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. 2014 Mar 27;6(6):1059-1072.
doi: 10.1016/j.celrep.2014.02.038. Epub 2014 Mar 6.

The Hippo transducer TAZ interacts with the SWI/SNF complex to regulate breast epithelial lineage commitment

Adam Skibinski  1 , Jerrica L Breindel  1 , Aleix Prat  2 , Patricia Galván  2 , Elizabeth Smith  3 , Andreas Rolfs  4 , Piyush B Gupta  5 , Joshua LaBaer  6 , Charlotte Kuperwasser  7
Affiliations

The Hippo transducer TAZ interacts with the SWI/SNF complex to regulate breast epithelial lineage commitment

Adam Skibinski et al. Cell Rep. .

Abstract

Lineage-committed cells of many tissues exhibit substantial plasticity in contexts such as wound healing and tumorigenesis, but the regulation of this process is not well understood. We identified the Hippo transducer WWTR1/TAZ in a screen of transcription factors that are able to prompt lineage switching of mammary epithelial cells. Forced expression of TAZ in luminal cells induces them to adopt basal characteristics, and depletion of TAZ in basal and/or myoepithelial cells leads to luminal differentiation. In human and mouse tissues, TAZ is active only in basal cells and is critical for basal cell maintenance during homeostasis. Accordingly, loss of TAZ affects mammary gland development, leading to an imbalance of luminal and basal populations as well as branching defects. Mechanistically, TAZ interacts with components of the SWI/SNF complex to modulate lineage-specific gene expression. Collectively, these findings uncover a new role for Hippo signaling in the determination of lineage identity through recruitment of chromatin-remodeling complexes.

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Figures

Figure 1
Figure 1. Screen for transcription factors involved in MEC lineage commitment
A, Schematic of the screening approach used to identify novel regulators of MEC fate. B and C, a list and dot plot representing the 46 identified TFs enriched greater than the two-fold cutoff (indicated by the dotted line in C). Enrichment scores were calculated as a fold increase over transduced but unscreened control cells (“pre-screen”). D, Box-and-whisker plot showing enrichment scores of the complete set of screen hits in gene expression profiles from purified mammary epithelial subsets as reported by Lim et al. (2010). E, MECs were isolated and sorted as in A, transduced with lentivirus containing TAZ cDNA, and subjected to a colony-forming assay. F, Unsorted MECs were transduced with TAZ cDNA and subjected to a colony-forming assay as in E. The colonies were co-immunostained with KRT14 (brown) and KRT18 (purple) to evaluate differentiation state (depicted in the representative images on the left). G, TAZ-transduced MECs were plated on 1 mg/ml collagen gels for colony formation and the colony types were quantified (classified as ductal, round/acinar, or flat as indicated in the representative images). In all panels, error bars represent the SEM. Significance values were computed by Student's t-test; a single asterisk represent a significance level of p < 0.05, double asterisks indicate p < 0.01. All colony-forming assays were performed using cells isolated from at least three tissue donors.
Figure 2
Figure 2. TAZ controls MEC differentiation state
A-F, TAZ cDNA or lacZ cDNA was expressed in MCF10F cells using lentiviral vectors (N = 3 experiments). A, Representative image of control MCF10F cells and MCF10F-TAZ cells. B, 3D colony formation in MCF10F-TAZ cells. C, Flow cytometry of MCF10F cells resolves EpCAMhigh/CD44low luminal-like and EpCAMlow/-/CD44high basal-like subpopulations. D, The relative proportion of luminal and basal cells in MCF10F-TAZ was quantified by the gating strategy indicated in C. S89A is a Hippo-refractory mutant form of TAZ. E, mRNA expression of known TAZ target genes (dark grey bars), luminal markers (red bars) and basal/ME markers (blue bars) in MCF10F-TAZ cells. Values are represented as a log2 fold-change over LacZ control cells. F-K, TAZ was depleted in MCF10A cells using shRNAs (N = 6 experiments). F, TAZ depletion caused many cells to become non-adherent and detach from the substrate (arrows). G, TAZ protein levels following transduction with shRNA constructs. H, Growth kinetics of MCF10A-shTAZ over 7 days. I, Mammospheres (> 30 μm diameter) formed by MCF10A-shTAZ cells. J, 3D morphogenesis assay after MCF10A-shTAZ cells were seeded on collagen gels as in C. K, qRT-PCR analysis of gene expression in MCF10A cells following TAZ knockdown. Gene expression values are represented as a log2 fold change over the control cell line. In all panels, error bars represent the SEM, a single asterisk indicates p < 0.05 and a double asterisk represents p < 0.01 as determined by Student's t-test (pairwise against the control cell line). See also Figure S1.
Figure 3
Figure 3. Lineage-specific Hippo signaling and TAZ expression in breast tissues
A, Schematic of sorting strategy used to purify luminal and basal subsets from breast reductions tissues using EpCAM and CD10 immunomagnetic beads. B, qRT-PCR analysis of CD10 and EpCAM expression following sorting demonstrating enrichment of the appropriate marker in the basal vs. luminal sorted cells (N = 4 tissue donors). C, Representative Western blot analysis of phospho-LATS1, total LATS1, and TAZ protein levels in purified luminal and basal cells. D, Low and high-power images of formalin fixed, paraffin-embedded human breast tissue specimens immunostained with an antibody reactive against both YAP and TAZ, demonstrating nuclear TAZ expression in basal cells. E, Co-immunofluorescence staining with YAP/TAZ and KRT14, a marker of basal cells, showing punctate nuclear for YAP/TAZ in K14+ cells vs. cytoplasmic staining in K14- cells (white arrows). F, Quantitation of the percent of cells with nuclear YAP/TAZ expression in large-diameter ducts vs. TDLUs. G, mRNA expression of TAZ targets CTGF and ANKRD1 in sorted subpopulations. H, Enrichment analysis of the TAZ target gene signature in microarray datasets of purified mouse and human MEC subpopulations. In all panels, error bars represent the SEM, a single asterisk indicates p < 0.05 and double asterisks represents p < 0.01 as determined by Student's t-test. See also Figure S2.
Figure 4
Figure 4. Developmental defects and lineage imbalance in Taz/Wwtr1-deficient mice
A, Whole-mount images and B, quantification of branching complexity in mammary glands from post-pubertal 16-week old Wwtr1lacZ mice (N = 6 per genotype). C, Quantification of the average percentage of cross-sectional gland area occupied by epithelium in 16-week old mice. D, Co-immunostaining of epithelia from 16-week old mice for EpCAM (red) and SMA (green) revealed a reduced number of SMA+ cell bodies in Wwtr1-deficient glands (arrows in bottom panels, N = 4 per genotype). E, Ratio of luminal to basal cells as identified by staining in D. F-G, MECs were isolated as a single-cell suspension and analyzed by flow cytometry. Representative biaxial plots (F) and mean proportions (G) of Lin-CD24high/CD49low luminal and Lin-CD24+/CD49high basal cells within Wwtr1-deficient epithelia are shown (N = 6 per genotype). H, Quantitation of Ki67-positive cells in 16-week old mouse mammary tissues. I, Colony formation when MECs from 16-week old mice were plated at clonal density on plastic substrates (N = 3). In all panels, error bars represent SEM, a single asterisk indicates p < 0.05 and double asterisks represents p < 0.01 as determined by Student's t-test (pairwise against wild-type). See also Figure S3.
Figure 5
Figure 5. Chromatin-remodeling complexes mediate the function of TAZ
A, Schematic of canonical SWI/SNF subunits (left) with a list of the components identified by TAZ-FLAG co-IP/mass spectrometry (right). B, Co-immunoprecipitation of endogenous TAZ and BRG1, one of the two SWI/SNF ATPases, in nuclear lysates from MCF10A cells. C-D, FLAG immunoprecipitation of either wild-type TAZ, or a deletion mutant lacking the WW domain,n in 293T cells. E, Western blot demonstrating BRM or BRG1 depletion in MCF10F cells using lentiviral shRNA vectors. F, qRT-PCR showing the expression of TAZ targets CTGF and ANKRD1 upon BRM/BRG1 knockdown. G-I, TAZ cDNA was stably expressed in MCF10F cells, followed by stable knockdown of BRM or BRG1. G, The luminal-like and basal-like MCF10F subpopulations were assessed by flow cytometry and are quantified in H. I, The expression of basal markers VIM and CD44 was also assessed in MCF10F-TAZ cells with or without BRM or BRG1 knockdown. J, Mean-centered gene expression of BRM or BRG1 in mammary epithelial subsets as reported by Lim et al. (2010). K, ChIP analysis of BRM and BRG1 at the CTGF promoter or RPL30 exon 3 in MCF10A cells. Data are expressed as a fold enrichment over the IgG negative control. In all panels, error bars indicate SEM, a single asterisk indicates p < 0.05 and a double asterisk represents p < 0.01 as determined by Student's t-test. See also Figure S4.
Figure 6
Figure 6. TAZ is associated with basal-like breast cancer
A, Analysis of TCGA data reveals that TAZ copy number is amplified in 44% of basal-like breast tumors (either low- or high-level amplification). B, TAZ gene expression is highest in basal-like tumors (error bars indicate SEM). C, Correlation between TAZ gene expression and the protein expression of various biomarkers in TCGA dataset (Pearson's R statistic is shown). D, Western blot showing TAZ protein levels in various breast cancer cell lines and normal human MECs. E, Kaplan-Meier curves showing relapse-free survival probability of patients with high or low TAZ gene expression in various breast cancer subtypes (log-rank p-values are shown).
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