Research ArticleCancer Metabolism

The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ

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Science Signaling  05 Dec 2017:
Vol. 10, Issue 508, eaan4667
DOI: 10.1126/scisignal.aan4667
  • Fig. 1 EphA2 activates the transcriptional coactivators YAP and TAZ in vitro.

    (A and B) Immunofluorescence of YAP (A) or TAZ (B) (both red) in MMTV-Neu cells infected with Ad-GFP or Ad-EphA2 for 72 hours. DAPI-stained nuclei (blue) were counted in ImageJ, and YAP- or TAZ-positive nuclei were counted using three fields of view in three independent experiments. Error bars are SEM. Scale bars, 10 μm. ***P < 0.005, Student’s t test. (C) Western blot of EphA2, YAP, and TAZ in MMTV-Neu cells infected with Ad-GFP or Ad-EphA2. (D) Super-resolution microscopy of YAP immunofluorescence from (A). Displayed image was compiled from 10 consecutive z-stack images. Signal intensity of YAP (red) or DAPI (blue) was determined per pixel moving across the y axis using ImageJ software. Scale bar, 10 μm. a.u., arbitrary units. (E) Relative mRNA expression was measured in MMTV-Neu cells retrovirally transduced with pBABE (“Control”) or pBABE-EphA2 (“EphA2”) by qRT-PCR from three independent experiments. Data are means ± SEM. *P < 0.05 and ***P < 0.005, Student’s t test.

  • Fig. 2 EphA2 activates YAP and TAZ in an MMTV-Neu mouse model.

    (A) Immunohistochemistry of YAP, TAZ, and EphA2 (brown staining) in tumors collected from wild-type (EFNA1+/+) and EFNA1 knockout (EFNA1−/−) mice. Scale bar, 10 μm. Percentages of YAP- or TAZ-positive nuclei and EphA2 intensity/cell were calculated on the basis of hematoxylin-stained nuclei (blue) using CellProfiler software from four fields per tumor sample (n = 4). *P < 0.05 and ***P < 0.005, Student’s t test. (B) Western blot of tumors collected from wild-type (EFNA1+/+) and EFNA1 knockout (EFNA1−/−) mice. The dotted line denotes noncontiguous lanes within a single blot. (C to E) Quantitation of Western blot data shown in (B) from EFNA1+/+ (n = 3) and EFNA1−/− (n = 3) tumors. (C) Phospho-YAP (Ser127 or Ser381) to total YAP, phospho-TAZ (Ser89) to total TAZ, (D) relative EFNA1 and EphA2 protein, and (E) phospho-EphA2 (Ser897 or Tyr588) are shown. Data are means ± SEM. *P < 0.05 and **P < 0.01, Student’s t test.

  • Fig. 3 EphA2 depends on Rho signaling to activate YAP and TAZ.

    (A to C) Immunofluorescence analysis of YAP (A) or TAZ (B) (red) and DAPI (blue) in MMTV-Neu cells infected with Ad-EphA2 and treated with PBS (control) or Rho inhibitor (CT04; 3 μg/ml for 4 hours). Scale bars, 10 μm. DAPI-stained nuclei were counted in ImageJ, and YAP- or TAZ-positive nuclei were counted using three fields of view in three independent experiments. Data (C) are means ± SEM. *P < 0.05 and **P < 0.01, Student’s t test. (D) Super-resolution microscopy of YAP immunofluorescence in control or CT04-treated cells from (A), compiled from 10 consecutive z-stacks. Scale bar, 10 μm. (E) Western blot analysis of cells in (A) and (B), treated with CT04 (3 μg/ml) for 6 hours. (F to H) As described for (A) to (C) with ROCK inhibitor (Y-27632; 10 μM for 4 hours). (I) Western blot analysis of cells in (F) and (G), treated with Y-27632 (10 μM) for 6 hours. (J) Western blot analysis of MMTV-Neu cells demonstrating ROCK inhibition by Y-27632.

  • Fig. 4 YAP and TAZ promote glutamine metabolism in a HER2-positive breast cancer model.

    (A) Intracellular glutamate concentration was measured 20 min after addition of EGF (20 ng/ml) + l-glutamine (2.5 mM) in MCF10A (“Parental”), MCF10A-EphA2 (“+EphA2”), MCF10A-HER2 (“+HER2”), or MCF10A-HER2-EphA2 (“+HER2/EphA2”) cells. Data were calculated as fold change from parental cells. Data are means ± SEM from three independent experiments. *P < 0.05 and **P < 0.01, one-way analysis of variance (ANOVA) and Tukey’s post hoc test. (B) Western blot of cells described in (A). (C) Intracellular glutamate concentration was determined as described in (B) in MCF10A-HER2 (“Control”) or MCF10A-HER2-EphA2 (“EphA2”) cells with YAP or TAZ knockdown and calculated as fold change from Control/siCtrl cells. Data are means ± SEM from three independent experiments. *P < 0.05 and **P < 0.01, two-way ANOVA and Tukey’s post hoc test. (D) Growth assay of YAP or TAZ knockdown MCF10A-HER2-EphA2 cells treated with vehicle (−DKG) or DKG (+DKG) for 3 days. Fold change in cell number was calculated on the basis of controls in each respective treatment group. Data are means ± SEM from three independent experiments. ***P < 0.005, two-way ANOVA and Tukey’s post hoc test.

  • Fig. 5 GLS and SLC1A5 gene expression is enhanced by EphA2-YAP/TAZ-TEAD4 signaling.

    (A to F) Abundance of the indicated mRNA (by qRT-PCR) and protein (by Western blotting) in MCF10A-HER2 cells after overexpression of EphA2 (A and B), overexpression of EphA2 and knockdown of YAP (C and D), or overexpression of EphA2 and knockdown of TAZ (E and F) relative to control cells. Data are means ± SEM from three independent experiments. **P < 0.01 and ***P < 0.005, Student’s t test (A) or one-way ANOVA (C and E) and Dunnett’s post hoc test. (G) ChIP-seq for TEAD4 at GLS (top) and SLC1A5 (bottom), downloaded from the University of California, Santa Cruz (UCSC) ENCODE Genome Browser. TEAD4-associated regions (hatched box) correlate with H3K4Me3 and H3K27Ac near exon 1 (black). (H and I) ChIP of MCF10A-HER2 cells transduced with shCtrl, shEphA2#1, or shEphA2#2. Relative immunoprecipitated genomic DNA was determined by qRT-PCR and normalized to IgG controls. EphA2 knockdown was confirmed by Western blotting (I). Data are means ± SEM from four independent experiments. #P < 0.005, two-way ANOVA and Tukey’s post hoc test. n.s., nonsignificant.

  • Fig. 6 EphA2 and YAP/TAZ expression correlates with increased glutaminolysis gene expression in human breast cancer patient data.

    (A and B) Log2 (mRNA) expression from the Minn Breast 2 database (n = 121). Pearson correlations (r) and t distribution (P) coefficients are shown. (C and D) Kaplan-Meier analysis of OS in HER2-positive breast cancer patients (C, n = 73; D, n = 117) exhibiting low (black) or high (red) (C) EPHA2, YAP, TAZ, and TEAD4 or (D) GLS and SLC1A5 expression. (E to G) Immunohistochemical analysis of YAP in HER2-positive samples from a human breast carcinoma TMA. Samples were categorized as metastatic or nonmetastatic, and images [represented in (E)] were assessed for YAP staining intensity per cell (F) and per sample (G). Scale bar, 10 μm. ***P < 0.005, Student’s t test. (G) Samples from (F) further stratified according to the amount of nuclear localization of YAP using the median percentage as the cutoff. χ2 analysis was calculated. (H and I) BPTES sensitivity in MMTV-Neu (H) or MCF10A-HER2 (I) cells retrovirally transduced with pBABE (“Control”) or pBABE-EphA2 (“EphA2”) and treated with vehicle or BPTES (10 μM) for 24 hours. Cell counts were calculated as fold change from vehicle controls from three independent experiments. ***P < 0.005, two-factor ANOVA (H) or one-way ANOVA (I) and Tukey’s post hoc test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/508/eaan4667/DC1

    Fig. S1. EphA2 activates YAP/TAZ in MCF10A-HER2 cells.

    Fig. S2. EphA2–ephrin-A1 interactions do not significantly contribute to YAP/TAZ activation.

    Fig. S3. Rho and ROCK inhibition did not enhance LATS1 phosphorylation in MMTV-Neu cells.

    Fig. S4. EphA2-mediated YAP/TAZ activation requires Rho catalytic activity.

    Fig. S5. YAP and TAZ promote glutamine metabolism in MCF10A-HER2-EphA2 cells.

    Fig. S6. YAP and TEAD4 are associated with GLS and SLC1A5 promoters.

    Fig. S7. YAP/TAZ and EphA2 expression strongly correlates with decreased patient survival in HER2-positive breast cancer.

    Table S1. ChIP primers.

    Movie S1. EphA2 overexpression leads to intranuclear accumulation of YAP.

    Movie S2. Nuclear exclusion of YAP upon Rho inhibition.

  • Supplementary Materials for:

    The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ

    Deanna N. Edwards, Verra M. Ngwa, Shan Wang, Eileen Shiuan, Dana M. Brantley-Sieders, Laura C. Kim, Albert B. Reynolds, Jin Chen*

    *Corresponding author. Email: jin.chen{at}vanderbilt.edu

    This PDF file includes:

    • Fig. S1. EphA2 activates YAP/TAZ in MCF10A-HER2 cells.
    • Fig. S2. EphA2–ephrin-A1 interactions do not significantly contribute to YAP/TAZ activation.
    • Fig. S3. Rho and ROCK inhibition did not enhance LATS1 phosphorylation in MMTV-Neu cells.
    • Fig. S4. EphA2-mediated YAP/TAZ activation requires Rho catalytic activity.
    • Fig. S5. YAP and TAZ promote glutamine metabolism in MCF10A-HER2-EphA2 cells.
    • Fig. S6. YAP and TEAD4 are associated with GLS and SLC1A5 promoters.
    • Fig. S7. YAP/TAZ and EphA2 expression strongly correlates with decreased patient survival in HER2-positive breast cancer.
    • Table S1. ChIP primers.
    • Legends for movies S1 and S2

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.30 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). EphA2 overexpression leads to intranuclear accumulation of YAP.
    • Movie S2 (.mov format). Nuclear exclusion of YAP upon Rho inhibition.

    Citation: D. N. Edwards, V. M. Ngwa, S. Wang, E. Shiuan, D. M. Brantley-Sieders, L. C. Kim, A. B. Reynolds, J. Chen, The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ. Sci. Signal. 10, eaan4667 (2017).

    © 2017 American Association for the Advancement of Science

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