Deuterium depletion and mitochondrial NADPH production: The link for epigenetic control of oncogenesis

Átírás

1 Science Friday Seminar December 13, 2013 Deuterium depletion and mitochondrial NADPH production: The link for epigenetic control of oncogenesis László G. Boros, M.D. Associate Professor of Pediatrics Harbor-UCLA Medical Center, Torrance, CA INVESTIGATOR - LABIOMED SIDMAP, LLC, Scientist - doi: /SCIENCEFRDEC132014LGB

2 Pro-/Eukaryotic Cell Structures doi: /SCIENCEFRDEC132014LGB

3 Pro-/Eukaryotic Cell Structures doi: /SCIENCEFRDEC132014LGB

4 Animal cell Internal membrane (nuclear membrane, Golgi apparatus) Mitochondria Cytoskeletal structures Microtubules Actin and actin binding proteins, e.g., α-actinin, fimbrin, filamin Microtubules, myosins provide dynamic character - doi: /SCIENCEFRDEC132014LGB

5 Mitochondria 1) Inner membrane 2) Outer membrane 3) Crista 4) Matrix doi: /SCIENCEFRDEC132014LGB

6 TCA, citric acid, Krebs cycle doi: /SCIENCEFRDEC132014LGB

7 - doi: /SCIENCEFRDEC132014LGB

8 X doi: /SCIENCEFRDEC132014LGB

9 Hereditary leiomyomatosis and renal cell cancer syndrome I. Reed s syndrome (familial leiomyomatosis cutis et uteri) Rare inherited condition Multiple cutaneous leiomyomas - doi: /SCIENCEFRDEC132014LGB

10 Hereditary leiomyomatosis and renal cell cancer syndrome II. Cutaneous piloleiomyomata Gideon Smith MD PhD, Noushin Heidary MD, Rishi Patel MD, Karla Rosenman MD, Shane A Meehan MD, Hideko Kamino MD, Miguel Sanchez MD Dermatology Online Journal 15 (8): doi: /SCIENCEFRDEC132014LGB

11 Clear cell renal cell carcinoma doi: /SCIENCEFRDEC132014LGB

12 Clear cell renal cell carcinoma Cortical tumor Malignant epithelial cells with clear cytoplasm Compact-alveolar (nested) Acinar growth pattern Intricate, arborizing vasculature Granular eosinophilic cytoplasm may be present doi: /SCIENCEFRDEC132014LGB

13 Clear cell renal cell carcinoma doi: /SCIENCEFRDEC132014LGB

14 Hereditary leiomyomatosis and renal cell cancer syndrome The syndrome is caused by a mutation in the fumarate hydratase gene Accumulation of fumarate Autosomal dominant disease - doi: /SCIENCEFRDEC132014LGB

15 X doi: /SCIENCEFRDEC132014LGB

16 Restored fumarate hydratase Restoring FH activity by inserting the fumarate hydratase gene into kidney tumor cells (UOK262WT) has been previously shown not to be tumorigenic These cells fail to produce tumors in nude mice Tong, et al. The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels. Cancer Cell Sep 13;20(3): doi: /j.ccr doi: /SCIENCEFRDEC132014LGB

17 X doi: /SCIENCEFRDEC132014LGB

18 Fumarate accumulation and epigenetic events I. High mitochondrial concentrations of fumarate may cross into the cytosol via dicarboxylate carriers to inhibit prolyl hydroxylases These target HIF-1α for VHL-dependent degradation under normoxia, thus rendering HIF-1α constitutively active Non-specific succination of cysteine residues of proteins Elevated levels of superoxide and peroxide (ROS), which is another important mediator of HIF-1α stabilization Yang Y, et al. Metabolic reprogramming for producing energy and reducing power in fumarate hydratase null cells from hereditary leiomyomatosis renal cell carcinoma. PLoS One Aug 15;8(8):e doi: /journal.pone doi: /SCIENCEFRDEC132014LGB

19 The question asked by biochemists! What does fumarase do? - doi: /SCIENCEFRDEC132014LGB

20 FH adds metabolic (matrix) water to TCA cycle intermediates doi: /SCIENCEFRDEC132014LGB

21 1937 Nobel Prize in Physiology and Medicine for his studies of biological oxidations, fumarate hydratase and for discovering ascorbic acid in adrenal glands. - doi: /SCIENCEFRDEC132014LGB

22 He proposed hydrogen-carrying catalytic role of succinate and fumarate in the 1920s Malate doi: /SCIENCEFRDEC132014LGB

23 The cycle consumes net two molecules of water in mammals doi: /SCIENCEFRDEC132014LGB

24 Nature 150, (04 July 1942) doi: /150021a0 Metabolic Water and Desiccation KENNETH MELLANBY THE utilization by the body of ingested food substances and of tissue reserves yields among other things quantities of metabolic water. As the complete combustion of 100 gm. of fat produces about 110 gm. of metabolic water, whereas 100 gm. of carbohydrate yields only 55 gm. of water, fat reserves and fatty foods are believed to be particularly valuable as a protection against desiccation. This contention would appear to be supported by the fact that many animals which exist in deserts have large reserves of fat. - doi: /SCIENCEFRDEC132014LGB

25 Natural fat with low deuterium The cycle consumes net two molecules of water in mammals doi: /SCIENCEFRDEC132014LGB

26 Metabolic water with low deuterium content Malate doi: /SCIENCEFRDEC132014LGB

27 Deuterium content in free water and food I. Measured using isotope-ratio mass spectrometry as VSMOW - Vienna Standard Mean Ocean Water (~155 ppm) Flour ~150 ppm (parts per million) Table sugar ~146 ppm Cottege cheese (dry) ~136 ppm Olive oil ~130 ppm Butter ~124 ppm Fatty acid animal fat ~118 ppm ER Kerstel, et al. Simultaneous determination of the (2)h/(1)h, (17)o/(16)o, and (18)o/(16)o isotope abundance ratios in water by means of laser spectrometry. Anal Chem Dec 1;71(23): doi: /ac990621e - doi: /SCIENCEFRDEC132014LGB

28 Deuterium content in free water and food II. Measured using isotope-ratio mass spectrometry as VSMOW - Vienna Standard Mean Ocean Water (~155 ppm) Unsaturated fat ~110 ppm Unsaturated plant fat at double bonds ~102 ppm I Billault, et al. Quantitative deuterium isotopic profiling at natural abundance indicates mechanistic differences for delta 12-epoxidase and delta 12-desaturase in Vernonia galamensis. J Biol Chem May 6;280(18): doi: /SCIENCEFRDEC132014LGB

29 Enzymes of photosynthesis and fat synthesis/desaturation drop deuterium Enzymes dropping deuterium H D doi: /SCIENCEFRDEC132014LGB

30 Fatty acids (~118 ppm) Glucose - free water (150 ppm) Fig. 1 CS and Aconitase a) Glycolysis c) Hexose/pentose cycling c) Oxidative pentose cycle G6PDH and 6PGD NADP + D (150 ppm) Fumarate hydratase b) Citrate NADP + D (118 ppm) H 2 O 2 GSH-D (150 ppm) X NADP + dependent IDH X d) FAS S Light DNA and nuclear plasma membrane fatty acids (118 ppm)

31 What is wrong with deuterium? It is twice as heavy as hydrogen Chemically identical Deuterium participates in reductive synthesis Changes DNA methylation kinetics Alters gene expression Likely induces aneuploidy - doi: /SCIENCEFRDEC132014LGB

32 What is wrong with deuterium? Hydrogen doi: /SCIENCEFRDEC132014LGB

33 What is wrong with deuterium? Deuterium doi: /SCIENCEFRDEC132014LGB

34 How does deuterium get into DNA? doi: /SCIENCEFRDEC132014LGB

35 X Tedeschi PM, et al. Contribution of serine, folate and glycine metabolism to the ATP, NADPH and purine requirements of cancer cells. Nature - Cell Death Dis Oct 24;4:e877. doi: /cddis

36 Glycolysis NADH NAD LDH ATP ADP ATP ADP DHAP X [ 13 C 2 ]-Lactate [1,2-13 C 2 ]-glucose glucose-6p PGI fructose-6p PFK fructose-1,6bip NAD NADH 1,3-biphosphoglycerate ADP PGK ATP 3pg ADP ATP HK FBPA TPI NADP NADPH NADP NADPH + 13 CO 2 TPI 6P-gluconolactone TPI 6P-gluconate G6PDH ribulose-5p GA3P GAPDH 2-phosphoglycerate ENO phosphoenolpyruvate NAD NADH + 13 CO 2 PGK PK pyruvate PDH Pentose phosphate pathway NAD TPI TA [2,3-13 C 2 ]-Acetyl-CoA oxaloacetate Serine synthesis glut PHGDH 3php PSAT pser PSPH ser NADH CS NADH TCA cycle xylulose-5p CoA erythrose-4p glyc H ox citrate NAD KG ACO 13 CO 2 P GC L NAD NADH + 13 CO 2 TK isocitrate IDH KG TK H am T H red ATP + HCO 3 AcCoA ACC ribose-5p sedoheptulose-7p NH 3 GLDH SHMT FAS NADPH + NH 3 NAD TK SOG pathway ADP ATP + for thf mlthf MalCoA One carbon cycle NADP C1ST C1ST [4,5-13 C 2 ]-glutamate ADP [ 13 C]-palmitate 10fthf C1ST methf NADPH Fatty acid synthesis - doi: /SCIENCEFRDEC132014LGB

37 Fatty acids (~118 ppm) Glucose - free water (150 ppm) Fig. 2 CS and Aconitase Glycolysis c) Hexose/pentose cycling c) Oxidative pentose cycle G6PDH and 6PGD X a) NADP + D (150 ppm) Fumarate hydratase b) Citrate NADP + D (118 ppm) H 2 O 2 GSH-D (150 ppm) NADP + dependent IDH X X d) FAS S Heavy/sticky DNA and nuclear plasma membrane fatty acids (150 ppm)

38 RNA ribose TCA Glycolysis Fatty acid Tracer WT-pFH +262 EV-FH -262 FH -262 FH -268 R 2 Correl [7] - Glucose tracer consumption (mg/24h) [12] - 13 CO 2 Glucose oxidation complete (D 13 C/ 12 C) [17] - Lactate 13 C labeled fraction (Sm) [22] - G6PDH flux NADPH production (m1/m2) [22B] - Lactate concentration (peak area) [76] - Glutamate 13 C labeled fraction (Sm) [77] - Glutamate 13 C Content (Smn) [79] - Glutamate via PDH (m2/sm) [80] - Glutamate via OA recycling (m3/sm) [81] - Glutamate via PC and PDH (m4/sm) [87B] - Glutamate-concentration (peak area) [143] - Lignocerate (C24:0) 13 C labeled fraction (Sm) [144] - Lignocerate (C24:0) 13 C Content (Smn) [150B] - Lignocerate (C24:0) concentration (peak area) [294] - RNA-ribose 13 C labeled fraction (Sm) [295] - RNA-ribose 13 C content (Smn) [296] - RNA-ribose via G6PDH/NADPH (m1/sm) [297] - RNA-ribose via Transketolase (m2/sm) [305B] - RNA-ribose concentration (peak area) Percent of Control: < 64 % 65 % - 78 % 79 % - 92 % 93 % % 107 % % 121 % % 135 % % > 150 %

39 Fatty acids (~118 ppm) Glucose - DDW water (100 ppm) Fig. 3 CS and Aconitase Glycolysis c) Hexose/pentose cycling c) Oxidative pentose cycle G6PDH and 6PGD X a) NADP + D (100 ppm) Fumarate hydratase FAS S b) Citrate NADP + D (118 ppm) NADP + dependent IDH X X H 2 O 2 GSH-D (100 ppm) Light DNA and nuclear plasma membrane fatty acids (100 ppm) d)

40

41 13 C labeled fatty acid fractions (% of total) De novo fatty acid and cholesterol synthesis MIA-PaCa lignocerate C:24 synthesis P<0.05 MIA-PaCa palmitate C:16 synthesis P<0.05 MCF-7-Breast cholesterol synthesis P< Deuterium ( 2 H) in water (ppm) doi: /SCIENCEFRDEC132014LGB

42 What is lignocerate? Saturated rigid phospholipid component of the nuclear membrane Helps maintaining aneuploidy - doi: /SCIENCEFRDEC132014LGB

43 Take-Home Capsule I. Impaired mitochondria are involved in cell transformation by limiting the low natural deuterium containing complete fatty acid oxidation product, metabolic water, to enter nuclear membranes and nucleotides via reductive synthesis - doi: /SCIENCEFRDEC132014LGB

44 Take-Home Capsule II. In turn, heavy natural water and sugar dependent NADPH production taken over by the oxidative branch of the pentose cycle, as well as the SOGC-pathway, are deuterium loading ticking time bombs with a strong isotope effect and thus oncogenic epigenetic events that include unstable hydrogen bonds, aneuploidy in DNA structures and chemically altered methylation sites with severely disrupted gene expression patterns - doi: /SCIENCEFRDEC132014LGB

45 Take-Home Capsule III. Nevertheless, extra-mitochondrial NADPH synthesis opens a therapeutic window for deuterium depleted water to maintain and/or restore normal cellular functions. - doi: /SCIENCEFRDEC132014LGB

46 Take-Home Capsule IV. Our approach provides a novel mechanism regarding lipid based ketogenic diets in the presence of hyperbaric oxygen treatment to decrease metastasis formation by producing low deuterium metabolic water via complete oxidation to prevent DNA, histone and nuclear membrane deuteration during NADPH dependent reductive synthesis. - doi: /SCIENCEFRDEC132014LGB

47 Acknowledgments 1 Dr. Gabor Somlyai HYD, LLC Tracy Lagunero - SiDMAP Analyses Dr. W-N Paul Lee UCLA Dr. W. Marston Linehan, Cris Rickets and Youfeng Yang NCI - doi: /SCIENCEFRDEC132014LGB

48 Acknowledgments 2 This work was supported by the Hirshberg Foundation for Pancreatic Cancer Research to LGB The UCLA Clinical & Translational Science Institute (UL1TR000124) and UCLA Center for Excellence in Pancreatic Diseases - Metabolomics Core (1-P01 AT A1). - doi: /SCIENCEFRDEC132014LGB

49 "Research is to see what everybody else has seen and to think what nobody else has thought." -- Albert Szent-Györgyi - doi: /SCIENCEFRDEC132014LGB - Teaching Evaluation - doi: /SCIENCEFDEC132014EVAL

50 Science-Fridays-LABiomed02a-DDW_English 1 00:00:06,009 --> 00:00:11,000 Introduction by Dr. Kevin W. Bruhn, Principal Investigator, Molecular Medicine, LABIOMED 2 00:00:11,009 --> 00:00:18,000 (Medical Diploma) Szeged, Hungary, in He came to the United States in 1990, 3 00:00:18,009 --> 00:00:27,000 spent 8 years at Ohio State, as a Research Scientist there... before coming to Harbor-UCLA in :00:27,009 --> 00:00:32,000 He spent some time with Dr. Paul Lee, who is here today, 5 00:00:32,009 --> 00:00:36,000 learning mass spec(trometry) and stable isotope applications 6 00:00:36,009 --> 00:00:43,000 Dr. Boros is currently an Associate Professor working at UCLA :00:43,009 --> 00:00:51,000 And he is actually the Chief Scientific Officer at an LABIOMED spin-off company called SiDMAP. 8 00:00:51,009 --> 00:00:58,000 SiDMAP is a pharmaceutical service provider for all phase metabolomics. 9 00:00:58,009 --> 00:01:04,000 Laszlo is going to talk to us about some of his work, metabolomics, and beyond, and, so :01:04,009 --> 00:01:07,000 I turn it over to you..., thank you doctor! 11 00:01:07,009 --> 00:01:11,000 And, it is a privilege, and thank you for the very kind introduction :01:11,009 --> 00:01:20,000 This is the last presentation of 2013, so I was trying to come up with a very interesting topic :01:20,009 --> 00:01:27,000 Which, I believe is a very.., truly.., a very intriguing topic..., Page 1

51 Science-Fridays-LABiomed02a-DDW_English 14 00:01:27,009 --> 00:01:38,000...just to talk about deuterium..., its depletion..., as part of cancer's epigenetic drivers :01:38,009 --> 00:01:45,000 This is a new theory, as well as a new model, that we have studied and now published (doi: /journal.pone ) 16 00:01:45,009 --> 00:01:50,000 in the medical literature, in collaboration with the National Cancer Institute, who, actually..., 17 00:01:50,009 --> 00:01:59,000...provided the model for the study. So..., just to start..., 18 00:01:59,009 --> 00:02:03,000...let's refresh our memories about how cells, 19 00:02:03,009 --> 00:02:09,000 in particular, how prokaryotic and eukaryotic cells, function? 20 00:02:09,009 --> 00:02:18,000 Eukaryotic cells form nuclei, and these nuclei contain the DNA material, or..., 21 00:02:18,009 --> 00:02:27,000...the nucleic acid material, surrounded by membranes..., It's a more complex structure, with a larger size, 22 00:02:27,009 --> 00:02:32,000 as far as the cell goes, and the nucleus has many more functions built into it..., 23 00:02:32,009 --> 00:02:41,000 e.g.:...to be able to differentiate, and to specialize in various functions :02:41,009 --> 00:02:47,000 And..., what is really interesting, and what captured me back in high school, and in :02:47,009 --> 00:02:53,000..early college is that: If you (the cell) want to form nuclei contained with a membrane..., 26 00:02:53,009 --> 00:02:58,000 you know.. the cell needs also all these other cellular particles :02:58,009 --> 00:03:07,000 And what we've got most interested in, is the mitochondria..., which means, that, really..., there is never an eukaryotic cell Page 2

52 Science-Fridays-LABiomed02a-DDW_English 28 00:03:07,009 --> 00:03:16,000 with nucleic acid surrounded by a membrane, without mitochondria! 29 00:03:16,009 --> 00:03:23,000 And..., there are no exceptions to this rule..., while cells, that do not have nuclear membranes, 30 00:03:23,009 --> 00:03:28,000 or have no solid or eukaryotic-like nuclear membranes, never have mitochondria :03:28,009 --> 00:03:35,000 So.., the link between cell morphology, function and energy production, is where we started :03:35,009 --> 00:03:40,000 to look into mitochondria in more details..., and started using models :03:40,009 --> 00:03:48,000 Where the mitochondrion was not functioning, and cancer has..., cancer developed! 34 00:03:48,009 --> 00:03:55,000 Just to review more background information..., the internal membrane system, which includes 35 00:03:55,009 --> 00:04:01,000 the nuclear membrane and the Golgi apparatus, host a lot of functional proteins :04:01,009 --> 00:04:09,000 Which actually produce fatty acids and membranes, using specialized enzymes :04:09,009 --> 00:04:15,000 And the mitochondrion is the site of energy production, yet, it's really not only an energy producing cell, 38 00:04:15,009 --> 00:04:21,000...or cell organelle, if we review the evolution of mammalian cells! 39 00:04:21,009 --> 00:04:27,000 Because, mitochondria first produce water..., which is one basic role of mitochondria :04:27,009 --> 00:04:30,000 And we will talk about this..., a bit more, in details :04:30,009 --> 00:04:37,000 The cytoskeleton has structures, which include micro-tubes, actin and binding proteins and..., Page 3

53 Science-Fridays-LABiomed02a-DDW_English 42 00:04:37,009 --> 00:04:41,000...so on. So that, a cell, that is eukaryotic and differentiated, 43 00:04:41,009 --> 00:04:49,000 can perform a number of additional functions, which also originate.., some of the cell's regulatory 44 00:04:49,009 --> 00:04:56,000 elements originate from mitochondrial functions. A mitochondrion has an inner membrane, an outer membrane, 45 00:04:56,009 --> 00:05:00,000 has cristae and also has the matrix! 46 00:05:00,009 --> 00:05:05,000 And..., the matrix is where the citric acid cycle :05:05,009 --> 00:05:11,000...takes place. The citric acid cycle, or Krebs cycle, is a set of 48 00:05:11,009 --> 00:05:18,000 biochemical reactions that produces... aaa :05:18,009 --> 00:05:29,000 that produce reducing equivalent, NADH and the proton is actually carried through the cytochrome system 50 00:05:29,009 --> 00:05:36,000 and, eventually, through cytochrome-c (complex-iv). The product of the proton released 51 00:05:36,009 --> 00:05:42,000 from citric acid cycle intermediates is water..., if there is oxygen in the 52 00:05:42,009 --> 00:05:47,000 mitochondria in order to produce water..., and this is how animals actually survive in the desert..., 53 00:05:47,009 --> 00:05:51,000 where they do not drink! They use their mitochondria, instead, to actually produce 54 00:05:51,009 --> 00:05:53,000 water! and...aaa :05:53,009 --> 00:06:02,000 that's how the lizards, the snakes and sea fish, and the... marine animals do not need to 56 Page 4

54 Science-Fridays-LABiomed02a-DDW_English 00:06:02,009 --> 00:06:06,000 drink water as they produce water in their mitochondria :06:06,009 --> 00:06:10,000 what the substrates are? I am going to talk about in more details :06:10,009 --> 00:06:18,000 There are two water molecules produced from..., when the Cycle is turned over and with 59 00:06:18,009 --> 00:06:22,000 the addition of oxygen water production is enhanced :06:22,009 --> 00:06:29,000 Yet, if the cells need ATP, what they do is, they actually shovel the protons 61 00:06:29,009 --> 00:06:37,000 to the inter-membrane matrix (space), and they actually use this complex-v, or ATP :06:37,009 --> 00:06:43,000...synthase. And, as they actually channel protons back into the matrix, they also produce ATP :06:43,009 --> 00:06:48,000...or energy. So the hydrogen comes from citric :06:48,009 --> 00:06:54,000...acid cycle metabolites and intermediates, so they are very critical to drive the whole process of :06:54,009 --> 00:07:00,000...water production, and also the proton channeling back and forth :07:00,009 --> 00:07:03,000...into the matrix. So..., 67 00:07:03,009 --> 00:07:10,000 Our hypothesis was from the very beginning, when I was preparing this talk and was getting :07:10,009 --> 00:07:16,000...ready to present, was that..., how does the weight of hydrogen affects the :07:16,009 --> 00:07:22,000...kinetics of these pumps, and, in overall, the biochemical reactions associated Page 5

55 Science-Fridays-LABiomed02a-DDW_English 00:07:22,009 --> 00:07:33,000 with mitochondrial failure, in cancer especially, and what're key components of this system, which are responsible :07:33,009 --> 00:07:34,000 for epigenetic events! So :07:34,009 --> 00:07:40,000 This is, in more details, the chain or set of reactions that happen in the mitochondria 73 00:07:40,009 --> 00:07:47,000 or the TCA cycle, and, as you can see, citrate is formed, which 74 00:07:47,009 --> 00:07:52,000 is why it is called the citrate cycle. And this citrate is formed from a two carbon metabolite..., 75 00:07:52,009 --> 00:07:56,000...which is acetyl-coa and an oxaloacetate, which is in the Cycle :07:56,009 --> 00:08:00,000 There is this six carbon citrate, which turns over..., 77 00:08:00,009 --> 00:08:07,000...two carbons are lost in the form of CO2, and as CO2 is produced, these hydrogens are :08:07,009 --> 00:08:15,000...actually taken out from the intermediates of the TCA cycle :08:15,009 --> 00:08:20,000 What is really interesting is that not identical carbons get oxidized in the first round, which :08:20,009 --> 00:08:25,000...have been entering the Cycle! And it is also very interesting that there :08:25,009 --> 00:08:30,000...is water consumed, while the cycle is spinning over :08:30,009 --> 00:08:36,000 So..., it is kind of interesting, and, it is really intriguing to know, why water is consumed, when, in 83 00:08:36,009 --> 00:08:40,000 the meantime, oxygen is reduced, and why water is necessary, which is Page 6

56 Science-Fridays-LABiomed02a-DDW_English 00:08:40,009 --> 00:08:44,000 matrix water, the one that is produced by cytochrome-c (and complex-iv)..., 85 00:08:44,009 --> 00:08:51,000 why this water is reused in the mammalian system, instead of using it as a water source..., 86 00:08:51,009 --> 00:08:54,000...just like the animals in the desert would do! 87 00:08:54,009 --> 00:09:03,000 Obviously, using matrix water for reductive synthesis, is where I am going to lead the topic...! 88 00:09:03,009 --> 00:09:11,000 Yet, it's a very intriguing system, as finding out, how water is exchanged and how the hydrogen :09:11,009 --> 00:09:15,000...is channeled into DNA, eventually..., which I am going to show you! 90 00:09:15,009 --> 00:09:23,000 Because, what is very interesting is that there is a cancer model, which we have studied in :09:23,009 --> 00:09:29,000...collaboration with the National Cancer Institute, where one of the enzymes of the Cycle, called :09:29,009 --> 00:09:36,000...fumarate hydratase, or fumarase, is mutated! So kidney cells that do not have this fumarate :09:36,009 --> 00:09:44,000...hydratase or fumarase enzyme, are not able to use water to hydrate fumarate into malic acid! 94 00:09:44,009 --> 00:09:50,000 And, as the result of that, the Cycle would turn back around, and actually, through reductive :09:50,009 --> 00:09:56,000 carboxylation, the Cycle would actually become the consumer of hydrogen of some other source..., 96 00:09:56,009 --> 00:09:59,000...other than water of the matrix! So..., 97 00:09:59,009 --> 00:10:06,000 with this total turning back around of TCA cycle metabolism, cancer readily develops :10:06,009 --> 00:10:10,000 Page 7

57 Science-Fridays-LABiomed02a-DDW_English...and, it is actually a very aggressive cancer, which is called the :10:10,009 --> 00:10:19,000...clear renal cell carcinoma syndrome, which is associated with hereditary leiomyomatosis..., :10:19,009 --> 00:10:31,000 after years of this fumarate hydratase deficiency, and also these skin bumps. These patients :10:31,009 --> 00:10:38,000...will actually develop cancer! It's a rare disease, it's called the Reeds syndrome..., :10:38,009 --> 00:10:41,000...based on the investigator who described it :10:41,009 --> 00:10:45,000 And, what we see in the beginning of the diseases is that there are these skin bumps..., :10:45,009 --> 00:10:54,000...histologically they look like a wide broad keratin layer between the sub-cutaneous :10:54,009 --> 00:11:00,000...structures. And this is the pre-exposition state, or this the :11:00,009 --> 00:11:07,000...preliminary stage before kidney cancer develops in these patients! :11:07,009 --> 00:11:13,000 When kidney cancer develops, the cancer is really unique in the sense that it is called :11:13,009 --> 00:11:18,000...clear cell renal cell carcinoma... It used to be called a type of hypernephroma..., :11:18,009 --> 00:11:23,000...but it is actually developing from the kidney tissue! :11:23,009 --> 00:11:30,000 And, what you see here is that there are clear cells with nuclear morphology characteristic :11:30,009 --> 00:11:33,000...of tumor cells! It is a very aggressive cancer -- and here :11:33,009 --> 00:11:38,000...is the normal kidney tissue, and because of this characteristics..., with the accumulation... Page 8

58 Science-Fridays-LABiomed02a-DDW_English :11:38,009 --> 00:11:46,000...of this cystic material in the cells, the cells' nuclei are pushed to the side, and..., :11:46,009 --> 00:11:51,000...because of this pathological feature, they call this type of cancer :11:51,009 --> 00:11:57,000...clear cell kidney (renal) cell carcinoma. And it develops based on this fumarate hydratase :11:57,009 --> 00:12:00,000...mutation, which is in the TCA cycle! :12:00,009 --> 00:12:05,000 So..., the cancer is a cortical tumor :12:05,009 --> 00:12:15,000...and it is a malignant epithelial cell - type of tumor, and it is clear, but I am not going to talk here about morphology..., :12:15,009 --> 00:12:17,000...in more details... But, yet..., just :12:17,009 --> 00:12:24,000...let's go to the syndrome, which is actually caused by a mutation in :12:24,009 --> 00:12:28,000...the fumarate hydratase gene, and as a result of that, there is an accumulation :12:28,009 --> 00:12:31,000 of water with fumaric acid in the TCA cycle and the mitochondria! :12:31,009 --> 00:12:39,000 And, it is an autosomal dominant disease. What we see here, is that once the mutation occurs..., :12:39,009 --> 00:12:44,000...over a longer period of time, there is fumaric acid buildup :12:44,009 --> 00:12:52,000...and there is decreasing malic acid concentration... And the question is, how these changes actually :12:52,009 --> 00:12:59,000...contribute to cancer development? Because, just looking at this scenario, you... Page 9