Loss Of Intercellular Adhesion Research Articles

Translational Aspects in Metaplastic Breast Carcinoma.

Breast cancer is the most common cancer among women. Metaplastic breast carcinoma (MpBC) is a rare, heterogeneous group of invasive breast carcinomas, which are classified as predominantly triple-negative breast carcinomas (TNBCs; HR-negative/HER2-negative). Histologically, MpBC is classified into six subtypes. Two of these are considered low-grade and the others are high-grade. MpBCs seem to be more aggressive, less responsive to neoadjuvant chemotherapy, and have higher rates of chemoresistance than other TNBCs. MpBCs have a lower survival rate than expected for TNBCs. MpBC treatment represents a challenge, leading to a thorough exploration of the tumor immune microenvironment, which has recently opened the possibility of new therapeutic strategies. The epithelial-mesenchymal transition in MpBC is characterized by the loss of intercellular adhesion, downregulation of epithelial markers, underexpression of genes with biological epithelial functions, upregulation of mesenchymal markers, overexpression of genes with biological mesenchymal functions, acquisition of fibroblast-like (spindle) morphology, cytoskeleton reorganization, increased motility, invasiveness, and metastatic capabilities. This article reviews and summarizes the current knowledge and translational aspects of MpBC.

Human organoids are superior to cell culture models for intestinal barrier research.

Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death.

Plakophilin 2 regulates intestinal barrier function by modulating protein kinase C activity in vitro

ABSTRACT Previous data provided evidence for a critical role of desmosomes to stabilize intestinal epithelial barrier (IEB) function. These studies suggest that desmosomes not only contribute to intercellular adhesion but also play a role as signaling hubs. The contribution of desmosomal plaque proteins plakophilins (PKP) in the intestinal epithelium remains unexplored. The intestinal expression of PKP2 and PKP3 was verified in human gut specimens, human intestinal organoids as well as in Caco2 cells whereas PKP1 was not detected. Knock-down of PKP2 using siRNA in Caco2 cells resulted in loss of intercellular adhesion and attenuated epithelial barrier. This was paralleled by changes of the whole desmosomal complex, including loss of desmoglein2, desmocollin2, plakoglobin and desmoplakin. In addition, tight junction proteins claudin1 and claudin4 were reduced following the loss of PKP2. Interestingly, siRNA-induced loss of PKP3 did not change intercellular adhesion and barrier function in Caco2 cells, while siRNA-induced loss of both PKP2 and PKP3 augmented the changes observed for reduced PKP2 alone. Moreover, loss of PKP2 and PKP2/3, but not PKP3, resulted in reduced activity levels of protein kinase C (PKC). Restoration of PKC activity using Phorbol 12-myristate 13-acetate (PMA) rescued loss of intestinal barrier function and attenuated the reduced expression patterns of claudin1 and claudin4. Immunostaining, proximity ligation assays and co-immunoprecipitation revealed a direct interaction between PKP2 and PKC. In summary, our in vitro data suggest that PKP2 plays a critical role for intestinal barrier function by providing a signaling hub for PKC-mediated expression of tight junction proteins claudin1 and claudin4.

Dsg2 Upregulation as a Rescue Mechanism in Pemphigus.

In pemphigus vulgaris (PV), autoantibodies directed against the desmosomal cadherin desmoglein (Dsg) 3 cause loss of intercellular adhesion. It is known that Dsg3 interactions are directly inhibited by autoantibody binding and that Dsg2 is upregulated in epidermis of PV patients. Here, we investigated whether heterophilic Dsg2-Dsg3 interactions occur and would modulate PV pathogenesis. Dsg2 was upregulated in PV patients’ biopsies and in a human ex vivo pemphigus skin model. Immunoprecipitation and cell-free atomic force microscopy (AFM) experiments demonstrated heterophilic Dsg2-Dsg3 interactions. Similarly, in Dsg3-deficient keratinocytes with severely disturbed intercellular adhesion Dsg2 was upregulated in the desmosome containing fraction. AFM revealed that Dsg2-Dsg3 heterophilic interactions showed binding frequency, strength, Ca2+-dependency and catch-bond behavior comparable to homophilic Dsg3-Dsg3 or homophilic Dsg2-Dsg2 interactions. However, heterophilic Dsg2-Dsg3 interactions had a longer lifetime compared to homophilic Dsg2-Dsg2 interactions and PV autoantibody-induced direct inhibition was significantly less pronounced for heterophilic Dsg2-Dsg3 interactions compared to homophilic Dsg3 interactions. In contrast, a monoclonal anti-Dsg2 inhibitory antibody reduced heterophilic Dsg2-Dsg3 and homophilic Dsg2-Dsg2 binding to the same degree and further impaired intercellular adhesion in Dsg3-deficient keratinocytes. Taken together, the data demonstrate that Dsg2 undergoes heterophilic interactions with Dsg3, which may attenuate autoantibody-induced loss of keratinocyte adhesion in pemphigus.

The role of cell adhesion molecule in cancer progression and its application in cancer therapy.

Multiple and diverse cell adhesion molecules take part in intercellular and cell-extracellular matrix interactions of cancer. Cancer progression is a multi-step process in which some adhesion molecules play a pivotal role in the development of recurrent, invasive, and distant metastasis. A growing body of evidence indicates that alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of cancer. Loss of intercellular adhesion and the desquamation of cells from the underlying lamina propria allows malignant cells to escape from their site of origin, degrade the extracellular matrix, acquire a more motile and invasion phenotype, and finally, invade and metastasize. In addition to participating in tumor invasiveness and metastasis, adhesion molecules regulate or significantly contribute to a variety of functions including signal transduction, cell growth, differentiation, site-specific gene expression, morphogenesis, immunologic function, cell motility, wound healing, and inflammation. Cell adhesion molecule (CAM), a diverse system of transmembrane glycoproteins has been identified that mediates the cell-cell and cell-extracellular matrix adhesion and also serves as the receptor for different kinds of virus. We summarize recent progress regarding the role of CAM, particularly, immunoglobulin-CAMs and cadherins in the progression of cancer and discuss the potential application of CAMs in the development of cancer therapy mainly on urogenital cancer.

Nfatc1 Promotes Interstitial Cell Formation During Cardiac Valve Development in Zebrafish.

The transcription factor NFATC1 (nuclear factor of activated T-cell 1) has been implicated in cardiac valve formation in humans and mice, but we know little about the underlying mechanisms. To gain mechanistic understanding of cardiac valve formation at single-cell resolution and insights into the role of NFATC1 in this process, we used the zebrafish model as it offers unique attributes for live imaging and facile genetics. To understand the role of Nfatc1 in cardiac valve formation. Using the zebrafish atrioventricular valve, we focus on the valve interstitial cells (VICs), which confer biomechanical strength to the cardiac valve leaflets. We find that initially atrioventricular endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the ECM (extracellular matrix) between the 2 endocardial cell monolayers, undergo endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a promigratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. With high-speed microscopy and echocardiography, we show that reduced VIC formation correlates with valvular dysfunction and severe retrograde blood flow that persist into adulthood. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b-a well-known regulator of epithelial-to-mesenchymal transition. Our study sheds light on the function of Nfatc1 in zebrafish cardiac valve development and reveals its role in VIC formation. It also further establishes the zebrafish as a powerful model to carry out longitudinal studies of valve formation and function.

Intercellular adhesion in testicular germ cell tumors

Testicular germ cell tumors (TGCT) is one of the main causes of oncological mortality in young men. One of the factors determining the prognosis of cancer patients is the determination of molecular biological markers which correlate with a poor prognosis. The purpose of the work. To determine the state of intercellular adhesion in testicular postpubertal-type yolk sac tumor (YSTPT), postpubertal-type teratoma (TPT), spermatocytic tumor (ST) and teratoma with somatic-type malignancy (TSTM). Material and methods. The study was performed on the material of TGCT of patients who have been examined and treated at the Kharkiv Regional Clinical Center of Urology and Nephrology named after V.I Shapoval for the period covering 1998-2017. Groups of investigation were formed according to the pTNM classification of WHO. For assessment of the state of intercellular adhesion in TGCT the relative area (S) and intensity (L) of E-cadherin and β-catenin expression were studied. Results. During the tumorous progression S of E-cadherin expression in YSTPT and TPT was gradually decreasing right up to the complete reduction of this marker expression. These changes were combined with increasing of β-catenin expression. In the case of positive immunohistochemical reaction with E-cadherin and β-catenin in all investigated TGCT instead of the «typical» membrane localization there were «heterogeneities» which were also considered as a loss of intercellular adhesion. In ST the S and L of E-cadherin expression were higher than those in the corresponding groups of investigated TGCT but marker localization was also atypical. As for β-catenin, S and L of its expression were at the level of mean values in YSTPT and higher than analogous indexes in the corresponding group of TPT. Localization of β-catenin was atypical. In TSTM of both groups the reaction with E-cadherin was either negative or marker had an atypical localization. The same situation was observed at investigation of β-catenin. Conclusion. In YSTPT and TPT at tumorous progression a loss of intercellular adhesion occurs what leads to the dissociation of tumor cells, development of vascular invasion and metastatic disease.

Abstract A47: Reducing actin contraction in E-cadherin inactivated mammary epithelial cells leads to the development of invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of ILC is the loss of intercellular adhesion, which occurs as a consequence of inactivation of E-cadherin (CDH1). Inactivation of E-cadherin often occurs through truncating mutations followed by loss of heterozygosity. E-cadherin is a master regulator of cell-cell interactions and thereby functions as an important tumor suppressor. Nevertheless, loss of E-cadherin by itself does not induce mammary tumor formation in mice. Moreover, somatic inactivation of E-cadherin in mouse mammary epithelium resulted in outgrowth mammary ducts with only E-cadherin proficient mammary epithelial cells, suggesting that loss of E-cadherin by itself is not tolerated. Why the inactivation of E-cadherin is not tolerated in mammary epithelial cells remains unclear. To uncover the fate of mammary epithelial cells upon inactivation of E-cadherin, a WapCre;Cdh1F/F;mTmg mouse model was generated. The introduction of the mTmG reporter allows for the identification and tracking of Cre-driven E-cadherin inactivated mammary epithelial cells. Analysis of the mammary ducts of these mice revealed that at least a portion of E-cadherin inactivated mammary epithelial cells survive. At 12 weeks of age clusters of E-cadherin inactivated mammary epithelial cells were found nested between the myoepithelial cells and the basal membrane. Despite the fact that they survive, these cells appear dormant since these clusters do not increase in size over time. Using in vivo imaging of WapCre;Cdh1F/F;mTmg mice, it becomes clear that the E-cadherin inactivated cells are motile and show a high degree of membrane blebbing. The E-cadherin inactivated cells have increased cortical accumulation of MYH9 and myosin light chain phosphorylation, which indicate increased actin contraction. Recently we identified PPP1R12A/B, TP53BP2, and MYH9 as drivers of ILC in an in vivo insertional mutagenesis screen. The identified drivers did not have any effect on the PI3 kinase and MAP kinase pathways nor did they alter P53 activation. They did, however, allow survival of E-cadherin inactivated mammary cells in vitro and in vivo. PPP1R12A/B and MYH9 are primarily known for their critical role in actin contractility. Interestingly, overexpression of the TRP53BP2 truncation variant identified in the screen reduced myosin light chain phosphorylation in a similar manner as the PPP1R12A truncation variant. To provide more evidence that the reduction of actin contraction allows rescue of E-cadherin inactivated cells, we inhibited actin contraction pharmacologically. Both exposure to a Rho kinase inhibitor as well as blebbistatin (inhibits non-muscle myosin ATPase activity) allowed for the growth of E-cadherin inactivated primary epithelial cells. Overall our data suggest that the inactivation of E-cadherin in mammary epithelial cells leads to aberrant actin contraction preventing oncogenic transformation. By reducing actin contraction, it allows for the many benefits of losing E-cadherin to drive the development of ILC. We are currently investigating through which signaling routes the increase in actin contraction prevents growth. Citation Format: Koen Schipper, Micha Nethe, Sjors M. Kas, Julian R. de Ruiter, Anne Paulien Drenth, Eline van der Burg, Ute Boon, Eva Schut, Jos Jonkers. Reducing actin contraction in E-cadherin inactivated mammary epithelial cells leads to the development of invasive lobular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A47.

Clinical and molecular implications of structural changes to desmosomes and corneodesmosomes.

Desmosomes provide the main intercellular adhesive properties between epidermal keratinocytes. Their distribution becomes uneven in severe dermatitis, multiple allergies and metabolic wasting syndrome due to desmoglein 1 deficiency and the loss of intercellular adhesion or acantholysis. When keratinocytes differentiate from granular cells into cornified cells, desmosomes are transformed into corneodesmosomes and can provide stronger intercellular adhesion. Degradation of corneodesmosomes is a tightly regulated process involving a number of proteases and their inhibitors. Peripheral corneodesmosomes are protected from proteolytic degradation by the tight junction-related structures around them, and this construction provides the basis for the normal basket weave-like structure of the stratum corneum. In Netherton syndrome, which is caused by an absence of the protease inhibitor lymphoepithelial Kazal-type-related inhibitor, premature degradation of corneodesmosomes occurs due to the overactivation of proteases involved in corneodesmosome degradation. Inflammatory peeling skin disease is caused by the absence of corneodesmosin, a unique component of corneodesmosomes. In this disease, corneodesmosomes are structurally abnormal, and their adhesiveness is compromised, which leads to intercellular splitting between the stratum corneum and stratum granulosum. The better we understand desmosome and corneodesmosome ultrastructure in normal and diseased skin, the clearer the physiological and pathological mechanisms of epidermal integrity become.

Abstract 846: A novel high-throughput 3D screening system for EMT inhibitors

Abstract EMT (epithelial-mesenchymal transition) is a significant event in tumor metastasis and malignancy. Therefore, inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we introduce an innovative three-dimensional (3D) high-throughput (HTS) system that leads to an identification of EMT inhibitors. At first, we established 3D-HTS EMT model with NanoCulture Plate (NCP) that provided a gel-free micro-patterned scaffold for cells forming spheroids naturally. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs. Citation Format: Kazuya Arai, M Mamunur Rahman, Manabu Itoh, Norio Masuda, Takanori Eguchi, Tetsuya Nakatsura, Stuart K. Calderwood. A novel high-throughput 3D screening system for EMT inhibitors [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 846. doi:10.1158/1538-7445.AM2017-846

A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.

Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 μM and 1.21 μM, respectively, while IC50 of SB431542 was 2.38 μM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

Abstract 673: The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of this type of cancer is the loss of intercellular adhesion which is facilitated by inactivating mutations in E-cadherin. However the loss of E-cadherin alone is not enough to induce ILC. An in vivo Sleeping Beauty insertional mutagenesis screen was performed to identify genes that together with E-cadherin loss contribute to ILC development in mice. In around 85% of the tumors that were analyzed during this screen one of four hits were observed, namely MYPT1, MYPT2, ASPP2 and MYH9. Interestingly these four hits appear to be mutually exclusive indicating a shared mechanism of action. To investigate how these genes might affect tumorigenesis we first looked at the location of the transposon insertions. This revealed that for MYPT1, MYPT2 and ASPP2 the transposons appeared to localize to a specific region in the gene indicating that these insertions could result in a truncation variant. In the case of MYH9 no clear localization of insertions was found which in combination with the recently published data indicates a loss of function. Northern blot analysis revealed the presence of truncation variants for MYPT1/2 and ASPP2 which for MYPT1 were confirmed to also result in truncated protein variants. In order to identify the tumorigenic potential of truncated MYPT1 and ASPP2 we overexpressed them in spontaneously immortalized mammary epithelial cell lines HC11 and NMuMG. We are using these cell lines to analyse differences in cell proliferation, anchorage independent growth, sensitivity to apoptosis in vitro and tumor formation in vivo. To further analyse the hits in an E-cadherin negative setting we made use of primary mammary epithelial cells (MECs) isolated from genetically engineered mice with mammary gland specific E-cadherin loss. These MECs normally do not grow past several passages in vitro. However, these MECs when we overexpress truncated MYPT1 or ASPP2, or use shRNA mediated knockdown of MYH9 can be passaged and grown in vitro consistently. Finally we are generating conditional mouse models with mammary gland specific E-cadherin loss in combination of expression of the truncated variants of MYPT1 or ASPP2, or loss of MYH9. We will to monitor whether these mice are prone to developing ILC. Citation Format: Koen Schipper, Julian de Ruiter, Sjors Kas, Eva Schut, Marco Koudijs, David Addams, Jos Jonkers. The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 673.

Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma.

In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC.

Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing.

Twist expression in dentigerous cyst, odontogenic keratocyst, and ameloblastoma.

Epithelial-mesenchymal transition (EMT) is a process which is associated with a loss of intercellular adhesion, acquired mesenchymal shape, and increased motility by epithelial cells. Twist is one of the key regulators of EMT.In view of the distinct clinical behavior of odontogenic lesions, the objective of the present study was to investigate the immunohistochemical expression of Twist in these lesions. In this study, 70 formalin-fixed, paraffin-embedded tissue blocks of odontogenic lesion consisting of 16 unicystic ameloblastomas (UA), 17 solid ameloblastomas (SA), 18 odontogenic keratocysts (OKC), and 19 dentigerous cysts (DC) were reviewed using immunohistochemistry for Twist staining. In this study, Twist immunostaining was evident in all groups of the specimens except the dentigerous cyst group. Twist expression was seen in 58.8% (10/17) of SA, 50% (8/16) of UA, and 44.4% (8/18) of OKCs. 23.5% of SA, 18.8% of UA, and 16.7% of OKCs showed Twist expression in more than 50% of cells. Statistical analysis showed that Twist expression levels were significantly higher in ameloblastomas (SA and UA) and OKCs than dentigerous cysts (P = 0.002). There were no significant differences between Twist expression in SAs, UAs, and OKCs (P > 0.05). The results of this study propose that the high expression rate of Twist plays a role in the pathogenesis of ameloblastomas and OKCs and might be one of the reasons for the aggressive behavior of ameloblastomas and high recurrence of OKCs and could reinforce the classification of OKC as an odontogenic tumor.

Investigating the roles of phenylpropanoids in the growth and development of Zea mays L.

The Poaceae includes some of the most important food, fiber, and bio-fuel crops. While there have been many studies investigating the function of phenylpropanoids in this family, most of our understanding is based on correlative data rather than experimental evidence. The current study was conducted to evaluate the roles of phenylpropanoids in the growth and development of Zea mays and to develop an experimental model for further investigations. Z. mays seedlings were grown in vitro with various concentrations of the competitive phenylalanine ammonia lyase inhibitor, 2-aminoindane-2-phosphonic acid (AIP). Ferulic acid, a downstream biosynthetic product, was added to determine if it could rescue the induced phenotypes. At lower concentrations of AIP, plants exhibited elongated roots and shoots, but at higher concentrations, growth was extremely stunted. At the cellular level, the epidermal cells of roots cultured with AIP exhibited a loss of intercellular adhesion and organization, and their cell walls were more readily degraded by enzymatic digestion. These characteristics were accompanied by significant reductions in primary cell wall autofluorescence, indicating that less ferulic acid and other phenolics were incorporated in the cell wall. The majority of these symptoms could be partially or entirely rescued by ferulic acid, providing further evidence that these differences were due to the inhibition of phenylpropanoid biosynthesis. This study provides experimental evidence supporting and expanding upon hypothesized functions of phenylpropanoids in the growth and development of Z. mays and provides an experimental system for further investigations in the Poaceae and other taxonomic groups.

Integrin-Linked Kinase (ILK) Expression Correlates with Tumor Severity in Clear Cell Renal Carcinoma

Integrin-linked kinase (ILK) is an unique intracellular serine/threonine kinase and adapter protein. When dysregulated, it has been associated with increased cell proliferation, anchorage-independent cell growth, evasion of apoptosis, angiogenesis, invasion of surrounding tissues, downregulation of E-cadherin expression, nuclear translocation of β-catenin and metastasis, all features of tumoral malignancy. The objective of the present work was to evaluate the expression of ILK in clear cell renal carcinomas (CCRC) as a possible prognostic indicator. ILK immunoexpression was evaluated in a tissue microarray (TMA) with 45 human CCRCs. In addition, the apoptotic and proliferative indices and the immuno-expression of β-catenin and E-cadherin were also evaluated. E-cadherin expression was significantly decreased in tumors with positive ILK expression in relation to those with negative immunoexpression (p = 0.011). ILK immunostaining was significantly increased in high-grade in comparison to low-grade CCRCs (p = 0.0008). ILK expression was also associated with increased proliferative index (p = 0.020), tumor size >7.0 cm (p = 0.018) and with renal vein and capsule invasion (p = 0.003 and p = 0.00). Finally, tumors stage I and II (noninvasive) presented significantly reduced ILK immunoexpression when compared to stage III (locally invasive) (p = 0.0028). ILK immunoexpression in CCRC increases with loss of intercellular adhesion, nuclear grading, increased proliferative index and Robson stage. Altogether, our data suggest a possible role for ILK in the progression of CRCC.

Dishonorable Discharge: The Oncogenic Roles of Cleaved E-Cadherin Fragments

Strong cell-cell interactions represent a major barrier against cancer cell mobility, and loss of intercellular adhesion by E-cadherin is a fundamental change that occurs during the progression of cancer to invasive disease. However, some aggressive carcinomas retain characteristics of differentiated epithelial cells, including E-cadherin expression. Emerging evidence indicates that proteolysis of E-cadherin generates fragments that promote tumor growth, survival, and motility, suggesting that E-cadherin cleavage converts this tumor suppressor into an oncogenic factor. In this review we discuss the emerging roles of cleaved E-cadherin fragments as modulators of cancer progression, and explore the translational and clinical implications of this research.

Altered expression of E-cadherin and β-catenin in malignant transformation of sinonasal inverted papillomas

E-cadherin and β-catenin are molecules that mediate cell-cell adhesion in normal epithelium. Aberrant expression of these adhesion molecules results in the loss of intercellular adhesion, with possible cell transformation and tumour progression. We determined the role of E-cadherin and β-catenin in the pathogenesis of sinonasal inverted papilloma (IP) and its malignant transformation. We determined the expression of E-cadherin and β-catenin by immunohistochemistry in paraffin-embedded tissue of 21 subjects with nasal polyps, 56 with IPs, 7 IPs with dysplasia and 18 IPs with squamous cell carcinoma (SCC). The clinicopathological variables of the IPs with SCC correlated with the degree of expression of E-cadherin and β-catenin. The degree of expression of E-cadherin and β-catenin in the cell membrane was significantly lower in IPs with SCC than in nasal polyps and IPs. The degree of expression of β-catenin was significantly lower in IPs with SCC with a malignant proportion > 50% compared to a malignant proportion ≤ 50%. However, there was no significant association between the degree of expression of E-cadherin and β-catenin and clinicopathological variables, such as age, gender, T stage, tumour differentiation, or SCC type (metachronous vs. synchronous). In addition, there was no significant relationship between recurrence or survival rate in IPs with SCC and the degree of expression of E-cadherin or β-catenin in the cell membrane or nuclear β-catenin. Decreased expression of E-cadherin and β-catenin in the cell membrane may be associated with carcinogenesis of IPs and help predict malignant transformation in sinonasal IPs.

The Extent of Desmoglein 3 Depletion in Pemphigus Vulgaris Is Dependent on Ca 2+-Induced Differentiation : A Role in Suprabasal Epidermal Skin Splitting?

Pemphigus vulgaris (PV) is an autoimmune disease of the skin and mucous membranes and is characterized by development of autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 and formation of intraepidermal suprabasal blisters. Depletion of Dsg3 is a critical mechanism in PV pathogenesis. Because we did not detect reduced Dsg3 levels in keratinocytes cultured for longer periods under high-Ca(2+) conditions, we hypothesized that Dsg depletion depends on Ca(2+)-mediated keratinocyte differentiation. Our data indicate that depletion of Dsg3 occurs specifically in deep epidermal layers both in skin of patients with PV and in an organotypic raft model of human epidermis incubated using IgG fractions from patients with PV. In addition, Dsg3 depletion and loss of Dsg3 staining were prominent in cultured primary keratinocytes and in HaCaT cells incubated in high-Ca(2+) medium for 3 days, but were less pronounced in HaCaT cultures after 8 days. These effects were dependent on protein kinase C signaling because inhibition of protein kinase C blunted both Dsg3 depletion and loss of intercellular adhesion. Moreover, protein kinase C inhibition blocked suprabasal Dsg3 depletion in cultured human epidermis and blister formation in a neonatal mouse model. Considered together, our data indicate a contribution of Dsg depletion to PV pathogenesis dependent on Ca(2+)-induced differentiation. Furthermore, prominent depletion in basal epidermal layers may contribute to the suprabasal cleavage plane observed in PV.