YAP induces an EMT. (A) YAP induces a morphology change on monolayer cultures. Representative phase-contrast images of MCF10A-YAP and vector control cells growing in monolayer cultures are shown. (Scale bars, 100 μm.) (B) YAP induces an invasive three-dimensional morphology. MCF10A-YAP and vector control cells were cultured on Matrigel for 8 days. Representative phase-contrast images are shown, increasing in magnification from left to right. (Scale bars, 100 μm.) (C) Expression of YAP results in loss of epithelial markers and gain of mesenchymal markers. Immunoblotting analysis reveals a decrease in E-cadherin and occludin (epithelial markers) and concomitant increase in N-cadherin, vimentin, and fibronectin (mesenchymal markers). β-Tubulin is used to show equal loading. (D) YAP overexpression results in loss of membrane E-cadherin and cortical actin. Immunofluorescence analysis shows loss of plasma membrane E-cadherin (magnification, ×50) and loss of localization of actin (stained with phalloidin; magnification, ×100) at cortical sites adjacent to cell–cell interfaces in MCF10A-YAP cells. (E) YAP induces Transwell migration. Control and YAP-expressing MCF10A cells were plated onto 8-μm Transwell filters and allowed to migrate for 24 h either in the presence (+EGF) or in the absence (−EGF) of EGF. Data are the mean number of migrated cells per ×20 field of four fields. Error bars equal ±SD of three independent experiments.

YAP overexpression inhibits apoptosis and transforms MCF10A cells. (A) YAP overexpression inhibits apoptosis in MCF10A cells. Control and MCF10A-YAP monolayer cells were treated with STS, Taxol, UV light, or cisplatin (Cisplat), or they were detached from matrix (anoikis) to induce apoptosis. (A Left) Data are the mean percentages of sub-G₁ DNA content cells determined by flow-cytometric analysis of propidium iodide-stained samples collected after the indicated treatments. (A Right) Cell-death data were quantified by using the cell-death detection ELISA kit, which measures DNA fragmentation; the data are the mean absorbance readings at 405–490 nm relative to vector control cells after 48 h of anoikis. att, attached control monolayer cells. All error bars equal ±SD of three independent experiments. (B) YAP overexpression inhibits apoptosis in HMEC. HMECtert-vector cells and HMECtert-YAP cells were treated with STS and cisplatin to induce apoptosis. Data are the mean percentages of the sub-G₁ content cells determined by flow cytometry as in A. Error bars equal ±SD of three independent experiments. (C) YAP overexpression induces anchorage-independent growth in soft agar. MCF10A-YAP cells and vector control cells were plated in soft-agar assays and allowed to grow for 21 days. (Left) Data are the mean number of colonies per six-well culture of either 10,000 cells (10k) or 50,000 cells (50k). Error bars equal ±SD of three independent experiments. (Right) Representative wells stained with 0.02% iodonitrotetrazolium chloride are shown.

Yap is a candidate “driver” gene in the mouse chromosome 9 amplicon. (A) Whole-genome profile of an individual tumor (CX4) showing ratio of tumor DNA signal vs. normal DNA control from the same mouse; x-axis coordinates represent oligonucleotide probes ordered by genomic map position, with the whole-genome filtered median (three nearest neighbors) data set plotted. High-level amplifications are labeled 1–3 and correspond to: 1, 2.6-Mb amplicon on chromosome 6, centering on Met (2); 2, 350-kb amplicon on chromosome 9, centering on Yap; 3, 4.6-Mb amplicon on chromosome 10, containing a large number of genes. (B) Mouse chromosome 9 amplicon. Filled diamonds denote the positions of probes detecting genomic amplification in CX4. Open diamonds indicate the positions of closest-neighbor probes detecting normal DNA copy number. Circles represent data from C. Filled circles denote amplification, and open circles indicate normal DNA content. (C) Independent confirmation of the amplicon boundaries by using qPCR.

YAP overexpression promotes proliferation. (A) YAP overexpression does not affect growth rate in the presence of EGF. MCF10A-vector cells were grown in parallel to MCF10A-YAP cells, and their growth was assessed over an 8-day time course. (B) YAP overexpression activates EGF-independent growth. MCF10A-YAP cells and vector control cells were grown on Matrigel in medium without EGF for 30 days. Representative phase-contrast images are shown from one of three independent experiments on day 12 on the Left, and both a high-power (Center) and low-power (Right) magnification of day 30. (Scale bars, 100 μm.) (C) YAP overexpression results in activation of ERK1/2 and AKT pathways. Immunoblotting analysis with antibodies to phosphorylated ERK1/2 (Thr-202/Tyr-204) and AKT (Ser-473) shows increased activation in MCF10A-YAP cells in the absence of EGF and serum.