The effect of the PKM2-EGFR axis on glioblastoma multiforme growth

Abstract

Despite promising progress in treating various cancers with targeted therapy against the epidermal growth factor receptor (EGFR), its therapeutic effect in glioblastoma multiforme (GBM) is still poor. EGFR signalling has been shown to be susceptible to regulation by pyruvate kinase M2 (PKM2). Moreover, EGFR was revealed as a potential target of the PKM2 protein kinase. This suggests that PKM2 may interact with EGFR and EGFR-binding proteins. The aim of this work is to elucidate whether and by which molecular mechanisms PKM2 may regulate the activation of EGFR signalling and tumour growth. For this purpose, we generated stable PKM2 loss-of-function GBM cell lines by CRISPR-mediated knockout and performed rescue experiments in vitro with various PKM2 overexpression constructs. Western blots and sphere formation assays revealed that PKM2 overexpression enhances both EGFR phosphorylation and tumour cell growth upon EGF stimulation. To check whether the PKM2-induced tumour cell growth is mediated through EGFR signalling, we inhibited EGFR activity with its specific inhibitor erlotinib. To determine which post-translational modifications (PTMs) and features of PKM2 are essential for its function on EGFR, we generated a variety of PKM2 mutants using site-directed mutagenesis, and investigated their effect on EGFR activation. The results show that the PHD3 hydroxylation site on PKM2 (proline 403 and 408) and acetylation of PKM2 on lysine 433 is required for enhancing EGFR activation. This indicates that these modifications, known to be associated with the protein kinase activity of PKM2, may promote EGFR activation and tumour growth. To test this hypothesis, we investigated whether the deacetylase inhibitors trichostatin A (TSA) or nicotinamide (NAM) cause a PKM2-mediated upregulation of EGFR signalling. Unexpectedly, we found that a small fraction of acetylated PKM2 correlated with the TSA-induced activation of EGFR. The results of this work strengthen the notion that PKM2 is an important determinant of tumour growth in EGFR-driven cancer cells, and they suggest that PKM2 promotes EGFR activation via a positive feedback-loop mechanism. From a therapeutic point of view, our results highlight the PKM2 lysine 433 acetylation as a potential therapeutic target, indicating that deacetylase activators might in part act through the PKM2-EGFR axis in cancer cells.