Targeting SIRT3 sensitizes glioblastoma to ferroptosis by promoting mitophagy and inhibiting SLC7A11

Abstract

Glioblastoma (GBM) cells require large amounts of iron for tumor growth and progression, which makes these cells vulnerable to destruction via ferroptosis induction. Mitochondria are critical for iron metabolism and ferroptosis. Sirtuin-3 (SIRT3) is a deacetylase found in mitochondria that regulates mitochondrial quality and function. This study aimed to characterize SIRT3 expression and activity in GBM and investigate the potential therapeutic effects of targeting SIRT3 while also inducing ferroptosis in these cells. We first found that SIRT3 expression was higher in GBM tissues than in normal brain tissues and that SIRT3 protein expression was upregulated during RAS-selective lethal 3 (RSL3)-induced GBM cell ferroptosis. We then observed that inhibition of SIRT3 expression and activity in GBM cells sensitized GBM cells to RSL3-induced ferroptosis both in vitro and in vivo. Mechanistically, SIRT3 inhibition led to ferrous iron and ROS accumulation in the mitochondria, which triggered mitophagy. RNA-Sequencing analysis revealed that upon SIRT3 knockdown in GBM cells, the mitophagy pathway was upregulated and SLC7A11, a critical antagonist of ferroptosis via cellular import of cystine for glutathione (GSH) synthesis, was downregulated. Forced expression of SLC7A11 in GBM cells with SIRT3 knockdown restored cellular cystine uptake and consequently the cellular GSH level, thereby partially rescuing cell viability upon RSL3 treatment. Furthermore, in GBM cells, SIRT3 regulated SLC7A11 transcription through ATF4. Overall, our study results elucidated novel mechanisms underlying the ability of SIRT3 to protect GBM from ferroptosis and provided insight into a potential combinatorial approach of targeting SIRT3 and inducing ferroptosis for GBM treatment.

Fig. 1. High expression of SIRT3 in GBM predicted poor prognosis.

A UALCAN online analysis (https://ualcan.path.uab.edu/) revealed higher expression of SIRT3 in GBM tissues compared with normal brain tissues. B UALCAN online analysis revealed that high expression of SIRT3 in GBM predicts poor prognosis. C Representative results of tissue microarray assay showing that SIRT3 expression was higher in GBM tissues. D Quantitative results for SIRT3 expression in tissue microarray. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2. SLC7A11 expression was higher in GBM tissues than in normal brain tissues and highly positively correlated with SIRT3 expression.

A Representative results of tissue microarray showing that SLC7A11 expression was higher in GBM patients. B Quantitative results of SLC7A11 expression from tissue microarray. *p < 0.05, ****p < 0.0001. C Correlation analysis revealed SIRT3 expression was highly correlated with SLC7A11 expression in GBM tissues.

Fig. 3. Erastin and RSL3 treatment led to increased SIRT3 protein expression during GBM cell ferroptosis.

A Erastin and RSL3 treatment induced GBM cell death in a dose-dependent manner. B Erastin and RSL3 treatment induced typical ferroptosis-related morphology changes in mitochondria, as shown by transmission electron microscopy in GBM cells, including smaller mitochondria, higher density of the mitochondrial membrane, and reduced mitochondrial cristae numbers. C Erastin and RSL3 treatment increased expression of ferroptosis-related proteins in GBM cells. D Erastin and RSL3 treatment increased SIRT3 protein expression. E Erastin and RSL3 treatment did not affect SIRT3 mRNA expression. IRP1, iron regulatory protein 1; HO-1, heme oxygenase 1; TFR1, transferrin 1; SLC7A11, solute family 7 member 11.

Fig. 4. Inhibition of SIRT3 promoted RSL3-induced U251 and GBM4# cell ferroptosis in vitro.

A Inhibition of SIRT3 expression and activity in U251 cells. B Inhibition of SIRT3 expression and activity promoted RSL3-induced U251 cell death, which was partially rescued by DFO and totally rescued by NAC in comparison with control cells. C Inhibition of SIRT3 expression and activity promoted RSL3-induced U251 cell death, which was partially rescued by Fer-1. D Inhibition of SIRT3 expression and activity promoted LPO accumulation in U251 cells. E Inhibition of SIRT3 expression and activity promoted MDA accumulation in U251 cells. F Inhibition of SIRT3 expression and activity in GBM#4 cells. G Inhibition of SIRT3 expression and activity promoted RSL3-induced GBM#4 cell death. H Inhibition of SIRT3 expression and activity promoted LPO accumulation in U251 cells. I Inhibition of SIRT3 expression and activity promoted MDA accumulation in U251 cells. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig. 5. Inhibition of SIRT3 promoted RSL3-induced U251 cell ferroptosis in vivo.

A 3-TYP + RSL3 treatments impeded GBM growth in xenograft mice, in comparison with the control and 3-TYP groups. B Quantitative results for tumor weights between the control, 3-TYP and 3-TYP + RSL3 treated groups. C MDA level was increased in 3-TYP and 3-TYP + RSL3 treated groups. D GSH/GSSG ratio was decreased in 3-TYP and 3-TYP + RSL3 treated groups. E Western blotting showed that 3-TYP treatment reduced SLC7A11 expression, while RSL3 treatment induced an adaptive increase in SLC7A11 in GBM tumor tissues in xenograft mice. F Immunohistochemical results showed that 3-TYP treatment had no effect on SIRT3 expression, but decreased SLC7A11 expression in tumor tissues in xenograft mice. Combination 3-TYP + RSL3 treatment increased both SIRT3 and SLC7A11 expression compared with 3-TYP treatment only.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 6. Inhibition of SIRT3 induced mitochondrial ROS and Fe²⁺ accumulation in U251 cells.

A Mitochondrial Fe²⁺ level increased after SIRT3 inhibition. B FACS analysis of Mito-FerroGreen and quantitative results after SIRT3 inhibition. C Mitochondrial ROS level increased after SIRT3 inhibition.

Fig. 7. Mitophagy was increased in U251 cells upon SIRT3 knockdown.

A Inhibition of SIRT3 induced mitophagy in U251 cells, as shown by transmission electron microscopy. B RNA-Seq revealed that mitophagy pathway genes were upregulated in U251 cells upon SIRT3 knockdown. C Heat map showing altered expression of genes in the mitophagy pathway. D Quantitative real-time PCR validation of altered expression of mitophagy-related genes in U251 cells upon SIRT3 knockdown. E Blocking mitophagy alleviated SIRT3-promoted, RSL3-induced U251 cell ferroptosis. ***p < 0.001, ****p < 0.0001.

Fig. 8. SIRT3 protected GBM cells from RSL3-induced ferroptosis partially through SLC7A11.

A RNA-Seq showed that targeting of SIRT3 inhibited SLC7A11 expression. B Quantitative real-time PCR also demonstrated that targeting of SIRT3 inhibited SLC7A11 expression at the mRNA level. C Targeting of SIRT3 also inhibited SLC7A11 expression at the protein level. D Targeting of SIRT3 inhibited cystine uptake of GBM cells. E Forced expression of SLC7A11 in U251 cells with SIRT3 knockdown. F Forced expression of SLC7A11 in U251 cells with SIRT3 knockdown restored cystine uptake. G Forced expression of SLC7A11 in U251 cells with SIRT3 knockdown restored the cellular GSH level. H Forced expression of SLC7A11 in U251 cells with SIRT3 knockdown partially recovered cell viability following RSL3 treatment. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig. 9. SIRT3 regulated SLC7A11 expression through ATF4.

A Targeting of SIRT3 reduced ATF4 expression but not NRF2 and ATF3 expression. B Forced expression of ATF4 in U251 cells with SIRT3 knockdown rescued SLC7A11 expression at the mRNA level. C Forced expression of ATF4 in U251 cells with SIRT3 knockdown rescued SLC7A11 expression at the protein level. *p < 0.05, ****p < 0.0001.

Fig. 10. Working model depicting how targeted reduction of SIRT3 expression sensitizes GBM cells to RSL3-induced ferroptosis and the underlying mechanisms.

SIRT3 is upregulated during RSL3-induced GBM cell ferroptosis, which protects GBM cells from ferroptosis by inhibiting mitophagy, and increasing SLC7A11 expression through ATF4 and IRP-1.