Research ArticleCancer

The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation

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Science Signaling  08 Dec 2015:
Vol. 8, Issue 406, pp. ra124
DOI: 10.1126/scisignal.aac6479
  • Fig. 1 PKCε-ATF2 signaling transcriptionally represses FUK and cellular fucosylation.

    (A) qRT-PCR analysis for FUK or PKCε expression in WM793 cells transfected with (left) EV, ATF2-targeted shRNA (shATF2), or shATF2 and ATF2WT, ATF2T52A, or ATF2T52E; or (right) control (siCTL) or PKCε-targeted siRNA (siPKCε). (B) qRT-PCR for FUK mRNA expression (left) and immunoblotting for FUK protein abundance (right) in primary human melanocytes (HEMn), VGP melanoma cell lines (WM793, WM1346, and WM1366), metastatic melanoma cell lines (LU1205, 501Mel, and YUGASP), and transformed HEK293 cells. pATF2, phosphorylated ATF2; tATF2, total ATF2. (C) Top: FUK 5′ promoter with ATF2 binding sites (E1, E2, and E3), ChIP targets, and transcriptional start site. Bottom: ATF2 ChIP of the FUK 5′ promoter E1 and E2/3 targets in 501Mel (left) and LU1205 (right) cells. IgG, immunoglobulin G. (D) Wild-type (WT) or E1, E2, or E3 single-mutant FUK promoter luciferase activities in (left) WM793 cells transfected with EV (black) or ATF2T52E (gray) or in (right) 501Mel cells. (E) Fluorescence-activated cell sorting (FACS) analysis of UEA1 lectin binding of cell lines in (B). Graphic inset represents a glycan recognized by UEA1 (red triangles: α-1,2/1,3-fucose; dark gray squares: N-acetylglucosamine; light gray circles: galactose). (F) UEA1 FACS analysis in WM793 cells transfected with (left) EV, ATF2T52E, or caPKCε or (right) control or FUK-targeted shRNA (shFUK). (G) FACS analysis of LCA and PSA in WM793 cells transfected as indicated. Graphic inset represents a glycan recognized by LCA or PSA (red triangles: α-1,4/1,6-fucose). (H) A schematic of the fucose salvage and de novo synthesis pathways. All data are means ± SD from three experiments. *P < 0.05, **P < 0.005, ***P < 0.0005 by a standard t test compared with controls.

  • Fig. 2 FUK expression reduces melanoma motility and invasiveness and increases adhesion.

    (A) Migration of LU1205 (left) or 501Mel (right) cells transfected with EV or FUK, assessed by scratch assays. (B) Migration of WM793 or 501Mel cells assessed by scratch assays in cultures supplemented with control water (5 μl) or 50 μM l-fucose (left) or 25 μM ac-GDP-l-fucose (right) for 36 hours. (C) Migration of WM793 cells transfected with control shRNA (shEV), one of two FUK-targeted shRNAs, ATF2T52E, or ATF2T52E and FUK, assessed by scratch assays. (D) The number of EV- or FUK-expressing HEK293 cells that remained attached (per 10× field) after wash-off at the indicated time points was quantified (four 10× fields per time point were counted). (E) Representative EV- or FUK-expressing LU1205 (left) or 501Mel (right) spheroids from three independent experiments. (F) Representative image (left) and quantitation (right) of the area of GFP-EV– or GFP-FUK–expressing 501Mel spheroids grown in Matrigel plugs. Data in (A) to (D) and (F) are means ± SD from three experiments. *P < 0.05, **P < 0.005, ***P < 0.0005 by a standard t test compared with controls or starting time point. Scale bars, 100 μm.

  • Fig. 3 Increased FUK expression attenuates melanoma development and metastasis and correlates with increased survival in human patient samples.

    (A) qRT-PCR analysis of PKCε and mFuk expression in SW1 and K1735 cells. (B and C) Scratch assays assessing the migration of SW1 cells (B) in which cultures were supplemented with control water (Control; 5 μl) or l-fucose (L-Fuc; 50 μM) for 36 hours or (C) in which cells were transfected with EV or mFuk. (D) Two representative spheroids (n = 3 experiments) formed by EV- or FUK-expressing SW1 cells in culture. Scale bars, 10 μm. (E and F) Tumor volume curves (left) and lung metastasis counts (right) of (E) C3H/HeJ mice bearing implanted SW1 tumors or (F) mice bearing implanted EV- or mFuk-expressing SW1 tumors provided with control water (Control) or water supplemented with l-fucose (Fucose, 100 mM). Data are means ± SD from six mice per group. (G) Representative UEA1-low (upper) and UEA1-high (lower) histospots from a human melanoma TMA (n = 320) immunostained with UEA1 lectin (green), S100/HMB45 (red), and 4′,6-diamidino-2-phenylindole (DAPI, blue). (H) Distribution of UEA1 scores within S100/HMB45-positive melanoma cells of individual histospots from primary or metastatic melanoma samples. Means, SD, and n as indicated; P = 0.0002 by unpaired t test. Diamonds depict means ± 1 SD. (I) Kaplan-Meier survival curves according to UEA1 abundance in a melanoma patient cohort. Data in (A) to (C) are means ± SD from three experiments. *P < 0.05, **P < 0.005 by a standard t test compared with parental K1735 cell lines, controls, or starting time point.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/406/ra124/DC1

    Methods

    Fig. S1. Transcriptional regulation of FUK by ATF2 and l-fucose uptake in melanoma cells.

    Fig. S2. Increasing melanoma fucosylation slows motility and invasiveness.

    Fig. S3. Increasing melanoma tumor fucosylation increases intratumoral CD45+ and NKp46+ immune cell infiltrates.

    Table S1. Primers.

  • Supplementary Materials for:

    The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation

    Eric Lau,* Yongmei Feng, Giuseppina Claps, Michiko N. Fukuda, Ally Perlina, Dylan Donn, Lucia Jilaveanu, Harriet Kluger, Hudson H. Freeze, Ze'ev A. Ronai*

    *Corresponding author. E-mail: eric.lau{at}moffitt.org (E.L.); zeev{at}ronailab.net (Z.A.R.)

    This PDF file includes:

    • Methods
    • Fig. S1. Transcriptional regulation of FUK by ATF2 and L-fucose uptake in melanoma cells.
    • Fig. S2. Increasing melanoma fucosylation slows motility and invasiveness.
    • Fig. S3. Increasing melanoma tumor fucosylation increases intratumoral CD45+ and NKp46+ immune cell infiltrates.
    • Table S1. Primers.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.43 MB


    Citation: E. Lau, Y. Feng, G. Claps, M. N. Fukuda, A. Perlina, D. Donn, L. Jilaveanu, H. Kluger, H. H. Freeze, Z. A. Ronai, The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation. Sci. Signal. 8, ra124 (2015).

    © 2015 American Association for the Advancement of Science

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