14 Feb 2023
by Richa

Summary- paper 15: Ferroptosis inhibition by lysosome-dependent catabolism of extracellular protein

David A. Armenta, Nouf N. Laqtom, Grace Alchemy, Wentao Dong, Danielle Morrow, Carson D. Poltorack, David A. Nathanson, Monther Abu-Remaileh, Scott J. Dixon

Cell Chemical Biology, 2022

Questions/gaps addressed:

  • Cysteine is conditionally essential for survival and proliferation of many cancer cells. Cys is required for synthesis of proteins, glutathione (GSH), coenzyme A. GSH and coenzyme A are needed to prevent ferroptosis, a non-apoptotic, oxidative form of cell death. How do cancer cells manage cysteine metabolism to inhibit ferroptosis?

  • GSH is a co-substrate for glutathione peroxidase 4 (GPX4), an essential enzyme that prevents ferroptosis by reducing potentially toxic membrane lipid hydroperoxides to non-toxic lipid alcohols. Mechanism of CoA in ferroptosis inhibition unclear.

  • Cysteine is typically present at low abundance in the cell and in the fluid surrounding tumors in vivo. Cystine, the disulfide of cysteine is typically present outside the cell. Complete cystine starvation can induce ferroptosis in cultured cancer cell lines. How do cancer cells cope with limited extracellular amino acids, especially cystine?

  • Cystine deprivation in vivo can slow tumor growth but does notcause consistent tumor regression. Are there compensatory mechanisms (besides transporter-mediated uptake of free amino acids) that limit ferroptosis in response to cystine deprivation?

  • mTORC1 inhibition promotes lysosomal protein degradation. mTORC1 inhibition prevents depletion of intracellular GSH and attenuated ferroptosis in cultured cells deprived of cystine. Does this modulate cancer cell death by ferroptosis?

Major hypotheses:

  • mTORC1 inhibition, together with uptake and catabolism of extracellular proteins, is sufficient to compensate for the loss of transporter-mediated cystine uptake and inhibit ferroptosis mediated cell death of cancer cells.

Key methods:

  • Deprived HT-1080 fibrosarcoma cells of cystine and measured cell proliferation and cell death over time. These cells express a live cell marker, nuclear-localized mKate2 and were incubated with the dead cell dye SYTOX Green allowing monitoring cell proliferation and death to be measured simultaneously. Used inhibitors to test if the death is due to general apopotosis (pan-caspase inhibitor Q-VD-OPh), or ferroptosis (ferrostatin-1 (Fer-1)). Deprivation of cystine, but not other amino acids Met, Arg, and Leu, led to ferroptosis mediated death.

  • Added serum protein albumin at a final concentration (3% [w/v]) in the media mimicking concentration in human serum, and deprived cystine in the media. Treatment with the ATP-competitive mTOR inhibitor INK128 (or other mTOR inhibitors Torin 1 and rapamycin or shRNA targeting the mTORC1 subunit RPTOR) was sufficient to attenuate cystine deprivation-induced cell death.

  • Saw similar effects of combination treatment in cystine-deprived cancerous cell lines: H1299 and H23Cas9 non-small cell lung carcinoma cells and U-2 OS osteosarcoma, A375 melanoma, T98G glioblastoma, and PaTu 8988T pancreatic adenocarcinoma cell lines.

  • Albumin + INK128 treatment protected equally well against ferroptosis in control and ATG7 KO PaTu 8988T cell lines, suggesting that autophagy does not mediate this effect. Treatment did not increase expression of the anti-ferroptotic proteins GPX4 or FSP1, or decrease intracellular iron levels, as assessed by expression of the transferrin receptor (TFRC) or IRP2, which are induced by iron starvation.

  • Monitored membrane lipid peroxidation in cystine-deprived cells using the sensor C11 BODIPY 581/591. Reduced oxidized signal when also treated with albumin. Albumin treatment alone weakly inhibited ferroptosis in response to low (but not high) doses of the GPX4 inhibitors FIN56 and ML162, Albumin can possibly act as a direct antioxidant to some extent. The effect was a lot more when combined with cystine deprivation.

  • Albumin + INK128 treatment + Inhibition of lysosomal proteases with deacidifying agents (e.g., chloroquine) or using a cocktail of the protease inhibitors pepstatin A, leupeptin- cells died again (A375, H1299, and T98G cancer cells, but no effect in U-2 OS, unclear why). Also lysosomal function by microscopy using fluorescent probe DQ-BSA.

  • Albumin + INK128 treatment + Cystine deprivation + treatment with cathepsin inhibitors: CTSB inhibitor CA074 methyl ester but not the cathepsin L and S inhibitor R11-OEt: led to DQ-BSA fluorescence quenching, i.e. Cathepsin B CTSB predominantly responsible for albumin breakdown in lysosome. No defect in endocytosis or internalization of tetramethylrhodamine (TMR)-dextran. Same effect observed upon genetic disruption of CTSB, CTSL, CTSD.

  • Examined the role pf Cystinosin (CTNS) in exporting cystine produced by Albumin breakdown by CTSB from the lysosomal lumen to the cytosol. Generated CTNS KO HT-1080 cells. Albumin + INK128 treatment + Cystine deprivation + CTNSKO cells, cells died from ferroptosis and had high lipid peroxidation.

  • Established HT-1080 spheroids to mimic tumor microenvironment over 3 days in ultra-low adherence vessels. vehicle + system xc− inhibitor erastin2 cells died from ferroptosis. Albumin + system xc− inhibitor erastin2 cells lived.

Major takeaways:

  • Endocytosis and lysosomal degradation of extracellular albumin, provides enough cysteine, and hence GSH to cancer cells to prevent death by ferroptosis, even in the absence of cystine uptake from the environment. This protective mechanism requires the lysosomal enzyme cathepsin B and the lysosomal cystine exporter cystinosin.