More proof that Warburg never claimed cancer was caused from a lack of oxygen

 Commentary by Daniel D. Billadeau, PhD, and Andrei V. Ougolkov, MD, PhD, Mayo Clinic, Rochester, Minn., on:

Metabolic sensitivity of pancreatic tumour cell apoptosis to glycogen phosphorylase inhibitor treatment

W.-N.P. Lee, P. Guo, S. Lim, S. Bassilian, S.T. Lee, J. Boren, M. Cascante, V.L.W. Go, L.G. Boros
Br J Cancer, 2004;91:2094-2100

PancreasWeb 25/02/05 

More than seven decades ago the German biochemist Otto Warburg, M.D., made the seminal observation that most tumors relied on anaerobic glycolysis even in the presence of abundant oxygen, a phenomenon now referred to as the "Warburg effect" (1). In fact, tumor cells are notorious for their consumption of glucose, a requirement to sustain their high proliferative rate and increased need for macromolecular synthesis. This characteristic of tumor cells has been confirmed and exploited by clinical radiologists using 2-fluoro-2-deoxy-D-glucose-positron emission tomography (PET) imaging. The switch from aerobic to anaerobic metabolism, while not being at all energy efficient (anaerobic glycolysis only realizes 2 ATP molecules per glucose molecule, whereas an additional 36 molecules could be generated during aerobic oxidative phosphorylation by oxidizing pyruvate to HCO3), is compensated by an increase in glucose uptake by tumor cells (2). Interestingly, while the glycolytic phenotype would appear to confer a significant competitive disadvantage on the tumor cells, recent mathematical modeling of the tumor-host interface suggests that the switch to anaerobic metabolism actually favors tumor cell invasion into the surrounding normal parenchyma where the tumor can outcompete normal cells for available resources (3). Moreover, the anaerobic glycolytic phenotype decreases the extracellular pH surrounding the tumor (due to the conversion of pyruvate to lactate by lactate dehydrogenase and increased excretion of protons through upregulated Na+/H+ antiport and other membrane transporters) resulting in increased p53-dependent apoptosis of normal cells (most tumor cells are p53 mutant and can survive in such a low pH environment), enhanced angiogenesis, decreased immune cell responses, degradation of the interstitial matrix and loss of intercellular gap junctions. Taken together, all of these events contribute to and favor tumor proliferation and metastasis.

In addition to D-glucose, multiple other carbohydrates can ultimately "feed" the glycolytic sequence to undergo energy-yielding degradation, or to become part of the ribose phosphate (DNA and RNA generation) and acetyl-CoA precursor synthesis pathways leading to fatty acid metabolism. One of these polysaccharides, glycogen, is the main storage polysaccharide comprised of branched D-glucose. The breakdown of glycogen through the action of glycogen phosphorylase (GP) and inorganic phosphate yields glucose-1-phosphate that can be converted to glucose-6-phosphate (G6P). G6P can then be shunted in to either the ribose pathway, acetyl-CoA-fatty acid synthesis pathways or used directly for glycolytic metabolism. Boros and colleagues reasoned that limiting glycogen breakdown in times of need (i.e. for the synthesis of macromolecules such as DNA, RNA, and fatty acids during S-phase progression) might lead to cell cycle arrest and apoptosis (4).

Using the GP specific inhibitor CP-320626, the Authors demonstrate a dose-dependent decrease in cellular proliferation and increase in apoptosis in the MIA pancreatic tumor cell line. Furthermore, CP-320626 was significantly better in inhibiting proliferation and oxidative metabolism in MIA cells compared to 2-deoxy-D-glucose, a glucose metabolite that poisons glycolysis and pentose cycle substrate flow. In addition, using [1,2-13C2]glucose stable isotope-based dynamic metabolic profiling (SIDMAP), which allows the quantification and tracking of carbon atoms among major metabolic and macromolecular synthesis pathways, the authors demonstrate a dose-dependent decrease in CO2 release, pentose and de novo fatty acid synthesis. Moreover, the inhibition of GP was found to affect the synthesis of macromolecules required for cell cycle entry and thus, cellular proliferation. Importantly, these decreases occurred at drug doses that did not significantly induce apoptosis, but did alter cell growth. Significantly, glucose uptake and lactate release were not inhibited by CP-320626 treatment, suggesting that MIA cells respond to this drug while maintaining both glucose uptake and viability.

From these data the Authors contend that the defective ribose and fatty acid synthesis due to GP inhibition are the likely causes of cell cycle arrest and apoptosis in MIA cells (and may be extended to other rapidly proliferating tumor cells). While the SIDMAP data would support this hypothesis, the authors do acknowledge that many tumor cells have a depleted glycogen pool. In fact, in previous studies, the Authors have shown that glycogen synthesis from media glucose was not detected in cultured MIA cells (5). These observations are hard to reconcile in light of the fact that increased glycogen was not demonstrated in CP-320626-treated MIA cells. While the Authors claim that the GP inhibitor CP-320626 is more specific than flavopiridol in specifically targeting GP, it was never demonstrated which GP MIA cells express (brain, liver or muscle), if they express GP at all. This question is crucial, because in the study of 59 cell lines, Schnier et al., 2003 showed that only cells expressing high levels of brain GP were growth inhibited by a GP inhibitor, whereas cells expressing low levels of brain GP were not affected by the drug (6). In fact, "off-targeting" is always a concern when using pharmacological inhibitors, and thus, the use of RNA interference toward the GP expressed in MIA cells would be invaluable to confirm the drug effect identified using SIDMAP. Lastly, it has been reported that the expression of glycogen is significantly decreased in poorly differentiated cancer cells that show the highest proliferation rate. In fact, MIA cells are a poorly differentiated and highly proliferative pancreatic cancer cell line. In contrast, high glycogen levels were reported in slow growing well-differentiated gastric cancers where brain GP is commonly expressed (7) and a negative correlation was demonstrated between the glycogen level and proliferation index in human colorectal carcinomas (8). Thus it becomes an important specificity issue to determine how the GP inhibitor is affecting these metabolic pathways in the MIA cells.

We agree that the use of GP inhibitors in the therapy of pancreatic cancer may improve the host glucose status via skeletal muscle glycogen storage because GP is active in the muscle of diabetic pancreatic cancer patients, but this remains to be investigated. However, while the exact mode of specific inhibition of GP in pancreatic cancer cells remains to be determined, the Authors provide compelling evidence that GPs play an important role in tumor cell proliferation by regulating macromolecular and fatty acid synthesis required for cell cycle progression. Since glucose uptake is not impaired in GP-inhibited cells, it remains feasible that glycogen, although limited in tumor cells, is a critical glucose reservoir required for optimal tumor cell proliferation. Although Dr. Warburg's seminal observations have gone unheeded for several decades, many groups, and biotechnology companies, have become interested in the altered metabolic pathways that tumor cells exploit, and see an 'Achilles heel' that can be targeted for therapeutic intervention, and quite possibly, tumor cell eradication.


References

1. Warburg, O. The metabolism of tumors. London: Constable Press, 1930

2. Garber, K. Energy boost: The Warburg effect returns in a new theory of cancer. JNCI 2004;96:1805-180

3. Gatenby, R.A. and Gawlinski, E.T. The glycolytic phenotype in carcinogenesis and tumor invasion: insights through mathematical models. Cancer Res 2003;63:3847-3854

4. Lee, W.-N.P., Guo, P., Lim, S., Bassilian, S., Lee, S.T., Boren, J., Cascante, M., Go, V.L.W., and Boros, L.G. Metabolic sensitivity of pancreatic tumor cell apoptosis to glycogen phosphorylase inhibitor treatment. Br J Cancer 2004;91:2094-2100.

5. Boros L.G., Bassilian S., Lim S., Lee, W.N. Genistein inhibits nonoxidative ribose synthesis in MIA pancreatic adenocarcinoma cells: a new mechanism of controlling tumor growth. Pancreas 2001;22:1-7.

6. Schnier, J.B., Nishi, K., Monks, A., Gorin, F.A., Bradbury, E.M. Inhibition of glycogen phosphorylase (GP) by CP-91,149 induces growth inhibition correlating with brain GP expression. Biochem Biophys Res Commun 2003;309:126-134

7. Namiot, Z., Stasiewicz, J., Szalaj, W., Skwarski, L., Jodczyk, J., Gorski, J. Gastric cancer with special references to WHO and Lauren's classifications: glycogen and triacylglycerol concentrations in the tumor. Neoplasma 1989;36:363-368

8. Takahashi, S., Satomi, A., Yano, K., Kawase, H., Tanimizu, T., Tuji, Y., Murakami, S., Hirayama, R. Estimation of glycogen levels in human colorectal cancer tissue: relationship with cell cycle and tumor outgrowth. J Gastroenterol 1999;34:474-480