2012 Dec 21
β-Catenin-driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis
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β-Catenin-driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis
Cell. 2013 Mar 28;153(1):267-70
Wnt/β-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of β-catenin in transformation, we classified β-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that β-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with β-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of β-catenin-dependent cancers in both cell lines and animal models. These observations define a β-catenin-YAP1-TBX5 complex essential to the transformation and survival of β-catenin-driven cancers.
Copyright © 2012 Elsevier Inc. All rights reserved.
Figure 1. Identification of genes essential for β-catenin active cell lines
(A) Strategy to identify genes required in β-catenin active cell lines. (B) β-catenin expression in cells expressing β-catenin-specific shRNAs. IB indicates immunoblotting. (C) Activity of the β-catenin/TCF4 reporter following suppression of β-catenin. LacZ was used for normalization. (D) β-catenin/TCF4 reporter activity in 85 cell lines. (E) TCF4 expression following introduction of
TCF4-specific shRNAs. (F) β-catenin/TCF4 activity following suppression of TCF4 or expression of DN-TCF4 (G) Proliferation or (H) AI growth following suppression of TCF4. Data presented as mean ± SD for 3 independent experiments. See also Figure S1.
Figure 2. YAP1 is essential for tumorigenicity of β-catenin dependent cancer cell lines
(A) Proliferation and (B) AI growth of the indicated cancer cell lines following suppression of
YAP1. Classification of β-catenin activity in each cell line is noted. (C) Proliferation, (D) AI growth and (E) expression of YAP1 in HuTu80 cell lines overexpressing wild type (WT) YAP1 and the indicated YAP1-specific or control (shLacZ) shRNAs. (F) Effects of suppressing YAP1 on orthotopic colon tumors. (G) YAP1 expression in tumors shown in (F). (H-J) HA1EM cells expressing β-catenin or YAP1 and YAP1- or β-catenin-specific shRNAs. (H) AI growth, (I) proliferation or (J) protein levels. In (H) inset shows representative images from AI growth assay. Data presented as mean ± SD for 3 independent experiments. See also Figure S2 and S3.
Figure 3. YAP1, β-catenin and TBX5 interact
(A) β-catenin or control IgG immune complexes were isolated from SW480 lysates and the indicated proteins were analyzed by immunoblotting. (B)
TBX5 mRNA levels measured by quantitative PCR in cells expressing TBX5-specific or control (shLacZ) shRNAs. Data presented as mean ± SD for 3 independent experiments. (C) Proliferation or (D) AI growth of the indicated cells following suppression of TBX5. (E) The indicated expression vectors were introduced into 293T cells, FLAG immune complexes were isolated and analyzed by immunoblotting with an anti-HA antibody. (F) FLAG-immune complexes were isolated from DLD1 cells stably expressing a FLAG-epitope tagged TBX5 protein and analyzed by immunoblotting with the indicated antibodies.
BCL2L1 and BIRC5 are transcriptional targets of the β-catenin-YAP1-TBX5 complex
(A) Proliferation or (B) AI growth following suppression of
BCL2L1 or BIRC5 in the indicated cell lines. (C, D) mRNA levels of BCL2L1 and BIRC5 in HT55 or HCT116 cells expressing β-catenin-specific, YAP1-specific or control shRNAs. (E) β-catenin, YAP1 or control immune complexes were isolated from HuTu80 cells and subjected to ChIP analysis with primers derived from the promoter regions of BCL2L1 (1-1000 bp) and BIRC5 (−952-0 bp). (F) β-catenin or (G) YAP1 immune complexes were derived from HuTu80 cells expressing TBX5-specific shRNAs were subjected to ChIP analysis using primers for BIRC5. (H) mRNA levels of BCL2L1 and BIRC5 in HCT116 cells expressing TBX⍰-specific shRNAs. (I) Immunoblot analysis of BCL-XL or BIRC5 in HuTu80 cells overexpressing BCL-XL, BIRC5 or LacZ and YAP1-specific, β-catenin-specific or control shRNAs. (J) Proliferation of the cell lines described in (I). Data presented as mean ± SD for 3 independent experiments. See also Figure S4.
Figure 5. YES1 is essential for tumorigenicity of β-catenin active cells
(A) AI growth or (B) proliferation following suppression of
YES1 expression. (C) Effects of suppressing YES1 on orthotopic HCT116 colon tumors. (D) YES1 expression in tumors shown in (C). (E) YAP1 immune complexes were isolated from SW480 cells, and the indicated proteins were analyzed by immunoblotting. (F) V5-epitope tagged YAP1 and WT or activated (Y537F) YES1 or SRC expression constructs were introduced into 293T cells. V5 immune complexes were analyzed by immunoblotting with the PY99 phosphotyrosine-specific antibody. (G) FLAG-epitope tagged WT or mutated (Y357F) YAP1 were transfected into 293T cells together with activated (Y537F) YES1. FLAG-epitope immune complexes were isolated and analyzed by immunoblotting with a phosphotyrosine antibody. (H) V5 immune complexes were isolated from HuTu80 or SW480 cells stably expressing WT or Y357F V5-epitope tagged YAP1 or control V5-LacZ. Immune complexes were analyzed by immunoblotting with a phosphotyrosine antibody. (I) HuTu80 or SW480 cells stably expressing V5-epitope tagged WT YAP1 were treated for 6 h with 0.5 or 2 μM of dasatinib. V5 immune complexes were analyzed by immunoblotting with a phosphotyrosine antibody. (J) Transgenic IFABP:RFP zebrafish were injected with 50 μM of YAP1- or YES1-specific morpholinos, and red fluorescence was assessed 4 d post fertilization (dpf) or IFABP expression was assessed 3 dpf using whole mount in situ hybridization with an IFABP-specific probe. (K) Embryos were exposed to 2 μM of dasatinib at 2 dpf, and IFABP expression was assessed after 24 h using whole mount in situ hybridization. See also Figure S5-S7.
Figure 6. Expression of YES1 is essential for formation of the YAP1-β-catenin-TBX5 complex
(A) YAP1 immune complexes were isolated from HuTu80 cells expressing
YES1-specific or control shRNAs and β-catenin abundance was analyzed by immunoblotting. (B) SW480 cells were treated for 6 h with increasing concentrations of dasatinib and β-catenin-YAP1 complexes were assessed as in (A). (C) HuTu80 cells expressing dasatinib resistant YES1 or SRC mutants were treated with 2μM of dasatinib for 6 h and the β-catenin-YAP1 interaction was assessed as in (A). (D) 293T cells were transfected with FLAG-epitope tagged WT or Y357F YAP1 (5 μg) with or without HA-epitope tagged β-catenin. FLAG immune complexes were assessed for the presence of HA tagged proteins. (E) V5 immune complexes were isolated from DLD1 or HuTu80 colon cancer cell lines stably expressing V5-epitope tagged WT or Y357F YAP1 or control LacZ and the presence of β-catenin was assessed by immunoblotting. (F) β-catenin or YAP1 immune complexes from HCT116 cells treated with 2 μM of dasatinib or vehicle (DMSO) were subjected to ChIP analysis. (G) mRNA levels of BCL2L1 and BIRC5 in HCT116 or HuTu80 cells treated for 1 h with 2 μM of dasatinib.
Figure 7. Dasatinib impairs the proliferation of β-catenin active cell lines
(A) Proliferation (7 d) following dasatinib treatment. Proliferation of (B) HCT116 or (C) HuTu80 cells following dasatinib treatment of cells stably expressing dasatinib resistant YES1 (T348I) or SRC (T341I) mutants. Data presented as mean ± SD for 4 independent experiments. (D) Representative images of colon organoids derived from WT or APC null mice treated for 6 d with 100 nM of dasatinib. (E) Quantification of results in D. Error bars represent SD from 4 replicates. Blank mark wells where no organoids were added. (F) WT or AXIN1 mutant (Masterblind) zebrafish were treated with 2 μM of dasatinib from 6-8 dpf. Arrow indicates developing gut. (G) H&E staining of zebrafish in (F). Width of epithelium noted by bars. (H) The number of epithelial cells/section was measured in WT or AXIN1 mutant zebrafish treated with 2 μM of dasatinib or DMSO. Error bars represent the SD from 20 different sections from 5 treated fish. P value was calculated using student’s t test.
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Adaptor Proteins, Signal Transducing / metabolism
Cell Transformation, Neoplastic
Colonic Neoplasms / metabolism
Colonic Neoplasms / pathology
Inhibitor of Apoptosis Proteins / genetics
Phosphoproteins / metabolism
Proto-Oncogene Proteins c-yes / antagonists & inhibitors
Proto-Oncogene Proteins c-yes / metabolism
T-Box Domain Proteins / metabolism
beta Catenin / metabolism
src-Family Kinases / antagonists & inhibitors
Adaptor Proteins, Signal Transducing
Inhibitor of Apoptosis Proteins
T-box transcription factor 5
YAP1 (Yes-associated) protein, human
Proto-Oncogene Proteins c-yes
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