Disruption Of Cell-cell Contacts Research Articles

Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) plays an important role in development and also in initiation of metastasis during cancer. Disruption of cell-cell contacts during EMT allowing cells to detach from and migrate away from their neighbors remains poorly understood. Using immunofluorescent staining and live-cell imaging, we analyzed early events during EMT induced by epidermal growth factor (EGF) in IAR-20 normal epithelial cells. Control cells demonstrated stable adherens junctions (AJs) and robust contact paralysis, whereas addition of EGF caused rapid dynamic changes at the cell-cell boundaries: fragmentation of the circumferential actin bundle, assembly of actin network in lamellipodia, and retrograde flow. Simultaneously, an actin-binding protein EPLIN was phosphorylated, which may have decreased the stability of the circumferential actin bundle. Addition of EGF caused gradual replacement of linear E-cadherin–based AJs with dynamic and unstable punctate AJs, which, unlike linear AJs, colocalized with the mechanosensitive protein zyxin, confirming generation of centripetal force at the sites of cell-cell contacts during EMT. Our data show that early EMT promotes heightened dynamics at the cell-cell boundaries—replacement of stable AJs and actin structures with dynamic ones—which results in overall weakening of cell-cell adhesion, thus priming the cells for front-rear polarization and eventual migration.

Piperazine Derivatives Enhance Epithelial Cell Monolayer Permeability by Increased Cell Force Generation and Loss of Cadherin Structures.

A major obstacle for topical and enteral drug delivery is the poor transport of macromolecular drugs through the epithelium. One potential solution is the use of permeation enhancers that alter epithelial structures. Piperazine derivatives are known permeation enhancers that modulate epithelial structures, reduce transepithelial electrical resistance, and augment the absorption of macromolecular drugs. The mechanism by which piperazine derivatives disrupt the structures of epithelial monolayers is not well understood. Here, the effects of 1-phenylpiperazine and 1-methyl-4-phenylpiperazine are modeled in the epithelial cell line NRK-52E. Live-cell imaging reveals a dose-dependent gross reorganization of monolayers at high concentrations, but reorganization differs based on the piperazine molecule. Results show that low concentrations of piperazine derivatives increase myosin force generation within the cells and do not disrupt the cytoskeletal structure. Also, cytoskeletally attached cadherin junctions are disrupted before tight junctions. In summary, piperazines appear to increase myosin-mediated contraction followed by disruption of cell-cell contacts. These results provide new mechanistic insight into how transient epithelial permeation enhancers act and will inform of the development of future generations of transepithelial delivery systems.

Inhibition of Meprins Reduces Pulmonary Edema in LPS-Induced Acute Lung Damage.

Pulmonary edema is the major factor of tissue hypoxia in acute lung injury. Disruption of cell-cell contacts and lung interstitium increases permeability of the vascular endothelium and alveolar epithelium, which leads to the development of pulmonary edema. Meprin metalloproteases cleave extracellular matrix proteins, thus aggravating pulmonary edema. Meprin inhibitor actinonin was administered to rats with LPS-induced acute lung injury. Damaged lungs looked spotted and had multiple hemorrhage focuses, protein concentration in lavage fluid was increased, and lung weight coefficient was high. Administration of meprin inhibitor actinonin considerably reduced protein content in the bronchoalvelolar lavage and lung coefficient; only solitary lung hemorrhages were seen after this treatment. Thus, inhibition of meprins potentially alleviates LPS-induced disorders in the lung tissue permeability and reduces pulmonary edema.

PO-194 Novel potential targets for hakai oncogene during tumour progression

IntroductionThe transition from a benign lesion or adenoma to carcinoma begins with the disruption of cell-cell contacts. The loss of E-cadherin at cell-cell contacts is a hallmark of the epithelial-to-mesenchymal transition (EMT) process. During EMT, cells lose their epithelial phenotype and acquire motility and invasive capacities, due to decreased expression of E-cadherin. Hakai is an E3 ubiquitin-ligase for E-cadherin, which induces its ubiquitination and degradation and it is an oncogene that induces transformation in epithelial cells to a mesenchymal and invasive phenotype both in vitro and in vivo. Moreover, Hakai expression is markedly higher in human colon adenocarcinoma compared to normal tissues. However, not all cellular changes observed when Hakai is overexpressed can be attributed to its action on E-cadherin. Here, we aim to identify new molecular targets for Hakai involved in cancer cell plasticity during tumour progression.Material and methodsWe employed iTRAQ technique followed by LC-MALDI-MS analysis in Hakai overexpresing epithelial cells compared to normal cells. Data obtained from the MS/MS acquisitions were quantified using ProteinPilot software and analysed by bioinformatics. Western blot was performed for validation of several proteins identified. Finally, by confocal analysis it was determined the subcellular localization of specific regulated proteins, such as Galectin-3 and Annexin A1.Results and discussionsOur results show that Hakai influences cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. We confirmed the regulation by Hakai of several specific proteins including Galectin-3 and Annexin A1. Confocal images show a total disappearance of Annexin A1 and a colocalization of Hakai and Galectin-3 in the cytoplasm in Hakai overexpressing cells. Interestingly, a profound decreased expression in several proteasome subunits was highlighted.ConclusionHakai influences cellular metabolism, cytoskeleton-related proteins, RNA-related proteins extracellular exosome-associated proteins. It is also evidenced a profound decreased expression in many 26S proteasome subunits during Hakai-driven EMT. Since in clinical trials the use of proteasome inhibitors has failed for solid tumours, our findings suggest that E3 ubiquitin-ligases, such as Hakai, may be a better target for using novel specific inhibitors in tumour subtypes.

Hakai overexpression effectively induces tumour progression and metastasis in vivo

At early stages of carcinoma progression, epithelial cells undergo a program named epithelial-to-mesenchymal transition characterized by the loss of the major component of the adherens junctions, E-cadherin, which in consequence causes the disruption of cell-cell contacts. Hakai is an E3 ubiquitin-ligase that binds to E-cadherin in a phosphorylated-dependent manner and induces its degradation; thus modulating cell adhesions. Here, we show that Hakai expression is gradually increased in adenoma and in different TNM stages (I-IV) from colon adenocarcinomas compared to human colon healthy tissues. Moreover, we confirm that Hakai overexpression in epithelial cells drives transformation in cells, a mesenchymal and invasive phenotype, accompanied by the downregulation of E-cadherin and the upregulation of N-cadherin, and an increased proliferation and an oncogenic potential. More importantly, for the first time, we have studied the role of Hakai during cancer progression in vivo. We show that Hakai-transformed MDCK cells dramatically induce tumour growth and local invasion in nude mice and tumour cells exhibit a mesenchymal phenotype. Furthermore, we have detected the presence of micrometastasis in the lung mice, further confirming Hakai role during tumour metastasis in vivo. These results lead to the consideration of Hakai as a potential new therapeutic target to block tumour development and metastasis.

Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF.

Proteomic Analysis of the E3 Ubiquitin-Ligase Hakai Highlights a Role in Plasticity of the Cytoskeleton Dynamics and in the Proteasome System.

Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell-cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell-cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, our findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT.

TGF-β-independent CTGF induction regulates cell adhesion mediated drug resistance by increasing collagen I in HCC.

Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapeutic agents and remains an unmet medical need. Here, we demonstrate a mechanism of cell adhesion-mediated drug resistance using a variety of HCC spheroid models to overcome environment-mediated drug resistance in HCC. We classified spheroids into two groups, tightly compacted and loosely compacted aggregates, based on investigation of dynamics of spheroid formation. Our results show that compactness of HCC spheroids correlated with fibroblast-like characteristics, collagen 1A1 (COL1A1) content, and capacity for chemoresistance. We also showed that ablation of COL1A1 attenuated not only the capacity for compact-spheroid formation, but also chemoresistance. Generally, connective tissue growth factor (CTGF) acts downstream of transforming growth factor (TGF)-β and promotes collagen I fiber deposition in the tumor microenvironment. Importantly, we found that TGF-β–independent CTGF is upregulated and regulates cell adhesion-mediated drug resistance by inducing COL1A1 in tightly compacted HCC spheroids. Furthermore, losartan, which inhibits collagen I synthesis, impaired the compactness of spheroids via disruption of cell-cell contacts and increased the efficacy of anticancer therapeutics in HCC cell line- and HCC patient-derived tumor spheroids. These results strongly suggest functional roles for CTGF-induced collagen I expression in formation of compact spheroids and in evading anticancer therapies in HCC, and suggest that losartan, administered in combination with conventional chemotherapy, might be an effective treatment for liver cancer.

Essential role of Cdc42 in cardiomyocyte proliferation and cell-cell adhesion during heart development

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/β-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

High-mobility group box 1 impairs airway epithelial barrier function through the activation of the RAGE/ERK pathway.

Recent studies have indicated that high-mobility group box 1 protein (HMGB1) and the receptor for advanced glycation end-products (RAGE) contribute to the pathogenesis of asthma. However, whether the activation of the HMGB1/RAGE axis mediates airway epithelial barrier dysfunction remains unknown. Thus, the aim of this study was to examine the effects of HMGB1 and its synergistic action with interleukin (IL)-1β on airway epithelial barrier properties. We evaluated the effects of recombinant human HMGB1 alone or in combination with IL-1β on ionic and macromolecular barrier permeability, by culturing air-liquid interface 16HBE cells with HMGB1 to mimic the differentiated epithelium. Western blot analysis and immunofluorescence staining were utilized to examine the level and structure of major junction proteins, namely E-cadherin, β-catenin, occludin and claudin-1. Furthermore, we examined the effects of RAGE neutralizing antibodies and mitogen-activated protein kinase (MAPK) inhibitors on epithelial barrier properties in order to elucidate the mechanisms involved. HMGB1 increased FITC-dextran permeability, but suppressed epithelial resistance in a dose-and time-dependent manner. HMGB1-mediated barrier hyperpermeability was accompanied by a disruption of cell-cell contacts, the selective downregulation of occludin and claudin-1, and the redistribution of E-cadherin and β-catenin. HMGB1 in synergy with IL-1β induced a similar, but greater barrier hyperpermeability and induced the disruption of junction proteins. Furthermore, HMGB1 elicited the activation of the RAGE/extracellular signal-related kinase (ERK)1/2 signaling pathway, which correlated with barrier dysfunction in the 16HBE cells. Anti-RAGE antibody and the ERK1/2 inhibitor, U0126, attenuated the HMGB1-mediated changes in barrier permeability, restored the expression levels of occludin and claudin-1 and pevented the redistribution of E-cadherin and β-catenin. Taken together, the findings of our study demonstrate that HMGB1 is capable of inducing potent effects on epithelial barrier function and that RAGE/ERK1/2 is a key signaling pathway involved in the crosstalk between formations of junction proteins and epithelial barrier dysfunction.

Abstract 16137: Essential Role of Cdc42 in Cardiomyocyte Proliferation and Cell-cell Adhesion During Heart Development

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/β-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

Abstract 3259: Giant obscurins: Novel tumor and metastasis suppressors in breast cancer

Abstract Obscurins, encoded by the single OBSCN gene, are giant cytoskeletal proteins containing tandem adhesion and signaling domains, including an active RhoGEF motif that directly binds and activates RhoA. The OBSCN gene is highly mutated in breast cancer resulting in a 2-fold reduction of its mRNA levels. Consistent with this, obscurin proteins are nearly lost from breast cancer cell lines and human biopsies, independently of their hormonal status or molecular differentiation. Loss of giant obscurins from breast epithelial cells confers them with a survival and growth advantage, following exposure to current chemotherapies. Moreover, obscurin-depleted breast epithelial cells fail to form adhesion junctions, undergo epithelial-to-mesenchymal transition and generate primary and secondary mammospheres bearing markers of cancer-initiating cells. In line with these phenotypic alterations, obscurin-deficient cells display markedly increased motility as a sheet in 2-dimensional (2D) substrata and individually in confined spaces, and invasion in 3D matrices. More importantly, loss of giant obscurins from breast epithelial cells promotes primary tumor formation and lung colonization in vivo. Obscurin depletion leads to >50% reduction in RhoA activity, as well as decreased phosphorylation of RhoA effectors, including myosin light chain phosphatase, myosin light chain, lim kinase, and cofilin, in both attached and suspended cells. These molecular alterations manifest as decreased actomyosin contractility, allowing suspended cells to escape detachment-induced apoptosis. Moreover, ∼40% of obscurin-depleted cells extend microtentacles, tubulin-based projections that mediate the attachment of circulating tumor cells to endothelium, an advantage that persists even after paclitaxel exposure. Collectively, our findings reveal that loss of giant obscurins from breast epithelium results in disruption of cell-cell contacts and acquisition of a mesenchymal phenotype that leads to enhanced tumorigenesis, migration and invasiveness in vitro and in vivo by affecting RhoA-mediated processes. Moreover, our data suggest that loss of obscurins may represent a substantial selective advantage for breast epithelial cells during metastasis, and that treatment with paclitaxel may exacerbate this advantage by preferentially allowing obscurin-deficient, stem-like cells to attach to the endothelium of distant sites, a first step towards colonizing metastatic tumors. Citation Format: Aikaterini Kontrogianni-Konstantopoulos, Marey Shriver, Nicole Perry, Kimberly Stroka, Michele Vitole, David Huso, Stuart Martin, Konstantinos Konstantopoulos. Giant obscurins: Novel tumor and metastasis suppressors in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3259. doi:10.1158/1538-7445.AM2015-3259

Abstract 1347: The role of cell-cell contacts in the survival of extracellular matrix detached mammary epithelial cells

Abstract Normal epithelial cells require attachment to the extracellular matrix (ECM) in order to survive. When detached from the ECM, normal cells will undergo apoptosis through a process known as anoikis. The ErbB2 receptor tyrosine kinase is well known to antagonize anoikis although the precise molecular mechanisms are not entirely known. Interestingly, we have discovered that the overexpression of ErbB2 in non-malignant mammary epithelial cells (MCF-10A) results in substantial aggregation of cells that coincides with protection from anoikis. Therefore, we were interested in understanding if these aggregates could be involved in the evasion of anoikis induction. We found that the disruption of cell-cell contacts in ErbB2-overexpressing MCF-10A cells (using methylcellulose or by antagonizing the formation of adherens junctions) induces caspase activation. Furthermore, in cells that form large aggregates, ErbB2 can physically interact with E-cadherin and EGFR in a fashion that prevents the internalization of EGFR and ultimately its lysosome mediated degradation. The disruption of aggregation causes the ErbB2/EGFR/E-Cadherin complex to fall apart and subsequently results in EGFR degradation in the lysosome. This degradation of EGFR diminishes signaling through the MAPK pathway which prevents the induction of anoikis. In summary, these data suggest that oncogenic signaling through ErbB2 promotes the formation of cellular aggregates that function to prevent EGFR from degradation and subsequently to block the induction of anoikis. Citation Format: Raju Rayavarapu, Nicholas Pagani, Brendan Heiden, Zachary T. Schafer. The role of cell-cell contacts in the survival of extracellular matrix detached mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2014-1347

House dust mite‐induced calcium signaling instigates epithelial barrier dysfunction and CCL20 production

House dust mite (HDM) affects the immunological and physical barrier function of airway epithelium, leading to allergic sensitization, airway remodeling, and eosinophilic inflammation in mouse models, although the mechanisms are still largely unknown. Given the implications for adenosine triphosphate (ATP)-dependent Ca(2+) signaling in allergic sensitization in mice, we sought to determine the role of intracellular Ca(2+) concentration ([Ca(2+)](i)) in HDM-induced barrier dysfunction and pro-inflammatory activity of bronchial epithelium. We investigated the effect of HDM on accumulation of [Ca(2+)](i) levels, barrier function, and CCL20 release in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from healthy subjects and asthma patients. Involvement of ATP-dependent activation of purinergic receptors and downstream Ca(2+) influx was studied, using the ATP hydrolyzing agent apyrase, the purinergic receptor agonist PPADS, the calcium chelator BAPTA-AM, and calpain inhibitors. Asthma PBECs were more susceptible to HDM-induced barrier dysfunction, CCL20 secretion, and Ca(2+) influx than healthy PBECs. Furthermore, we show that the HDM-induced increase in CCL20 in PBECs and 16HBE cells and the HDM-induced barrier dysfunction in 16HBE cells are dependent on [Ca(2+)](i) accumulation. Additionally, we demonstrate that [Ca(2+)](i) accumulation is initiated partly through the activation of purinergic receptors, which contributes to HDM-induced epithelial barrier dysfunction by disruption of cell-cell contacts, but not CCL20 secretion. Our data show for the first time that Ca(2+) signaling plays a crucial role in barrier dysfunction and the pro-inflammatory response of bronchial epithelium upon HDM exposure and may thus have important implications for the development of allergic asthma.

Posttranscriptional regulation by RNA-binding proteins during epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT), one of the crucial steps for carcinoma cells to acquire invasive capacity, results from the disruption of cell–cell contacts and the acquisition of a motile mesenchymal phenotype. Although the transcriptional events controlling EMT have been extensively studied, in recent years, several posttranscriptional mechanisms have emerged as critical in the regulation of EMT during tumor progression. In this review, we highlight the regulation of posttranscriptional events in EMT by RNA-binding proteins (RBPs). RBPs are responsible for controlling pre-mRNA splicing, capping, and polyadenylation, as well as mRNA export, turnover, localization, and translation. We discuss the most relevant aspects of RBPs controlling the metabolism of EMT-related mRNAs, and describe the implication of novel posttranscriptional mechanisms regulating EMT in response to different signaling pathways. Novel insight into posttranscriptional regulation of EMT by RBPs is uncovering new therapeutic targets in cancer invasion and metastasis.

Cell-type-specific crosstalk between p38 MAPK and Rho signaling in lung micro- and macrovascular barrier dysfunction induced by Staphylococcus aureus-derived pathogens

Lung inflammation and alterations in endothelial cell (EC) micro- and macrovascular permeability are key events to development of acute lung injury. Using ECs derived from human pulmonary artery and lung microvasculature, we investigated the interplay between p38 stress mitogen-activated protein kinase (MAPK) and Rho guanosine triphosphatase signaling in inflammatory and hyperpermeability responses. Both cell types were treated with Staphylococcus aureus-derived peptidoglycan (PepG) and lipoteichoic acid (LTA) with or without pretreatment with p38 MAPK or Rho kinase inhibitors. LTA and PepG increased permeability markedly in both pulmonary macrovascular and microvascular ECs. Agonist-induced hyperpermeability was accompanied by cytoskeletal remodeling, disruption of cell-cell contacts, formation of paracellular gaps, and activation of p38 MAPK, nuclear factor kappa-B (NFκB), and Rho/Rho kinase signaling. In macrovascular ECs, pharmacologic inhibition of Rho kinase with Y27632 suppressed p38 MAP kinase cascade activation significantly, whereas inhibition of p38 MAPK with SB203580 had no effect on Rho activation. In contrast, inhibition of p38 MAPK in microvascular ECs suppressed LTA/PepG-induced activation of Rho, whereas the Rho inhibitor suppressed activation of p38 MAPK. Inhibition of either p38 MAPK or Rho kinase attenuated activation of NFκB signaling substantially. These results demonstrate cell-type-specific differences in signaling induced by Staphylococcus aureus-derived pathogens in pulmonary endothelium. Thus, although Gram-positive bacterial compounds caused barrier dysfunction in both EC types, it was induced by a different pattern of crosstalk between Rho, p38 MAPK, and NFκB signaling. These observations may have important implications in defining microvasculature-specific therapeutic strategies aimed at the treatment of sepsis and acute lung injury induced by Gram-positive bacterial pathogens.

Abstract 73: Bone Morphogenetic Protein Activity Modulates Endothelial Barrier Function

Introduction: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are associated with high morbidity and mortality in patients. During the progression of ALI, the endothelial cell barrier of the pulmonary vasculature becomes compromised, leading to pulmonary edema, a characteristic feature of ALI. It is well-established that EC barrier dysfunction is initiated by cytoskeletal remodeling, which leads to disruption of cell-cell contacts and formation of paracellular gaps, allowing penetration of protein-rich fluid and inflammatory cells. Bone morphogenetic proteins (BMPs) are important players in endothelial dysfunction and inflammation but their effects on endothelial permeability in ALI have not been investigated until now. Methods and Results: As a first approach to assess the role of BMPs in acute lung injury we analysed BMP4 and BMPER expression in an infectious (LPS) and a non-infectious (bleomycin) mouse models of acute lung injury. In both models BMP4 and BMPER protein expression levels were reduced demonstrated by western blots, suggesting that BMPs are involved in progression ALI. To assess the role of BMPs on vascular leakage, a key feature of ALI, BMP activity in mice was inhibited by i.p. administration of LDN193189, a small molecule that blocks BMP signalling. After 3 days Evans blue dye (EVB) was administered i.v. and dye extravasation into the lungs was quantified as a marker for vascular leakage. Interestingly, LDN193189 significantly increased endothelial permeability compared to control lungs, indicating that BMP signaling is involved in maintenance of endothelial barrier function. To quantify effects of BMP inhibition on endothelial barrier function in vitro, HUVECs were seeded onto transwell filters and were exposed to LDN193189. After 3 days FITC-dextrane was added and passage into the lower chamber was quantified as a marker for endothelial barrier function. Thrombin served as a positive control. As expected from our in vivo experiments inhibition of BMP signaling by LDN193189 enhanced FITC-dextrane passage. To study specific effects of BMPs on endothelial barrier function, two protagonist of the BMP family, BMP2 and BMP4, or BMP modulator BMPER were tested in the transwell assay in vitro. Interestingly BMP4 and BMPER, but not BMP2, reduced FITC-dextrane passage demonstrating that BMP4 and BMPER improved endothelial barrier function. Vice versa, specific knock down of BMP4 or BMPER increased leakage in transwell assays. Im immuncytochemistry silencing of BMPER or BMP4 induced hyperpermeability as a consequence of a pro-inflammatory endothelial phenotype characterised by reduced cell-cell contacts and increased actin stress fiber formation. Additionally, the pro-inflammatory endothelial phenotype was confirmed by real-time revealing increased expression of adhesion molecules ICAM-1 or proinflammatory cytokines such as IL-6 and IL-8 in endothelial cells after BMPER or BMP4 knock down. Confirming these in vitro results BMPER +/- mice exhibit increased extravasation of EVB into the lungs, indicating that partial loss of BMPER impairs endothelial barrier function in vitro and in vivo. Conclusion: We identify BMPER and BMP4 as local regulators of vascular permeability. Both are protective for endothelial barrier function and may open new therapeutic avenues in the treatment of acute lung injury.

Abstract 2082: The role of cell-cell contacts in the survival of extracellular matrix detached mammary epithelial cells

Abstract Normal epithelial cells require attachment to the extracellular matrix (ECM) in order to proliferate and survive. Binding to the ECM facilitates the activation of pathways responsible for ATP production and cell survival. When detached from the ECM, normal cells will upregulate apoptotic pathways and will be systematically removed. This detachment-induced apoptosis is known as anoikis. ECM detachment in normal epithelial cells disrupts cellular signaling derived from integrin-mediated basal attachment as well as cadherin assisted signaling from lateral cell contacts. Cadherins, primarily E-cadherin, have been shown to be classic tumor suppressor genes and can prevent the proliferation of cells that fail to assume normal epithelial morphology and cell-cell contacts. The loss of E-cadherin endows an increased invasion capacity to breast tumors and regulation of E-cadherin levels are directly mediated by factors (i.e. Slug) that promote epithelial-to-mesenchymal transition (EMT) and metastasis. Cadherin molecules have previously been reported to interact with other receptor tyrosine kinases, such as EGFR (epithelial growth factor receptor), in order to suppress the ability of the receptor to bind ligand, dimerize and elicit downstream signaling. HER2 is an orphan member of the EGFR family of tyrosine kinases and is a potent oncogene found in 30% of all breast cancer tissue. Previous studies in our laboratory have found a direct correlation between upregulation of survival pathways in ECM detached cells and expression of HER2. In this study, we find that the overexpression of HER2 in non-malignant mammary epithelial cells (MCF-10A) results in substantial aggregation of detached cells, the inhibition of caspase activation and a rescue of ATP production. These data suggest that HER2 plays a role in protecting detached cells from anoikis and this activity may be linked to cell-cell adhesion in suspension. We have found that disruption of cell-cell contacts in HER2-expressing MCF-10A cells utilizing methylcellulose can induce caspase activity and diminish global ATP levels. Preliminary studies also reveal a previously uncharacterized interaction between HER2 and E-cadherin that takes place when E-cadherin dimers are disrupted. Thus, the disruption of E-cadherin dimers in cellular aggregates may facilitate the binding of E-cadherin to HER2 in a fashion that compromises downstream survival signaling. These data together suggest a potential role for E-cadherin as a modulator of HER2 activity and further suggest that the loss of E-cadherin via mechanisms like EMT may function as a means to promote ECM-detached tumor cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2082. doi:1538-7445.AM2012-2082

Abstract LB-14: Raf-1 in epithelial-mesenchymal transition: a novel therapeutic target for ovarian and endometrial cancers

Abstract Ovarian and endometrial cancers are the 5th and 8th overall leading causes of female cancer related deaths in the US, respectively. According to the American Cancer Society, 21,990 new ovarian cancer cases and 46,470 new endometrial cancer cases were diagnosed in 2011. These cancers share a common mechanism of pathogenesis, epithelial-mesenchymal transition (EMT). EMT increases the invasiveness and progression of epithelial tumor cells into more aggressive and metastatic disease. Amplification of growth factor signaling is also common in malignancy. Epidermal growth factor receptor (EGFR) is amplified in 60-80% of ovarian and endometrial cancer and its overexpression is associated with increased invasiveness and poor prognosis. Studies have shown that specific cytokines, including transforming growth factor-β (TGFβ) and epidermal growth factor (EGF), interact with the intracellular MEK/ERK signaling cascade, a pathway involved in several malignant processes including EMT. Raf-1 is an intracellular MAPK signaling molecule that is recruited to membrane bound Ras upon activation by growth factors. Activation of Raf-1 includes phosphorylation at ser 338, which in turn activates the downstream MEK/ERK signaling cascade. Our data showed that EGF phosphorylates Raf-1 at ser 338 and ERK in endometrial cancer cells, which is inhibited by the specific Raf-1 antagonist GW5074. Little is known on the involvement of Raf-1 in the pathogenesis of ovarian or endometrial cancer. Our studies show that inhibition of Raf-1 by GW5074 significantly decreases TGFβ and EGF induced invasiveness of immortalized endometrial epithelial cells (EECs). EGF and to a lesser degree TGFβ, enhanced the survival of EECs under serum starvation. GW5074 inhibited this effect as evidenced by increased apoptosis and caspase 3/7 activity, suggesting that Raf-1 mediates EEC survival. While EGF had minimal effects on inducing ovarian cancer cell line SKOV3, the addition of GW5074 significantly reduced SKOV3 cell growth by half. Our data shows that the addition of EGF, but not TGF-β, to endometrial and ovarian epithelial cells induced EMT as evidenced by the disruption of cell-cell contact, loss of the characteristic “cobblestone” appearance of epithelial cells, increased motility and induction of the mesenchymal marker vimentin. Treatment with GW5074 of cells exposed to EGF reversed these changes. Our FACS analysis showed that EGF decreases the cell surface detection of EPCAM, an epithelial cell surface adhesion molecule, by more than half, further suggesting that EGF is involved in EMT in endometrial cancer cells. Our data suggests that multiple steps in the pathogenesis of both ovarian and endometrial cancers are mediated by Raf-1. Raf-1's involvement in inducing EMT suggests that Raf-1 may be a novel therapeutic target for ovarian and endometrial cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-14. doi:1538-7445.AM2012-LB-14

Obscurins Invade Breast Cancer Research

Obscurins are giant multidomain proteins, encoded by the single OBSCN gene, which play important roles in the structural organization and contractile activity of striated muscles. Recently, genetic linkage analysis identified OBSCN and TP53 as the only common and most frequently mutated genes in breast and colorectal cancers among 13,023 genes studied. Our laboratory set forth to study the expression profile of the OBSCN gene in breast cancer. Our data revealed that obscurins are readily expressed in normal breast epithelial cells (MCF10A), but their amounts are significantly diminished in breast cancer cells (MCF7 & MDA‐MB‐ 231). Down‐regulation of obscurins in MCF10A cells via siRNA technology resulted in decreased levels of β‐catenin and Ecadherin, both of which are major components of adherens junctions (AJ) and are heavily implicated in the formation and metastasis of breast tumors. Residual β‐catenin showed a preferential nuclear accumulation in obscurin‐null MCF10A cells, and a concomitant loss from cell‐cell junctions. Interestingly, TGF‐β1 treatment of normal MCF10A cells resulted in down‐regulation of obscurins, β‐catenin, and E‐cadherin. In summary, our findings indicate that TGF‐β1 may negatively regulate the expression of obscurins in breast epithelium, loss of which results in diminished levels and altered distribution of major AJ proteins, and thus disruption of cell‐cell contacts.