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. 2021 Jun:42:101949.
doi: 10.1016/j.redox.2021.101949. Epub 2021 Mar 19.

Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer

Laurie Freire Boullosa  1 , Jinthe Van Loenhout  2 , Tal Flieswasser  2 , Jorrit De Waele  2 , Christophe Hermans  3 , Hilde Lambrechts  2 , Bart Cuypers  4 , Kris Laukens  5 , Esther Bartholomeus  6 , Vasiliki Siozopoulou  7 , Winnok H De Vos  8 , Marc Peeters  9 , Evelien L J Smits  10 , Christophe Deben  2
Affiliations

Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer

Laurie Freire Boullosa et al. Redox Biol. 2021 Jun.

Abstract

Auranofin (AF) is an FDA-approved antirheumatic drug with anticancer properties that acts as a thioredoxin reductase 1 (TrxR) inhibitor. The exact mechanisms through which AF targets cancer cells remain elusive. To shed light on the mode of action, this study provides an in-depth analysis on the molecular mechanisms and immunogenicity of AF-mediated cytotoxicity in the non-small cell lung cancer (NSCLC) cell line NCI-H1299 (p53 Null) and its two isogenic derivates with mutant p53 R175H or R273H accumulation. TrxR is highly expressed in a panel of 72 NSCLC patients, making it a valid druggable target in NSCLC for AF. The presence of mutant p53 overexpression was identified as an important sensitizer for AF in (isogenic) NSCLC cells as it was correlated with reduced thioredoxin (Trx) levels in vitro. Transcriptome analysis revealed dysregulation of genes involved in oxidative stress response, DNA damage, granzyme A (GZMA) signaling and ferroptosis. Although functionally AF appeared a potent inhibitor of GPX4 in all NCI-H1299 cell lines, the induction of lipid peroxidation and consequently ferroptosis was limited to the p53 R273H expressing cells. In the p53 R175H cells, AF mainly induced large-scale DNA damage and replication stress, leading to the induction of apoptotic cell death rather than ferroptosis. Importantly, all cell death types were immunogenic since the release of danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation occurred irrespective of (mutant) p53 expression. Finally, we show that AF sensitized cancer cells to caspase-independent natural killer cell-mediated killing by downregulation of several key targets of GZMA. Our data provides novel insights on AF as a potent, clinically available, off-patent cancer drug by targeting mutant p53 cancer cells through distinct cell death mechanisms (apoptosis and ferroptosis). In addition, AF improves the innate immune response at both cytostatic (natural killer cell-mediated killing) and cytotoxic concentrations (dendritic cell maturation).

Keywords: Auranofin; Cancer cell death; Mutant p53; Non-small cell lung cancer; Oxidative stress; Thioredoxin reductase.

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Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Expression of Trx system in NSCLC patients. (A) Expression of the thioredoxin pathway genes, thioredoxin (TXN), thioredoxin reductase 1–2 (TXNRD1-2) and peroxiredoxin 1-6 (PRDX1-6) represented as fragments per kilobase million (FPKM). FPKM values were extracted from the LUAD cohort of the cancer genome atlas (TCGA) RNA-seq dataset using the Human Protein Atlas. (B) Kaplan-Meier survival curves showing the correlation of TXN, TXNRD1-2 and PRDX4 and 6 gene expression with the survival of LUAD patients. Curves were plotted based on the best expression cut-off, i.e. the FPKM value that yields maximal difference with regard to survival using the Human Protein Atlas. Number of LUAD patients with low or high gene expression were included between brackets. (C) Representative sections of NSCLC tumor samples classified as negative (IRS0), weak (IRS1), moderate (IRS2) and strong (IRS3) TrxR protein expression using immunohistochemistry staining. (D) Pie chart representing the percentage of NSCLC patients with IRS0 to IRS3 for TrxR protein expression. (E) Kaplan-Meier survival curves for overall survival (OS) and progression-free survival (PFS) based on IRS of TrxR protein. Crosses signify censored events where a patient's life ended. Significance was reached when p < 0.05 determined by log rank. IRS: immunoreactivity scoring.
Fig. 2
Fig. 2
Cytotoxic response to AF in NSCLC and PDAC cell lines and their p53 expression levels. (A) Dose-response survival curves after 72 hours of AF treatment (0–8 μM) in eight NSCLC and PDAC cell lines. (B) IC50 values (μM) after 72 hours of AF treatment in eight NSCLC and PDAC cell lines with different p53 backgrounds. (C) Spearman's r correlation between baseline p53 protein expression (determined by Western blotting) and AF IC50 values (μM). (D) Western blots representing the baseline protein expression of NRF2, catalase, TrxR1, p53, SLC7A11, GPX4, SOD1 and Trx in eight NSCLC and PDAC cell lines. β-actin was used as internal control. Molecular weights of the proteins are represented in kilodalton (kDa). Quantification of triplicates is presented in Supplementary Fig. 2. (E) Spearman's r correlation coefficient between baseline mutant p53 protein and AF IC50 values, and baseline expression of TrxR1, Trx, GPX4, SLC7A11, NRF2, SOD1, catalase (determined by Western blotting), GSH (determined by luminescence) and ROS (determined by IncuCyte ZOOM analysis) in eight NSCLC and PDAC cell lines. (F) p53 protein expression in the isogenic mutant p53 R175H and R273H cells (determined by Western blotting). (G) IC50 values (μM) after 72 hours of AF treatment in the isogenic NCI–H1299 cell panel (determined by SRB assay). (H) Fold change of Trx protein expression, relative to NCI–H1299 Null control cells (determined by Western blotting using two different anti-Trx antibodies, red dots), in the isogenic NCI–H1299 cell line panel. Experiments were performed at least in triplicate. Error bars represent the standard deviation. *p ≤ 0.05 significant differences. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
Effect of AF on mRNA and protein targets related to antioxidant mechanisms, ferroptosis and DNA damage. (A) TrxR activity in μMol/Minute/mgProtein after treatment of the isogenic NCI–H1299 cell lines with a cytostatic (1 μM) and cytotoxic (5 μM) AF concentration for six and 24 hours. (B) Relative GSH protein content after treatment of the isogenic NCI–H1299 cells with a cytostatic (1 μM) and cytotoxic (5 μM) AF concentration for six and 24 hours, relative to untreated. Error bars represent the standard deviation. *p ≤ 0.05 significant differences compared with untreated control. (C) Ingenuity pathway analysis of affected pathways after treatment with 1 μM AF for 24 hours in the isogenic NCI–H1299 cell lines. Color key legend represents -log (p-value). (D) Differentially expressed genes after AF treatment, relative to untreated, in the isogenic NCI–H1299 cell lines. Color key legend represents expression log2 ratio (red = upregulation; green = downregulation). (E) Western blot was used to determine the protein levels of NRF2, HMOX1, GPX4, SOD1 and Trx in the isogenic NCI–H1299 cells after treatment with 1 μM AF for 24 hours of two independent replicates (REP1 and REP2). β-actin was used as internal control. Molecular weights of the proteins are represented in kilodalton (kDa). (F–G) Box plot representing γH2AX spot occupancy (F) and pRPA32 nuclear intensity (G) calculated using an algorithm on Image J software, in the isogenic NCI–H1299 cell lines treated with PBS, 1 μM and 2 μM AF for 24 hours. Representative images are presented in Supplementary Fig. 8. Box boundaries represent the upper and lower quartiles and the black line within represents the median. The whiskers display the 95% confidence interval, while the dots outside the whiskers represent outliers. Experiments were performed at least in triplicate. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
Induction of apoptotic and ferroptotic cell death after AF treatment of mutant p53 NSCLC cells. (A–C) Percentage of cell death after treatment with 2 μM AF for 72 hours in the absence or presence of apoptosis, necroptosis and ferroptosis inhibitors and ROS scavenger NAC in the isogenic p53 Null (A), p53 R175H (B) and p53 R273H mutated NCI–H1299 cells (C). (D) Fold change of the CellROX Green Calibration Units (GCU), relative to untreated, of the three isogenic NCI–H1299 cell lines after treatment with 2 μM AF for 16 h. (E–F) Percentage of Caspase-3/7 green positive cells (E) and Caspase-3/7 total green objected integrated intensity (GCU x μm2/image) (F) in the isogenic NCI–H1299 cell lines after treatment with 2 μM AF for 72 hours. Error bars represent the standard error of the mean. (G) Overlay histograms of C11 BODIPY Green 581/591 signal after treatment of the isogenic NCI-H1299 cell lines with PBS, 2 μM AF (48 hours) or cumene hydroperoxide (positive control, 2 hours). (H) Relative ratio of red over green mean fluorescent intensity (MFI) signal of the C11-BODIPY 581/591 reagent after treatment with 2 μM AF (48 hours) or cumene hydroperoxide (positive control, 2 hours) of the isogenic NCI–H1299 cell lines. Error bars represent the standard deviation. Experiments were performed at least in triplicate. *p ≤ 0.05 significant differences compared to untreated control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5
Fig. 5
AF treatment of mutant p53 NSCLC cells induces the release of ICD markers and DC maturation. (A) Percentage of surface-exposed ecto-calreticulin (ecto-CALR) positive NCI–H1299 Null, p53 R175H and p53 R273H cells after treatment with 2 μM AF for 48 hours. Gating strategy of CALR+ cells represented in Supplementary Fig. 13. (B) Secretion of ATP in the supernatant of the isogenic NCI–H1299 cell lines 24 hours after treatment with 2 μM AF (nM range). (C) HMGB1 secretion induced 48 hours after treatment with 2 μM AF in the isogenic NCI–H1299 cells (ng/mL range). (D) Percentage of MHC-II and CD86 double positive DCs after 48 hours of co-culture with AF-treated H1299 null, p53 R175H and p53 R273H cells (E:T ratio 1:1) using flow cytometry, relative to untreated. Gating strategy represented in Supplementary Fig. 14. Error bars represent the standard deviation. (E) Contour plots of the DC population double positive for MHC-II and CD86 in co-culture with either PBS- or AF-treated mutant p53 R175H cells. (F–H) Fold change of the cytokines TNF-α (F), IL-6 (G) and TGF-β (H) released by DCs in supernatant of co-cultures with PBS- or AF-treated NCI–H1299 cells. Error bars represent the standard error of the mean with every dot representing a different healthy donor. Experiments were performed in triplicate. *p ≤ 0.05 significant differences compared with untreated control. DCs: dendritic cells.
Fig. 6
Fig. 6
Effect of AF treatment on co-cultures of mutant p53 NSCLC cells and natural killer cells. (A) Percentage of death NSCLC cells in co-cultures of (IL-15) NK cells and NCI–H1299 Null, p53 R175H and p53 R273H cells exposed to a non-toxic concentration of 0.55 μM AF for 72 hours. Every dot represents NK cells isolated from a different healthy donor added to the co-culture in 5:1 E:T ratio. Error bars represent the standard error of the mean with every dot representing a different healthy donor. *p ≤ 0.01, **p ≤ 0.001. (B) Representative IncuCyte ZOOM images show the number of NucLight Red positive viable p53 R175H cells and Cytotox Green positive dead p53 R175H cells. (C) Caspase-3/7 total green objected integrated intensity (GCU x μm2/image) in co-cultures of (IL-15) NK cells and p53 R175H cells exposed to Pan-caspase inhibitor Z-VAD-FMK (50 μM) and a non-toxic concentration of 0.55 μM AF for 48 hours. *p ≤ 0.05 (D) Percentage of death NSCLC cells in co-cultures of (IL-15) NK cells and p53 R175H cells exposed to Pan-caspase inhibitor Z-VAD-FMK (50 μM) and a non-toxic concentration of 0.55 μM AF for 72 hours. Error bars represent the standard error of the mean with every dot representing a different healthy donor. (E) Overton percentage of CD107a, IFNγ, CD69 and GZMA positive unstimulated NK cells in co-culture with AF-treated NCI–H1299 Null, p53 R175H and p53 R273H cells, relative to unstimulated NK cells in co-culture with PBS-treated cells. (F) Overton percentage of CD107a, IFNγ, CD69 and GZMA positive IL-15 stimulated NK cells in co-culture with AF-treated NCI–H1299 Null, p53 R175H and p53 R273H cells, relative to IL-15 stimulated NK cells in co-culture PBS-treated cells. IL-15 NK cells were stimulated with 10 ng/mL IL-15 overnight. Gating strategy represented in Supplemental Fig. 17. Error bars represent the standard deviation with every dot representing a different healthy donor. NK cells: natural killer cell. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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