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The Warburg Effect: Role in Cancer

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1 The Warburg Effect: Role in Cancer
Paul Bansal, Robert Calvaruso, Hemangi Dave & Henry Pun Sept PHM Fall 2015 Instructor: Dr. Jeffrey Henderson

2 Overview Discovery & Description of the Warburg Effect Mechanism
Glycolysis pathway NADPH/ROS Occurs in aerobic conditions (Warburg effect = aerobic glycolysis) Detection FDG-PET Therapeutics Drugs that target specifically target glycolytic pathway to selectively dest

3 The Discovery of the Warburg Effect
Cellular phenomenon in cancer cells discovered by Otto Warburg in 1924 Initially measured lactate production and glucose consumption in rat liver carcinoma and normal liver tissue Warburg determined that cancer tissue consumed 10x more glucose than accounted for by respiration, and produced up to 100X more lactic acid than in normal tissue Cancerous cells preferentially use glycolysis for energy production rather than oxidative phosphorylation Initially believed to be the cause of cancer but recent evidence shows it as a byproduct of cancer Initial explanation for Warburg effect - dysfunction of mitochondrial cells medicine/laureates/1931/ The warburg effect believed to b a result of mitochondrial dysfunction but now proven to not be so From Bensinger and Chistofk (2012):

4 What is the Warburg Effect?
Utilization of aerobic glycolysis as the major source of ATP Activates pentose phosphate cycle Produces NADPH Protects cells against ROS Involves massive increase in glucose uptake, reliance on glycolysis, and inhibition of oxidative phosphorylation Vidugiriene (2013) Increased cycles of glycolysis result in increased production of NADPH which effectively lowers ROS levels in the cell environment, allowing cancer cells to continue proliferating

5 What factors push cells to enter aerobic glycolysis?
Genomic regulation Phosphoglycerate dehydrogenase (PHGDH) Transcriptional regulation HIF1 MYC P53 Metabolic isoform switching Pyruvate kinase M2 (PKM2) 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) Post-translational regulation Activation of PI3K/AKT pathway signaling PKM2 PHGDH increased in melanoma and breast cancer catalyzes first step in serine biosynthesis pathway (glycolytic carbon → serine/glycine) suppression of PHGDH in breast cancer cell lines with multiple gene copies impairs cell proliferation HIF1 (HIF1α + HIF1β) upregulates expression of glucose transporters and most glycolytic enzymes upregulates expression of pyruvate dehydrogenase kinases (PDK) PDKs inactivate mitochondrial PDH complex PDH controls entry of glycolysis-derived pyruvate into TCA cycle reduces pyruvate flux into TCA, oxidative phosphorylation rates, oxygen consumption in NORMOXIA → HIF1α degraded (via ubiquitination) in oxygen-dependant manner in HYPOXIA → HIF1α stabilized increased HIF1 activity commonly observed in tumours: non-hypoxic stabilization of HIF1α transcriptional upregulation of HIF1α from activation of PI3K/AKT/mTOR pathway MYC upregulates thousands of genes, including those that would promote Warburg Effect glucose transporters glycolytic enzymes PDK1 lactate dehydrogenase A glutamine metabolism (can provide glutathione) MYC overexpression is molecular alteration most associated with high FDG uptake in breast cancer p53 tumour suppressor commonly associated with DNA damage response, apoptosis loss-of-function in cancers suppresses glycolysis by upregulating TIGAR (fructose-2,6-bisphophatase) promotes oxidative phosphorylation by enhancing expression of cyt c oxidase-2 PKM2 tetrameric protein, one of four variants of PK catalyzes final rate-limiting step of glycolysis (phosphoenolpyruvate + ADP → pyruvate + ATP) PKM1 is in many tissues PKM2 is only found in self-renewing cells (stem cells, TUMOUR cells) PKM2 promotes Warburg effect and tumorigenesis PKM2 is a transcriptional coactivator of HIF1, and HIF1 targets amplification of PKM2 gene PKM2 promotes Warburg effect through a positive feedback loop PFKFB controls levels of fructose-2,6-bisphosphate (F2,6BP), which is an allosteric activator of phosphofructokinase-1 (PFK-1) PFK-1 is a rate-limiting enzyme in glycolysis PFKFB can phosphorylate, or dephosphorylate, F6P to and from F2,6BP PFKFB1,2,4 have equal kinase and phosphatase activity PKFKB3 is an inducible kinase/phosphatase, and induced by inflammation and HYPOXIA 740:1 kinase:phosphatase ratio will highly favour F2,6BP formation, and activation of PFK-1 elevated levels of PKFKB3 in human tumours PKFKB3 expression promoted by mutations in JAK/STAT signalling pathway

6 Many factors promote Warburg Effect
PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation. When cytosolic PKM2 is activated by F1,6BP, glycolytic intermediates are diverted into the TCA cycle (blue arrows). However, PKM2 can be inhibited by receptor tyrosine kinase activation, direct phosphorylation by FGFR1, acetylation in the presence of high glucose concentrations, and oxidation upon elevated ROS levels (red arrows). PKM2 inhibition promotes redirection of glycolytic intermediates into biosynthetic pathways important for proliferation (solid red arrows indicate proven flux changes, dotted red arrows indicate hypothesized flux changes). PKM2 also functions as a transcriptional coactivator to promote aerobic glycolysis and proliferation in cancer cells. Nuclear PKM2 enhances HIF1 binding to hypoxia response elements (HRE) promoting transcription of HIF1 target genes and interacts with tyrosine-phosphorylated -catenin to promote transcription of cyclin D1. Thus both cytosolic and nuclear PKM2 contribute to altered metabolism and proliferation in cancer. Bensinger (2012) Vander Heiden (2009)

7 Detection using FDG-PET
Non-invasive assay for visualizing rate of glucose uptake in cell Uses radiotracer: 2-deoxy-2[18F]fluoro-D-glucose (fluorodeoxyglucose) Approved for diagnosis and monitoring in many cancers Not useful for some cancers (prostate, pancreatic, hepatocellular carcinoma) No Warburg effect Poor probe perfusion into tumour Low tumour cell density High background High G6P expression Kelloff (2005) This is why some medical imaging techniques can help us locate tumours when they reach a certain size. Radio-active glucose is injected in patients. The Positron Emission Tomography (PET) scan tool is sensible to radio-active material. Since cancer cell will consume 18 to 19 times more glucose than normal cells, they will accumulate more radio-active material as illustrated in the picture below. Shown in the left is a Positron Emission Tomography (PET) scan of a 62 year old man with a brain tumour. The irregular bright yellow and orange area in the lower left portion of the brain indicates the location of the tumour, which metabolizes glucose faster than normal cells. A PET scan uses a small amount of a radioactive drug, or tracer, to show differences between healthy tissue and diseased tissue. The most commonly used tracer is called FDG (fluorodeoxyglucose), so the test is sometimes called an FDG-PET scan. Flourine 18 substituted in place of an OH Bensinger (2012)

8 FDG-PET as a monitoring tool for anticancer therapy
Decreased metabolism of glucose by tumors, visualized by PET with the glucose analog FDG, predicts response to anticancer therapy. Shown are fused coronal images of FDG-PET and computerized tomography (CT) obtained on a hybrid PET/CT scanner after the infusion of FDG in a patient with a form of malignant sarcoma (gastrointestinal stromal tumor) before and after therapy with a tyrosine kinase inhibitor (sunitinib). The tumor (T) is readily visualized by FDG-PET/CTbefore therapy (left). After 4 weeks of therapy (right), the tumor shows no uptakeof FDG despite persistent abnormalities on CT. Excess FDG is excreted in the urine,and therefore the kidneys (K) and bladder (B) are also visualized as labeled. [Image courtesy of A. D. Van den Abbeele, Dana-Farber Cancer Institute, Boston] Vander Heiden (2009)

9 Therapeutics Warburg's work helped to show the potential for beneficial pharmaceuticals that could be developed by inhibiting certain chemical mechanisms of glycolysis to specifically target and kill cancer cells. -Targetting of specific metabolic pathways to kill cancer cells Need to add reference to bottom of slide Pelicano (2006)

10 Drug Therapy Therapeutics that exploit the Warburg effect Elicano 2006
Compound status Mechanisms of action Drug development 2-Deoxyglucose Inhibits phosphorylation of glucose by hexokinase Clinical trials (I/II) Lonidamine Inhibits glycolysis and mitochondrial respiration Clinical trials (II/III) Inhibits HK; dis-associating HK from mitochondria 3-Bromopyruvate Inhibits HK; acts as an alkylating agent Pre-clinical Imatinib Inhibit Bcr-Abl tyrosine kinase; causes a decrease in HK and G6PD activity Approved for clinical use Oxythiamine Suppresses PPP by inhibiting transketolase; inhibits pyruvate dehydrogenase Pelicano (2006) Elicano 2006

11 3-BrPA use in-vivo while the in vivo experiment in f shows that when 3-BrPA is injected at the site of a large hepatocellular carcinoma growing in the upper back of the rat “R4” the tumor disappears within 3 weeks. Ko (2004)

12 Lonidamine (LND) in tumorigenic GL15 cells
Figure 3. Autophagic response of GL15 cells to LND. Cells were incubated with 400μM LND for 1–2hours (A) or with 50–400μM LND for 48hours (B). Protein expression was evaluated by Western blotting and analyzed by densitometry. Data are the mean ± SD of three independent experiments (*p<0.05 vs control). Representative blots are shown. (C) Cells were treated with 400μM LND for 48hours and stained with acridine orange for 15min. The arrow indicates acidic vesicular organelles. Original magnification 400X. (D) Cells were treated with 400μM LND for 48hours and immunostained with a LC3 antibody. The arrows indicate bright puncta. Original magnification 600X. (E) Cells were treated with 400μM LND for 48hours and immunostained with an α-tubulin antibody. LND caused cytoskeleton disorganization. Arrows indicate α-tubulin condensed in the perinuclear zone. Original magnification 400X. The energy blockers bromopyruvate and lonidamine lead GL15 glioblastoma cells to death by different p53-dependent routes. Davidescu (2015)

13 Oxythiamine in mice lung carcinoma
Immunohistochemical (IHC) staining of Antiproliferating cellular nuclear antigen of proliferating cellular nuclear antigen (PCNA) in lung tissues of C57BL/6 mice. One day after subcutaneous injection with Lewis lung carcinoma (LLC) ( /100 ll), the mice were supplemented with OT 250, and OT 500 for 5 wk. b Percentage of PCNA-positive tumor cells in lung tissues of C57BL/6 mice. Values are means ± SD, n = 3–7; means without a common letter differ, P \ 0.05 Yang (2010)

14 Summary The Warburg effect was discovered in 1924 by Otto Warburg
The Warburg effect is the reliance of cancer cells on aerobic glycolysis as opposed to oxidative phosphorylation Features: ATP generation via glycolysis Increased NADPH through the pentose phosphate shunt Generates protection against reactive oxidative species – allows continuous cell proliferation Promotes generation of macromolecules required for proliferation Multiple causes PKM2 isoform switching Overexpression of HIF1, Myc P53 underexpression Detected using FDG-PET Uses radiolabelled glucose analog (FDG) to visualize areas of increased glucose uptake Can detect location of tumours as well as monitor the progression of cancer therapy Therapeutic applications Drugs (ex. 3-BrPA, 2-DG, oxythiamine) target enzymes at various points in the glycolytic pathway Thereby selectively inhibiting cancer cell proliferation Drug examples: 3-BrPA, 2-DG, oxythiamine target various

15 Summary Warburg's work helped to show the potential for beneficial pharmaceuticals that could be developed by inhibiting certain chemical mechanisms of glycolysis to specifically target and kill cancer cells. -Targetting of specific metabolic pathways to kill cancer cells Need to add reference to bottom of slide Pelicano (2006)

16 References
Cancer cell metabolism. (2008). Retrieved from Davidescu, M. et al. (2015). “The energy blockers bromopyruvate and lonidamin lead GL15 glioblastoma cells to death by different p53-dependent routes.” Nature: Scientific Reports. 5:14343, p Pedersen, P.L. (2007). “Warburg, me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancers’ most common phenotypes, the “Warburg Effect”, i.e., elevated glycolysis in the presence of oxygen”. Journal of Bioenergy and Biomembranes. 39: Yang, CM et al. (2010). “The in vitro and in vivo anti-metastatic efficacy of oxythiamine and the possible mechanisms of action.” Clinical Experimental Metastasis. 27: Heiden, M.G.V. et al. (2009). “Understanding the Warburg effect: the metabolic requirements of cell proliferation” Science. 324(5930): Kim J. and Dang C. (2006) Cancer’s Molecular Sweet Tooth and the Warburg Effect. Cancer Res. 66: Bensinger S.J. and Christofk H.R. (2012). “New aspects of the Warburg effect in cancer cell biology.” Seminars in Cell & Developmental Biology. 23: Hsu P.P. and Sabatini D.M. (2008). “Cancer Cell Metabolism: Warburg and Beyond.” Cell. 134: Kelloff G.J. et al. (2005). “Progress and Promist of FDG-PET Imaging for Cancer Patient Management and Oncologic Drug Development.” Clin Cancer Res. 11(8): Pelicano H. et al. (2006). “Glycolysis inhibition for anticancer treatment.” Oncogene. 25: Ko Y.H. et al (2004). “Advanced cancers: eradication in all cases using 3-bromopyruvate therapy to deplete ATP. Biochem Biophys Res Commun. 324(1) Vidugiriene J. (2013) “Tools for cell metabolism: Bioluminescent NAD(P)/NAD(P)H-Glo Assays”. Promega Corp. Heiden, M.G.V. et al. (2009). “Understanding the Warburg effect: the metabolic requirements of cell proliferation” Science. 324(5930):

17 Mechanism Glycolysis generates 2 ATP, while oxidative phosphorylation generates 36-38ATP. Despite that, most cancer cells have been found to exclusively produce their energy via the glycolytic pathway regardless of the level of oxygen in the surroundings. A possible reason for this is that the glycolytic pathway enables the production of specific metabolites (NADPH) that decrease the presence of ROS species/oxidative stress. This is crucial for tumour cells as it allows them to proliferate indefinitely and survive in “unfavourable conditions: NADPH -- how? Show diagram of glycolysis Show entry of glucose and end products (in normal vs tumour cells) Oncogenes that promote the Warburg effect: Pyruvate kinase M1 and M2 → M2 higher in Warburg effect Normally, glycolysis occurs in anaerobic conditions

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