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Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells.

ABSTRACT: BACKGROUND:Focal adhesion kinase (FAK) is a cytoplasmatic protein tyrosine kinase that associates with both integrins and growth factor receptors toward the adhesion, migration and invasion of cancer cells. The G-protein coupled estrogen receptor (GPER) has been involved in the stimulatory action of estrogens in breast tumor. In this study, we have investigated the engagement of FAK by GPER signaling in triple negative breast cancer (TNBC) cells. METHODS:Publicly available large-scale database and patient data sets derived from "The Cancer Genome Atlas" (TCGA; www.cbioportal.org ) were used to assess FAK expression in TNBC, non-TNBC tumors and normal breast tissues. MDA-MB 231 and SUM159 TNBC cells were used as model system. The levels of phosphorylated FAK, other transduction mediators and target genes were detected by western blotting analysis. Focal adhesion assay was carried out in order to determine the focal adhesion points and the formation of focal adhesions (FAs). Luciferase assays were performed to evaluate the promoters activity of c-FOS, EGR1 and CTGF upon GPER activation. The mRNA expression of the aforementioned genes was measured by real time-PCR. Boyden chamber and wound healing assays were used in order to evaluate cell migration. The statistical analysis was performed by ANOVA. RESULTS:We first determined by bioinformatic analysis that the mRNA expression levels of the gene encoding FAK, namely PTK2, is higher in TNBC respect to non-TNBC and normal breast tissues. Next, we found that estrogenic GPER signaling triggers Y397 FAK phosphorylation as well as the increase of focal adhesion points (FAs) in TNBC cells. Besides, we ascertained that GPER and FAK activation are involved in the STAT3 nuclear accumulation and gene expression changes. As biological counterpart, we show that FAK inhibition prevents the migration of TNBC cells upon GPER activation. CONCLUSIONS:The present data provide novel insights regarding the action of FAK in TNBC. Moreover, on the basis of our findings estrogenic GPER signaling may be considered among the transduction mechanisms engaging FAK toward breast cancer progression.

SUBMITTER: Rigiracciolo DC

PROVIDER: S-EPMC6364402 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells.

Rigiracciolo Damiano Cosimo DC Santolla Maria Francesca MF Lappano Rosamaria R Vivacqua Adele A Cirillo Francesca F Galli Giulia Raffaella GR Talia Marianna M Muglia Lucia L Pellegrino Michele M Nohata Nijiro N Di Martino Maria Teresa MT Maggiolini Marcello M

Journal of experimental & clinical cancer research : CR 20190206 1

<h4>Background</h4>Focal adhesion kinase (FAK) is a cytoplasmatic protein tyrosine kinase that associates with both integrins and growth factor receptors toward the adhesion, migration and invasion of cancer cells. The G-protein coupled estrogen receptor (GPER) has been involved in the stimulatory action of estrogens in breast tumor. In this study, we have investigated the engagement of FAK by GPER signaling in triple negative breast cancer (TNBC) cells.<h4>Methods</h4>Publicly available large-s ...[more]

PMID: 30728047

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Project description:PurposeRegulation of lens development involves an intricate interplay between growth factor (e.g. FGF and TGFbeta) and extracellular matrix (ECM) signaling pathways. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays key roles in transmitting ECM signals by integrins. In this study, we delineated patterns of FAK expression and tyrosine phosphorylation (Y397) in the developing lens and investigated its regulation by FGF2. We also examined FAK expression and activation during disrupted fiber differentiation in mice expressing a dominant-negative TGFbeta receptor.MethodsFAK expression and activation (phosphorylation on Y397) was studied in embryonic and postnatal rodent lenses by in situ hybridization, immunofluorescence, and western blotting. Rat lens explants were used to investigate the effects of FGF2 on FAK expression and activation. Immunofluorescence and western blotting were used to examine FAK expression and phosphorylation in transgenic mice that express a dominant-negative TGFbeta receptor.ResultsFAK is widely expressed and phosphorylated during embryonic stages of lens morphogenesis and differentiation. However, in postnatal lenses its expression and activation becomes restricted to the posterior germinative zone and the transitional zone at the lens equator. While both NH2- and COOH-terminal antibodies revealed cytoplasmic and membrane-associated staining in lens cells, the NH2-terminal antibody also showed FAK was present in fiber cell nuclei. In vitro, FAK expression and phosphorylation on Y397 were increased by concentrations of FGF2 that initiate lens epithelial cell migration (10 ng/ml) and differentiation (50 ng/ml) but not proliferation (5 ng/ml). Moreover, reactivity for Y397 phosphorylated FAK is prominent in the nuclei of differentiating fibers both in vivo and in vitro. Disruption of TGFbeta-like signals by ectopic expression of a dominant-negative TGFbeta receptor (TbetaRII(D/N)) results in abnormal lens fiber differentiation in transgenic mice. While FAK expression is initiated normally in the posterior germinative zone of TbetaRII(D/N) transgenic lenses, as fiber differentiation proceeds, FAK becomes localized to a perinuclear compartment, decreases its association with the cytoskeleton and is poorly phosphorylated on Y(397).ConclusionsFAK is widely expressed and activated during early lens morphogenesis. During secondary lens fiber differentiation, FAK is expressed and phosphorylated on Y397 as epithelial cells exit the cell cycle, initiate migration at the equator, and undergo differentiation in the transitional zone. During terminal fiber differentiation an NH2-terminal fragment of FAK, including Y397, is translocated to the nucleus. The expression, activation, and nuclear localization of FAK are regulated, at least partly, by FGF2. FAK activity and subcellular localization are also modulated by TGFbeta-like signals. In fiber cells of TbetaRII(D/N) transgenic lenses, FAK is abnormally retained in a perinuclear compartment, loses its association with the cytoskeleton, and is poorly phosphorylated. These data suggest that integrin signaling via FAK plays important roles during lens differentiation, mediated by FGFs and TGFbeta-superfamily signals.

Project description:BackgroundUnderstanding the intricate signaling network involved in triple-negative breast cancer (TNBC) represents a challenge for developing novel therapeutic approaches. Here, we aim to provide novel mechanistic insights on the function of the S100A8/A9-RAGE system in TNBC.MethodsTNM plot analyzer, Kaplan-Meier plotter, Meta-analysis, GEPIA2 and GOBO publicly available datasets were used to evaluate the clinical significance of S100A8/A9 and expression levels of S100A8/A9, RAGE and Filamin family members in breast cancer (BC) subtypes. METABRIC database and Cox proportional hazard model defined the clinical impact of high RAGE expression in BC patients. Multiple bioinformatics programs identified the main enriched pathways within high RAGE expression BC cohorts. By lentiviral system, TNBC cells were engineered to overexpress RAGE. Western blotting, immunofluorescence, nucleus/cytoplasm fractionation, qRT-PCR, gene silencing and luciferase experiments were performed to identify signal transduction mediators engaged by RAGE upon stimulation with S100A8/A9 in TNBC cells. Proliferation, colony formation and transwell migration assays were carried out to evaluate the growth and migratory capacity of TNBC cells. Statistical analysis was performed by ANOVA and independent t-tests.ResultsWe found a remarkable high expression of S100A8 and S100A9 in BC, particularly in HER2-positive and TNBC, with the latter associated to worst clinical outcomes. In addition, high RAGE expression correlated with a poor overall survival in BC. Next, we determined that the S100A8/A9-RAGE system triggers FAK activation by engaging a cytoskeleton mechanosensing complex in TNBC cells. Through bioinformatics analysis, we identified the Hippo pathway as the most enriched in BC patients expressing high RAGE levels. In accordance with these data, we demonstrated the involvement of S100A8/A9-RAGE-FAK signaling in the control of Hippo/YAP activities, and we established the crucial contribution of RAGE-FAK-YAP circuitry in the growth and migratory effects initiated by S100A8/A9 in TNBC cells.ConclusionsThe present study provides novel mechanistic insights on RAGE actions in TNBC. Moreover, our findings suggest that RAGE-FAK-YAP transduction pathway could be exploited as a druggable system halting the aggressive TNBC subtype.

Project description:Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis.To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible) system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD), and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors.Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p < 0.05) by FAKsiRNA.Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal specific expression of genes affected by silencing of FAK.

Project description:Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase-dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb-iCreert -driven endothelial cell (EC)-specific, tamoxifen-inducible homozygous mutant mouse lines: FAK wild-type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT/WT , ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT/WT and ECCre-;FAKWT/WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice, as compared with Cre - littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice. Furthermore, although ECCre+;FAKKD/KD mice showed reduced blood vessel leakage, ECCre+;FAKDM/DM and ECCre-;FAKDM/DM mice showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD/KD ECs as compared with Cre+;FAKWT/WT cells, with no change in phosphorylation of the known Src targets FAK-Y577, FAK-Y861, FAK-Y925, paxillin-Y118, p130Cas-Y410. Cre+;FAKDM/DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE-cadherin-pY658 levels in Cre+;FAKKD/KD ECs were rescued in Cre+FAKDM/DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM/DM mice. We show that EC-specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD-dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells.

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Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells.

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