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SILAC identifies LAD1 as a filamin-binding regulator of actin dynamics in response to EGF and a marker of aggressive breast tumors

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Science Signaling  30 Jan 2018:
Vol. 11, Issue 515, eaan0949
DOI: 10.1126/scisignal.aan0949
  • Fig. 1 SILAC-based phosphoproteome analysis of EGF-stimulated mammary cells.

    (A) MCF10A cells (0.5 × 105) were seeded in Transwells without or with epidermal growth factor (EGF; 10 ng/ml) in the presence of either untreated or dialyzed serum (D. serum). Sixteen hours later, cells were fixed in paraformaldehyde (3%), washed, and stained using crystal violet. Histograms are means ± SE of three experiments. ***P < 0.001. Scale bars, 1 mm. n.s., nonsignificant. (B) MTT assay of MCF10A cells (2 × 103) seeded in a 96-well plate and grown for 4 days with or without EGF (10 ng/ml). Data are means ± SE of quadruplicate experiments (***P < 0.001). (C) A schematic representation of the experimental layout of stable isotope labeling by amino acids in tissue culture (SILAC)–based analysis. Arrowheads mark time points of cell lysate preparation. (D) Relative abundances of phosphorylated residues in the proteomic data set (PXD008100 in ProteomeXchange). (E) Heatmap representation of the abundance of specific phosphopeptides in MCF10A cells stimulated with either EGF or serum for the indicated time intervals. Signals were normalized to the unstimulated state and sorted according to the timing of their peak (see the color bar for reference; missing values are shown in white).

  • Fig. 2 Proteomic and biochemical data identifies LAD1 as a downstream target of active EGFRs.

    (A) Listed are proteins exhibiting phosphorylation changes in response to EGF or serum treatment. The relevant phosphorylated amino acids are indicated. The color bar represents signal intensities. TMEM131, transmembrane protein 131. (B) A heatmap of all ladinin-1 (LAD1) phosphopeptides identified using SILAC. The relevant phosphorylated amino acid, either serines or threonines, is indicated. (C) Serum-starved MCF10A cells were stimulated with EGF or serum for different time intervals, and extracts were immunoblotted (IB) with the indicated antibodies (AB). The blot is representative of three replicates. (D) MCF10A cells overexpressing LAD1-V5 were serum-starved and then preincubated with the indicated inhibitors before EGF stimulation. Wort, Wortmannin; GAPDH; glyceraldehyde 3-phosphate dehydrogenase. Cell extracts were as indicated. Blot is representative of three replicates. (E) Serum-starved MCF10A cells were stimulated for 60 min with EGF. Extracts were subjected to immunoprecipitation (IP) with an antibody to LAD1 and incubated without or with calf intestinal alkaline phosphatase (CIP) before immunoblotting as indicated. Data are from three experiments. (F) MDA-MB-231 cells ectopically expressing the indicated mutants of V5-tagged LAD1 were serum-starved and preincubated with U0126 before stimulation with EGF. Cell extracts were analyzed as in (D).

  • Fig. 3 LAD1 is partly colocalized with the actin cytoskeleton and its C-terminal region associates with F-actin.

    (A) MCF10A cells were stained for LAD1 (red), actin (green), and nuclei (blue). LAD1 and actin colocalization revealed in the merged images (yellow). Scale bar, 10 μm. (B) Serum-starved MCF10A cells ectopically expressing V5-LAD1 were treated with EGF for the indicated time intervals, fractionated, and immunoblotted as indicated. The black and gray arrowheads mark the endogenous and ectopic LAD1-V5, respectively. Data are representative of three replicates. (C) MCF10A cells were visualized using the indicated antibodies, phalloidin, LifeAct-cherry (red), or LAD1-GFP (green). Scale bar, 10 μm. (D) Serum-starved MCF10A cells were treated with EGF, fixed, and probed for LAD1 (green), F-actin (red), and 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 30 μm. (E) Extracts from MCF10A and HCC70 cells (2 × 104) were preincubated (10 min) in F-actin–stabilizing buffer, in the absence or presence of phalloidin. Thereafter, soluble and filamentous actin forms were separated using an ultracentrifuge, and the fractions were probed with the indicated antibodies. Sup, supernatant. (F) MDA-MB-231 expressing V5-tagged forms of wild-type (WT) or deletion mutants of LAD1 [N terminal (N-term.) amino acids 1 to 276 and C-terminal (C-term.) amino acids 277 to 517] were treated and processed as in (E). Data are representative of two experiments. Cont., control.

  • Fig. 4 LAD1 regulates the migratory and invasive potential of mammary cells.

    (A) Extracts from MCF10A cells stably expressing shRNAs specific to LAD1 (or a scrambled shRNA) were immunoblotted using the indicated antibodies. Quantification shows normalized LAD1 abundance. Data are means ± SE of three independent replicates. (B) MCF10A cells expressing shLAD1 were incubated for 24 hours in Transwell chambers or in Matrigel-coated chambers. Migration and invasion were quantified after staining with crystal violet. The experiments were repeated thrice. Scale bar, 1 mm. *P < 0.05, **P < 0.01. (C) The indicated cells were plated on fibronectin, and 60 min later, cells that did not adhere to the substrate were removed. The numbers of adherent cells were determined after staining (in triplicates). *P < 0.05. (D) The indicated derivatives of MCF10A cells (1 × 105) were incubated in EGF-containing medium on plates precoated with fluorescein isothiocyanate (FITC)–labeled gelatin. After 12 hours of incubation, the cells were fixed and counterstained with rhodamine-phalloidin and DAPI. The broken line insets were magnified (×2) and presented (upper right corners). Quenched spots of gelatin were quantified in at least eight nonoverlapping fields. *P < 0.05, **P < 0.01 [one-way analysis of variance (ANOVA) with Bonferroni’s correction]. Scale bars, 10 μm. Data are means ± SEM. Each experiment was performed in duplicates.

  • Fig. 5 Mammary cell proliferation is inhibited when LAD1 is depleted.

    (A) MTT analysis of viability in MCF10A cells expressing shLAD1 after 4 days in culture. Data are means ± SEM of six replicates. *P < 0.05, ***P < 0.001 (one-way ANOVA with Bonferroni’s correction). (B) Extracts of the indicated breast cancer lines were probed as indicated. (C) Serum-starved MDA-MB-231 cells expressing LAD1-V5 and shLAD1 MCF10A cells were stimulated with EGF (12 hours). Bromodeoxyuridine (BrdU) was added, and 60 min later, cells were stained. DAPI- and BrdU-stained nuclei were counted (in >5 fields). Data are means ± SEM of three experiments. *P < 0.05, ***P < 0.001. (D) MCF10A cells (5 × 103) stably expressing shLAD1 (or shScramble) were grown in EGF-containing Matrigel (2 weeks), acini were photographed, and ImageJ was used to determine volumes. ***P < 0.0001. Scale bar, 100 μm. Data are from three experiments. (E) MCF10A cells were incubated with RO-3306 (10 nM; 15 hours), washed, cultured (<90 min), and stained for DAPI, LAD1 (green), and acetylated tubulin (red). (F) Cells pretreated as in (D) were fixed and probed at metaphase. Scale bar, 10 μm. (G) Cells ectopically expressing LAD1-GFP and LifeAct-cherry were pretreated as in (D). Frames taken from live cell movies are shown. Scale bar, 10 μm.

  • Fig. 6 LAD1 physically interacts and colocalizes with FLNA.

    (A) Images of MCF10A cells probed for LAD1 (red), filamin A (FLNA; green), and nuclei (DAPI, blue). Arrowheads mark colocalization. Scale bar, 10 μm. (B) MCF10A cells were probed with antibodies recognizing LAD1 and FLNA and processed for proximity ligation assay (PLA) with a tetramethylrhodamine-5-isothiocyanate (TRITC) probe (red). Counterstaining used DAPI (blue) and phalloidin-FITC (green). Single-antibody control experiments are shown. Scale bar, 10 μm. (C) Extracts of MDA-MB-231 cells overexpressing the indicated forms of LAD1 were subjected to coimmunoprecipitation using beads conjugated to an anti-V5 antibody. The eluates were blotted as indicated, and bands were quantified in biological duplicates. Both short (short exp.; 10 s) and longer film exposure (long exp.) are shown. (D) MCF10A cells underwent transfection with siFLNA oligonucleotides. Serum-starved cells were stimulated with EGF for the indicated time intervals, and extracts were immunoblotted as indicated. The experiment was repeated thrice. (E) A model depicting the putative signaling events leading to association of LAD1 with actin treadmilling. LAD1 undergoes phosphorylation at multiple sites downstream to the MEK pathway. This augments binding of LAD1 with 14-3-3σ, a cytoskeletal solubility cofactor. Another ligand of LAD1 is FLNA, which cross-links actin filaments. By receiving extracellular signals, LAD1 might regulate actin polymerization, thereby controlling cell migration and proliferation during cancer progression.

  • Fig. 7 LAD1 depletion retards basal-like breast tumors in animals, and high LAD1 associates with poor patient prognosis.

    (A) HCC70 cells stably expressing shLAD1 were analyzed using immunoblotting and polymerase chain reaction (PCR). PCR signals were quantified relative to β2-microglobulin. Blots are representative of three experiments. (B) HCC70 cells were incubated in the presence of BrdU (60 min). BrdU- and DAPI-stained nuclei were counted in several fields. Shown are histograms. Data are means ± SEM from three experiments. *P < 0.05, ***P < 0.001 by one-way ANOVA with Tukey’s correction. (C) HCC70 cells (3.5 × 106) expressing shLAD1 were implanted in six CD1/nude mice, and tumorigenic growth was monitored. Data are mean tumor volumes (±SEM). Growth rate effects of shLAD1.1 versus shLAD1.2 compared to shScramble were P < 0.05 and P < 0.01, respectively, by two-way ANOVA of repeated-measures with Dunnett’s test. (D) Extracts prepared from the xenografts presented in (C) were analyzed using immunoblotting as indicated. Note that two tumors were analyzed per group. (E) Analysis of LAD1 in the 10 integrative subtypes of breast cancer (n = 1980). (F) Patients were divided into three groups: 15% upper and lower LAD1 mRNA abundance and the rest. Breast cancer–related deaths are shown (brackets), along with survival curves (P < 0.001).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/515/eaan0949/DC1

    Fig. S1. LAD1 is an intracellular protein that undergoes MEK-dependent phosphorylation inresponse to EGF.

    Fig. S2. Genomic, proteomic, and transcriptional characteristics of LAD1.

    Fig. S3. Knockdown of LAD1 impairs invadopodia formation by mammary cells.

    Fig. S4. Phosphorylation of LAD1 on specific serine sites moderately regulates cell migration and viability, without markedly affecting FLNA binding.

    Fig. S5. LAD1 increases EGF-inducible ERK phosphorylation and physically interacts with14-3-3σ (SFN).

    Fig. S6. Effects of LAD1 on specific transcripts of breast cancer cells.

    Fig. S7. Like LAD1, SFN expression correlates with poor prognosis in breast cancer patients;FLNA expression displays distinct pathological patterns.

    Table S1. SILAC data.

    Table S2. EGF-induced phosphorylation changes.

    Table S3. Serum-induced phosphorylation changes.

    Table S4. Lists of putative partners of LAD1 revealed by using either Y2H screens or proteomicanalyses of coimmunoprecipitataed proteins.

    Table S5. RNA-seq data.

    Table S6. GOrilla analysis.

    Table S7. Primers.

  • Supplementary Materials for:

    SILAC identifies LAD1 as a filamin-binding regulator of actin dynamics in response to EGF and a marker of aggressive breast tumors

    Lee Roth, Swati Srivastava, Moshit Lindzen, Aldema Sas-Chen, Michal Sheffer, Mattia Lauriola, Yehoshua Enuka, Ashish Noronha, Maicol Mancini, Sara Lavi, Gabi Tarcic, Gur Pines, Nava Nevo, Ori Heyman, Tamar Ziv, Oscar M. Rueda, Davide Gnocchi, Eli Pikarsky, Arie Admon, Carlos Caldas, Yosef Yarden*

    *Corresponding author. Email: yosef.yarden{at}weizmann.ac.il

    This PDF file includes:

    • Fig. S1. LAD1 is an intracellular protein that undergoes MEK-dependent phosphorylation in response to EGF.
    • Fig. S2. Genomic, proteomic, and transcriptional characteristics of LAD1.
    • Fig. S3. Knockdown of LAD1 impairs invadopodia formation by mammary cells.
    • Fig. S4. Phosphorylation of LAD1 on specific serine sites moderately regulates cell migration and viability, without markedly affecting FLNA binding.
    • Fig. S5. LAD1 increases EGF-inducible ERK phosphorylation and physically interacts with 14-3-3σ (SFN).
    • Fig. S6. Effects of LAD1 on specific transcripts of breast cancer cells.
    • Fig. S7. Like LAD1, SFN expression correlates with poor prognosis in breast cancer patients; FLNA expression displays distinct pathological patterns.
    • Legends for tables S1 to S3
    • Table S4. Lists of putative partners of LAD1 revealed by using either Y2H screens or proteomic analyses of coimmunoprecipitataed proteins.
    • Legends for tables S5 and S6
    • Table S7. Primers.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.76 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). SILAC data.
    • Table S2 (Microsoft Excel format). EGF-induced phosphorylation changes.
    • Table S3 (Microsoft Excel format). Serum-induced phosphorylation changes.
    • Table S5 (Microsoft Excel format). RNA-seq data.
    • Table S6 (Microsoft Excel format). GOrilla analysis.

    [Download Tables S1 to S3 and S5 and S6]


    Citation: L. Roth, S. Srivastava, M. Lindzen, A. Sas-Chen, M. Sheffer, M. Lauriola, Y. Enuka, A. Noronha, M. Mancini, S. Lavi, G. Tarcic, G. Pines, N. Nevo, O. Heyman, T. Ziv, O. M. Rueda, D. Gnocchi, E. Pikarsky, A. Admon, C. Caldas, Y. Yarden, SILAC identifies LAD1 as a filamin-binding regulator of actin dynamics in response to EGF and a marker of aggressive breast tumors. Sci. Signal. 11, eaan0949 (2018).

    © 2018 American Association for the Advancement of Science

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