CANCER GENOMICS & PROTEOMICS 11 : 239-250 (2014)

Modulation of Liver-Intestine Cadherin (Cadherin 17) Expression, ERK Phosphorylation and WNT Signaling in EPHB6 Receptor-expressing MDA-MB-231 Cells LOKESH BHUSHAN 1, NADIA TAVITIAN 1, DILIP DEY 1, ZOHRA TUMUR 2, CYRUS PARSA 3 and RAJ P. KANDPAL 1

Departments of 1Basic Medical Sciences, 2Dental Medicine and 3Pathology Western University of Health Science, Pomona, CA, U.S.A.

Abstract. Aberrant expression of -producing inverse relationship between the levels of phospho-ERK and hepatocellular carcinoma cell (EPH) receptors has been the abundance of cadherin 17, β- catenin and phospho- reported in a variety of human cancer types. In addition to GSK3 β in EPHB6-expressing MDA-MB-231 cells. From modulating cell proliferation and migration, EPH receptors these data we conclude that EPHB6-mediated alterations are also involved in tumor progression. The transcriptional arise due to changes in abundance and localization of activation and silencing of EPH receptors are also cadherin 17 and activation of . associated with tumorigenesis. However, the mechanisms Transcriptional silencing of EPHB6 in native MDA-MB-231 underlying the involvement of EPH receptors in cells and consequent effects on cadherin 17 and WNT tumorigenesis have not been completely deciphered. We have pathway may, thus, be responsible for the invasive behavior investigated and described the role of EPHB6, a kinase- of these cells. deficient receptor, in modulating the abundance of cadherin 17 and activation of other intracellular signaling . Erythropoietin-producing hepatocellular carcinoma cell We previously showed that EPHB6 alters the tumor receptor B6 (EPHB6) is a member of the largest family of phenotype of breast carcinoma cells. However, the receptor tyrosine kinases (1, 2). EPHB6 and EPHA10 are the mechanisms underlying these phenotypic changes had not only kinase-deficient members of this family. These kinase- previously been investigated. Herein we demonstrated the deficient receptors are likely to transduce signals by downstream effects of EPHB6 expression on the abundance associating with other kinase-sufficient receptors (3).The of cadherin 17, mitogen-activated kinase (MEK2), modulation of cell attachment and cell migration by EPH extracellular signal-regulated kinase (ERK), phospho-ERK, receptors has been correlated to invasiveness of tumor cells β- catenin, phospho- glycogen synthase kinase 3 beta (4-6). The decrease in the expression of EPHB6 receptor has (GSK3 β) (ser21/9), cell morphology and actin cytoskeleton. been linked to aggressiveness and invasiveness in melanoma These comparisons were made between EPHB6-deficient (7), neuroblastoma (8) and non-small cell lung carcinoma MDA-MB-231 cells transfected with an empty pcDNA3 (9). The invasiveness of MDA-MB-231 cells has been vector and cells stably transfected with an expression attributed, in part, to loss of EPHB6 (10), and ectopic construct of EPHB6. The results indicate elevated levels of expression of EPHB6 has been shown to suppress in vitro MEK2 and phospho-ERK. While there was no change in the invasiveness of MDA-MB-231 cells (11). However, the amount of ERK, the abundance of cadherin 17, β- catenin mechanism by which EPHB6 receptor suppresses the and phospho-GSK3 β was significantly reduced in EPHB6- invasive phenotype of MDA-MB-231 cells remains unclear. transfected cells. These studies clearly demonstrate an In addition to transducing signals from the cell surface to the nucleus, EPH receptors also contribute to cell adhesion. Similarly, a superfamily of cell surface molecules, which contribute to cell adhesion and signal transduction, is Correspondence to: Raj Kandpal, Department of Basic Medical comprised of cadherins. Cadherins are transmembrane Sciences, Western University of Health Sciences, Pomona, CA 91766, U.S.A. Tel: +1 9097063520, e-mail: [email protected] receptors that mediate calcium-dependent homophilic or heterophilic adhesion between cells. Members of the Key Words: EPHB6 , cadherin 17, CDH17 , ERK, WNT, β- catenin, cadherin superfamily include classical cadherins, GSK3 β, breast carcinoma. desmosomal cadherins, protocadherins and products of

1109 -6535 /2014 239 CANCER GENOMICS & PROTEOMICS 11 : 239-250 (2014) tumor-suppressor such as c-rearranged during catenin and phosphorylated glycogen synthase kinase 3 beta transfection ( RET ) and FAT (12). Classical cadherins (GSK3 β). We discussed these molecular changes in the enable adhesion between cells, maintain cell polarity and context of morphological and cytoskeletal rearrangements in preserve tissue integrity (13). Cadherins are linked with the native and EPHB6-expressing MDA-MB-231 cells. Our actin cytoskeleton via α, β, and γ isoforms of cytoplasmic studies suggest that EPHB6 down-regulates the levels of catenins (14-16), and facilitate a variety of molecular cadherin 17, and possibly also alters its cellular localization. changes during cell development and morphogenesis (17, 18). Aberrant expression of cadherins has been associated Materials and Methods with a variety of tumors (19, 20), and oncogenic properties of some cadherins have been confirmed (21, 22). Altered Cell culture. MCF10A, MCF7, BT20, MDA-MB-435, and MDA- expression of cadherin 17, in particular, has been implicated MB-468 cells were grown as described previously (10, 11). Native in several types of cancers (21, 23), and it has also been MDA-MB-231 cells transfected with pcDNA3 vector and EPHB6- considered a useful marker for gastrointestinal carcinomas transfected MDA-MB-231 cells (11) were cultured at 37˚C/7% CO 2 in Dulbecco’s modified eagle’s medium (DMEM) supplemented (24). The association of cadherin 17 expression with caudal- with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml type homeobox protein 2 (CDX2) transcription factor in PenStrep, and 10% fetal bovine serum (all Life Technologies, Grand ovarian cancer (23), and epidermal Island, NY, USA). and nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) signaling proteins in gastric cancer (21, 25) Total RNA isolation and reverse transcription polymerase chain suggests important roles of cadherin 17 in signal reaction (RT PCR). Cells were grown until 85-95% confluence in 25- transduction. Although EPH receptors and cadherins have mm cell culture flasks. RNA was isolated from various cell lines using TRI reagent (Molecular Research Center, Cincinnati, OH, been investigated extensively, detailed investigations of USA) according to the manufacturer’s protocol. The amount of RNA interactions between these two families of cell surface was determined by NanoDrop (Thermo Scientific, Waltham, MA, molecules have not been performed. Some recent USA), and its purity checked by determining the ratio of absorbance investigations, however, demonstrate modulation of signal at 260 nm and 280 nm. The RNA preparation was treated with transduction by cadherin and EPH receptor (26, 27). RNase-free DNase and 1.5 μg RNA was reverse transcribed with The altered regulation of various EPH receptors has been oligo dT using the Superscript III First-Strand RNA synthesis observed in a variety of cancer types (28). However, the (Life Technologies) according to the manufacturer’s protocol. The abundance of cadherin 17 ( CDH17 ) transcript was quantified by PCR downstream intracellular effector molecules have not been using 5’ACAATCGACCCACGTTTCTC3’ and characterized completely. Some EPH receptor signaling 5’ATATTGTGCACCGGGATCAT3’ oligonucleotides (Integrated events include EPHB4 dependent modulation of estrogen DNA Technologies, Inc., Coralville, IA, USA) as forward and receptor-alpha in breast cancer (29), activation of reverse primers, respectively. The reaction mixture (20 μl) containing mammalian target of rapamycin complex 1 and extracellular- 10 ng cDNA, 6.0 pmol each of forward and reverse primers, 4.0 regulated kinase pathways by EPHA2 (30), interaction of nmol dATP, dCTP, dGTP and dTTP, 0.4 units Taq DNA polymerase EPHB1 receptor with tumor-suppressor phosphatase and and 1X reaction buffer was amplified for 35 cycles of denaturation at 95˚C for 1 min, annealing at 65˚C for 1 min and extension at 72˚C tensin homolog (31), and EPHB3-mediated suppression of for 1 min. The amplified product was separated by electrophoresis metastasis via protein phosphatase 2(PP2A)/RAC/AKT on a 1% agarose gel and visualized by staining with ethidium signaling pathway in non-small cell lung carcinoma (32). bromide. PCR amplifications were also carried out with primers Morphological changes mediated by EPH have been corresponding to housekeeping gene β-actin transcript to normalize characterized by co-localization of EPHA2 and integrin α3 the amounts of cDNA templates used from various cell lines. at cell edges and protrusions in a glioblastoma cell line (33). The modulation of cell proliferation, migration and apoptosis Western blotting. MDA-MB-231 cells, stably transfected with an EPHB6 construct (MDA-MB-231-EPHB6) or an empty pcDNA3 has been attributed to specific activation of EPH receptors vector (MDA-MB-231-pCDNA), were grown in DMEM to 85-90% by their cognate ligands (34-39). Given the diversity confluence. The culture medium was aspirated and the dish washed of intracellular proteins affected by EPH receptors, it is not with 3 ml of ice-cold phosphate buffered saline (PBS). Cells were surprising that EPHB4 has been designated as both a tumor lysed by incubating with 250 μl of RIPA buffer (10 mM Tris, pH promoter and a tumor suppressor (40). 7.4, 150 mM NaCl, 5 mM EDTA, 8% glycerol, 1% Triton X-100, In light of the background described above, we 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 10 investigated the association of cadherin 17 expression with mM Na3VO4, 1 mM sodium fluoride) and complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN, USA) for kinase-deficient EPHB6 receptor in the breast cancer cell 15 min with gentle shaking. The lysates were cleared by line MDA-MB-231, and demonstrated the effects of EPHB6 centrifugation at 16,000 g for 10 min at 4˚C, and the concentration on cytoskeleton components and the levels of intracellular of protein in the supernatant was determined using BCA protein signaling proteins such as mitogen-activated estimation kit (Thermo Scientific, Waltham, MA, USA). Forty (MEK2), extracellular signal-regulated kinase (ERK), β- micrograms of total protein per well were separated by SDS-

240 Brushan et al : EPHB6 Modulates Cadherin 17 and ERK-WNT Pathways polyacrylamide gel electrophoresis (PAGE) and the separated CDH17 transcript was detectable only in the MDA-MB-231 proteins were electroblotted onto methanol-presoaked cell line (Figure 1A). In light of our earlier observation that polyvinylidene fluoride membranes. The membrane was blocked for EPHB6 suppresses invasiveness of MDA-MB-231 cells (10, 60 min with 5% bovine serum albumin (BSA) containing 0.1% 11) and our current results demonstrating the expression of Tween 20 in Tris-buffered saline at room temperature and then incubated overnight at 4˚C with monoclonal antibodies to phospho- cadherin 17 selectively in MDA-MB-231, we investigated the ERK1/2 (Thr202/Tyr204, 1:1,000; Cell Signaling Technology, relationship between EPHB6 and cadherin 17 by Danvers, MA, USA), ERK1/2 (1:2,000; Cell Signaling Technology), characterizing the abundance of CDH17 transcript in MDA- β- catenin (1:1,000; Cell Signaling Technology), phospho-GSK3 MB-231 cells stably transfected with either an empty (ser 21/9, 1:1,000; Cell Signaling Technology) or cadherin 17 (1: pcDNA3 vector or EPHB6 -containing pcDNA3. The levels 1000; R&D, Minneapolis, MN, USA). Antigen–antibody complexes of CDH17 transcript were found to be higher in MDA-MB- were detected by enhanced chemiluminescence (Amersham/GE 231-pcDNA3 cells than MDA-MB231-EPHB6 cells (Figure Healthcare, Piscataway, NJ, USA) after incubating with the corresponding horseradish peroxidaseHRP-conjugated secondary 1B and C). antibodies. The amounts of protein in individual lanes were normalized by probing the membrane with antibody to Abundance of cadherin 17 protein in breast carcinoma cells . glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10000 As described above, the levels of CDH17 transcript were dilution; Millipore, Billerica, MA, USA). The intensity of specific lower in EPHB6 -expressing MDA-MB-231 cells, compared to bands corresponding to cadherin 17, MEK2, phosphor-ERK and cells transfected with empty pcDNA3 vector. The abundance GAPDH were quantified using ImageJ software (National Institutes of transcript in these cell lines was, therefore, compared to the of Health, Bethesda, MD, USA). Intensity ratios of cadherin 17/GAPDH, MEK2/GAPDH, phospho-ERK/GAPDH, phospho- levels of corresponding protein by western blotting. The GSK3 β and β- catenin in MDA-MB-231-pcDNA3 and MDA-MB- amount of cadherin 17 protein in EPHB6- expressing cells was 231-EPHB6 cells were plotted using GraphPad software (GraphPad lower than the corresponding empty vector-transfected cells Software Inc., La Jolla, CA, USA). (Figure 1D and E). It warrants mentioning that all quantitations were performed in three biological replicates Immunofluroscence. Cells were grown in a glass-bottom dish in with three technical replicates of each experiment. DMEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate (Life Technologies), 100 U/ml of PenStrep, and 10% fetal Cellular localization of cadherin 17 and its EPHB6- bovine serum (all Life Technologies) at 37˚C/7% CO 2 for 72 h. Culture medium was changed every 24 h. The dishes were washed dependent down-regulation. Semi-quantitative PCR revealed twice with ice cold PBS (pH 7.4) and treated with Histochoice MB a decrease in the abundance of CDH17 transcript in MDA- tissue fixative (Amresco, Solon, OH, USA) for 10 min. Fixative MB-231-EPHB6 cells compared to empty vector-transfected solution was aspirated, cells were permeabilized with 0.3 Triton X- cells. This result prompted us to examine the in situ 100 made in PBS for 10 min, and incubated with antibody to expression of cadherin 17 protein in MDA-MB231-EPHB6 cadherin 17 (1:100 dilution, R&D) overnight at 4˚C. Cells were and MDA-MB-231-pcDNA3 cell lines. Figure 2 shows washed five times with ice-cold PBS (pH 7.4), incubated with alexa flour 546 donkey antibody to mouse IgG (1: 500 dilution) for 2 h at localization and abundance of cadherin 17 protein in these room temperature, and treated with 4’,6-diamidino-2-phenylindole cell lines. MCF10A cells, which have no detectable levels of (DAPI)-containing mounting solution. The fluorescence-labeled cadherin 17 protein, were used as a negative control for these cells were analyzed on a Nikon A1 3 color confocal microscope experiments. Figure 2A and C clearly show that MDA-MB- (Nikon Inc., Melville, NY, USA) using ×20 objective. 231-pcDNA cells express significantly higher amounts of the protein as compared to EPHB6- expressing cells (Figure 2D Phalloidin labeling. To study the cytoskeletal re-arrangement, cells and F). Although the distinction between membranous and were labeled with Phalloidin-fluorescein isothiocyanate (FITC) (Enzo Life Sciences, Farmingdale, NY, USA) and treated with subcellular localization of cadherin 17 was not obvious from DAPI-containing mounting solution. The fluorescence was analyzed the images shown, optical sectioning of fluorescent images on Nikon A1 3-color confocal microscope using a ×100 objective. (data not shown) in other experiments tentatively suggested the localization of cadherin 17 on the cell surface in empty Results pcDNA3 vector-transfected MDA-MB-231 cells and around the nucleus in EPHB6 -transfected cells. Semi-quantitative PCR of cadherin 17 transcript in breast carcinoma cells. To address the involvement of cell adhesion EPHB6 activates ERK/MAPK pathway in MDA-MB-231 cell molecules in invasiveness of breast carcinoma cells, we line. In order to delineate the signal transduction pathways investigated the expression pattern of cadherin 17 transcripts mediated by EPHB6, expression of ERK/MAPK was in MCF10A, MCF7, BT-20, MDA-MB-231, MDA-MB-435 investigated in vector-transfected and EPHB6 -transfected and MDA-MB-468 cell lines. We had previously observed a MDA-MB-231 cell lines as described in Materials and differential expression of a variety of cadherin transcripts in Methods. As shown in Figure 3, the amount of MEK2 these cell lines (data not shown). However, the expression of protein was significantly higher in MDA-MB-231-EPHB6

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Figure 1. Cadherin 17 expression in breast carcinoma cell lines. A: Total RNA was isolated from various cell lines and cadherin 17 (CHD17) transcript was amplified by PCR with CHD17-specific primers as described in the Materials and Methods section. The amplified product from MCF10A (lane 2), MCF7 (lane 3), BT20 (lane 4), MDA-MB-231 (lane 5), MDA-MB-435 (lane 6) and MDA-MB-468 (lane 7) cells was separated on an agarose gel. Lane 1 represents a no-template control and lane 8 shows a molecular weight marker. The arrow indicates the presence of 480- basepair (bp) amplicon corresponding to CHD17 transcript. B: Total RNA was isolated and cadherin 17 transcript was amplified as described above. The amplified products from pcDNA3 vector-transfected cell line MDA-MB-231-pcDNA3 (lane 1) and EPHB6-transfected cell line MDA- MB-231-EPHB6 (lane 2) were separated on an agarose gel. The amounts of cDNA used for amplification were normalized by amplifying actin transcript in each sample. The arrows indicate the amplified products of expected size. C: The amounts of amplified products corresponding to MDA-MB-231-pcDNA3 and MDA-MB-231-EPHB6 were determined by densitometry using imageJ program and the ratios of CHD17 and actintranscripts in cell lines were compared as a histogram. The data are presented as the mean and standard deviation of three experiments and triplicate amplifications in each experiment. D: Total protein was isolated from MDA-MB-231-pcDNA3 and MDA-MB-231-EPHB6 cell lines, separated by electrophoresis, probed with antibody against cadherin 17 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the antigen–antibody complexes detected by chemiluminescence. E: The amounts of protein corresponding to cadherin 17 and GAPDH in MB-231- pcDNA3 and MDA-MB-231-EPHB6 were determined by densitometry using the imageJ program and the data presented as the mean and standard deviation of three experiments.

242 Brushan et al : EPHB6 Modulates Cadherin 17 and ERK-WNT Pathways

Figure 2. In situ expression of cadherin 17 in pcDNA3 vector-transfected MDA-MB-231-pcDNA3 and EPHB6-transfected MDA-MB231-EPHB6 cell lines. MDA-MB-231-pcDNA3 (panels A, B and C) and MDA-MB-231-EPHB6 (panels D, E and F) cell lines were grown on glass slides, fixed and incubated with fluorescence-tagged cadherin 17 antibody and 4’, 6’ diamino-2-phenylindole (DAPI). The stained cells were imaged on a Nikon A1 3-color confocal microscope using a ×100 objective. Panels A and D show cadherin 17 expression, panels B and E indicate DAPI-stained nuclei, and panels C and F are merged images. All images were taken at the same magnification as indicated by the line scale at the bottom of each panel.

cells than in MDA-MB-231-pcDNA3 cells (Figure 3A and EPHB6 induces re-organization of actin cytoskeleton. B). We subsequently quantified the levels of total ERK and EPHB6-mediated suppression of invasiveness of MDA-MB- phospho-ERK in these two cell lines. As shown in Figure 231 cells prompted us to examine actin organization in 3C, the levels of total ERK were found to be comparable in EPHB6-transfected MDA-MB-231 cells. MDA-MB-231- MDA-MB-231-pcDNA3 and MDA-MB-231-EPHB6 cells. EPHB6 and MDA-MB-231-pcDNA3 cell lines were grown Although we did not observe any 44-kDa isoform of as described in the Materials and Methods, and stained with phospho-ERK under these conditions, the abundance of the FITC-conjugated phalloidin. The comparison of the actin 42-kDa phosphorylated form of ERK ( Thr202/Tyr204 ) was cytoskeleton indicated morphological as well as size elevated in MDA-MB-231-EPHB6 cells compared to MDA- alterations in EPHB6 -transfected cells. The pronounced MB-pCDNA3 cells (Figure 3C and D). contours of the cytoskeleton in MDA-MB-231-EPHB6 cells are clearly evident in Figure 5. Activated ERK alters WNT signaling in MDA-MB-231 cells. To investigate the downstream effects of signals transduced Discussion by EPHB6 receptor, we examined GSK3 β as an intracellular target protein for phosphorylation. The levels of EPH receptors are involved in regulating cell development phoshorylated (serine 21/9) GSK3 β were significantly higher and differentiation (41, 42), and aberrant expression of these in MDA-MB231-pcDNA3 cells compared to EPHB6- molecules has been also associated with a variety of human transfected MDA-MB-231 cells (Figure 4A and B). A cancers (43, 44). We have previously reported transcriptional concomitant decrease in the amount of β- catenin was also silencing of kinase-deficient EPHB6 receptor in invasive observed in MDA-MB-231-EPHB6 cells (Figure 4C and D). breast carcinoma cell lines (45), and demonstrated EPHB6-

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Figure 3. Mitogen-activated protein kinase (MEK2) and extracellular signal-regulated kinase (ERK) expression in EPHB6-transfected MDA-MB-231- EPHB6 and pcDNA3 vector-transfected MDA-MB231-pcDNA3 cell lines. A: Total protein was isolated from MDA-MB-231-pcDNA3 and MDA-MB- 231-EPHB6 cell lines, separated by electrophoresis, probed with antibody against MEK2 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the antigen–antibody complex detected by chemiluminescence. The arrow indicates the protein corresponding to 45 kDa MEK2 in MDA-MB- 231-pcDNA3 (lane 1) and MDA-MB231-EPHB6 (lane 2) cells. The bottom portion shows the band corresponding to GAPDH. B: The histogram shows the ratios of MEK2 and GAPDH proteins in MDA-MB-231-pcDNA3 and MDA-MB-231-EPHB6 cell lines. The results are the mean of three experiments, and error bars indicate the standard deviation. C: Protein blots were probed with antibodies indicated as above. The arrows indicate the bands corresponding to phospho-(p)-ERK (Thr202/Tyr204), GAPDH and total (t)-ERK in MDA-MB-231-pcDNA3 (lane 1) and MDA-MB-231- EPHB6 (lane 2) cells. D: The intensity of bands in panel C was determined by densitometry and ratios of p-ERK and GAPDH proteins in MDA-MB- 231-pcDNA3 and MDA-MB-231-EPHB6 cells are shown in the histogram. Experiments were repeated three times and each experiment contained three lanes for each sample. Error bars indicate standard deviation.

mediated phenotypic changes in these cells (11). In light of cells are mediated by MAPK and WNT signaling pathways. our unpublished observations on differential expression of To our knowledge, this is the first report showing expression various cadherin transcripts in breast carcinoma cell lines of liver-intestine cadherin in a breast carcinoma cell line and and in order to characterize the functional relationship of correlating its expression with EPHB6 levels. Based on the EPHB6 with cadherin family of cell adhesion molecules, we intracellular proteins linked to EPHB6 signaling in MDA- investigated the abundance of cadherin 17 transcript in a MB-231 cells (3) and a report suggesting expression of variety of breast cell lines. Although these cell lines were EPHB6 in breast tumors (46), it is reasonable to explain distinguishable by the expression of a combination of various these results as context-dependent consequences of EPHB6 cadherin transcripts, cadherin 17 was uniquely present in in normal and tumor cells. MDA-MB-231 cells. Combined with our earlier results (10, The phosphorylation-dependent signal transduction by the 11), MDA-MB-231 cells can thus be recognized by the kinase-deficient EPHB6 receptor can be interpreted by the presence of cadherin 17 and the absence of EPHB6. These ability of other receptors of this family to dimerize with and two observations set the basis for investigating any possible cross-phosphorylate EPHB6. In fact, it has been shown that cross-talk between EPHB6 and cadherin 17 and to EPHB6 can be phosphorylated upon stimulation of cells with characterize EPHB6-modulated changes in the abundance of EPHB2 (47), and its phosphorylation has also been observed some intracellular signaling proteins. Herein we showed that in a complex with EPHB1. Our recent results demonstrate EPHB6 modulates the expression of cadherin 17, and the the interaction of EPHB6 with kinase-sufficient receptors phenotypic changes in EPHB6 -expressing MDA-MB-231 EPHB2 and EPHA2 (48). Both receptors have been

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Figure 4. The levels of phosphorylated glycogen synthase kinase 3 beta (p-GSK3 β) and β- catenin in pcDNA3 vector transfected MDA-MB-pcDNA3 and EPHB6-transfected MDA-MB-231-EPHB6 cell lines. A: Total protein was isolated from MDA-MB-231-pcDNA3 and MDA-MB-231-EPHB6 cells, separated by electrophoresis, probed with an antibody against GSK3 β or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as indicated, and the antigen–antibody complexes were detected by chemiluminescence. The arrows indicate the bands corresponding to pGSK3 β and GAPDH in MDA-MB-231-pcDNA3 (lane 1) and MDA-MB-231-EPHB6 (lane 2) cells. B: The intensity of bands in panel A was determined by densitometry and ratios of pGSK3 β and GAPDH proteins in MDA-MB-231-pcDNA3 and MDA-MB-231-EPHB6 cells are shown in the histogram. Experiments were repeated three times and each experiment contained three lanes for each sample. Error bars indicate standard deviation. C: Protein blots were probed with antibody against β- catenin or GAPDH as indicated, and the antigen–antibody complexes were detected by chemiluminescence. The arrows indicate the bands corresponding to β- catenin and GAPDH in MDA-MB-231-pcDNA3 (lane 1) and MDA-MB-231-EPHB6 (lane 2) cells. D: The intensity of bands in panel C was determined by densitometry and the ratios of β- catenin and GAPDH proteins in MDA-MB-231-pcDNA3 and MDA-MB-231-EPHB6 cells are shown in the histogram. Experiments were repeated three times and each experiment contained three lanes for each sample. Error bars indicate standard deviation.

implicated in a variety of cancer types (26, 34, 49-52). The likely suppresses invasiveness of MDA-MB-231 cells. These above observations provide a clear rationale for the altered observations are supported by the reports demonstrating levels of phosphorylated intracellular proteins in response to cadherin 17-mediated transformation of premalignant liver altered expression of a kinase-deficient receptor. These progenitor cells to liver carcinomas in mice (54), and its changes in intracellular proteins are likely to have arisen by altered expression in colorectal (22, 55), pancreatic (56) and the binding of kinases or regulator proteins to gastric (57-59) cancer. Thus breast carcinoma can be added phosphorylated amino acid residues on EPHB6 receptor. to the growing list of human cancer types that show aberrant Although detailed mechanisms of EPHB6 effects have not expression of cadherin 17. It warrants mention that aberrant been worked out, it has been shown to activate MAPK expression of cadherin 17, a cell surface molecule, is pathway in lung adenocarcinoma (53). associated with tumor cell metastasis (19, 55, 57, 60, 61), To explain the EPHB6-mediated invasiveness phenotype and thus is indicative of its involvement in cell adhesion. The of MDA-MB-231 cells, we reasoned that EPHB6 receptor altered cellular localization of cadherin 17 in EPHB6- may affect adhesion between two cells. Thus, the expression transfected cells suggests that cadherin 17 may play a dual profiling of cell adhesion molecules appeared to be a logical role in adhesion and cell signaling. Based on these choice. Based on the unique expression of cadherin 17 in observations, we postulate that cadherin 17 modulates the MDA-MB-231 cells, we investigated the effect of EPHB6 on activation of EPHB6, and a reciprocal association likely the abundance of cadherin 17. As described in the results exists between the amounts of these two proteins. Such a section, the amount of cadherin 17 was significantly lower linkage is supported by the observation that EPH and in cells stably transfected with EPHB6 . The decrease in cadherin interaction facilitates the recruitment of a cadherin 17 levels in EPHB6 -transfected cells strengthens disintegrin and metalloproteinase domain-containing protein the argument that cadherin 17 is oncogenic and EPHB6 10 (ADAM10) protease at the plasma membrane for

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Figure 5. Staining of pcDNA3 vector-transfected MDA-MB-231-pcDNA3 and EPHB6-transfected MDA-MB-EPHB6 cells with fluorescien isothiocyanateFITC-phalloidin. MDA-MB-231-pcDNA3 (panels A, B and C) and MDA-MB-231-EPHB6 (panels D, E and F) cells were grown on glass slides, fixed and incubated with fluorescence-tagged phalloidin and 4’, 6’ diamino-2-phenylindole (DAPI). The stained cells were imaged on a Nikon A1 3-color confocal microscope using a ×100 objective. Panels A and D show phalloidin signal, panels B and E indicate DAPI-stained nuclei, and panels C and F are merged images. All images were taken at the same magnification as indicated by the line scale at the bottom of each panel.

dampening signal transduction (27). Our observations, proliferating cells (62, 63), and its activity is modulated by therefore, suggest a novel dimension to the regulation of phosphorylation of tyrosine residue 216 and serine residue 9 EPH receptors and cadherins. (64, 65). GSK3 β is also known to suppress as well as The increased amount of MEK2 and the 42 kDa isoform promote tumor growth, and these activities are related to its of phospho-ERK (p42) in MDA-MB-231-EPHB6 cells phosphorylation status. While the kinase-inactive clearly demonstrates EPHB6-mediated modulation of MAPK promotes mammary tumorigenesis (66), a GSK3 inhibitor pathway in breast carcinoma cells. These results are caused cell death through cyclin D1 depletion in breast supported by the observations made in lung adenocarcinoma cancer cells (67). The effects of GSK inhibitor have been cells (53). The signals received by EPHB6 appear to be attributed to an increase in the inactivating phosphorylation relayed by activation of MAPK pathway. We have therefore of GSK3 α at Ser 21 and GSK3 β at Ser 9, and a decrease in explored the downstream targets of MAPK pathway the activating phosphorylation of GSK3 β at Tyr216 (67). The mediated by EPHB6 receptor. Based on the reports that overexpression of GSK3 β has been reported in human down-regulation of cadherin 17 inactivates WNT signaling ovarian, colon and pancreatic carcinoma (68, 69), and and inhibits tumor growth (54), we reasoned GSK3 β and β- GSK3 β has also been linked to cell survival and proliferation catenin as likely downstream targets of EPHB6 receptor. in ovarian cancer cells (70). After establishing an inverse relationship between EPHB6 Our previous observations indicating suppression of and cadherin expression, we investigated the abundance of invasiveness of EPHB6 -transfected MDA-MB-231 cells can phospho-GSK3 β and β- catenin in MDA-MB-231 cells be explained by implicating cytoskeleton rearrangements, transfected with EPHB6 . The levels of both phospho-GSK3 β modulation of intracellular proteins and altered transcription and β- catenin were lower in EPHB6- expressing cells, thus of genes. Cytoskeletal arrangements play a central role in suggesting a collective decrease in the amounts of cadherin migration and invasiveness of tumor cells (71, 72). In line 17, phospho-GSK3 β and β- catenin. However, the cause and with the involvement of the cytoskeleton in these processes, effect linkage between these proteins remains unclear. we observed distinct and extensive cell protrusions in GSK3 β is known to be constitutively active in non- EPHB6 -transfected MDA-MB-231 cells. Such protrusions

246 Brushan et al : EPHB6 Modulates Cadherin 17 and ERK-WNT Pathways were particularly visible in cells sharing close proximity, and 8 Tang XX, Evans AE, Zhao H, Cnaan A, London W, Cohn SL, thus indicated EPHB6 mediated interactions between cells. Brodeur GM and Ikegaki N: High-level expression of EPHB6, These observations have further clarified the likely role of EFNB2, and EFNB3 is associated with low tumor stage and high TRKA expression in human neuroblastomas. Clin Cancer Res 5: EPHB6 receptor in modulating the invasiveness of MDA- 1491-1496, 1999. MB-231 cells. It is noteworthy that effects of EPHB3 on 9 Muller-Tidow C, Diederichs S, Bulk E, Pohle T, Steffen B, tumor suppression and cell adhesion in colon cancer have Schwäble J, Plewka S, Thomas M, Metzger R, Schneider PM, also been attributed to cytoskeleton rearrangement (73). Brandts CH, Berdel WE and Serve H: Identification of We believe that the tumor-suppressor activities of EPHB6 metastasis-associated receptor tyrosine kinases in non-small cell are mediated by dampening tumor-promoting activities of lung cancer. Cancer Res 65 : 1778-1782, 2005. GSK3 β and β- catenin. However, our data are insufficient to 10 Fox BP and Kandpal RP: Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as suggest a mechanism for altered phosphorylation and molecular markers of potential diagnostic and prognostic decreased abundance of GSK3 β and β- catenin in EPHB6 - application. Biochem Biophys Res Commun 318 : 882-892, 2004. transfected MDA-MB-231 cells. Our data are in agreement 11 Fox BP and Kandpal RP: EPHB6 receptor significantly alters with biological activities of intracellular proteins that appear invasiveness and other phenotypic characteristics of human to be modulated by differential stimulation of EPHB6 breast carcinoma cells. Oncogene 28 : 1706-1713, 2009. receptor. The phenotypic changes in MDA-MB-231 breast 12 Takeichi M: Cadherins in cancer: implications for invasion and carcinoma cell lines presented here and EPHB6 expression metastasis. Curr Opin Cell Biol 5: 806-811, 1993. observed in breast tumors (46) suggest that EPHB6-mediated 13 Takeichi M: Cadherins: a molecular family important in selective cell–cell adhesion. Annu Rev Biochem 59 : 237-252, 1990. alterations are context dependent. Such context dependence 14 Gumbiner BM and McCrea PD: Catenins as mediators of the of specific receptors and regulatory molecules can, in large cytoplasmic functions of cadherins. J Cell Sci Suppl 17 : 155- part, be attributed to redundancy of receptors, as well as 158, 1993. intracellular signaling proteins. 15 Kemler R: From cadherins to catenins: cytoplasmic protein In conclusion, we presented the first report describing the interactions and regulation of cell adhesion. Trends Genet 9: expression of cadherin 17, a liver-intestine cadherin, in a 317-321, 1993. breast carcinoma cell line. We have also demonstrated 16 Van den Bossche J, Malissen B, Mantovani A, De Baetselier P and Van Ginderachter JA: Regulation and function of the E- modulation of its expression by EPHB6 receptor. Furthermore, cadherin/catenin complex in cells of the monocyte-macrophage our results have allowed us to suggest a biological association lineage and DCs. Blood 119 : 1623-1633, 2012. of EPHB6 receptor with intracellular signaling proteins that 17 Halbleib JM and Nelson WJ: Cadherins in development: cell mediate MAPK and WNT signaling pathways in normal and adhesion, sorting, and tissue morphogenesis. Genes Dev 20 : carcinoma cells. These results provide adequate basis for 3199-3214, 2006. predicting an association of cadherin 17 with a subset of 18 Dusek RL, Getsios S, Chen F, Park JK, Amargo EV, Amargo EV, invasive breast tumors. Such an association of cadherin 17 Cryns VL and Green KJ: The differentiation-dependent with breast tumors, if confirmed, can potentially be applied to desmosomal cadherin desmoglein 1 is a novel caspase-3 target that regulates apoptosis in keratinocytes. J Biol Chem 281: 3614-3624, diagnostic and therapeutic purposes. 2006. 19 Dong W, Yu Q and Xu Y: Altered expression of a Li-cadherin in References gastric cancer and intestinal metaplasia. Dig Dis Sci 52 : 536- 542, 2007. 1 Wilkinson DG: Multiple roles of EPH receptors and in 20 Agiostratidou G, Li M, Suyama K, Badano I, Keren R, Keren R, neural development. Nat Rev Neurosci 2: 155-164, 2001. Chung S, Anzovino A, Hulit J, Qian B, Bouzahzah B, Eugenin 2 Himanen JP and Nikolov DB: Eph receptors and ephrins. Int J E, Loudig O, Phillips GR, Locker J and Hazan RB: Loss of Biochem Cell Biol 35 : 130-134, 2003. retinal cadherin facilitates mammary tumor progression and 3 Truitt L, Freywald T, DeCoteau J, Sharfe N and Freywald A: The metastasis. Cancer Res 69: 5030-5038, 2009. EphB6 receptor cooperates with c-Cbl to regulate the behavior of 21 Wang J, Kang WM, Yu JC, Liu YQ, Meng QB and Cao ZJ: breast cancer cells. Cancer Res 70 : 1141-1153, 2010. Cadherin-17 induces tumorigenesis and lymphatic metastasis in 4 Vaught D, Brantley-Sieders DM and Chen J: Eph receptors in gastric cancer through activation of NFkappaB signaling breast cancer: roles in tumor promotion and tumor suppression. pathway. Cancer Biol Ther 14 : 262-270, 2013. Breast Cancer Res 10 : 217, 2008. 22 Kwak JM, Min BW, Lee JH, Choi JS, Lee SI, Park SS, Kim J, 5 Chen J, Zhuang G, Frieden L and Debinski W: Eph receptors Um JW, Kim SH and Moon HY: The prognostic significance of and ephrins in cancer: common themes and controversies. E-cadherin and liver intestine-cadherin expression in colorectal Cancer Res 68 : 10031-10033, 2008. cancer. Dis Colon Rectum 50 : 1873-1880, 2007. 6 Castano J, Davalos V, Schwartz S Jr. and Arango D: EPH 23 Huang LP, Yu YH, Sheng C and Wang SH: Up-regulation of receptors in cancer. Histol Histopathol 23: 1011-1023, 2008. cadherin 17 and down-regulation of homeodomain protein 7 Hafner C, Bataille F, Meyer S, Becker B, Roesch A, Landthaler CDX2 correlate with tumor progression and unfavorable M, Vogt T: Loss of EPHB6 expression in metastatic melanoma. prognosis in epithelial ovarian cancer. Int J Gynecol Cancer 22 : Int J Oncol 23 : 1553-1559, 2003. 1170-1176, 2012.

247 CANCER GENOMICS & PROTEOMICS 11 : 239-250 (2014)

24 Panarelli NC, Yantiss RK, Yeh MM, Liu Y and Chen YT: Tissue- 40 Xiao Z, Carrasco R, Kinneer K, Sabol D, Jallal B, Coats S and specific cadherin CDH17 is a useful marker of gastrointestinal Tice DA: EphB4 promotes or suppresses RAS/MEK/ERK adenocarcinomas with higher sensitivity than CDX2. Am J Clin pathway in a context-dependent manner: Implications for Pathol 138 : 211-222, 2012. EPHB4 as a cancer target. Cancer Biol Ther 13 : 630-637, 2012. 25 Sakamoto N, Oue N, Sentani K, Anami K, Uraoka N, Naito Y, 41 Nikolov DB, Xu K and Himanen JP: Eph/ephrin recognition and Oo HZ, Hinoi T, Ohdan H, Yanagihara K, Aoyagi K, Sasaki H the role of Eph/ephrin clusters in signaling initiation. Biochim and Yasui W: Liver-intestine cadherin induction by epidermal Biophys Acta 1834 : 2160-2165, 2013. growth factor receptor is associated with intestinal differentiation 42 Hogerheyde TA, Stephenson SA, Harkin DG, Bray LJ, Madden of gastric cancer. Cancer Sci 103 : 1744-1750, 2012. PW, Woolf MI and Richardson NA: Evaluation of EPH receptor 26 Miura K, Nam JM, Kojima C, Mochizuki N and Sabe H: EphA2 and ephrin expression within the human cornea and limbus. Exp engages Git1 to suppress Arf6 activity modulating epithelial Eye Res 107 : 110-120, 2013. cell–cell contacts. Mol Biol Cell 20 : 1949-1959, 2009. 43 Ferguson BD, Liu R, Rolle CE, Tan YH, Krasnoperov V, Kanteti 27 Solanas G, Cortina C, Sevillano M and Batlle E: Cleavage of E- R, Tretiakova MS, Cervantes GM, Hasina R, Hseu RD, Iafrate cadherin by ADAM10 mediates epithelial cell sorting downstream AJ, Karrison T, Ferguson MK, Husain AN, Faoro L, Vokes EE, of EPHB signalling. Nat Cell Biol 13 : 1100-1107, 2011. Gill PS and Salgia R: The EphB4 receptor 28 Lisabeth EM, Falivelli G and Pasquale EB: Eph receptor promotes lung cancer growth: a potential novel therapeutic signaling and ephrins. Cold Spring Harb Perspect Biol 5: doi: target. PLoS One 8: e67668, 2013. 10.1101/cshperspect, 2013. 44 Pasquale ES: Eph receptors and ephrins in cancer: bidirectional 29 Schmitt F, Nguyen PH, Gupta N and Mayer D: Eph receptor B4 signaling and beyond. Nature Rev Cancer 10 : 165-180, 2010. is a regulator of estrogen receptor alpha in breast cancer cells. J 45 Fox BP and Kandpal RP: Transcriptional silencing of EphB6 Recept Signal Transduct Res 33 : 244-248, 2013. in invasive breast carcinoma cells and 30 Cui XD, Lee MJ, Kim JH, Hao PP, Liu L, Yu GR and Kim DG: detection of methylated promoter by methylation-specific PCR. Activation of mammalian target of rapamycin complex 1 Biochem Biophys Res Commun 340 : 268-276, 2006. (mTORC1) and RAF/PYK2 by growth factor-mediated EPH 46 Brantley-Sieders DM, Jiang A, Sarma K, Badu-Nkansah A, receptor 2 (EPHA2) is required for cholangiocarcinoma growth Walter DL, Shyr Y and Chen J: EPH/ephrin profiling in human and metastasis. Hepatology 57 : 2248-2260, 2013. breast cancer reveals significant associations between expression 31 Rodriguez S and Huynh-Do U: Phosphatase and tensin homolog level and clinical outcome. PLoS One 6: e24426, 2011. regulates stability and activity of EphB1 receptor. FASEB J 27 : 47 Matsuoka H, Obama H, Kelly ML, Matsui T and Nakamoto M: 632-644, 2013. Biphasic functions of the kinase-defective Ephb6 receptor in cell 32 Li G, Ji XD, Gao H, Zhao JS, Xu JF, Sun ZJ, Deng YZ, Shi S, adhesion and migration. J Biol Chem 280 : 29355-29363, 2005. Feng YX, Zhu YQ, Wang T, Li JJ and Xie D: EphB3 suppresses 48 Fox BP and Kandpal RP: A paradigm shift in EPH receptor non-small-cell lung cancer metastasis via a PP2A/RACK1/AKT interaction: biological relevance of EPHB6 interaction with signalling complex. Nat Commun 3: 667, 2012. EPHA2 and EPHB2 in breast carcinoma cell lines. Cancer 33 Makarov A, Ylivinkka I, Nyman TA, Hyytiainen M and Keski- Genomics Proteomics 8: 185-193, 2011. Oja J: Ephrin-As, EPH receptors and integrin alpha3 interact and 49 Wykosky J and Debinski W: The EphA2 receptor and ephrinA1 co-localise at membrane protrusions of U251MG glioblastoma ligand in solid tumors: function and therapeutic targeting. Mol cells. Cell Biol Int 37 : 1080-1088, 2013. Cancer Res 6: 1795-1806, 2008. 34 Wang B: Cancer cells exploit the Eph-ephrin system to promote 50 Ireton RC and Chen J: EphA2 receptor tyrosine kinase as a invasion and metastasis: tales of unwitting partners. Sci Signal promising target for cancer therapeutics. Curr Cancer Drug 4: pe28, 2011. Targets 5: 149-157, 2005. 35 Astin JW, Batson J, Kadir S, Charlet J, Persad RA, Gillatt D, 51 Oricchio E, Nanjangud G, Wolfe AL, Schatz JH, Mavrakis KJ, Oxley JD and Nobes CD: Competition amongst Eph receptors Jiang M, Liu X, Bruno J, Heguy A, Olshen AB, Socci ND, regulates contact inhibition of locomotion and invasiveness in Teruya-Feldstein J, Weis-Garcia F, Tam W, Shaknovich R, prostate cancer cells. Nat Cell Biol 12 : 1194-1204, 2010. Melnick A, Himanen JP, Chaganti RS and Wendel HG: The Eph- 36 Dohn M, Jiang J and Chen X: Receptor tyrosine kinase EphA2 receptor A7 is a soluble tumor suppressor for follicular is regulated by p53-family proteins and induces apoptosis. lymphoma. Cell 147 : 554-64, 2011. Oncogene 20 : 6503-6515, 2001. 52 Oshima T, Akaike M, Yoshihara K, Shiozawa M, Yamamoto N, 37 Miao H, Wei BR, Peehl DM, Li Q, Alexandrou T, Schelling JR, Sato T, Akihito N, Nagano Y, Fujii S, Kunisaki C, Wada N, Rino Rhim JS, Sedor JR, Burnett E and Wang B: Activation of EPHA Y, Tanaka K, Masuda M and Imada T: Overexpression of EphA4 receptor tyrosine kinase inhibits the RAS/MAPK pathway. Nat gene and reduced expression of EphB2 gene correlates with liver Cell Biol 3: 527-530, 2001. metastasis in colorectal cancer. Int J Oncol 33 : 573-577, 2008. 38 Duxbury MS, Ito H, Zinner MJ, Ashley SW and Whang EE: 53 Yu J, Bulk E, Ji P, Hascher A, Koschmieder S, Berdel WE and Ligation of EphA2 by -Fc inhibits pancreatic Müller-Tidow C: The kinase defective EPHB6 receptor tyrosine adenocarcinoma cellular invasiveness. Biochem Biophys Res kinase activates MAP kinase signaling in lung adenocarcinoma. Commun 320 : 1096-1102, 2004. Int J Oncol 35 : 175-179, 2009. 39 Noblitt LW, Bangari DS, Shukla S, Knapp DW, Mohammed S, 54 Liu LX, Lee NP, Chan VW, Xue W, Zender L, Zhang C, Mao Kinch MS and Mittal SK: Decreased tumorigenic potential of M, Dai H, Wang XL, Xu MZ, Lee TK, Ng IO, Chen Y, Kung EphA2-overexpressing breast cancer cells following treatment HF, Lowe SW, Poon RT, Wang JH and Luk JM: Targeting with adenoviral vectors that express ephrin A1. Cancer Gene cadherin-17 inactivates Wnt signaling and inhibits tumor growth Ther 11 : 757-766, 2004. in liver carcinoma. Hepatology 50 : 1453-1463, 2009.

248 Brushan et al : EPHB6 Modulates Cadherin 17 and ERK-WNT Pathways

55 Takamura M, Ichida T, Matsuda Y, Kobayashi M, Yamagiwa S, 65 Park CH, Lee BH, Ahn SG, Yoon JH and Oh SH: Serine 9 and Genda T, Shioji K, Hashimoto S, Nomoto M, Hatakeyama K, tyrosine 216 phosphorylation of GSK-3 beta differentially Ajioka Y, Sakamoto M, Hirohashi S and Aoyagi Y: Reduced regulates autophagy in acquired cadmium resistance. Toxicol Sci expression of liver-intestine cadherin is associated with 135 : 380-389, 2013. progression and lymph node metastasis of human colorectal 66 Farago M, Dominguez I, Landesman-Bollag E, Xu X, Rosner A, carcinoma. Cancer Lett 212 : 253-259, 2004. Cardiff RD and Seldin DC: Kinase-inactive glycogen synthase 56 Takamura M, Sakamoto M, Ino Y, Shimamura T, Ichida T, kinase 3 beta promotes Wnt signaling and mammary Asakura H and Hirohashi S: Expression of liver-intestine tumorigenesis, Cancer Res 65 : 5792-5801, 2005. cadherin and its possible interaction with galectin-3 in ductal 67 Kim HM, Kim CS, Lee JH, Jang SJ, Hwang JJ, Ro S and Choi adenocarcinoma of the pancreas. Cancer Sci 94 : 425-430, 2003. J: CG0009, a novel glycogen synthase kinase 3 inhibitor, induces 57 Ko S, Chu KM, Luk JM, Wong BW, Yuen ST, Leung SY and cell death through cyclin D1 depletion in breast cancer cells. Wong J: Overexpression of Li-cadherin in gastric cancer is PLoS One 8: e60383, 2013. associated with lymph node metastasis. Biochem Biophys Res 68 Ougolkov AV and Billadeau DD: Targeting GSK-3: A promising Commun 319 : 562-568, 2004. approach for cancer therapy? Future Oncol 2: 91-100, 2006. 58 Ito R, Oue N, Yoshida K, Kunimitsu K, Nakayama H, Nakachi K 69 Kitano A, Shimasaki T, Chikano Y, Nakada M, Hirose M, Higashi and Yasui W: Clinicopathological significant and prognostic T, Ishigaki Y, Endo Y, Takino T, Sato H, Sai Y, Miyamoto K, influence of cadherin-17 expression in gastric cancer. Virchows Motoo Y, Kawakami K and Minamoto T: Aberrant glycogen Arch 447 : 717-722, 2005. synthase kinase 3 beta is involved in pancreatic cancer cell 59 Hippo Y, Taniguchi H, Tsutsumi S, Machida N, Chong JM, invasion and resistance to therapy. PLoS One 8: e55289, 2013. Fukayama M, Kodama T and Aburatani H: Global gene 70 Cao Q, Lu X and Feng YJ: Glycogen synthase kinase-3 beta expression analysis of gastric cancer by oligonucleotide positively regulates the proliferation of human ovarian cancer microarrays. Cancer Res 62 : 233-240, 2002. cells. Cell Res 16 : 671-677, 2006. 60 Yu QF, Dong WG and Ren JL: Knockdown of Li-cadherin 71 Vignjevic D and Montagnac G: Reorganisation of the dendritic increases metastatic behaviors of LoVo cells. J Cancer Res Clin actin network during cancer cell migration and invasion. Semin Oncol 136 : 1641-1649, 2010. Cancer Biol 18 : 12-22, 2008. 61 Ko S, Chu KM, Luk JM, Wong BW, Yuen ST, Leung SY and 72 Yamaguchi H and Condeelis J: Regulation of the actin Wong J: CDX2 co-localizes with liver-intestine cadherin in cytoskeleton in cancer cell migration and invasion. Biochim intestinal metaplasia and adenocarcinoma of the stomach. J Biophys Acta 1773 : 642-652 2007. Pathol 205 : 615-622, 2005. 73 Chiu ST, Chang KJ, Ting CH, Shen HC, Li H and Hsieh FJ: 62 Pap M and Cooper GM: Role of glycogen synthase kinase-3 in Overexpression of EPHB3 enhances cell-cell contacts and the phosphatidylinositol 3-kinase/Akt cell survival pathway. J suppresses tumor growth in HT-29 human colon cancer cells. Biol Chem 273 : 19929-19932, 1998. Carcinogenesis 30 : 1475-1486, 2009. 63 Grimes CA and Jope RS: The multifaceted roles of glycogen synthase kinase 3 beta in cellular signaling. Prog Neurobiol 65 : 391-426, 2001. 64 Dajani R, Fraser E, Roe SM, Young N, Good V, Dale TC and Pearl LH: Crystal structure of glycogen synthase kinase 3 beta: Received June 26, 2014 structural basis for phosphate-primed substrate specificity and Revised July 17, 2014 autoinhibition. Cell 105 : 721-732, 2001. Accepted July 18, 2014

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