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Smad signaling is required for maintenance of epigenetic gene silencing during breast cancer progression

ABSTRACT: In this study, we took advantage of a previously established breast cancer progression cell line model system, which consists of a parental MCF10A (MI) spontaneously immortalized mammary epithelial cell line and two of its derivatives: 1) MCF10ATk.cl2 (MII), a MCF10A H-Ras transformed cell line and 3) MCF10CA1h (MIII), derived from a xenograft of the MII cells in nude mice that progressed to carcinoma (1, 2). These cell lines were previously reported to exhibit distinct tumorigenic properties when re-implanted in nude mice; MI is non-tumorigenic, MII forms benign hyperplastic lesions and MIII forms low-grade, well differentiated carcinomas (2, 3). The advantage of this system is that these cell lines were derived from a common genetic background (MCF10A) and accumulated distinct genetic/epigenetic alterations in vivo enabling them to acquire a range of non-tumorigenic to carcinogenic properties. Our initial studies showed that MIII cells, but not MI or MII, exhibit an EMT phenotype, promoter DNA hypermethylation of epithelial genes and highly invasive properties in vitro. To investigate the role of TGFβ pathway in these processes, we disrupted the TGFβ downstream signaling events in MIII, by stably overexpressing the inhibitory Smad7, and analyzed the gene expression profiles of MII, MIII and MIIISmad7 cells using microarray analysis. Total RNA was isolated from three biological replicates corresponding to MIIpB, MIIIpB and MIIIpB-Smad7 cells using Trizol (Invitrogen) according to the manufacturer’s protocol, and the RNeasy mini-kit (Qiagen) was used to clean-up the RNA. Labeled cRNA fragments derived from the samples were hybridized onto human genome U133 plus 2.0 arrays (Affymetrix). Gene-expression estimates and a measure of sequence-specificity of the hybridization intensities were both determined using standard settings in MAS5 (Affymetrix). Probesets that did not exhibit sequence-specific hybridization in any sample were excluded from subsequent analysis. Differential expression between MIIpB and MIIIpB as well as between MIIIpB and MIIIpBSmad7 was assessed using Student’s t-test. Genes with a false discovery rate (FDR) < 0.05 and a greater than 2-fold difference in expression between the two cell lines were considered to be differentially expressed.

ORGANISM(S): Homo sapiens

PROVIDER: GSE18070 | GEO | 2009/12/31

SECONDARY ACCESSION(S): PRJNA119337

REPOSITORIES: GEO

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Project 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.

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Smad signaling is required for maintenance of epigenetic gene silencing during breast cancer progression

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