Antioxidant and oncogene rescue
of metabolic defects caused by
loss of matrix attachment
Schafer ZT, Grassian AR, Song L,
Jiang Z, Gerhart-Hines Z, Irie HY, Gao
S, Puigserver P, and Brugge JS
Excess proliferation displaces cells from
their niches
autophagy
Vargo-Gogola T, Nat Rev Cancer (2007).
Determine the basis for the observed metabolic defect in matrix deprived cells
and provide evidence that an oncogene can rescue this defect.
EGFR expression is reduced after detachment in MCF10A cells
and overexpression can block anoikis
EGFR levels decrease upon matrix detachment and forced expression
rescues downstream signaling and allows cell survival
Reginato MJ, et al Nat Cell Biol (2003)
Glucose metabolism yields more than just energy
+
Fatty acid oxidation (FAO)
• I’m not fat I just have a lot
of potential energy!
• Each cycle trims 2 C from
the chain and produces 1
acetyl-CoA, 1 FADH2, and
1 NADH
ATP
ATP
Adapted from Mira A (2009)
ATP
The Take-Home Messages
• Detachment from ECM causes an ATP
deficiency that occurs as a result of reduced
glucose uptake
• This metabolic deficiency can be rescued by
overexpression of the oncogene ErbB2, via
constitutive signaling through EGFR and PI3K,
and it depends on flux through the PPP
• Antioxidants can reverse the metabolic defect,
independent of glucose uptake, by increasing
flux through the PPP
• Antioxidants can enhance the transforming
activity of oncogenic cells.
MCF10A cells that are in contact with ECM
Glucose
EGFR
ECM
PI3K
Glucose
PPP
ATP
ATP
NADPH
NADPHNADPH
ATP
ROS
FAO
MCF10A cells that are matrix deprived
Glucose
X
PI3K
Glucose
PPP
ATP
NADPH
ROS
Bim
FAO
Expression of ErbB2 removes the need for ECM signaling
ErbB2
Glucose
PI3K
Glucose
PPP
ATP
ATP
NADPH
NADPHNADPH
ATP
ROS
FAO
Figure 1. Loss of matrix attachment causes reduction in
cellular ATP that is rescued by ERBB2
• Depriving MCF10A cells of matrix leads to a drop in ATP
levels that is not seen in cells that overexpress ErbB2.
Supplementary Figures 4 & 5. Drop in ATP after detachment
is not affected by inhibition of apoptosis
24 hrs
Inhibition of apoptosis
does not affect the drop
in ATP after detachment
48 hrs
ATP has dropped by 24
hours of matrix detatchment
but cells are not apoptotic
Supplementary Figure 6. Drop in ATP after detachment is
not affected by inhibition of apoptosis or autophagy
Beclin-1 and ATG5 are key autophagy proteins
Inhibition of autophagy does not affect drop in ATP
Metabolic defect at 24 hours in detached MCF10A cells that is not a
result of apoptosis or autophagy
EGFR expression is reduced after detachment in MCF10A cells
and overexpression can block anoikis
EGFR levels decrease upon matrix detachment and forced expression
rescues downstream signaling and allows cell survival
Reginato MJ, et al Nat Cell Biol (2003)
Figure 1. Loss of matrix attachment causes reduction in
cellular ATP that is rescued by ERBB2
• EGFR is markedly stabilized in cells overexpressing ErbB2
• Targeted knockdown of EGFR in ErbB2-MCF10A cells leads
to a drop in ATP following matrix deprivation
• EGFR expression allows maintenance of ERK signaling and
an enhancement of PI3K signaling
Figure 1. Loss of matrix attachment causes reduction in
cellular ATP that is rescued by ERBB2 through PI3K pathway
activation
• Sustained ATP is lost after
inhibition of the PI3K
pathway but not the ERK
pathway
• Constitutive activation of
PI3K or AKT allows a
sustained level of ATP
Figure 1. Loss of matrix attachment causes reduction in
cellular ATP that is rescued by ERBB2 through PI3K pathway
activation
• Sustained ATP is lost after
inhibition of the PI3K
pathway but not the ERK
pathway
• Constitutive activation of
PI3K or AKT allows a
sustained level of ATP
ErbB2 sustains ATP levels by preventing
downregulation of EGFR, which maintains PI3K
pathway signaling.
Figure 2. Matrix detachment causes a reduction in glucose
uptake
The increase in glucose uptake seen in ErbB2-MCF10As is
important for sustaining ATP levels after detachment and is
dependent upon PI3K signaling
Figure 2. Matrix detachment causes a reduction in glucose
uptake
• Addition of pyruvate can rescue the
drop in ATP in detached MCF10A cells.
• Pyruvate rescue is not seen when the
mitochondria is inhibited.
• Addition of a toxic glucose analog
(2DG) does not allow sustained levels
of ATP in detached ErbB2-MCF10As
The increase in glucose uptake seen in ErbB2MCF10As is important for the elevated level of
ATP in these cells after detachment.
Pentose phosphate pathway is an alternative mode of
glucose oxidation
G6PD
• Major products
are NADPH and
ribose-5phosphate
• Controlled by
glucose 6
phosphate
dehydrogenase
(G6PD)
Figure 2. Matrix detachment causes a reduction in glucose
uptake and ERBB2 rescue of this defect is dependent on PPP
flux
ErbB2-MCF10A
MCF10A
• ROS is elevated and GSH drops
in detached cells
• Not seen in ErbB2-MCF10A cells
• Antioxidant NAC rescues GSH
reduction in MCF10A cells
Figure 2. Matrix detachment causes a reduction in glucose
uptake and ERBB2 rescue of this defect is dependent on
PPP flux
ErbB2-MCF10A
DHEA & 6AN
• ATP levels drop and ROS
levels increase in ErbB2MCF10A cells following PPP
inhibition
Pentose phosphate pathway is an alternative mode of
glucose oxidation
G6PD
• Major products
are NADPH and
ribose-5phosphate
• Controlled by
glucose 6
phosphate
dehydrogenase
(G6PD)
Figure 2. Matrix detachment causes a reduction in glucose
uptake and ERBB2 rescue of this defect is dependent on
PPP flux
ErbB2-MCF10A
• G6PD levels increase after
matrix detachment
• Knockdown increases ROS
and does not allow sustained
ATP in ErbB2-MCF10A cells
Suggests that sustained ATP levels
in ErbB2-MCF10A cells is dependent
on flux through the PPP
Figure 3. Antioxidants rescue low ATP levels in detached
cells
MCF10A
• Cells treated with the
antioxidants NAC and Trolox do
not experience an ATP drop
upon detachment
• Rescue is independent of
changes in glucose uptake
Figure 3. Antioxidants rescue low ATP levels in detached
cells by permitting fatty acid oxidation
• MCF10A detachment
causes a drop in FAO that
can be reversed by Trolox
treatment
• Antioxidant rescue of FAO
is reversed by inhibiting
fatty acid transport into the
mitochondria
Figure 3. Antioxidants rescue low ATP levels in detached
cells by permitting fatty acid oxidation
• Restoration of NADPH to detached
MCF10A cells rescues their metabolic
defects
• Blocking FAO in detached ErbB2MCF10A cells reduces ATP
significantly implicating this pathway in
the maintenance of ATP in these cells.
Figure 4. Analysis of antioxidant effects on acinar morphogenesis
and colony formation in soft agar
day 7
• Visualization of NAD(P)H and ROS demonstrates the metabolic
dichotomy between matrix-attached and matrix-starved cells in a
more physiologic setting.
Figure 4. Analysis of antioxidant effects on acinar morphogenesis
and colony formation in soft agar
• Treatment of acini formed in
3D culture with Trolox
diminishes the metabolic
dichotomy between the
matrix-attached and matrixstarved cells
ROS is contributing to the observed metabolic dichotomy
Figure 4. Analysis of antioxidant effects on acinar morphogenesis
and colony formation in soft agar
• Neutralization of ROS can reduce luminal clearing alone or in
combination with an apoptotic block.
Matrix-independent survival requires circumventing both metabolic
defects and apoptosis
Figure 4. Analysis of antioxidant effects on acinar morphogenesis
and colony formation in soft agar
• Neutralization of ROS can reduce luminal clearing alone or in
combination with an apoptotic block.
Can antioxidants promote the transforming activity of mammary
epithelial cells?
Figure 4. Analysis of antioxidant effects on acinar morphogenesis
and colony formation in soft agar
ErbB2-MCF10A
BT-474
• Colony formation was increased by treatment with Trolox in E7/BCL2MCF10As, ErbB2-MCF10As, and and ErbB2-expressing breast cancer
cell line
MCF10A cells that are in contact with ECM
Glucose
EGFR
ECM
PI3K
Glucose
PPP
ATP
ATP
NADPH
NADPHNADPH
ATP
ROS
FAO
Expression of ErbB2 removes the need for ECM signaling
ErbB2
Glucose
PI3K
Glucose
PPP
ATP
ATP
NADPH
NADPHNADPH
ATP
ROS
Antioxidants
FAO
The Take-Home Messages
• Detachment from ECM causes an ATP
deficiency that occurs as a result of reduced
glucose uptake
• This metabolic deficiency can be rescued by
overexpression of the oncogene ErbB2, via
constitutive signaling through EGFR and PI3K,
and it depends on flux through the PPP
• Antioxidants can reverse the metabolic defect,
independent of glucose uptake, by increasing
flux through the PPP
• Antioxidants can enhance the transforming
activity of oncogenic cells.
Final thoughts and possible
therapeutic implications
• Provides and explanation of how tumor
cells can adapt to glucose deprivation
• Are antioxidants more harmful than
previously thought?
– Prevent DNA damage vs. promote
anchorage-independent survival
• Does this lend support to the relevance of
PI3K inhibition?