Ferroptosis inhibition by lysosome-dependent catabolism of extracellular protein

Abstract

Cancer cells need a steady supply of nutrients to evade cell death and proliferate. Depriving cancer cells of the amino acid cystine can trigger the non-apoptotic cell death process of ferroptosis. Here, we report that cancer cells can evade cystine deprivation-induced ferroptosis by uptake and catabolism of the cysteine-rich extracellular protein albumin. This protective mechanism is enhanced by mTORC1 inhibition and involves albumin degradation in the lysosome, predominantly by cathepsin B (CTSB). CTSB-dependent albumin breakdown followed by export of cystine from the lysosome via the transporter cystinosin fuels the synthesis of glutathione, which suppresses lethal lipid peroxidation. When cancer cells are grown under non-adherent conditions as spheroids, mTORC1 pathway activity is reduced, and albumin supplementation alone affords considerable protection against ferroptosis. These results identify the catabolism of extracellular protein within the lysosome as a mechanism that can inhibit ferroptosis in cancer cells.

Keywords: ROS; albumin; cancer; cathepsin; cell death; cysteine; ferroptosis; glutathione; lysosome; mTOR.

Figure 1.. Albumin promotes cell survival in response to cystine deprivation.

(A) Cell proliferation determined by live cell (nuclear-mKate2 positive objects; mKate2⁺ Obj.) counts over time in complete medium (CM) or medium lacking the indicated amino acid ± apoptosis (Q-VD-OPh) or ferroptosis (Fer-1) inhibitors. Cys₂: cystine. (B) Cell death in populations from (A) determined by integration of live (mKate2⁺) and dead (SYTOX Green-positive) counts over time into the lethal fraction score (Forcina et al., 2017). A lethal fraction score of 0 equals no cell death and 1 equals complete population cell death. (C) Representative images from three independent experiments of HT-1080^N cells cultured over time in complete medium (CM) or medium lacking cystine. Medium contains 20 nM SYTOX Green to mark dead cells. INK128: 1 µM. Scale bar = 30 µm. (D) Quantification of cell death in (C). BSA: bovine serum albumin. (E) Cell death in different cell lines in medium lacking cystine. (F) Cell death in response to different inducers of cell death. (G) Cell death and proliferation over time. Note that some of the lethal fraction data shown here for comparison purposes is also depicted in (D). These data were from the same experiment. Results in (A, B, D, and G) are mean ± SD from three independent experiments. Datapoints from independent experiments are shown in (E) and (F). See also Figure S1 and Figure S2.

Figure 2.. Albumin increases glutathione levels during cysteine deprivation.

(A) C11 BODIPY 581/591 (C11) oxidation assessed by confocal microscopy in HT-1080 cells. BSA: bovine serum albumin (3% w/v). INK128: 1 µM. Scale bar = 20 µm. (B) Cell death in HT-1080^N cells pre-treated with BSA (24 h) then treated as indicated at time 0 and examined 48 and 96 h later. Era2 (erastin2): 1 µM. ML162: 2 µM. Fer-1 (ferrostatin-1): 1 µM. (C) Cell death over time. INK128: 1 µM. (D) Protein levels following 10 h treatment. Blot is representative of three independent experiments. Mean ATF4/Actin protein level ratios (normalized to the +cystine condition) determined from densitometry of three independent blots are indicated. (E) Total glutathione (GSH + GSSG) following 8 h treatment. INK128: 1 µM. (F) Cell death following 24 h pretreatment ± BSO (buthionine sulfoximine) then treated as indicated alongside BSO. BSA: 3% w/v. Results in (B) and (E) show datapoints from independent experiments. Results in (C) and (F) are mean ± SD from three independent experiments. See also Figure S3.

Figure 3.. Lysosomal function is necessary for albumin to protect from ferroptosis.

(A) Cell death over time. A+I: bovine serum albumin (BSA, 3% w/v) + INK128 (1 µM). Prot. inhibs: protease inhibitor cocktail (leupeptin: 10 µM, pepstatin A: 2 µM, E-64: 2 µM). Data represent mean ± SD from three independent experiments. (B) DQ-BSA fluorescence assessed by fluorescence microscopy in HT-1080^N cells treated. Chloroquine (30 µM), protease inhibitor cocktail (Prot. inhibs, as in (A)). Images are representative of three independent experiments. Scale bar = 30 µm. (C) Quantification of DQ-BSA puncta or area (in square pixels) per cell. Datapoints represent counts of puncta from all images within a condition divided by number of cells in those images, from three independent experiments (minimum 30 cells/experiment and condition). (D) ATF4 protein levels in response to different treatment conditions (10 h). CQ: chloroquine. Blots are representative of three independent experiments. Mean ATF4/Actin protein level ratios (normalized to the +cystine condition) determined from densitometry of three independent blots are indicated. (E) Cell death in various cell lines ± chloroquine (30 µM). (F) Cell death in various cell lines ± protease inhibitor cocktail as in (A). Datapoints from independent experiments are shown in (E) and (F). See also Figure S4.

Figure 4.. CTSB is required for ferroptosis suppression by extracellular albumin.

(A) LysoTracker Red, a marker for lysosomes, and self-quenched bovine serum albumin (DQ-BSA) fluorescence in PaTu 8988T cells. Scale bar = 5 µm. (B) Activity of the mTORC1 signaling pathway in PaTu 8988T cells starved of leucine for 2 h, then restimulated with medium containing leucine (Leu), bovine serum albumin (BSA, 5% w/v), or γ-globulin (3% w/v) for 4 h. Raptor was used as a loading control. (C) Analysis of lysosomal uptake and degradation of self-quenched bovine serum albumin (DQ-BSA) in PaTu 8988T cells. The macropinocytic cargo 70 kDa tetramethylrhodamine dextran (TMR-dextran) is used to assess any effect on uptake. On the right, each datapoint represents ≥ 3 fields of view with ≥ 10 cells in total, with the median indicated by the horizontal bar. Scale bar = 5 μm. CTSB^KO1: cathepsin B knockout, CTSB^KO1+CTSB: CTSB knockout reconstituted with CTSB cDNA. (D) Cell death determined by counting SYTOX Green-positive (SG⁺) dead cells. BSA: 3% w/v, INK128: 1 µM, Fer-1 (ferrostatin-1): 1 µM. NTC: non-targeting CRISPR control. (E) Total glutathione (GSH + GSSG) measured using Ellman’s reagent following 24 h of treatment. INK128: 1 µM, BSA: 3% w/v. (F) Protein levels determined by western blot. Blots are representative of three independent experiments. Mean ATF4/Actin protein level ratios (normalized to the +cystine condition) determined from densitometry of three independent blots are indicated. See also Figure S5.

Figure 5.. Cystinosin is required for ferroptosis suppression by extracellular albumin.

(A) Cell death in HT-1080^N Control or cystinosin (CTNS) gene-disrupted (knockout, KO) cell lines. INK128: 1 µM, bovine serum albumin (BSA): 3% w/v. Data represent mean ± SD from three independent experiments. (B) C11 BODIPY 581/591 (C11) oxidation ratio (oxidized C11/[reduced C11 + oxidized C11]). INK128: 1 µM, BSA: 3% w/v. Data acquired from 59–512 individual cells per condition were analyzed. Representative data from one of three independent experiments is shown. (C) Total glutathione (GSH + GSSG) measured using Ellman’s reagent. INK128: 1 µM, BSA: 3% w/v. (D) Protein levels determined by western blot. C: Control, KO: knockout. Blots are representative of three independent experiments. See also Figure S6.

Figure 6.. Albumin promotes spheroid viability in response to cystine deprivation.

(A) HT-1080^N spheroids established over three days (day 0) prior to treatment for three days (Day 3). BSA: bovine serum albumin, 3% w/v. Scale bar = 100 µm. (B) Viability of HT-1080^N spheroids as established and treated as in (A) determined using CellTiter-Glo. (C) mTOR pathway activity assessed by western blot following 24 h treatment. M: monolayer, S: spheroid. INK128: 1 µM. (D) Gliomaspheres (line: GS187) established over three days (day 0) prior to treatment for five days. BSA: 3% w/v. Scale bar = 100 µm. (E) Viability of GS187 spheroids as established and treated as in (D) determined using CellTiter-Glo. (F) mTOR pathway activity assessed by western blot in HT-1080^N cells grown in monolayer (control) or GS187 cells grown as spheroids and treated ± INK128 (1 µM) for 24 h. In (A) and (D), images are representative of three independent experiments. In (B) and (E), data are mean ± SD from three independent experiments. In (C) and (F), blots are representative of three independent experiments. p-RPS6 is phosphorylated at Ser235/236. See also Figure S7.

Figure 7.. Model for how exogenous protein protects from ferroptosis.

CTNS, cystinosin. Note that while in some cases albumin likely enters the cell via macropinocytosis other routes of entry and trafficking to lysosomes is possible.