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No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis - PubMed

No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis

Katharina Bluemlein et al. Oncotarget. 2011 May.

Abstract

The Warburg effect describes the circumstance that tumor cells preferentially use glycolysis rather than oxidative phosphorylation for energy production. It has been reported that this metabolic reconfiguration originates from a switch in the expression of alternative splice forms (PKM1 and PKM2) of the glycolytic enzyme pyruvate kinase (PK), which is also important for malignant transformation.However, analytical evidence for this assumption was still lacking. Using mass spectrometry, we performed an absolute quantification of PKM1 and PKM2 splice isoforms in 25 human malignant cancers, 6 benign oncocytomas, tissue matched controls, and several cell lines. PKM2 was the prominent isoform in all analyzed cancer samples and cell lines. However, this PKM2 dominance was not a result of a change in isoform expression, since PKM2 was also the predominant PKM isoform in matched control tissues. In unaffected kidney, lung, liver, and thyroid, PKM2 accounted for a minimum of 93% of total PKM, for 80% - 96% of PKM in colon,and 55% - 61% of PKM in bladder. Similar results were obtained for a panel of tumor and non-transformed cell lines, where PKM2 was the predominant form.Thus, our results reveal that an exchange in PKM1 to PKM2 isoform expression during cancer formation is not occurring, nor do these results support conclusions that PKM2 is specific for proliferating, and PKM1 for non-proliferating tissue.

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Figures

Figure 1
Figure 1. Absolute quantification of PKM1 and PKM2 splice forms in tissue extracts

a. Yeast expressing human PKM1 (PKM1-yeast, left panel) and human PKM2 (PKM2-yeast, right panel) were analyzed by nanoflow liquid chromatography/multiple reaction monitoring (LC-MRM) to quantify a PKM1 and a PKM2 specific peptide as well as a peptide which is specific for both isoform (PKMall) (lower chromatograms). Matching heavy isotope labeled peptides (AQUA peptides) were included in every sample and used for quantification (upper chromatograms, please note that they are displaced on the Y axis for better illustration). The determined concentrations were 3.3 fmol/μg protein for PKM1, and 19.3 fmol/μg protein for PKM2 in yeast. b. Exemplary chromatogram for a human tissue sample, quantification of PKM1 and PKM2 in bladder tissue by LC-MRM. The analysis was performed as in (a). Absolute and relative values determined in human tissue are given in Table 1. c. Plot of the concentrations obtained for PKM1 plus PKM2 against the concentration of a peptide specific for both isoforms [PKMall]. The obtained concentrations show linear correlation (R² > 0.97)

Figure 2
Figure 2. Accuracy of PKM1 and PKM2 quantification

Differently concentrated digests of PKM1-yeast (n = 11) and PKM2-yeast (n = 12) were injected, and the peptides PKM1, PKM2, and PKMall quantified as well as their corresponding AQUA standards analyzed by LC-MRM. Shown is a correlation plot of the concentration of the specific peptide (PKM1 or PKM2) and the PKMall peptide, concentrations are given in absolute values (fmol).

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