Research ArticleCancer

Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion

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Science Signaling  07 Mar 2017:
Vol. 10, Issue 469, eaal2464
DOI: 10.1126/scisignal.aal2464
  • Fig. 1 VAV2 is an SF-1 transcriptional target in ACC cells.

    (A) ChIP-seq data (17) showing enrichment of SF-1 binding in correspondence with upstream and intronic sites of the VAV2 gene after Dox treatment of H295R-TR SF-1 cells. Peaks of SF-1 binding are framed. (B) Time course of VAV2 mRNA abundance after Dox treatment of H295R-TR SF-1 cells. (C) Time course of SF-1, VAV2, and β-tubulin protein abundance after Dox treatment of H295R-TR SF-1 cells. (D and E) Quantification of VAV2 (D) and SF-1 (E) protein abundance represented in (C) after normalization to that of β-tubulin. ctrl, control. (F) Time course of VAV2 and phospho-VAV2 (Tyr174) (p-VAV2) protein abundance after Dox treatment of H295R-TR SF-1 cells. (G) Quantification of (F) as a ratio of phospho-VAV2 to total VAV2. Data are means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, analysis of variance (ANOVA) with Dunnett’s posttest for multiple comparisons.

  • Fig. 2 Increased SF-1 dosage in ACC cells induces an increase in Cdc42 and Rac1, but not RhoA, activity.

    (A and B) Representative images (A) and quantification (B) of CDC42 activity measured by FRET analysis in H295R-TR SF-1 cells treated with either vehicle or Dox. (C and D) As in (A) and (B) for RAC1 activity. (E and F) As in (A) and (B) for RHOA activity. Scale bars, 10 μm. (G) Quantification of CDC42 (green histogram), RAC1 (black histogram), and RHOA (red histogram) activities by a biochemical assay (G-LISA) in Dox-treated H295R-TR SF-1 cells. FRET data are means ± SEM of 20 cells analyzed per sample. G-LISA data are means ± SEM of at least three independent experiments performed in duplicate. *P < 0.05; ***P < 0.001, Mann-Whitney test.

  • Fig. 3 Increased SF-1 dosage in ACC cells induces cytoskeleton remodeling.

    (A) SF-1 (green) and actin cytoskeleton labeled by phalloidin (red) in H295R-TR SF-1 cells treated with either vehicle or Dox. Scale bars, 5 μm. Insets show filopodia and lamellipodia-ruffles present only in the cell treated with Dox. (B to E) Time course of quantification of filopodia-forming cells (B) and lamellipodia-ruffles–forming cells (D) after Dox treatment in H295R-TR SF-1 cells and in the parental H295R-TR cell line (C and E). (F) Percentage of filopodia-forming cells and (G) number of filopodia per cell in H295R-TR SF-1 cells treated with either vehicle (white histogram) or Dox for 48 hours. The Dox-treated cell population was subdivided into cells with low (gray histogram) or high (black histogram) SF-1 abundance, as measured by immunofluorescence. Data are means ± SEM of at least three independent experiments, each analyzing more than 250 cells per condition. *P < 0.05; **P < 0.01; ***P < 0.001, Fisher’s exact test (B to E) and ANOVA with Dunnett’s posttest for multiple comparisons (F and G).

  • Fig. 4 VAV2 is critical for cytoskeleton remodeling triggered by an increased SF-1 dosage in ACC cells.

    (A) Cytoskeletal morphology revealed by phalloidin staining (red) in H295R-TR SF-1 cells transfected with vectors encoding either GFP or GFP-VAV2 (green). Scale bars, 5 μm. (B) VAV2 protein knockdown by nucleofection of specific siRNA (siVAV2) compared to control siRNA (siC) in H295R-TR SF-1 cells treated with either vehicle or Dox. β-Tubulin is shown as a control. (C) Quantification of VAV2 knockdown. White histograms, cells nucleofected with siC; gray histogram, cells nucleofected with siVAV2. (D to F) Effect of VAV2 knockdown on the percentage of filopodia-forming (D) and lamellipodia-ruffle–forming cells (F) and the number of filopodia per cell (E) in Dox-treated H295R-TR SF-1 cells. Data are means ± SEM of at least three independent experiments, each analyzing more than 250 cells per condition. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA with Tukey’s posttest for multiple comparisons.

  • Fig. 5 VAV2 is critical for the increase in cell invasion triggered by an increased SF-1 dosage in ACC cells.

    (A) H295R-TR SF-1 cells migrated to the bottom side of a Matrigel filter during an invasion assay in cells treated with vehicle or Dox. Scale bars, 50 μm. (B) Quantification of the Matrigel invasion assay. Percentage of invading cells compared to control (vehicle-treated) for the parental H295R-TR clone (black histogram), H295R-TR SF-1 cells expressing wild-type (WT) SF-1 (red histogram), and H295R-TR SF-1 cells expressing the AF-2 SF-1 mutant (AF-2 mut) (green histogram). (C) Diagram of the CAM invasion assay. Cancer cells (green) are grafted on the upper part of the chorionic epithelium (upper CAM) of the chicken embryo. They can invade through the basement membrane (blue) of the chorionic epithelium and into vascular structures (pink) and diffuse to distant structures and organs, including the lower part of the chorionic epithelium (lower CAM). This method was reproduced with permission from (29). (D) Quantification of invasion of H295R-TR SF-1 cells in the CAM assay in samples treated with either vehicle or Dox. Ten eggs per condition were analyzed. (E) VAV2 protein knockdown 6 days after nucleofection of siVAV2 compared to siC in H295R-TR SF-1 cells cultured in the presence of vehicle or Dox. (F) Quantification of VAV2 knockdown. White histograms, siC; gray histogram, siVAV2. (G) Rescue of VAV2 protein abundance by cotransfection of GFP, WT GFP-VAV2, or GEF-inactive GFP-VAV2 mutant together with siC and siVAV2, as indicated. (H) Quantification of the Matrigel invasion assay in H295R-TR SF-1 cells treated with either vehicle or Dox and cotransfected with siC-siVAV2 and vectors for GFP, WT GFP-VAV2, or GEF-inactive GFP-VAV2 mutant, as indicated. Results represent means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA with Dunnett’s posttest for multiple comparisons (B), Mann-Whitney test (D), and ANOVA with Tukey’s posttest for multiple comparisons (F and H).

  • Fig. 6 High VAV2 levels are a negative prognostic marker in ACC.

    (A) Correlation between SF-1–NR5A1 and VAV2 mRNA levels in the Cochin cohort. r = 0.59, P = 1.26 × 10−5. (B) Correlation between SF-1–NR5A1 and VAV2 mRNA expression in the TCGA cohort. r = 0.32, P = 0.00364. (C) Relative abundance of VAV2 mRNA in the low (green) and high (red) groups of the Cochin cohort. P = 2.31 × 10−12, Mann-Whitney test. (D) Relative abundance of VAV2 mRNA in the low (green) and high (red) groups of the TCGA cohort. P = 8.6 × 10−14, Mann-Whitney test. (E) Graph showing overall survival for the groups of ACC patients in the Cochin cohort with low (green curve) and high (red curve) VAV2 mRNA. HR, 4.97; P = 0.002, Kaplan-Meier method. (F) Graph showing overall survival for the groups of ACC patients in the TCGA cohort with low (green curve) and high (red curve) VAV2 mRNA. HR, 2.35; P = 0.033, Kaplan-Meier method. (G) Examples of VAV2 IHC staining in ACC TMA samples presenting different H-scores (0 to 3), cortisol-producing adrenocortical adenoma (CPA), and colon carcinoma. (H) Heatmap of H-scores for SF-1 and VAV2 in German ACC cases. P < 0.00001, χ2 test. (I) Left: Recurrence-free survival for the groups of ACC patients in the German cohort displaying low (green curve) and high (red curve) VAV2 IHC staining (univariate analysis). HR, 3.25; P = 0.004. Right: Overall survival for the groups of ACC patients in the German cohort displaying low (green curve) and high (red curve) VAV2 IHC staining (univariate analysis). HR, 1.04; P = 0.042, Kaplan-Meier method. (J) Left: Recurrence-free survival for the groups of ACC patients in the German cohort displaying low (green curve) and high (red curve) VAV2 IHC staining (multivariate analysis). HR, 5.84, P < 0.0001. Right: Overall survival for the groups of ACC patients in the German cohort displaying low (green curve) and high (red curve) VAV2 IHC staining (multivariate analysis). HR, 2.24; P = 0.01, Kaplan-Meier method.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/469/eaal2464/DC1

    Fig. S1. SF-1 dosage–dependent effects in H295R-TR SF-1 cells.

    Fig. S2. High VAV2 abundance is a negative prognostic marker in ACC patients after complete tumor resection.

    Fig. S3. Distribution of Ki67 LI in patient tumors grouped by VAV2 abundance.

    Table S1. Statistical data of the Cochin and TCGA cohorts of ACC patients.

    Table S2. Statistical data of the German cohort of ACC patients.

  • Supplementary Materials for:

    Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion

    Carmen Ruggiero, Mabrouka Doghman-Bouguerra, Silviu Sbiera, Iuliu Sbiera, Maddy Parsons, Bruno Ragazzon, Aurélie Morin, Estelle Robidel, Judith Favier, Jérôme Bertherat, Martin Fassnacht, Enzo Lalli*

    *Corresponding author. Email: ninino{at}ipmc.cnrs.fr

    This PDF file includes:

    • Fig. S1. SF-1 dosage–dependent effects in H295R-TR SF-1 cells.
    • Fig. S2. High VAV2 abundance is a negative prognostic marker in ACC patients after complete tumor resection.
    • Fig. S3. Distribution of Ki67 LI in patient tumors grouped by VAV2 abundance.
    • Legends for tables S1 and S2

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Statistical data of the Cochin and TCGA cohorts of ACC patients.
    • Table S2 (Microsoft Excel format). Statistical data of the German cohort of ACC patients.

    [Download Tables S1 and S2]


    Citation: C. Ruggiero, M. Doghman-Bouguerra, S. Sbiera, I. Sbiera, M. Parsons, B. Ragazzon, A. Morin, E. Robidel, J. Favier, J. Bertherat, M. Fassnacht, E. Lalli, Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion. Sci. Signal. 10, eaal2464 (2017).

    © 2017 American Association for the Advancement of Science

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