Role Of E-cadherin Research Articles

E-cadherin inhibits tumor cell growth by suppressing PI3K/Akt signaling via β-catenin-Egr1-mediated PTEN expression

E-cadherin is a cell-cell adhesion protein and tumor suppressor that is silenced in many malignancies. E-cadherin is thought to suppress tumor cell growth by antagonizing β-catenin signaling. However, the role of E-cadherin in ovarian cancer progression is still controversial. In this study, we showed that loss of E-cadherin induced ovarian cancer cell growth and constitutive activation of phosphoinositide 3-kinase (PI3K)/Akt signaling by the inhibition of phosphatase and tensin homolog (PTEN) transcription through the downregulation of early growth response gene 1 (Egr1). In addition, immunofluorescence microscopy and T-cell factor promoter/luciferase reporter assays showed that E-cadherin loss was associated with enhanced nuclear β-catenin signaling. Constitutive activation of PI3K/Akt signaling reinforced nuclear β-catenin signaling by inactivating glycogen synthase kinase-3β indicating cross-talk between the PI3K/Akt and β-catenin signaling pathways. Finally, we found that E-cadherin negatively regulates tumor cell growth, in part, by positively regulating PTEN expression via β-catenin-mediated Egr1 regulation, thus influencing PI3K/Akt signaling. In summary, endogenous E-cadherin inhibits PI3K/Akt signaling by antagonizing β-catenin-Egr1-mediated repression of PTEN expression. Thus, the loss of E-cadherin itself may contribute to dysregulated PI3K/Akt signaling through its effects on PTEN, or it may exacerbate the frequent activation of PI3K/Akt signaling that occurs as a result of overexpression, mutation and/or amplification.

Role of E-cadherin in the Pathogenesis of Gastroesophageal Reflux Disease

Role of E-cadherin in the Pathogenesis of Gastroesophageal Reflux Disease

Abstract P5-05-04: E-Cadherin and Its Proteolytic Fragments Trump Epithelial-Mesenchymal Transition and Function as Dominant Oncogenes in Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) and its lymphovascular embolic signature seem to challenge the dogma of prerequisite epithelialmesenchymal transition (EMT) in human breast cancer progression by the apparent maintenance of an epithelial state in all stages of IBC. Previous studies with a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated that overexpressed E-cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super-high density in vitro. Although slug, snail and twist are increased in MARY-X spheroids and may decrease E-cadherin transcription to some extent, their effects are neutralized and frankly overridden by the post-transcriptional regulation of E-cadherin which includes its altered degradation and trafficking which, in turn, maintain the epithelial state. Materials and Methods: Because of the uniqueness of the MARY-X model and the observation that E-cadherin was not functioning as a tumor suppressor gene whose loss was triggering EMT but rather whose overexpression was mediating increased survival and resistance to apoptosis, we decided to examine the fate and role of E-cadherin more closely. Results: We discovered that E-cadherin underwent extensive proteolytic processing which resulted in fragments also mediating increased survival and resistance to apoptosis. In both MARY-X and the MARY-X spheroids, Western blot revealed 5 E-cadherin bands: one full length (120 kDa) and four fragments: E-cad/NFT1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa), E-cad/NTF4 (80kDa). Corresponding E-cad/CTF1-4 fragments were also generated. Compared to MARY-X, NFT1 was dramatically increased in the MARY-X spheroids whereas NFT2-4 were significantly decreased. These E-cadherin fragments were produced by specific proteolytic cleavage based on inhibitor studies: NFT1 was produced by calpain, NFT2 by γ-secretase, NFT3 by a matrix metalloproteinase and NFT4 by an unknown mechanism. Predictably levels of calpain activity were significantly increased in the MARY-X spheroids. NFT1 generatedby increased calpain activity retained the p120ctn binding site but lost both the β-catenin and α-binding sites, facilitating its 360° distribution around the cell. Calpain inhibition decreased NFT1 without any affect on full length E-cadherin yet induced disadherence of intact spheroids and prevented the assembly of de novo spheroids. Identical proteolytic fragments of E-cadherin were observed in 3 other human IBC cell lines as well as in lymphovascular emboli of human IBC cases obtained by laser capture microdissection. Discussion: The selective proteolytic processing of E-cadherin ultimately then is responsible for the increased survival of the lymphovascular embolus of IBC and its resistance to chemotherapy-induced apoptosis. Select NT proteolytic fragments may function as adhesion molecules whereas select CT proteolytic fragments may function as transcription factors which confer this survival advantage. E-cadherin and its proteolytic fragments then both trump EMT as well as function as dominant oncogenes in IBC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-04.

Inhibitory effects of miRNA-200c on chemotherapy-resistance and cell proliferation of gastric cancer SGC7901/DDP cells

miRNA-200c can not only inhibit the aggressiveness of cancer cells but also increase the sensitivity of cells to antitumor drugs. However, some mechanisms are still unclear. Recent researches revealed that E-cadherin is more than an inhibitor of metastasis, and it also plays important roles in reversing drug resistance. We had previously found that miRNA-200c could not only induce the expression of E-cadherin but also increase the sensitivity of gastric cancer SGC7901/DDP cells to cisplatin (DDP). This study aimed to explore the effects of miRNA-200c on biological characteristics of SGC7901/DDP cells and the roles of E-cadherin in the regulatory pathway of miRNA-200c. SGC7901/DDP cells and its parental cell line SGC7901 cells were transfected with miRNA-200c precursor (Pre-200c) and E-cadherin siRNA, respectively. Real-time RT-PCR was used to detect miRNA-200c expression after transfection with Pre-200c in SGC7901/DDP cell line. Drug sensitivities to DDP, 5-fluorouracil (5-FU), paclitaxel, and adriamycin (ADR) after transfection were tested using MTT assay. The proliferation of SGC7901/DDP cells was also detected after transfection. The protein changes of E-cadherin, Bax, and Bcl-2 after transfection were detected by Western blot. The miRNA-200c expression in SGC7901/DDP cells after transfection of Pre-200c was 7.128 ± 0.159 times of that in negative control (P < 0.05). The IC50 of DDP, 5-FU, paclitaxel, and ADR in Pre-200c-transfected group were significantly lower than that in negative control group (P < 0.05). Compared to the control group, cell proliferation was significantly decreased (P < 0.05). The relative protein expressions of E-cadherin and Bax in Pre-200c-transfected group were significantly higher than those in negative control group (P < 0.05), whereas Bcl-2 was significantly lower than that in control (P < 0.05). Additionally, E-cadherin protein expression was significantly inhibited after transfected with E-cadherin siRNA in SGC7901 cells. The Bax protein expression was significantly down-regulated by E-cadherin siRNA (P < 0.05), whereas the Bcl-2 expression was significantly up-regulated (P < 0.05). miRNA-200c can indirectly regulate apoptosis through E-cadherin in SGC7901/DDP cells, which may be a possible mechanism of miRNA-200c in reversing drug resistance and inhibiting proliferation.

Clinical and Functional Relevance of Interactions Between the Bone Marrow Stormal Microenvironment and Primary Human Acute Myeloid Leukemia (AML) Cells

AbstractAbstract 2756 Objectives:Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment confers protection from chemotherapy cytotoxicity. We sought to extend our knowledge of the molecular mechanisms by which the BM stroma support AML by identifying all functionally relevant adhesion receptors that were associated with clinical outcomes, identifying new functional AML-stromal interactions, and examining for aberrant cytokine production by stroma derived from patient AML BM. Methods:We prospectively analyzed by multicolor flow cytometry the expression of 17 different adhesion receptors by AML blasts derived from peripheral blood or BM of 42 adult patients, ages 19–80. Analyses included percent expression, mean fluorescence intensity (MFI), and in 7 patients, the adhesion receptors expressed by the CD34+CD38−CD123+ putative leukemia stem cell (LSC) population. We correlated percent expression and MFI with the likelihood of complete remission (CR) using Wilcoxon two-sample testing with Monte Carlo estimation of the exact p value. Guided by data from the AML blast adhesion receptor analyses, we designed a functional screen to assay primary AML blast adhesion to a normal BM stromal cell line (HS27a). For 10 patients, we assessed the capacity of function blocking monoclonal antibodies directed against a panel of 11 adhesion molecules to inhibit adhesion of purified primary AML blasts to HS27a, allowing functional identification of previously undescribed AML-stromal adhesion determinants not only on AML cells but also on BM stroma. Lastly, we successfully grew primary BM stroma from 5 AML patients, assessed production of 10 cytokines important for normal hematopoiesis, and measured the same cytokines directly in plasma of AML patient BM aspirate specimens. Results:Adhesion receptor analysis demonstrates that the mean expression by all AML patients was >90% for CD11a, β1, PECAM-1, CD44, α4, and α5. Comparison of the adhesion receptor profile of the LSC population to the blast population from which they derived showed a 23.1% +/− 7.7% (mean ± standard error) increase in the expression of α6, and a decrease by 0.4% ± 2.5% for CD34+ blasts compared with all blasts (p = 0.04, Student t-test). We found that in our sample of 18 pretreatment BM samples the 15 patients having CR had a higher MFI for αL (p = 0.021), a higher % blasts expressing α4 (p = 0.039) and PECAM1 (p = 0.012), and a lower MFI for α2 (p = 0.029), α6 (p = 0.028), and CXCR4 (p = 0.005). The data for α4 and CXCR4 agree with prior publications demonstrating improved outcomes for high α4β1 expression and poorer outcomes with high CXCR4 expression (Blood 2009; 113:866–74, J Clin Oncol 2010; 28:2831–8, Blood 2007; 109:786–91). Analyzing the functional importance of adhesion receptors for AML-stromal interactions, we found that function blocking antibodies to β1 (p = 0.0003, Student two-sided t-test), CXCR4 (p < 0.0001), and E-cadherin (p < 0.00002) most strongly inhibited AML blast adhesion to HS27a, with mean inhibitions of 71%, 67%, and 63% respectively (Fig 1). [Display omitted] While β1 and CXCR4 represent known factors in AML-stromal adhesion, we propose a novel role for E-cadherin in AML as a potentially novel therapeutic target whose known biology in other malignancies spans cell-cell adhesion and canonical β-catenin/Wnt signaling. The magnitude of the β1 blocking effect proved consistent with its partnering role with multiple adhesion receptors (e.g. α2, α4, α5, and α6). Analyzing the microenvironment cytokine milieu we found normal BM produced G-CSF, SDF-1, and SCF (6, 3001, and 4857 pg/mL respectively) as expected while some AML specimens produced undetectable levels of G-CSF (4/5 plasmas, 4/5 stromas), SCF (4/5 plasmas), or SDF-1 (2/5 plasmas, 3/5 stromas). Conclusions:Among the 6 receptors correlated with response to induction chemotherapy, high α6 (VLA6 laminin receptor) and CXCR4 MFI correlated with poor outcome. The putative LSC subpopulation showed significantly more α6 expression compared with the entire AML blast population.Antibodies against β1, CXCR4, and E-cadherin exhibited the highest function blocking (60–75% range) of AML-stromal adhesion and our AML-stroma functional assay offers a tool to screen for more novel interactions.Cytokine profiling of the AML microenvironment showed deficiencies in cytokines necessary for normal hematopoiesis which may contribute to the clinical cytopenias in AML. Disclosures:No relevant conflicts of interest to declare.

E-cadherin mediated cellular effects and molecular partners. Gastric cancer as an invasive model

Germline mutations of E-cadherin gene (CDH1) occur in 30% of families with Hereditary Diffuse Gastric Cancer (HDGC); of these, 23% are missense mutations. The CDH1 missense mutations described to date span the entire gene and most of them are functionally relevant and constitute a invaluable model do study E-cadherin dependent signaling pathways and find new molecular targets for therapy. Recently, we explored the hypothesis that mutations affecting different E-cadherin protein domains have distinct effects on cell motility. To verify this hypothesis we characterized the effect of eleven HDGC CDH1 germline missense mutations (T118R, L214P, G239R, A298T, T340A, P373L, R749W, E757K, E781D, P799R and V832M) on cell motility. Further, we studied their effect on the activation of signaling pathways known to be relevant for cell motility, such as the EGFR signaling. We found that all cell lines expressing extracellular mutant forms of E-cadherin (T118R, L214P, G239R, A298T and P373L) displayed higher EGFR activation than cells expressing wild-type E-cadherin. This phenotype is similar to what we observed for cells negative for E-cadherin, which also displayed increased EGFR phosphorylation in comparison to the wild-type expressing cells. The same pattern was observed for the cells expressing the intracellular mutations within the juxtamembrane region, R749W and E757K, which is in accordance with the motility analysis results. Our results allowed the identification of the E-cadherin domains pivotal for cell motility, further demonstrated a genotype–phenotype correlation, and defined a subset of HDGC cases which may benefit from EGFR inhibitors. The role of E-cadherin in tumor development is well established, with many human carcinomas exhibiting E-cadherin loss at the invasive front. In many invasive carcinomas, the mechanisms leading to the loss of E-cadherin remains elusive. We recently hypothesize that trafficking deregulation has a role in the regulation of E-cadherin missense mutations. We first analyzed the intracellular trafficking of two HDGC-associated missense mutations, R749W and E757K, localized in the justamembranar domain of E-cadherin, know to have higher impact in E-cadherin trafficking. We found that these mutations frequently accumulate in the endoplasmic reticulum. Moreover, treatment of the mutant cells with specific chemical chaperones, restored mutant E-cadherin to the cell membrane and rescued its function. The protein-stabilizing power of chemical chaperones has been show to be effective on rescuing different disease-associated mutant proteins, but the mechanism is not understood. In order to understand and further explore the therapeutic power of such organic molecules in the context of HDGC, be believe its crucial to dissect their mode of action in the regulation of E-cadherin mutants. We are now investigating the mechanism by which the chemical chaperone DMSO rescues mutant E-cadherin expression by analyzing its effect on different known E-cadherin trafficking regulators.

Lateral Cell Membranes and Shunt Resistance in Rabbit Esophageal Epithelium

The structures that contribute to shunt resistance (Rs) in esophageal epithelium are incompletely understood, with 35-40% of Rs known to be calcium-dependent, reflecting the role of e-cadherin. Two calcium-independent candidates for the remaining approximately 60% of Rs have been identified: the glycoprotein matrix (GPM) within stratum corneum of esophageal epithelium, and the lateral cell membranes (LCMs) from neighboring cells. To determine the contribution of GPM and LCMs to Rs, rabbit esophageal epithelium was mounted in Ussing chambers so that transepithelial resistance (R(T)), a marker of Rs, could be monitored during luminal exposure to either glycosidases for disruption of the GPM or to hypertonic urea for separation of the LCMs. Glycosidases had no effect on R(T). In contrast, hypertonic urea reduced R(T), increased fluorescein flux and widened the intercellular spaces. That urea reduced R(T), and so Rs, by widening the intercellular spaces, and not by altering the e-cadherin-dependent apical junctional complex, was supported by the ability of: (a) calcium-free solution to reduce R(T) beyond that produced by urea, (b) hypertonic urea to reduce R(T) beyond that produced by calcium free solution, (c) hypertonic sucrose to collapse the intercellular spaces and raise R(T), and (d) empigen, a zwitterionic detergent, to non-osmotically widen the intercellular spaces and reduce R(T). These data indicate that the LCMs from neighboring cells are a major contributor to shunt resistance in esophageal epithelium. As resistor, they are distinguishable from the apical junctional complex by their sensitivity to (luminal) hypertonicity and insensitivity to removal of calcium.

Abstract 2336: E-cadherin re-expression affects the growth and survival of breast cancer cells in metastatic colonization of the liver

Abstract Numerous studies have shown that loss of E-cadherin through an epithelial to mesenchymal transition (EMT) contributes to tumor invasiveness. However, these studies focus only on the role of E-cadherin in initial dissemination. Interestingly, histopathology of metastases often appear more differentiated than the primary tumor, and several studies have shown that metastases often express E-cadherin despite loss from the primary tumor. We hypothesize that breast cancer cells re-express E-cadherin through a mesenchymal to epithelial transition (MET) to enable colonization within the liver, a common site of breast cancer metastases. We have previously shown that coculture of E-cadherin-negative MDA-MB-231 breast cancer cells with hepatocytes results in re-expression of E-cadherin, which leads to both ligation between cancer cells and hepatocytes as well as activation of the Erk and Akt signaling pathways. To explore the role of E-cadherin in hepatic colonization, we stably transfected full-length E-cadherin and E-cadherin shRNA constructs into MDA-MB-231 (231) cells. Wild-type 231 and single cell clones of 231-Ecad and 231-shEcad cells were seeded onto a hepatocyte monolayer and cultured for 5 days. Cancer cells were RFP-labeled and the number of cancer cells in each coculture was counted daily. Expression of E-cadherin affected the initial attachment of cancer cells to the hepatocyte monolayer, as twice as many 231-Ecad cells were counted 1 day after seeding compared to their E-cadherin-negative counterparts. In addition, wild-type 231 and 231-shEcad cells initially exhibited similar growth curves but began to diverge after 3 days of coculture, with 231 cells expanding twice as fast as 231-shEcad cells. The divergence coincides with a time course of E-cadherin re-expression. After 5 days of coculture, E-cadherin expression was observed by immunofluoresence in 231 and 231-Ecad cells but not in 231-shEcad cells as expected. Immunostaining also revealed that despite E-cadherin re-expression, vimentin expression persisted in 231 cells, suggesting that E-cadherin re-expression may not result in a full MET. Because E-cadherin-based adhesions have been shown to protect against cell death, all three cell lines were treated with staurosporine in the absence of hepatocytes and assayed for cell survival with calcein AM staining. 231-Ecad cells were more chemoresistant compared to 231 cells, and addition of an E-cadherin antibody abrogated this protection. Future studies will evaluate whether E-cadherin re-expression has a chemoprotective role in hepatocyte coculture and in vivo animal models. The role of E-cadherin down-regulation as a key initiator of metastasis has been clearly established. However, our work suggests that phenotypic plasticity of the metastatic cancer cell through E-cadherin re-expression may also be important for colonization and survival at distant sites. 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 2336.

Abstract 1107: Mutant p53 increases epithelial mesenchymal transition, estrogen receptor alpha and erbB2/Her2/neu

Abstract OBJECTIVES.- Progression of carcinomas involves transition from an epithelial cobblestone-like cytoarchitecture to an elongated, mesenchymal phenotype., leading to an epithelial to mesenchymal transdifferentiation (EMT) (1). Since the p53 tumor suppressor pathway is inactivated in most human cancers due to gene mutations or dysfunction, we now investigated in human wt p53 breast carcinoma cells, whether EMT and cell proliferation are influenced by introducing a dominant-negative (DN) R175H mutant p53 (2). This mutation occurring in the p53 zinc-binding domain, is common to breast cancer patients poorly responsive to therapy (2) METHODS.- .- Fluorescence microscopy was used to determine EMT-linked decreases in E-cadherin and cytokeratin 18. These changes and others associated with EMT, were verified by immune blotting. Laser scanning cytometry was used to measure expression of ErbB2 and estrogen receptor alpha, cell cycle profile and proliferation (2). RESULTS.- Cells transduced with the DN-R175H mutant p53, increased expression of the mesenchymal marker vimentin and proliferation-associated cyclin A, also showing a greater G2 population which correlated with increased c-neu/Her2/ErbB2. Joint cytometric analysis of the Ki67proliferation marker and estrogen receptor alpha revealed a greater number of estrogen receptor-positive proliferating cells (3) among the mesenchymal population harbouring the DN-R175H mutant p53 .CONCLUSIONS.- These results suggest that EMT induced by the DN-R175H mutant p53 promotes a gain of pro-oncogenic function by increasing the % of proliferating cells expressing estrogen receptor alpha (3) and the expression of erbB2 (4) REFERENCES 1.-Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009 119: 1420-8. 2.- Rieber M, Strasberg Rieber M. DN-R175H p53 mutation is more effective than p53 interference in inducing epithelial disorganization and activation of proliferation signals in human carcinoma cells : role of E-cadherin. Int J Cancer. 2009; 125:1604-12. 3.- Shoker BS, Jarvis C, Clarke RB, Anderson E, Hewlett J, Davies MP, Sibson DR, Sloane JP. Estrogen receptor-positive proliferating cells in the normal and precancerous breast. Am J Pathol 1999, 155:1811-1815. 4.- Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B, Brisken C, Minucci S, Di Fiore PP, Pelicci PG. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell. 2009; 138:1083-95. 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 1107.

Abstract 3379: Role of E-cadherin in radiation resistance of human breast cancer cells

Abstract E-cadherin is the major adhesion protein associated with epithelial cells loss of its expression is diagnostic of the epithelial to mesenchymal transition (EMT). Our preliminary data suggest that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which typically are relatively radiosensitive. To definitively test this hypothesis we compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) were more sensitive to increasing doses of radiation and had high expression of E-cadherin. Treatment of MCF-7 cells with 100nM siRNA to E-cadherin for 72 hrs made the cells more resistant to radiation with the surviving fraction changing from 42.3% in control cells to 56.4% in siRNA treated cells. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 cells with a CDH1-expression vector and isolated stable clones. These clones were selected and tested for radiosensitivity. MDA-MB-231 cells transfected with a control vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized the cells compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. Thus, there was a general correlation between EMT, based on loss of E-cadherin expression, and radioresistance. Overall, our results suggest that E-cadherin interacts with radiation and enhances the radioresponse of human breast cancer cells. A detailed investigation of the mechanism is ongoing. 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 3379.

Abstract 1776: Lymphovascular tumor emboli exhibit a quiescent state refractory to chemotherapy due to the selective proteolytic processing of E-cadherin

Abstract Previous studies with a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated that overexpressed E-cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super-high density in vitro. In vitro, cells in MARY-X spheroids grow very slowly and can not be cultured as attached monolayers. Our previous data showed that exogenous growth factors could not stimulate the growth of the spheroids in vitro. Whereas treatment with EGF, IGF-1 or TGF-β increases the level of phosphorylated Akt in numerous cell lines which grow as monolayers, treatment of intact MARY-X spheroids with these growth factors actually decreases the levels of phosphorylated Akt. With EGF treatment, while the levels of phosphorylated EGFR increased in the spheroids, indicating that the treatment with EGF results in phosphorylation of its receptor, there is no downstream signaling through Akt. However when the cells within the MARY-X spheroids are dissociated into single cells by a number of different maneuvers, EGF treatment both phosphorylates its receptor and increases the levels of phosphorylated Akt. The results with the other growth factors are similar. It had been reported that E-cadherin-mediated adhesion may inhibit ligand-dependent activation of diverse growth factor receptor tyrosine kinases by decreasing receptor mobility and / or ligand-binding affinity. Because of the uniqueness of the MARY-X model and the role of E-cadherin in possibly regulating this growth factor signaling, we decided to examine E-cadherin more closely. In both MARY-X and the MARY-X spheroids, Western blot revealed 5 E-cadherin bands: one full length (120 kDa) and four fragments: E-cad/NFT1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa), E-cad/NTF4 (80kDa). Compared to MARY-X, NFT1 was dramatically increased in the MARY-X spheroids whereas NFT2-4 were significantly decreased. These E-cadherin fragments were produced by specific proteolytic cleavage based on inhibitor studies: NFT1 was produced by calpain, NFT2 by γ-secretase, NFT3 by a matrix metalloproteinase and NFT4 by an unknown mechanism. Predictably levels of calpain activity were significantly increased in the MARY-X spheroids. NFT1 generated by increased calpain activity retained the p120ctn binding site but lost both the β-catenin and α-binding sites, facilitating its 360° distribution around the cell. Calpain inhibition decreased NFT1 without any affect on full length E-cadherin yet induced disadherence of intact spheroids and prevented the assembly of de novo spheroids. Both of these manipulations converted the cells from being growth factor refractory to growth factor sensitive. The selective proteolytic processing of E-cadherin ultimately then is responsible for the state of quiescence exhibited by the MARY-X spheroids and the associated lymphovascular emboli. 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 1776.

Abstract 5650: Silibinin inhibits epithelial to mesenchymal transition in prostate cancer cells: Role of E-cadherin and beyond

Abstract Prostate cancer (PCA) is second leading cause of cancer-related deaths in US men. High morbidity and mortality in PCA patients are due to its metastatic spread to distant organs. Epithelial to mesenchymal transition (EMT) in PCA cells is considered a pre-requisite for acquiring advanced migratory/invasive phenotype, and for subsequent metastasis. Even though multiple signaling mechanisms and cellular changes are involved in EMT, loss of E-cadherin is considered as the single most important biological event in PCA cells for acquiring an invasive mesenchymal phenotype. Accordingly, we hypothesized that promoting E-cadherin expression in PCA cells using non-toxic phytochemicals, such as silibinin, would prevent EMT and subsequent metastasis. Recently, we have established strong efficacy of silibinin against PCA cells and currently it is in phase II clinical trial in PCA patients, but its efficacy and mechanism against EMT in PCA cells are largely unknown. Results of present study showed that silibinin strongly inhibits migratory potential of quiescent PC3 and PC3MM2 PCA cells in wound healing assay (P less than 0.001); and invasive potential of PC3 (by 11-66%), PC3MM2 (by 11-61%) and C4-2B (by 32-71%) cells in transwell matrigel invasion assay. Importantly, cell viability assay confirmed that anti-invasive effect of silibinin was not due to its cytotoxicity. Immunoblot analysis showed that silibinin increases E-cadherin expression in PC3 cells in a dose and time dependent manner, which was at cellular membrane as evidenced by sub-cellular fractional and confocal analyses. Silibinin treatment also decreased the level of transcriptional repressors (Snail1, Slug and ZEB1) and increased E-cadherin mRNA expression. Silibinin treatment also decreased the level of phosphorylated Src and Hakai (an E-3 ubiquitin ligase) suggesting post-translational stabilization of E-cadherin. In other studies, silibinin decreased nuclear and cytoplasmic level of beta-catenin but not in cellular membrane. Morphological examination supported these molecular alterations as silibinin treated PC3 cells were more adherent. Similar E-cadherin increase, beta-catenin decrease and morphological changes with silibinin were also observed in PC3MM2 and C4-2B cells. Next, we generated PC3 cells with stable knock-down of E-cadherin expression (ShEC-PC3) and respective control cells (Sh-PC3). Employing these cells, we found that the anti-migratory/anti-invasive potential of silibinin is only partially dependent on E-cadherin. Since molecules, other than E-cadherin, affected by silibinin treatment (beta-catenin, Snail1, Slug and ZEB1) could also directly influence EMT-related changes, our findings suggest multiple molecular targets of silibinin in its anti-EMT and anti-migratory/anti-invasive potential. Overall, the results of present study suggest silibinin usefulness against PCA metastasis. 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 5650.

Abstract 5179: Inflammatory breast cancer cell motility: Understanding the role of E-cadherin and gli1 in IBC migration and the development of a novel high content assay to assess cell migration

Abstract Inflammatory Breast Cancer (IBC) is a highly aggressive subtype of breast cancer and is now regarded as clinicopathologically distinct from neglected locally advanced breast cancer (LABC). In addition, African-Americans, native Americans and younger women are disproportionately affected with IBC having the worse survival. The molecular mechanisms underlying the invasiveness of IBC are not clear. Interestingly, upregulation of the integral membrane protein E-cadherin is the marker reported to be seen most consistently in IBC. In contrast to IBC, E-cadherin is typically reduced in most cancers and the subsequent loss in cell-cell adhesion results in the rapid progression to an invasive and metastatic phenotype. Inactivation of the E-cadherin cell adhesion system can occur by a number of genetic and epigenetic mechanisms. Gli1, the main transcriptional activator of hedgehog signaling, can promote loss of E-cadherin. Gli1 induces expression of Snail which is then found to downregulate E-cadherin expression. In an IBC cellular model, SUM149, we have identified elevated levels of Gli1. Further, inhibition of Gli1 using siRNA caused apoptosis in these cells. We hypothesize that the invasiveness of IBC cells is modulated in part by a connection between E-cadherin and Gli1 levels in IBC and that modulating this pathway will lead to changes in IBC cell motility. This project has two main objectives: 1), Investigate the expression levels of E-cadherin and Gli1 in IBC cells and their roles in IBC motility, and 2), Develop and validate a high content microscope-based assay for measuring motility of IBC cells. Cell motility assays were performed in six-well plates with Gli1 siRNA treated SUM149 cells that resulted in reduced motility and viability of these cells compared to control siRNA treated cells. These results suggest that targeting Gli1 can reduce IBC's invasiveness. This pathway provides opportunities for identifying molecules that might affect tumor invasiveness. We have also developed a novel live cell high content phenotypic screen for modulators of IBC cell motility that may potentially lead to new therapeutics for IBC. An understanding of how any of these molecules affect the Gli-Snail-E-cadherin system would provide useful tools for better probing the role of this pathway in IBC. Furthermore, the high content migration phenotypic assay should be generally applicable to investigating a range of invasive cancer types. 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 5179.

Role of E-Cadherin in Epithelial Architecture Maintenance

Morphogenesis and architecture of a developing epithelium is controlled by both cell shape and contacts, mediated by spatially and temporally regulated cell adhesion molecules. The authors study if E-cadherin functions as a key factor of epithelial adhesion and epidermal architecture in vivo. They apply whole-mount digital deconvolution microscopy to evaluate three-dimensional (3D) E-cadherin expression during skin morphogenesis of Rhinella arenarum and in a cell adhesion alteration model. Results show morphogenetic changes in the 3D E-cadherin spatiotemporal expression pattern correlated with the increase of E-cadherin and in the number of cells with hexagonal geometry. Alterations in junction-protein phosphorylation showed drastic loss of E-cadherin and beta-catenin in cell-cell contacts and the increase of cytoplasm and nuclear beta-catenin in epidermis, suggesting the activation of the beta-catenin signal pathway. Surprisingly, no changes in cell shape and skin architecture were registered, suggesting that epidermal E-cadherin appears to be involved in signaling rather than cell contact maintenance in vivo.

Nature meets nurture: molecular genetics of gastric cancer

The immensity of genes and molecules implicated in gastric carcinogenesis is overwhelming and the relevant importance of some of these molecules is too often unclear. This review serves to bring us up-to-date with the latest findings as well as to look at the larger picture in terms of how to tackle the problem of solving this multi-piece puzzle. In this review, the environmental nurturing of intestinal cancer is discussed, beginning with epidemiology (known causative factors for inducing molecular change), an update of H. pylori research, including the role of inflammation and stem cells in premalignant lesions. The role of E-cadherin in the nature (genotype) of diffuse gastric cancer is highlighted, and finally the ever growing discipline of SNP analysis (including IL1B) is discussed.

The role of N-acetylglucosaminyltransferase III and V in the post-transcriptional modifications of E-cadherin

It has long been recognized that E-cadherin dysfunction is a major cause of epithelial cell invasion. However, very little is known about the post-transcriptional modifications of E-cadherin and its role in E-cadherin mediated tumor progression. N-acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, and has been pointed as a metastasis suppressor. N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6 GlcNAc branching of N-glycans, and has been associated to increase metastasis. The regulatory mechanism between E-cadherin expression and the remodeling of its oligosaccharides structures by GnT-III and GnT-V were explored in this study. We have demonstrated that wild-type E-cadherin regulates MGAT3 gene transcription resulting in increased GnT-III expression. We also showed that GnT-III and GnT-V competitively modified E-cadherin N-glycans. The GnT-III knockdown cells revealed a membrane de-localization of E-cadherin leading to its cytoplasmic accumulation. Further, the GnT-III knockdown cells also caused modifications of E-cadherin N-glycans catalyzed by GnT-III and GnT-V. Altogether our results have clarified the existence of a bidirectional crosstalk between E-cadherin and GnT-III/GnT-V that was, for the first time, reproduced in an in vivo model. This study opens new insights into the post-transcriptional modifications of E-cadherin in its biological function, in a tumor context.

Presenilin 1/γ-Secretase Is Associated with Cadmium-Induced E-Cadherin Cleavage and COX-2 Gene Expression in T47D Breast Cancer Cells

Cadmium is a heavy metal that has multiple toxic effects on human health and has been classified as a human carcinogen. E-cadherin is a major target of cadmium; however, the roles of E-cadherin and cadmium and the mechanisms of tumor progression remain to be defined. Here, we demonstrate that cadmium increases E-cadherin processing via a gamma-secretase in the T47D breast cancer cell lines. This presenilin 1 (PS1)/gamma-secretase-dependent cleavage of E-cadherin was accompanied by changes in reactive oxygen species or calcium. E-cadherin cleavage was blocked by a PS1 dominant-negative mutant, gamma-secretase inhibitors [N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and L-685,486], antioxidants (N-acetylcysteine and Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride), or a calcium chelating drug 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester. Immunofluorescence analysis confirmed the disappearance of E-cadherin staining at the cell surface. Those inhibitors attenuated cadmium-induced cytotoxicity. Additionally, cadmium treatment increased cell motility and invasion ability, which was abated by DAPT. Interestingly, cyclooxygenase-2 (COX-2) expression induced by cadmium was also inhibited by DAPT. The cadmium-induced cell motility and invasion ability were inhibited by a COX-2 inhibitor, NS398. Our data indicate a novel molecular mechanism that links cytotoxicity of cadmium and disrupted E-cadherin processing to adherens junctions; cadmium induces COX-2 expression via gamma-secretase, which increases cell motility and invasion ability. Understanding the downstream signaling cascades of cadmium that promote tumor progression might be a key to the development of novel therapeutic strategies.

Molecular prognostic markers in squamous cell carcinoma of the head and neck: the role of E-cadherin

6040 Background: E-cadherin is a transmembrane glycoprotein which is localized on the surface of epithelial cells and mediates adhesion through Ca2+-dependent homotypic binding. Downregulation of E-cadherin expression correlates with increased invasiveness and metastasis in carcinomas of several anatomic sites. In the present study we investigated the relationship between membranous E-cadherin expression and outcome in patients with head and neck squamous cell carcinoma (HNSCC). Methods: A tissue microarray including 102 specimens from primary HNSCC cases was constructed. Protein expression levels for E-cadherin were analyzed using a system of in-situ protein expression analysis (AQUA). AQUA uses molecular methods to define subcellular compartments. It then quantifies the amount of protein expressed within the compartment by co-localization. Primary endpoints of this study were progression-free survival (PFS) and overall survival (OS). Survival analysis was performed by Kaplan-Meier method with log-rank for assessing statistical significance. Results: Mean follow-up time for the cohort was 36 months. Seventy of 102 (68.6%) cases had sufficient tissue for AQUA analysis. In univariate analysis, the top quartile of membranous E-cadherin expression was associated with improved PFS (p=0.017) and OS (p=0.05). In multivariable Cox regression, high membranous E-cadherin expression was an independent predictor for better OS at 5 years. Conclusions: Our results indicate that high membranous E-cadherin expression levels as assessed by AQUA are predictive of improved survival rates. No significant financial relationships to disclose.

Absence of E-Cadherin Expression Distinguishes Noncohesive from Cohesive Pancreatic Cancer

The role of E-cadherin in carcinogenesis is of great interest, but few studies have examined its relevance to pancreatic carcinoma. We evaluated E-cadherin protein expression by immunohistochemistry in pancreatobiliary cancers having a noncohesive histologic phenotype (21 undifferentiated adenocarcinomas and 7 signet ring carcinomas), comparing the results with pancreatic cancers having a cohesive phenotype (25 moderately differentiated and 14 poorly differentiated adenocarcinomas). Twenty of 21 undifferentiated cancers had complete absence of E-cadherin expression, as did two signet ring carcinomas. In contrast, cohesive cancers (n = 39) had E-cadherin labeling at the plasma membrane (P < 0.001). Subsets of cancers were also evaluated for beta-catenin expression. All of the cohesive lesions (n = 28) showed a membranous beta-catenin expression pattern, whereas noncohesive foci (n = 7) were characterized by either cytoplasmic labeling or complete absence of beta-catenin protein expression, suggestive of a deficient zonula adherens in noncohesive cancers. E-cadherin promoter hypermethylation was observed in an undifferentiated pancreatic cancer cell line, MiaPaCa-2, whereas two pancreatic cancer cell lines derived from differentiated lesions lacked any evidence of E-cadherin promoter methylation. No pattern of E-cadherin promoter methylation could be determined in three primary cancers having mixed histologic patterns (contained both cohesive and noncohesive foci). No somatic mutations in E-cadherin were identified in noncohesive pancreatic cancers having inactivated E-cadherin. Noncohesive pancreatic cancers were characterized by the loss of E-cadherin protein expression. Promoter hypermethylation is a possible mechanism of E-cadherin gene silencing in a subset of these cancers.

Role of E-cadherin in the induction of apoptosis of HPV16-positive CaSki cervical cancer cells during multicellular tumor spheroid formation

Multicellular tumor spheroids (MCTS) are three dimensional cell culture systems induced by suspension culture. MCTS are widely used in cancer research because of their similarity to solid tumors. CaSki cells are derived from a metastatic cervical cancer containing human papillomavirus 16 (HPV16). Cell death of CaSki cells in MCTS has been previously reported, and our model is used to better characterize the mechanisms of cell death of HPV16-positive keratinocytes. In this study, we found that apoptosis of CaSki cells was induced by suspension culture along with the formation of MCTS after 24 h of incubation. In suspended CaSki cells, monoclonal antibodies blocking E-cadherin function inhibited MCTS formation and suppressed suspension-induced apoptosis in a dose-dependent manner. Western blot for E-cadherin detected upregulation of the authentic 120 kDa band from MCTS of CaSki cells as well as a shorter 100 kDa band. Addition of EGF, whose receptor is known to form a complex with E-cadherin, abrogated apoptosis of suspended CaSki cells in a dose-dependent manner. These findings suggest that E-cadherin-dependent cell-cell contact, directly or indirectly, mediates the signal to undergo apoptosis of CaSki cells during MCTS formation, and thus provides new information on the role of E-cadherin in cervical cancer cell apoptosis.