Disruption Of Cell-cell Adhesion Research Articles

Ultrastructural effects of modulating calcium handling in the cardiac intercalated disc

Decreased extracellular calcium in the heart reduces cell-cell adhesion and increases intermembrane spacing in the intercalated disc (ID), particularly within the gap junction adjacent nanodomain known as the perinexus. Disruption of cell-cell adhesion within the perinexus slows cardiac conduction and can lead to arrhythmias. It is unknown whether the perinexus contains calcium-handling proteins or what mechanism(s) may locally regulate calcium levels within these ID nanodomains. L-type calcium channels (Cav1.2) located in T-tubules are the primary mediators of calcium influx and subsequent calcium-induced calcium release from the sarcoplasmic reticulum.

Re-Evaluation of Initial Bone Mineralization from an Engineering Perspective.

Bone regeneration was one of the earliest fields to develop in the context of tissue regeneration, and currently, repair of small-sized bone defects has reached a high success rate. Future researches are expected to incorporate more advanced techniques toward achieving rapid bone repair and modulation of the regenerated bone quality. For these purposes, it is important to have a more integrative understanding of the mechanisms of bone formation and maturation from multiple perspectives and to incorporate these new concepts into the development and designing of novel materials and techniques for bone regeneration. This review focuses on the analysis of the earliest stages of bone tissue development from the biology, material science, and engineering perspectives for a more integrative understanding of bone formation and maturation, and for the development of novel biology-based engineering approaches for tissue synthesis in vitro. More specifically, the authors describe the systematic methodology that allowed the understanding of the different nucleation sites in intramembranous and endochondral ossification, the space-making process for mineral formation and growth, as well as the process of apatite crystal cluster growth in vivo in the presence of suppressing biomolecules.Impact StatementA detailed understanding of the developmental process of bone tissue leads to the acquisition of useful information for the bone tissue fabrication. This review summarizes the study of the calcification process of the calvaria and epiphyses from an engineering perspective and provides useful information for the realization of bone tissue biofabrication. Here, we describe the new mechanism of space formation for mineralization such as rupture of chondrocytes and disruption of cell-cell adhesion. We also describe the roles of nucleation site such as cell membrane nanofragments and matrix vesicles.

Arrhythmogenic Cardiomyopathy: Mechanisms, Genetics, and Their Clinical Implications

Arrhythmogenic cardiomyopathy (ACM) represents a collection of cardiomyopathies with etiologies convergent upon highly arrhythmogenic clinical presentations not explained by ischemic, hypertensive, or valvular heart disease. This review describes the current state of knowledge regarding molecular mechanisms and genetic diagnosis in ACM. Clinical manifestations, specifically structural presentations, of ACM may span the right and left side of the heart. The umbrella definition of ACM encompasses disease mechanisms involving disruption of cell-cell adhesion, mechanotransduction, transcription, calcium handling, and post-transcriptional regulators. As such, the underlying genetics are themselves wide ranging but generally map to perturbations of proteins comprising the desmosome and regulating downstream cellular signaling. The recognition that many genetic forms of ACM share the feared outcomes of life-threatening ventricular arrhythmia and heart failure motivates the identification of causative genetic variants for family screening and in some cases precision therapy. In turn, contemporary clinical practice has shifted towards broadened genetic testing strategy and the discovery of complex gene-specific clinical ramifications. The molecular mechanisms of ACM are complex, and the genetic architecture is ever evolving. Given this, multidisciplinary patient care for the incorporation of genetics and family cardiovascular care is critical. Future work will refine the understanding of gene-specific outcomes in ACM and implications for tailored therapy.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Transcriptional suppression of AMPKα1 promotes breast cancer metastasis upon oncogene activation

AMP-activated protein kinase (AMPK) functions as an energy sensor and is pivotal in maintaining cellular metabolic homeostasis. Numerous studies have shown that down-regulation of AMPK kinase activity or protein stability not only lead to abnormality of metabolism but also contribute to tumor development. However, whether transcription regulation of AMPK plays a critical role in cancer metastasis remains unknown. In this study, we demonstrate that AMPKα1 expression is down-regulated in advanced human breast cancer and is associated with poor clinical outcomes. Transcription of AMPKα1 is inhibited on activation of PI3K and HER2 through ΔNp63α. Ablation of AMPKα1 expression or inhibition of AMPK kinase activity leads to disruption of E-cadherin-mediated cell-cell adhesion in vitro and increased tumor metastasis in vivo. Furthermore, restoration of AMPKα1 expression significantly rescues PI3K/HER2-induced disruption of cell-cell adhesion, cell invasion, and cancer metastasis. Together, these results demonstrate that the transcription control is another layer of AMPK regulation and suggest a critical role for AMPK in regulating cell-cell adhesion and cancer metastasis.

Transcription Factors and Downstream Genes in Cadmium Toxicity.

Cadmium (Cd) is a harmful heavy metal widely present in the environment which can cause severe kidney damage. The proximal tubular cells are the main target of renal Cd toxicity. The consequences of Cd cytotoxicity involve apoptosis and necrosis. Recently, we and others have focused on how Cd affects transcription factors and the regulation of their target genes. Those studies showed that transcription factors initiate numerous pathways upon Cd exposure, leading to apoptosis, autophagic cell death, disruption of cell-cell adhesion, and generation of mitochondrial reactive oxygen species. Of particular note, Cd induces endoplasmic reticulum stress, resulting in not only apoptosis but also autophagic dysregulation, which can trigger cell damage. In some cases, however, Cd-regulated transcription factors can induce cell survival signaling. This review centers on our own research to elucidate the transcription factor-downstream gene cascades that are central to Cd-induced renal toxicity.

Protein Phosphatase 2A: More Than a Passenger in the Regulation of Epithelial Cell-Cell Junctions.

Cell–cell adhesion plays a key role in the maintenance of the epithelial barrier and apicobasal cell polarity, which is crucial for homeostasis. Disruption of cell–cell adhesion is a hallmark of numerous pathological conditions, including invasive carcinomas. Adhesion between apposing cells is primarily regulated by three types of junctional structures: desmosomes, adherens junctions, and tight junctions. Cell junctional structures are highly regulated multiprotein complexes that also serve as signaling platforms to control epithelial cell function. The biogenesis, integrity, and stability of cell junctions is controlled by complex regulatory interactions with cytoskeletal and polarity proteins, as well as modulation of key component proteins by phosphorylation/dephosphorylation processes. Not surprisingly, many essential signaling molecules, including protein Ser/Thr phosphatase 2A (PP2A) are associated with intercellular junctions. Here, we examine how major PP2A enzymes regulate epithelial cell–cell junctions, either directly by associating with and dephosphorylating component proteins, or indirectly by affecting signaling pathways that control junctional integrity and cytoskeletal dynamics. PP2A deregulation has severe consequences on the stability and functionality of these structures, and disruption of cell–cell adhesion and cell polarity likely contribute to the link between PP2A dysfunction and human carcinomas.

Delphinidin inhibits epidermal growth factor‐induced epithelial‐to‐mesenchymal transition in hepatocellular carcinoma cells

Epithelial-to-mesenchymal transition (EMT), important cellular process in metastasis of primary tumors, is characterized by loss of their cell polarity, disruption of cell-cell adhesion, and gain certain properties of mesenchymal phenotype that enable migration and invasion. Delphinidin is a member of anthocyanidin belong to flavonoid groups, known as having pharmacological and physiological effects including anti-tumorigenic, antioxidative, anti-inflammatory, and antiangiogenic effects. However, the effects of delphinidin on EMT is rarely investigated. Epidermal growth factor (EGF) is known as a crucial inducer of EMT in various cancer including hepatocellular carcinoma (HCC). To determine whether delphinidin inhibits EGF-induced EMT in HCC cells, antiproliferative effect of delphinidin on Huh7 and PLC/PRF/5 cells were measured by Cell Counting Kit-8 assay. As a result, delphinidin inhibited cell proliferation in a dose-dependent manner. Based on the result of proliferation, to measure the effects of delphinidin on EGF-induced EMT, we designated a proper concentration of delphinidin, which is not affected to cell proliferation. We found that delphinidin inhibits morphological changes from epithelial to mesenchymal phenotype by EGF. Moreover, delphinidin increased the messenger RNA and protein expression of E-cadherin and decreased those of Vimentin and Snail in EGF-induced HCC cells. Also, delphinidin prevented motility and invasiveness of EGF-induced HCC cells through suppressing activation of matrix metalloproteinase 2, EGF receptor (EGFR), AKT, and extracellular signal-regulated kinase (ERK). Taken together, our findings demonstrate that delphinidin inhibits EGF-induced EMT by inhibiting EGFR/AKT/ERK signaling pathway in HCC cells.

The function of WNK1/OSR1 in cell migration and angiogenesis

Deletion of the WNK1 (with no lysine kinase-1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial-specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive-1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to be stimulated by TGF-β/Smad signaling which induces epithelial-mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF-βII receptor at tight junctions which mediates ubiquitination of GTP-RhoA and dissolution of tight junction upon activation by TGF-β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for the expression of TGF-β/Smad-induced EMT factors, suppression of RhoA activity and disruption of cell-cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 siRNA-mediated knockdown decreased the expression of TGF-β-induced EMT mediators such as Slug, the receptor tyrosine kinase Axl and the transcription factor p63 (ΔNp63α) (normalized densitometry values to control; siWNK1-Slug: 0.5 ± 0.25, Axl: 0.2 ± 0.05, p63: 0.35 ± 0.15 vs. siControl: 1, n = 2) in MCFDCIS cancer cell line. Similarly, WNK1 pharmacological inhibition decreased Axl expression (normalized densitometry values to control; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n = 3, *p<0.05) in primary HUVECs (Human Umbilical Vein Endothelial Cells). This suggests that WNK1 is involved in Slug/p63/Axl network in endothelial and cancer cells. We found that knockdown of WNK1 increased F-actin content (normalized arbitrary fluorescence units to control; siWNK1: 1.6 ± 0.3 vs. siControl: 1, n = 2, 4–5 replicates per experiment) in HeLa cells. Because RhoA activity regulates actin dynamics, RhoA activity was measured in WNK1 depleted cells and the ratio of GTP-bound RhoA to total RhoA was found to be significantly increased (siWNK1: 3.08 ± 0.75 vs. siControl: 0.34 ± 0.05, n = 3, *p<0.035). Finally, we determined that OSR1 and Occludin, a tight junction protein are co-localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co-localization during TGF-β signaling in primary HUVECs (Pearson's coefficient; Control: 0.08 ± 0.2 vs. TGF-β: 0.61 ± 0.15 vs. TGF-β+WNK1i: 0.12 ± 0.25, n = 2, 2 replicates per experiment). These data suggest WNK1-dependent function of OSR1 in cell migration and angiogenesis through effects on GTP-RhoA degradation. Combined, these data indicate the importance of WNK1 function in endothelial and cancer cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis, ischemia-reperfusion injury as well as cancer. Support or Funding Information Early part of this work was supported by NIH 5R01GM053032-16 grant. Current work is being supported by the Welch Foundation grant I-1243. Co-localization of OSR1 and Occludin in primary HUVECs This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Simple and Robust Method for Culturing Human-Induced Pluripotent Stem Cells in an Undifferentiated State Using Botulinum Hemagglutinin.

Clinical and industrial applications of human-induced pluripotent stem cells (hiPSCs) is hindered by the lack of robust culture strategies capable of sustaining a culture in an undifferentiated state. Here, a simple and robust hiPSC-culture-propagation strategy incorporating botulinum hemagglutinin (HA)-mediated selective removal of cells deviating from an undifferentiated state is developed. After HA treatment, cell-cell adhesion is disrupted, and deviated cells detached from the central region of the colony to subsequently form tight monolayer colonies following prolonged incubation. The authors find that the temporal and dose-dependent activity of HA regulated deviated-cell removal and recoverability after disruption of cell-cell adhesion in hiPSC colonies. The effects of HA are confirmed under all culture conditions examined, regardless of hiPSC line and feeder-dependent or -free culture conditions. After routine application of our HA-treatment paradigm for serial passages, hiPSCs maintains expression of pluripotent markers and readily forms embryoid bodies expressing markers for all three germ-cell layers. This method enables highly efficient culturing of hiPSCs and use of entire undifferentiated portions without having to pick deviated cells manually. This simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures.

Abstract A30: Characterizing the non-linear dependency of the CDK5-Rb axis in non-small cell lung cancer

Abstract In spite of recent therapeutics advances and early detection, lung cancer is still the leading cause of cancer-associated deaths worldwide. The five-year survival rates for its two major subtypes, small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) are estimated to be 6% and 18%, respectively. This high mortality is due to its aggressive nature even when detected at an early stage. Besides its aggressive nature and tendency for early metastasis, another feature of lung cancer is the inactivation of the retinoblastoma protein (Rb) that is observed in both lung cancer subtypes. NSCLC exhibits Rb inactivation by hyper-phosphorylation. Rb is phosphorylated by cyclin dependent kinases (CDKs) and their cyclin regulatory partners. CDK5 is a unique CDK because its regulatory partners, p35 and p39, are not cyclins, hence its function is not cell cycle related. This work aims to employ empirical modeling and statistical experimental design techniques to gain a comprehensive mechanistic understanding of metastasis. Previous mass spectroscopy experiments performed in our laboratory on a panel of NSCLC cell lines identified five-residues (S249, S612, S807, S811, and T821) whose phosphorylation is associated with lack of E-cadherin expression, and therefore may be associated with metastasis. Our hypothesis is that Rb inactivation via phosphorylation due to CDK5 leads to the acquisition of the epithelial-to-mesenchymal transition (EMT) phenotype and the disruption of cell-cell adhesion that characterizes metastatic cells. As a first step to establish a mathematical model the dependency of the controllable variables, Rb residues and CDK5 expression was assessed. To this end, we used siRNA to knockdown CDK5 expression, which caused a decrease in phosphorylation of Rb in the five residues mentioned above and a subsequent decrease in E-cadherin expression. These data suggest that Rb phosphorylation in the five residues is dependent on CDK5 expression. This dependency in our controllable variables points to a nested experimental design. Our next step is to perform invasion assays and lung acini formation to quantify the invasion capacity and the malignant transformation. In the long term, at a clinical setting, a lung cancer tumor may be extracted from a biopsy and immunohistochemistry staining will be performed with antibodies of interest such as Rb phosphorylation residues, E-cadherin, and CDK5/p35 or CDK5/p39. This mathematical model would help provide a prognosis about the invasion capacity and a malignancy prediction of the lung tumor. Citation Format: Jaileene Perez-Morales, Mauricio Cabrera-Rios, Jonathan Gonzalez-Flores, Pedro Santiago-Cardona. Characterizing the non-linear dependency of the CDK5-Rb axis in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A30.

Loss of MLCK leads to disruption of cell-cell adhesion and invasive behavior of breast epithelial cells via increased expression of EGFR and ERK/JNK signaling.

Myosin light chain kinase (MLCK) expression is downregulated in breast cancer, including invasive ductal carcinoma compared with ductal breast carcinoma in situ and metastatic breast tumors. However, little is known about how loss of MLCK expression contributes to tumor progression. MLCK is a component of the actin cytoskeleton and its known role is the phosphorylation of the regulatory light chain of myosin II. To gain insights into the role of MLCK in breast cancer, we perturbed its function using small interfering RNA (siRNA) or pharmacological inhibition in untransformed breast epithelial cells (MCF10A). Loss of MLCK by siRNAs led to increased cell migration and invasion, disruption of cell-cell adhesions and enhanced formation of focal adhesions at the leading edge of migratory cells. In addition, downregulation of MLCK cooperated with HER2 in MCF10A cells to promote cell migration and invasion and low levels of MLCK is associated with a poor prognosis in HER2-positive breast cancer patients. Associated with these altered migratory behaviors were increased expression of epidermal growth factor receptor and activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways in MLCK downregulated MCF10A cells. By contrast, inhibition of the kinase function of MLCK using pharmacological agents inhibited cell migration and invasion, and did not affect cellular adhesions. Our results show that loss of MLCK contributes to the migratory properties of epithelial cells resulting from changes in cell-cell and cell-matrix adhesions, and increased epidermal growth factor receptor signaling. These findings suggest that decreased expression of MLCK may have a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

A hybrid model for cell proliferation and migration in glioblastoma

Glioblastoma is the most aggressive type of brain cancer with themedian survival time of one year. A particular microRNA, miR-451, and its counterpart,AMPK complex are known to play a key role in controlling a balance between rapid proliferationand aggressive invasion in response to metabolic stress in the microenvironment.The present paper develops a hybrid model of glioblastomathat identifiesa key mechanism behind the molecular switches between proliferative phase and migratoryphase in response to metabolic stress and biophysical interaction between cells.We focus on the core miR-451-AMPK control system and show how up- ordown-regulation of components in these pathways affects cell proliferation and migration.The model predicts the larger window of bistable systems when there exists a timedelay in the inhibitory pathway from CAB39/LKB1/STRAD/AMPK to miR-451.Delayed down-regulation of miR-451 along this pathway would let glioma cells staylonger in the proliferative stage despite relatively low glucose levels, making it a possibletherapeutic target.Analysis of the model predicts theexistence of a limit cycle with two time delays. We thenstudy a hybrid model for the biomechanical interaction between invasive and proliferative cells, inwhich all cells are modeled individually, and show how biophysical properties of cells andcore miR-451-AMPK control system affect the growth/invasion patterns of glioma spheroidsin response to various glucose levels in the microenvironment.The model predicts that cell migration not only depends on glucose availabilitybut also on mechanical constraints between cells. The model suggests that adhesion strengthbetween cells plays an important role in cell shedding from the main core and the disruptionof cell-cell adhesion is a pre-requisite for glioma cell invasion.The model also suggests that injection of glucose after surgery will increase visibility of individualmigratory cells and the second surgery may eradicate the remaining cancer cells, preventingregrowth of the invisible migratory glioma cells.

Cell Adhesion and Its Endocytic Regulation in Cell Migration during Neural Development and Cancer Metastasis

Cell migration is a crucial event for tissue organization during development, and its dysregulation leads to several diseases, including cancer. Cells exhibit various types of migration, such as single mesenchymal or amoeboid migration, collective migration and scaffold cell-dependent migration. The migration properties are partly dictated by cell adhesion and its endocytic regulation. While an epithelial-mesenchymal transition (EMT)-mediated mesenchymal cell migration requires the endocytic recycling of integrin-mediated adhesions after the disruption of cell-cell adhesions, an amoeboid migration is not dependent on any adhesions to extracellular matrix (ECM) or neighboring cells. In contrast, a collective migration is mediated by both cell-cell and cell-ECM adhesions, and a scaffold cell-dependent migration is regulated by the endocytosis and recycling of cell-cell adhesion molecules. Although some invasive carcinoma cells exhibit an EMT-mediated mesenchymal or amoeboid migration, other cancer cells are known to maintain cadherin-based cell-cell adhesions and epithelial morphology during metastasis. On the other hand, a scaffold cell-dependent migration is mainly utilized by migrating neurons in normal developing brains. This review will summarize the structures of cell adhesions, including adherens junctions and focal adhesions, and discuss the regulatory mechanisms for the dynamic behavior of cell adhesions by endocytic pathways in cell migration in physiological and pathological conditions, focusing particularly on neural development and cancer metastasis.

WAVE2 Protein Complex Coupled to Membrane and Microtubules

E-cadherin is one of the key molecules in the formation of cell-cell adhesion and interacts intracellularly with a group of proteins collectively named catenins, through which the E-cadherin-catenin complex is anchored to actin-based cytoskeletal components. Although cell-cell adhesion is often disrupted in cancer cells by either genetic or epigenetic alterations in cell adhesion molecules, disruption of cell-cell adhesion alone seems to be insufficient for the induction of cancer cell migration and invasion. A small GTP-binding protein, Rac1, induces the specific cellular protrusions lamellipodia via WAVE2, a member of WASP/WAVE family of the actin cytoskeletal regulatory proteins. Biochemical and pharmacological investigations have revealed that WAVE2 interacts with many proteins that regulate microtubule growth, actin assembly, and membrane targeting of proteins, all of which are necessary for directional cell migration through lamellipodia formation. These findings might have important implications for the development of effective therapeutic agents against cancer cell migration and invasion.

Human mesenchymal stem cells induce E-cadherin degradation in breast carcinoma spheroids by activating ADAM10.

Mesenchymal stem cells (MSCs) have been shown to communicate with tumor cells. We analyzed the effect of human MSCs (hMSCs) on breast cancer cells in three-dimensional cultures. By using GFP expression and immunohistochemistry, we show that hMSCs invade 3D breast cancer cell aggregates. hMSCs caused breast cancer spheroids to become disorganized which was accompanied by a disruption of cell-cell adhesion, E-cadherin cleavage, and nuclear translocation of E-cadherin, but not by epithelial/mesenchymal transition or by an increase in ERK1/2 activity. In addition, hMSCs enhanced the motility of breast cancer cells. Inhibition of ADAM10 (a disintegrin and metalloprotease 10), known to cleave E-cadherin, prevented both hMSC-mediated E-cadherin cleavage and enhanced migration. Our data suggest that hMSCs interfere with cell-cell adhesion and enhance migration of breast cancer cells by activating ADAM10.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction - a model for cross-modulation.

BackgroundA feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC.MethodsPMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome.ResultsWhen PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk.ConclusionST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

The Transforming Functions of PI3‐kinase‐γ Are Linked to Disruption of Intercellular Adhesion and Promotion of Cancer Cell Invasion

The involvement of phosphoinositide 3-kinases class IA (PI3K-alpha and -beta) in cancer cell proliferation, survival, motility, and invasiveness is now well established. However, the possible contribution of the class IB PI3Kgamma in cancer cell transformation remains to be explored. In this study, we have stably transfected the PI3Kgamma-deficient human colon cancer cell line HCT8/S11 with expression vectors encoding either wild-type PI3Kgamma, its plasma membrane targeted form CAAX-PI3Kgamma, or the PI3Kgamma lipid and protein kinase-dead mutant (CAAX-K832R). We provide evidence that the constitutively active CAAX-PI3Kgamma variant induced collagen type I invasion in HCT8/S11 cells through disruption of cell-cell adhesion, with no apparent impact on cell proliferation and motility. The proinvasive activity of CAAX-PI3K-gamma was abolished by pharmacological inhibitors targeting PI3-K activities (wortmannin), Rho-GTPases, and the Rho-Rho kinase axis (C3T exoenzyme and Y27632, respectively). Conversely, the wild-type PI3Kgamma and its double mutant CAAX-K832R were ineffective on cancer cell invasion measured under control or stimulated conditions operated with the proinvasive agents leptin and intestinal trefoil factor. Taken together, our data indicate that PI3Kgamma exerts transforming functions via several mechanisms in human colon epithelial cancer cells, including alterations of homotypic cell-cell adhesion and induction of collagen type I invasion through canonical proinvasive pathways.

Increased Proliferation and Altered Growth Factor Dependence of Human Mammary Epithelial Cells Overexpressing the Gab2 Docking Protein

The docking protein Gab2 is a proto-oncogene product that is overexpressed in primary breast cancers. To determine the functional consequences of Gab2 overexpression, we utilized the immortalized human mammary epithelial cell line MCF-10A. In monolayer culture, expression of Gab2 at levels comparable with those detected in human breast cancer cells accelerated epidermal growth factor (EGF)-induced cell cycle progression and was associated with increased basal Stat5 tyrosine phosphorylation and enhanced and/or more sustained EGF-induced Erk and Akt activation. Three-dimensional Matrigel culture of MCF-10A cells resulted in the formation of polarized, growth-arrested acini with hollow lumina. Under these conditions, Gab2 increased cell proliferation during morphogenesis, leading to significantly larger acini, an effect dependent on Gab2 binding to Grb2 and Shp2 and enhanced by recruitment of the p85 subunit of phosphatidylinositol 3-kinase. Pharmacological inhibition of MEK revealed that, in addition to direct activation of phosphatidylinositol 3-kinase, increased Erk signaling also contributed to Gab2-mediated enhancement of acinar size. In addition, Gab2 overcame the proliferative suppression that normally occurs in late stage cultures and conferred independence of the morphogenetic program from exogenous EGF. Finally, higher levels of Gab2 expression led to the formation of large disorganized structures with defective luminal clearance. These findings support a role for Gab2 in mammary tumorigenesis.

EphA2 Phosphorylates the Cytoplasmic Tail of Claudin-4 and Mediates Paracellular Permeability

Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell-cell interaction. EphA2 is expressed in various cancer tissues and cell lines. Although the mechanism of action of EphA2 is unknown, its expression correlates with progression of the malignant phenotype of cancerous tissues. Here, we have shown that EphA2 modulates the localization and function of claudin-4, a constituent of tight junctions. EphA2 associates with claudin-4 via their extracellular domains. This association, in turn, leads to phosphorylation of the cytoplasmic carboxyl terminus of claudin-4 at Tyr-208. The tyrosine phosphorylation of claudin-4 attenuates association of claudin-4 with ZO-1, decreasing integration of claudin-4 into sites of cell-cell contact and enhancing paracellular permeability. These results indicate that EphA2 moderates the function of tight junctions via phosphorylation of claudin-4.