The non-metabolic functions of folate enzyme MTHFD2 in glioblastoma multiforme pathogenesis

Abstract

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour in adults. Despite the aggressive standard of care, patients with GBM only have a median survival of less than two years. The recurrence of this incurable disease is almost inevitable. A better understanding of the molecular basis of GBM pathogenesis and the discovery of novel therapeutic targets are needed. Reprogrammed metabolism, an emerging hallmark of cancers, supports tumour cell proliferation and survival even under unfavourable conditions. Dysregulation of metabolic enzymes has been implicated in cancer initiation and progression. Among the various metabolic pathways, the one-carbon folate metabolism is a critical one and has been exploited as an anti-cancer target for decades. In several cancer types, studies have demonstrated the aberrant expression and oncogenic feature of an essential mitochondrial folate enzyme, namely, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Interestingly, this enzyme has recently been considered to have non-metabolic “moonlighting” functions in cancer biology. However, the functional roles of MTHFD2 in GBM pathogenesis remain unclear. In the present study, by analysing microarray databases and clinical specimens, it was demonstrated that MTHFD2 was frequently upregulated in different grades of glioma including GBM, and was particularly higher in the G-CIMP GBM subtype. Knockdown of MTHFD2 significantly suppressed malignant features of GBM cells in vitro – including cell proliferation, clonogenicity, migration and invasion – and induced cellular apoptosis. MTHFD2 depletion impeded GBM growth in both subcutaneous and orthotopic in vivo models. Overexpression of MTHFD2 promoted malignant behaviours of GBM cells in vitro and accelerated tumour growth in vivo. To gain mechanistic insights, this study interrogated the consequences of MTHFD2 depletion in GBM cells from both the metabolic and the putative non-metabolic perspectives. Central-carbon metabolites profiling showed that knockdown of MTHFD2 broadly suppressed essential metabolism pathways, including glycolytic activity, the pentose phosphate pathway, and the tricarboxylic acid cycle. Gene set enrichment analysis (GSEA) indicated that MTHFD2 might be associated with the unfolded protein response (UPR) process and translation initiation. Immunoblotting assays confirmed that MTHFD2 loss-of-function significantly activated a UPR arm, namely the PERK/eIF2α pathway. Nascent protein labelling assay showed that MTHFD2 depletion significantly suppressed protein synthesis. PERK inhibitor rescued the apoptosis induction and translation inhibition caused by MTHDF2 depletion, indicating an essential role of the PERK/eIF2α pathway in MTHFD2-mediated anti-cancer effects. In addition, ChIP-PCR assay showed that MTHFD2 might be transcriptionally upregulated by a master transcription factor c-Myc. The present study investigates the functions of MTHFD2 in GBM pathogenesis with a primary focus on the non-enzymatic aspect. The putative oncogenic role of MTHFD2 is validated. Apart from the influence on metabolic re-programming, this enzyme also has essential non-metabolic functions in GBM. The role of MTHFD2 in modulating UPR and protein translation provides novel insights into its “moonlighting” functions. The potent anti-tumour effects upon MTHFD2 depletion implicates it as a promising therapeutic target. Further efforts may be devoted to the evaluation of the translational implication of MTHFD2-targeted therapy in GBM, the mechanism of MTHFD2-mediated cancer transformation or progression, and its broader non-metabolic functions.