Maintenance Of Cell-cell Adhesion Research Articles

αE-Catenin Is a Positive Regulator of Pancreatic Islet Cell Lineage Differentiation

SUMMARYThe development and function of epithelia depend on the establishment and maintenance of cell-cell adhesion and intercellular junctions, which operate as mechanosensor hubs for the transduction of biochemical signals regulating cell proliferation, differentiation, survival, and regeneration. Here, we show that αE-catenin, a key component of adherens junctions, functions as a positive regulator of pancreatic islet cell lineage differentiation by repressing the sonic hedgehog pathway (SHH). Thus, deletion of αE-catenin in multipotent pancreatic progenitors resulted in (1) loss of adherens junctions, (2) constitutive activation of SHH, (3) decrease in islet cell lineage differentiation, and (4) accumulation of immature Sox9+ progenitors. Pharmacological blockade of SHH signaling in pancreatic organ cultures and in vivo rescued this defect, allowing αE-catenin-null Sox9+ pancreatic progenitors to differentiate into endocrine cells. The results uncover crucial functions of αE-catenin in pancreatic islet development and harbor significant implications for the design of β cell replacement and regeneration therapies in diabetes.

Lipopolysaccharide-induced α-catenin downregulation enhances the motility of human colorectal cancer cells in an NF-κB signaling-dependent manner

α-Catenin is an important molecule involved in the maintenance of cell–cell adhesion and a prognostic marker in cancer since its expression is essential for preventing cancer metastasis. However, the mechanism that leads to the downregulation of α-catenin in cancer progression remains unclear. The present study revealed that lipopolysaccharide (LPS)-induced NF-κB signaling activation suppressed α-catenin expression and motility in SW620 colorectal cancer (CRC) cells, using real-time polymerase chain reaction, Western blotting, and transwell migration assays. LPS treatment reduced both the mRNA and protein expression of α-catenin and thereby enhanced cell motility. Conversely, incubating cells with an NF-κB inhibitor disrupted these effects. Furthermore, the ectopic expression of p65 alone mimicked the effects of LPS stimulation. In CRC tissues, the presence of enteric bacterial LPS-related neutrophil-enriched foci was correlated with α-catenin downregulation. Collectively, these findings suggest that LPS-induced NF-κB signaling is related to α-catenin suppression and enhanced cell motility in CRC. Therefore, NF-κB is a novel potential therapeutic target for CRC metastasis.

Abstract 4988: Clues for targeted therapies in inflammatory breast cancer (IBC)

Abstract Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development, and poor prognosis. The peculiar clinical presentation of IBC characterized by breast erythema and swelling is caused by the invasion of aggregates of tumor cells (tumor emboli) into the dermal lymphatics, causing an obstruction of the lymph channels. Although, tumor emboli are also occasionally demonstrated in non-IBC tumors, they are more frequently detected and more numerous in IBC. Tumor emboli expressed cell-cell adhesion molecules that maintain the tumor cells together. The high risk of disease recurrence in soft tissue and lymph nodes shortly after completion of multidisciplinary treatment particularly in patients with residual disease supports the hypothesis that these tumor emboli are critically related to the development of micro metastatic disease. We developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters in suspension that were strongly positive for E-cadherin, β-catenin, and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. The maintenance of cell-cell adhesions allow the migration of tumor cells through the lymphatic and blood vessels as clusters. FC-IBC02 cells shown a partial or incomplete epithelial-mesenchymal transition (EMT); these cells express some epithelial markers (EpCAM, E-cadherin) and also mesenchymal markers (VIM, FN1, Snai2, Twist1). Furthermore, circulating tumor cells (CTC) from IBC patients are present in the blood as single cells and clusters supporting the collective migration of tumor cells in IBC. FC-IBC02 cells were highly metastatic when injected in the fat mammary pad of SCID mice and these cells were able to produce brain metastasis in mice by intracardiac or intra-peritoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5-7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6-4 copies in IBC cells. FAK1 is amplified, upregulated and phosphorylated (active) in inflammatory breast cancer. Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. Our results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC. We are currently evaluating an ALK/FAK inhibitor using the novel established IBC model. Citation Format: Sandra V. Fernandez, Fredika M. Robertson, Sankar Addya, Zhaomei Mu, Massimo Cristofanilli. Clues for targeted therapies in inflammatory breast cancer (IBC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4988. doi:10.1158/1538-7445.AM2014-4988

Changes in regulation of cell—cell adhesion during tumor transformation

Cadherin-mediated cell-cell adhesion defines the integrity of most tissues. Cell-cell adherens junctions are dynamic structures whose functional state is regulated by interactions of cadherin with beta-catenin, p120, and actin cytoskeleton structures. Small GTPases of the Rho family and GTPase Rap1 play the central role in the formation and maintenance of cell-cell adhesion. Aberrant activation of signaling pathways, transcriptional repression of the E-cadherin gene, ectopic expression of N-cadherin, and disturbances in regulation of adhesive and transcriptional functions of beta-catenin stimulate tumor progression.

E-cadherin but not β-catenin expression is decreased in laryngeal biopsies from patients with laryngopharyngeal reflux

Abnormal exposure of acid refluxate on the esophageal mucosa has been shown to decrease the epithelial barrier function through an alteration in the intercellular junctional complex. However, only few studies have examined the molecular effects caused by abnormal exposure of gastric refluxate on the laryngeal epithelium. E-cadherin and beta-catenin are cell membrane-associated proteins playing a major role in the maintenance of cell-cell adhesion in epithelial tissues. In this study we tried to analyse the molecular effect of laryngopharyngeal reflux (LPR) on the cellular expression of these proteins. Therefore, we compared the expression of E-caherin and beta-catenin in laryngeal biopsies from patients with and without pH-documented laryngopharyngeal reflux. Paraffin-embedded archival laryngeal biopsies taken from 21 patients, who had undergone rigid laryngoscopy under general anaesthesia and postoperative 24-h pH monitoring, were evaluated immunohistochemically with antibodies to E-cadherin and beta-catenin. The membrane expression of the two proteins was categorized in no expression, mild, moderate and strong (grade 0-3). In LPR patients (n=14) the mean grade of E-cadherin and beta-catenin expression was 1.57 and 1.21, while in specimens of patients without pH-documented LPR it was 2.57 and 1.29. The difference in E-cadherin expression was statistically significant (P=0.011). From our findings we conclude that LPR can cause a decrease in the laryngeal expression of E-cadherin but not of beta-catenin. The reduction of E-cadherin-mediated adhesion could contribute to the development of laryngeal neoplasms. E-cadherin immunostaining of laryngeal biopsies could be a further diagnostic tool to confirm the diagnosis in patients with suspected LPR.

Increased Internalization of p120-uncoupled E-cadherin and a Requirement for a Dileucine Motif in the Cytoplasmic Domain for Endocytosis of the Protein

E-cadherin is a member of the cadherin family of Ca2+-dependent cell-cell adhesion molecules. E-cadherin associates with beta-catenin at the membrane-distal region of its cytosolic domain and with p120 at the membrane-proximal region of its cytoplasmic domain. It has been shown that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. Further, p120 knockdown by small interference RNA resulted in dose-dependent elimination of cell surface E-cadherin. Consistent with these observations, we found that selective uncoupling of p120 from E-cadherin by introduction of amino acid substitutions in the p120-binding site increased the level of E-cadherin endocytosis. The increased endocytosis was clathrin-dependent, because it was blocked by expression of a dominant-negative form of dynamin or by hypertonic shock. A dileucine motif in the juxtamembrane cytoplasmic domain is required for E-cadherin endocytosis, because substitution of these residues to alanine resulted in impaired internalization of the protein. The alanine substitutions in the p120-uncoupled construct reduced endocytosis of the protein, indicating that this motif was dominant to p120 binding in the control of E-cadherin endocytosis. Therefore, these results are consistent with the idea that p120 regulates E-cadherin endocytosis by masking the dileucine motif and preventing interactions with adaptor proteins required for internalization.

Change of E-Cadherin by Hepatocyte Growth Factor and Effects on the Prognosis of Hypopharyngeal Carcinoma

Hepatocyte growth factor (HGF) is known to induce scattering in various epithelial cells, and E-cadherin plays important roles in the maintenance of cell-cell adhesion. However, the mechanisms surrounding these actions are not fully understood. Therefore, we examined how HGF affects the expression and distribution of E-cadherin. In addition, we observed the relationship between prognosis and modulation of E-cadherin by HGF in hypopharyngeal carcinoma. Tumor tissues from 66 patients with hypopharyngeal squamous cell carcinoma were evaluated for the expression of HGF, its receptor (c-Met), and E-cadherin. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot test were performed on hypopharyngeal cancer tissues. The association and changes of E-cadherin with HGF treatment in a hypopharyngeal cancer cell line were investigated by RT-PCR, Western blot analysis, inhibition assay, immunofluorescence staining, and invasion assay. E-cadherin expression was found in 87.9% of squamous cell carcinomas; these could be further classified as membranous type (46.9%) or nonmembranous type (53.1%). The expression of HGF in tumors with nonmembranous type E-cadherin expression was far higher than in tumors with membranous expression. Nonmembranous type E-cadherin expression correlated significantly with lymph node metastasis, distant metastasis, and recurrence (P < .05). HGF decreased the expression of E-cadherin and induced the translocation of E-cadherin to the cytoplasm. HGF and E-cadherin neutralizing antibody stimulated dispersion, and HGF significantly enhanced the invasion of hypopharyngeal cancer cells in a dose-dependent manner (P < .05). These results suggest that HGF can modulate the expression and intracellular localization of E-cadherin in hypopharyngeal cancer cells. In addition, these results indicate that changes in E-cadherin by HGF can affect the prognosis of hypopharyngeal carcinoma.

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.

E‐cadherin promoter polymorphism (C‐160A) and risk of recurrence in patients with superficial bladder cancer

Tumor recurrence is a hallmark of superficial bladder cancer. Currently, a molecular marker for bladder cancer recurrence is lacking. E-cadherin plays an important role in epithelial development and in the establishment and maintenance of cell-cell adhesion and tissue architecture. The purpose of this study is to investigate the association of an E-cadherin promoter polymorphism (CDH1c-160a) with the risk of bladder cancer recurrence. This study included 302 patients with superficial bladder cancer. Genomic DNA was extracted from peripheral blood lymphocytes and genotyping was performed using Taqman assay. Clinical data were collected by medical chart review. Cox proportional hazard model was used to estimate the hazard ratios (HRs) associated with genotypes while adjusting for age, gender, smoking status, tumor stage and grade where appropriate. During a median follow-up of 27.65 months, 151 patients experienced disease recurrence. Subsequent analyses were restricted to Caucasians only due to the small sample size of other ethnic groups (13 in recurrence group and 15 in non-recurrence group). Among the 274 Caucasian patients, 138 developed recurrence during the same length of follow-up time. In Caucasian patients, having at least one variant A allele conferred a 32% reduction in recurrence risk (adjusted HR: 0.68; 95% CI: 0.48-0.96). The median recurrence-free survival for patients carrying at least one variant A allele was significantly longer than that for patients with a homozygous CC genotype (40.4 vs 12.5 months, p=0.04). Our findings suggest that the E-cadherin promoter polymorphism may be a valuable molecular marker for bladder cancer recurrence.

Involvement of protein phosphatase 2A in the maintenance of E‐cadherin‐mediated cell–cell adhesion through recruitment of IQGAP1

Serine/threonine protein phosphatase (PP) 2A regulates many biological processes, however it remains unclear whether PP2A participates in cadherin-mediated cell-cell adhesion. We show here that the core enzyme of PP2A (PP2A-AC) is localized in the cell-cell adhesion sites between adjacent cells and associated with the E-cadherin-catenins complex in non-malignant human mammary epithelial (HME) cells at confluence. Treatment of the cells with either okadaic acid (OA), an inhibitor of PP2A, or siRNA for the regulatory subunit A of PP2A (PP2A-A) caused disruption of cell-cell adhesion and F-actin assembly, without affecting the complex formation of E-cadherin with beta- and alpha-catenins. While a small GTPase Rac and its effector IQGAP1 were associated with the E-cadherin-catenins complex, either OA or PP2A-A siRNA concomitantly induced the dissociation of IQGAP1, but not Rac, from the complex and the internalization of E-cadherin from the cell surface. We therefore propose that PP2A plays a crucial role in the maintenance of cell-cell adhesion through recruitment of IQGAP1 to the Rac-bound E-cadherin-catenins complex.

Cdc42 Effector Protein 2 (XCEP2) is required for normal gastrulation and contributes to cellular adhesion in Xenopus laevis.

BackgroundRho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis.ResultsIn a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis, a gene encoding a Rho GTPase effector protein (Xenopus Cdc42 effector protein 2, or XCEP2) was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell-cell adhesion in early embryos. This loss of cell adhesion could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA.ConclusionsXCEP2 appears to be an essential component in the early developmental program in Xenopus laevis. XCEP2 is involved in maintenance of cell-cell adhesion, and as such may constitute a regulatory component that could help to balance the need for tissue integrity and plasticity during the dynamic cellular rearrangements of gastrulation.

α-Catenin expression is decreased in human gastric cancers and in the gastric mucosa of first degree relatives

BACKGROUND AND AIMSThe role of altered cell adhesion is critical for the development of epithelial cancers. E-cadherin plays an important role in the maintenance of cell-cell adhesion and its function...

Expression of E-cadherin adhesion molecule in vocal cord carcinomas.

E-cadherin is a 120 kDa transmembrane protein that plays a major role in the maintenance of cell-cell adhesion in epithelial tissues. In the present study its expression was examined immunohistochemically for the first time using paraffin-embedded archival tissues of 38 vocal cord carcinomas. Ten cases of vocal cord polyps were used as positive controls. According to our criteria, results showed that all but one of the polyps, in which staining was lost, had strong or moderate E-cadherin expression, whereas 32% of the cases with tumors showed preserved staining, and 68% of cases showed a reduced staining. E-cadherin expression was significantly correlated with histological stages, with a greater expression in carcinoma in situ and microinvasive carcinoma than in invasive carcinoma (P = 0.010). A significant correlation was also found between E-cadherin expression and the degree of tumor differentiation (P = 0.018). Some of these findings were consistent with previously published data using fresh tissues. Our study provides evidence that E-cadherin may be linked to progression and differentiation of laryngeal cancer and could play a role as a tumor-suppressor gene in this cancer.

Immunohistochemical evaluation of alpha-catenin expression in human gastric cancer.

E-cadherin (E-cad) plays a major role in the maintenance of cell-cell adhesion in epithelial tissues, and impaired E-cad expression correlates with tumour invasion and metastasis. Alpha-catenin (alpha-cat), an undercoat protein of adherens junctions, binds to the cytoplasmic domain of E-cad and is essential for linking E-cad to actin-based cytoskeleton. We investigated E-cad and alpha-cat expression in 60 human gastric cancers immunohistochemically. The 60 gastric cancers were classified into 18 (30%) in which alpha-cat expression was preserved, and 42 (70%) reduced cases. The reduction of alpha-cat expression was significantly related to dedifferentiation, depth of invasion, infiltrative growth and lymph node metastasis. We also examined the co-expression of alpha-cat and E-cad. Seventeen (28%) tumours preserved both molecules [alpha-cat(+)/E-cad(+)] and 33 (55%) tumours reduced both [alpha-cat(-)/E-cad(-)], whereas 9 (15%) tumours exhibited alpha-cat(-)/E-cad(+). The frequency of lymph node metastasis in alpha-cat(-)/E-cad(+) tumour (67%) was significantly higher than that in alpha-cat(+)/E-cad(+) tumours (24%) and was close to that in alpha-cat(-)/E-cad(-) tumours (82%). The frequency of haematogenous liver metastasis in alpha-cat(-)/E-cad(+) tumours (44%) was significantly higher than that in alpha-cat(+)/E-cad(+) tumours (6%) or alpha-cat(-)/E-cad(-) tumours (9%). Thus, in all E-cad(+) tumours, the frequency of lymph node and liver metastasis was higher in alpha-cat(-) tumours than in alpha-cat(+) tumours. alpha-Cat expression is apparently better at predicting tumour invasion and metastasis than E-cad expression.