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Brugge metabolismfinal
Brugge metabolismfinal
Brugge metabolismfinal
Brugge metabolismfinal
Brugge metabolismfinal
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  • One of the most fascinating aspects of cancer research is the uncharted path that we follow in tracking down the mechanisms underlying this complex disease --Five years ago, no two years ago, I certainly would never had predicted that I would be invited to give a talk in a TUmor metabolism session -- indeed it has been 40 year since my last lecture on intermediatry metabolism and needless to say, the depth of my understanding of metabolic processes was indeed shallow! I wouldn’t go so far as to say that we went into this field kicking and screaming, but I will say that it took me four years to convince a postdoc in the lab to take on the metabolic questions that begged to be addresses.
  • Our foray into metabolism was trigged through studies of the mechanisms that regulate tumor cell survival. More specifically, we were investigating the mechanisms that trigger the death of normal cells when they lose attachment to matrix and how tumor cells escape these death mechanisms. that mechanism that drive tumor cell death when cultured with out attachment to extracelular matrix and the mechanism whereby tumor cells could escape this death. Martin Schwartz and Steve Frisch first described this requirement of normal cells matrix attachment for short term survival, but this work followed from what would now be considered ancient studies fo Macpherson and Montagnier showing that only tumor cells could make colonies in soft agar whereas their normal counterparts don’t?
    What is the relevance of these properties of anchorage dependence or independence in culture to tumor cells in humans. Truth is, we don’ know for certain; however Colony formation in soft agar has been found to correlate most closely with in tumorigenesis in animal models and we would predict...

  • Our foray into metabolism was trigged through studies of the mechanisms that regulate tumor cell survival. More specifically, we were investigating the mechanisms that trigger the death of normal cells when they lose attachment to matrix and how tumor cells escape these death mechanisms. that mechanism that drive tumor cell death when cultured with out attachment to extracelular matrix and the mechanism whereby tumor cells could escape this death. Martin Schwartz and Steve Frisch first described this requirement of normal cells matrix attachment for short term survival, but this work followed from what would now be considered ancient studies fo Macpherson and Montagnier showing that only tumor cells could make colonies in soft agar whereas their normal counterparts don’t?
    What is the relevance of these properties of anchorage dependence or independence in culture to tumor cells in humans. Truth is, we don’ know for certain; however Colony formation in soft agar has been found to correlate most closely with in tumorigenesis in animal models and we would predict...

  • Our foray into metabolism was trigged through studies of the mechanisms that regulate tumor cell survival. More specifically, we were investigating the mechanisms that trigger the death of normal cells when they lose attachment to matrix and how tumor cells escape these death mechanisms. that mechanism that drive tumor cell death when cultured with out attachment to extracelular matrix and the mechanism whereby tumor cells could escape this death. Martin Schwartz and Steve Frisch first described this requirement of normal cells matrix attachment for short term survival, but this work followed from what would now be considered ancient studies fo Macpherson and Montagnier showing that only tumor cells could make colonies in soft agar whereas their normal counterparts don’t?
    What is the relevance of these properties of anchorage dependence or independence in culture to tumor cells in humans. Truth is, we don’ know for certain; however Colony formation in soft agar has been found to correlate most closely with in tumorigenesis in animal models and we would predict...

  • During early stages of tumorigenesis, excess proliferation eventually displaces cells from their normal niches
























  • Conclude that oncogenes would need to rescue both --
  • Conclude that oncogenes would need to rescue both --
  • Not possible to measure ATP in situ, so to got a preliminary idea of metabolic activity of cells within the 3D structures by evaluate the levels of NADH and NADPH by two-photon microscopy -- NADH, as opposed to NAD, has a native fluorescece that canb e activated at
  • dichlorodihydrofluorescein diacetate

  • Add someting on senthils results



  • The ability of ERbB2 to rescue ATP in suspension involves increase in glucose uptake i because blockage of glucose metabolism with 2DG prevented ErbB2 rescue.




  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.
  • Quiescent cells such as those that are detached from matrix, have a basal rate of glycolysis, converting glucose (glc) to pyruvate (pyr), which is then oxidized in the TCA cycle. Cells can also oxidize other substrates like amino acids and fatty acids obtained from either the environment or the degradation of cellular macromolecules. As a result, the majority of ATP (yellow stars) is generated by oxidative phosphorylation. Craig Thompson had found that FA oxidation can provide energy under conditions of glucose starvation.




































  • Transcript

    • 1. 1
    • 2. Extracellular Matrix Control of Tumor Cell Metabolism Joan Brugge Department of Cell Biology Harvard Medical School
    • 3. Anchorage Dependence of Normal Cells and Independence of TUmor Cells Normal Epithelial Cells Detach from matrix Martin Schwartz Adapted from Geiger and Peeper, Cancer Monolayer Steve Frisch Res 2005; 65: 7033-36. Suspension
    • 4. Anchorage Dependence of Normal Cells and Independence of TUmor Cells Normal Epithelial Cells Tumor Cells Detach from matrix Martin Schwartz Adapted from Geiger and Peeper, Cancer Monolayer Steve Frisch Res 2005; 65: 7033-36. Suspension
    • 5. Anchorage Dependence of Normal Cells and Independence of TUmor Cells Normal Epithelial Cells Tumor Cells Detach from matrix Martin Schwartz Adapted from Geiger and Peeper, Cancer Monolayer Steve Frisch Res 2005; 65: 7033-36. Suspension Normal Cells Tumor Cells MacPherson and Montagnier 1964 Soft agar Soft agar
    • 6. In Early Tumorigenesis, Excess Proliferation Displaces Cells from Their Normal Niches Laminin DCIS Laminin 4
    • 7. Cells from non-glandular tumors also displaced from natural matrix attachment 5
    • 8. Tumors Cells Encounter Foreign Matrix Environments during Invasion and Metastasis Text (Image from Merck Biosciences www.youtube.com/watch?v=5L6lHfgL10Y
    • 9. Tumors Cells Encounter Foreign Matrix Environments during Invasion and Metastasis Text The pathways that allow tumor cells to survive outside their niches provide attractive targets for therapeutic intervention (Image from Merck Biosciences www.youtube.com/watch?v=5L6lHfgL10Y
    • 10. Models Employed to Study Anchorage Independence MCF-10A cells/HMECs Full medium + serum, Suspension EGF, insulin, culture hydrocortisone Polyhema Mauricio Reginato
    • 11. Models Employed to Study Anchorage Independence Caspase3 MCF-10A cells/HMECs Full medium + serum, Suspension EGF, insulin, culture hydrocortisone Polyhema Apoptosis Mauricio Reginato
    • 12. Models Employed to Study Anchorage Independence Caspase3 MCF-10A cells/HMECs Full medium + serum, Suspension EGF, insulin, culture hydrocortisone Polyhema Apoptosis Integrin EGFR Erk Mauricio Akt Reginato
    • 13. Models Employed to Study Anchorage Independence Caspase3 MCF-10A cells/HMECs Full medium + serum, Suspension EGF, insulin, culture hydrocortisone Polyhema Apoptosis Integrin EGFR pErk Mauricio pAkt Reginato
    • 14. Models Employed to Study Anchorage Independence Caspase3 MCF-10A cells/HMECs Full medium + serum, Suspension EGF, insulin, culture hydrocortisone Polyhema Apoptosis Integrin EGFR pErk Mauricio pAkt Reginato
    • 15. Models Employed to Study Anchorage Independence Caspase3 MCF-10A cells/HMECs Full medium + serum, Suspension EGF, insulin, culture hydrocortisone Polyhema Apoptosis Integrin EGFR Mauricio Reginato
    • 16. Models Employed to Study Anchorage Independence Caspase3 MCF-10A cells/HMECs Full medium + serum, Suspension EGF, insulin, culture hydrocortisone Polyhema Apoptosis Proapototic Bim Integrin Anti-apototic Bcl-2 EGFR Mauricio Reginato
    • 17. Models Employed to Study Anchorage Independence MCF-10A cells 3D basement membrane cultures Mauricio Reginato
    • 18. Models Employed to Study Anchorage Independence MCF-10A cells 3D basement membrane cultures Mauricio Jay Reginato Debnath
    • 19. Models Employed to Study Anchorage Independence MCF-10A cells 3D basement membrane cultures Laminin Mauricio Jay Reginato Debnath
    • 20. Models Employed to Study Anchorage Independence MCF-10A cells 3D basement membrane cultures phosphoAKT (phospho mTor, Laminin Mauricio Jay phosphoFKHD) Reginato Debnath
    • 21. Models Employed to Study Anchorage Independence MCF-10A cells 3D basement membrane cultures phosphoAKT Erk (phospho mTor, Laminin Mauricio Jay Akt phosphoFKHD) Reginato Debnath
    • 22. Models Employed to Study Anchorage Independence MCF-10A cells 3D basement membrane cultures phosphoAKT Laminin Caspase3 Erk (phospho mTor, Mauricio Jay Akt phosphoFKHD) Reginato Debnath
    • 23. Models Employed to Study Anchorage Independence Caspase3 Suspension culture MCF-10A cells Apoptosis 3D basement membrane cultures phosphoAKT Laminin Caspase3 Erk (phospho mTor, Mauricio Jay Akt phosphoFKHD) Reginato Debnath
    • 24. Models Employed to Study Anchorage Independence Caspase3 Suspension culture MCF-10A cells Apoptosis 3D basement membrane Bcl-2 cultures Mauricio Jay Reginato Debnath
    • 25. Models Employed to Study Anchorage Independence Caspase3 Suspension culture MCF-10A cells Apoptosis 3D basement membrane Bcl-2 cultures Mauricio Jay Reginato Debnath
    • 26. Models Employed to Study Anchorage Independence Caspase3 Suspension culture MCF-10A cells Apoptosis Autophagy 3D basement membrane Bcl-2 cultures Mauricio Jay Reginato Debnath
    • 27. Is autophagy associated with a reduction in ATP? Cells suspended in complete medium with EGF, serum, etc. Alex Grassian Zach Schafer
    • 28. Is autophagy associated with a reduction in ATP? Cells suspended in complete medium with EGF, serum, etc. Alex Grassian Zach Schafer
    • 29. Is autophagy associated with a reduction in ATP? Cells suspended in complete medium with EGF, serum, etc. Alex Grassian Zach Schafer
    • 30. Detached Cells Show Severe Reduction in Glucose Uptake and Exogenous Pyruvate Can Rescue the ATP Defiency 30000 Glucose Uptake 25000 p=1.08 x 10-5 20000 15000 10000 5000 0 Detached Attached Zach Schafer
    • 31. Detached Cells Show Severe Reduction in Glucose Uptake and Exogenous Pyruvate Can Rescue the ATP Defiency 30000 Glucose Uptake 25000 p=1.08 x 10-5 20000 15000 10000 5000 0 Detached Attached 12 Zach Schafer
    • 32. Detached Cells Show Severe Reduction in Glucose Uptake and Exogenous Pyruvate Can Rescue the ATP Defiency glucose X 30000 Glucose Uptake 25000 p=1.08 x 10-5 20000 15000 10000 glucose-6P X 5000 0 Detached Attached Glycolysis X methyl methyl pyruvate pyruvate pyruvate TCA TCA cycle 12 cycle Zach Schafer
    • 33. Fate of Glucose glucose Glucose transporters glucose-6P G6P Isomerase Glycolysis ATP Lactate Pyruvate + NADH TCA cycle 13 ATP
    • 34. Fate of Glucose glucose Glucose transporters glucose-6P G6P Dehydrogenase G6P Isomerase Pentose Phosphate Glycolysis Pathway ATP Lactate Pyruvate + NADH NADPH- anti-oxidant TCA cycle 13 ATP
    • 35. Loss of PPP generation of NADPH Could Contribute to ROS Production glucose X glucose-6P G6P Isomerase Glycolysis X X G6P Dehydrogenase Pentose Phosphate Pathway ATP ROS? Zach Schafer
    • 36. Loss of PPP generation of NADPH Could Contribute to ROS Production glucose X glucose-6P G6P Isomerase Glycolysis X X G6P Dehydrogenase Pentose Phosphate Pathway ATP ROS? Zach Schafer
    • 37. Loss of PPP generation of NADPH Could Contribute to ROS Production 13000 ROS Levels - carboxy- H2 DCF-DA 12000 11000 ROS levels (a.u.) 10000 9000 8000 glucose X 7000 6000 5000 10A Bcl-2 Attached Detached glucose-6P G6P Isomerase Glycolysis X X G6P Dehydrogenase Pentose Phosphate Pathway ATP ROS? Zach Schafer
    • 38. Loss of PPP generation of NADPH Could Contribute to ROS Production 13000 ROS Levels - carboxy- H2 DCF-DA 12000 11000 ROS levels (a.u.) 10000 9000 8000 glucose X 7000 6000 5000 10A Bcl-2 Attached Detached glucose-6P Also detected increase in X X oxidized glutathione G6P Dehydrogenase G6P Isomerase Pentose Phosphate Glycolysis Pathway ATP ROS? Zach Schafer
    • 39. Multiple Death Processes Ass’d with Matrix Detachment LC3-GFP Metabolic Impairment Apoptosis ATP 15 ROS?
    • 40. Matrix-Deprived Cells in Center of 3D Structures MCF-10 cells laminin
    • 41. Matrix-Deprived Cells in Center of 3D Structures MCF-10 cells laminin Apoptosis Caspase3*
    • 42. Matrix-Deprived Cells in Center of 3D Structures MCF-10 cells laminin Apoptosis Autophagy Caspase3*
    • 43. Two Photon NAD(P)H Fluorescence Loling Song 17 Since NADH is the principal electron donor in glycolytic and oxidative energy metabolism, it represents a non-invasive fluorescent reporter of the metabolic state
    • 44. Oxidative Stress in Acini DCF-DA d7 carboxy-H2DCFDA cell-permeant indicator for reactive oxygen species that is retained in the cell after deacetylation and is nonfluorescent until oxidation occurs within the cell
    • 45. Oxidative Stress in Acini DCF-DA d7 carboxy-H2DCFDA cell-permeant indicator for reactive oxygen species that is retained in the cell after deacetylation and is nonfluorescent until oxidation occurs within the cell 18
    • 46. Similar Features of Matrix Detached Cells and Inner Cells of 3D Structures Autophagy Elevated Elevated Metabolic Impairment Low ATP Lower NADH ROS Elevated Elevated Apoptosis ++++ ++++ 19 Entosis +++++ +
    • 47. Similar Features of Matrix Detached Cells and Inner Cells of 3D Structures Autophagy Elevated Elevated What are the implications of Metabolic Impairment Low ATP Lower NADH these findings for ROS Elevated Elevated tumorigenesis? Apoptosis ++++ ++++ 19 Entosis +++++ +
    • 48. Survival of HyperproliferativeTumor Cells Apoptosis Anti-apoptotic activity Text Rescue Metabolic Metabolic Impairment Impairment Low ATP High ROS 20
    • 49. Survival of HyperproliferativeTumor Cells Apoptosis Anti-apoptotic activity Text Rescue Metabolic Metabolic Impairment Impairment Low ATP High ROS How? 20
    • 50. ErbB2 Rescue of ATP Deficiency in Suspended Cells ext 21
    • 51. ErbB2 Rescue of ATP Deficiency in Suspended Cells ext Glucose uptake 21
    • 52. ErbB2 Rescue of ATP Deficiency in Suspended Cells ext ROS Glucose uptake 21
    • 53. ErbB2 Rescue of ATP Deficiency in Suspended Cells + ErbB2 ext ROS Glucose uptake Ras PI3K Mek Akt Erk 21
    • 54. ErbB2 Rescue of ATP in Suspension is Suppressed by 2-DG glucose 2-deoxyglucose glucose 2-deoxyglucose Glycolysis X pyruvate TCA cycle Zach Schafer
    • 55. ErbB2 Rescue of ATP in Suspension is Suppressed by 2-DG glucose 2-deoxyglucose glucose 2-deoxyglucose Glycolysis X pyruvate TCA cycle 22 Zach Schafer
    • 56. Summary -- Alternate Rescue of Metabolic Defect of Matrix-Deprived Cells glucose uptake ATP ROS ErbB2 Rescues glucose uptake • Restores glycolysis • Restores PPP - anti-Ox 23
    • 57. Effects of Loss of Glucose Uptake glucose X glucose-6P G6P Isomerase Glycolysis X X G6P Dehydrogenase Pentose Phosphate Pathway ATP ROS? Zach Schafer
    • 58. Effects of Loss of Glucose Uptake glucose X glucose-6P G6P Isomerase Glycolysis X X G6P Dehydrogenase Pentose Phosphate Pathway ATP ROS? WHAT IF ADD ANTI-OXIDANT TO SUSPENDED CONTROL CELLS? Zach Schafer
    • 59. Antioxidant Treatment Leads to Enhanced ATP Levels in Detached Cells anti-oxidant Low ATP Elevated ATP 10A 1 mM 50 uM Superoxide Vitamin E Vitamin E dismutase analog analog mimic
    • 60. Antioxidant Treatment Leads to Enhanced ATP Levels in Detached Cells anti-oxidant Low ATP Elevated ATP 10A 1 mM 50 uM Superoxide Vitamin E Vitamin E dismutase analog analog mimic
    • 61. Summary -- Alternate Rescue of Metabolic Defect of Matrix-Deprived Cells glucose uptake ATP ROS ErbB2 Anti-Oxidant Rescues glucose uptake Does not rescue glucose uptake • Restores glycolysis • Restores PPP - anti-Ox 26
    • 62. Summary -- Alternate Rescue of Metabolic Defect of Matrix-Deprived Cells glucose uptake ATP ROS ErbB2 Anti-Oxidant Rescues glucose uptake Does not rescue glucose uptake • Restores glycolysis How do anti- • Restores PPP - anti-Ox oxidants rescue ATP levels?26
    • 63. Alternate Energy Pathways Glucose Glycolysis ATP pyruvate PDH Acetyl CoA Ciitrate OAA TCA cycle Adapted from DeRobertis et al Cell Metabolism 2008200 27 A
    • 64. Alternate Energy Pathways Glucose Oxidizable substrates Macromolecular Glycolysis degradation ATP Oxidizable substrates pyruvate Oxidizable PDH substrates Acetyl CoA Ciitrate OAA TCA cycle Adapted from DeRobertis et al Cell Metabolism 2008200 27 A
    • 65. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates pyruvate Glucose deprivation Oxidizable PDH substrates Acetyl Upregulate CoA FATTY ACID Ciitrate OXIDATION OAA TCA cycle Adapted from DeRobertis et al Cell Metabolism 2008200 27 Craig Thompson A
    • 66. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates pyruvate Glucose deprivation Oxidizable PDH substrates Acetyl Upregulate CoA FATTY ACID Ciitrate OXIDATION OAA TCA cycle ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 27 Craig Thompson A
    • 67. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates pyruvate Glucose deprivation Oxidizable PDH substrates Acetyl Upregulate CoA FATTY ACID Ciitrate OXIDATION OAA TCA cycle ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 27 Craig Thompson A
    • 68. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates pyruvate Glucose deprivation Oxidizable PDH substrates •Loss of glucose transport Acetyl Upregulate CoA FATTY ACID Ciitrate OXIDATION OAA TCA cycle ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 27 Craig Thompson A
    • 69. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates pyruvate Glucose deprivation Oxidizable PDH substrates •Loss of glucose transport Acetyl Upregulate CoA FATTY ACID Ciitrate • Upregulation of FAO mRN OXIDATION program OAA TCA cycle ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 27 Craig Thompson A
    • 70. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates pyruvate Glucose deprivation Oxidizable PDH substrates •Loss of glucose transport Acetyl Upregulate CoA FATTY ACID Ciitrate • Upregulation of FAO mRN OXIDATION program OAA • FAO not sustained in TCA suspended cells cycle ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 27 Craig Thompson A
    • 71. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates pyruvate Glucose deprivation Oxidizable PDH substrates •Loss of glucose transport Acetyl Upregulate CoA FATTY ACID Ciitrate • Upregulation of FAO mRN OXIDATION program OAA • FAO not sustained in TCA suspended cells cycle ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 ATP 27 Levels Fall! Craig Thompson A
    • 72. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates pyruvate Glucose deprivation Oxidizable PDH substrates •Loss of glucose transport Acetyl Upregulate CoA FATTY ACID Ciitrate • Upregulation of FAO mRN OXIDATION program OAA • FAO not sustained in TCA suspended cells cycle Anti-oxidants rescue FAO ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 ATP 27 ATP Levels Fall! Restored! Craig Thompson A
    • 73. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates X ROS pyruvate Glucose deprivation Oxidizable PDH substrates •Loss of glucose transport Acetyl Upregulate CoA FATTY ACID Ciitrate • Upregulation of FAO mRN OXIDATION program OAA • FAO not sustained in TCA suspended cells cycle Anti-oxidants rescue FAO ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 ATP 27 ATP Levels Fall! Restored! Craig Thompson A
    • 74. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates X ROS Anti-Ox pyruvate Glucose deprivation Oxidizable PDH substrates •Loss of glucose transport Acetyl Upregulate CoA FATTY ACID Ciitrate • Upregulation of FAO mRN OXIDATION program OAA • FAO not sustained in TCA suspended cells cycle Anti-oxidants rescue FAO ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 ATP 27 ATP Levels Fall! Restored! Craig Thompson A
    • 75. Alternate Energy Pathways Glucose Oxidizable substrates Glycolysis X Macromolecular degradation ATP Oxidizable substrates Anti-Ox pyruvate Glucose deprivation Oxidizable PDH substrates •Loss of glucose transport Acetyl Upregulate CoA FATTY ACID Ciitrate • Upregulation of FAO mRN OXIDATION program OAA • FAO not sustained in TCA suspended cells cycle Anti-oxidants rescue FAO ATP maintained Adapted from DeRobertis et al Cell Metabolism 2008200 ATP 27 ATP Levels Fall! Restored! Craig Thompson A
    • 76. Summary -- Alternate Rescue of Metabolic Defect of Matrix-Deprived Cells glucose uptake ATP ROS ErbB2 Anti-Oxidant Rescues glucose uptake Does not rescue glucose uptake • Restores glycolysis • Restores fatty acid oxidation • Restores PPP - anti-Ox 28
    • 77. Can Anti-Oxidant Affect Morphogenesis In 3D Cultures? Apoptosis NAD(P)H ROS DFC-DA Autophagy 29
    • 78. Trolox Reduces NAD(P)H Dichotomy Between the Inner and Outer Cells 30
    • 79. Anti-Oxidants Increase Luminal Filling Clear Mostly Clear Mostly Filled Filled Blue: DAPI Red: Laminin V Green: Cleaved Caspase-3 Control 56.7% 26.7% 13.3% 3.3% ROS NAc 38.1% 4.8% 4.8% 52.4% Trolox 7.4% 14.8% 48.1% 29.6% Zach Schafer
    • 80. Trolox Also Increases Anchorage-Independent Colony Formation in Soft Agar MCF10A HPV-E7 Bcl-2 Rb Blocks p27, p21 apoptosis Hyperproliferation 32
    • 81. Trolox Also Increases Anchorage-Independent Colony Formation in Soft Agar MCF10A HPV-E7 Bcl-2 Rb Blocks p27, p21 apoptosis Hyperproliferation 32
    • 82. Potential Implications: Dichotomous Activity of Anti-Oxidants Oxidative stress Mutations in tumor suppressors and oncogenes Lead to hyperproliferation
    • 83. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants Protective against DNA, protein and lipid damage Oxidative stress Mutations in tumor suppressors and oncogenes Lead to hyperproliferation
    • 84. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k ec protein and lipid ch damage Oxidative stress Mutations in tumor suppressors and oncogenes Lead to hyperproliferation
    • 85. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k ec protein and lipid ch damage Oxidative stress Mutations in tumor suppressors and oncogenes Lead to hyperproliferation
    • 86. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k ec protein and lipid ch damage Oxidative stress Mutations in tumor suppressors and oncogenes Lead to hyperproliferation
    • 87. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k ec protein and lipid ch damage If anti-apop checkpoints lost Oxidative stress Mutations in tumor suppressors and oncogenes Lead to hyperproliferation
    • 88. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k ec protein and lipid ch damage If anti-apop checkpoints lost Oxidative stress Mutations in Loss of glucose tumor uptake suppressors and Increase in ROS oncogenes Lead to hyperproliferation
    • 89. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k ec protein and lipid ch damage If anti-apop checkpoints lost Oxidative stress Mutations in Loss of glucose tumor uptake suppressors and Increase in ROS oncogenes Ros inhibits use of other substrates like Lead to fatty acids by hyperproliferation suppression FAO
    • 90. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k ec protein and lipid ch damage If anti-apop checkpoints lost Oxidative stress Mutations in Loss of glucose tumor uptake suppressors and Increase in ROS oncogenes Ros inhibits use of other substrates like Lead to fatty acids by hyperproliferation suppression FAO
    • 91. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k es ec cu ch res protein and lipid ha t ke e t upta damage gen e If anti-apop O nco lucos g checkpoints lost Oxidative stress Mutations in Loss of glucose tumor uptake suppressors and Increase in ROS oncogenes Ros inhibits use of other substrates like Lead to fatty acids by hyperproliferation suppression FAO
    • 92. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k es ec cu ch res protein and lipid ha t ke e t upta damage gen e If anti-apop O nco lucos g checkpoints lost Oxidative stress Anti-oxidant Mutations in program Loss of glucose tumor uptake suppressors and Increase in ROS oncogenes Ros inhibits use of other substrates like Lead to fatty acids by hyperproliferation suppression FAO
    • 93. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k es ec cu ch res protein and lipid ha t ke e t upta damage gen e If anti-apop O nco lucos g checkpoints lost Oxidative stress Anti-oxidant Mutations in program Loss of glucose tumor uptake Cell generate ATP suppressors and Increase in ROS through FAO oncogenes Ros inhibits use of other substrates like Lead to fatty acids by hyperproliferation suppression FAO
    • 94. Potential Implications: Dichotomous Activity of Anti-Oxidants Anti-oxidants ic t ot tac pt in Protective po ts a in against DNA, If po k es ec cu ch res protein and lipid ha t ke e t upta damage gen e If anti-apop O nco lucos g checkpoints lost Oxidative stress Anti-oxidant Mutations in program Loss of glucose tumor uptake Cell generate ATP suppressors and Increase in ROS through FAO oncogenes Ros inhibits use of other substrates like Lead to fatty acids by hyperproliferation suppression FAO
    • 95. ROS As Secondary Checkpoint to Eliminate Abnormal, Hyperproliferative Cells APOPTOSIS Displaced cells lose survival signals from ECM 34
    • 96. ROS As Secondary Checkpoint to Eliminate Abnormal, Hyperproliferative Cells APOPTOSIS Displaced cells lose survival signals from If anti-apop ECM checkpoints lost Cells survive but metabolically impaired due to lack of nutrients 34
    • 97. ROS As Secondary Checkpoint to Eliminate Abnormal, Hyperproliferative Cells APOPTOSIS ROS INCREASE Displaced cells lose survival signals from If anti-apop ECM checkpoints lost Cells survive but metabolically impaired due to lack of nutrients 34
    • 98. ROS As Secondary Checkpoint to Eliminate Abnormal, Hyperproliferative Cells APOPTOSIS ROS INCREASE ABNORMAL CELLS DIE Displaced cells lose survival signals from If anti-apop ECM checkpoints lost Cells survive but metabolically impaired due to lack of nutrients 34
    • 99. ROS As Secondary Checkpoint to Eliminate Abnormal, Hyperproliferative Cells APOPTOSIS ROS INCREASE ABNORMAL CELLS DIE Displaced cells lose survival signals from If anti-apop ECM checkpoints lost Cells survive but metabolically impaired due to lack of nutrients ROS IS BACK UP CHECKPOINT TO PREVENT OUTGROWTH OF ABNORMAL 34 CELLS!
    • 100. ROS As Secondary Checkpoint to Eliminate Abnormal, Hyperproliferative Cells APOPTOSIS ROS INCREASE ABNORMAL CELLS DIE Displaced cells lose survival signals from If anti-apop ECM checkpoints lost Cells survive but metabolically impaired due to lack of nutrients Anti-oxidants prevent ROS killing 34
    • 101. ROS As Secondary Checkpoint to Eliminate Abnormal, Hyperproliferative Cells APOPTOSIS ROS INCREASE ABNORMAL CELLS DIE Displaced cells lose survival signals from If anti-apop ECM checkpoints lost Cells survive but metabolically impaired due to lack of nutrients Anti-oxidants prevent ROS killing Anti-oxidants have been shown to prevent other types of death involving ROS -- e.g. radiation, chemotherapy, etc. 34
    • 102. ROS As Secondary Checkpoint to Eliminate Abnormal, Hyperproliferative Cells APOPTOSIS ROS INCREASE ABNORMAL CELLS DIE Displaced cells lose survival signals from If anti-apop ECM checkpoints lost Cells survive but metabolically impaired due to lack of nutrients Anti-oxidants prevent ROS killing Anti-oxidants have been shown to prevent other types of death involving ROS -- e.g. radiation, chemotherapy, etc. Rigorous controlled studies in mouse models and humans 34 required to understand effects of anti-oxidants.
    • 103. SOD2 and TXN Elevated in Grade3 Tumors from Oncomine 35
    • 104. SOD2 Differentially Expressed in High Grade Breast Tumors Thioredoxin looks almost identical 36 From Oncomine
    • 105. Human Protein Atlas - SOD2 IHC Normal 37
    • 106. Selection to Rescue Oxidative Stress Increased ROS Loss of matrix DNA damage Loss of nutrients/O2 etc etc 38
    • 107. Selection to Rescue Oxidative Stress Increased ROS Anti-oxidant program Loss of matrix DNA damage Loss of nutrients/O2 etc etc 38
    • 108. Selection to Rescue Oxidative Stress Increased ROS Inhibit anti-oxidants X Anti-oxidant program Loss of matrix DNA damage Loss of nutrients/O2 etc etc 38
    • 109. Selection to Rescue Oxidative Stress Increased ROS Inhibit anti-oxidants X Anti-oxidant program Loss of matrix DNA damage Loss of nutrients/O2 etc etc 38
    • 110. Acknowledgments METABOLISM TWO-PHOTON Zach Schafer Alex Grassian Loling Song U. Notre Dame APOPTOSIS/AUTOPHAGY LAB MANAGER Mauricio Yoko Irie Grace Gao Jay Debnath Mike Reginato Overholtzer UCSF Drexel MSKCI
    • 111. Acknowledgments Fatty Acid Oxidation - Pere Puigserver Zach Gerhart-Hines Nikon Imaging Center Harvard Medical School 40
    • 112. We studied the effect of -carotene supplementation on colorectal adenoma recurrence among subjects in a multicenter double-blind, placebo-controlled clinical trial of antioxidants for the prevention of colorectal adenomas. A total of 864 subjects who had had an adenoma removed and were polyp-free were randomly assigned (in a factorial design) to receive -carotene (25 mg or placebo) and/or vitamins C and E in combination (1000 mg and 400 mg, respectively, or placebo), and were followed with colonoscopy for adenoma recurrence 1 year and 4 years after the qualifying endoscopy. 41
    • 113. We studied the effect of -carotene supplementation Among subjects who neither smoked cigarettes nor drank alcohol, -carotene was associated with subjects decrease in on colorectal adenoma recurrence among a marked thearisk of one or more recurrent adenomas! clinical in multicenter double-blind, placebo-controlled trial of antioxidants for the prevention of colorectal adenomas. A total of 864 subjects who had had an adenoma removed and were polyp-free were randomly assigned (in a factorial design) to receive -carotene (25 mg or placebo) and/or vitamins C and E in combination (1000 mg and 400 mg, respectively, or placebo), and were followed with colonoscopy for adenoma recurrence 1 year and 4 years after the qualifying endoscopy. 41
    • 114. We studied the effect of -carotene supplementation Among subjects who neither smoked cigarettes nor drank alcohol, -carotene was associated with subjects decrease in on colorectal adenoma recurrence among a marked thearisk of one or more recurrent adenomas! clinical in multicenter double-blind, placebo-controlled trial of antioxidants for the prevention of colorectal adenomas. For participants who smoked cigarettes and also A total of 864 subjects who had had an adenoma removed drank more than one alcoholic drink per day, -carotene and were polyp-free were randomly assigned (in a doubled design) to of adenoma recurrence (RR = 2.07, 95% factorial the risk receive -carotene (25 mg or placebo) CI= 1.39 to 3.08;and E in combination (1000 mg and 400 and/or vitamins C P for difference from nonsmoker/ mg, respectively, or placebo), and were followed with colonoscopy nondrinker RR < .001).year and 4 years after the for adenoma recurrence 1 qualifying endoscopy. 41
    • 115. Extracellular Matrix Protein IHC in Human and Mouse Tumors Human Breast Tumors Mouse Mammary Tumors Text 42 Mike Overholtzer; Arnaud Mailleux; Stuart Schnitt

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