Cell-cell Adhesion Formation Research Articles

Interplay between the plasma membrane and cell-cell adhesion maintains epithelial identity for correct polarised cell divisions.

Polarised epithelial cell divisions represent a fundamental mechanism for tissue maintenance and morphogenesis. Morphological and mechanical changes in the plasma membrane influence the organisation and crosstalk of microtubules and actin at the cell cortex, thereby regulating the mitotic spindle machinery and chromosome segregation. Yet, the precise mechanisms linking plasma membrane remodelling to cell polarity and cortical cytoskeleton dynamics to ensure accurate execution of mitosis in mammalian epithelial cells remain poorly understood. Here, we manipulated the density of mammary epithelial cells in culture, which led to several mitotic defects. Perturbation of cell-cell adhesion formation impairs the dynamics of the plasma membrane, affecting the shape and size of mitotic cells and resulting in defects in mitotic progression and the generation of daughter cells with aberrant architecture. In these conditions, F- actin-astral microtubule crosstalk is impaired, leading to mitotic spindle misassembly and misorientation, which in turn contributes to chromosome mis-segregation. Mechanistically, we identify S100 Ca2+-binding protein A11 (S100A11) as a key membrane-associated regulator that forms a complex with E-cadherin (CDH1) and the leucine-glycine-asparagine repeat protein LGN (also known as GPSM2) to coordinate plasma membrane remodelling with E-cadherin-mediated cell adhesion and LGN-dependent mitotic spindle machinery. Thus, plasma membrane-mediated maintenance of mammalian epithelial cell identity is crucial for correct execution of polarised cell divisions, genome maintenance and safeguarding tissue integrity.

Study of cell-cell adhesion formation and dynamics in cancer cells using a hybrid cell-lipid bilayer system.

The formation and regulation of connections between cells play a crucial role in maintaining tissue homeostasis and tumour repression. In many tissues, particular importance comes to adherens junctions that are formed by the binding of E-cadherin proteins at the surface of adjacent cells, followed by their clustering and recruitment of catenins and other adaptor proteins on the intracellular site linking the protein complex to the actomyosin cytoskeleton. This process depends on multiple parameters such as the mobility, density, type of cadherins, and the mechanical forces acting on the cadherin-catenin complexes and can change dramatically during developmental stages or in diseases such as cancer. To study the initial steps of adherens junction formation in a controlled environment in cancer cells, we have established a reconstituted system of cells adhering to supported lipid bilayers decorated with extracellular domains of E-cadherin proteins. This reconstituted system allowed us to capture the initiation and dynamics of cadherin interactions, the real-time localisation and clustering of adaptor proteins and the maturation of adherens junction in cancer cells over time using live cell imaging with confocal and total internal reflection microscopy. To understand the importance of the different parameters and to mimic the cell/tissue abnormalities occurring in cancer scenarios, we then perturb the controlled system by varying cadherin density, membrane mobility and inducing mechanical stress by confining the cell system over time. We find, for example, that reduced E-cadherin mobility leads to the formation of stable regions showing actin enrichment and that confinement increases the steady state adhesion zone. This approach provides new insights about cell surface dynamics during cell-cell adhesion and can improve our understanding of the role of cell mechanics and E-cadherin organisation patterns in cancer progression.

Computational model of E-cadherin clustering under force

E-cadherins play a critical role in the formation of cell-cell adhesions for several physiological functions, including tissue development, repair, and homeostasis. The formation of clusters of E-cadherins involves extracellular adhesive (trans-) and lateral (cis-) associations between E-cadherin ectodomains and stabilization through intracellular binding to the actomyosin cytoskeleton. This binding provides force to the adhesion and is required for mechanotransduction. However, the exact role of cytoskeletal force on the clustering of E-cadherins is not well understood. To gain insights into this mechanism, we developed a computational model based on Brownian dynamics. In the model, E-cadherins transit between structural and functional states; they are able to bind and unbind other E-cadherins on the same and/or opposite cell(s) through trans- and cis-interactions while also creating dynamic links with the actomyosin cytoskeleton. Our results show that actomyosin force governs the fraction of E-cadherins in clusters and the size and number of clusters. For low forces (below 10 pN), a large number of small E-cadherin clusters form with less than five E-cadherins each. At higher forces, the probability of forming fewer but larger clusters increases. These findings support the idea that force reinforces cell-cell adhesions, which is consistent with differences in cluster size previously observed between apical and lateral junctions of epithelial tissues.

P0041CHARACTERISTICS OF URINE-DERIVED RENAL EPITHELIAL CELLS (UREC) FROM CHILDREN WITH POLYCYSTIC KIDNEY DISEASE (PKD)

Abstract Background and Aims In hereditary PKD, epithelial cell defects are mostly caused by mutation of proteins of the cilia-centrosome complex. Use of primary cells from patients allows to study specific cell characteristics and altered activation of signal pathways in epithelial cell function-related culture models. In this ex vivo approach, comparison of cell properties is based on defined genetic origin and can be related to kidney function at the time of UREC collection, revealing consequences of genetic disposition and kidney-stress related changes on cell function. Method The study considers patients with genetically confirmed causes of PKD, autosomal recessive polycystic kidney disease (ARPKD), nephronophtisis (NPH), Bardet-Biedl syndrome (BBS), and age-matched controls with normal kidney function. UREC are grown from cellular urine sediment using selective growth conditions. Primary cells are cultured up-to 21 days (passages 2 & 3) and tested with respect to their proliferation, tubular origin and epithelial properties in 2D/3D culture conditions. To allow pathway analysis and improve control conditions, immortalized UREC lines are also included. Results UREC preparations of cohorts from ARPKD, NPH, and BBS patients, and controls were studied quantitatively to determine specific epithelial cell properties. Considering basic characteristics of each primary culture including cell morphology, expression of epithelial markers, and formation of cell-cell adhesions, the potential of UREC cells to polarize and generate liquid-filled epithelial spheroids was measured. The capacity of lumen formation in 3D culture strongly varies among individual cell preparations and appears to reflect the genetic origin of UREC cells, whereas no correlation to (remaining) kidney function (eGFR) is observed. Conclusion Determining specific, quantitative UREC cell properties related to genetics and kidney function, we expect to gain a better mechanistic understanding of cellular and renal epithelial defects in PKD patients. Ex vivo analysis based on UREC cells may provide options for testing of personalized pharmaceutical intervention.

Role for Asef in the Maturation and Maintenance of Endothelial Cell‐Cell Adhesions

The formation of cell‐cell adhesions involves the spatiotemporal regulation of protein complexes, including cadherins and reorganization of the actin cytoskeleton by Rho GTPases. Previous work identified a cooperative role between cadherins and nectins to induce and rapidly suppress Rac1 GTPase activity during de novocell adhesion in epithelial cells. In this study, the role of Asef (guanine nucleotide exchange factor (GEF) for Rac1) on the assembly and maturation of cell‐cell contacts in human umbilical vein endothelial cell (HUVEC) is examined. The Rho GTPases, Rac1 and RhoA, have been previously shown to regulate endothelial cell permeability, but the proteins involved in controlling the activity of these Rho GTPases during endothelial junction assembly and maintenance remain relatively unknown. The current study shows that endogenous Asef is gradually recruited to cell‐cell contacts 3–5 hours after initiation of cell‐cell adhesion, and localizes to established endothelial cell‐cell contacts. Knockdown of Asef reduces the amount of VE‐cadherin and Asef’s only known binding partner, Adenomatous Polyposis Coli (APC) at cell‐cell contacts. Significantly, knockdown of Asef causes an increase in paracellular permeability of HUVEC monolayers and a decrease in HUVEC cell adhesiveness. These data indicate a role for Asef in maintaining the integrity of endothelial cell‐cell adhesions, and potentially a specific role at tight junctions. Future work aims to decipher whether Asef regulates the recruitment and maintenance of adherens junctions, tight junctions or both at cell‐cell contacts, and the signaling pathways involved in the spatiotemporal regulation of these protein complexes at cell‐cell adhesions in various adherent cell lines.

An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.

Cell Adhesion Structures in Epithelial Cells Are Formed in Dynamic and Cooperative Ways.

There are many morphologically distinct membrane structures with different functions at the surface of epithelial cells. Among these, adherens junctions (AJ) and tight junctions (TJ) are responsible for the mechanical linkage of epithelial cells and epithelial barrier function, respectively. In the process of new cell-cell adhesion formation between two epithelial cells, such as after wounding, AJ form first and then TJ form on the apical side of AJ. This process is very complicated because AJ formation triggers drastic changes in the organization of actin cytoskeleton, the activity of Rho family of small GTPases, and the lipid composition of the plasma membrane, all of which are required for subsequent TJ formation. In this review, the authors focus on the relationship between AJ and TJ as a representative example of specialization of plasma membrane regions and introduce recent findings on how AJ formation promotes the subsequent formation of TJ.

Nectin-1 Expression in Colorectal Cancer: Is There a Group of Patients with High Risk for Early Disease Recurrence?

Background: Despite improvements in therapy of colorectal cancer, some patients will present occurrence of recurrence either locally or distantly. Tumor metastasis constitutes the major cause of cancer-associated morbidity and mortality. Nectin-1 belongs to the family of immunoglobulin-like cell adhesion molecules that contribute to the formation of cell-cell adhesions and regulate a series of cellular activities including cell polarization, differentiation, movement, proliferation, and survival. Expression of Nectin-1 in malignant tumors has been associated with aggressive tumor phenotypes. Objectives: The aim of the present study was to assess Nectin-1 expression patterns in colorectal cancer and to investigate its clinical significance. Methods: Nectin-1 expression was assessed via immunohistochemistry in surgical specimens of a cohort comprised of 111 patients with primary resectable colorectal cancer. Results were correlated with clinicopathological characteristics and survival data. Progression-free survival was defined as the primary outcome of the present study. Results: Nectin-1 was strongly expressed in the cytoplasm of colorectal cancer cells. High Nectin-1 expression was associated with advanced stage of disease (p = 0.012) and lymph node metastasis (p = 0.007). Progression-free survival of patients exhibiting high expression of Nectin-1 in the first 36 months after surgery was significantly worse compared to patients with low expression of Nectin-1 (55.7%, 95% CI = 47–70, vs. 82.1%, 95% CI = 69–93, p = 0.014) and independent of other clinicopathological characteristics (HR = 0.389, 95% CI = 0.156–0.972, p = 0.043). Conclusion: Nectin-1 expression in colorectal cancer is associated with a significantly worse 3-year progression-free survival identifying therefore a group of patients with high risk for early disease recurrence.

The cadherin-catenin complex is necessary for cell adhesion and embryogenesis in Nematostella vectensis

The cadherin-catenin complex is a conserved, calcium-dependent cell-cell adhesion module that is necessary for normal development and the maintenance of tissue integrity in bilaterian animals. Despite longstanding evidence of a deep ancestry of calcium-dependent cell adhesion in animals, the requirement of the cadherin-catenin complex to coordinate cell-cell adhesion has not been tested directly in a non-bilaterian organism. Here, we provide the first analysis of classical cadherins and catenins in the Starlet Sea Anemone, Nematostella vectensis. Gene expression, protein localization, siRNA-mediated knockdown of α-catenin, and calcium-dependent cell aggregation assays provide evidence that a bonafide cadherin-catenin complex is present in the early embryo, and that α-catenin is required for normal embryonic development and the formation of cell-cell adhesions between cells dissociated from whole embryos. Together these results support the hypothesis that the cadherin-catenin complex was likely a complete and functional cell-cell adhesion module in the last common cnidarian-bilaterian ancestor. Summary statementEmbryonic manipulations and ex vivo adhesion assays in the sea anemone, Nematostella vectensis, indicate that the necessity of the cadherin-catenin complex for mediating cell-cell adhesion is deeply conserved in animal evolution.

Multiple S100 protein isoforms and C-terminal phosphorylation contribute to the paralog-selective regulation of nonmuscle myosin 2 filaments

Nonmuscle myosin 2 (NM2) has three paralogs in mammals, NM2A, NM2B, and NM2C, which have both unique and overlapping functions in cell migration, formation of cell-cell adhesions, and cell polarity. Their assembly into homo- and heterotypic bipolar filaments in living cells is primarily regulated by phosphorylation of the N-terminally bound regulatory light chain. Here, we present evidence that the equilibrium between these filaments and single NM2A and NM2B molecules can be controlled via S100 calcium-binding protein interactions and phosphorylation at the C-terminal end of the heavy chains. Furthermore, we show that in addition to S100A4, other members of the S100 family can also mediate disassembly of homotypic NM2A filaments. Importantly, these proteins can selectively remove NM2A molecules from heterotypic filaments. We also found that tail phosphorylation (at Ser-1956 and Ser-1975) of NM2B by casein kinase 2, as well as phosphomimetic substitutions at sites targeted by protein kinase C (PKC) and transient receptor potential cation channel subfamily M member 7 (TRPM7), down-regulates filament assembly in an additive fashion. Tail phosphorylation of NM2A had a comparatively minor effect on filament stability. S100 binding and tail phosphorylation therefore preferentially disassemble NM2A and NM2B, respectively. These two distinct mechanisms are likely to contribute to the temporal and spatial sorting of the two NM2 paralogs within heterotypic filaments. The existence of multiple NM2A-depolymerizing S100 paralogs offers the potential for diverse regulatory inputs modulating NM2A filament disassembly in cells and provides functional redundancy under both physiological and pathological conditions.

Steered Molecular Dynamics Simulation Study of Quantified Effects of Point Mutation Induced by Breast Cancer on Mechanical Behavior of E-Cadherin

E-cadherin is a member of the cadherin family that plays a key role in the formation of cell-cell adhesion among epithelial tissues. Point mutations are one of the structural abnormalities of E-cadherin in human carcinomas. Such abnormalities can alter mechanical properties of proteins that play an important role in their biological activities. To determine the impact of point mutations on protein mechanical properties, the second fragment of extracellular domain of E-cadherin was modeled using steered molecular dynamics simulations. The molecular dynamics modeling included application of tensile forces in both constant velocity and constant force modes to examine the effects of Met282 to He and Asn315 to Ser mutations on mechanical behavior of protein structure. The stabilities of the wild type and mutant structures were also obtained by the protein design foldX algorithm. Results confirmed the lower stability of the mutant domains compared to the wild type. The mutated proteins displayed softer behavior than the reference protein and their stiffness decreased by up to 34%. Our findings suggest that local changes in molecular structure due to mutations may lead to noticeable alterations in mechanical properties within the entire domain. Since the function of protein is related to its structure, these changes may influence the function of the protein.

Abstract 4466: The key role of Abi1 loss in dysregulating cell-cell adhesion during prostate cancer tumorigenesis

Abstract Prostate cancer accounts for 20-25% of cancer cases every year and is predicted at over 180,000 new cases in 2016. However, the mechanisms of prostate cancer initiation and castration resistance still remains poorly understood. Our previous research shows mutations and deletions in Abelson interactor 1 (Abi1), an integral part of WAVE complex, in prostate tumor samples. Moreover, WAVE complex levels are inversely correlated to prostate cancer recurrence and castration resistance, outlining their clinical significance. A common phenomenon found early on in most cancers, including prostate cancer, is dysregulated cell-cell adhesion. This points towards a possible tumor suppressor mechanism, since Abi1 and the WAVE complex are in fact the primary regulators of branched actin polymerization via the Arp2/3 complex, and are critical for the formation and maintenance of cell-cell adhesion complexes. Hence, to study Abi1 in prostate cancer, we generated a prostate-specific conditional Abi1 knock-out (KO) mouse. In these mice, we observed evidence of prostatic intraepithelial neoplasia (PIN), a precursor of invasive carcinoma, with changes in proliferation and cell-cell adhesion markers. The goal of this study is to elucidate the mechanism by which the loss of Abi1 leads to the development of PIN in these mice. We are leading this analysis by primary 3D cultures with cells from our Abi1 KO prostates, and 2D cultures with mouse embryonic fibroblasts (MEFs). Loss of Abi1 leads to increase in size of the cultured spheroids, which indicates increased proliferation and dysregulated cell-cell adhesion. Also, adherens junction (AJ) markers E-cadherin and β-catenin levels are lower in western blots and immunostaining, suggesting defects in cell-cell adhesion. In MEFs, we observe loss of membrane localization of AJ proteins in Abi1 KO cells. Loss of Abi1 is often associated with upregulation of Abi2, which may be a contributing factor to the phenotype. To validate our findings in a human system, we have recently developed a CRISPR-mediated KO of Abi1 in the RWPE-1 non-tumor prostate cell line. We will use this KO cell line to determine cell-cell adhesion formation and maintenance in 2D and 3D cultures. In conclusion, loss of Abi1 leads to dysregulation of cell-cell adhesion in prostate epithelial cells, which may be a key mechanism for tumor development and progression in prostate cancer. Supported by NCI R01 CA161018 (LK). Citation Format: Disharee Das, Anita Hryniewicz-Jankowska, Heidi Hehnly, Dawn Post, Gennady Bratslavsky, Leszek Kotula. The key role of Abi1 loss in dysregulating cell-cell adhesion during prostate cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4466. doi:10.1158/1538-7445.AM2017-4466

Altered Contractile Machinery in Airway Epithelial Cells in Response to Cigarette Smoke

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death in the US with cigarette smoke being the primary insult leading to disease progression. While the lung epithelium is the site that makes initial and primary contact with the inhaled cigarette smoke, we do not understand the role of cytoskeleton and cell mechanics in the airway response to cigarette smoke. We know that cytoskeletal proteins, such as myosin II, actin, alpha-actinin, the catenins, and E-cadherin, are involved in cell-cell adhesion formation and barrier function of the lung. Furthermore, E-cadherin levels decrease in primary epithelial cells from COPD patients. Therefore, we hypothesized that cigarette smoke drives key cytoskeletal protein changes that lead to alterations in barrier function and thereby influence the development of chronic lung disease. To test this, we studied major cytoskeletal (actin and nonmuscle myosin II isoforms) and cell adhesion proteins (E-cadherin) in normal human bronchial epithelial cells (NHBE, collected directly from human patients) or 16HBE cells (primary human bronchial epithelial cell line) grown on an air-liquid interface. Using a Vitrocell smoke chamber, we found that in response to acute smoke exposure (four cigarettes in 24 hrs), myosin IIB and actin assembled into apical stress fibers, which are not normally found in airway epithelial cells. Cigarette smoke exposure also resulted in decreased total E-cadherin levels as well as decreased E-cadherin at the basolateral surface. Thus, cigarette smoke promotes dramatic acute cytoskeletal changes that likely lead to altered cell mechanical properties (tension, elasticity), which in turn lead to reduced epithelial barrier function and our data suggest that epithelial barrier function is critical in dictating chronic tissue responses that contributes to COPD development and progression.

Quantification of the stiffness and strength of cadherin ectodomain binding with different ions

The stiffness and strength of extracellular (EC) region of cadherin are proposed to be two important mechanical properties both for cadherin as a mechanotransductor and for the formation of cell-cell adhesion. In this study, we quantitatively characterized the stiffness and strength of EC structure when it binds with different types of ions by molecular dynamics simulations. Results show that EC structure exhibits a rod-like shape with high stiffness and strength when it binds with the bivalent ions of calcium or magnesium. However, it switches to a soft and collapsed conformation when it binds with the monovalent ions of sodium or potassium. This study sheds light on the important role of the bivalent ions of calcium in the physiological function of EC.

ARHGAP21 Protein, a New Partner of α-Tubulin Involved in Cell-Cell Adhesion Formation and Essential for Epithelial-Mesenchymal Transition

Cell-cell adhesions and the cytoskeletons play important and coordinated roles in cell biology, including cell differentiation, development, and migration. Adhesion and cytoskeletal dynamics are regulated by Rho-GTPases. ARHGAP21 is a negative regulator of Rho-GTPases, particularly Cdc42. Here we assess the function of ARHGAP21 in cell-cell adhesion, cell migration, and scattering. We find that ARHGAP21 is localized in the nucleus, cytoplasm, or perinuclear region but is transiently redistributed to cell-cell junctions 4 h after initiation of cell-cell adhesion. ARHGAP21 interacts with Cdc42, and decreased Cdc42 activity coincides with the appearance of ARHGAP21 at the cell-cell junctions. Cells lacking ARHGAP21 expression show weaker cell-cell adhesions, increased cell migration, and a diminished ability to undergo hepatocyte growth factor-induced epithelial-mesenchymal transition (EMT). In addition, ARHGAP21 interacts with α-tubulin, and it is essential for α-tubulin acetylation in EMT. Our findings indicate that ARHGAP21 is a Rho-GAP involved in cell-cell junction remodeling and that ARHGAP21 affects migration and EMT through α-tubulin interaction and acetylation.

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.

HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner

BackgroundAcquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory.ResultsAn anoikis-resistant clone (HEK293ar), derived from anoikis-sensitive parental Human Embryonic Kidney 293 cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147 family) was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K/AKT) inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated with the extracellular signal-regulated kinase (ERK) inhibitor PD98059.ConclusionsOur results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar). Understanding of the role of HAb18G/CD147 cell-cell contacts in anoikis resistance may help in understanding the survival of cells in anchorage-independent growth, such as cells in tumor metastasis and suspension culture produced for biomedical engineering. Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.

Abstract 5592: Role of Afadin in Vascular Endothelial Growth Factor- and Sphingosine 1-Phosphate-Induced Angiogenesis

[Background] Better understanding of mechanism of angiogenesis and angiogenic signaling of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) will aid in developing therapeutic angiogenesis for ischemic heart disease and peripheral arterial disease. Recently, Rap1 small G protein was identified as a key angiogenic signaling molecule. However, mechanisms involved in the Rap1 regulation of angiogenesis and downstream effectors of Rap1 are not known. Afadin is an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1. Recently, we identified that, besides the role in cell-cell adhesion formation in epithelial cells, afadin plays a role in the regulation of directional cell movement of fibroblasts. We therefore attempted to investigate the role of endothelial afadin in angiogenesis and clarify the underlying molecular mechanism. [Method and Results] Knockdown of Rap1 or afadin by siRNA inhibited the VEGF- and S1P-induced tubulogenesis, migration and proliferation, and conversely, increased the serum deprivation-induced apoptosis of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation and immunofluorescence studies revealed that Rap1 regulated the interaction between afadin and phosphatidyl-inositol 3-kinase (PI3K) and recruitment of the afadin-PI3K complex to the leading edge, suggesting that Rap1 and afadin regulates the PI3K signaling. Indeed, knockdown of Rap1 or afadin by siRNA inhibited the VEGF- and S1P-induced phosphorylation of Akt. Thus, afadin regulated the PI3K-Akt signaling. To examine the role of afadin in angiogenesis in vivo, we developed endothelial cell-specific afadin-knockout mice using the Cre/loxP system. Majority (96%) of afadin-knockout mice were embryonically lethal, suggesting an essential role of endothelial afadin in embryonic vascular development and angiogenesis. We performed Matrigel plug assay in born adult afadin-knockout mice and observed that VEGF- and S1P-induced angiogenesis was impaired in afadin-knockout mice. [Conclusion] These results demonstrate that afadin regulates VEGF- and S1P-induced angiogenesis downstream of Rap1.

Rac-WAVE-mediated actin reorganization is required for organization and maintenance of cell-cell adhesion

During cadherin-dependent cell-cell adhesion, the actin cytoskeleton undergoes dynamic reorganization in epithelial cells. Rho-family small GTPases, which regulate actin dynamics, play pivotal roles in cadherin-dependent cell-cell adhesion; however, the precise molecular mechanisms that underlie cell-cell adhesion formation remain unclear. Here we show that Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE)-mediated reorganization of actin, downstream of Rac plays an important role in normal development of cadherin-dependent cell-cell adhesions in MDCK cells. Rac-induced development of cadherin-dependent adhesions required WAVE2-dependent actin reorganization. The process of cell-cell adhesion is divided into three steps: formation of new cell-cell contacts, stabilization of these new contacts and junction maturation. WAVE1 and WAVE2 were expressed in MDCK cells. The functions of WAVE1 and WAVE2 were redundant in this system but WAVE2 appeared to play a more significant role. During the first step, WAVE2-dependent lamellipodial protrusions facilitated formation of cell-cell contacts. During the second step, WAVE2 recruited actin filaments to new cell-cell contacts and stabilized newly formed cadherin clusters. During the third step, WAVE2-dependent actin reorganization was required for organization and maintenance of mature cell-cell adhesions. Thus, Rac-WAVE-dependent actin reorganization is not only involved in formation of cell-cell adhesions but is also required for their maintenance.

Functional Gene Expression Analysis Uncovers Phenotypic Switch in Aggressive Uveal Melanomas

Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.