Internalization Of E-cadherin Research Articles

The Scribble-SGEF-Dlg1 complex regulates E-cadherin and ZO-1 stability, turnover and transcription in epithelial cells.

SGEF (also known as ARHGEF26), a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of both E-cadherin and ZO-1 (also known as TJP1) protein levels. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble-SGEF-Dlg1 complex. Our results show that the three members of the ternary complex are required to maintain the stability of the apical junctions, ZO-1 protein levels and tight junction (TJ) permeability. In contrast, only SGEF is necessary to regulate E-cadherin levels. The absence of SGEF destabilizes the E-cadherin-catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, β-catenin signaling and the transcriptional repressor Slug (also known as SNAI2).

Abstract 4389: The role of Rab5 in epithelial growth factor stimulated E-cadherin internalization in spheroid growth of colon cancer cells

Abstract In epithelial based cancer formation, such as colon cancer, the tumor growth is accompanied with malfunction of cell-cell contact inhibition which is regulated by E-cadherin. However, the dynamics of E-cadherin is complicated, the underlying mechanism in tumor nest growth and collective cell migration is still not clear. Rab5 is the endocytic vesicle protein for the membrane receptor internalization into early endosome. Therefore, this study is to investigate the role of Rab5 in E-cadherin turnover of spheroid formation in colon cancer cells. HT-29 colon cancer cells were used in this study. The cells with less anchorage ability were isolated for spheroid formation. The distribution of Rab5, E-cadherin and focal adhesion kinase were observed by immuno-confocal microscopy. The molecular expressions were analyzed by western blot. HT-29 cells showed well defined epithelial phenotype. In the result of immunostaining, Rab5 was distributed in the cytosol and the cell-cell contacts where it colocalized with E-cadherin. Rab5 was also observed in the cell leading edge, however, not much associated with focal adhesions. When HT-29 cell clusters were stimulated with epidermal growth factor, Rab5 was found abundant in the cell-cell contacts. In cell spheroid that derived from HT-29 cells, Rab5 and E-cadherin co-localization at cell-cell contacts was also increased. Furthermore, when cells were treated with Nocodazole, the tubulin inhibitor blocking tubulin-mediated endocytosis, Rab5 accumulated and colocalized with E-cadherin in the cell leading edge. The results of this study demonstrated Rab5 plays the novel role in E-cadherin turnover upon epidermal growth factor stimulation in the progress of spheroid formation and Rab5 may mediate E-cadherin endocytosis through the cell leading edge. Citation Format: Yu-Chun Wang, Yu-Chien Ching, Hui-Chun Chen, Chun-Pu Cheng, Chia-En Ku, Shin-Ru Lee, Wei-Ting Chao. The role of Rab5 in epithelial growth factor stimulated E-cadherin internalization in spheroid growth of colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4389.

Abstract A115: Collective invasion facilitates liver metastasis in pancreatic ductal adenocarcinoma

Abstract Fifty percent of PDAC patients present with distant metastasis and face a dismal prognosis of 8 to 11 months. However, patients with lung metastases have better prognosis than patients with metastatic spread to other sites. In our PDAC metastasis cohort, patients with lung-only metastasis survived longer than patients with liver metastasis, despite sharing the same tumor subtype. To understand features of the primary tumor linked to metastatic site, we generated a novel gene signature for primary organotropism (pORG) that distinguishes primary tumors that developed liver metastasis from those that developed lung metastasis, independent of known tumor subtypes. Our pORG signature predicts survival independently of transcriptional subtype, genetic alterations and clinicopathological variables and in a validation cohort consisting of TCGA pancreatic cancer. Primary tumors with high pORG scores, i.e., liver-tropic primaries, have increased transcriptomic signatures of proliferation and replication stress whereas low pORG tumors have increased lymphocyte gene expression. To validate our findings, we generated cyclic immunofluorescence multiplex imaging data from 34 primary tumors. As anticipated, single cell image analysis revealed high B and T cell infiltration in low-pORG tumors, and increased proliferation and replication stress foci in high-pORG tumors. Intriguingly, malignant cells from high pORG tumors had an increased fraction KRT19-high tumor cells relative to low pORG tumors and were distinctly epithelial in appearance. To reconcile the metastatic behavior of high pORG tumors with their apparent enrichment for epithelial-state cells, we hypothesized that liver-tropism was associated with a partial epithelial-to-mesenchymal transition (EMT) state and metastasis via collective invasion. Bulk transcriptional analysis of 200 PDAC primary tumors revealed a distinct subset of high pORG tumors have increased expression of markers linked to partial EMT and collective invasion, including RAB11a, Rho-like GTPases, and proteases MMP7 and KLK7. Our results support a model of E-cadherin internalization facilitating partial EMT and collective invasion. Current efforts include in-vitro characterization of migration modes in patient-derived cell lines and organoids. Our work suggests unique and targetable tumor intrinsic features linked to organotropism in PDAC. Citation Format: Jennifer R. Eng, Carl Pelz, Koei Chin, Sam Sivagnanam, Link M. Jason, Dove Keith, Lisa Coussens, Rosalie C. Sears. Collective invasion facilitates liver metastasis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A115.

Multifaceted control of E-cadherin dynamics by Adaptor Protein Complex 1 during epithelial morphogenesis.

Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargoes. We examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. We found that AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell-extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. We discovered a distinct pool of AP-1 that localizes at the subapical adherens junctions. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. We then provide evidence that E-cadherin hyperinternalization acts upstream of cell death in a potential tumor-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell-cell adhesion.

Calcium signaling induces a partial EMT.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating invivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.

Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

ABSTRACT A cytoskeletal protein keratin 19 (K19) is highly expressed in breast cancer but its effects on breast cancer cell mechanics are unclear. In MCF7 cells where K19 expression is ablated, we found that K19 is required to maintain rounded epithelial-like shape and tight cell-cell adhesion. A loss of K19 also lowered cell surface E-cadherin levels. Inhibiting internalization restored cell-cell adhesion of KRT19 knockout cells, suggesting that E-cadherin internalization contributed to defective adhesion. Ultimately, while K19 inhibited cell migration and invasion, it was required for cells to form colonies in suspension. Our results suggest that K19 stabilizes E-cadherin complexes at the cell membrane to maintain cell-cell adhesion which inhibits cell invasiveness but provides growth and survival advantages for circulating tumor cells.

Proper E-cadherin membrane location in colon requires Dab2 and it modifies by inflammation and cancer.

We reported that Disabled-2 (Dab2) is located at the apical membrane in suckling rat intestine. Here, we discovered that, in colon of suckling and adult mouse and of adult human, Dab2 is only at lateral crypt cell membrane and colocalized with E-cadherin. Dab2 depletion in Caco-2 cells led to E-cadherin internalization indicating that its membrane location requires Dab2. In mice, we found that 3 days of dextran sulfate sodium-induced colitis increased Dab2/E-cadherin colocalization, which was decreased as colitis progressed to 6 and 9 days. In agreement, Dab2/E-cadherin colocalization increased in human mild and severe ulcerative colitis and in polyps, being reduced in colon adenocarcinomas, which even showed epithelial Dab2 absence and E-cadherin delocalization. Epithelial Dab2 decrement preceded that of E-cadherin. We suggest that Dab2, by inhibiting E-cadherin internalization, stabilizes adherens junctions, and its absence from the epithelium may contribute to development of colon inflammation and cancer.

Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposure.

Bromine (Br2) is an organohalide found in nature and is integral to many manufacturing processes. Br2 is toxic to living organisms, and high concentrations can prove fatal. To meet industrial demand, large amounts of purified Br2 are produced, transported, and stored worldwide, providing a multitude of interfaces for potential human exposure through either accidents or terrorism. To identify the key mechanisms associated with acute Br2 exposure, we have surveyed the lung proteomes of C57BL/6 male mice and human lung-derived microvascular endothelial cells (HMECs) at 24 h following exposure to Br2 in concentrations likely to be encountered in the vicinity of industrial accidents. Global discovery proteomics applications combined with systems biology analysis identified robust and highly significant changes in proteins associated with three biological processes: 1) exosome secretion, 2) inflammation, and 3) vascular permeability. We focused on the latter, conducting physiological studies on isolated perfused lungs harvested from mice 24 h after Br2 exposure. These experiments revealed significant increases in the filtration coefficient (Kf) indicating increased permeability of the pulmonary vasculature. Similarly, confluent monolayers of Br2 and Br-lipid-treated HMECs exhibited differential levels of zona occludens-1 that were found to be dissociated from cell wall localization, an increase in phosphorylation and internalization of E-cadherin, as well as increased actin stress fiber formation, all of which are consistent with increased permeability. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with physiological measurements of permeability, revealed both profound and novel biological changes that contribute to our current understanding of Br2 toxicity.

Prostaglandin E2 and its receptor EP2 trigger signaling that contributes to YAP-mediated cell competition.

Cell competition is a biological process by which unfit cells are eliminated from “cell society.” We previously showed that cultured mammalian epithelial Madin‐Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by “normal” MDCK cells. However, the molecular mechanism underlying the elimination of active YAP‐expressing cells was unknown. Here, we used high‐throughput chemical compound screening to identify cyclooxygenase‐2 (COX‐2) as a key molecule triggering cell competition. Our work shows that COX‐2‐mediated PGE2 secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase‐cyclic AMP‐PKA pathway that, in the presence of active YAP, induces E‐cadherin internalization leading to apical extrusion. Thus, COX‐2‐induced PGE2 appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.

ZEB1/NuRD complex suppresses TBC1D2b to stimulate E-cadherin internalization and promote metastasis in lung cancer

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, due in part to the propensity of lung cancer to metastasize. Aberrant epithelial-to-mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell-cell adhesion is reduced allowing cells to dissociate and invade. Of the EMT-associated transcription factors, ZEB1 uniquely promotes NSCLC disease progression. Here we apply two independent screens, BioID and an Epigenome shRNA dropout screen, to define ZEB1 interactors that are critical to metastatic NSCLC. We identify the NuRD complex as a ZEB1 co-repressor and the Rab22 GTPase-activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis.

Inhibition of Intestinal Epithelial Wound Healing through Protease-Activated Receptor-2 Activation in Caco2 Cells.

The mechanisms of epithelial wound healing are not completely understood, especially in the context of proteases and their receptors. It was recently shown that activation of protease-activated receptor-2 (PAR2) on intestinal epithelial cells induced the expression of cyclooxygenase-2 (COX-2), which has protective functions in the gastrointestinal tract. It was hypothesized that PAR2-induced COX-2 could enhance wound healing in intestinal epithelial cells. Caco2 cells were used to model epithelial wound healing of circular wounds. Cellular proliferation was studied with a 5-ethynyl-2'-deoxyuridine assay, and migration was studied during wound healing in the absence of proliferation. Immunofluorescence was used to visualize E-cadherin and F-actin, and the cellular transcription profile during wound healing and PAR2 activation was explored with RNA sequencing. PAR2 activation inhibited Caco2 wound healing by reducing cell migration, independently of COX-2 activity. Interestingly, even though migration was reduced, proliferation was increased. When the actin dynamics and cell-cell junctions were investigated, PAR2 activation was found to induce actin cabling and prevent the internalization of E-cadherin. To further investigate the effect of PAR2 on transcriptionally dependent wound healing, RNA sequencing was performed. This analysis revealed that PAR2 activation, in the absence of wounding, induced a similar transcriptional profile compared with wounding alone. These findings represent a novel effect of PAR2 activation on the mechanisms of epithelial cell wound healing that could influence the resolution of intestinal inflammation.

Aberrant endocytosis leads to the loss of normal mitotic spindle orientation during epithelial glandular morphogenesis

Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. Glandular epithelial tissues comprise cysts and tubules that are formed from a polarized, single-epithelial cell layer surrounding a central, fluid-filled lumen. The pathways regulating key processes in epithelial tissue morphogenesis such as mitotic spindle formation are incompletely understood, but are important to investigate, as their dysregulation is a signature of epithelial tumors. Here, we describe a signaling axis that manifests in a defect in mitotic spindle orientation during epithelial growth and cystogenesis. We found that activation of the small GTPase ADP-ribosylation factor 6 (ARF6) results in the sustained internalization of cell-surface components such as the cMet receptor and the cell-adhesion molecule E-cadherin. The spindle orientation defect arising from elevated levels of ARF6-GTP required an increase in cMet endocytosis, but was independent of E-cadherin internalization or elevated extracellular signal-regulated kinase (ERK) activity resulting from internalized receptor signaling on endosomes. Misorientation of the mitotic spindle resulted in the development of epithelial cysts with structural abnormalities, the most conspicuous of which was the presence of multiple intercellular lumens. Abnormal mitotic spindle orientation was necessary but insufficient to disrupt glandular development, as blocking the strong prosurvival signal resulting from ERK hyperactivation yielded structurally normal cysts despite continued manifestation of spindle orientation defects. Our findings highlight a previously unknown link between ARF6 activation, cMet receptor internalization, and mitotic spindle orientation during epithelial glandular morphogenesis.

Annexin A2 overexpression associates with colorectal cancer invasiveness and TGF-\xdf induced epithelial mesenchymal transition via Src/ANXA2/STAT3

Annexin A2 (ANXA2) is upregulated in several malignancies, including colorectal cancer (CRC). However, there is little knowledge on the molecular mechanisms involved to its upregulation. The aim of this study was to identify the mechanism through which ANXA2 overexpression leads to CRC progression and evaluate its potential prognostic value. We used human CRC samples to analyse the correlation between ANXA2 levels and tumour staging. ANXA2 expression was increased in CRC tissues compared to normal colon tissues. In addition, we observe increased ANXA2 levels in stage IV tumours and metastasis, when compared to stage I-III. Whereas E-cadherin, an epithelial marker, decreased in stage II-IV and increased in metastasis. We’ve also shown that TGF-β, a classic EMT inductor, caused upregulation of ANXA2, and internalization of both E-cadherin and ANXA2 in CRC cells. ANXA2 silencing hindered TGF-β-induced invasiveness, and inhibitors of the Src/ANXA2/STAT3 pathway reversed the EMT. In silico analysis confirmed overexpression of ANXA2 and association to the consensus moleculars subtypes (CMS) with the worst prognosis. Therefore, ANXA2 overexpression play a pivotal role in CRC invasiveness through Src/ANXA2/STAT3 pathway activation. The association of ANXA2 to distinct CMSs suggests the possible use of ANXA2 as a prognostic marker or directed target therapy.

Epithelial-to-mesenchymal transition of A549 lung cancer cells exposed to electronic cigarettes

ObjectivesEpithelial-to-mesenchymal transition (EMT) is the initial step enabling the metastasis of cancer cells, which often leads to death. Although smoking is a major risk factor for lung cancer, there is still widespread use of conventional cigarettes. Recently, the tobacco industry has been transformed by the introduction of electronic cigarettes (ECs), which have lower levels of carcinogens and may provide a safer alternative. Here, we investigate the ability of EC liquids and aerosols to induce an EMT in A549 lung cancer cells. Materials and MethodsHuman adenocarcinoma alveolar basal epithelial cells (A549) were exposed to EC liquids and aerosols from a popular product for 3–8 days. Live cell imaging, EMT biomarker analysis, and machine learning/image processing algorithms were used to characterize changes associated with EMT. ResultsLong-term exposure of A549 cells to menthol or tobacco-flavored EC liquids or aerosols induced an EMT that was characterized by acquisition of a fibroblast-like morphology, loss of cell-to-cell junctions, internalization of E-cadherin, increased motility, and upregulation of other EMT markers. The EMT was concurrent with plasma membrane to nuclear translocation of active β-catenin. ConclusionThis is the first known study to show an EMT of lung cancer cells during exposure to EC products. Because an EMT is an initial step leading to metastasis, an intractable problem that often leads to patient death, this critical finding has significant implications for former or heavy cigarette smokers who are using EC and may be at risk for lung cancer or who may already have a lung tumor.

Abstract A84: Annexin A2 as a regulator of colorectal tumorigenesis: Influence over epithelial-mesenchymal transition and invasive abilities

Abstract Annexins are calcium-dependent phospholipid binding proteins that act on several cellular functions such as cytoskeleton organization, ion transport, and cell signalling. Due to its relation to the actin structure and cell proliferation; alterations in its expression have known implications in the progression of distinct cancer types. Among the proteins of the family, annexin A2 (AnxA2) is known for promoting cell proliferation, migration, and invasion when overexpressed. A recent study suggested AnxA2 as a diagnostic and prognostic biomarker for colorectal cancer. However, there is little evidence of its regulation and role in cell signalling pathways involved in colorectal cancer tumorigenesis. Our goal is to unravel the role of AnxA2 in the tumor progression and the signalling pathways involved. The total and phosphorylated (Y23 residue) AnxA2 levels were assessed in three human colorectal cancer cell lines (Caco-2, HT-29 e HCT-116), through imunoblotting. HT-29 cells, control or silenced (siRNA) for AnxA2, were submitted to TGF-β; treatment to induce characteristic epithelyal-mesenchymal transition (EMT) events. Proliferation, migration, and invasion were also assessed for these cells. Our results show that Caco-2 cells have the lowest phosphorylation level among the analyzed cells. In face TGF-β; treatment, the HT-29 cell line showed morphologic alterations characteristic to EMT, reduced E-cadherin protein levels, and increased vimentin expression. AnxA2 (total and phospho) expression was also enhanced. The loss of cell-cell contacts due to treatment was observed with the internalization of E-cadherin and AnxA2. The analysis of such internalization through Structured Illumination Microscopy (SIM) allowed us to observe intracellular colocalization between these two proteins. Proliferation assays reveal that TGF-β; has antiproliferative effects and in a less drastic form so does AnxA2 siRNA. AnxA2 silencing also reduced cell migration in Wound Healing assay, in a manner independent of TGF-β. The EMT induction through treatment also led to incresead invasion, which was inhibted with AnxA2 silencing or STA21 pretreatment (STAT3 selective inhibtor). Results suggest that AnxA2 has a role in cellular junction disassembly and enhanced invasion ability, characteristics of EMT process, and in cellular proliferation and migration. Citation Format: Murilo Ramos Rocha, Annie Cristhine Moraes Sousa Squiavinato, Pedro Barcellos de Souza, Jose Andres Morgado Diaz. Annexin A2 as a regulator of colorectal tumorigenesis: Influence over epithelial-mesenchymal transition and invasive abilities [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A84.

Role of JNK during buccopharyngeal membrane perforation, the last step of embryonic mouth formation.

The buccopharyngeal membrane is a thin layer of cells covering the embryonic mouth. The perforation of this structure creates an opening connecting the external and the digestive tube which is essential for oral cavity formation. In humans, persistence of the buccopharyngeal membrane can lead to orofacial defects such as choanal atresia, oral synechiaes, and cleft palate. Little is known about the causes of a persistent buccopharyngeal membrane and, importantly, how this structure ruptures. We have determined, using antisense and pharmacological approaches, that Xenopus embryos deficient c-Jun N-terminal kinase (JNK) signaling have a persistent buccopharyngeal membrane. JNK deficient embryos have decreased cell division and increased cellular stress and apoptosis. However, altering these processes independently of JNK did not affect buccopharyngeal membrane perforation. JNK deficient embryos also have increased intercellular adhesion and defects in e-cadherin localization. Conversely, embryos with overactive JNK have epidermal fragility, increased E-cadherin internalization, and increased membrane localized clathrin. In the buccopharyngeal membrane, clathrin is colocalized with active JNK. Furthermore, inhibition of endocytosis results in a persistent buccopharyngeal membrane, mimicking the JNK deficient phenotype. The results of this study suggest that JNK has a role in the disassembly adherens junctions by means of endocytosis that is required during buccopharyngeal membrane perforation. Developmental Dynamics 246:100-115, 2017. © 2016 Wiley Periodicals, Inc.

Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile.

ABSTRACTShrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.

Desmoglein 3 regulates membrane trafficking of cadherins, an implication in cell-cell adhesion

ABSTRACTE-cadherin mediated cell-cell adhesion plays a critical role in epithelial cell polarization and morphogenesis. Our recent studies suggest that the desmosomal cadherin, desmoglein 3 (Dsg3) cross talks with E-cadherin and regulates its adhesive function in differentiating keratinocytes. However, the underlying mechanism remains not fully elucidated. Since E-cadherin trafficking has been recognized to be a central determinant in cell-cell adhesion and homeostasis we hypothesize that Dsg3 may play a role in regulating E-cadherin trafficking and hence the cell-cell adhesion. Here we investigated this hypothesis in cells with loss of Dsg3 function through RNAi mediated Dsg3 knockdown or the stable expression of the truncated mutant Dsg3ΔC. Our results showed that loss of Dsg3 resulted in compromised cell-cell adhesion and reduction of adherens junction and desmosome protein expression as well as the cortical F-actin formation. As a consequence, cells failed to polarize but instead displayed aberrant cell flattening. Furthermore, retardation of E-cadherin internalization and recycling was consistently observed in these cells during the process of calcium induced junction assembling. In contrast, enhanced cadherin endocytosis was detected in cells with overexpression of Dsg3 compared to control cells. Importantly, this altered cadherin trafficking was found to be coincided with the reduced expression and activity of Rab proteins, including Rab5, Rab7 and Rab11 which are known to be involved in E-cadherin trafficking. Taken together, our findings suggest that Dsg3 functions as a key in cell-cell adhesion through at least a mechanism of regulating E-cadherin membrane trafficking.

Loss of ezrin in human intrahepatic cholangiocarcinoma is associated with ectopic expression of E-cadherin

Ezrin connects proteins from the plasma membrane to the subcortical cytoskeleton, and contributes to epithelial integrity by interacting with the cell-cell adhesion molecule E-cadherin. In the liver, ezrin is restricted to cholangiocytes, where it regulates biliary secretory functions. During carcinogenesis, ezrin expression is impaired and associated with enhancement of cell migratory activity in cancer cells; therefore, we aimed to analyse ezrin in cholangiocarcinogenesis. Ezrin expression was evaluated by immunohistochemistry on tissue microarrays from 94 surgical specimens of intrahepatic cholangiocarcinoma (CCA), and correlated with clinicopathological factors and E-cadherin expression. Ezrin function was also analysed in human CCA cell lines. In CCA, ezrin was negative/weakly expressed in 49 cases (52%) and moderately/strongly expressed in 45 cases (48%), mostly in cell cytoplasm. The negative/weak expression of ezrin was more frequent in peripheral than in perihilar CCA (P = 0.002), and was associated with high tumour size (P = 0.001), low mucus secretion (P = 0.042), the presence of satellite nodules (P = 0.024), and ectopic cytoplasmic expression of E-cadherin (P = 0.005). In vitro, silencing of ezrin in CCA cells caused internalization of E-cadherin and favoured cell migration. Ezrin is down-regulated during cholangiocarcinogenesis, and its loss results in a more aggressive phenotype.

Abstract P5-03-09: Eribulin affects E-cadherin localization consistent with a reversal of the epithelial-to-mesenchymal transition

Abstract Microtubule targeting agents (MTAs) are some of the most important compounds used to treat breast cancer. While both microtubule stabilizers and destabilizers have utility against breast cancers, not all patients respond and there are differences among the agents. Eribulin is a microtubule depolymerizer used to treat patients with locally advanced or metastatic breast cancerwho have previously received at least two chemotherapeutic regimens, including an anthracycline and a taxane. Our goal was to compare the effects of eribulin to four other clinically relevant MTAs on signaling processes important for cancer cell metastasis that are known to be regulated by the cytoskeleton. One process is the appropriate localization of E-cadherin, which plays a critical role in maintaining an epithelial phenotype by facilitating the ability of cells to form cell-cell contacts. These contacts help prevent cell motility and epithelial to mesenchymal transition (EMT) by sequestering several proteins involved in EMT at the plasma membrane, including β-catenin. E-cadherin is absent from the cell membrane in cells with a mesenchymal phenotype. We hypothesized that eribulin could affect the internalization of E-cadherin and promote its retention at the cell periphery and that it might do so differently than other MTAs because eribulin has been shown to reverse EMT in breast cancer cells and in xenograft models. Mesenchymal breast cancer HCC1937 cells were treated for 2 hr with MTAs using a concentration that caused a similar degree of microtubule disruption and localization of E-cadherin was evaluated. The microtubule destabilizers eribulin and vinorelbine promoted a localization of E-cadherin to the plasma membrane. In contrast, the microtubule stabilizers paclitaxel, docetaxel and ixabepilone caused E-cadherin to localize to internal structures. β-catenin, a protein bound by E-cadherin, also became enriched at the cell periphery after microtubule destabilizer treatment. In the more epithelial MCF-7 cell line, E-cadherin was constitutively localized at the cell periphery and the MTAs had no effect on this localization, consistent with the epithelial phenotype. Src family kinases (SFKs) are known to promote the internalization and degradation of E-cadherin and to promote EMT. Accordingly, the SFK inhibitor dasatinib caused effects similar to eribulin and vinorelbine by promoting the localization of E-cadherin to the plasma membrane in the mesenchymal HCC1937 cell line. Subsequent studies supported this result and demonstrated the increased activity of the combination of eribulin and dasatinib in HCC1937 cells. The ability of eribulin to rapidly induce changes in E-cadherin localization is consistent with its ability to reverse EMT. Studies are ongoing to evaluate whether eribulin's effects are mediated through the ability of SFKs to regulate E-cadherin. Our results show differences between microtubule stabilizers and destabilizers and are consistent with the previously shown ability of eribulin to reverse EMT. Funding for this work provided by Eisai Inc. Citation Format: Dybdal-Hargreaves NF, Rohena CC, Risinger AL, Mooberry SL. Eribulin affects E-cadherin localization consistent with a reversal of the epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-09.