Isocitrate dehydrogenase mutations rescue lactate dehydrogenase-deficient cells from cell death under hypoxia

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  • Presentation type: IC - Undergraduate Students
  • Track: 5. Metabolism and Glycobiology
  • Keywords: Glioma; metabolism; Lactate dehydrogenase; isocitrate dehydrogenase; hypoxia;
  • 1 Universidade Federal do Rio de Janeiro

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Abstract

INTRODUCTION AND OBJECTIVES: Isocitrate Dehydrogenase (IDH) mutations play a significant role in glioma tumorigenesis by disrupting cellular metabolism. It is known that these mutations change the epigenetic landscape and generate cellular heterogeneity, which promotes tumor development. However, it is not yet fully understood how cells bearing mutated-IDH (mtIDH) copies are selected and favored during tumor progression in a pre-malignant context. Lactate is a metabolite generated by Lactate Dehydrogenase (LDH) from pyruvate and ensures cell survival under hypoxic conditions especially in the central nervous system. Interestingly, mtIDH gliomas lack LDH expression, which led us to hypothesize whether such mutations could confer these cells the ability to survive under hypoxic conditions even in the absence of lactate in a pre-malignant context. Therefore, we aim to demonstrate whether: (1) mtIDH gliomas lack LDH expression; (2) knock-down of LDH expression in wild-type (wtIDH) glioma cells and murine astrocytes leads to cell death under hypoxic conditions; (3) ectopic mtIDH expression in LDH-deficient cells can prevent cell death under hypoxia. MATERIAL AND METHODS: To demonstrate LDH expression in wt and mtIDH gliomas, we performed immunohistochemistry (IHC) and WB in patient biopsies. We knocked-down LDH expression in wtIDH glioma cell lines using siRNA or CRISPR. Ectopic mtIDH expression was induced by plasmid transfection in LDH-defficient cells. Cell death was measured by Trypan Blue counting and MTT assay. RESULTS AND CONCLUSION: We identified in a set of 12 patients that mtIDH gliomas lack LDH expression in contrast to wtIDH gliomas, confirmed by IHC and WB. Murine astrocytes and GBM95 cell line that had their LDH expression knocked-down through siRNA died after 24 and 48 hours under hypoxic conditions. This observation suggests that LDH expression and subsequent lactate production are essential for cell survival in hypoxia. We therefore introduced ectopic mtIDH expression into LDH-deficient cells and observed that this expression is sufficient to rescue them from cell death under hypoxic conditions. These preliminary results suggest that mtIDH rescues LDH-deficient cells from cell death under low-oxygen conditions.

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Author

Victor Wanderkoke

Agradecemos, 
Sobre as questões:
O estudo a respeito dessa via alternativa de metabolismo energético pode permitir tanto um aumento da especificidade do prognóstico, ao estimar a malignidade dos tumores baseando-se na presença ou não da mutação na IDH, ou futuramente no desenvolvimento de terapias que manipulem essa via e assim, o próprio metabolismo dos tumores, com o propósito de diminuir sua malignidade.

Author

Victor Wanderkoke

Agradecemos os elogios e os questionamentos!
A respeito das curiosidades:

1. Nas nossas células não foi verificada a expressão da isoforma B da lactato desidrogenase. A LDHB foi observada em análises de bioinformática apenas.

2.As células foram colocadas em estufa de hipóxia e manipuladas em fluxo laminar comum.

3.Desde já agradecemos a sugestão.
Sobre os resultados, ainda não é possível dizer exatamente a causa dessa variação. Todavia, pensamos que, os glioblastomas possuindo maior expressão de LDH tendem a responder de maneira mais evidente ao silenciamento dessa enzima. 

4.O nosso trabalho está mais focado num contexto mais pré-maligno do que no próprio tumor já desenvolvido onde encontramos poucos focos de hipóxia. Mas acreditamos que, a partir da descoberta dos mecanismos de como a mutação em IDH pode afetar o metabolismo energético, especialmente no que diz respeito à produção e secreção de lactato, poderemos explorar novos alvos terapêuticos no futuro utilizando estas vias