Comment[ArrayExpressAccession]	E-GEOD-56445
MAGE-TAB Version	1.1						
Public Release Date	2014-04-02
Investigation Title	The transcriptional regulators TAZ and YAP direct Transforming Growth Factor-beta-induced tumorigenic phenotypes in breast cancer cells						
Comment[Submitted Name]	The transcriptional regulators TAZ and YAP direct Transforming Growth Factor-beta-induced tumorigenic phenotypes in breast cancer cells
Experiment Description	Uncontrolled Transforming growth factor-beta (TGFβ) signaling promotes aggressive metastatic properties in late-stage breast cancers. However, how TGFβ-mediated cues are directed to induce late-stage 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 non-tumorigenic cells to overcome TGFβ repressive effects. Our work thus identifies crosstalk between nuclear TAZ/YAP and TGFβ signaling in breast cancer cells, revealing novel insight into late-stage disease-driving mechanisms. Expression profiling was conducted following the repression of the transcriptional regulators TAZ and YAP (TAZ/YAP), the TEAD family of transcription factors (TEAD1/2/3/4), or the TGFb signaling pathway (with SB-431542, an inhibitor of the TBRI recpeptor) in human MDA-MB-231-LM2 breast cancer cells treated with TGFβ1. Human MDA-MB-231-LM2-4 breast cancer cells were transfected with control siRNA, or siRNAs targeting TAZ/YAP or all four TEADs and were treated 24 hours later with 500pM TGFβ1 or 5mM SB-431542 for an additional 24 hours. Total RNA was isolated and twelve microarrays in total were performed, with each condition carried out three times on separate days. The Boston University Microarray Core generated the data using the Affymetrix Human Gene 1.0 St Array.						
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl					
Person Last Name	Gower	Hiemer	Varelas				
Person First Name	Adam	Samantha	Xaralabos				
Person Mid Initials	C	E					
Person Email	agower@bu.edu						
Person Affiliation	Boston University School of Medicine						
Person Address	Clinical and Translational Science Institute, Boston University School of Medicine, 72 East Concord Street, E631, Boston, MA, USA						
Person Roles	submitter						
Protocol Name	P-GSE56445-1	P-GSE56445-5	P-GSE56445-6	P-GSE56445-2	P-GSE56445-3	P-GSE56445-4	P-GSE56445-7
Protocol Description	Raw Affymetrix CEL files were normalized to produce Entrez Gene-identifier-specific expression values using the implementation of the Robust Multiarray Average (RMA) in the affy package in the Bioconductor software suite (version 2.7) and the Brainarray hugene10stv1hsentrezg R package (version 14.0.0) (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/14.0.0/entrezg.download/HuGene10stv1_Hs_ENTREZG_14.0.0.zip). ID_REF =  VALUE = log2-transformed, RMA-normalized Entrez Gene expression values	Biotin labeling was performed using the Ambion WT Expression Kit (Life Technologies, Grand Island, NY) according to the manufacturer's protocol, followed by the GeneChip WT Terminal Labeling and Controls Kit (Affymetrix, Santa Clara, CA).	The labeled, fragmented DNA was hybridized to the Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) for 18 hours in a GeneChip Hybridization oven 640 at 45oC with rotation (60 rpm). The hybridized samples were washed and stained using an Affymetrix fluidics station 450.	RNA interference was performed by transfecting the indicated siRNA using Dharmafect 1 (Thermo Scientific) according to manufacturer’s protocol.	MDA-MB-231-LM2-4 cells were cultured using RPMI media supplemented with 10% FBS.	Total RNA was isolated and purified by Quick-RNA MiniPrep (Zymogen Research) according to manufacturer’s protocol.	After staining, microarrays were immediately scanned using an Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix, Santa Clara, CA).
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	TREATMENT						
Experimental Factor Type	treatment						
Publication Title	The transcriptional regulators TAZ and YAP direct Transforming Growth Factor-beta-induced tumorigenic phenotypes in breast cancer cells.						
Publication Author List	Hiemer SE, Szymaniak AD, Varelas X						
PubMed ID	24648515						
Publication DOI	10.1074/jbc.M113.529115						
Comment[SecondaryAccession]	GSE56445						
Comment[GEOReleaseDate]	2014-04-02						
Comment[ArrayExpressSubmissionDate]	2014-04-02						
Comment[GEOLastUpdateDate]	2014-04-02						
Comment[AEExperimentType]	transcription profiling by array						
SDRF File	E-GEOD-56445.sdrf.txt