Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma

Abstract

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system X_c ^- transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy. SIGNIFICANCE: Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659.

Figure 1.

Identification of novel anoikis suppressors in oncogene-transformed fibroblasts. A, Soft-agar colony formation of NIH3T3 cells stably expressing MSCV vector control, oncogenic KRas^G12V, or oncogenic ETV6–NTRK3 (EN). Scale bar, 5 mm. B, Spheroid growth of the indicated cells in 3-D cultures. Scale bar, 500 μm. C, Caspase-3 activity of the indicated cells grown in 3-D cultures. D, Schematic showing the strategies for identifying a novel signature regulating anoikis resistance. E, Schematic demonstrating the experimental design for global proteome analysis in cells cultured in 3-D conditions during the adaptation phase (0–6 hours). F, Global proteome changes induced by different oncogenes in NIH3T3 cells. G, Schematic demonstrating the experimental design for acute translatomic analysis in cells cultured in 3-D conditions during the adaptation phase (0–6 hours). H, Global acute translatomic changes induced by different oncogenes in NIH3T3 cells. I, Integrated analyses of the global proteomic and acute translatomic data sets shown in F and H identifies 31 upregulated proteins in response to detachment that were shared between KRas^G12V and EN transformed cells compared with MSCV controls. For all panels, data, the mean ± SD. Statistical significance was determined using unpaired two-tailed Student t test; ***, P < 0.001.

Figure 2.

IL1RAP is highly expressed in Ewing sarcoma (EwS) and promotes anoikis resistance and in vivo metastasis. A,IL1RAP mRNA expression profile in human tumor samples. The plot is generated based on published RNA-sequencing data sets. B, IHC staining of IL1RAP in Ewing sarcoma (TC32) xenografts in mice and human Ewing sarcoma samples. Scale bars, 50 μm. C, IF staining of IL1RAP in the indicated cells analyzed by microscopy. Scale bar, 10 μm. D, Flow cytometry–based quantification of cell counts between TC32-shIL1RAP (tdTomato+) and shCtrl cells cultured in 2-D or 3-D conditions. E, Left, hematoxylin and eosin (H&E) staining in TC32-derived xenografts implanted in the renal subcapsular space in mice. Dashed lines indicate the boundaries between Ewing sarcoma tumors and the surrounding kidney tissues, and blue arrows indicate the invasive Ewing sarcoma tumor cells. Scale bar, 100 μm. Right, quantification of local invasion distance by tumor cells with different IL1RAP staining intensities, n = 7–10. F, H&E staining of the lung sections and quantification of lung macrometastases and metastasis burden (see Methods); n = 7–10. G, IHC staining (left) and quantification (right) of 4-HNE staining intensity (n = 15–16 fields). Scale bar, 100 μm. H, GSH/GSSG ratios in the Ewing sarcoma xenografts derived from TC32 cells (n = 6–8). In A, data are presented as median values and the interquartile range. In D, data, means ± SD. In E–G, data, means ± SEM. In H, data, mean values and 10–90 percentile. Statistical significance was determined using unpaired two-tailed Student t test in all panels except the right panel of F. **, P < 0.01; ***, P < 0.001; n.s., no significance.

Figure 3.

IL1RAP forms a complex with CD98 and xCT of the system X_c⁻ cystine transporter and promotes cystine uptake. A, Volcano plots of proteins identified in the IL1RAP interactome in A673. B, Representative immunoblots of the indicated co-IP samples in A673 cells. The experiments were repeated twice with similar results. C, Representative IF staining of IL1RAP and CD98 in A673 cells. D, PLA of IL1RAP and CD98 in A673 cells (n = 5; >100 cells were analyzed for each condition). E, PLA of IL1RAP–CD98 or IL1RAP–xCT in TC32-derived Ewing sarcoma (EwS) xenografts in the murine renal subcapsular implantation model. Scale bar, 30 μm; red arrows indicate positive PLA signals in Ewing sarcoma tumor cells. F, Representative immunoblots of the indicated co-IP samples in A673 cells expressing wild-type (WT) or mutant CD98 (C109S or C330S). Rabbit (Rb) IgG and goat (Gt) IgG were used as isotype controls for the co-IP experiments. The experiments were repeated twice with similar results. G, Representative immunoblots of the indicated co-IP samples in HEK293 cells transfected with WT or mutant IL1RAP (C362S) and WT HA-tagged CD98. H and I, Representative images showing the IF staining of IL1RAP and xCT (H) and PLA assay of IL1RAP and xCT (I) in A673 cells. Scale bars, 10 μm. For I, n = 5–7; ≥50 cells were analyzed for each condition. J, The impact of IL1RAP knockdown (by sh#39 or #40) on cystine uptake was measured by cystine consumption assay in media containing regular (200 μmol/L) or low (10 μmol/L) levels of cystine. K and L, Intracellular cysteine and GSH pools were measured in the indicated cell lines (K) or Ewing sarcoma xenografts derived from TC32 (L) with or without IL1RAP knockdown (by sh#39 or #40). M, Rescue of WT or mutant IL1RAP (C362S) in Ewing sarcoma cells with IL1RAP depletion was determined by immunoblot analysis. N, Cystine uptake and glutamate secretion were determined in the indicated Ewing sarcoma cells cultured in media containing 10 μmol/L cystine. Data presented are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001;P values were determined by two-tailed unpaired Student t test.

Figure 4.

The IL1RAP–CTH axis promotes the TSS pathway. A, Schematic showing the pathway that mediates de novo cysteine synthesis and GSH production via TSS. B, Schematic showing the global proteome analysis in A673 Ewing sarcoma cells with control knockdown (shCtrl) or IL1RAP knockdown (sh#39 and sh#40) grown in 3-D cultures. C, Global proteome changes induced by IL1RAP depletion with two independent shRNAs (n = 3 biologically independent samples). D, Immunoblot analysis of the indicated proteins. Densitometry analysis of CTH normalized to the vinculin loading control is shown. The experiment was repeated twice with similar results. E, Cell death and cell growth were measured by Incucyte in the indicated cells cultured in media containing different concentrations of cystine supplemented with/without 0.4 mmol/L homocysteine (Hcy). F, Intracellular levels of cystathionine and GSH were determined in cells cultured in media ± 200 μmol/L cystine or 0.4 mmol/L Hcy for 12 hours. G,De novo cysteine synthesis and GSH production in the indicated cells were determined in cystine-depleted cultures by flux analysis of ¹³C-serine, a key substrate for the TSS pathway as shown in A. ¹²C-labeled (M + 0) and 13C-labeled (M+1) metabolites were measured after 24 hours. H and I, The effects of CTH reexpression on GSH pools (12 hours; H), lipid ROS accumulation (22 hours; I, left), and cell death (48 hours; I, right) in IL1RAP-depleted cells with the indicated treatments. 2 and 5 μmol/L erastin was used in H and I, respectively; and 2 μmol/L Fer-1 was used in I. P values were determined by two-tailed unpaired Student t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

CTH promotes invasion and metastasis in Ewing sarcoma. A, Immunoblot analysis in lysates of TC32-derived Ewing sarcoma xenograft tumors in mice (n = 4 independent tumors). B, Volumes of TC32-derived Ewing sarcoma tumors in the renal subcapsular space of mice (n = 10). C, H&E staining of TC32-derived tumors in the renal subcapsular space of mice and quantification of local invasion distance (n = 10). Dashed lines indicate the boundaries between primary Ewing sarcoma tumors and the surrounding kidney tissues; arrows indicate invasive Ewing sarcoma cells. Scale bar, 50 μm. D, Left, H&E staining of lung sections from mice implanted with Ewing sarcoma (TC32) tumors in the renal subcapsular space. Arrows indicate Ewing sarcoma metastases. Scale bar, 300 μm. Right, quantifications of macrometastases in the lungs (n = 10). E, The metastatic burden in the lungs of each group (see Methods for details). In B, data, means ± SEM. In C and D, the lines in the violin plots show the median and interquartile range. P values were determined by two-tailed unpaired Student t test.

Figure 6.

EWS–FLI1 directly regulates IL1RAP expression via enhancer activation. A, Flow cytometry–based quantification of ROS (CellROX Deep Red) 96 hours after Dox treatment. B, Incucyte analysis of SYTOX orange staining (red) in 2-D and 3-D cultures to evaluate cell death. The ratio between SYTOX orange intensity and cell area was calculated (n = 6). Scale bar, 100 μm. C, Relative cell viability in the indicated cells was measured by CCK-8 assay after 24-hour treatment (n = 3). D,EWS–FLI1 and IL1RAP mRNA expression in the indicated samples determined by qPCR (n = 3). E, Immunoblotting analysis of the indicated proteins. The experiment was repeated over three times with similar results. F, ChIP-seq results of FLI1 in the indicated cells with shGFP control knockdown or shFLI1 knockdown (48 hours; GEO: GSE61953). G and H, ChIP-qPCR assays were performed to determine the occupancy of EWS–FLI1 and H3K27ac at the IL1RAP locus in A673 Ewing sarcoma cells with Dox-inducible knockdown of EWS–FLI1 (0.5 μmol/L Dox, 72 hours). I, ChIP-seq results of H3K27ac in the indicated cells with shGFP control knockdown or shFLI1 knockdown (48 hours; GEO: GSE61953). J, Representative immunoblots of the indicated samples. The experiment was repeated twice with similar results. K and L, ChIP-qPCR assays were performed to determine the occupancy of BAF155 and p300 at the IL1RAP locus in A673 Ewing sarcoma cells with Dox-inducible knockdown of EWS–FLI1 (0.5 μmol/L Dox, 72 hours). For all panels, data, means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; P values were determined by two-tailed unpaired Student t test.

Figure 7.

IL1RAP is a potential cell-surface therapeutic target in Ewing sarcoma (EwS). A, IHC analysis of IL1RAP in pediatric normal organ TMAs. Scale bars, 50 μm in high-power images and 200 μm in low-power images. B, Median asinh (signal/150) differences in IL1RAP intensity of the indicated stained cells from their corresponding FMO (fluorescence minus one) control. C and D, Binding of the IL1RAP antibody V_H–Fc X1 to a panel of human cancer cell lines was determined by flow cytometry (C) and IF analysis (D). Scale bar, 10 μm. E, The ADCC reporter assay was performed using the Jurkat T-NFAT-Luc-CD16A effector cells encoding the luciferase reporter gene driven by the nuclear factor of activated T cells response element (NFAT-RE). A673 Ewing sarcoma cells were used as target cells. Target:effector cell ratio, 1:6. An isotype control or no antibody was added in the control groups. Baseline reporter activity was determined by omitting the Ewing sarcoma target cells in the assay. F, ADCC assays using PBMCs as effector cells and a panel of Ewing sarcoma cells as target cells (n = 3–4). Target:effector cell ratio, 1:15. All data presented are means ± SD. In B, one-sample t test was performed, and two-tailed unpaired Student t test was used in other panels. *, P < 0.05; **, P < 0.01; ***, P < 0.001.