Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody.

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface. Molecular dynamics simulations and single-molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes: one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped ß-strand and its complementary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

The functional activity of E-cadherin controls tumor cell metastasis at multiple steps.

E-cadherin is a tumor suppressor protein, and the loss of its expression in association with the epithelial mesenchymal transition (EMT) occurs frequently during tumor metastasis. However, many metastases continue to express E-cadherin, and a full EMT is not always necessary for metastasis; also, positive roles for E-cadherin expression in metastasis have been reported. We hypothesize instead that changes in the functional activity of E-cadherin expressed on tumor cells in response to environmental factors is an important determinant of the ability of the tumor cells to metastasize. We find that E-cadherin expression persists in metastatic lung nodules and circulating tumor cells (CTCs) in two mouse models of mammary cancer: genetically modified MMTV-PyMT mice and orthotopically grafted 4T1 tumor cells. Importantly, monoclonal antibodies that bind to and activate E-cadherin at the cell surface reduce lung metastasis from endogenous genetically driven tumors and from tumor cell grafts. E-cadherin activation inhibits metastasis at multiple stages, including the accumulation of CTCs from the primary tumor and the extravasation of tumor cells from the vasculature. These activating mAbs increase cell adhesion and reduce cell invasion and migration in both cell culture and three-dimensional spheroids grown from primary tumors. Moreover, activating mAbs increased the frequency of apoptotic cells without affecting proliferation. Although the growth of the primary tumors was unaffected by activating mAbs, CTCs and tumor cells in metastatic nodules exhibited increased apoptosis. Thus, the functional state of E-cadherin is an important determinant of metastatic potential beyond whether the gene is expressed.

Reconstitution of the full transmembrane cadherin-catenin complex.

The dynamic regulation of epithelial adherens junctions relies on all components of the E-cadherin-catenin complex. Previously, the complexes have been partially reconstituted and composed only of α-catenin, ß-catenin, and the E-cadherin cytoplasmic domain. However, p120-catenin and the full-length E-cadherin including the extracellular, transmembrane, and intra-cellular domains are vital to the understanding of the relationship between extracellular adhesion and intracellular signaling. Here, we reconstitute the complete and full-length cadherin-catenin complex, including full-length E-cadherin, α-catenin, ß-catenin, and p120-catenin, into nanodiscs. We are able to observe the cadherin in nanodiscs by cryo-EM. We also reconstitute α-catenin, ß-catenin, and p120-catenin with the E-cadherin cytoplasmic tail alone in order to analyze the affinities of their binding interactions. We find that p120-catenin does not associate strongly with α- or ß-catenin and binds much more transiently to the cadherin cytoplasmic tail than does ß-catenin. Overall, this work creates many new possibilities for biochemical studies understanding transmembrane signaling of cadherins and the role of p120-catenin in adhesion activation.

Cell contact and Nf2/Merlin-dependent regulation of TEAD palmitoylation and activity.

The Hippo pathway is involved in regulating contact inhibition of proliferation and organ size control and responds to various physical and biochemical stimuli. It is a kinase cascade that negatively regulates the activity of cotranscription factors YAP and TAZ, which interact with DNA binding transcription factors including TEAD and activate the expression of target genes. In this study, we show that the palmitoylation of TEAD, which controls the activity and stability of TEAD proteins, is actively regulated by cell density independent of Lats, the key kinase of the Hippo pathway. The expression of fatty acid synthase and acetyl-CoA carboxylase involved in de novo biosynthesis of palmitate is reduced by cell density in an Nf2/Merlin-dependent manner. Depalmitoylation of TEAD is mediated by depalmitoylases including APT2 and ABHD17A. Palmitoylation-deficient TEAD4 mutant is unstable and degraded by proteasome through the activity of the E3 ubiquitin ligase CHIP. These findings show that TEAD activity is tightly controlled through the regulation of palmitoylation and stability via the orchestration of FASN, depalmitoylases, and E3 ubiquitin ligase in response to cell contact.

Enhanced endothelial barrier function by monoclonal antibody activation of vascular endothelial cadherin.

Excessive vascular permeability occurs in inflammatory disease processes. Vascular endothelial cadherin (VE-cadherin) is an adhesion protein that controls vascular permeability. We identified monoclonal antibodies (mAbs) to human VE-cadherin that activate cell adhesion and inhibit the increased permeability of endothelial cell monolayers induced by thrombin receptor activator peptide-6 (TRAP-6). Two mAbs, 8A12c and 3A5a, reduce permeability, whereas an inhibitory mAb, 2E11d, enhances permeability. Activating mAbs also reduce permeability induced by tumor necrosis factor-α (TNF-α) and vascular endothelial cell growth factor (VEGF). The activating mAbs also stabilize the organization of the adherens junctions that are disrupted by TRAP-6, VEGF, or TNF-α. The activating mAbs act directly on the adhesive function of VE-cadherin because they did not block the accumulation of actin filaments stimulated by TRAP-6 and enhance physical cell-cell adhesion of VE-cadherin-expressing tissue culture cells. Therefore, VE-cadherin function can be regulated at the cell surface to control endothelial permeability.NEW & NOTEWORTHY Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.

Allosteric Regulation of E-Cadherin Adhesion.

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation.

The Hippo-YAP signaling pathway and contact inhibition of growth.

The Hippo-YAP pathway mediates the control of cell proliferation by contact inhibition as well as other attributes of the physical state of cells in tissues. Several mechanisms sense the spatial and physical organization of cells, and function through distinct upstream modules to stimulate Hippo-YAP signaling: adherens junction or cadherin-catenin complexes, epithelial polarity and tight junction complexes, the FAT-Dachsous morphogen pathway, as well as cell shape, actomyosin or mechanotransduction. Soluble extracellular factors also regulate Hippo pathway signaling, often inhibiting its activity. Indeed, the Hippo pathway mediates a reciprocal relationship between contact inhibition and mitogenic signaling. As a result, cells at the edges of a colony, a wound in a tissue or a tumor are more sensitive to ambient levels of growth factors and more likely to proliferate, migrate or differentiate through a YAP and/or TAZ-dependent process. Thus, the Hippo-YAP pathway senses and responds to the physical organization of cells in tissues and coordinates these physical cues with classic growth-factor-mediated signaling pathways. This Commentary is focused on the biological significance of Hippo-YAP signaling and how upstream regulatory modules of the pathway interact to produce biological outcomes.

Deregulation of the Hippo pathway in mouse mammary stem cells promotes mammary tumorigenesis.

The Hippo-YAP pathway mediates organ size control, contact inhibition, and tumorigenesis. It is a kinase cascade that inhibits the nuclear localization and transcriptional activities of YAP and TAZ. E-cadherin, cell junctions, polarity proteins, and the merlin/NF2 tumor suppressor activate the pathway to inhibit YAP/TAZ activity, while growth factor signaling inhibits the pathway to activate YAP/TAZ in the nucleus. We examined its role in the development of mouse mammary glands and tumor formation using gland reconstitution by transplantation of genetically modified mammary stem cells (MaSCs). Knockdown of YAP and TAZ with shRNA in MaSCs did not inhibit gland reconstitution. In contrast, knockdown of ß-catenin blocked gland reconstitution, consistent with the known role of Wnt signaling in mammary gland development. However, we find that Hippo signaling is involved in mammary tumor formation. Expression of a constitutively active form of YAP caused rapid formation of large tumors. Moreover, knockdown of YAP/TAZ slowed the development of tumors in polyoma middle T transgenic mice, a well-studied mammary tumor model involving activation of several signaling pathways. YAP accumulated in nuclei of mammary glands in ErbB2/EGFR-transgenic mice, suggesting that EGFR signaling affects YAP in vivo similar to cell culture. ErbB2/EGFR-transgenic mice develop mammary tumors in 7-8 months, but surprisingly, MaSCs from these mice did not form tumors when transplanted into host mice. Nonetheless, expression of dominant-negative Lats, which inhibits Hippo signaling, leads to tumor formation in ErbB2-transgenic mice, suggesting that Hippo signaling is involved in EGFR-induced mammary tumorigenesis.

A phase I pharmacokinetics trial comparing PF-05280586 (a potential biosimilar) and rituximab in patients with active rheumatoid arthritis.

AIMS: Pharmacokinetic (PK) similarity was assessed among PF-05280586 (a proposed biosimilar) vs. rituximab sourced from the European Union (rituximab-EU) and the United States (rituximab-US). Pharmacodynamics (PD), overall safety and immunogenicity were also evaluated. METHODS: Patients with active rheumatoid arthritis on a background of methotrexate and inadequate response to one or more tumour necrosis factor antagonist therapies were randomized to intravenous PF-05280586, rituximab-EU or rituximab-US 1000 mg doses on study days 1 and 15. RESULTS: A total of 220 patients were randomized to receive study treatment as assigned. Of these, 198 met per-protocol population criteria for inclusion in the PK data analysis. PF-05280586, rituximab-EU and rituximab-US exhibited similar PK profiles following administration of assigned study drug on days 1 and 15. The 90% confidence intervals of test-to-reference ratios for Cmax , AUCT , AUC0-∞ and AUC2-week were within the bioequivalence margin of 80.00-125.00% for comparisons of PF-05280586 with rituximab-EU, PF-05280586 with rituximab-US, and rituximab-EU with rituximab-US. All treatments resulted in a rapid and profound reduction in CD19+ B cells and sustained profound B cell suppression up to week 25. The incidence of antidrug antibody (ADA) response (n = 7, 10 and 9 for PF-05280586, rituximab-EU and rituximab-US, respectively), time to ADA emergence and ADA titres were similar across treatments. None of the ADA-positive samples was positive for neutralizing activity. No clinically meaningful differences in adverse events were identified. CONCLUSIONS: The study demonstrated PK similarity among PF-05280586, rituximab-EU and rituximab-US. In addition, all treatments showed comparable CD19+ B cell depletion PD responses, as well as safety and immunogenicity profiles.

Comparative assessment of clinical response in patients with rheumatoid arthritis between PF-05280586, a proposed rituximab biosimilar, and rituximab.

AIMS: To evaluate potential differences between PF-05280586 and rituximab sourced from the European Union (rituximab-EU) and USA (rituximab-US) in clinical response (Disease Activity Score in 28 Joints [DAS28] and American College of Rheumatology [ACR] criteria), as part of the overall biosimilarity assessment of PF-05280586. METHODS: A randomised, double-blind, pharmacokinetic similarity trial was conducted in patients with active rheumatoid arthritis refractory to anti-tumour necrosis factor therapy on a background of methotrexate. Patients were treated with 1000 mg of PF-05280586, rituximab-EU or rituximab-US on days 1 and 15 and followed over 24 weeks for pharmacokinetic, clinical response and safety assessments. Key secondary end points were the areas under effect curves for DAS28 and ACR responses. Mean differences in areas under effect curves were compared against respective reference ranges established by observed rituximab-EU and rituximab-US responses using longitudinal nonlinear mixed effects models. RESULTS: The analysis included 214 patients. Demographics were similar across groups with exceptions in some baseline disease characteristics. Baseline imbalances and group-to-group variation were accounted for by covariate effects in each model. Predictions from the DAS28 and ACR models tracked the central tendency and distribution of observations well. No point estimates of mean differences were outside the reference range for DAS28 or ACR scores. The probabilities that the predicted differences between PF-05280586 vs. rituximab-EU or rituximab-US lie outside the reference ranges were low. CONCLUSIONS: No clinically meaningful differences were detected in DAS28 or ACR response between PF-05280586 and rituximab-EU or rituximab-US as the differences were within the pre-specified reference ranges. TRIAL REGISTRATION NUMBER: NCT01526057.

Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositide-dependent kinase-1.

The Hippo signaling pathway inhibits cell growth and regulates organ size through a kinase cascade that leads to the phosphorylation and nuclear exclusion of the growth-promoting transcriptional coactivator Yes-associated protein (YAP)/Yorkie. It mediates contact inhibition of cell growth downstream of cadherin adhesion molecules and other cell surface proteins. Contact inhibition is often antagonized by mitogenic growth factor signaling. We report an important mechanism for this antagonism, inhibition of Hippo pathway signaling by mitogenic growth factors. EGF treatment of immortalized mammary cells triggers the rapid translocation of YAP into the nucleus along with YAP dephosphorylation, both of which depend on Lats, the terminal kinase in the Hippo pathway. A small-molecule inhibitor screen of downstream effector pathways shows that EGF receptor inhibits the Hippo pathway through activation of PI3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1), but independent of AKT activity. The PI3K-PDK1 pathway also mediates YAP nuclear translocation downstream of lysophosphatidic acid and serum as a result of constitutive oncogenic activation of PI3K. PDK1 associates with the core Hippo pathway-kinase complex through the scaffold protein Salvador. The entire Hippo core complex dissociates in response to EGF signaling in a PI3K-PDK1-dependent manner, leading to inactivation of Lats, dephosphorylation of YAP, and YAP nuclear accumulation and transcriptional activation of its target gene, CTGF. These findings show that an important activity of mitogenic signaling pathways is to inactivate the growth-inhibitory Hippo pathway and provide a mechanism for antagonism between contact inhibition and growth factor action.

Cadherin-6B is required for the generation of Islet-1-expressing dorsal interneurons.

Cadherin-6B induces bone morphogenetic protein (BMP) signaling to promote the epithelial mesenchymal transition (EMT) in the neural crest. We have previously found that knockdown of Cadherin-6B inhibits both BMP signaling and the emigration of the early pre-migratory neural crest cells from the dorsal neural tube. In this study, we found that inhibition of BMP signaling in the neural tube, mediated by the ectopic expression of Smad-6 or Noggin, decreased the size of the Islet-1-positive dorsal cell population. Knockdown or loss of function of Cadherin-6B suppressed the generation of Islet-1-expressing cells in the dorsal neural tube, but not the Lim-1/2 positive dorsal cell population. Our results thus indicate that Cadherin-6B is necessary for the generation of Islet-1-positive dorsal interneurons, as well as the initiation of pre-migratory neural crest cell emigration.

N-cadherin mediates the migration of MCF-10A cells undergoing bone morphogenetic protein 4-mediated epithelial mesenchymal transition.

Epithelial-mesenchymal transition (EMT) of mammary epithelial cells is important in both normal morphogenesis of mammary glands and metastasis of breast cancer. Cadherin switching from E-cadherin to N-cadherin plays important roles in EMT. We found that cadherin switching is important in bone morphogenetic protein 4 (BMP4)-induced EMT in MCF-10A cells. BMP4 increased the phosphorylation of SMAD proteins in MCF-10A cells. Canonical BMP4 signaling decreased the expression of E-cadherin and disrupted the polarity of the tight junction protein ZO-1 in MCF-10A cells. However, the expression of N-cadherin and SNAI2 was up-regulated in BMP4-treated MCF-10A cells. MCF-10A cells that expressed N-cadherin migrated into type I collagen gels in response to BMP4 when evaluated using three-dimensional culture assays. Thus, active canonical BMP4 signaling is important for the migration and EMT of mammary epithelial cells. Moreover, the decrease in E-cadherin and/or increase in N-cadherin may be required for BMP4-induced migration and EMT.

E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components.

Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms, and the growth of tumors is characterized by a loss of contact inhibition of proliferation. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. The modulation of the phosphorylation and nuclear localization of Yes-associated protein (YAP) by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied. However, cell-surface receptors regulating the Hippo signaling pathway in mammals are not well understood. In this study, we show that Hippo signaling pathway components are required for E-cadherin-dependent contact inhibition of proliferation. Knockdown of the Hippo signaling components or overexpression of YAP inhibits the decrease in cell proliferation caused by E-cadherin homophilic binding at the cell surface, independent of other cell-cell interactions. We also demonstrate that the E-cadherin/catenin complex functions as an upstream regulator of the Hippo signaling pathway in mammalian cells. Expression of E-cadherin in MDA-MB-231 cells restores the density-dependent regulation of YAP nuclear exclusion. Knockdown of ß-catenin in densely cultured MCF10A cells, which mainly depletes E-cadherin-bound ß-catenin, induces a decrease in the phosphorylation of S127 residue of YAP and its nuclear accumulation. Moreover, E-cadherin homophilic binding independent of other cell interactions is sufficient to control the subcellular localization of YAP. Therefore, Our results indicate that, in addition to its role in cell-cell adhesion, E-cadherin-mediated cell-cell contact directly regulates the Hippo signaling pathway to control cell proliferation.

Strengthening E-cadherin adhesion via antibody-mediated binding stabilization.

E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8-mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site-directed mutagenesis, and single-molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped ß-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.

The acyclic CB1R inverse agonist taranabant mediates weight loss by increasing energy expenditure and decreasing caloric intake.

Cannabinoid 1 receptor (CB1R) inverse agonists are emerging as a potential obesity therapy. However, the physiological mechanisms by which these agents modulate human energy balance are incompletely elucidated. Here, we describe a comprehensive clinical research study of taranabant, a structurally novel acyclic CB1R inverse agonist. Positron emission tomography imaging using the selective CB1R tracer [(18)F]MK-9470 confirmed central nervous system receptor occupancy levels ( approximately 10%-40%) associated with energy balance/weight-loss effects in animals. In a 12-week weight-loss study, taranabant induced statistically significant weight loss compared to placebo in obese subjects over the entire range of evaluated doses (0.5, 2, 4, and 6 mg once per day) (p < 0.001). Taranabant treatment was associated with dose-related increased incidence of clinical adverse events, including mild to moderate gastrointestinal and psychiatric effects. Mechanism-of-action studies suggest that engagement of the CB1R by taranabant leads to weight loss by reducing food intake and increasing energy expenditure and fat oxidation.

Cadherin-6B stimulates an epithelial mesenchymal transition and the delamination of cells from the neural ectoderm via LIMK/cofilin mediated non-canonical BMP receptor signaling.

We previously provided evidence that cadherin-6B induces de-epithelialization of the neural crest prior to delamination and is required for the overall epithelial mesenchymal transition (EMT). Furthermore, de-epithelialization induced by cadherin-6B was found to be mediated by BMP receptor signaling independent of BMP. We now find that de-epithelialization is mediated by non-canonical BMP signaling through the BMP type II receptor (BMPRII) and not by canonical Smad dependent signaling through BMP Type I receptor. The LIM kinase/cofilin pathway mediates non-canonical BMPRII induced de-epithelialization, in response to either cadherin-6B or BMP. LIMK1 induces de-epithelialization in the neural tube and dominant negative LIMK1 decreases de-epithelialization induced by either cadherin-6B or BMP. Cofilin is the major known LIMK1 target and a S3A phosphorylation deficient mutated cofilin inhibits de-epithelialization induced by cadherin-6B as well as LIMK1. Importantly, LIMK1 as well as cadherin-6B can trigger ectopic delamination when co-expressed with the competence factor SOX9, showing that this cadherin-6B stimulated signaling pathway can mediate the full EMT in the appropriate context. These findings suggest that the de-epithelialization step of the neural crest EMT by cadherin-6B/BMPRII involves regulation of actin dynamics via LIMK/cofilin.

Cadherin 6B induces BMP signaling and de-epithelialization during the epithelial mesenchymal transition of the neural crest.

The development of neural crest cells involves an epithelial-mesenchymal transition (EMT) associated with the restriction of cadherin 6B expression to the pre-migratory neural crest cells (PMNCCs), as well as a loss of N-cadherin expression. We find that cadherin 6B, which is highly expressed in PMNCCs, persists in early migrating neural crest cells and is required for their emigration from the neural tube. Cadherin 6B-expressing PMNCCs exhibit a general loss of epithelial junctional polarity and acquire motile properties before their delamination from the neuroepithelium. Cadherin 6B selectively induces the de-epithelialization of PMNCCs, which is mediated by stimulation of BMP signaling, whereas N-cadherin inhibits de-epithelialization and BMP signaling. As BMP signaling also induces cadherin 6B expression and represses N-cadherin, cadherin-regulated BMP signaling may create two opposing feedback loops. Thus, the overall EMT of neural crest cells occurs via two distinct steps: a cadherin 6B and BMP signaling-mediated de-epithelialization, and a subsequent delamination through the basement membrane.

Crosstalk between different adhesion molecules.

Cell adhesion molecules mediate cell-cell and cell-extracellular matrix adhesions, and coordination between these molecules is essential for tissue formation and morphogenesis. Crosstalk between integrins and cadherins may result from a physical response to integrin-mediated adhesion, complex cell differentiation processes, or direct signaling pathways linking the two adhesion systems. Nectins have recently been shown to regulate the organization of cadherins into adherens junctions and the formation of tight junctions by several processes. Furthermore, protocadherins can interact with extracellular matrix proteins or function by regulating classical cadherins.

Strengthening E-cadherin adhesion via antibody mediated binding stabilization.

E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8 mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site directed mutagenesis and single molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped ß-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.