The Biology of Cancer:

Cancer Cell Metabolism

Jim Gould, PhD
Metabolism & Cancer Susceptibility Section, LCC
NCI-Frederick
October 18, 2010
Hallmarks of Cancer: Adaptations to Evade Death

Cell, Vol. 100, 57–70, January 7, 2000

 Tumor microenvironment plays an important role

•  Nutrient source
–  Glucose
–  Amino Acids
–  Oxygen
•  Growth factors
•  Structural support
–  Extracellular Matrix
•  Cell-Cell interactions

Nature Vol 455; 18 September 2008

What is Metabolism?
 Glucose is the body s major nutrient and energy source

•  The human body exquisitely maintains

the level of circulating glucose in the
range of 5 mM.
•  Nearly all carbohydrates ingested in
the diet are converted to glucose
following transport to the liver.
•  Breakdown of dietary or cellular
proteins generates carbon atoms that
can be utilized for glucose synthesis
via gluconeogenesis.
•  Additionally, skeletal muscle and
erythrocytes provide lactate that can
be converted to glucose via
gluconeogenesis.
•  Maintenance of glucose homeostasis
is of paramount importance to the
survival of the human organism.

The same could be said of cancer....

Cells alter their metabolism in response to stimuli

•  Microbes and cells from multicellular

organisms have similar metabolic
profiles under similar environmental
conditions.

•  During proliferation, these organisms

both metabolize glucose primarily
through glycolysis, excreting large
amounts of carbon in the form of
ethanol, lactate, or another organic
acid.

•  When starved of nutrients, both rely

primarily on oxidative metabolism.

•  Evolutionarily, there is an advantage

to oxidative metabolism during nutrient
limitation and an advantage in
glycolysis during cell proliferation.

Matthew G. Vander Heiden, et al. Science 324, 1029 (2009)

 Glycolysis in Normal Tissues

•  During aerobic glycolysis, pyruvate

in most cells is further metabolized via
the TCA cycle. Aerobic glycolysis
generates substantially more ATP per
mole of glucose oxidized than does
anaerobic glycolysis.
•  Under anaerobic conditions, pyruvate
is converted to lactate by the enzyme
lactate dehydrogenase (LDH), and
the lactate is transported out of the
cell into the circulation.
•  The conversion of pyruvate to lactate
provides the cell with a mechanism
for the oxidation of NADH to NAD+
without which glycolysis will cease.
•  The utility of anaerobic glycolysis, to
a muscle cell when it needs large
amounts of energy, stems from the
fact that the rate of ATP production
from glycolysis is approximately 100X
faster than from OxPhos.
•  Muscle cells derive almost all of the
ATP consumed during exertion from
anaerobic glycolysis allowing it to
generate the maximum amount of
ATP, for muscle contraction, in the
shortest time frame.
Michael W. King; themedicalbiochemistrypage.org
Cancer Metabolism
Aerobic Glycolysis: The Warburg Effect
•  In the presence of O2, nonproliferating tissues
first metabolize glucose to pyruvate via
glycolysis and then completely oxidize most of
that pyruvate in the mitochondria to CO2 during
the process of oxidative phosphorylation
(OxPhos).
•  Because oxygen is required as the final electron
acceptor to completely oxidize the glucose,
oxygen is essential for this process.
•  When oxygen is limiting, cells can redirect the
pyruvate generated by glycolysis away from
mitochondrial OxPhos by generating lactate.
•  Lactate production allows glycolysis to continue
(by cycling NADH back to NAD+), but results in
minimal ATP production when compared with
OxPhos.
•  Otto Warburg observed that cancer cells tend
to convert most glucose to lactate regardless of
whether oxygen is present (aerobic glycolysis).
This property is shared by normal proliferative
tissues.
•  Mitochondria remain functional and some
oxidative phosphorylation continues in both
cancer cells and normal proliferating cells.
•  Aerobic glycolysis is less efficient than OxPhos
for generating ATP. The cells make up for this
by consuming more glucose.

Matthew G. Vander Heiden, et al. Science 324, 1029 (2009)

 How cancer cells reprogram their metabolism
•  Metabolic cross-talk allows for both NADPH
production and Ac-CoA flux for lipid
synthesis.
•  These metabolic pathways can be
influenced by cell proliferation signaling
pathways.
•  Activation of growth factor receptors leads to
downstream signaling cascade activation.
•  PI3K/Akt activation stimulates glucose
uptake and flux through the early part of
glycolysis.
•  Tyrosine kinase signaling negatively
regulates flux through the late steps of
glycolysis, making glycolytic intermediates
available for macromolecular synthesis as
well as supporting NADPH production.
•  Myc drives glutamine metabolism, which
also supports NADPH production.
•  LKB1/AMPK signaling and p53 decrease
metabolic flux through glycolysis in
response to cell stress.
Vander Heiden, et al. Science 324, 1029 (2009)
How Warburg is
Advantageous
The Warburg effect gives tumor cells a growth advantage
through reduced oxygen consumption

By slowing the consumption

of O2 in the hypoxic cells, O2
diffuses further and fewer
cells reach anoxic levels that
are toxic. Mild hypoxia can
support cellular growth.

Denko, Nature Reviews: Cancer Vol 8; Sept 2008

 Molecular underpinnings of the Warburg effect
The Warburg effect describes the enhanced conversion of glucose to lactate by tumor cells, even in
the presence of adequate oxygen that would ordinarily be used for OxPhos.

•  Activation of the AKT oncogene results in

enhanced glycolytic rates.
•  MYC oncogene is activates glycolytic
genes and mitochondrial biogenesis,
which can result in reactive oxygen
species (ROS).
•  ROS could cause mtDNA mutations that
render mitochondria dysfunctional.
•  p53 stimulates respiration through
activation of a component of the
respiratory chain.
•  Hypoxic sensor HIF-1 is stabilized and
accumulates
•  HIF-1 transactivates glycolytic genes as
well as directly activates the PDK1 gene,
which in turn inhibits PDH that catalyzes
the conversion of pyruvate to acetyl-CoA.
•  Acetyl-CoA enters the TCA cycle, which
donates electrons to the mitochondrial
respiratory chain complexes I to IV.
•  Inhibition of PDH by PDK1 attenuates
mitochondrial function, resulting in the
shunting of pyruvate to lactate.

Cancer Res 2006; 66: (18). September 15, 2006

How Cancer Alters its
Metabolism
Global Changes in Cancer Metabolism:
Genetic Mutations

•  Mutations and epigenetic changes lead to

changes in the function of oncogenes and
tumor suppressor genes.
•  Genomic instability causes further changes
that upset the balance of oncogenes and
tumor suppressor genes.
•  These events lead to changes in the
function of 3 transcription factors: activation
of HIF-1 and MYC and loss of p53 function.
•  The changes in these transcription factors
cause a coordinated change in the
enzymes, transporters, regulators and
metabolites as well as changes in
mitochondrial function.
•  This brings about a characteristic metabolic
signature of cancer cells.
•  This metabolic reprogramming provides
growth and survival advantages for the
cancer cells in the tumor microenvironment.
Cell. Mol. Life Sci. 65 (2008) 3981 – 3999
 Regulation of Cancer Metabolism:
Protein Signaling Pathways
•  Activation of the AKT signaling may be
sufficient to bring about the switch to
glycolytic metabolism in cancer.
–  regulates glucose transporter 1 (GLUT1)
expression
–  activates HK2 which promotes
phosphorylation of glucose to glucose 6-
phosphate
–  regulates de novo fatty acid synthesis
and b-oxidation
•  mTOR is situated in the crossroads of
signaling pathways and is an integration
center of the signals to bring
coordinated regulation of nutrient
uptake, energy metabolism, cell growth,
proliferation, and cell survival.
•  mTOR is an upstream activator of
HIF-1a in cancer cells, which is a
subunit of a transcription factor that
upregulates the expression of nearly all
the genes involved in the glycolytic
pathway
Cell. Mol. Life Sci. 65 (2008) 3981 – 3999
c-MYC, HIF-1 and p53 Regulates Glycolytic Metabolism:
Transcription

•  The Warburg effect is partly

due to
–  increased activity of the
transcription factors MYC
and HIF-1 in cancer cells
–  Upregulation of genes
coding for glucose
transporters and glycolytic
and regulatory enzymes
mediated by, and
–  A coordinated loss of
regulatory proteins due to
loss of p53 function.
–  Loss of p53 function also
leads to activation of
GLUT-3 transcription via
NFkB.

Cell. Mol. Life Sci. 65 (2008) 3981 – 3999

Hypoxia Inducible Factor
(HIF)
 HIF has a global effect on metabolism

•  HIF upregulates glycolysis

–  Increased uptake of glucose through
glucose transporters GLUT1 and GLUT3.
–  Glucose metabolism by the increased
levels of the glycolytic enzymes
–  Increased pyruvate levels, which is largely
converted to lactate by LDHA
–  Pyruvate is removed from the cell by the
monocarboxylate transporter

•  HIF downregulates OxPhos in

mitochondria
–  decreased pyruvate flow into the TCA cycle
–  decreased mitochondrial biogenesis
–  Switch to high efficiency cytochrome
oxidase

Denko, Nature Reviews: Cancer Vol 8; Sept 2008

HIF1α protein structure is important in its regulation

Sites of proline hydroxylation (P402/P564) are indicated in the

O2-dependant degradation domain of the human protein.
Asparagine (N803) hydroxylation in the carboxy-terminal
transactivation domain (TAD) by factor inhibiting HIF (FIH)
regulates HIF1 activity but not stability.

Denko, Nature Reviews: Cancer Vol 8; Sept 2008

 Mechanisms of (HIF1α) stabilization

•  Oxygen levels are sensed through O2-

dependent proline hydroxylation on
HIF1α.
•  This modification is due to one of the
three prolyl hydroxylase (PHD), which
mediate proteasomal degradation.
•  Oncogenic activation, can also cause
HIF1α stabilization through unknown
mediators.
•  TCA intermediates such as succinate
and fumarate, or mitochondrial reactive
oxygen species (ROS), can inhibit the
activity of PHDs, also stabilizing HIF1α.
•  Stabilized HIF1α associates with HIF1β,
which binds to hypoxia-responsive
elements (HREs) in target genes.

Denko, Nature Reviews: Cancer Vol 8; Sept 2008 Kaelin & Thompson, Nature; Vol 465; 3 June 2010
 HIF1 targets that regulate glucose metabolism

cOX4I2, cytochrome oxidase isoform 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT,

glucose transporter; HIF, hypoxia-inducible factor; HK, hexokinase; LDHA, lactate dehydrogenase A;
McT, monocarboxylate trasporter; MXI, max interactor; PDK, pyruvate dehydrogenase kinase; PFK,
phosphofructokinase; PFKFB, 6-phospho-2-kinase/fructose 2,6 bisphosphatase; PGK, phosphoglycerate
kinase; PGI, phosphoglucose isomerase; PGM, phosphoglycerate mutase; PK, pyruvate kinase;
TPI, triosephosphate isomerase.

Denko, Nature Reviews: Cancer Vol 8; Sept 2008

Alternative Nutrients and
Preventative Agents
 Alternative Nutrients: Amino Acids

All 20 of the amino acids,

excepting leucine and lysine,
can be degraded to TCA cycle
intermediates. This allows the
carbon skeletons of the amino
acids to be eventually
converted to pyruvate. The
pyruvate thus formed can be
utilized by the gluconeogenic
pathway.
 Vitamin C
•  Vitamin C (L-ascorbic acid, ascorbate, VitC)
is one of the most abundantly produced
substances in plants and living organisms
(but not humans).
•  VitC is not part of any metabolic pathway in
humans but is an essential co-factor in
many enzymatic reactions.
•  Studies have suggested that insufficient
dietary intake levels of VitC may adversely
affect health and normal life span in man,
and could be one of the reasons for the
relatively high incidence of cancer in
humans.
•  Glucose and VitC are six-carbon sugars
whose over-consumption positively and
negatively affects cancer, respectively.
•  Glucose addiction is a hallmark of cancer
cells, promoting cell proliferation and
increasing the risk of cancer, BUT high
levels of VitC protect multi-cellular
organisms from ROS damage and
uncontrolled cell proliferation.

The Search for the Achilles Heel of Cancer, PeproTech, Inc; 2010
Targeting Metabolism for
Cancer Therapy
 Targeting Metabolism for Cancer Therapy

•  Small molecule drugs that disrupt

glucose metabolism or decrease
glucose uptake by tumors could
provide anti-cancer therapy
•  We can visualize altered glucose
metabolism in tumors by 18F-
deoxyglucose positron emission
tomography (FDG-PET)
•  The ability to inhibit tumor FDG
uptake correlates with tumor
regression
•  Seen here:
–  Malignant sarcoma (gastrointestinal
stromal tumor) before and after
therapy with a tyrosine kinase
inhibitor (sunitinib).

Matthew G. Vander Heiden, et al. Science 324, 1029 (2009)

 Glycolysis Can Promote Resistance to Cancer Therapy

•  Glycolysis provides the metabolites and energy for DNA repair and
chemotherapy drug inactivation/detoxification.
•  Glycolysis can provide ATP/NAD+ for DNA repair.
•  Glycolysis, pentose phosphate pathway and glutaminolysis can also
provide NADPH
•  These mechanisms can potentially contribute to resistance of the
cancer to therapy.

Cell. Mol. Life Sci. 65 (2008) 3981 – 3999

 Summary
•  How cancer changes •  Why these changes
metabolism are advantageous
–  Expression of –  Decreased O2
oncogenes and tumor consumption
suppressors –  Increased Redox
–  Expression/activity of potential
glycolytic enzymes –  Faster use of glucose
–  Interactions with –  Increased building
microenvironment blocks
–  Aerobic glycolysis –  Evasion of cell cycle
checkpoints
 How I Study Cancer
Metabolism: Proline Cycle
 We study how proline metabolism relates to
the cancer phenotype
Tools:
• O2 Consumption •  Enzymatic Assays
•  Lactate Assay •  Molecular Biology
•  Glucose Assay •  Gene silencing
•  ATP Assay •  2D and 3D culture
•  ROS Assay •  Nutrient Profiling

Phang et al, Annu. Rev. Nutr. 2010. 30:15.1–15.23

 Cell lines are vital to studying
cancer
•  UOK262: Fumarate hydratase deficient (FH-/FH-)
cell line
•  HLRCC: hereditary leiomyomatosis renal cell
carcinoma
•  Model cell line for the Warburg Effect
–  Pseudohypoxic HIF1-α stabilization
–  Highly glucose-dependent growth
–  Compromised OxPhos and increased anaerobic
glycolysis
–  Elevated lactate efflux and GLUT1 expression
 Activity
•  Read questions 1-6, 8-9, 11, 15-16 from
Nature Q&A article Clues from cell
metabolism
•  Write a one sentence summary of the
answer…so that a 6th grader could
understand it.
•  We will share your answers with the rest of
the class