Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Sep 11;109(37):E2441-50.
doi: 10.1073/pnas.1212021109. Epub 2012 Aug 13.

The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain

John M Lamar  1 , Patrick Stern, Hui Liu, Jeffrey W Schindler, Zhi-Gang Jiang, Richard O Hynes
Affiliations

The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain

John M Lamar et al. Proc Natl Acad Sci U S A. .

Abstract

The transcriptional coactivator Yes-associated protein (YAP) is a major regulator of organ size and proliferation in vertebrates. As such, YAP can act as an oncogene in several tissue types if its activity is increased aberrantly. Although no activating mutations in the yap1 gene have been identified in human cancer, yap1 is located on the 11q22 amplicon, which is amplified in several human tumors. In addition, mutations or epigenetic silencing of members of the Hippo pathway, which represses YAP function, have been identified in human cancers. Here we demonstrate that, in addition to increasing tumor growth, increased YAP activity is potently prometastatic in breast cancer and melanoma cells. Using a Luminex-based approach to multiplex in vivo assays, we determined that the domain of YAP that interacts with the TEAD/TEF family of transcription factors but not the WW domains or PDZ-binding motif, is essential for YAP-mediated tumor growth and metastasis. We further demonstrate that, through its TEAD-interaction domain, YAP enhances multiple processes known to be important for tumor progression and metastasis, including cellular proliferation, transformation, migration, and invasion. Finally, we found that the metastatic potential of breast cancer and melanoma cells is strongly correlated with increased TEAD transcriptional activity. Together, our results suggest that increased YAP/TEAD activity plays a causal role in cancer progression and metastasis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
YAPS127A enhances tumor growth and promotes metastasis. (A and B) The indicated cells (5 × 105) stably expressing control vector or YAPS127A were transplanted into the mammary fat pads (A) or delivered s.c. (B) into mice. After 3–5 wk, tumor masses were compared using box and whisker plots. n = 8 mice. (C–E) 67NR cells (2 × 105), 4T1 cells (5 × 104), or A375-GFP cells (1 × 106) expressing either control vector or YAPS127A were assayed by tail-vein metastasis assays. (C) (Left) Representative H&E-stained sections at 19 d postinjection; arrows indicate metastases. (Right) Graph shows percentage of total lung area that was metastatic burden (± SEM) for all mice. n = 3 mice. (D and E) The numbers of metastases were counted in H&E-stained sections (D) or in whole lungs (E) at 7 d (D) or 34 d (E) postinjection. n = 3–5 mice in D and 5 mice in E. (F and G) NMuMG cells expressing control vector or YAPS127A (5 × 105 for each condition) were assayed by either orthotopic mammary transplant or tail-vein metastasis assays. (F) Tumor masses were compared using box and whisker plots. n = 4 mice. (G) The numbers of metastases were counted in H&E-stained sections at 10 d postinjection. n = 5 mice. n.s., not significant (P = 0.15); +P = 0.06; #P = 0.09; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, two-tailed paired t test.
Fig. 2.
Fig. 2.
YAPS127A promotes metastasis through a cell-autonomous mechanism. The indicated cell lines were stably transduced with vectors expressing mCherry, ZSgreen, or YAPS127A-2A-ZSgreen. Two separate cell mixtures were generated by mixing equal numbers of mCherry cells with either ZSgreen cells (Control Mix) or with YAPS127A-2A-ZSgreen cells (YAPS127A Mix). Then 5 × 105 cells of the indicated mixtures were assayed by tail-vein metastasis assay. (Left) Fluorescent images of whole lungs after 14 d. (Right) Lung lobes were digested to single-cell suspensions, and the relative numbers of ZSgreen- and mCherry-positive cells were counted using flow cytometry. Graphs show fold increase in ZSgreen-positive cells compared with mCherry-positive cells for each mix (± SEM). n = at least 6 mice for each condition; n.s., not significant; ***P ≤ 0.001; two-tailed paired t test.
Fig. 3.
Fig. 3.
YAPS127A-mediated tumor growth and metastasis are dependent on the TEAD-interaction domain of YAP. (A) Schematic of human YAP protein showing the proline-rich N terminus (P-rich), the TEAD-interaction domain (TEAD-ID), WW domains (WW1 and WW2), the SH3-binding motif (SH3bm), the coiled-coil domain (CC), the transactivation domain, and the PDZ-binding motif (PDZbm). Several point mutations were introduced into a YAPS127A construct with an N-terminal Flag-tag. (B) Immunoblots of lysates from 67NR and NMuMG cells stably expressing barcoded versions of either control vector or the indicated mutant forms of YAPS127A. (C–G) 67NR or NMuMG cells from B were mixed in equal numbers and then either 5 × 105 cells (C, F, and G), or 2.5 × 105 cells (D) were used in Luminex-based tail-vein metastasis (C) or orthotopic mammary transplant and spontaneous metastasis assays (D–G). After 8 (C), 28 (D and E), or 86 (F and G) d, lungs and primary tumors were isolated, and the relative numbers of cells expressing each vector were quantified using Luminex. Graphs show mean signal (± SEM) for each cell line relative to the signal for cells expressing control vector 1. Results similar to those in B–F were obtained in a second independent experiment. (H and I) A375 cells (5 × 105) expressing the indicated constructs were injected s.c. into mice, and tumor masses and the numbers of lung metastases were compared. Graphs show mean (± SEM) of 14 mice (C); 10 mice (D and E); 7 mice (F); 5 of the mice from F that developed metastases (G); or 7 mice (H and I); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; one-way ANOVA with Dunnett’s post test.
Fig. 4.
Fig. 4.
A Luminex-based approach for multiplexing in vivo tumor growth and metastasis assays. (A) Schematic of our Luminex-based approach for multiplexing in vivo tumor growth and metastasis assays (Methods). Individual cell lines stably expressing barcoded (BC) versions of each YAPS127A mutant were mixed in equal numbers and either injected into the tail veins (TV) or transplanted orthotopically into the mammary fat pads of mice (MT). Genomic DNA was isolated from the resulting tumors and metastasis-containing lungs, and the relative amount of each barcode was quantified using streptavidin-conjugated APC (Strep.-APC) and the Luminex FlexMap 3D system. (B) 4T1 mammary carcinoma cells were stably transduced with uniquely barcoded retroviral vectors expressing miR30-based shRNAs targeting either RPA3 or control firefly luciferase (FF) and were mixed in equal numbers. Then 2 × 105 cells were used in Luminex-based tail-vein metastasis assays and orthotopic mammary transplant assays. The starting population also was collected at the time of injection as a reference. After 14 d (B) and 28 d (C) lungs and primary tumors were isolated, and the relative numbers of cells expressing each vector were quantified using Luminex. Graphs show mean signal (± SEM) for each RPA3-shRNA–expressing cell line in the lungs or primary tumor (normalized to the signal for firefly luciferase) relative to the signal in the starting population. n = 6 mice in B and 4 mice in C.
Fig. 5.
Fig. 5.
YAPS127A-mediated proliferation, migration, and invasion are dependent on the TEAD-interaction domain of YAP. (A and B) NMuMG cells stably expressing the indicated constructs were mixed together in equal numbers and used in Luminex-based soft agar assays (A) or were assayed for their ability to grow in 3D Matrigel (B). (C) 67NR or NMuMG cells expressing barcoded versions of the indicated constructs were assayed for proliferation in vitro using Luminex-based assays. Graphs show mean relative signal for each cell population relative to the control vector-expressing population (± SEM). n = 6 wells per cell line. (D–G) A375 (D and E), 67NR (F), or NMuMG (G) cells expressing the indicated constructs were assayed by transwell migration or invasion assays; graphs show either the numbers of cells on the bottom of the filter or mean fold invasion (relative to control cells) (± SEM). n = 3 triplicate wells in D and E; n = 3 independent experiments done in duplicate in F; and n = 3 independent experiments done in triplicate in G. n.s., not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; repeated measures ANOVA with Dunnett’s post test (C) or paired t test (D–G).
Fig. 6.
Fig. 6.
TEAD transcriptional activity is strongly correlated with metastatic potential. (A) 67NR or NMuMG cells expressing the indicated constructs were assayed for TEAD transcriptional activity using a TEAD-dependent promoter-driven firefly luciferase reporter construct. Graphs show normalized luciferase activity (relative to control cells, ± SEM); n = 3 independent experiments done in duplicate. (B) The indicated cell lines were assayed for TEAD transcriptional activity as above. The graphs show normalized luciferase activity (relative to MCF10A cells) for each cell line (± SEM). n = at least 6 separate transfections assayed in duplicate; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n.s., not significant; one-way ANOVA with Dunnett’s post test.
Fig. 7.
Fig. 7.
Loss of Hippo pathway-mediated repression of YAP leads to increased TEAD transcriptional activity and enhanced metastasis. (A) 67NR cells were stably transduced with barcoded control vector or vectors expressing wild-type YAP (YAP-SS), YAPS127A (YAP-AS), YAPS381A (YAP-SA), or YAPS127A,S381A (YAP-AA) and were assayed by Western blot for the indicated proteins or for TEAD transcriptional activity. Graphs show normalized luciferase activity (relative to control cells; ± SEM); n = 6 independent experiments done in duplicate. (B) Cells from A were mixed in equal numbers, and then 5 × 105 cells were used in Luminex-based orthotopic mammary transplant and spontaneous metastasis assays. After 29 d, lungs and primary tumors were isolated, and the relative numbers of cells expressing each vector were quantified using Luminex. Graphs show mean signal (± SEM) for each cell line relative to the signal for cells expressing control vector; n = 10 mice. *P ≤ 0.05; ***P ≤ 0.001; one-way ANOVA with Dunnett’s post test.
Fig. P1.
Fig. P1.
Working hypothesis for the role of YAP and the Hippo pathway during tumor progression and metastasis. The activation of the Hippo pathway by changes in cell density, cell shape, and cell adhesion leads to phosphorylation of the Hippo kinases (MST1/2 in mammals), which in combination with the adaptor protein Sav phosphorylate LATS1/2 kinases and their partner, MOB. The LATS/MOB complex then phosphorylates the transcriptional coactivator YAP and thereby represses YAP activity by promoting both cytoplasmic sequestration via 14-3-3 proteins and proteasomal degradation. Our results show that inhibiting the ability of the Hippo pathway to repress YAP results in increased YAP/TEAD-dependent gene expression, which influences both tumor growth and metastasis by enhancing processes that occur at both the primary tumor and at the metastatic site. A close homolog of YAP, TAZ, is regulated in a similar fashion by the Hippo pathway and likely plays a similar role.
See this image and copyright information in PMC

References

    1. Zhao B, et al. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 2007;21:2747–2761. - PMC - PubMed
    1. Kim NG, Koh E, Chen X, Gumbiner BM. E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components. Proc Natl Acad Sci USA. 2011;108:11930–11935. - PMC - PubMed
    1. Zhao B, et al. Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev. 2012;26:54–68. - PMC - PubMed
    1. Wada K, Itoga K, Okano T, Yonemura S, Sasaki H. Hippo pathway regulation by cell morphology and stress fibers. Development. 2011;138:3907–3914. - PubMed
    1. Dupont S, et al. Role of YAP/TAZ in mechanotransduction. Nature. 2011;474:179–183. - PubMed

Publication types

MeSH terms