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, 289 (19), 13461-74

The Transcriptional Regulators TAZ and YAP Direct Transforming Growth Factor β-Induced Tumorigenic Phenotypes in Breast Cancer Cells

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The Transcriptional Regulators TAZ and YAP Direct Transforming Growth Factor β-Induced Tumorigenic Phenotypes in Breast Cancer Cells

Samantha E Hiemer et al. J Biol Chem.

Abstract

Uncontrolled transforming growth factor-β (TGFβ) signaling promotes aggressive metastatic properties in late-stage breast cancers. However, how TGFβ-mediated cues are directed to induce tumorigenic events is poorly understood, particularly given that TGFβ has clear tumor suppressing activity in other contexts. Here, we demonstrate that the transcriptional regulators TAZ and YAP (TAZ/YAP), key effectors of the Hippo pathway, are necessary to promote and maintain TGFβ-induced tumorigenic phenotypes in breast cancer cells. Interactions between TAZ/YAP, TGFβ-activated SMAD2/3, and TEAD transcription factors reveal convergent roles for these factors in the nucleus. Genome-wide expression analyses indicate that TAZ/YAP, TEADs, and TGFβ-induced signals coordinate a specific pro-tumorigenic transcriptional program. Importantly, genes cooperatively regulated by TAZ/YAP, TEAD, and TGFβ, such as the novel targets NEGR1 and UCA1, are necessary for maintaining tumorigenic activity in metastatic breast cancer cells. Nuclear TAZ/YAP also cooperate with TGFβ signaling to promote phenotypic and transcriptional changes in nontumorigenic cells to overcome TGFβ-repressive effects. Our work thus identifies cross-talk between nuclear TAZ/YAP and TGFβ signaling in breast cancer cells, revealing novel insight into late-stage disease-driving mechanisms.

Keywords: Breast Cancer; Cell Migration; Cell Signaling; Coregulator Transcription; Hippo Pathway; Signal Transduction; Signaling Cross-talk; Transforming Growth Factor β (TGFβ); YAP/TAZ.

Figures

FIGURE 1.
FIGURE 1.
TAZ/YAP are required for TGFβ-induced tumorigenic events. A, panel of breast cancer cell lines was divided by TGFβ-induced tumor suppression and promoting responses and examined by immunofluorescence for endogenous TAZ and YAP localization. B, LM2-4 cells were transiently transfected with control siRNA (siCTL) or siRNA targeting TAZ (siTAZ), YAP (siYAP), or TAZ and YAP (siTAZ/YAP). Cells were left untreated, treated with TGFβ or SB-431542 + TGFβ, and grown in anchorage-independent conditions. Primary mammospheres were examined for knockdown or were passaged into secondary spheres. Secondary mammospheres following SB-431542 (SB) treatment, or transfection with siTAZ, siYAP, or siTAZ/YAP, were unable to be determined due to low numbers. Representative images are shown, and three independent experiments from each condition were quantitated, measuring the number of colonies formed and the size of each colony. Black error bars represent the average + S.E., and red error bars represent the average ± S.E., *, p < 0.025; **, p < 0.005; ***, p < 0.0001 (t test). C, LM2-4 cell lysates were immunoblotted to examine endogenous levels of the indicated proteins upon TGFβ or SB-431542 treatment compared with GAPDH (loading control). D, LM2-4 cells were transiently transfected with siCTL or siTAZ/YAP. Cells were left untreated, treated with TGFβ, or SB-431542 + TGFβ. Monolayers were wounded and analyzed for cell migration. E, LM2-4 cells stably expressing control shRNA (shCTL) or shRNA targeting TAZ and YAP (shTAZ/YAP) were treated with TGFβ or SB-431542 + TGFβ and incubated in three-dimensional Matrigel culture conditions. Representative images from three independent experiments are shown.
FIGURE 2.
FIGURE 2.
TAZ/YAP, TEADs, and SMAD2/3 interact endogenously. A, HEK293T cells expressing the indicated proteins were lysed and subjected to immunoprecipitation (IP) with a FLAG antibody followed by immunoblotting with the indicated antibodies. B, LM2-4 cells were left untreated or treated with SB-431542 (SB) or TGFβ and examined by immunofluorescence for endogenous TAZ or YAP localization. C and D, LM2-4 cells left untreated or treated with TGFβ were probed with primary antibodies recognizing TAZ/YAP and SMAD2/3 (C), TEAD1 and TAZ (D), or TEAD1 and SMAD2/3 (E). In situ PLA followed by confocal microscopy were performed using mouse and rabbit secondary probes. Red dots indicate endogenous interactions, and nuclei were visualized with Hoechst stain. Representative images are shown, and three fields from each condition were quantitated, measuring the nuclear-cytoplasmic localization of the interactions and the number of interactions per nucleus. Black error bars either represent the average + S.E. or the average ± S.E.
FIGURE 3.
FIGURE 3.
TAZ/YAP, TEADs, and TGFβ direct different and overlapping transcriptional events. A, LM2-4 cells were transfected with control siRNA (siCTL), siRNA targeting TAZ and YAP (siTAZ/YAP), or siRNA targeting all four TEADs (siTEAD1–4), and then treated with TGFβ or SB-431542 + TGFβ. RNA from cell lysates was harvested, and global gene expression profiles were examined using Affymetrix microarrays. The Venn diagram highlights the number of genes with significant expression changes occurring for the indicated condition relative to the siCTL + TGFβ sample. Hierarchical clustering was performed on the significantly changing genes, which revealed four major clusters as indicated. Top significantly changing genes of interest are highlighted in each of the four clustered groups. B–F, LM2-4 cells were transiently transfected with siCTL, siTAZ, siYAP, siTAZ/YAP, or siTEADs and treated with or without TGFβ or SB-431542 + TGFβ. Relative expression of genes indicated in the microarray analysis was determined by qPCR. All data are shown as the average of three independent experiments + S.E. B, confirmation of knockdown. C, group 1, genes repressed by siTAZ/YAP, siTEAD1–4, and SB-431542 treatment. D, group 2, genes repressed by siTAZ/YAP and siTEAD1–4 but induced by SB-431542 treatment. E, group 3, genes induced by siTAZ/YAP, siTEAD1–4, and SB-431542 treatment. F, group 4, genes induced by siTAZ/YAP and siTEAD1–4 but repressed by SB-431542 treatment.
FIGURE 4.
FIGURE 4.
NEGR1 and UCA1 are direct transcriptional targets of TAZ/YAP, TEADs, and SMADs. LM2-4 cells treated with TGFβ or SB-431542 (SB) were subjected to ChIP analysis using control rabbit IgG, TAZ/YAP, TEAD4, or SMAD2/3 antibodies. Samples were analyzed by qPCR using primers recognizing the indicated regions in the promoter of NEGR1 (A), UCA1 (B), or CTGF (C). Normalized values are shown as the average of three independent experiments + S.E.
FIGURE 5.
FIGURE 5.
NEGR1 and UCA1 are necessary for TGFβ-induced tumorigenic events. A, LM2-4 cells were transiently transfected with control siRNA (siCTL) or siRNA targeting NEGR1 (siNEGR1) or UCA1 (siUCA1) and treated with TGFβ. Monolayers were wounded and analyzed for cell migration. Representative images of three independent experiments are shown. B, LM2-4 cells transfected with siCTL, siNEGR1, or siUCA1 were plated on transwell filters to assess cell migration. Migrated cells are shown as the average number in 10 random fields over two independent experiments + S.E. C, LM2-4 cells were transfected with siCTL, siNEGR1, or siUCA1 and then grown under anchorage-independent conditions in the presence of TGFβ to examine primary mammosphere formation. Representative images are shown, and three independent experiments from each condition were quantitated, measuring the number of colonies formed and the size of each colony. Black error bars represent the average + S.E., and red error bars represent the average ± S.E., **, p < 0.005; ***, p < 0.0001 (t test).
FIGURE 6.
FIGURE 6.
TAZ and YAP synergize with TGFβ to promote distinct morphological changes and gene transcription. A, doxycycline (Dox)-inducible MCF10A cells expressing 3×FLAG-TAZ(4SA) or 3×FLAG-YAP(5SA) were treated with increasing levels of doxycycline with or without TGFβ. Expression of TAZ or YAP was determined by immunoblotting along with GAPDH (loading control). B, doxycycline-inducible MCF10A control cells or cells expressing 3×FLAG-TAZ(4SA) or 3×FLAG-YAP(5SA) were treated with doxycycline with or without TGFβ and examined for cell morphology. Representative images of three independent experiments are shown. C, doxycycline-inducible MCF10A cells expressing 3×FLAG-TAZ(4SA) or 3×FLAG-YAP(5SA) were treated with or without doxycycline and/or TGFβ. Monolayers were wounded and analyzed for cell migration. Representative images are shown, and three independent experiments were quantitated. Error bars represent the average + S.E., *, p < 0.05; **, p < 0.01; ***, p < 0.0005 (t test). D–F, doxycycline-inducible MCF10A cells expressing 3×FLAG-TAZ(4SA) or 3×FLAG-YAP(5SA) were treated with increasing levels of doxycycline with or without TGFβ. Relative expression of group 1 genes (D), group 2 genes (E), and group 4 genes (F) was analyzed by qPCR and is shown as the average of three independent experiments + S.E.
FIGURE 7.
FIGURE 7.
Nuclear TAZ and YAP overcome TGFβ-induced cell cycle arrest. A, doxycycline-inducible MCF10A control cells or cells expressing 3×FLAG-TAZ(4SA) or 3×FLAG-YAP(5SA) were treated with doxycycline (Dox) with or without TGFβ. Cells were counted over 6 days and graphed to determine their rate of proliferation. Cell number counts are shown as the average of three independent experiments ± S.E. B, doxycycline-inducible MCF10A control cells or cells expressing 3×FLAG-TAZ(4SA) or 3×FLAG-YAP(5SA) were treated with doxycycline with or without TGFβ. Cells were subject to propidium iodide staining and flow cytometry analysis to determine DNA content. Data from a representative experiment are shown. C, cell cycle phase quantitation from the data in B is represented as the ratio of cells in S + G2 to cells in G1. The average of three independent experiments + S.E. is shown, *, p < 0.015 (t test).
FIGURE 8.
FIGURE 8.
Model for how TAZ/YAP direct TGFβ-induced tumorigenic events. We propose that increased nuclear TAZ/YAP, resulting from defects in upstream Hippo pathway signals, overcome TGFβ-mediated tumor suppressive functions (e.g. cytostasis) and concomitantly drive tumorigenic transcriptional events by promoting the activity of TEAD·SMAD complexes.

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