Overexpression Of E-cadherin Research Articles

Abstract P5-04-05: E-Cadherin is Required for In Vivo Growth of Inflammatory Breast Cancer: Importance of Mesenchymal-Epithelial Transition (MET) and Role of HIF1α

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and deadly form of breast cancer and is phenotypically distinct from other forms of locally advanced breast cancer. The 5-year survival for IBC patients (40%) has not improved even with the implementation of multi-disciplinary care and targeted therapies (e.g. anti-HER2 or anti-estrogen therapies). Lymph node involvement is common at first diagnosis and the accelerated metastasis program that characterizes IBC account for a disproportionate number of the 40,000 women who will die from breast cancer. The most common molecular marker for IBC is the cell adhesion surface glycoprotein, E-Cadherin. Classically, E-Cadherin is thought to be a tumor suppressor and its loss is associated with the epithelial-mesenchymal transition (EMT) and acquisition of an invasive phenotype. The importance of EMT in metastasis is well accepted in numerous tumor models although the presence of EMT in patient samples is rarely demonstrated. Given the propensity of IBC to metastasize, one would expect a strong EMT signature in IBC cell lines, however the persistent robust expression of E-Cadherin in IBC patient tumors and in IBC cell lines, with a lack of expression of ZEB1, a transcriptional repressor of E-cadherin and diminished expression of genes within the transforming growth factor beta signaling pathway, suggests otherwise. Methods: To further define the role of E-Cadherin in IBC, we used the SUM149 IBC cell line to manipulate E-Cadherin via shRNA knockdown and by overexpression of ZEB1 a known transcriptional repressor of E-cadherin. Results: While the lack of E-Cadherin or ZEB1 overexpression in SUM149 cells led to an EMT phenotype in vitro, E-Cadherin was required for in vivo growth of SUM149 tumor cells. Gene profiling identified novel E-Cadherin signaling pathways including an hypoxia gene signature through HIF1a as well as cytokine responsive pathways. The requirement of HIF-1a for in vivo growth of SUM149 cells was confirmed by shRNA knockdown of HIF-1a. Conclusions: The absolute requirement for E-Cadherin and maintenance of the mesenchymal-epithelial transition in IBC tumor growth is demonstrated. Furthermore, a novel functional link between E-Cadherin and HIF-1a and in their critical role in survival of IBC tumors is identified for the first time and warrants further investigation. Supported in part by The Susan G Komen Organization Promise Grant KG081287 (FMR and MC) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-05.

IS2-1 - Molecular Changes and Targeted Therapy in Inflammatory Breast Cancer

ABSTRACT Inflammatory breast cancer (IBC) is one of the most aggressive subtypes of breast cancer. IBC represents 2%–4% of breast cancer, but it represents 10% of breast cancer-related mortality. Despite multimodality approaches, the prognosis for patients with this disease remains poor. Although we are making efforts to improve treatment for IBC, IBC research remains to be limited because this is a rare disease and there are few defined targets for IBC due to undefined IBC biology. To improve this situation, we require insights from clinical trials and epidemiologic studies investigating IBC biology. Molecular targets such as vasculolymphatic, proliferation, and metastatic process has been identified, but all of these unique targets require clinical validation by clinical trials. Molecular targets in cell proliferation processes have been investigated the most and have yielded promising clinical results leading to improved outcomes for patients with IBC. These cell proliferation targets include HER2 and EGFR. As was the case in non-IBC, the HER2 antibody trastuzumab dramatically changed the prognosis of patients with HER2-overexpressing IBC. Lapatinib, which inhibits both HER2 and EGFR, also showed benefit in patients with HER2-positive breast cancer, including IBC, in clinical trials. Additional cell proliferation targets—WISP3, RhoC GTPase, and p27kip1—have also been studied in IBC. Molecules involved in metastasis processes may have a unique role in IBC owing to the aggressiveness of this disease. E-cadherin has been observed to be overexpressed in IBC, and loss of E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT). Although this E-cadherin overexpression might be a transient phenomenon, we may be able to target this unique feature. Other potential candidate is the EGFR pathway. We have been investigating the role of this pathway and the possibility of EGFR-targeted therapy for IBC. We are conducting a clinical trial with panitumumab, a human EGFR antibody, plus chemotherapy (nab-paclitaxel and carboplatin) as a preoperative regimen in patients with HER2-negative IBC. In summary, further extensive preclinical and clinical work based on molecular findings is needed to improve outcomes in patients with IBC.

Targeting a Novel Onco-glycoprotein Antigen at Tumoral Pancreatic Cell Surface by mAb16D10 Induces Cell Death

The mAb16D10 was raised against a pathological onco-glycoform of bile salt-dependent lipase isolated from the pancreatic juice of a patient suffering from a pancreatic adenocarcinoma. We previously showed that mAb16D10 specifically discriminates human pancreatic tumor tissues from other cancer and nontumor tissues. In this study, we report that mAb16D10 inhibited the proliferation of only human pancreatic tumor cells expressing 16D10 plasma membrane Ag. Interaction of mAb16D10 with its cognate surface Ag on pancreatic cells promoted cell death by activation of the p53- and caspase-dependent apoptotic pathway, and silencing of p53 decreased cell death. The decreased proliferation was also partly due to cell cycle arrest in G1/S phase, mAb16D10 triggering of glycogen synthase kinase-3β (GSK-3β) activation, degradation of β-catenin, and decreased expression of cyclin D1. GSK-3β positively affected p53 expression in pancreatic tumor cells after mAb16D10 binding. Inhibition of GSK-3β activity reversed the effects induced by mAb16D10 in SOJ-6 cells, supporting the pivotal role of GSK-3β signaling in the mechanisms of action induced by mAb16D10. Also, mAb16D10 cell treatment led to membrane overexpression of E-cadherin. Both E-cadherin and tumor Ag were localized in membrane lipid cholesterol-rich microdomains and are thought to belong to signaling platforms involved in the induction of cell cycle arrest and cell death. Overall, this study reveals that mAb16D10 holds great potential to prevent pancreatic tumor proliferation by apoptotic cell death, thus promising therapeutic prospects for treatment of pancreatic adenocarcinoma, a highly lethal disease.

E-cadherin promotor methylation and mutation are inversely related to motility capacity of breast cancer cells

Inactivation of the tumor suppressor E-cadherin is an important event during breast tumorigenesis, as its decreased expression is linked to aggressiveness and metastasis. However, the relationship between the different modes of E-cadherin inactivation (mutation versus promotor hypermethylation) and breast cancer cell behavior is incompletely understood. The high correlation between E-cadherin inactivation status and cell morphology in vitro suggests different biological roles for the two inactivation modes during breast tumorigenesis. Because E-cadherin has been linked to cell invasion and metastasis, and cell motility is a crucial prerequisite to form metastases, we here compared the cell motility capacities of breast cancer cell lines with known E-cadherin status. Using barrier migration assays and time-lapse microscopy, we analyzed the migratory capacity of nine well-characterized human breast cancer cell lines (MDA-MB-231, MCF-7, T47D, BT549, MPE600, CAMA-1, SUM159PT, SUM52PE, and SK-BR-3). This subset was chosen based on E-cadherin gene status (wild-type, mutated, and promotor hypermethylated): three cell lines of each group. In addition, cell proliferation assays were performed for all conditions, to dissect migratory from proliferative effects. In this study, we demonstrate an overt association between the mode of E-cadherin inactivation and cell migration. Promotor hypermethylated E-cadherin cell lines showed a higher migration capacity, while cell lines with mutated E-cadherin were less motile compared to wild-type E-cadherin cell lines. Migration induction by fibronectin and basic fibroblast growth factor did not alter the cell motility association differences. Cell proliferation assays showed that the associations found were not caused by proliferation differences. Inhibition and overexpression of E-cadherin as well as DNA demethylation confirmed the relationship between E-cadherin and breast cancer cell motility. Our results demonstrate an association between the mode of E-cadherin inactivation and migration of breast cancer cells, which justifies more detailed research on the role of E-cadherin inactivation in cell migration and metastasis.

Spherical Bullet Formation via E-cadherin Promotes Therapeutic Potency of Mesenchymal Stem Cells Derived From Human Umbilical Cord Blood for Myocardial Infarction

The beneficial effects of stem cells in clinical applications to date have been modest, and studies have reported that poor engraftment might be an important reason. As a strategy to overcome such a hurdle, we developed the spheroid three dimensional (3D) bullet as a delivery method for human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) through the maintenance of cell-cell interactions without additional xenofactors, cytokines, or matrix. We made spheroid 3D-bullets from hUCB-MSCs at 24 hours' anchorage-deprived suspension culture. To investigate the in vivo therapeutic efficacy of 3D-bullets, we used rat myocardial infarction (MI) model. Transplantation of 3D-bullet was better than that of single cells from monolayer culture or from 3D-bullet in improving left ventricular (LV) contractility [LV ejection fraction (LVEF) or LV fractional shortening (LVFS)] and preventing pathologic LV dilatation [LV end-systolic diameter (LVESD) or LV end-diastolic diameter (LVEDD)] at 8 weeks. In the mechanism study of 3D-bullet formation, we found that calcium-dependent cell-cell interaction was essential and that E-cadherin is a key inducer mediating hUCB-MSC 3D-bullet formation among several calcium-dependent adhesion molecules which were nominated as candidates after cDNA array analysis. In more specific experiments with E-cadherin overexpression using adenoviral vector or with E-cadherin neutralization using blocking antibody, we found that E-cadherin regulates vascular endothelial growth factor (VEGF) secretion via extracellular signal-regulated kinase (ERK)/v-akt murine thymoma viral oncogene homolog1 (AKT) pathways. During formation of spheroid 3D-bullets, activation of E-cadherin in association with cell-cell interaction turns on ERK/AKT signaling pathway that are essential to proliferative and paracrine activity of MSCs leading to the enhanced therapeutic efficacy.

Preventive effect of Curcumin on AD through increasing PS1/E-cadherin/beta-catenin complex mediated by E-cadherin

Background The deprivation or abnormality of the molecular function of Wnt/b-catenin triggers the genesis and development of AD, and b-catenin is an important positive mediator in the Wnt/b-catenin signaling pathway. In our previous study, we found that Curcumin could inhibit the expression of b-catenin and prevent AD, but the mechanisms were not fully understood. E-cadherin is a negative mediator in the Wnt/b-catenin signaling pathway, and interacts with b-catenin and PS1 form a trimeric complex, so, we hypothesized that Curcumin prevented AD through increasing PS1/E-cadherin/bcatenin complex by overexpression of E-cadherin.

E-cadherin inhibits nuclear accumulation of Nrf2: implications for chemoresistance of cancer cells

Nrf2 has an anti-carcinogenic effect. However, an increase in Nrf2 activity is also implicated in cancer chemoresistance. A switch from E-cadherin to N-cadherin affects the transdifferentiation and metastasis of cancer cells. In view of the key role of this switch in cancer malignancy, we investigated the regulatory effect of E-cadherin on Nrf2. In HEK293 cells, overexpression of E-cadherin inhibited the nuclear accumulation of Nrf2, and prevented Nrf2-dependent gene induction. GST pull-down and immunocytochemical assays verified the interaction between E-cadherin and Nrf2: E-cadherin bound the C-terminus of Nrf2, but not its N-terminus, which comprises the Neh2 domain responsible for phosphorylation of Ser40. Our finding that the mutation of Ser40 to alanine in Nrf2 did not affect the ability of E-cadherin to bind Nrf2 and repress target gene transactivation suggests that E-cadherin might not disturb the phosphorylation. Studies using mutant constructs of E-cadherin suggested that the β-catenin-binding domain contributes to the inhibitory effect of E-cadherin on Nrf2. Consistently, knockdown of β-catenin attenuated not only the effect of E-cadherin binding to Nrf2, but also Keap1-dependent ubiquitylation of Nrf2, and thereby increased Nrf2 activity, supporting the involvement of β-catenin in the interactions. Collectively, E-cadherin recruits Nrf2 through β-catenin, and assists the function of Keap1 for the inhibition of nuclear localization and transcriptional activity of Nrf2. In HepG2 cells, the loss of E-cadherin by either siRNA knockdown or treatment with TGFβ1 enhanced the constitutive or inducible activity of Nrf2, implying that chemoresistance of cancer cells upon the loss of E-cadherin might be associated with Nrf2.

S100A4 silencing blocks invasive ability of esophageal squamous cell carcinoma cells

To investigate a potential role of S100A4 in esophagus squamous cell carcinoma metastasis (ESCCs). Expression of S100A4 and E-cadherin were analyzed in frozen sections from ESCCs (metastasis, n = 28; non-metastasis, n = 20) by reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction and immunohistochemistry. To explore the influence of S100A4 on esophageal cancer invasion and metastasis, S100A4 was overexpressed or silenced by S100A4 siRNA in TE-13 or Eca-109 cells in vitro and in vivo. We found the mRNA and protein levels of S100A4 expression in ESCCs was significantly upregulated, and more importantly, that expression of S100A4 and E cadherin are strongly negatively correlated in patients who had metastasis. It was indicated that overexpression of S100A4 in TE-13 and Eca-109 cells downregulates the expression of E-cadherin, leading to increased cell migration in vitro, whereas knockdown of S100A4 inhibited cell migration and upregulation of E-cadherin expression. Moreover, the loss of cell metastatic potential was rescued by overexpression of E-cadherin completely. In addition, nude mice inoculated with S100A4 siRNA-transfected cells exhibited a significantly decreased invasion ability in vivo. S100A4 may be involved in ESCC progression by regulate E-cadherin expression, vector-based RNA interference targeting S100A4 is a potential therapeutic method for human ESCC.

MDR-1-overexpression in HT 29 colon cancer cells grown in SCID mice

The multidrug-resistance 1 (MDR-1) P-glycoprotein (Pgp) is a transmembrane transporter system, which actively pumps cytotoxic drugs out of the cell. MDR-1 acquired in vitro differs from MDR-1 acquired in vivo, but has important consequences on the cellular phenotype and metastatic behavior. Here we report that the human colonic cancer cell line HT29 (MDR-1 negative) is more malignant than its MDR-1 overexpressing variant (HT29 MDR-1 positive). HT29 MDR-1 negative cells produce undifferentiated signet ring carcinomas when implanted subcutaneously into SCID mice, while HT29 MDR-1 positive cells form tumors with tubular structures, but without signet ring cells. Immunohistochemical proliferation marker analysis revealed that the MDR-1 positive cells proliferate much more slowly than the MDR-1 negative cells. MDR-1 overexpression results in a less differentiated phenotype at the cellular level (absence of mucin producing cells) but in a more differentiated phenotype at the tissue level (tubule formation). In addition, lectin binding patterns including that of Helix pomatia agglutinin (HPA), an indicator of metastatic potential, differed between the two cell lines. HT29 MDR-1 positive cells had less HPA binding sites than HT29 MDR-1 negative counterparts and metastasized less frequently in SCID mice. As slow proliferation, low degree of differentiation and multidrug-resistance is a hallmark of cancer stem cells and all were present in MDR-1 positive tumors, it is attractive to speculate that they represent a stem cell rich tumor. As shown by global gene expression analyses, genes involved, e.g. in cell adhesion, glycosylation and signal transduction, were deregulated in MDR-1 positive tumors compared to MDR-negative tumors. Overexpression of E-cadherin and carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1) may provide clues to the mechanisms responsible for the reduced metastatic potential of MDR-1 overexpressing tumors. Since drug treatment shifted the cells towards a less metastatic phenotype in this in vivo model, it seems conceivable to achieve this using drug treatment also in a clinical situation.

E-cadherin impairment increases cell survival through Notch-dependent upregulation of Bcl-2

The role of E-cadherin in tumorigenesis has been attributed to its ability to suppress invasion and metastization. However, E-cadherin impairment may have a wider impact on tumour development. We have previously shown that overexpression of mutant human E-cadherin in Drosophila produces a phenotype characteristic of downregulated Notch. Hence, we hypothesized that Notch signalling may be influenced by E-cadherin and may mediate tumour development associated with E-cadherin deficiency. De novo expression of wild-type E-cadherin in two cellular models led to a significant decrease in the activity of the Notch pathway. In contrast, the ability to inhibit Notch-1 signalling was lost in cells transfected with mutant forms of E-cadherin. Increased Notch-1 activity in E-cadherin-deficient cells correlated with increased expression of Bcl-2, and increased resistance to apoptotic stimuli. After Notch-1 inhibition, E-cadherin-deficient cells were re-sensitized to apoptosis in a similar degree to wild-type E-cadherin cells. We also show that Notch-inhibiting drugs are able to significantly inhibit the growth of E-cadherin-deficient cells xenografted into nude mice. This effect was comparable with the one observed in animals treated with the chemotherapeutic agent taxol, a chemical inducer of cell death. In conclusion, our results show that aberrant Notch-1 activation, Bcl-2 overexpression and increased cell survival are likely to play a crucial role in neoplastic transformation associated with E-cadherin impairment. These findings highlight the possibility of new targeted therapeutical strategies for the treatment of tumours associated with E-cadherin inactivation.

E-Cadherin and β-Catenin Expression in Patients with Nasopharyngeal Carcinoma (NPC) undergoing Curative Intent Radiation ± Platinum Chemotherapy

Aberrant expression of the E-cadherin-catenin cell adhesion complex has been described in various cancers. E-cadherin expression may be up- or down-regulated, depending on tumor type and/or stage of progression. Reduced intercellular adhesion is most frequently associated with tumor invasiveness and metastases; however, amplification of the adhesion axis has been described with lymphovascular emboli/invasion. In human squamous cell carcinoma cell lines, E-cadherin overexpression may also mediate anchorage-independent cell growth and resistance to apoptosis.

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT‐like phenotype change and MMP‐9‐mediated degradation of E‐cadherin

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.

Abstract 4228: Ganoderma lucidum (Reishi) reduces inflammatory breast cancer progression via protein synthesis inhibition

Abstract The anti-cancer effects of the medicinal mushroom Ganoderma lucidum (Reishi) were tested as a potential therapeutic for Inflammatory Breast Cancer (IBC). IBC is a rare, lethal and aggressive form of locally advanced breast cancer that manifests itself without a typical tumor mass. Studies suggest that the mammalian target of rapamycin (mTOR) pathway is a key regulator of cell growth via the regulation of protein synthesis. The mTOR effectors for protein synthesis are eukaryotic initiation factor 4E-binding protein 1 (4EBP-1) and the ribosomal protein S6 kinase (p70S6K). The 4EBP-eIF4E complex exerts control over cap-dependent translation, while p70S6K stimulates eIF4F translation of specific mRNAs with highly structured 5′UTRs, promoting cell growth, and tumor progression. In IBC the eukaryotic initiation factor, eIF4GI is overexpressed, which reprograms protein synthesis for cap-independent translation via increased initiation of internal ribosome entry site (IRES)-dependent translation. eIF4GI increased mRNA translation is partly responsible for the unique IBC pathological properties: overexpression of E-cadherin, strong homotypic IBC cell interaction, and formation of tumor emboli to promote IBC cell invasion. Using the patient derived IBC cell line, SUM 149, we previously demonstrated that Reishi extract selectively inhibits IBC cell growth and invasion by downregulating the expression of genes involved in invasion, survival and proliferation, and the protein expression of E-cadherin. The present study demonstrates that Reishi extract reduces the expression of AKT, which signals to activate the translational machinery by mTOR. Reishi extract reduced the expression of p70s6K and eIF4G, and diminished the expression of the IRES dependent mRNA containing proteins: c-Myc, p120 catenin, vascular endothelial growth factor (VEGF), BCL-2 and BCL-XL. Furthermore, Reishi extract reduced the expression of eIF4E and the phosphorylation of its inhibitor, 4EBP-1. Our results provide evidence that Reishi suppresses protein synthesis potentially by both cap-dependent and cap independent mechanisms. Our findings suggest that Reishi is a potential natural therapeutic that can be used in combination with traditional therapy to treat this intractable and deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4228. doi:10.1158/1538-7445.AM2011-4228

Abstract 5008: Epidermal growth factor downregulates expression of neutrophil gelatinase-associated lipocalin (NGAL) through E-cadherin in pancreatic cancer cells

Abstract Background: Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, is a secreted protein that is upregulated in multiple cancer types. Our group previously reported that NGAL overexpression significantly blocked invasion and angiogenesis in pancreatic ductal adenocarcinoma (PDAC). We and others have also demonstrated loss NGAL expression in advanced stages of PDAC. However, little is known about mechanisms that regulate NGAL expression in PDAC. As EGF-EGFR axis is significantly upregulated in PDAC, we examined EGF-mediated NGAL regulation in these cells. Methods: NGAL-positive AsPC-1 and BxPC-3 cells were used as model system. Quantitative RT-PCR, western blot analysis, and immunofluorescence studies were used to investigate EGF-mediated effects on NGAL expression. E-cadherin expression was manipulated using lentiviral overexpression or shRNA constructs. NGAL promoter activity was assessed by luciferase-reporter assay and electrophoretic mobility shift assay (EMSA). Results: NGAL expression was positively associated with tumor differentiation and was significantly downregulated after EGF treatment along with a concomitant reduction of E-cadherin expression in PDAC cells. E-cadherin downregulation was partly through the EGF receptor (EGFR)-dependent MEK1/2-ERK1/2 signaling pathway, which was demonstrated using pharmacologic inhibitors of EGFR (AG1478) and MEK1/2 (U0126 and PD98059). In addition, E-cadherin downregulation reduced NGAL expression in PDAC cells, whereas overexpression of E-cadherin led to increased NGAL expression and partly rescued inhibition of NGAL expression by EGF. Furthermore, EGF in part through E-cadherin reduced NGAL promoter activity by blocking NF-κB activation. Conclusion: We demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect is partly mediated through activation of the EGFR-MEK1/2-ERK1/2 signaling pathway, which in turn downregulated E-cadherin with a subsequent reduction in NF-κB activation. Our findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2011-5008

E‐cadherin antagonizes TGFbeta1 gene induction in hepatic stellate cells by inhibiting RhoA‐dependent Smad3/2 phosphorylation

Cadherins mediate cell‐cell adhesion and catenin (ctn)‐related signaling pathways. Liver fibrosis is accompanied by the loss of E‐cadherin that promotes the process of epithelial mesenchymal transition. This study investigated whether E‐cadherin overexpression inhibits transforming growth factor‐beta1 (TGFbeta1) gene induction and, if so, what molecular basis. Forced expression of E‐cadherin decreased the level of mesenchymal markers, and the constitutive or inducible expression of the TGFbeta1 gene and its downstream genes. E‐cadherin overexpression decreased Smad3/2 phosphorylations, and consequent its transcriptional activity. The ability of E‐cadherin to inhibit TGFbeta1‐inducible Smad activity was reversed by RhoA activation. Molecular approaches revealed that the p120‐ctn binding domain of E‐cadherin was responsible for TGFbeta1 repression. E‐cadherin was capable of binding p120‐ctn, which recruited RhoA, preventing the ability of TGFbeta1 to increase RhoA‐mediated Smad3 phosphorylation. In the liver tissues of patients with liver fibrosis, E‐cadherin expression reciprocally correlated with the severity of fibrosis. In conclusion, E‐cadherin inhibits Smad3/2 phosphorylations by recruiting RhoA to p120‐ctn, supporting the notion that the loss of E‐cadherin due to cadherin switch promotes TGFbeta1 and target gene induction, and facilitates liver fibrosis.

Chemopreventative Potential of the Cruciferous Vegetable Constituent Phenethyl Isothiocyanate in a Mouse Model of Prostate Cancer

This study was undertaken to determine the chemopreventative efficacy of phenethyl isothiocyanate (PEITC), a bioactive constituent of many edible cruciferous vegetables, in a mouse model of prostate cancer, and to identify potential biomarker(s) associated with PEITC response. The chemopreventative activity of dietary PEITC was investigated in Transgenic Adenocarcinoma of Mouse Prostate mice that were fed a control diet or one containing 3 μmol PEITC/g (n = 21 mice per group) for 19 weeks. Dorsolateral prostate tissue sections were stained with hematoxylin and eosin for histopathologic evaluations and subjected to immunohistochemistry for analysis of cell proliferation (Ki-67 expression), autophagy (p62 and LC3 protein expression), and E-cadherin expression. Autophagosomes were visualized by transmission electron microscopy. Apoptotic bodies were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Plasma proteomics was performed by two-dimensional gel electrophoresis followed by mass spectrometry to identify potential biomarkers of PEITC activity. All statistical tests were two-sided. Administration of PEITC (3 μmol/g diet) decreased incidence (PEITC diet vs control diet, mean = 21.65 vs 57.58%, difference = -35.93%, 95% confidence interval = -45.48% to -13.10%, P = .04) as well as burden (affected area) (PEITC diet vs control diet, mean = 18.53% vs 45.01%, difference = -26.48%, 95% confidence interval = -49.78% to -3.19%, P = .02) of poorly differentiated tumors in the dorsolateral prostate of transgenic mice compared with control mice, with no toxic effects. PEITC-mediated inhibition of prostate carcinogenesis was associated with induction of autophagy and overexpression of E-cadherin in the dorsolateral prostate. However, PEITC treatment was not associated with a decrease in cellular proliferation, apoptosis induction, or inhibition of neoangiogenesis. Plasma proteomics revealed distinct changes in the expression of several proteins (eg, suppression of clusterin protein) in the PEITC-treated mice compared with control mice. In this transgenic model, dietary PEITC suppressed prostate cancer progression by induction of autophagic cell death. Potential biomarkers to assess the response to PEITC treatment in plasma were identified.

Epidermal growth factor down‐regulates the expression of neutrophil gelatinase‐associated lipocalin (NGAL) through E‐cadherin in pancreatic cancer cells

The authors previously reported that neutrophil gelatinase-associated lipocalin (NGAL) overexpression significantly blocked invasion and angiogenesis of pancreatic ductal adenocarcinoma (PDAC). They also demonstrated a loss of NGAL expression in the advanced stages of PDAC. However, little is known regarding the mechanisms of NGAL regulation in PDAC. Because the epidermal growth factor (EGF)-EGF receptor (EGFR) axis is up-regulated significantly in PDAC, they examined EGF-mediated NGAL regulation in these cells. The NGAL-positive cell lines AsPC-1 and BxPC-3 were used as a model system. Quantitative real-time polymerase chain reaction (RT-PCR), Western blot analysis, and immunofluorescence studies were used to investigate EGF-mediated effects on NGAL expression. E-cadherin expression was manipulated using lentiviral overexpression or small hairpin RNA constructs. NGAL promoter activity was assessed by luciferase-reporter assay and electrophoretic mobility shift assay. NGAL expression was positively associated with tumor differentiation and was down-regulated significantly after EGF treatment along with a concomitant reduction of E-cadherin expression in PDAC cells. E-cadherin down-regulation was partly through the EGFR-dependent mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) (MEK-ERK) signaling pathway. In addition, E-cadherin down-regulation reduced NGAL expression in PDAC cells, whereas overexpression of E-cadherin led to increased NGAL expression and partly rescued the inhibition of NGAL expression by EGF. Furthermore, EGF, in part through E-cadherin, reduced NGAL promoter activity by blocking nuclear factor κB (NF-κB) activation. The current study demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect was mediated in part through activation of the EGFR-MEK-ERK signaling pathway, which, in turn, down-regulated E-cadherin with a subsequent reduction in NF-κB activation. These findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC.

Involvement of HDAC1 in E-cadherin expression in prostate cancer cells; its implication for cell motility and invasion

In this study, we investigate the molecular mechanism by which histone deacetylase (HDAC) inhibitors exert anti-invasiveness effect against prostate cancer cells. We first evaluate the growth inhibition effect of HDAC inhibitors in prostate cancer cells, which is accompanied by induction of p21 WAF1 expression and accumulation of acetylated histones. And we found that the migration and invasion of prostate cancer cells is strongly inhibited by treatment with HDAC inhibitors. In parallel, E-cadherin level is highly up-regulated in HDAC inhibitor-treated prostate cancer cells. And siRNA knockdown of E-cadherin significantly diminishes the anti-invasion effect of HDAC inhibitors, indicating that E-cadherin overexpression is one of possible mechanism for anti-invasion effect of HDAC inhibitors. Furthermore, specific downregulation of HDAC1, but not HDAC2, causes E-cadherin expression and subsequent inhibition of cell motility and invasion. Collectively, our data demonstrate that HDAC1 is a major repressive enzyme for E-cadherin expression as well as HDAC inhibitor-mediated anti-invasiveness.

Mechanical Force Affects Expression of an In Vitro Metastasis-Like Phenotype in HCT-8 Cells

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21–47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.

Slug regulates proliferation and invasiveness of esophageal adenocarcinoma cells in vitro and in vivo

Slug is a transcription factor and E-cadherin repressor, which has recently been demonstrated to be important for cancer cells to down-regulate epithelial markers and up-regulate mesenchymal markers in order to become motile and invasive. In the present study, we assessed the relevance of Slug for invasion and growth potential of esophageal adenocarcinoma (EA) cells in vitro and in vivo. Slug expression was detected in nine human esophageal cancer cell lines. OE33 cell line was infected with Slug siRNA to knockdown of Slug; TE7 cell line was infected with full Slug cDNA to increase Slug expression. Then, Bcl-2 and E-cadherin expression and Caspase-3 activity were analyzed. MTT assay was applied to detect growth curve. The flow cytometric and Hoechst33258 staining was performed to detect apoptosis. The cells invasion in vitro was detected with a Boyden chamber. A pseudometastatic model of OE33 and TE7 in immunodeficient mice was used to assess the effects of knockdown of Slug and Slug overexpression on metastasis development. A subcutaneously nude mice xenograft model of OE33 and TE7 was used to assess the effects of knockdown of Slug and Slug overexpression on tumor growth. Immunohistochemical staining was used to analyze the expression of Slug, bcl-2 and E-cadherin, and TUNEL was used to detected apoptosis in vivo. Western blotting and RT-PCR showed that Slug expression was detectable in 7 of 9 human esophageal cancer cell lines. Bcl-2 was down-regulated and E-cadherin was up-regulated significantly in Slug siRNA-infected OE33 cell line (P<0.01). Bcl-2 was upregulated and E-cadherin was downregulated significantly in Slug cDNA-infected TE7 cells (P<0.05). OE33 cells with Slug knockdown were shown to possess markedly decreased invasiveness (P<0.05) and markedly increased apoptosis (P<0.05). Slug cDNA-infected TE7 cells were shown only to possess markedly increased invasiveness (P<0.05). There was significant relationship between Slug knockdown or Slug overexpression and cells proliferation (respectively, P<0.05). Animals injected with Slug-silenced OE33 cells had fewer seeded tumor (P<0.01), more apoptosis cells (P<0.05) and significantly xenograft tumor growth regression (P<0.05). But in Slug cDNA-infected TE7 cells, more seeded tumor number and significantly xenograft tumor growth were found in xenograft tumor (respectively, P<0.05). It was showed in the subcutaneously nude mice xenograft model tumor tissue, bcl-2 expression was reduced followed by the decrease of Slug expression in Slug-silenced tumor, and bcl-2 expression was increased followed by the increase of Slug expression. In pseudometastatic model, E-cadherin overexpression was found in Slug siRNA tumor tissue, but less E-cadherin expression was found in Slug cDNA tissue. Slug is an important modulator of apoptosis, growth and invasion in EA in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of EA.