Reduced E-cadherin Levels Research Articles

Farnesoid X receptor activation protects against renal fibrosis via modulation of β-catenin signaling

ObjectiveActivation of farnesoid X receptor (FXR), a bile acid nuclear receptor, may be implicated in the pathophysiology of diabetic nephropathy. We explored a possible role for FXR activation in preventing renal fibrosis in high fat diet (HFD)-fed mice. MethodsWe investigated the effects of HFD on mouse kidney and renal tubular epithelial cells both in vivo and in vitro, and observed the changes of FXR and β-catenin pathway. FXR agonist was also used to alleviate this HFD-induced effect, and the interaction between FXR and β-catenin was further verified. ResultsMice were fed by a 60% kcal fat diet for 20 weeks developed the typical traits of metabolic syndrome with subsequent renal lipid accumulation and renal injury. Treatment with the FXR agonist CDCA or GW4064 decreased body weight, renal lipid accumulation, as well as renal injury. Moreover, renal β-catenin signaling was activated and improved with FXR-agonist treatment in HFD-fed mice. To examine whether FXR affected β-catenin signaling, and was involved in tubulo-interstitial fibrosis, we explored the FXR expression and function in ox-LDL induced-renal tubular injury. In rat proximal tubular epithelial cells (NRK-52E) stimulated by ox-LDL, FXR protein was decreased compared to control group, and phosphorylated (Ser675) β-catenin was activated by ox-LDL in a dose- and time-dependent manner. Ox-LDL enhanced α-SMA and fibronectin expressions and reduced E-cadherin levels, whereas FXR agonism or FXR overexpression inhibited fibronectin and α-SMA expressions and restored E-cadherin. Moreover, FXR agonist treatment also decreased phosphorylated (Ser675) β-catenin, nuclear translocation and β-catenin-mediated transcription induced by ox-LDL in NRK-52E cells. We showed that FXR could bind with β-catenin via the AF1 domain, and disrupt the assembly of the core β-catenin/TCF4 complex. ConclusionThese experimental data suggest that FXR activation, via modulating β-catenin signaling, may contribute to attenuating the development of lipid-mediated tubulo-interstitial fibrosis.

C-Mannosylated tryptophan-containing WSPW peptide binds to actinin-4 and alters E-cadherin subcellular localization in lung epithelial-like A549 cells

The Trp-x-x-Trp (W-x-x-W) peptide motif, a consensus site for C-mannosylation, is the functional motif in cytokine type I receptors or thrombospondin type I repeat (TSR) superfamily proteins. W-x-x-W motifs are important for physiological and pathological functions of their parental proteins, but effects of C-mannosylation on protein functions remain to be elucidated. By using chemically synthesized WSPW peptides and C-mannosylated WSPW peptides (C-Man-WSPW), we herein investigated whether C-mannosylation of WSPW peptides confer additional biological functions to WSPW peptides. C-Man-WSPW peptide, but not non-mannosylated WSPW, reduced E-cadherin levels in A549 cells. Via peptide mass fingerprinting analysis, we identified actinin-4 as a C-Man-WSPW-binding protein in A549 cells. Actinin-4 partly co-localized with E-cadherin or β-catenin, despite no direct interaction between actinin-4 and E-cadherin. C-Man-WSPW reduced co-localization of E-cadherin and actinin-4; non-mannosylated WSPW had no effect on localization. In actinin-4-knockdown cells, E-cadherin was upregulated and demonstrated a punctate staining pattern in the cytoplasm, which suggests that actinin-4 regulated cell-surface E-cadherin localization. Thus, C-mannosylation of WSPW peptides is required for interaction with actinin-4 that subsequently alters expression and subcellular localization of E-cadherin and morphology of epithelial-like cells. Our results therefore suggest a regulatory role of C-mannosylation of the W-x-x-W motif in interactions between the motif and its binding partner and will thereby enhance understanding of protein C-mannosylation.

Investigating Helicobacter pylori-related pyloric hypomotility: functional, histological, and molecular alterations.

Multiple theories have been proposed describing the pathogenic mechanisms of Helicobacter pylori (H. pylori)-associated gastric motility disorders. We assessed ex vivo pyloric activity in H. pylori-infected rats, and tried to explore the associated ghrelin hormone alteration and pyloric fibrogenesis. In addition, miR-1 was assessed in pyloric tissue samples, being recently accused of having a role in smooth muscle dysfunction. Ninety adult male Wistar albino rats were assigned into nine groups: 1) control group, 2) sterile broth (vehicle group), 3) amoxicillin control, 4) omeperazole control, 5) clarithromycin control, 6) triple therapy control, 7) H. pylori- group, 8) H. pylori-clarithromycin group, and 9) H. pylori-triple therapy group. Urease enzyme activity was applied as an indicator of H. pylori infection. Ex vivo pyloric contractility was evaluated. Serum ghrelin was assessed, and histological tissue evaluation was performed. Besides, pyloric muscle miR-1 expression was measured. The immunological epithelial to mesenchymal transition (EMT) markers; transforming growth factor β (TGFβ), α-smooth muscle actin (α-SMA), and E-cadherin-3 were also evaluated. By H. pylori infection, a significant (P < 0.001) reduced pyloric contractility index was recorded. The miR-1 expression was decreased (P < 0.001) in the H. pylori-infected group, associated with reduced serum ghrelin, elevated TGFβ, and α-SMA levels and reduced E-cadherin levels. Decreased miR-1 and disturbed molecular pattern were improved by treatment. In conclusion, H. pylori infection was associated with reduced miR-1, epithelial to mesenchymal transition, and pyloric hypomotility. The miR-1 may be a target for further studies to assess its possible involvement in H. pylori-associated pyloric dysfunction, which might help in the management of human H. pylori manifestations and complications.NEW & NOTEWORTHY This work is investigating functional, histopathological, and molecular changes underlying Helicobacter pylori hypomotility and is correlating these with miR-1, whose disturbance is supposed to be involved in smooth muscle dysfunction and cell proliferation according to literature. Epithelial to mesenchymal transition and reduced ghrelin hormone may contribute to H. pylori infection-associated hypomotility. H. pylori infection was associated with reduced pyloric miR-1 expression. Targeting miR-1 could be valuable in the clinical management of pyloric hypofunction.

Heparanase Loosens E-Cadherin-Mediated Cell-Cell Contact via Activation of Src.

Activity of heparanase, responsible for cleavage of heparan sulfate (HS), is strongly implicated in tumor metastasis. This is due primarily to remodeling of the extracellular matrix (ECM) that becomes more prone to invasion by metastatic tumor cells. In addition, heparanase promotes the development of blood and lymph vessels that mobilize disseminated cells to distant organs. Here, we provide evidence for an additional mechanism by which heparanase affects cell motility, namely the destruction of E-cadherin based adherent junctions (AJ). We found that overexpression of heparanase or its exogenous addition results in reduced E-cadherin levels in the cell membrane. This was associated with a substantial increase in the phosphorylation levels of E-cadherin, β-catenin, and p120-catenin, the latter recognized as a substrate of Src. Indeed, we found that Src phosphorylation is increased in heparanase overexpressing cells, associating with a marked decrease in the interaction of E-cadherin with β-catenin, which is instrumental for AJ integrity and cell-cell adhesion. Notably, the association of E-cadherin with β-catenin in heparanase overexpressing cells was restored by Src inhibitor, along with reduced cell migration. These results imply that heparanase promotes tumor metastasis by virtue of its enzymatic activity responsible for remodeling of the ECM, and by signaling aspects that result in Src-mediated phosphorylation of E-cadherin/catenins and loosening of cell-cell contacts that are required for maintaining the integrity of epithelial sheets.

Alterations at the Synthesis and Degradation of E-cadherin in the Human Lungs with Emphysema

Pulmonary emphysema leads to a cascade of events starting with enlarged alveoli, loss of alveoli and, subsequently to the damage and disruption of pulmonary epithelium. The integrity of the pulmonary epithelium, which is constituted by pneumocytes linked to each other through E-cadherin proteins, is important for respiration. The aim of the present study was to detect the content and destruction of E-cadherin protein and to investigate the contribution of E-cadherin to pulmonary emphysema pathogenesis. The structural changes, reparative capacity of the pulmonary epithelium, amount of E-cadherin protein and, the immunoreactivity of neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9) were evaluated in emphysematous (n=7) and non-emphysematous (n=6) areas of lung samples taken from patients with chronic obstructive pulmonary disease. Emphysematous areas are characterized by enlarged alveoli, disrupted alveolar walls and epithelium, increased type 2 pneumocytes and NEDD9 immunoreactivity, and reduced E-cadherin proteins. Our data shows that E-cadherin levels are decreased in emphysematous areas due to its degradation by NEDD9. Decreased E-cadherin levels also lead to the disintegration of the pulmonary epithelium by causing the presence of weakness intercellular connections or the absence of intercellular connections. The repair of the pulmonary epithelium could not complete due to the reduced E-cadherin, because type 2 pneumocytes could not differentiate into type 1 pneumocytes. In conclusion, the reduced E-cadherin levels lead to emphysematous alterations in human lungs and contributes to pulmonary emphysema pathogenesis.

Glycoprotein 90K Promotes E-Cadherin Degradation in a Cell Density-Dependent Manner via Dissociation of E-Cadherin-p120-Catenin Complex.

Glycoprotein 90K (also known as LGALS3BP or Mac-2BP) is a tumor-associated protein, and high 90K levels are associated with poor prognosis in some cancers. To clarify the role of 90K as an indicator for poor prognosis and metastasis in epithelial cancers, the present study investigated the effect of 90K on an adherens junctional protein, E-cadherin, which is frequently absent or downregulated in human epithelial cancers. Treatment of certain cancer cells with 90K significantly reduced E-cadherin levels in a cell-population-dependent manner, and these cells showed decreases in cell adhesion and increases in invasive cell motility. Mechanistically, 90K-induced E-cadherin downregulation occurred via ubiquitination-mediated proteasomal degradation. 90K interacted with the E-cadherin–p120-catenin complex and induced its dissociation, altering the phosphorylation status of p120-catenin, whereas it did not associate with β-catenin. In subconfluent cells, 90K decreased membrane-localized p120-catenin and the membrane fraction of the p120-catenin. Particularly, 90K-induced E-cadherin downregulation was diminished in p120-catenin knocked-down cells. Taken together, 90K upregulation promotes the dissociation of the E-cadherin–p120-catenin complex, leading to E-cadherin proteasomal degradation, and thereby destabilizing adherens junctions in less confluent tumor cells. Our results provide a potential mechanism to explain the poor prognosis of cancer patients with high serum 90K levels.

Abstract 5530: Evidence linking aquaporin-3 loss to increased invasiveness in bladder cancer

Abstract Bladder cancer (BCa) is the most common type of urothelial cancer (UC). According to American Cancer Society, in 2016, diagnoses of 76,960 new cases of bladder cancer (BCa) and 16,390 BCa associated deaths have been estimated. More than 90% of bladder cancer starts in the innermost transitional epithelium (urothelium) of the bladder. Aquaporin 3 (AQP3), one of 13 members of a transmembrane channel forming protein family (AQP0-12), has an N-terminal basolateral sorting signal and localizes to the basolateral membrane in epithelial tissues. Recent studies have shown that AQP3 expression is decreased in UC thus supporting a new hypothesis that alteration of the expression of AQP3 plays a role in the pathogenesis of UC. However, it remains to be determined whether AQP3 is involved in the invasion and metastasis of UC cells. In the present study, human bladder cancer cell lines of different grades, SW780 (G1) and TCCSUP (G4) were studied. Transcript and protein levels of AQP3 expression were significantly higher in low grade, SW780 cells when compared to the higher-grade TCCSUP cells. Inhibition of AQP3 by CuSO4 or siRNA specific knockdown of AQP3 resulted in increased migration in the wound-healing scratch assay. Over-expression of AQP3 plasmid resulted in reduced migration. It is known that epithelial cell polarity contributes to tumor suppression and that loss of E-cadherin is a crucial step in epithelial-to-mesenchymal transition (EMT). Our results indicate that AQP3 overexpression induced expression of E-cadherin, consistent with a role for AQP3 as a tumor suppressor by maintaining epithelial cell polarity and inhibiting EMT in BCa. Conversely, reduced AQP3 levels leading to reduced E-cadherin levels in high-grade BCa cells may result in loss of cell polarity, increased invasiveness and increased EMT. Citation Format: Arpita Roy, Dinuka M. DeSilva, Donald P. Bottaro. Evidence linking aquaporin-3 loss to increased invasiveness in bladder cancer [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 5530. doi:10.1158/1538-7445.AM2017-5530

Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm

CDC14 is an essential dual-specificity phosphatase that counteracts CDK1 activity during anaphase to promote mitotic exit in Saccharomyces cerevisiae Surprisingly, human CDC14A is not essential for cell cycle progression. Instead, it regulates cell migration and cell adhesion. Little is known about the substrates of hCDC14A and the counteracting kinases. Here, we combine phospho-proteome profiling and proximity-dependent biotin identification to identify hCDC14A substrates. Among these targets were actin regulators, including the tumor suppressor eplin. hCDC14A counteracts EGF-induced rearrangements of actin cytoskeleton by dephosphorylating eplin at two known extracellular signal-regulated kinase sites, serine 362 and 604. hCDC14APD and eplin knockout cell lines exhibited down-regulation of E-cadherin and a reduction in α/β-catenin at cell-cell adhesions. Reduction in the levels of hCDC14A and eplin mRNA is frequently associated with colorectal carcinoma and is correlated with poor prognosis. We therefore propose that eplin dephosphorylation by hCDC14A reduces actin dynamics to restrict tumor malignancy.

Calreticulin Is Required for TGF-β-Induced Epithelial-to-Mesenchymal Transition during Cardiogenesis in Mouse Embryonic Stem Cells.

SummaryCalreticulin, a multifunctional endoplasmic reticulum resident protein, is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) and subsequent cardiomyogenesis. Using embryoid bodies (EBs) derived from calreticulin-null and wild-type (WT) embryonic stem cells (ESCs), we show that expression of EMT and cardiac differentiation markers is induced during differentiation of WT EBs. This induction is inhibited in the absence of calreticulin and can be mimicked by inhibiting TGF-β signaling in WT cells. The presence of calreticulin in WT cells permits TGF-β-mediated signaling via AKT/GSK3β and promotes repression of E-cadherin by SNAIL2/SLUG. This is paralleled by induction of N-cadherin in a process known as the cadherin switch. We show that regulated Ca2+ signaling between calreticulin and calcineurin is critical for the unabated TGF-β signaling that is necessary for the exit from pluripotency and the cadherin switch during EMT. Calreticulin is thus a key mediator of TGF-β-induced commencement of cardiomyogenesis in mouse ESCs.

Abstract 3233: Macrophages orchestrate early dissemination of HER2+ cancer cells

Abstract Metastatic dissemination was proposed to occur only in invasive cancer. However, detection of cancer cells in the bone marrow of patients with pre-invasive breast ductal carcinoma in situ (DCIS) showed that dissemination could, through unknown mechanisms, take place earlier. Late evolved invasive breast cancer cells disseminated by recruiting circulating monocyte-derived tumor associated macrophages (TAMs). We therefore investigated whether macrophages might actively participate in early dissemination. We show that pre-malignant cancer cells in MMTV-HER2 mice produced CCL2 in an NFkB dependent manner and thereby recruited resident mammary tissue macrophages into the duct, months before monocyte-derived TAMs arrived. Intra-epithelial mammary tissue macrophages induced an epithelial-to-mesenchymal transition in early HER2+ cells in a Wnt-dependent manner and thereby drove early dissemination. Consequently, depletion of macrophages from pre-malignant MMTV-HER2 mice significantly reduced early dissemination of pre-malignant cancer cells as measured by reduction in levels of circulating cancer cells and disseminated cancer cells in lungs. Interestingly, macrophage depletion during pre-malignant stages alone was sufficient to reduce the onset of late metastasis in MMTV-HER2 mice, indicating that early disseminating cancer cells contributed to late metastasis formation. In humans DCIS samples we frequently found intra-epithelial macrophages inside of ducts and their presence correlated with reduced E-Cadherin levels. Finally, a first pilot study indicated that those patients whose DCIS lesions contained macrophage +/E-Cadherin low microenvironments frequently had DCCs in the bone marrow. Taken together, we reveal that resident mammary tissue macrophages can promote early dissemination, and unravel a mechanism how early cancer spread might proceed in breast cancer patients and in other cancers where metastasis are diagnosed and the primary tumor is never detected (cancer of unknown primary). We also propose that early DCCs play a long-term causal role in metastasis development when patients are diagnosed in late stages, implying that the heterogeneity of metastases is higher than we might anticipate. Citation Format: Nina Linde, Arthur Mortha, Nicole Saenger, Maria Soledad Sosa, Kathryn Harper, Ethan Tardio, Tanja Fehm, Thomas Karn, Miriam Merad, Julio A. Aguirre-Ghiso. Macrophages orchestrate early dissemination of HER2+ cancer cells. [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 3233.

DNA methylation-induced E-cadherin silencing is correlated with the clinicopathological features of melanoma.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, has an important role in epithelial cell function, maintenance of tissue architecture and cancer suppression. Loss of E-cadherin promotes tumor metastatic dissemination and predicts poor prognosis. The present study investigated the clinicopathological significance of E-cadherin expression in cutaneous, mucosal and uveal melanoma related to epigenetic mechanisms that may contribute to E-cadherin silencing. E-cadherin expression was reduced in 55/130 cutaneous (42.3%), 49/82 mucosal (59.7%) and 36/64 uveal (56.2%) melanoma samples as compared to normal skin controls and was inversely associated with promoter methylation. Of the 10 different CpG sites studied (nt 863, 865, 873, 879, 887, 892, 901, 918, 920 and 940), two sites (nt 892 and 940) were 90-100% methylated in all the melanoma specimens examined and the other ones were partially methylated (range, 53-86%). In contrast, the methylation rate of the E-cadherin gene was low in normal tissues (range, 5-24%). In all the three types of melanoma studied, a significant correlation was found between reduced levels of E-cadherin and reduced survival, high mitotic index and metastasis, accounting for the predilection of lymph nodal localization. In cutaneous and mucosal melanoma, low E-cadherin expression was positively correlated also with head/neck localization and ulceration. A high frequency of reduced E-cadherin levels occurred in choroid melanomas. In vitro experiments showed that E-cadherin transcription was restored following 5-aza-2'-deoxycytidine (5-aza-dC) treatment or DNMT1 silencing and was negatively correlated with the invasive potential of melanoma cells. The significant relationship between E-cadherin silencing and several poor prognostic factors indicates that this adhesion molecule may play an important role in melanomagenesis. Therefore, the inverse association of E-cadherin expression with promoter methylation raises the intriguing possibility that reactivation of E-cadherin expression through promoter demethylation may represent a potential therapeutic strategy for the treatment of melanoma.

Actin cytoskeleton regulation of epithelial mesenchymal transition in metastatic cancer cells.

Epithelial-mesenchymal transition (EMT) is associated with loss of the cell-cell adhesion molecule E-cadherin and disruption of cell-cell junctions as well as with acquisition of migratory properties including reorganization of the actin cytoskeleton and activation of the RhoA GTPase. Here we show that depolymerization of the actin cytoskeleton of various metastatic cancer cell lines with Cytochalasin D (Cyt D) reduces cell size and F-actin levels and induces E-cadherin expression at both the protein and mRNA level. Induction of E-cadherin was dose dependent and paralleled loss of the mesenchymal markers N-cadherin and vimentin. E-cadherin levels increased 2 hours after addition of Cyt D in cells showing an E-cadherin mRNA response but only after 10-12 hours in HT-1080 fibrosarcoma and MDA-MB-231 cells in which E-cadherin mRNA level were only minimally affected by Cyt D. Cyt D treatment induced the nuclear-cytoplasmic translocation of EMT-associated SNAI 1 and SMAD1/2/3 transcription factors. In non-metastatic MCF-7 breast cancer cells, that express E-cadherin and represent a cancer cell model for EMT, actin depolymerization with Cyt D induced elevated E-cadherin while actin stabilization with Jasplakinolide reduced E-cadherin levels. Elevated E-cadherin levels due to Cyt D were associated with reduced activation of Rho A. Expression of dominant-negative Rho A mutant increased and dominant-active Rho A mutant decreased E-cadherin levels and also prevented Cyt D induction of E-cadherin. Reduced Rho A activation downstream of actin remodelling therefore induces E-cadherin and reverses EMT in cancer cells. Cyt D treatment inhibited migration and, at higher concentrations, induced cytotoxicity of both HT-1080 fibrosarcoma cells and normal Hs27 fibroblasts, but only induced mesenchymal-epithelial transition in HT-1080 cancer cells. Our studies suggest that actin remodelling is an upstream regulator of EMT in metastatic cancer cells.

Abstract 4068: Overexpression of anillin in colorectal cancer promotes the cell proliferation, cell mobility and cell invasion

Abstract Colorectal cancer (CRC) is the third most prevalent cancer in the developed countries, accounting for more than 50,000 cancer deaths per year. We used microarray to analyze gene expression in patients with different stages of colorectal cancer. Among the 157 metastasis-related genes, RT-PCR and quantitative real-time PCR (qPCR) were used to verify 11 genes. The roles of anillin (ANLN), one of 11 metastasis-associated genes, in colorectal cancer are not clear. Therefore, we focused on ANLN in this study. We found that the expression levels of anillin are higher in tumor specimens, even in metastasis tumor grade compared with those of adjacent normal tissues by real-time PCR. In order to understand the roles of anillin in tumorigenesis and metastasis of colorectal cancer, we used anillin overexpressed HT29 and shRNA-knockdowned SW480. Our data indicated that anillin overexpressed HT29 showed faster replication kinetics probably due to prolonged G2/M phase. Next, we found that the number and size of colony were increased in anillin over expressed cells in soft agar assay suggesting a role of anillin in tumorgenesis of colorectal cancer. Notably, anillin overexpressed SW480 and HT29 lines demonstrated increased migration and invasion. This increased mobility in anillin overexpressed SW480 and HT29 lines partially correlated with reduced E-cadherin levels. By confocal analysis of philloidin stained cells, we also found increased F-actin bundles accumulation in anillin overexpressed HT29 cells. We hypothesize that anillin may promote partial EMT and cell migration through induced cytoskeleton remodeling. Taken together, we suggest that anillin is involved in enhancement of proliferation, mobility, invasion of colorectal cancer cell lines, which may lead to enhanced tumorgenesis and metastasis potential. Citation Format: Hui Yu Chuang, Yu Hsuan Ou. Overexpression of anillin in colorectal cancer promotes the cell proliferation, cell mobility and cell invasion. [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 4068. doi:10.1158/1538-7445.AM2014-4068

Reactivation of NCAM1 Defines a Subpopulation of Human Adult Kidney Epithelial Cells with Clonogenic and Stem/Progenitor Properties

The nephron is composed of a monolayer of epithelial cells that make up its various compartments. In development, these cells begin as mesenchyme. NCAM1, abundant in the mesenchyme and early nephron lineage, ceases to express in mature kidney epithelia. We show that, once placed in culture and released from quiescence, adult human kidney epithelial cells (hKEpCs), uniformly positive for CD24/CD133, re-express NCAM1 in a specific cell subset that attains a stem/progenitor state. Immunosorted NCAM1(+) cells overexpressed early nephron progenitor markers (PAX2, SALL1, SIX2, WT1) and acquired a mesenchymal fate, indicated by high vimentim and reduced E-cadherin levels. Gene expression and microarray analysis disclosed both a proximal tubular origin of these cells and molecules regulating epithelial-mesenchymal transition. NCAM1(+) cells generated clonal progeny when cultured in the presence of fetal kidney conditioned medium, differentiated along mesenchymal lineages but retained the unique propensity to generate epithelial kidney spheres and produce epithelial renal tissue on single-cell grafting in chick CAM and mouse. Depletion of NCAM1(+) cells from hKEpCs abrogated stemness traits in vitro. Eliminating these cells during the regenerative response that follows glycerol-induced acute tubular necrosis worsened peak renal injury in vivo. Thus, higher clone-forming and developmental capacities characterize a distinct subset of adult kidney-derived cells. The ability to influence an endogenous regenerative response via NCAM1 targeting may lead to novel therapeutics for renal diseases.

Contactin-1 Reduces E-Cadherin Expression Via Activating AKT in Lung Cancer

Contactin-1 has been shown to promote cancer metastasis. However, the underlying mechanisms remain unclear. We report here that knockdown of contactin-1 in A549 lung cancer cells reduced A549 cell invasion and the cell's ability to grow in soft agar without affecting cell proliferation. Reduction of contactin-1 resulted in upregulation of E-cadherin, consistent with E-cadherin being inhibitive of cancer cell invasion. In an effort to investigate the mechanisms whereby contactin-1 reduces E-cadherin expression, we observed that contactin-1 plays a role in AKT activation, as knockdown of contactin-1 attenuated AKT activation. Additionally, inhibition of AKT activation significantly enhanced E-cadherin expression, an observation that mimics the situation observed in contactin-1 knockdown, suggesting that activation of AKT plays a role in contactin-1-mediated downregulation of E-cadherin. In addition, we were able to show that knockdown of contactin-1 did not further reduce A549 cell's invasion ability, when AKT activation was inhibited by an AKT inhibitor. To further support our findings, we overexpressed CNTN-1 in two CNTN-1 null breast cancer cell lines expressing E-cadherin. Upon overexpression, CNTN-1 reduced E-cadherin levels in one cell line and increased AKT activation in the other. Furthermore, in our study of 63 primary lung cancers, we observed 65% of primary lung cancers being contactin-1 positive and in these carcinomas, 61% were E-cadherin negative. Collectively, we provide evidence that contactin-1 plays a role in the downregulation of E-cadherin in lung cancer and that AKT activation contributes to this process. In a study of mechanisms responsible for contactin-1 to activate AKT, we demonstrated that knockdown of CNTN-1 in A549 cells did not enhance PTEN expression but upregulated PHLPP2, a phosphatase that dephosphorylates AKT. These observations thus suggest that contactin-1 enhances AKT activation in part by preventing PHLPP2-mediated AKT dephosphrorylation.

Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins

E-cadherin (CDH1) loss occurs frequently in carcinogenesis, contributing to invasion and metastasis. We observed that mouse and human epithelial cell lines overexpressing the replication licensing factor Cdc6 underwent phenotypic changes with mesenchymal features and loss of E-cadherin. Analysis in various types of human cancer revealed a strong correlation between increased Cdc6 expression and reduced E-cadherin levels. Prompted by these findings, we discovered that Cdc6 repressed CDH1 transcription by binding to the E-boxes of its promoter, leading to dissociation of the chromosomal insulator CTCF, displacement of the histone variant H2A.Z, and promoter heterochromatinization. Mutational analysis identified the Walker B motif and C-terminal region of Cdc6 as essential for CDH1 transcriptional suppression. Strikingly, CTCF displacement resulted in activation of adjacent origins of replication. These data demonstrate that Cdc6 acts as a molecular switch at the E-cadherin locus, linking transcriptional repression to activation of replication, and provide a telling example of how replication licensing factors could usurp alternative programs to fulfill distinct cellular functions.

Anthrax toxins cooperatively inhibit endocytic recycling by the Rab11/Sec15 exocyst

Bacillus anthracis is the causative agent of anthrax in humans and other mammals1, 2. In lethal systemic anthrax, proliferating bacilli secrete large quantities of the toxins lethal factor (LF) and edema factor (EF), leading to widespread vascular leakage and shock. While host targets of LF (MAPKKs) and EF (cAMP-dependent processes)3 have been implicated in the initial phase of anthrax1, 2, less is understood about toxin action during the final stage of infection. Here, we use Drosophila to identify the Rab11/Sec15 exocyst, which acts at the last step of endocytic recycling, as a novel target of both EF and LF. EF reduces levels of apically localized Rab11, and indirectly blocks vesicle formation by its binding partner and effector Sec15 (Sec15-GFP), while LF acts more directly to reduce Sec15-GFP vesicles. Convergent effects of EF and LF on Rab11/Sec15 inhibit expression of and signaling by the Notch ligand Delta and reduce DE-cadherin levels at adherens junctions (AJ). In human endothelial cells, the two toxins act in a conserved fashion to block formation of Sec15 vesicles, inhibit Notch signaling, and reduce cadherin expression at AJ. This coordinated disruption of the Rab11/Sec-15 exocyst by anthrax toxins may contribute to toxin-dependent barrier disruption and vascular dysfunction during B. anthracis infection.

Abstract 2032: MicroRNA control of tumor cell plasticity in a new tumor metastasis model

Abstract We have developed a new prostate cancer model to discover the molecular changes underlying metastasis. A series of cell lines were developed by isolating metastatic cells from lymph nodes of nude mice following orthotopic injection of DU145 human prostate cancer cells and expression changes analyzed using gene chips. We found that these DU145-LN cells exhibit a mesenchymal-epithelial transition (MET). Repeated rounds of in vivo metastatic selection progressively increased the expression of epithelial markers including E-cadherin, cytokeratin-18 and EpCAM, and decreased mesenchymal markers such as vimentin. Despite being more epithelial, DU145-LN cells are more migratory and show reduced proliferation in vitro. However, they show increased tumorigenesis when reinjected into nude mice. Interestingly, clinical metastases are frequently epithelial, despite strong evidence that EMT enhances metastasis. To explain this, current models of metastasis invoke EMT with subsequent MET at the secondary site. Our cells appear to demonstrate this plasticity making them an ideal model system for studying MET regulation. The MET changes in the DU145-LN cells suggested a coordinated mechanism of regulation. MicroRNA arrays revealed several candidates which correlated with the epithelial phenotype. DU145-LN cells showed increased expression of the miR-200 family (a, b, c and miR-141), and treatment with synthetic miR-200 inhibitors resulted in reduced cell-cell interactions and decreased E-cadherin levels. Our data complement recent evidence showing down-regulation of miR-200 by EMT. miR-200 acts in a feedback loop with the transcription factor ZEB1 to regulate EMT. We also observed significantly reduced expression of an additional microRNA not previously linked to cell morphology in the DU145-LN cells. Transfection of precursor miRNA to this target altered the epithelial morphology and reduced E-cadherin levels, suggesting an additional pathway of MET/EMT control. We are currently determining the mechanism of action of this new microRNA target. Our data support a role for MET in tumor metastasis, and indicate that microRNAs are an important mechanism mediating tumor cell plasticity and EMT/MET. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2032.

Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization.

Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar- and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney.

RalA suppresses early stages of Ras-induced squamous cell carcinoma progression

Ras proteins activate Raf and PI-3 kinases, as well as exchange factors for RalA and RalB GTPases. Many previous studies have reported that the Ral signaling cascade contributes positively to Ras-mediated oncogenesis. Here, utilizing a bioengineered tissue model of early steps in Ras-induced human squamous cell carcinoma of the skin, we found the opposite. Conversion of Ras-expressing keratinocytes from a premalignant to malignant state induced by decreasing E-cadherin function was associated with and required a knockdown of RalA to a similar degree by shRNA expression in these cells decrease in RalA expression. Moreover, direct ∼2-3 fold knockdown of RalA by shRNA expression in these cells reduced E-cadherin levels and also induced progression to a malignant phenotype. Knockdown of the Ral effector, Exo84, mimicked the effects of decreasing RalA levels in these engineered tissues. These phenomena can be explained by our finding that the stability of E-cadherin in Ras-expressing keratinocytes depends upon this RalA signaling cascade. These results imply that an important component of the early stages in squamous carcinoma progression may be a modest decrease in RalA gene expression that magnifies the effects of decreased E-cadherin expression by promoting its degradation.