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
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?
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
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
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
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
Hinweis der Redaktion
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&#x2019;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&#x2019;t?
What is the relevance of these properties of anchorage dependence or independence in culture to tumor cells in humans. Truth is, we don&#x2019; 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&#x2019;t?
What is the relevance of these properties of anchorage dependence or independence in culture to tumor cells in humans. Truth is, we don&#x2019; 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&#x2019;t?
What is the relevance of these properties of anchorage dependence or independence in culture to tumor cells in humans. Truth is, we don&#x2019; 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.