MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma

Our plain-English summary of MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma, by M. Tomasetti, F. Monaco, N. Manzella, et al (Oncotarget, 2016).

The study

• Authors: M. Tomasetti, F. Monaco, N. Manzella, J. Rohlena, K. Rohlenova, S. Staffolani, S. Gaetani, V. Ciarapica, M. Amati, M. Bracci, M. Valentino, J. Goodwin, M. Nguyen, J. Truksa, M. Sobol, P. Hozak, L.F. Dong, L. Santarelli, J. Neuzil
• Journal: Oncotarget
• Published: 2016;7(24):36338-36352
• Cited by: 40 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Autophagy favors both cell survival and cancer suppression, and increasing evidence reveals that microRNAs (MIRs) regulate autophagy. Previously we reported that MIR126 is downregulated in malignant mesothelioma (MM). Therefore, we investigated the role of MIR126 in the regulation of cell metabolism and autophagy in MM models. We report that MIR126 induces autophagic flux in MM cells by downregulating insulin receptor substrate-1 (IRS1) and disrupting the IRS1 signaling pathway. This was specific to MM cells, and was not observed in non-malignant cells of mesothelial origin or in MM cells expressing MIR126-insensitive IRS1 transcript. The MIR126 effect on autophagy in MM cells was recapitulated by IRS1 silencing, and antagonized by IRS1 overexpression or antisense MIR126 treatment. The MIR126-induced loss of IRS1 suppressed glucose uptake, leading to energy deprivation and AMPK-dependent phosphorylation of ULK1. In addition, MIR126 stimulated lipid droplet accumulation in a hypoxia-inducible factor-1α (HIF1α)-dependent manner. MIR126 also reduced pyruvate dehydrogenase kinase (PDK) and acetyl-CoA-citrate lyase (ACL) expression, leading to the accumulation of cytosolic citrate and paradoxical inhibition of pyruvate dehydrogenase (PDH) activity. Simultaneous pharmacological and genetic intervention with PDK and ACL activity phenocopied the effects of MIR126. This suggests that in MM MIR126 initiates a metabolic program leading to high autophagic flux and HIF1α stabilization, incompatible with tumor progression of MM. Consistently, MIR126-expressing MM cells injected into immunocompromised mice failed to progress beyond the initial stage of tumor formation, showing that increased autophagy has a protective role in MM.

In plain English

This laboratory study tested what the microRNA MIR126 does to cell metabolism and autophagy in malignant mesothelioma (MM). The authors used human MM cell lines (sarcomatoid H28 and epithelioid IstMes2) and non-malignant mesothelial Met5A cells, transfected with a MIR126-carrying plasmid or an empty plasmid, plus a xenograft experiment in Balb-c nu/nu mice (6 per group). MIR126 increased acid vesicle formation and autophagic flux in H28 cells but not in Met5A cells or in IstMes2 cells, which carry a truncated IRS1 lacking the MIR126 binding site. Silencing IRS1 reproduced the effect; IRS1 overexpression or antisense MIR126 reversed it. In H28 cells MIR126 raised GLUT-4 yet reduced glucose uptake, activated AMPK and ULK1 phosphorylation at Ser-555, produced lipid droplets (eliminated by HIF1alpha silencing), lowered PDK with paradoxically reduced PDH activity and raised pyruvate. In H28 cells, combining ACL silencing with the PDK inhibitor DCA likewise lowered PDH activity and raised pyruvate, alongside acid vesicle formation, lipid droplet accumulation and reduced proliferation and colony formation. In mice, empty-plasmid H28 tumours grew while MIR126 tumours were resorbed by day 10.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study using human mesothelioma and non-malignant mesothelial cell lines (transfection, imaging, western blotting, gene expression microarray) with a mouse xenograft experiment
• Who was studied: Human cell lines: non-malignant mesothelial Met5A, sarcomatoid MM H28, and epithelioid IstMes2; plus Balb-c nu/nu mice, 6 per experimental group
• Exposure: Transfection with a MIR126-carrying plasmid versus empty plasmid; also IRS1 silencing or overexpression, antisense MIR126, HIF1alpha and ACL shRNA, and drug treatments 3MA (50 µM), chloroquine (10 µM) and dichloroacetate (20 mM)
• Main results:
  • In MM H28 cells transfected with MIR126, acid vesicles increased compared with empty-plasmid cells; this was reversed by anti-MIR126, and no such effect occurred in non-malignant Met5A cells
  • The ratio of red to green punctae (lysosomal delivery of autophagosomes) rose in MIR126-expressing H28 cells but was unchanged in Met5A cells
  • In MIR126-transfected MM cells, SQSTM1 was significantly upregulated while BECN1 showed no significant change, and LC3II was reduced rather than accumulated
  • IstMes2 cells, which carry truncated IRS1 lacking the MIR126 binding site, were not sensitive to MIR126-induced autophagy
  • MIR126 raised GLUT-4 in Met5A and more so in H28 cells, but reduced GLUT-4 in IstMes2 cells; in H28 cells glucose uptake fell despite high GLUT-4, and anti-MIR126 restored uptake
  • In MIR126-transfected H28 cells AMPK phosphorylation increased, with increased ULK1 phosphorylation at Ser-555 and decreased phosphorylation at Ser-757; this was not seen in Met5A or IstMes2 cells
  • MIR126-transfected H28 cells accumulated lipid droplets (Oil Red O and TEM) with swollen mitochondria; this was not seen in IstMes2 cells and was prevented by anti-MIR transfection
  • Silencing HIF1alpha in MIR126-transfected H28 cells removed the lipid droplets but did not change acid vesicle formation or the autophagy signalling pathway
  • MIR126 downregulated PDK roughly 50-fold in MM cells yet PDH activity fell and pyruvate rose in H28 cells
  • In H28 cells, ACL silencing reduced PDH activity, as did ACL silencing with DCA, and pyruvate rose, resembling MIR126-transfected H28 cells
  • These metabolic changes were accompanied by acid vesicle formation and lipid droplet accumulation, with reduced proliferation and colony formation; the authors reported that combined PDK and ACL inhibition recapitulated MIR126's effects on tumorigenic potential and autophagy while single treatment had little or no effect
  • In mice, empty-plasmid H28pRS cells formed progressively growing tumours while MIR126-transfected H28 cells formed small tumours that were resorbed by day 10 (6 mice per group)
• The authors' conclusion: The authors concluded that MIR126 drives a complex metabolic reprogramming of MM cells, including autophagy activation after disruption of IRS signalling, downregulation of PDK and ACL activity, citrate accumulation and HIF1alpha-dependent lipid droplet formation, and that these effects inhibit tumour progression, indicating a protective role for enhanced autophagic flux in MM
• Limitations the authors state:
  • The authors stated that the mechanism by which ACL inhibition keeps the PDH complex inactive despite loss of PDK control is not clear, and offered only a possible explanation
  • The authors noted that 3MA, used as an autophagy inhibitor, can itself promote autophagic flux under certain conditions, complicating interpretation of that experiment

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, Hypoxia.

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