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)

Vander Heiden, et al. S

cience 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.

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

•   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

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.

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

metabolism –   Expression of

oncogenes and tumor suppressors

–   Expression/activity of glycolytic enzymes

–   Interactions with microenvironment

–   Aerobic glycolysis

•   Why these changes are advantageous –   Decreased O2

consumption –   Increased Redox

potential –   Faster use of glucose –   Increased building

blocks –   Evasion of cell cycle

checkpoints

How I Study Cancer Metabolism: Proline Cycle

We study how proline metabolism relates to the cancer phenotype

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

Tools: •  O2 Consumption •   Lactate Assay •   Glucose Assay •   ATP Assay •   ROS Assay

•   Enzymatic Assays •   Molecular Biology •   Gene silencing •   2D and 3D culture •   Nutrient Profiling

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