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. 2019 Dec 5;20(12):e48375.
doi: 10.15252/embr.201948375. Epub 2019 Oct 31.

Class I HDAC inhibitors enhance YB-1 acetylation and oxidative stress to block sarcoma metastasis

Amal M El-Naggar  1   2   3 , Syam Prakash Somasekharan  4 , Yemin Wang  2 , Hongwei Cheng  4 , Gian Luca Negri  5 , Melvin Pan  2 , Xue Qi Wang  2 , Alberto Delaidelli  1   2 , Bo Rafn  2 , Jordan Cran  2 , Fan Zhang  4 , Haifeng Zhang  1   2 , Shane Colborne  5 , Martin Gleave  4 , Anna Mandinova  6 , Nancy Kedersha  7 , Christopher S Hughes  2 , Didier Surdez  8 , Olivier Delattre  8 , Yuzhuo Wang  4 , David G Huntsman  1   2 , Gregg B Morin  5 , Poul H Sorensen  1   2
Affiliations

Class I HDAC inhibitors enhance YB-1 acetylation and oxidative stress to block sarcoma metastasis

Amal M El-Naggar et al. EMBO Rep. .

Erratum in

Abstract

Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS-275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing its binding to 5'-UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS-275 promotes rapid acetylation of the YB-1 RNA-binding protein at lysine-81, blocking binding and translational activation of NFE2L2, as well as known YB-1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS-275 dramatically reduces sarcoma metastasis in vivo, but an MS-275-resistant YB-1K81-to-alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275-treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.

Keywords: HDAC inhibitors; NRF2; YB-1; metastasis; sarcoma.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Class 1 HDAC inhibitors are potent oxidative stress sensitizers in sarcoma cells

  1. Cell‐based screens for ROS sensitizers in U2OS cells (see Materials and Methods). Average z‐scores for activated caspase‐3/7 from three independent experiments are plotted.

  2. Time course of treatment with 1 μM class I HDAC inhibitor MS‐275 for 24 h alone or in combination with 100 μM NaAsO2 for 1 h to assess U2OS viability by NucView‐488 fluorescence. DMSO was used as a vehicle control. Error bars indicate SEM (n = 3 independent experiments, each performed in triplicate).

  3. U2OS cells were treated without (vehicle) or with MS‐275 (1 μM, 24 h), and without (−NaAsO2) or with NaAsO2 (+NaAsO2) (500 μM, 1 h) and assessed for ROS levels using CM‐H2DCFDA. ROS levels were normalized to protein content. Error bars indicate SEM for n = 3 independent experiments, each performed in triplicate.

  4. U2OS cells +/− MS‐275 (1 μM, 24 h) were exposed to vehicle (white bars) or NaAsO2 (red bars; 500 μM, 1 h) and assessed for redox stress using intracellular GSH/GSSG ratios as a readout. Data are expressed as mean ± SEM. for n = 3 independent experiments, each performed in triplicate.

  5. Immunoblotting showing effects of the antioxidant N‐acetylcysteine (NAC) on PARP cleavage at the indicated time points in U2OS cells subjected to treatment with or without MS‐275 (1 μM, 24 h) and NaAsO2 (100 μM, 1 h). GRB2 was used as a loading control.

  6. Immunoblotting showing effects of MS‐275 on NRF2 expression in U2OS cells treated with NaAsO2 (500 μM, 1 h) as indicated. β‐actin was used as a loading control.

  7. Luciferase reporter assays showing antioxidant response element (ARE) activity in U2OS cells (left panel) and CHLA‐10 cells (right panel) in the presence or absence (vehicle) of MS‐275 (1 μM, 24 h) and either untreated (UT; white bars) or treated with NaAsO2 (100 μM, 1 h; red bars). Error bars indicate SEM for n = 3 independent experiments, each performed in triplicate.

  8. U2OS was transfected with empty vector or NRF2‐expressing vector. Two days post‐transfection, cells were treated without (vehicle) or with MS‐275 (1 μM, 24 h), and without (−NaAsO2) or with NaAsO2 (+NaAsO2) (100 μM, 1 h) and assessed for ROS levels using CM‐H2DCFDA. Data are presented as fold change over vehicle control (−NaAsO2) for each group. Error bars indicate SEM for n = 3 independent experiments, each performed in triplicate.

  9. Total RNA (white bars) and polysome fractionated RNA (red bars) were isolated from U2OS cells treated +/− NaAsO2 (100 μM, 1 h), and assayed for NFE2L2 expression by RT–PCR using NFE2L2 primers. Data were normalized against GAPDH and expressed as fold change ± SEM of two independent experiments, each performed in triplicate.

  10. Total and polysomal RNA from U2OS cells treated without (vehicle) or with MS‐275 (1 μM, 24 h) and further treated with NaAsO2 (100 μM, 1 h) were subjected to polysomal fractionation followed by RT–PCR using primers for NFE2L2. Data were normalized against GAPDH and expressed as fold change ± SEM of 2 independent experiments, each performed in triplicate.

Data information: Unpaired two‐tailed Student's t‐test, *P < 0.05; **P < 0.005; ***P < 0.0005; n.s. = non‐significant.Source data are available online for this figure.
Figure EV1
Figure EV1. Increased ROS production in 3D culture of sarcoma cells
  1. A

    Effects of MS‐275 treatment at the indicated doses on U2OS cell proliferation as detected by Incucyte. The graph represents fold change relative to day 0, with error bar  =  standard deviation for n = 3 independent experiments.

  2. B

    U2OS cells treated +/− MS‐275 and NaAsO2 (100 μM, 1 h) were assessed for ROS levels using CM‐H2DCFDA. ROS levels were normalized to protein content. Error bars indicate SEM for n = 3 independent experiments, each performed in triplicate.

  3. C

    CHLA‐10 cells were treated without (vehicle) or with MS‐275 (1 μM, 24 h), and without (−NaAsO2) or with NaAsO2 (+NaAsO2) (500 μM, 1 h) and assessed for ROS levels using CM‐H2DCFDA. Data are presented as fold change over vehicle control. Error bars indicate SEM for n = 3 independent experiments; each performed in triplicate

  4. D

    U2OS cells treated +/− MS‐275 and NaAsO2 were assessed for ROS levels using the fluorogenic probe, CellRox. Data are presented as fold change over vehicle control. Error bars indicate SEM for n = 3 independent experiments; each performed in triplicate.

  5. E

    U2OS cells grown in monolayer cultures (2D) were treated with vehicle alone or antioxidants NAC (5 mM) and catalase (2,000 units/ml), prior to transfer to new 2D plates or ultra‐low attachment surface plates (3D) and cultured for a further 18 h with continuation of treatment as indicated. ROS levels were then assessed using CM‐H2DCFDA. Data are presented as fold change over vehicle control. Error bars indicate SEM for n = 3 independent experiments; each performed in triplicate.

  6. F

    U2OS cells grown in monolayers (3D−) or ultra‐low attachment plates (3D+) +/− MS‐275 and the antioxidant NAC were assessed for ROS levels using CM‐H2DCFDA. Data are presented as fold change over vehicle control. Error bars indicate SEM for n = 3 independent experiments; each performed in triplicate.

  7. G

    Effects of antioxidants on the viability of U2OS cells grown in 3D cultures in the presence of MS‐275 as above, determined by immunoblotting for cleaved caspase‐3 as indicated. GRB2 was used as a loading control.

  8. H

    Fractionation of polysomes extracted from U2OS cells +/− MS‐275 (1 μM, 24 h), and +/− NaAsO2 (100 μM, 1 h) on sucrose gradients displays marked differences in ribosome patterns dependent on displayed treatments. Total cytoplasmic polysomes were separated on sucrose gradients (10–45%) prepared using a BioComp Gradient Station unit and centrifuged using an SW60Ti rotor. Gradients were fractionated using a BioComp Gradient Fractionator equipped with a Triax module for acquisition of UV signal (260 nm).

  9. I, J

    Top panels: Immunoblots showing NRF2 protein decay after cycloheximide (CHX) addition in U2OS (I) and CHLA‐10 (J) cells +/− MS‐275 treatment. Cells were pre‐treated with the NRF2 inducing agent, SFN (L‐sulforaphane) at 20 μM conc for 4 h. Then, CHX was added along with SFN for the indicated time periods. GAPDH was used loading controls. Bottom panels: Graphical representation of NRF2 protein levels based on densitometry in U2OS (top panel I) and CHLA‐10 (top panel J) cells +/− MS‐275 treatment at the indicated time points after cycloheximide (CHX). Half‐lives are shown under the curves, representing results of two independent experiments ± SEM.

Data information: Unpaired two‐tailed Student's t‐test; *P < 0.05; **P < 0.005; ***P < 0.0005; n.s = non‐significant.Source data are available online for this figure.
Figure 2
Figure 2. MS‐275‐induced YB‐1 acetylation

  1. Scatterplot showing log2 transformed ratios of heavy amino acid (MS‐275 treated; +MS‐275) over light amino acid (vehicle; −MS‐275) conditions with the indicated NaAsO2 treatment (250 μM, 1 h). Dots are color coded to identify translation‐associated proteins (green) histones (orange) or other categories of proteins (gray).

  2. Analysis of YB‐1 acetylation in U2OS cells +/− MS‐275 (1 μM) over the indicated time course. Lysine‐acetylated proteins were affinity purified using anti‐acetyl‐lysine (α‐acK) antibodies and analyzed by immunoblotting using antibodies to YB‐1. IgG was used as negative antibody control and GAPDH as a loading control.

  3. Differential acetylation of FLAG‐tagged wt YB‐1 or the indicated FLAG‐tagged lysine (K) to alanine (A) YB‐1 mutant (K81A) in the presence of MS‐275. U2OS cells were transfected with FLAG‐tagged wt YB‐1 or FLAG‐tagged K81A. Cells were then treated with vehicle alone or MS‐275 (1 μM, 24 h). Lys‐acetylated proteins were immunoprecipitated using α‐acK antibodies, and acetylated FLAG‐tagged proteins were analyzed by anti‐FLAG, YB‐1, or acetylated histone H4 (Ac‐H4). IgG was used as a negative antibody control.

Source data are available online for this figure.
Figure EV2
Figure EV2. MS‐275‐induced acetylation of translation‐associated proteins

  1. Heatmap of translation‐associated proteins and histones from SILAC analysis, showing fold change represented as log2 transformed ratios of heavy amino acid (MS‐275 treated; +MS‐275) over light amino acid (vehicle; −MS‐275) conditions with or without NaAsO2 treatment (500 μM, 1 h), as described in the Materials and Methods. Color scale represents Log2 SILAC Ratio heavy/light (H/L).

  2. Analysis of YB‐1 and G3BP1 acetylation in U2OS cells +/− MS‐275 (1 μM, 24 h) and NaAsO2 (500 μM, 1 h) as indicated. Lysine‐acetylated proteins were affinity purified using anti‐acetyl‐lysine (α‐acK) antibodies and analyzed by immunoblotting, using antibodies to YB‐1 or G3BP. IgG was used as negative antibody control and GRB2 as a loading control.

  3. Immunoblotting showing time course analysis of Ac‐H4 (top) and total histone H4 acetylation (middle) in response to MS‐275 treatment (1 μM, 24 h) in U2OS cells. IgG was used as negative antibody control and GAPDH as a loading control.

  4. Analysis of YB‐1 acetylation in U2OS cells +/− the indicated class I HDAC inhibitor treatments for 24 h; MS‐275 (1 μM), Quisinostat, and Romidepsin (100 nM). Lysine‐acetylated proteins were affinity purified using anti‐acetyl‐lysine (α‐acK) antibodies and analyzed by immunoblotting, using antibodies to YB‐1. IgG was used as negative antibody control and GAPDH as a loading control.

  5. Immunoblotting showing expression levels of FLAG‐tagged wt YB‐1 or the indicated lysine (K) to alanine (A) (K‐to‐A) mutant (K81A) (top arrow) or endogenous YB‐1 in U2OS cells, as analyzed by antibodies to YB‐1. GRB2 was used as a loading control.

Source data are available online for this figure.
Figure 3
Figure 3. NRF2 is a novel YB‐1 downstream target

  1. Immunoblotting showing effects of YB‐1 KD on NRF2 expression in U2OS cells under the indicated oxidative stress inducing conditions: NaAsO2 (100 μM, 1 h) and H2O2 (200 μM, 1 h). GRB2 was used as a loading control

  2. In vitro cell‐free translation assay using reporter constructs with NFE2L2 or beta‐globin (HBB) 5′‐UTRs linked to the SP6 RNA polymerase promoter incubated with increasing amounts of recombinant YB‐1 protein and assessed for LUC activity. Results are displayed as means ± SD from two independent experiments, each performed in triplicate.

  3. Electrophoretic mobility gel shift assay (EMSA) to detect binding of recombinant YB‐1 to the NFE2L2 5′‐UTR. Biotin end‐tagged NFE2L2 5′‐UTR probe (Bio‐UTP‐NFE2L2 5′‐UTR) and unlabeled full‐length NFE2L2 5′‐UTR were incubated with recombinant GST‐YB‐1. Arrow shows supershifted Bio‐UTP‐RNA probe/YB‐1 complexes

  4. NFE2L2 mRNA binding to FLAG‐tagged wt or YB‐1‐K81A in CHLA‐10 cells +/− MS‐275 (1 μM, 24 h) and NaAsO2 (100 μM, 1 h), as measured by qRT–PCR of total RNA isolated from cell lysates. Data were normalized for each sample against the geometric mean of YBX1 mRNA binding and expressed as fold change ± SEM of two independent experiments, each performed in triplicate. Unpaired two‐tailed Student's t‐test;*P < 0.05; **P < 0.005.

  5. Acutely synthesized NRF2 in CHLA‐10 cells expressing wtYB‐1‐FLAG or YB‐1‐K81A‐FLAG. Cells were treated +/− MS‐275 (1 μM, 2 h). Then, cells were methionine starved and then pulsed with AHA and NaAsO2 (100 μM), with continuation of MS‐275 treatment for 1 h. Acutely synthesized NRF2 was identified by immunoblotting with NRF2 antibodies (top blot) and compared to total NRF2 levels (middle blot). Total GRB2 was used as a loading control (lower blot).

Source data are available online for this figure.
Figure 4
Figure 4. MS‐275‐induced YB‐1 acetylation inhibits YB‐1 translational regulation activity

  1. Immunoblotting showing time course analysis of HIF1α expression in response to MS‐275 treatment (1 μM) in CHLA‐10, incubated at 1% O2 for the indicated times. GRB2 was used as a loading control.

  2. Immunoblotting showing G3BP protein levels in U2OS cells treated with or without MS‐275 (1 μM) and NaAsO2 (100 μM) for the indicated time periods. GRB2 was used as a loading control.

  3. Total and polysomal RNA isolated from CHLA‐10 cells treated without (vehicle) or with MS‐275 (1 μM, 24 h) and further incubated under hypoxia (1% O2, 4 h) as indicated, was subjected to RT–PCR using HIF1A primers. Data were normalized against GAPDH expression. Mean values ± SEM (error bars) are shown for two independent experiments, each performed in triplicate.

  4. Total RNA from CHLA‐10 cells +/− MS‐275 treatment (1 μM, 24 h) and NaAsO2 (100 μM, 1 h) was subjected to polysomal fractionation. Total (red bars), and polysome‐bound mRNA levels (white bars) were determined by qRT–PCR using primers for G3BP1. Data were normalized against GAPDH expression. Mean values ± SEM. (error bars) are shown for two independent experiments, each performed in triplicate.

  5. HIF1A mRNA binding to FLAG‐tagged wt or YB‐1‐K81A in CHLA‐10 cells, subjected to the indicated treatments as described in (C), and as measured by qRT–PCR. Data were normalized for each sample against the geometric mean of YBX1 mRNA binding and expressed as fold change ± SEM of two independent experiments, each performed in triplicate.

  6. G3BP1 mRNA binding to FLAG‐tagged wt or YB‐1‐K81A in CHLA‐10 cells +/− MS‐275 (1 μM, 24 h) and NaAsO2 (100 μM, 1 h), as measured by qRT–PCR of total RNA isolated from cell lysates. Data were normalized for each sample against the geometric mean of YBX1 mRNA binding and expressed as fold change ± SEM of two independent experiments, each performed in triplicate.

Data information: Unpaired two‐tailed Student's t‐test;*P < 0.05; **P < 0.005; ***P < 0.0005; n.s = non‐significant.Source data are available online for this figure.
Figure EV3
Figure EV3. MS‐275‐mediated downregulation of YB‐1 targets

  1. Acutely synthesized G3BP in CHLA‐10 cells expressing wtYB‐1‐FLAG or YB‐1‐K81A‐FLAG. Cells were treated +/− MS‐275 (1 μM, 2 h). Then, cells were methionine starved and then pulsed with AHA and NaAsO2 (100 μM), with continuation of MS‐275 treatment for 1 h. Acutely synthesized G3BP was identified by immunoblotting with G3BP antibodies (top blot) and compared to total G3BP levels (middle blot). Total GAPDH was used as a loading control (lower blot).

  2. Acutely synthesized HIF1α in CHLA‐10 cells expressing wtYB‐1‐FLAG or YB‐1‐K81A‐FLAG. Cells were methionine starved and then treated with +/− MS‐275 (1 μM) along with AHA for 4 h under hypoxia (1% O2). Acutely synthesized HIF1α was identified by immunoblotting with HIF1α antibodies (top blot) and compared to total HIF1α levels (middle blot). Total GRB2 was used as a loading control (lower blot).

  3. Left panel: U2OS‐expressing FLAG‐tagged empty vector (EV) or the indicated vectors; G3BP1, wtYB‐1, or YB‐1 K81A mutant, respectively, were treated with MS‐275 and NaAsO2 (250 μM), prior to fixation and processing for IF and SG detection. Scale bars = 10 μm. Right panel: SGs were quantified in 20 high‐power fields (HPFs) using unpaired two‐tailed Student's t‐test, and results are represented by bar graphs. Error bars indicate SEM. ***P < 0.0005; n.s = non‐significant.

Source data are available online for this figure.
Figure EV4
Figure EV4. MS‐275 inhibits tumor metastasis in vivo

  1. (i‐ii) H&E‐stained representative sections of CHLA‐10 xenografts, representing vehicle‐treated and MS‐275‐treated tumors. Arrows show highly invasive growth patterns of vehicle tumor xenografts (i) and non‐invasive borders of MS‐275 treated xenografts (ii). (iii–iv) H&E staining of metastatic lung lesions (arrows) in mice with renal subcapsular tumor xenografts in the indicated mouse groups. (v–vi) Immunohistochemical staining (brown) of the EwS marker CD99 in lung tissues to highlight metastatic lesions.

  2. Local invasion in CHLA‐10 xenografts was assessed in 5 low power fields per tumor (3 tumors/group) using unpaired two‐tailed Student's t‐test and graphically represented. Error bars indicate SEM.

  3. Total number of mice bearing xenografts of the indicated CHLA‐10 tumor groups that developed lung metastases, determined using a Fisher's exact test.

  4. Analysis of YB‐1 acetylation in CHLA‐10 tumor lysates −/+ MS‐275 as indicated.

  5. Left panel: representative IHC images for the oxidative stress marker 4‐hydroxynonenal (4‐HNE) in renal subcapsular implantation site tumors (tumor #1–4) of the indicated CHLA‐10 tumor groups (vehicle and MS‐275 treated) using scale bars = 100 μm. Right panel: Quantification of 4‐HNE staining intensity in the indicated groups was assessed in 12 HPFs per tumor (4 tumors/group) using ImageJ software, statistically analyzed using unpaired two‐tailed Student's t‐test, and graphically represented. Error bars indicate SEM.

  6. Relative tumor sizes in EwS patient‐derived xenograft (PDX) model +/− MS‐275 treatment (8 mice/group). Tumor measurements were conducted three times/week, and mice were euthanized when humane endpoints were reached. Error bars indicate SEM.

  7. Kaplan–Meier survival curves, with P values computed with a log‐rank test, of ± MS‐275 groups of mice showing relative survival in both groups for a total of 55 days starting from the initiation of MS‐275 treatment. The numbers of mice are shown in brackets.

Data information: * indicates significant differences between the two groups, P < 0.05, **P < 0.005.Source data are available online for this figure.
Figure 5
Figure 5. MS‐275 inhibits sarcoma metastasis in vivo

  1. Left panel: Frozen sections of the indicated CHLA‐10 tumor groups (vehicle treated and MS‐275 treated) were assessed for ROS levels using dihydroethidium (DHE) staining (red). Right panel: Quantification of DHE staining in each group was assessed in 10 high‐power fields per tumor (four tumors/group) using ImageJ, analyzed with unpaired two‐tailed Student's t‐test, and graphically represented. Error bars indicate SEM.

  2. Left panel (Top): H&E‐stained representative sections of (IC‐pPDX‐3) EwS PDX xenografts in NRG mice, +/− MS‐275 treatment. Arrows show highly invasive growth patterns of vehicle tumor xenografts and non‐invasive borders of MS‐275‐treated xenografts. Left panel (Bottom): immunohistochemical staining (brown) of the EwS marker CD99. Right panel: quantification of local invasion in EwS PDX xenografts in vehicle‐treated and MS‐275‐treated groups as conducted using unpaired two‐tailed Student's t‐test. Average distances of invasion of single or tumor cell clusters at the tumor/kidney interface in 15 high‐power fields per tumor (three tumors/group) were assessed, normalized to vehicle‐treated group, and graphically represented. Error bars indicate SEM.

  3. Top panels: H&E staining of metastatic lung lesions (arrows) in mouse xenografts in the indicated tumor groups. Bottom panels: immunohistochemical staining (brown) of the Ewing sarcoma marker CD99 in lung metastases.

  4. Total number of mice bearing xenografts of the indicated PDX tumor groups that developed lung metastases, determined using a Fisher's exact test.

  5. Relative mean size of lung metastases developed in NRG mice (8/group) bearing EwS PDX (IC‐pPDX‐3) +/− MS‐275 treatment as conducted using unpaired two‐tailed Student's t‐test. Error bars indicate SEM.

  6. Immunoblotting showing YB‐1 acetylation in vivo. Tumor lysates from EwS PDX tumors +/− MS‐275 treatment were subjected to immunoprecipitation with α‐acK antibody as described in Fig EV4D. Acetylated YB‐1 was analyzed by immunoblotting using antibodies to YB‐1. IgG was used as negative antibody control, and GRB2 was used as a loading control to assess input loading.

Data information: *P < 0.05; ***P < 0.0005. All scale bars = 100 μm.Source data are available online for this figure.
Figure EV5
Figure EV5. MS‐275‐mediated inhibition of NRF2

  1. Left panel: IHC of NRF2 in CHLA‐10 xenografts from the indicated tumor groups, showing three representative tumors (tumor #1–3) per group. Scale bars = 50 μm. Right panel: Quantitation of staining intensity, normalized to control, was conducted using the color deconvolution plug‐in of ImageJ. Error bars indicate SEM for n = 15 representative images of three different tumors/group.

  2. IHC of total NRF2 at the tumor–normal kidney interface in CHLA‐10 primary implantation site tumors from the indicated tumor groups.

  3. Left panel: representative IHC images of NRF2 in tumor xenografts (tumor #1–3) of the indicated EwS IC‐pPDX‐3 tumor groups (vehicle treated and MS‐275 treated). Scale bars = 50 μm. Right panel: Quantification of staining intensity conducted using ImageJ. Error bars indicate SEM for n = 15 representative images of three different tumors/group.

  4. Immunoblot showing NRF2 expression in EwS PDX tumor lysates +/− MS‐275 treatment from three independent mouse tumors (vehicle or MS‐275 #1‐3) per group. GAPDH was used as a loading control.

  5. Immunoblot showing G3BP expression in CHLA‐10 tumor lysates +/− MS‐275 treatment from three independent mouse tumors (vehicle or MS‐275 #1–3) per group. GRB2 was used as a loading control.

  6. Immunoblot showing HIF1α expression in CHLA‐10 tumor lysates +/− MS‐275 treatment from three independent mouse tumors (vehicle or MS‐275 #1–3) per group. GRB2 was used as a loading control.

  7. Left panel: viable tumors areas were cryosectioned and subjected to IF with antibodies to the SG markers, FMRP (green) and TIA1 (red), to identify SGs. Scale bars = 10 μm. Right panel: quantification of stress granules (SGs) in each tumor group shown as a bar graph, in which 15 high‐power fields of representative tumor sections from each group (n = 3) were used for SG quantification using ImageJ software.

Data information: Unpaired two‐tailed Student's t‐test; **P < 0.005; ***P < 0.0005.Source data are available online for this figure.
Figure 6
Figure 6. YB‐1‐K81A rescues sarcoma cells’ metastatic phenotype in vivo

  1. Comparison of relative tumor sizes in CHLA‐10 tumor xenografts of the indicated groups (8 mice/group), 6 weeks post‐xenotransplantation +/− MS‐275 treatment was conducted using unpaired two‐tailed Student's t‐test. Error bars indicate SEM.

  2. H&E staining of metastatic lung lesions (arrows) in mice with renal subcapsular tumor xenografts of CHLA‐10 cells expressing empty vector (EV), wtYB‐1, or YB‐1K81A, and treated with vehicle or MS‐275 as described in Fig 5B. Scale bars = 100 μm.

  3. Total number of mice bearing xenografts of the indicated CHLA‐10 tumor groups that developed lung metastases, determined using a Fisher's exact test.

  4. Immunoblotting showing YB‐1 acetylation in vivo. Tumor lysates from the indicated tumor groups, +/− MS‐275 treatment were subjected to immunoprecipitation with α‐acK antibody as described in Fig EV4D. Acetylated YB‐1 was analyzed by immunoblotting using antibodies to YB‐1. IgG was used as negative antibody control, and Vinculin was used as a loading control to assess input loading.

  5. Immunoblot showing HIF1α, NRF2, G3BP, and YB‐1 expression in tumor lysates of CHLA‐10 cells expressing wtYB‐1, +/− MS‐275 treatment from three independent mouse tumors (vehicle or MS‐275 #1‐3) per group. GRB2 was used as a loading control. Note that HIF1α, NRF2, and G3BP are all downregulated in tumor lysates from MS‐275 treated mice.

  6. Immunoblot showing HIF1α, NRF2, G3BP, and YB‐1 expression in tumor lysates of CHLA‐10 cells expressing YB‐1‐K81A, +/− MS‐275 treatment from three independent mouse tumors (vehicle or MS‐275 #1‐3) per group. GAPDH was used as a loading control. Note the equal expression of HIF1α, NRF2, and G3BP in tumor lysates +/− MS‐275 treatment.

Data information: *P < 0.05; **P < 0.005; n.s = non‐significant.Source data are available online for this figure.
Figure 7
Figure 7. Correlation between YB‐1 expression and HIF1α, NRF2, and G3BP1 levels in EwS and OS
  1. A, B

    Scatterplots showing correlations between G3BP1‐HIF1A (left panel), G3BP1‐NFE2L2 (middle panel), and HIF1A‐NFE2L2 (right panel) mRNA expression in Ewing sarcoma (A, top panels) and osteosarcoma (B, bottom panels), quantified with Spearman's correlation, using publicly available cohorts of EwS (GSE63157) (A; top panel), and OS (GSE42352) (B; bottom panel), respectively. rho: Spearman's correlation coefficient.

Figure 8
Figure 8. Co‐expression of YB‐1 targets in clinical samples
  1. A

    Kaplan–Meier plots, with P‐values computed with a log‐rank test, showing relapse free survival in Ewing sarcoma (top panel) and overall survival in osteosarcoma patients (bottom panel) based on G3BP1 mRNA expression. Clinical data were obtained from the GSE63157 publicly available gene expression dataset for EwS and from http://r2.amc.nl for osteosarcoma.

  2. B, C

    Top panels: Immunohistochemistry (IHC) was conducted on serial sections of EwS (B) and OS (C) TMAs using antibodies against YB‐1, G3BP1, NRF2, and HIF1α. Spearman's rank correlation coefficient (rho; brown color), computed on H‐scores (staining intensity × percentage) for the different markers, along with relative P‐values in black color in the lower left quadrants are shown. Bottom panels: representative IHC images for YB‐1, G3BP1, NRF2, and HIF1α as indicated, on serial histological sections of EwS (bottom panel B; comparing primary to recurrent paired specimens) and OS (bottom panel C; comparing Stage IA to Stage IIB paired specimens). Scale bars = 100 μm.

Source data are available online for this figure.
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