growth Schell, J. C., Olson, K. A., Jiang, L., Hawkins, A. J., Van Vranken, J. G., Xie, J., Egnatchik, R. A., Earl, E. G., Deberardinis, R. J., and Rutter, J. (2014) A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth. Mol Cell. 56, 400–413 25 Course leading Questions 26 Will it inhibit or accelerate the cancer cells’ growth? Warburg Effect What is the molecular mechanism that regulates the WA? Metabolism and cancer Can metabolism be the driver of cancers? 27 Human tumor suppressor genes that have been cloned Evading growth suppressors Metabolic gene Mitochondria Citrate Isocitrate 𝛼-Ketoglutarate Succinyl-CoA Succinate Fumarate L-Malate Oxaloacetate IDH SDH Mutated metabolic enzymes in cancer FH 28 Mitochondria Citrate Isocitrate 𝛼-Ketoglutarate Succinyl-CoA Succinate Fumarate L-Malate Oxaloacetate Citrate Isocitrate 𝛼-Ketoglutarate IDH2 IDH3 IDH1 Isocitrate dehydrogenase (IDH) Exists as three isoform: 29 IDH mutation in cancer IDH1 is mutated in 80% of grade II–III gliomas Dang L, et al. (2009) Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462(7274):739–744. IDH mutation are selected at early stages of tumorigenesis What does this mutation do to the enzymatic activity? 30 IDH1 Isocitrate 𝛼-Ketoglutarate IDH1-R132H Isocitrate 2-hydroxyglutarate IDH1 mutations produce 2-hydroxyglutarate 31 How do 2HG affect the cells? IDH movie 2-hydroxyglutarate 32 Succinate Dehydrogenase Mitochondria Citrate Isocitrate 𝛼-Ketoglutarate Succinyl-CoA Succinate Fumarate L-Malate Oxaloacetate SDH Figure 16 Lehninger33 SDH mutation SDH was the first mitochondrial enzyme found mutated in cancer Mutations in SDH are found in familial paraganglioma and pheochromocytoma, renal carcinomas, T-Cell leukemia, and gastrointestinal stromal tumors. SDH mutants lead to Succinate accumulation What is the function of succinate in cancer? 34 Fumarate hydratase Mitochondria Citrate Isocitrate 𝛼-Ketoglutarate Succinyl-CoA Succinate Fumarate L-Malate Oxaloacetate FH FH converts fumarate to malate 35 FH mutation Heterozygous FH mutations followed by the loss of heterozygosity of the second allele cause Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). FH mutants leads to fumarate accumulation What is the function of fumarate in cancer? Figure 16 Lehninger 36 Fumarate and EMT Vimentin-mesenchymal marker Sciacovelli M, et al. (2016) Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition. Nature 537(7621):544–547. E-cadherin- epithelial marker FH-deficient cells display mesenchymal features 37 Epigenetic Reprogramming by Oncometabolites Yang M, Pollard PJ (2013) Succinate: A New Epigenetic Hacker. Cancer Cell 23(6):709–711. Modification in the function of all three enzymes leads to epigenetic changes 38 Course leading Questions Will it inhibit or accelerate the cancer cells’ growth? Warburg Effect What is the molecular mechanism that regulates the WA? Metabolism and cancer Can metabolism be the driver of cancers? Amino acid metabolism in cancer What is the role of AA metabolism in cancer Amino acid and cancer 40 The most abundant amino acid in the plasma Major carrier of nitrogen between the organs Proliferating cells use glutamine as the nitrogen donor for the biosynthesis of nucleotides, nonessential amino acids, and hexosamines. Glutamine Glutamine can fuel the TCA cycle 41 -1 ATP -1 ATP +2 ATP +2 ATP Lactate Glutamine Glutamine Glutamate 𝛼-Ketoglutarate Deberardinis RJ, Cheng T (2009) Q's next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene 29(3):313. GLS 2x Pyruvate 3-PG PHGDH NAD+ NADH PSAT1 PSPH Serine Glu 𝛼-KG H2O Possemato, R., Marks, K. M., Shaul, Y. D.,et. al. (2011) Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature. 476, 346–350 Serine Synthesis Pathway (SSP) Three step reaction 42 DHF THF Serine Glycine N5, N10 methyl THFN5, methyl THF DHFR Purine and pyrimidine metabolism Folate Serine conversion to glycine, transfer of one carbon to N5, methyl-THF This carbon is essential for nucleotide metabolism Serine and one-carbon metabolism SHMT 43 Staining of PHGDH in different breast cancer samples PHGDH amplification in cancer PHGDH expression is associated with aggressive breast cancer markers PHGDH is also overexpressed in gliomas 44 Possemato, R., Marks, K. M., Shaul, Y. D., Pacold, M. E., Kim, D., Birsoy, K., Sethumadhavan, S., Woo, H.-K., Jang, H. G., Jha, A. K., Chen, W. W., Barrett, F. G., Stransky, N., Tsun, Z.-Y., Cowley, G. S., Barretina, J., Kalaany, N. Y., Hsu, P. P., Ottina, K., Chan, A. M., Yuan, B., Garraway, L. A., Root, D. E., Mino-Kenudson, M., Brachtel, E. F., Driggers, E. M., and Sabatini, D. M. (2011) Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature. 476, 346–350 Course leading Questions 45 Will it inhibit or accelerate the cancer cells’ growth? Warburg Effect What is the molecular mechanism that regulates the WA? Metabolism and cancer Can metabolism be the driver of cancers? Amino acid metabolism in cancer What is the role of AA metabolism in cancer Oncogenic signaling and metabolic pathways in cancer mTOR pathway as an example Metabolic and signaling pathway 46 Signaling pathwaysMetabolic pathways The interaction between metabolic and signaling pathways Metabolic and signaling pathway Signaling pathways Transcription regulation Post-transcriptional modification Metabolic and signaling pathway 48 Signaling pathwaysMetabolites activate signaling pathway The interaction between metabolic and signaling pathways Upstream Downstream The mTORC1 and mTORC2 complexes mTOR complex 1- mTOR interacts with raptor mTOR complex 2- mTOR interacts with rictor Sabatini DM (2006) mTOR and cancer: insights into a complex relationship. Nature Reviews Cancer 6(9):729–734. mTOR contains is serine–threonine protein kinase that can form two complexes The Amino Acid Sensing Pathway Upstream of mTORC1 Wolfson RL, Sabatini DM (2017) The Dawn of the Age of Amino Acid Sensors for the mTORC1 Pathway. 26(2):301–309. CASTORSestrin2 SLC38A9V-ATPase Metabolic and signaling pathway Signaling pathwaysMetabolites activate signaling pathway The interaction between metabolic and signaling pathways Upstream Downstream 52 CAD is a direct substrate of S6K1 Ben-Sahra I, Howell JJ, Asara JM, Manning BD (2013) Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science 339(6125):1323–1328. Pyrimidines biosynthesis pathway mTORC1 S6K S6K 4EBP CAD Course leading Questions 53 How can we exploit metabolic dependency for tumor inhibition? Antimetabolites 54 Proliferating cells Building blocks Support rapid growth All improve overall survival Metabolic enzymes as drug targets However, many of these drugs also target normal proliferating cells Antimetabolites 5-fluorouracil Methotrexate Hydroxyurea 55 DHF THF Methotrexate Serine Glycine N5, N10 methyl THFN5, methyl THF DHFR Methotrexate Purine and pyrimidine metabolism Folate Serine conversion to glycine, transfer of one carbon to N5, methyl-THF This carbon is essential for nucleotide metabolism Methotrexate inhibits the ability of cells to synthesize nucleotides 56 5-fluorouracil dUMP dTMP 5-FLUN5, N10 methyl THF DHF TYMS • 5-FLU inhibits TYMS, an important enzyme in pyrimidine synthesis 57 Metabolic liabilities Several tumor types are auxotrophic for 1 or more amino acids owing to deficiencies in a corresponding endogenous biosynthesis or salvage pathway. Thus, Cancer cells auxotrophic Normal cells auxotrophic 58 Metabolic liabilities Cancer cells auxotrophic Normal cells Non-auxotrophic Amino Acid Amino AcidX X X 59 L-asparaginase in the treatment of acute lymphoblastic leukemia (ALL) Aspartate Asparagine ASNS ALL-low expression of ASNS Aspartate Asparagine ASNS Normal cells -high expression of ASNS AspartateEscherichia coli L-asparaginase II Serum supply of Asparagine Serum supply of Asparagine Selective starvation of ALL Asparagine-non essential in normal cells essential in ALL Bioinformatic tools to study cancer metabolism To which metabolic pathway does my gene belong ? In which cell and tissues it is expressed? Is this gene important for the cancer? Does my enzyme undergo posttranslation modification? Is my gene mutated in cancer? Metastatic Cascade 1. S. Brabletz, H. Schuhwerk, T. Brabletz, M. P. Stemmler, Dynamic EMT: a multi‐tool for tumor progression. Embo J, e108647 (2021). 61 62 What makes the cell leave the tumor? 63 In order to execute the journey the cells needs to change Epithelial Partially Mesenchymal Drug Response Sensitive Epithelial-Mesenchymal Transition (EMT) Resistant X EMT Created with BioRender.com Epithelial Partially Mesenchymal Cell Migration Epithelial-Mesenchymal Transition (EMT) This Photo by Unknown Author is licensed under CC BY-SA Slow Fast Created with BioRender.com EMT 66 A picture taken in our lab The migrating cells contains “hands” that help them migrate Metastatic Cascade 1. S. Brabletz, H. Schuhwerk, T. Brabletz, M. P. Stemmler, Dynamic EMT: a multi‐tool for tumor progression. Embo J, e108647 (2021). Currently, as far as we know, there are no available anti-EMT drugs. My lab interest is to identify metabolic processes that push cancer cells to become aggressive 69 X How do we study? Active Enzyme Non-active Enzyme X Transwell migration assayActive Enzyme Non-active Enzyme Cell migration competition Results Active Enzyme Non-active Enzyme Slow Fast EMT 71 My Team Email: yoavsh@ekmd.huji.ac.il