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Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation.

ABSTRACT: A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the MEK-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. We recently identified A-Raf as a potent inhibitor of the proapoptotic mammalian sterile 20-like kinase (MST2) tumor suppressor pathway in several cancer entities including head and neck, colon, and breast. Independent of kinase activity, A-Raf binds to MST2 thereby efficiently inhibiting apoptosis. Here, we show that the interaction of A-Raf with the MST2 pathway is regulated by subcellular compartmentalization. Although in proliferating normal cells and tumor cells A-Raf localizes to the mitochondria, differentiated non-carcinogenic cells of head and neck epithelia, which express A-Raf at the plasma membrane. The constitutive or induced re-localization of A-Raf to the plasma membrane compromises its ability to efficiently sequester and inactivate MST2, thus rendering cells susceptible to apoptosis. Physiologically, A-Raf re-localizes to the plasma membrane upon epithelial differentiation in vivo. This re-distribution is regulated by the scaffold protein kinase suppressor of Ras 2 (KSR2). Downregulation of KSR2 during mammary epithelial cell differentiation or siRNA-mediated knockdown re-localizes A-Raf to the plasma membrane causing the release of MST2. By using the MCF7 cell differentiation system, we could demonstrate that overexpression of A-Raf in MCF7 cells, which induces differentiation. Our findings offer a new paradigm to understand how differential localization of Raf complexes affects diverse signaling functions in normal cells and carcinomas.

SUBMITTER: Rauch J

PROVIDER: S-EPMC4947672 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation.

Rauch J J Vandamme D D Mack B B McCann B B Volinsky N N Blanco A A Gires O O Kolch W W

Cell death and differentiation 20160219 8

A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the MEK-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. We recently identified A-Raf as a potent inhibitor of the proapoptotic mammalian sterile 20-like kinase (MST2) tumor suppressor pathway in several cancer entities including head and neck, colon, and breast. Independent of kinase activity, A-Raf binds to MST2 thereby efficient ...[more]

PMID: 26891695

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Project description:BackgroundMyocardin-related transcription factors (MRTF) A and B link actin dynamics and mechanotransduction to gene expression. In mice, MRTF-A is involved in mammary gland differentiation, but its role in human mammary epithelial cells remains unclear.MethodsThree-dimensional cultures of human mammary epithelial MCF10A cells were used to model acinar morphogenesis. Stable MRTF-A knockdown, MRTF-A/B rescue and MRTF-A/B overexpression was established to characterize the functional role during morphogenesis using confocal microscopy and expression analysis. Breast cancer patient databases were analyzed for MRTF-A expression.ResultsWe showed that a precise temporal control of MRTFs is required for normal morphogenesis of MCF10A mammary acini. MRTF transcriptional activity, but not their protein amounts, is transiently induced during 3D acini formation. MRTF-A knockdown dramatically reduces acini size and prevents lumen formation. These effects are rescued by re-expression of MRTF-A, and partially by MRTF-B. Conversely, overexpression of MRTF-A and MRTF-B increases acini size, resulting in irregular spheroids without lumen and defective apico-basal polarity. These phenotypes correlate with deregulated expression of cell cycle inhibitors p21/Waf1, p27/Kip1 and altered phosphorylation of retinoblastoma protein. In MRTF overexpressing spheroids, proliferation and apoptosis are simultaneously increased at late stages, whilst neither occurs in control acini. MRTFs interfere with anoikis of the inner cells and cause an integrin switch from ?6 to ?5, repression of E-cadherin and induction of mesenchymal markers vimentin, Snai2 and Zeb1. Moreover, MRTF-overexpressing spheroids are insensitive to alteration in matrix stiffness. In two breast cancer cohorts, high expression of MRTF-A and known target genes was associated with decreased patient survival.ConclusionMRTF-A is required for proliferation and formation of mammary acini from luminal epithelial cells. Conversely, elevated MRTF activity results in pre-malignant spheroid formation due to defective proliferation, polarity loss and epithelial-mesenchymal transition.

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Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation.

Publications

Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation.

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