1. The Warburg Effect And Cancer
Thomas Fung, Sam Hasheminejad, Jesse Ropat, Sherief Saleh
PHM Fall 2017
Instructor: Dr. Jeffrey Henderson

2. Introduction and Origins
Term comes from the Otto Warburg
German physiologist and medical doctor
Original research on oxygen consumption in sea urchins
Awarded nobel prize in 1931
The term Warburg effect comes from the name of the scientist who discovered it, Otto Warburg. He was a German physiologist and medical doctor who originally conducted research on oxygen consumption in sea urchins. In 1931, he was awarded the nobel prize for his research on tumors and the respiration mechanism of cancer cells in them.

3. The Original Experiments
Manometric techniques to measure CO2 emissions
First to use thin tissue slices
Cancer cells vs Normal tissue
CO2 emissions indicative of lactic acid formation
Readings
Sample
Warburg’s original experiments on these cancer cells involved the use of manometry on thin tissue slices. These manometric techniques allowed him to measure the CO2 emissions of cancer cells compared to normal cells and therefore indirectly determine their O2 consumption and lactic acid formation. To describe how the manometer works, samples are placed at the bottom right chamber and readings are made on the left side.

4. What is Cancer? Uncontrolled cellular growth
initiated through mutations in genomes accumulating into several hallmarks
Self-sufficient proliferation, insensitivity to antiproliferative signals, evasion of apoptosis, limitless replicative potential, maintenance of vascularization, tissue invasion and metastasis
exhibit altered behaviours in cellular processes and signalling pathways compared to normal cells
Just as a quick reminder: What is cancer?
Cancer is the disease which is characterized by uncontrolled cellular growth of abnormal cells in the body
It is a result of a mutations in the genome that accumulate and lead to several hallmarks:
Self sufficient proliferation
Insensitivity to antiproliferative signals
Evasion of apoptosis
Limitless replicative potential
Maintenance of vascularization
Tissue invasion and metassis
These behaviours lead to several distinctions between cancer cells and normal cells and give them a proliferative advantage. Eventually, their abnormal growth cause a number of pathological effects in the body that can eventually lead to death
Now let’s see how the Warburg Effect ties into cancer itself

5. Warburg effect and cancer
Warburg hypothesis: Cancer cells undergo higher than usual rates of intracellular glycolysis despite presence of oxygen – “aerobic glycolysis”
Increased glycolysis is followed by increased cellular fermentation decreasing oxidative phosphorylation (2ATP vs 38ATP)
Warburg’s research revolutionized the way cancer cells are differentiated from normal cells
Increased fermentation: positive feedback
Lactic acid builds up -> intracellular pH decrease
Continues to increase glycolysis + cancer development

6. Warburg effect – biochemical pathways
pyruvate eventually converted to lactate through activation of lactate dehydrogenase (LDH), to regenerate NAD+, allowing these cells to continue with high rates of glycolysis.

7. Warburg effect - glucose
Aerobic glycolysis
Increased glucose uptake
Glycogen synthesis
Lactate production
Induced acidosis --> acid mediated tumor invasion and impairment of mitochondrial function
Fermentation is markedly inefficient
Cells require greater flux of glucose in cells
Glucose is transported into cells via multiple transporters (i.e. GLUT family)
GLUT family receptors are often dysregulated in cancer
Stored as glycogen when excess
Cancerous cells display increased glucose into PPP that occurs in parallel to glycolysis generating large volumes of NADPH

8. ROS (Reactive Oxygen Species)
Damage to mitochondrial function
ROS produced by oxidative phosphorylation and other cellular processes damage cells and intracellular pathways
Damage to mitochondrial function can further glycolysis in mitochondrial cells
Production of reactive oxygen species and detrimental effects
H202 enhances Na+/H+ ATPase pump and heightens expression of proto-oncogenes
ROS contributes to cancer growth and glycolysis, decline in mitochondrial function

9. Advantages for cancer cell – Warburg Effect
Greater energy yields than oxidative phosphorylation by greatly increasing glycolysis
Large amount of glycolytic products for intermediates in biosynthetic pathways
Glycolysis produces less ROS than mitochondrial OXPHOS
Net effect: aids cancerous cells in cell division and further tumour growth
Associated with increase in anabolic processes (eg. PPP) = more resistant to oxidative stress
Cancer cells typically display what is known as the “Warburg phenotype,” characterized by excessive glycolytic activity. Typically, cells in this state overexpress glycolytic enzymes and oncogenes, leading to increased rates of proliferation and increased cellular metabolism.9 A common instigator of glycolytic overexpression in several cancer cell types is the protein HIF-1a, which also plays a role in tumour angiogenesis and metastasis.10 Additionally, an increase in glycolysis is associated with an increase in anabolic processes potentially allowing these cells to become more resistant to increasing oxidative stress, as well as supporting their needs for cellular growth and division.2 There are advantages to such a method of energy production. By forcing most energy production through the glycolytic pathway, the cells are able to experience greater energy yields than oxidative phosphorylation by greatly increasing the rate of glycolysis.11 This yields large amounts of glycolytic products which can be used as intermediates in biosynthetic pathways. Combining these two factors may aid cancerous cells in cell division and can result in further tumour growth.12

10. Reverse Warburg effect
Cancer cells may induce aerobic glycolysis in surrounding fibroblasts (carcinoma-associated fibroblasts)
Result ^ [lactate], converted to pyruvate, used for mito OXPHOS in cancer cells
Tumor cells in normoxic pockets vs hypoxic pockets of tumor microenvironment
Tumour
oxidative tumor cells in normoxic microenvironment use mitochondrial OXPHOS to spare glucose, which can be utilized by glycolytic tumor cells located in hypoxic microenvironment
(1) enhanced glycolysis and suppressed mitochondrial OXPHOS with a hypoxic condition; and (2) relatively suppressed glycolysis and restoration of mitochondrial OXPHOS with nutrient shortage because of high proliferation rates

11. Warburg effect – therapeutic strategies
TARGETS:
Glycolytic Pathway:
3- bromopyruvate (3-BP)
- most potent glycolytic inhibitor

12. ⟝ 3-Bromopyruvate 3-BP Potent Hexokinase II inhibitor
-significantly reduces energy production in cells dependent on glycolysis

13. 3-Bromopyruvate not 100% effective
Low ability to suppress mito OXPHOS/redox potential
Combinatorial treatment?
Oxidative Stress targeting:
ROS-inducing chemotherapeutics
Cancer cells operate at higher resting ROS levels than normal cells
Mitochondrial targeting:
Selective targeting electron transport system

14. Warburg effect – molecular basis
Mitochondrial dysfunction/damage
Originally proposed as the reason for Warburg Effect, though it is not present in all cells,
There are a number of cancers that exhibit Warburg Effect and still have functional mitochondria
Dysregulated mitochondrial function is common in cancer but is not necessarily the only reason for Warburg Effect
Although we’ve talked about what is the warburg effect, and the reasons cancer cells have developed it, we haven’t talked much about the exact mechanisms and molecular changes in cancer cells that have led to these advantages.
Originally, both Warburg and initial researchers proposed that mitochondrial dysfunction and damage was the reason for the Warburg effect as it may shift the energy production towards aerobic glycolysis
-however, a number of cancers still exhibit the Warburg effect and have function mitochondrial
-although there is reduced levels of mitochondrial function and it is dysregulated in cancer, its not necessarily the only reason for the Warburg effect

15. Warburg effect – molecular basis
PKM2 is an isoform of normal pyruvate kinase
PKM2 is anabolic:
Upregulated in cancer cells
Increases levels of PEP and shifts towards PPP
One important principle is that the enzymes involved in the glycolysis pathway are dysregulated
-one example is Pyruvate kinase, one of the rate limiting steps in glycolysis
One isoform of PKM is PKM2, which shifts glycolysis towards the PPP
IN cancer cells, PKM2 expression is increased in tumor cells and upgrelated.
This is just one example of cancer cells altering specific enzymes in the glycolysis pathway to favour the Warburg effect

16. Warburg effect – molecular basis⟝
p53
PKM2
For the reasons this happen we need to look at some common signalling pathways that are dysregulated in cancers. One of such is PI3K/Akt/MTOR pathway that regulates a large number of cellular processes. Now I cannot stress how simplified this diagram but let me highlight some sections you may know.
below we have the glycolysis pathway leading to lactic acid fermentation and oxidative phosphorylation. We can see that oncogenes that are commonly mutated and upregulated in cancer: Akt: PI3K, MTOR, Myc, and HIF all have promoting effects on glucose transport with increasing transporters, increasing the production of pyruvate and specifically promoting lactic acid fermentation by upregulating lactate dehydrogenase and decreasing pyruvate dehydrogenase.
Also note, PKM2 falls under this pathway as well. A number of cancer mutations induce increased PKM2 signalling
Something else you might have heard of is p53. Now p53 plays a large role in inhibiting many parts of this pathway such as the transporters, AKT, mTOR, PTEN. As you may know, cancer cells often have mutated or dysfunction p53 leading to further contribution towards warburg
Now the molecular mechanisms behind this is much more complicated however what we can see is that the Warburg effect is a result of oncogenic activation and tumor suppression
Warburg effect is a result of oncogenic activation/tumor suppression

17. The molecular basis behind the Warburg Effect
Redefining the mechanism of Warburg Effect
Cancer cells exhibit several mutations that contribute and accumulate to the standard hallmarks (sustained cellular proliferation, evasion of growth suppressors, etc.)
Many of the genes that are mutated (onco-genes, tumor suppressors) control cellular metabolism and energetic mechanisms
On top of inducing the hallmarks, the advantages that a cancer cell exhibiting Warburg bioenegetics provides it with a selective advantage

18. Summary Slide
Warburg Effect was initially observed by Otto Warburg: Manometric experiments demonstrated cancer tissues exhibit anaerobic breakdown of glucose in presence of oxygen vs normal tissues
Warburg effect:
Cancer cells display “aerobic glycolysis”, high levels of glycolysis despite O2 followed by cellular fermentation
Increased glucose uptake + utilization to account for consumption
Cancer cells have an advantage with the Warburg Effect:
Reduced ROS production + Protection through NADPH production
Higher energy yields
Increased anabolic processes
Therapeutic strategies against cancer cells
Targeting specific steps in glycolytic pathway could have therapeutic benefits
3 Bromo-pyruvate targeting hexokinase II stops glucose -> glucose 6 phosphate
Combinatorial therapy targeting multiple pathways for increased efficacy
Molecular basis of Warburg Effect
Warburg effect is a result of oncogenic activation and tumor suppressor inhibition found commonly in cancer cells
These mutations induce signalling changes in various pathways (i.e. AKT-PI3K-mTOR) that promote Warburg phenotype

19. References:
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4. Chandel, N. S. et al. Reactive oxygen species generated at mitochondrial Complex III stabilize hypoxia-inducible factor-1?? during hypoxia: A mechanism of O2 sensing. J. Biol. Chem. 275, 25130–25138 (2000).
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6. WARBURG, O. On the origin of cancer cells. Science 123, 309–14 (1956).
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8. Courtnay, Rupert, et al. "Cancer metabolism and the Warburg effect: the role of HIF-1 and PI3K." Molecular biology reports 42.4 (2015):
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10. Yang, Weiwei, et al. "ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect." Nature cell biology 14.12 (2012): 1295.
11. Hitosugi, Taro, et al. "Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth." Science signaling2.97 (2009): ra73.