Gab2 and Src co-operate in human mammary epithelial cells to promote growth factor independence and disruption of acinar morphogenesis

Bennett, Haley L.; Brummer, Tilman; Jeanes, Angela; Yap, Alpha S.; Daly, Roger J.

doi:10.1038/sj.onc.1210928

Cited by 40 publications

(35 citation statements)

References 42 publications

(72 reference statements)

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“…Typically, untransformed MCF-10A cells form hollow, spherical acini when plated in Matrigel (16), whereas transformed MCF-10A cells form lumina-filled acini due to the inhibition of apoptosis. Moreover, the transformed acini are not surrounded by an intact basement membrane, and there is invasion of cells into the Matrigel that requires Src activity (6,10). Wild-type SIAH2 is not known to be a driving oncogene.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Src Tyrosine Kinase/SIAH2 E3 Ubiquitin Ligase Pathway That Regulates C/EBPδ Expression and Contributes to Transformation of Breast Tumor Cells

Sarkar

Sharan

Wang

et al. 2012

Molecular and Cellular Biology

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The transcription factor CCAAT/enhancer-binding protein delta (C/EBP␦, CEBPD) is a tumor suppressor that is downregulated during breast cancer progression but may also promote metastasis. Here, we have investigated the mechanism(s) regulating C/EBP␦ expression and its role in human breast cancer cells. We describe a novel pathway by which the tyrosine kinase Src downregulates C/EBP␦ through the SIAH2 E3 ubiquitin ligase. Src phosphorylates SIAH2 in vitro and leads to tyrosine phosphorylation and activation of SIAH2 in breast tumor cell lines. SIAH2 interacts with C/EBP␦, but not C/EBP␤, and promotes its polyubiquitination and proteasomal degradation. Src/SIAH2-mediated inhibition of C/EBP␦ expression supports elevated cyclin D1 levels, phosphorylation of retinoblastoma protein (Rb), motility, invasive properties, and survival of transformed cells. Pharmacological inhibition of Src family kinases by SKI-606 (bosutinib) induces C/EBP␦ expression in an SIAH2-dependent manner, which is necessary for "therapeutic" responses to SKI-606 in vitro. Ectopic expression of degradation-resistant mutants of C/EBP␦, which do not interact with SIAH2 and/or cannot be polyubiquitinated, prevents full transformation of MCF-10A cells by activated Src (Src truncated at amino acid 531 [Src-531]) in vitro. These data reveal that C/EBP␦ expression can be regulated at the protein level by oncogenic Src kinase signals through SIAH2, thus contributing to breast epithelial cell transformation.

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Section: Resultsmentioning

confidence: 99%

Identification of a Src Tyrosine Kinase/SIAH2 E3 Ubiquitin Ligase Pathway That Regulates C/EBPδ Expression and Contributes to Transformation of Breast Tumor Cells

Sarkar

Sharan

Wang

et al. 2012

Molecular and Cellular Biology

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“…25 Aside from a putative STAT5-binding site, Gab2 possesses multiple protein-binding motifs such as Src homology (SH)2 and SH3 domains, proline-rich repeats suitable for interaction with the p85a moiety of PI3K, as well as multiple tyrosine phosphorylation sites that may be targeted by kinases including Src family members. 35,36 Use of …”

Section: Discussionmentioning

confidence: 99%

Interferon-γ-induced increases in intestinal epithelial macromolecular permeability requires the Src kinase Fyn

Smyth

Phan

Wang

et al. 2011

Laboratory Investigation

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Interferon-g (IFNg) is an important immunoregulatory cytokine that can also decrease intestinal epithelial barrier function. Little is known about the intracellular signalling events immediately subsequent to IFNg/IFNg receptor interaction that mediate increases in epithelial permeability; data that could be used to ablate this effect of IFNg while leaving its immunostimulatory effects intact. This study assessed the potential involvement of Src family kinases in IFNg-induced increases in epithelial permeability using confluent filter-grown monolayers of the human colon-derived T84 epithelial cell line. Inhibition of Src kinase with the pharmacologic PP1 and use of Fyn kinase-specific siRNA significantly reduced IFNg-induced increases in epithelial permeability as gauged by translocation of noninvasive E. coli (HB101 strain) and flux of horseradish peroxidase (HRP) across monolayers of T84 cells. However, the drop in transepithelial resistance elicited by IFNg was not affected by either treatment. Immunoblotting revealed that IFNg activated the transcription factor STAT5 in T84 cells, and immunoprecipitation studies identified an IFNg-inducible interaction between STAT5b and the PI3K regulatory subunit p85a through formation of a complex requiring the adaptor molecule Gab2. siRNA targeting STAT5b and Gab2 reduced IFNg-induced increases in epithelial permeability and phosphorylation of PI3K(p85a). PP1 and Fyn siRNA reduced IFNg-induced PI3K activity (indicated by decreased phospho-Akt) and the formation of the STAT5b/PI3K(p85a) complex. Collectively, the results suggest the formation of a Fyn-dependent STAT5b/Gab2/PI3K complex that links IFNg to PI3K signalling and the regulation of macromolecular permeability in a model enteric epithelium.

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“…It is of particular interest that Gab2-sustained growth in suspension was impaired when cells were cultured in the absence of EGF, indicating that Gab2 can only overcome the lack of adhesive consensus in the presence of an upstream signal from GFs. Recently, Gab2 was found to promote GF independence in cooperation with oncogenic Src (Bennett et al, 2007). Therefore, Gab2 can rescue cells from the need for two concomitant proliferative stimuliactivated GF receptors and activated Src-when at least one of the two is present at sufficiently high levels.…”

Section: Gab2 Promotes Anchorage-independent Growthmentioning

confidence: 99%

The GAB2 signaling scaffold promotes anchorage independence and drives a transcriptional response associated with metastatic progression of breast cancer

et al. 2009

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Acquisition of independence from anchorage to the extracellular matrix is a critical event for onset and progression of solid cancers. To identify and characterize new genes conferring anchorage independence, we transduced MCF10A human normal breast cells with a retroviral cDNA expression library and selected them by growth in suspension. Microarray analysis targeted on library-derived transcripts revealed robust and reproducible enrichment, after selection, of cDNAs encoding the scaffolding adaptor Gab2. Gab2 was confirmed to strongly promote anchorageindependent growth when overexpressed. Interestingly, downregulation by RNA interference of endogenous Gab2 in neoplastic cells did not affect their adherent growth, but abrogated their growth in soft agar. Gab2-driven anchorage independence was found to specifically involve activation of the Src-Stat3 signaling axis. A transcriptional 'signature' of 205 genes was obtained from GAB2-transduced, anchorageindependent MCF10A cells, and found to contain two main functional modules, controlling proliferation and cell adhesion/migration/invasion, respectively. Extensive validation on breast cancer data sets showed that the GAB2 signature provides a robust prognostic classifier for breast cancer metastatic relapse, largely independent from existing clinical and genomic indicators and from estrogen receptor status. This work highlights a pivotal role for GAB2 and its transcriptional targets in anchorage-independent growth and breast cancer metastatic progression.

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Gab2 and Src co-operate in human mammary epithelial cells to promote growth factor independence and disruption of acinar morphogenesis

Cited by 40 publications

References 42 publications

Identification of a Src Tyrosine Kinase/SIAH2 E3 Ubiquitin Ligase Pathway That Regulates C/EBPδ Expression and Contributes to Transformation of Breast Tumor Cells

Identification of a Src Tyrosine Kinase/SIAH2 E3 Ubiquitin Ligase Pathway That Regulates C/EBPδ Expression and Contributes to Transformation of Breast Tumor Cells

Interferon-γ-induced increases in intestinal epithelial macromolecular permeability requires the Src kinase Fyn

The GAB2 signaling scaffold promotes anchorage independence and drives a transcriptional response associated with metastatic progression of breast cancer

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