Paula J. McKeown-Longo

Krüppel-Like Factor 8 Induces Epithelial to Mesenchymal Transition and Epithelial Cell Invasion

Tumor invasion and metastasis are the main causes of death from cancer. Epithelial to mesenchymal transition (EMT) is a determining step for a cancer cell to progress from a noninvasive to invasive state. Krüppel-like factor 8 (KLF8) plays a key role in oncogenic transformation and is highly overexpressed in several types of invasive human cancer, including breast cancer. To understand the role of KLF8 in regulating the progression of human breast cancer, we first established stable expression of KLF8 in an immortalized normal human breast epithelial cell line. We found that KLF8 strongly induced EMT and enhanced motility and invasiveness in the cells, by analyzing changes in cell morphology and epithelial and mesenchymal marker proteins, and using cell migration and Matrigel invasion assays. Chromatin immunoprecipitations (ChIP), oligonucleotide precipitations, and promoter-reporter assays showed that KLF8 directly bound and repressed the promoter of E-cadherin independent of E boxes in the promoter and Snail expression. Aberrant elevation of KLF8 expression is highly correlated with the decrease in E-cadherin expression in the invasive human breast cancer. Blocking KLF8 expression by RNA interference restored E-cadherin expression in the cancer cells and strongly inhibited the cell invasiveness. This work identifies KLF8 as a novel EMT-regulating transcription factor that opens a new avenue in EMT research and suggests an important role for KLF8 in human breast cancer invasion and metastasis.

Regulation of HEF1 Expression and Phosphorylation by TGF-β1 and Cell Adhesion

Transforming growth factor-β1 (TGF-β1) is a multipotential cytokine, which regulates remodeling of tissue extracellular matrix during early tumorigenesis and wound healing. Human enhancer of filamentation-1 (HEF1), a multifunctional docking protein, is involved in integrin-based signaling, which affects cell motility, growth, and apoptosis. Our studies reveal that TGF-β1 is a potent inducer of HEF1 gene transcription in human dermal fibroblasts. TGF-β1 promoted HEF1 expression in a dose-dependent manner and resulted in a 16-fold increase in HEF1 protein level. TGF-β1 had no effect on the stability of either HEF1 protein or mRNA. The TGF-β1-induced HEF1 expression was independent of cell adhesion and resistant to cytoskeleton disruption. TGF-β1 increased levels of both p105 and p115 HEF1 in adherent fibroblasts. Digestion with specific phosphatases indicated that the p115HEF1 resulted from serine/threonine phosphorylation of p105HEF1. The appearance of the p115HEF1 as well as tyrosine phosphorylation of p105HEF1 required cell adhesion and/or an organized cytoskeleton. Anin vitro kinase assay indicated that p105HEF1 was a substrate for Src. PP1, a specific Src kinase inhibitor, was able to block adhesion-dependent tyrosine phosphorylation of p105HEF1. These findings suggest that TGF-β1 regulatesHEF1 gene expression and that HEF1 phosphorylation is dependent on cell adhesion and Src kinase activity.

Urokinase-type Plasminogen Activator Receptor Regulates a Novel Pathway of Fibronectin Matrix Assembly Requiring Src-dependent Transactivation of Epidermal Growth Factor Receptor

Previous studies have indicated that the urokinase-type plasminogen activator receptor (uPAR) can functionally interact with integrins thereby modulating integrin activity. We have previously demonstrated that treatment of fibroblasts with the uPAR ligand, P25, results in an increase in the activation of the β1 integrin and a 35-fold increase in fibronectin matrix assembly (Monaghan, E., Gueorguiev, V., Wilkins-Port, C., and McKeown-Longo, P. J. (2004) J. Biol. Chem. 279, 1400-1407). Experiments were conducted to address the mechanism of uPAR regulation of matrix assembly. Treatment of fibroblasts with P25 led to an increase in the activation of the epidermal growth factor receptor (EGFR) and a colocalization of activated EGFR with β1 integrins in cell matrix contacts. The effects of P25 on matrix assembly and β1 integrin activation were inhibited by pretreatment with EGFR or Src kinase inhibitors, suggesting a role for both Src and EGFR in integrin activation by uPAR. Phosphorylation of EGFR in response to P25 occurred on Tyr-845, an Src-dependent phosphorylation site and was inhibited by PP2, the Src kinase inhibitor, consistent with Src kinase lying upstream of EGFR and integrin activation. Cells null for Src kinases also showed a loss of P25-induced matrix assembly, integrin activation, and EGFR phosphorylation. These P25-induced effects were restored following Src re-expression. The effects of P25 were specific for uPAR as enhanced matrix assembly by P25 was not seen in uPAR-/- cells, but was restored upon uPAR re-expression. These data provide evidence for a novel pathway of fibronectin matrix assembly through the uPAR-dependent sequential activation of Src kinase, EGFR, and β1 integrin.

Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin

The fibronectin matrix contains cryptic sites which are thought to modulate cellular biological responses. One of these sites, located in fibronectins first type III repeat (III1c), influences signaling pathways that are relevant to cytoskeletal organization and cell cycle progression. The purpose of this study was to identify possible mechanisms responsible for the effects of III1c on cell behavior. Recombinant peptides representing various type III repeats of fibronectin were compared for their effects on fibronectin matrix organization and activation of intracellular signaling pathways. III1c and III13 but not III11c or III10 bound to monolayers of human skin fibroblasts in a dose- and time-dependent manner and were localized to the extracellular matrix. Binding of III13, but not III1c, to matrix was sensitive to heparitinase, suggesting that the association of III1c with the matrix was not dependent on heparan sulfate proteoglycans. Quantitative and morphological assessment indicated that, in contrast to previously published reports, the binding of III1c to cell layers did not result in the loss or disruption of matrix fibronectin. Binding of III1c but not III13 to the extracellular matrix did result in the loss of a conformationally sensitive epitope present within the EDA type III module of cellular fibronectin. III1c-induced loss of the EDA epitope did not require the presence of cells, occurred within 1 hour and was associated with the activation of p38 mitogen-activated protein kinase (MAPK) followed by the formation of filopodia. Maximal phosphorylation of p38 MAPK occurred within 1 hour, whereas cytoskeletal changes did not appear until 12 hours later. These findings are consistent with a model in which the binding of III1c to the extracellular matrix results in a conformational remodeling of the fibronectin matrix, which has both short- and long-term effects on cell physiology.

Inhibition of Fibronectin Matrix Assembly by the Heparin-binding Domain of Vitronectin

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the amino-terminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin’s heparin-binding domain inhibited both β1 and non-β1 integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin-associated proteins α-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton.

The First Type III Repeat in Fibronectin Activates an Inflammatory Pathway in Dermal Fibroblasts

Remodeling of the fibronectin matrix occurs during a variety of pathological and regenerative processes. Cellular generated tensional forces can alter the secondary and tertiary structure of the fibronectin matrix and regulate the exposure of cryptic activities that directly impact cell behavior. In the present study, we evaluated the effect of the partially unfolded Type III fibronectin module, FnIII-1c, on gene expression in dermal fibroblasts. Microarray and PCR analysis indicated that the addition of FnIII-1c to human dermal fibroblasts induced the expression of several inflammatory genes including the cytokines, IL-8 and TNF-α. ELISA analysis indicated that the increased gene expression was accompanied by the secretion of IL-8 and TNF-α protein. FnIII-1c-induced gene expression was preceded by increased phosphorylation of IκB kinase (IKK) and IκBα as well as the nuclear translocation of NFκB. PCR and ELISA analysis showed that inhibition of the NFκB signaling pathway completely blocked the induction of IL-8 and TNF-α. Blocking antibodies to Toll-like receptor 4 inhibited both the activation of the NFκB signaling pathway as well as cytokine expression in response to FnIII-1c. These data suggest that fibronectin matrix remodeling can induce the expression of cytokines by stromal cells present in the tissue microenvironment.

Cell adhesion regulates Ser/Thr phosphorylation and proteasomal degradation of HEF1

Human enhancer of filamentation 1 (HEF1), a multifunctional docking protein of the Cas family, participates in integrin and growth factor signaling pathways that regulate global cellular processes including growth, motility and apoptosis. HEF1 consists of two isoforms, p105 and p115, the larger molecular weight form resulting from Ser/Thr phosphorylation of p105HEF1. The molecular mechanisms that regulate the interconversion of the two HEF1 species as well as the function of HEF1 Ser/Thr phosphorylation are unknown. Our study reveals that cell adhesion and detachment regulate the interconversion of the two HEF1 isoforms. Experiments using various inhibitors of cytoskeletal organization indicated that disruption of actin microfilaments but not intermediate filaments or microtubules resulted in a complete conversion of p115HEF1 to p105HEF1. The conversion of p115HEF1 to p105HEF1 was prevented by inhibition of protein phosphatase 2A (PP2A), suggesting that cytoskeletal regulation of PP2A activity controlled the dephosphorylation of p115HEF1. Degradation of endogenous HEF1 was dependent on proteasomes with the p115 species of HEF1 being preferentially targeted for turnover. Dephosphorylation of HEF1 by suspending cells or disrupting actin filaments protected HEF1 from degradation. These results suggest that the adhesion-dependent actin organization regulates proteasomal turnover of HEF1 through the activity of PP2A.

Regulation of the Innate Immune Response by Fibronectin: Synergism between the III-1 and EDA Domains

Fibronectin is a critical component of the extracellular matrix and alterations to its structure will influence cellular behavior. Matrix fibronectin is subjected to both mechanical and biochemical regulation. The Type III domains of fibronectin can be unfolded in response to increased cellular contractility, included or excluded from the molecule by alternative splicing mechanisms, or released from the matrix by proteolysis. Using Inflammatory Cytokine microarrays we found that the alternatively spliced fibronectin Type III domain, FnEDA, and the partially unfolded III-1 domain, FnIII-1c, induced the expression of a multitude of pro-inflammatory cytokines in human dermal fibroblasts, most notably CXCL1-3, IL-8 and TNF-α. FnIII-1c, a peptide representing an unfolded intermediate structure of the first Type III domain has been shown to initiate the toll-like receptor-4 (TLR4)-NFκB-dependent release of cytokines from human dermal fibroblasts (You, et al., J. Biol. Chem., 2010). Here we demonstrate that FnIII-1c and the alternatively spliced FnEDA domain induce a TLR4 dependent activation of p38 MAP kinase and its downstream effector, MAPKAP Kinase-2 (MK-2), to regulate cytokine expression in fibroblasts. RT-qPCR analysis indicated that the p38-MK-2 pathway regulates IL-8 mRNA stability. Interestingly, addition of FnIII-1c and FnEDA synergistically enhanced TLR4-dependent IL-8 release. These data indicate that Fn contains two Type III domains which can activate TLR signaling to induce an inflammatory response in fibroblasts. Furthermore, our data identifies the NF-κB and p38/MK2 signaling pathways as transducers of signals initiated in response to structural changes in fibronectin.

Epidermal Growth Factor (EGF) Regulates α5β1 Integrin Activation State in Human Cancer Cell Lines through the p90RSK-dependent Phosphorylation of Filamin A

Background: Regulation of integrin activation has important implications for tumor cell invasion and metastasis. Results: EGF activates ERK/p90RSK and Rho/Rho kinase signaling in A431 and DiFi colon cancer cells, leading to phosphorylation of filamin A (FLNa) and inactivation of the α5β1 integrin receptor. Conclusion: EGF promotes α5β1 inactivation through the p90RSK-dependent phosphorylation of FLNa. Significance: We have identified a novel EGF-dependent mechanism controlling the α5β1 integrin activation state. Cell adhesion, motility, and invasion are regulated by the ligand-binding activity of integrin receptors, transmembrane proteins that bind to the extracellular matrix. Integrins whose conformation allows for ligand binding and appropriate functional activity are said to be in an active state. Integrin activation and subsequent ligand binding are dynamically regulated by the association of cytoplasmic proteins with integrin intracellular domains. In this study, we evaluated the role of EGF in the regulation of the activation state of the α5β1 integrin receptor for fibronectin. The addition of EGF to either A431 squamous carcinoma cells or DiFi colon cancer cells resulted in loss of α5β1-dependent adhesion to fibronectin but no loss of integrin from the cell surface. EGF activated the EGF receptor/ERK/p90RSK and Rho/Rho kinase signaling pathways. Blocking either pathway inhibited EGF-mediated loss of adhesion, suggesting that they work in parallel to regulate integrin function. EGF treatment also resulted in phosphorylation of filamin A (FLNa), which binds and inactivates β1 integrins. EGF-mediated FLNa phosphorylation was completely blocked by an inhibitor of p90RSK and partially attenuated by an inhibitor of Rho kinase, suggesting that both pathways converge on FLNa to regulate integrin function. A431 clonal cell lines expressing non-phosphorylated dominant-negative FLNa were resistant to the inhibitory effects of EGF on integrin function, whereas clonal cell lines overexpressing wild-type FLNa were more sensitive to the inhibitory effect of EGF. These data suggest that EGF-dependent inactivation of α5β1 integrin is regulated through FLNa phosphorylation and cellular contractility.

Anastellin, the Angiostatic Fibronectin Peptide, Is a Selective Inhibitor of Lysophospholipid Signaling

Angiogenesis is regulated by integrin-dependent cell adhesion and the activation of specific cell surface receptors on vascular endothelial cells by angiogenic factors. Lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P) are bioactive lysophospholipids that activate G protein–coupled receptors that stimulate phosphatidylinositol 3-kinase (PI3K), Ras, and Rho effector pathways involved in vascular cell survival, proliferation, adhesion, and migration. Previous studies have shown that anastellin, a fragment of the first type III module of fibronectin, functions as an antiangiogenic peptide suppressing tumor growth and metastasis. We have previously shown that anastellin blocks serum-dependent proliferation of microvessel endothelial cells (MVEC) by affecting extracellular signal-regulated kinase (ERK)–dependent G1-S transition. However, the mechanism by which anastellin regulates endothelial cell function remains unclear. In the present study, we mapped several lysophospholipid-mediated signaling pathways in MVEC and examined the effects of anastellin on LPA- and S1P-induced MVEC proliferation, migration, and cytoskeletal organization. Both LPA and S1P activated PI3K, Ras/ERK, and Rho/Rho kinase pathways, leading to migration, G1-S cell cycle progression, and stress fiber formation, respectively. Stimulation of proliferation by LPA/S1P occurred through a Gi-dependent Ras/ERK pathway, which was independent of growth factor receptors and PI3K and Rho/Rho kinase signaling. Although LPA and S1P activated both PI3K/Akt and Ras/ERK signaling through Gi, anastellin inhibited only the Ras/ERK pathway. Stress fiber formation in response to LPA was dependent on Rho/Rho kinase but independent of Gi and unaffected by anastellin. These results suggest that lysophospholipid mediators of Gi activation leading to PI3K/Akt and Ras/ERK signaling bifurcate downstream of Gi and that anastellin selectively inhibits the Ras/ERK arm of the pathway. (Mol Cancer Res 2009;7(2):255–65)