Isotype‐specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation

Abstract

Tumor cells express the glycolytic regulator pyruvate kinase subtype M2 (M2-PK), which can occur in a tetrameric form with high affinity to its substrate phosphoenolpyruvate (PEP) and a dimeric form with a low PEP affinity. The transition between both conformations contributes to the control of glycolysis and is important for tumor cell proliferation and survival. Here we targeted M2-PK by synthetic peptide aptamers, which specifically bind to M2-PK and shift the isoenzyme into its low affinity dimeric conformation. The aptamer-induced dimerization and inactivation of M2-PK led to a significant decrease in the PK mass-action ratio as well as ATP:ADP ratio in the target cells. Furthermore, the expression of M2-PK-binding peptide aptamers moderately reduced the growth of immortalized NIH3T3 cell populations by decelerating cell proliferation, but without affecting apoptotic cell death. Moreover, the M2-PK-binding peptide aptamers also reduced the proliferation rate of human U-2 OS osteosarcoma cells. In the present study, we developed the first specific inhibitors of the pyruvate kinase isoenzyme type M2 and present evidence that these inhibitors moderately decelerate tumor cell proliferation. ' 2008 Wiley-Liss, Inc.Key words: tumor metabolism; peptide aptamer; proliferation; pyruvate kinase Tumorigenesis is characterized by an increase in glycolytic enzyme activities as well as distinct changes in the glycolytic isoenzyme equipment. 1-3 A key glycolytic enzyme which is consistently altered in tumor cells is pyruvate kinase (EC 2.7.1.40), catalyzing the dephosphorylation of phosphoenolpyruvate (PEP) to pyruvate, which significantly contributes to net adenosine triphosphate (ATP) production within the glycolytic pathway. Depending on the metabolic functions of various tissues, different isoenzymes of pyruvate kinase are expressed. 4 The pyruvate kinase isoenzyme M1 (M1-PK) is characteristic for tissues with high energy turnover (muscle and brain), whereas the pyruvate kinase isoform L (L-PK) is found in tissues with gluconeogenesis, such as liver. When quiescent cells re-enter the cell cycle, as during tumorigenesis, the tissue-specific isoenzymes disappear and the pyruvate kinase isoenzyme type M2 (M2-PK) is expressed. 3,4 According to the different metabolic functions, M1-PK has the highest affinity to its substrate PEP, whereas the PEP affinity of M2-PK depends on its quaternary structure. M2-PK occurs as a tetrameric form characterized by a high affinity to its substrate PEP which is highly active at physiological PEP concentrations and as a dimeric form with low PEP affinity which is nearly inactive under physiological conditions. 3 The tetrameric but not the dimeric form of M2-PK is associated with other glycolytic enzymes as well as with lactate dehydrogenase (LDH), nucleotide diphosphate kinase and adenylate kinase, within a large complex, referred to as the glycolytic enzyme complex. 5-7 A high amount of the tetrameric form of M2-PK correlates with a relative high PK mass-action ratio ([pyruvate] AE[ATP])...

Targeting cancer metabolism: a therapeutic window opens

“…Selection for a less active form of pyruvate kinase may help divert glucose metabolites upstream of pyruvate kinase into biosynthetic pathways 5,97,100 . Efforts have been made to selectively inhibit PKM2 101,102 . Peptide aptamers that promote the less active form of pyruvate kinase have been shown cause energy stress and cell death in cultured cancer cells 101 , and more modest effects were observed using small molecule inhibitors of PKM2 102 .…”

“…Efforts have been made to selectively inhibit PKM2 101,102 . Peptide aptamers that promote the less active form of pyruvate kinase have been shown cause energy stress and cell death in cultured cancer cells 101 , and more modest effects were observed using small molecule inhibitors of PKM2 102 . Targeting PKM2 with shRNA can slow cell proliferation in cell culture 99 , however these cells retain the ability to proliferate even with the near complete absence of pyruvate kinase activity.…”

Death-associated protein kinase increases glycolytic rate through binding and activation of pyruvate kinase

“…Altogether we suggest that DAPk can only bind and activate a sub-population of PKM2 associated with the cytoskeleton. Multi-protein complexes containing glycolytic proteins are known to be formed in order to increase the flux through consecutive enzymatic reactions, and PK is known to be localized to such glycolytic enzyme complex (Mazurek et al, 2001;Spoden et al, 2008). Additionally, localization to F-actin has been correlated with activation of other glycolytic enzymes (Real-Hohn et al, 2010).…”

“…As biosynthesis of nucleotides, through the pentose phosphate pathway, and of amino acids, is required for cell growth (Deberardinis et al, 2008), activation of PK by DAPk could potentially impede cellular proliferation. Notably, the correlation between PKM2 activity and cell proliferation is still debated, as inhibition of PKM2 was shown to have both pro-and anti-proliferative effects (Spoden et al, 2008;Hitosugi et al, 2009). Here expression of inactive DAPk led to a reduction in cell proliferation rate.…”

“…Cells were harvested after 48 h, stained with trypan blue and counted with an automated cell counter (Countess, Invitrogen) to determine total cell count, and live and dead cell counts. Population doubling time (PDT) was calculated according to the equation (Spoden et al, 2008):…”