Tightly controlled MRTF-A activity regulates epithelial differentiation during formation of mammary acini

Abstract

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 α6 to α5, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0860-3) contains supplementary material, which is available to authorized users.

Micropatterning as a tool to identify regulatory triggers and kinetics of actin-mediated endothelial mechanosensing

“…In line with our present data for primary endothelial cells, Cui et al have reported that MRTF-A translocates much faster than YAP when fibroblasts are subjected to cyclic stretching forces (Cui et al, 2015). More recently, a study from the Posern group has highlighted that a precise temporal control of MRTF-A is required for the formation of mammary acini (Seifert and Posern, 2017).…”

Feeling Things Out: Bidirectional Signaling of the Cell–ECM Interface, Implications in the Mechanobiology of Cell Spreading, Migration, Proliferation, and Differentiation

“…The role of matrix stiffness on control of MRTF activation appears to be essential in promoting contact‐mediated proliferation and metastasis. Forced overexpression of MRTF overcomes the requirement of stiffness‐mediated G‐actin recruitment and leads to protection from anoikis in mammary epithelial cells, regardless of matrix stiffness, implying that mechanical cues strictly regulate MRTF activation, not endogenous MRTF signaling functions 199. Further, mouse models of peritoneal fibrosis have shown that lysophosphatidic acid (LPA) signaling at sites of tissue injury induces MRTF nuclear activation and upregulation of connective tissue growth factor (CTGF) expression in mesothelial cells 200.…”

“…Proper function of the mechanically sensitive proliferation pathway is required for regulating the proliferation requirements of anchorage‐dependence and contact‐inhibition. Upregulated activation or expression of FAK, ERK pathway members, and/or the Rho‐actin‐MRTF axis are responsible for loss of proliferation control mechanisms that underly pathological hyperproliferation 199,201…”

EMT/MET at the Crossroad of Stemness, Regeneration and Oncogenesis: The Ying-Yang Equilibrium Recapitulated in Cell Spheroids

“…EMT and its opposite MET (mesenchymal-to-epithelial transition) are significantly involved in stemness balance in both normal and malignant cell spheroids, and their modulation is strategic for the achievement of specific cell phenotypes [ 3 , 4 , 5 , 6 ]. At molecular level, EMT is mediated by the activation of several transcription factors (TFs), including those belonging to the Snail superfamily, such as SNAI1 and SNAI2 [ 7 ], by the loss of cell-junction molecules, such as E-cadherin (encoded by CDH1 ), and the acquisition of mesenchymal markers, such as vimentin.…”