A critical review of the role of M2PYK in the Warburg effect

Abstract

It is becoming generally accepted in recent literature that the Warburg effect in cancer depends on inhibition of M₂PYK, the pyruvate kinase isozyme most commonly expressed in tumors. We remain skeptical. There continues to be a general lack of solid experimental evidence for the underlying idea that a bottle neck in aerobic glycolysis at the level of M₂PYK results in an expanded pool of glycolytic intermediates (which are thought to serve as building blocks necessary for proliferation and growth of cancer cells). If a bottle neck at M₂PYK exists, then the remarkable increase in lactate production by cancer cells is a paradox, particularly since a high percentage of the carbons of lactate originate from glucose. The finding that pyruvate kinase activity is invariantly increased rather than decreased in cancer undermines the logic of the M₂PYK bottle neck, but is consistent with high lactate production. The "inactive" state of M₂PYK in cancer is often described as a dimer (with reduced substrate affinity) that has dissociated from an active tetramer of M₂PYK. Although M₂PYK clearly dissociates easier than other isozymes of pyruvate kinase, it is not clear that dissociation of the tetramer occurs in vivo when ligands are present that promote tetramer formation. Furthermore, it is also not clear whether the dissociated dimer retains any activity at all. A number of non-canonical functions for M₂PYK have been proposed, all of which can be challenged by the finding that not all cancer cell types are dependent on M₂PYK expression. Additional in-depth studies of the Warburg effect and specifically of the possible regulatory role of M₂PYK in the Warburg effect are needed.

Figure 1.

Synthetic data for two isozymes with different K_M values as a demonstration that reduced substrate affinity of an isozyme alone is not sufficient to predict activity in the cell. If substrate concentrations are sufficiently high in the cell (indicated by gray column) two isozymes with different affinity for substrate can give rise to the same level of activity. The Michaelis Menten equation was used to generate synthetic data in the left panel here and in Figures 2, and 4. Data in the left panel are hyperbolic (consistent with M₁PYK data) on a linear x-axis, but appear sigmoidal in this figure due to the logarithmic scale of the x-axis. Data in the middle panel were derived from the Hill equation (with a n_H value equal to 3). The resulting sigmoidal response is more representative of that found for M₂PYK. The panel on the right compares a hyperbolic response with a higher substrate affinity (representing M₁PYK) to a sigmoidal response with a lower substrate affinity (representing M₂PYK). As exemplified in this figure, the ideas discussed in the text can be represented by the simpler comparison of hyperbolic data. Therefore, all other figures in this review include only data generated from the simpler Michaelis Menten equation. Nonetheless, the reader should keep in mind that M₂PYK has a sigmoidal response.

Figure 2.

Synthetic Data for two isozymes with altered K_M and enzyme increased enzyme concentration and/or increased V_max activity to demonstrate the possibility that an isozyme with lower substrate affinity can give rise to higher activity even at sub-saturating substrate concentrations, if it also has either higher specific activity or if it is expressed at higher concentrations.

Figure 3.

A role for altered affinity of cancer M₂PYK for PEP in creating a new steady-state in Warburg metabolism. The top panel represents the activity response curves of either normal PYK or cancer M₂PYK. “Normal” PYK can be M₁PYK, LPYK, RPYK, or M₂PYK isolated from normal tissue. Concentrations of PEP in the cell are represented by red boxes and the respective PYK activity at that PEP concentration is represented by black dashes intersecting the red box (also marked with letters A and B at the beginning and the two steady-state PYK activities) and the response curve. The blue arrows trace changes related to the progression to cancer: 1) The PYK isozyme changes to cancer M₂PYK, which has little activity at the PEP concentration present in normal tissue. 2) PEP concentrations build up due to reduced PYK activity. 3) Due to increased substrate availability caused by PEP build up, the cancer form of M₂PYK has activity at high PEP. This condition maintains high levels of glycolytic intermediates and allows for high flux to lactic acid production. Green arrows connect the two steady-state PYK activities (A and B) with panels representing the respective metabolism. In these two lower panels, the thickness of arrows is intended to represent the relative concentrations of intermediates in the pathway indicated by the respective arrow. Left) When normal PYK is expressed, normal metabolism is at a steady-state and in the presence of O₂, that steady-state flux of carbon from sugar into the mitochondria for oxidative phosphorylation. Right) When cancer M₂PYK is expressed, a new steady-state flux is established that includes higher concentrations of glycolytic intermediates. This includes high rates of flux through the cancer PYK reaction and increased production of lactic acid. We anticipate higher flux through the mitochondria as well, thus generating higher levels of reactive oxygen species (ROS) and oxidative stress. However, the increased concentration of glucose 6-phosphate in the new steady-state condition drives increased flux through the pentose phosphate pathway due to substrate availability. This change produces increased NADPH. NADPH is needed to regenerate glutathione, which counteracts the increased oxidative stress, thus allowing cancer cells to live with higher intracellular ROS concentrations.

Figure 4.

A theoretical response curve of normal PYK and cancer M₂PYK, if “inhibited” cancer M₂PYK involves reduced k_cat. Here 5% activity compared to Normal PYK is used as an example, although the points in the text are more relevant if inhibition removes all catalytic activity. “Normal” PYK can be M₁PYK, LPYK, RPYK, or M₂PYK isolated from normal tissue. In the absence of changes in M₂PYK expression, the only way to increase flux through the PYK reaction would be to activate the M₂PYK protein, thus requiring a dynamic control of M₂PYK to achieve both inhibition for build-up of glycolytic intermediates and to allow sufficient flux through the PYK reaction to generate high levels of lactic acid. The need for that dynamic regulation might predict oscillating flux of high glycolytic intermediate dispersed with periods of high lactic acid production.