Adhesion-Mediated Squamous Cell Carcinoma Survival through Ligand-Independent Activation of Epidermal Growth Factor Receptor
2004; Elsevier BV; Volume: 165; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63390-1
ISSN1525-2191
AutoresXiaodong Shen, Randall H. Kramer,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoThe survival and growth of squamous epithelial cells require signals generated by integrin-matrix interactions. After conversion to squamous cell carcinoma, the cells remain sensitive to detachment-induced anoikis, yet in tumor cell aggregates, which are matrix-deficient, these cells are capable of suprabasal survival and proliferation. Their survival is enhanced through a process we call synoikis, whereby junctional adhesions between neighboring cells generate specific downstream survival signals. Here we show that in squamous cell carcinoma cells, E-cadherin-mediated cell-cell contacts specifically induce activation of epidermal growth factor receptor (EGFR). EGFR activation in turn triggers the ERK/MAPK signaling module, leading to elevation of anti-apoptotic Bcl-2. After intercellular adhesion, formation of adherens junctions triggers the formation of E-cadherin-EGFR complexes, correlating with EGFR transactivation. Analysis of the process with a dominant-negative EGFR mutant indicated that activation of EGFR is ligand-independent. Our data implicate cell-cell adhesion-induced activation of EGFR as a cooperative mechanism that generates compensatory survival signaling, protecting malignant cells from detachment-induced death. The survival and growth of squamous epithelial cells require signals generated by integrin-matrix interactions. After conversion to squamous cell carcinoma, the cells remain sensitive to detachment-induced anoikis, yet in tumor cell aggregates, which are matrix-deficient, these cells are capable of suprabasal survival and proliferation. Their survival is enhanced through a process we call synoikis, whereby junctional adhesions between neighboring cells generate specific downstream survival signals. Here we show that in squamous cell carcinoma cells, E-cadherin-mediated cell-cell contacts specifically induce activation of epidermal growth factor receptor (EGFR). EGFR activation in turn triggers the ERK/MAPK signaling module, leading to elevation of anti-apoptotic Bcl-2. After intercellular adhesion, formation of adherens junctions triggers the formation of E-cadherin-EGFR complexes, correlating with EGFR transactivation. Analysis of the process with a dominant-negative EGFR mutant indicated that activation of EGFR is ligand-independent. Our data implicate cell-cell adhesion-induced activation of EGFR as a cooperative mechanism that generates compensatory survival signaling, protecting malignant cells from detachment-induced death. Survival of normal epithelial cells depends on signals generated by the interaction of these cells with a thin extracellular matrix called the basement membrane. In the absence of these signals, the cells die, exhibiting molecular characteristics of apoptosis.1Ruoslahti E Reed JC Anchorage dependence, integrins, and apoptosis.Cell. 1994; 77: 477-478Abstract Full Text PDF PubMed Scopus (981) Google Scholar, 2Frisch SM Ruoslahti E Integrins and anoikis.Curr Opin Cell Biol. 1997; 9: 701-706Crossref PubMed Scopus (1000) Google Scholar This form of apoptosis is also called anoikis, or "death of homelessness," because it is believed to preclude epithelial cells from reattachment and growth outside their proper tissue context.3Frisch SM Francis H Disruption of epithelial cell-matrix interactions induces apoptosis.J Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2854) Google Scholar, 4Meredith Jr, JE Winitz S Lewis JM Hess S Ren XD Renshaw MW Schwartz MA The regulation of growth and intracellular signaling by integrins.Endocr Rev. 1996; 17: 207-220Crossref PubMed Scopus (147) Google Scholar By contrast, many cancer cells of epithelial origin are anoikis resistant: they can survive in the absence of contact with the basement membrane inside three-dimensional tumor nests and in the absence of matrix attachment during metastasis.5Frisch SM Screaton RA Anoikis mechanisms.Curr Opin Cell Biol. 2001; 13: 555-562Crossref PubMed Scopus (1202) Google Scholar Therefore, acquisition of anoikis resistance plays an essential role in the progression of certain cancers. Some of the mechanisms that confer anoikis resistance on epithelial cells during tumor progression have been described.6Khwaja A Rodriguez-Viciana P Wennstrom S Warne PH Downward J Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (945) Google Scholar, 7Rosen K Rak J Leung T Dean NM Kerbel RS Filmus J Activated Ras prevents downregulation of Bcl-X(L) triggered by detachment from the extracellular matrix. A mechanism of Ras-induced resistance to anoikis in intestinal epithelial cells.J Cell Biol. 2000; 149: 447-456Crossref PubMed Scopus (122) Google Scholar, 8Jost M Huggett TM Kari C Rodeck U Matrix-independent survival of human keratinocytes through an EGF receptor/MAPK-kinase-dependent pathway.Mol Biol Cell. 2001; 12: 1519-1527Crossref PubMed Scopus (93) Google Scholar, 9Tran NL Adams DG Vaillancourt RR Heimark RL Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization.J Biol Chem. 2002; 277: 32905-32914Crossref PubMed Scopus (146) Google Scholar Our previous work implicated E-cadherin-mediated cell-cell adhesion in protecting oral squamous cell carcinoma (SCC) cells from anoikis.10Kantak SS Kramer RH E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells.J Biol Chem. 1998; 273: 16953-16961Crossref PubMed Scopus (209) Google Scholar However, the exact mechanism of E-cadherin-mediated anoikis resistance is unclear. Because cadherins lack enzymatic activity, their ability to induce cell survival signals may depend on their association with other signaling systems. E-cadherin can physically associate with a number of signaling effectors, such as PI3K and PTP1B, in adherens junctions.11Arregui C Pathre P Lilien J Balsamo J The nonreceptor tyrosine kinase fer mediates cross-talk between N-cadherin and beta1-integrins.J Cell Biol. 2000; 149: 1263-1274Crossref PubMed Scopus (109) Google Scholar, 12Kovacs EM Ali RG McCormack AJ Yap AS E-cadherin homophilic ligation directly signals through Rac and phosphatidylinositol 3-kinase to regulate adhesive contacts.J Biol Chem. 2002; 277: 6708-6718Crossref PubMed Scopus (268) Google Scholar In this study we focused on the potential role of the growth factor receptor tyrosine kinase, epidermal growth factor receptor (EGFR), because it is well known that in most oral SCCs and in cell lines established from these tumors EGFR is overexpressed13Todd R Wong DT Epidermal growth factor receptor (EGFR) biology and human oral cancer.Histol Histopathol. 1999; 14: 491-500PubMed Google Scholar and that EGFR can complex with E-cadherin14Hoschuetzky H Aberle H Kemler R Beta-catenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor.J Cell Biol. 1994; 127: 1375-1380Crossref PubMed Scopus (683) Google Scholar leading to activated EGFR.15Pece S Gutkind JS Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation.J Biol Chem. 2000; 275: 41227-41233Crossref PubMed Scopus (289) Google Scholar We hypothesized that E-cadherin-mediated cell-cell adhesion transactivates EGFR and that activation of EGFR and the downstream pathways promotes survival and resistance to anoikis. To test this hypothesis, we used a three-dimensional multicellular model of SCC in which cells are cultured in the absence of adhesive substrate yet form complex cell-cell adhesive interactions.10Kantak SS Kramer RH E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells.J Biol Chem. 1998; 273: 16953-16961Crossref PubMed Scopus (209) Google Scholar We first performed experiments to confirm that EGFR is activated on E-cadherin-mediated cell-cell contact formation in SCC cells and that this EGFR activation is mediated by E-cadherin. Using specific pharmacological inhibitors, we confirmed that activation of EGFR and the downstream MAPK pathway is required for E-cadherin-mediated anoikis resistance in SCC cells. In search of an underlying mechanism(s) by which E-cadherin transactivates EGFR, we found evidence that E-cadherin-mediated EGFR activation is ligand-independent and is related to E-cadherin/EGFR complex formation and clustering. Taken together, our results suggest that E-cadherin-mediated cell-cell adhesion promotes cell survival through ligand-independent activation of EGFR and the downstream MAPK pathway. Human recombinant epidermal growth factor (EGF) was from Invitrogen, Carlsbad, CA. Pharmacological inhibitors of EGFR (tyrphostin AG1478) were from Calbiochem (La Jolla, CA), MEK1/2 inhibitor (U0126) from Promega (Madison, WI), and PI3K inhibitor (LY294002) from Cell Signaling Technology, Beverly, MA. A mouse anti-E-cadherin monoclonal antibody (mAb) (HECD-1; obtained from M. Takeichi, Kyoto University, Japan) was used in immunoprecipitation, immunofluorescence staining, and antibody inhibition and clustering experiments. Anti-human E-cadherin (rat mAb, E9; obtained from C. Damsky, University of California, San Francisco, CA) was used in Western blotting. Anti-mouse E-cadherin (rat mAb ECCD-2; Zymed, South San Francisco, CA) was used in fluorescence-activated cell-sorting (FACS) analysis and immunofluorescent staining. Anti-EGFR and tyrosine-phosphorylated EGFR (activated EGFR) (mAb clones 13 and 74, respectively; Transduction Laboratories, Lexington, KY) were used in Western blotting and immunofluorescence staining. Antibodies raised against the extracellular domain of EGFR (Ab-11; NeoMarkers, Freemont, CA) were used in Western blotting. Antibodies against phospho-MAPK (phospho-p44/42-Thr202/Tyr204; Cell Signaling Technology) and MAPK (anti-pan-ERK mAb; Transduction Laboratories) were used in Western blotting. A mouse anti–Bcl-2 mAb (ascites; DAKO, Carpinteria, CA) was used in immunoprecipitation and Western blotting. A polyclonal antibody to Bax (Ab-1; Oncogene Research, Boston, MA) and to Bcl-xL (BD Biosciences) was used in Western blotting. For loading control, a mouse anti-tubulin mAb (Ab-4; NeoMarkers) was used in Western blotting. The human oral SCC cell lines HSC-3 and HOC-313, clones D1 and C8, have been described previously.16Kawano K Kantak SS Murai M Yao CC Kramer RH Integrin alpha3beta1 engagement disrupts intercellular adhesion.Exp Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gemini, Woodland, CA). HOC-313 cells were cloned by serial dilution of a single-cell suspension plated in 96-well plates. Single-cell clones were randomly selected for further culture. Cell monolayer cultures were prepared by seeding 6 × 105 cells in tissue culture dishes (10 cm; Falcon). For the culture of HSC-3 and C8 cells in suspension, monolayers were trypsinized the day before and then briefly treated with 5 mmol/L ethylenediaminetetraacetic acid (EDTA) to prepare single cells. To generate multicellular aggregates (MCAs), cells were then plated on polyhydroxylethyl-methacrylate (poly-HEMA)-coated 10-cm dishes (2 × 106 cells/dish) in the presence of Dulbecco's modified Eagle's medium supplemented with 0.5% fetal bovine serum. To produce suspended single-cell cultures, cells were suspended in semisolid medium consisting of 0.5% fetal bovine serum/Dulbecco's modified Eagle's medium containing 1.5% methylcellulose (Sigma, St. Louis, MO) at 6 × 105 cells per 10-cm poly-HEMA-coated dish. For harvesting of single cells in methylcellulose, cultures were diluted with two parts of phosphate-buffered saline (PBS) and centrifuged at 900 × g. The infection of HSC-3 cells by recombinant adenoviral vectors was performed as described previously.17Reardon DB Contessa JN Mikkelsen RB Valerie K Amir C Dent P Schmidt-Ullrich RK Dominant negative EGFR-CD533 and inhibition of MAPK modify JNK1 activation and enhance radiation toxicity of human mammary carcinoma cells.Oncogene. 1999; 18: 4756-4766Crossref PubMed Scopus (116) Google Scholar Briefly, the recombinant adenoviral vectors carrying EGFR-CD533 or green fluorescent protein (GFP) (gifts from K. Valerie, Virginia Commonwealth University, Richmond, VA) were added to HSC-3 cells at a multiplicity of infection of 100. Cells were then incubated for 24 hours before further experiments to ensure adequate expression of the genes of interest. We generated the E-cadherin or Bcl-2 expression vectors by taking the full-length mouse E-cadherin cDNA or human Bcl-2 cDNA and subcloning into the pcDNA3 vector (Invitrogen). For constitutive expression of E-cadherin or Bcl-2, 1 × 106 E-cadherin-negative C8 cells or HSC-3 cells were transfected with 4 μg of pcDNA3/E-cadherin vector or pcDNA3/Bcl-2 vector, respectively, using the Lipofectamine Plus kit (Life Technologies, Inc., Grand Island, NY) according to the manufacturer's protocol. Stable transfected cells were selected in 800 μg/ml of G418 (Life Technologies, Inc.) for 2 weeks. Because the Bcl-2-transfected cells exhibit a slightly higher rate of proliferation (data not shown), we were able to isolate a cell population after several more passages that expressed elevated levels of ectopically expressed Bcl-2, which was confirmed by immunoblot analysis. To enrich for E-cadherin expressing C8 cells, the population of transfected cells was processed for two cycles of FACS after labeling with ECCD-2 anti-E-cadherin mAb. HSC-3 cells were plated as MCA culture for the indicated times and then transferred onto poly-l-lysine-coated glass coverslips and incubated for 30 minutes at 37°C. After washing with 0.1 mmol/L Na3VO4 in PBS, cells were fixed and permeabilized for 15 minutes at room temperature with 4% paraformaldehyde and 0.1% Triton X-100. Cells were stained with primary antibodies and counterstained by either fluorescein- or rhodamine-labeled secondary antibodies. Analysis was performed with a confocal microscope (Bio-Rad, Richmond, CA). Immunoprecipitation and immunoblotting were performed as described previously.16Kawano K Kantak SS Murai M Yao CC Kramer RH Integrin alpha3beta1 engagement disrupts intercellular adhesion.Exp Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar Cells were lysed in RIPA buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 5 mmol/L EDTA, 2 mmol/L phenylmethyl sulfonyl fluoride, 1 mmol/L aprotinin, 1 mmol/L leupeptin, 10 mmol/L sodium pyrophosphate, 10 mmol/L sodium fluoride, and 1 mmol/L sodium vanadate). Cell lysates were processed for immunoprecipitation with specific antibody. The immunoprecipitates or total cell lysates were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and probed with appropriate antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). HSC-3 cells grown to near confluence were serum-starved overnight. For antibody-mediated clustering, cells were harvested from culture with 5 mmol/L EDTA, washed with PBS, and plated on poly-HEMA-coated dishes. The cells in suspension were incubated for 30 minutes at 4°C with a predetermined saturating concentration of HECD-1 antibody (20 μg/ml) in serum-free medium. The cells were then incubated with 5 μg/ml of goat anti-mouse IgG for various times at 37°C to induce clustering of E-cadherin. At the end of each time period, the cells were washed with ice-cold PBS containing 10 mmol/L NaF, 10 mmol/L Na4P2O7, and 0.5 mmol/L Na3VO4, and lysed with RIPA buffer, followed by immunoblotting. To assay for intranucleosomal DNA cleavage, cells were grown as monolayers, suspended single cells, or MCAs for the indicated times. Cells were then collected from poly-HEMA dishes by pipetting or from tissue culture dishes by scraping into the medium in which they had been incubated. In monolayer cultures, floating cells were collected and combined with the attached cells before DNA extraction. Harvested cells were then processed for the DNA-laddering assay and a FACS-based terminal dUTP nick-end labeling (TUNEL) assay. For intranucleosomal DNA laddering, genomic DNA was extracted using a Suicide Track DNA Ladder isolation kit (Oncogene Research). Samples were then analyzed in a 1.5% agarose gel. For the TUNEL assay, cells (2 × 106) were fixed for 15 minutes in 1% paraformaldehyde in PBS, followed by 30-minute fixation in 70% ethanol. The fixed cells were then washed, stained with FITC-dUTP, and treated with propidium iodide/RNase using an APO-Direct kit (Pharmingen, La Jolla, CA), followed by flow cytometric analysis using a FACScan Analyzer (Becton Dickinson, Mountain View, CA). To assess the proportion of cells that not only survived but also regained proliferative ability after suspension, cells from suspension cultures were replated at low cell density on tissue culture plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The clonogenic and proliferative ability of cells that reattached after plating was determined 3 days later by crystal violet staining and counting of colonies. We have previously shown that survival after loss of attachment to the extracellular matrix requires E-cadherin-mediated intercellular adhesion. In the current study, we first examined the dynamics of E-cadherin and EGFR distribution, because the two receptors have been reported to be associated as a complex in SCC cells.14Hoschuetzky H Aberle H Kemler R Beta-catenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor.J Cell Biol. 1994; 127: 1375-1380Crossref PubMed Scopus (683) Google Scholar, 15Pece S Gutkind JS Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation.J Biol Chem. 2000; 275: 41227-41233Crossref PubMed Scopus (289) Google Scholar Previously we had reported that when HSC-3 cells were plated on poly-HEMA-coated dishes, the cells gradually formed E-cadherin-mediated aggregates, beginning with single cells that subsequently formed small aggregates of a few cells by 3 hours that had begun to compact by 6 hours, collected into large, irregular clumps of cells by 12 hours, and condensed into spherical MCAs between 15 and 24 hours.10Kantak SS Kramer RH E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells.J Biol Chem. 1998; 273: 16953-16961Crossref PubMed Scopus (209) Google Scholar, 16Kawano K Kantak SS Murai M Yao CC Kramer RH Integrin alpha3beta1 engagement disrupts intercellular adhesion.Exp Cell Res. 2001; 262: 180-196Crossref PubMed Scopus (57) Google Scholar Analysis of the distribution of E-cadherin and EGFR by confocal microscopy showed that in freshly detached cells, both receptors were diffusely and uniformly distributed, with no evidence of patching (Figure 1A, a). As intercellular adhesion proceeded, the receptors began to co-localize at the cell-cell boundaries by as early as 3 hours (Figure 1A, b). However, there was a fraction of cells that had just started to form adhesions in which E-cadherin and EGFR remained evenly dispersed around the cell periphery (Figure 1A, c). In some cases it appeared that E-cadherin was more enriched at the junctional areas compared to EGFR (Figure 1A, b). By 6 hours much of E-cadherin and EGFR had co-distributed at sites of cell-cell adhesion, and had disappeared from cell surface areas that were devoid of cell-cell contact (Figure 1A, d and e). That EGFR was rapidly concentrated at cell-cell contacts in MCAs prompted us to investigate whether E-cadherin-mediated intercellular adhesion induces EGFR phosphorylation and activation. After cells started to form MCAs, phosphorylated EGFR (p-EGFR), visualized with specific antibodies, was detectable at cell-cell adhesion sites by 3 hours but was the intensity was weak and variable (Figure 1A, c). However, by 6 hours a strong signal for p-EGFR was detected in most cell aggregates (Figure 1A, e). To confirm that phosphorylated EGFR detected at cell-cell junctions represented transactivation of the receptor, we analyzed the level of activated EGFR during the formation of aggregates by immunoblotting with antibodies specific for tyrosine-phosphorylated EGFR (Figure 1B). At the time of plating, activated EGFR was at basal levels. After the gradual formation of cell aggregates, there was a parallel induction of EGFR activation evident at 6 hours and persisting at even higher levels at the 12- and 24-hour time points. In contrast, phosphorylated EGFR in suspended single cells remained at low levels throughout this time period. To test whether cell-cell adhesion was correlated with intrinsic receptor kinase activity, we used tyrphostin AG1478, a specific inhibitor of EGFR kinase activity.18Levitzki A Gazit A Tyrosine kinase inhibition: an approach to drug development.Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1638) Google Scholar Incubation with 1 μmol/L AG1478 abolished EGFR activation in aggregates at 24 hours (Figure 1B). Together, these results suggest that cell-cell adhesion leads to EGFR activation at sites of E-cadherin engagement and that adhesion-mediated EGFR activation requires receptor kinase activity. To establish the role of E-cadherin in EGFR activation, HSC-3 cells were treated with inhibiting anti-E-cadherin antibody HECD-1 (50 μg/ml) before plating and during MCA formation. In the presence of HECD-1, the formation of the large, compact aggregates was severely inhibited at 24 hours (Figure 1C). As shown in Figure 1D, hindering E-cadherin engagement with blocking antibodies ablated EGFR activation detected in immunoblots, whereas the control IgG was without effect. Therefore, the activation of EGFR after cell-cell adhesion formation is dependent on functional E-cadherin. MCAs of HSC-3 cells were resistant to apoptosis, whereas suspended single cells rapidly underwent anoikis, as detected by the extensive DNA laddering typical of apoptotic intranucleosomal cleavage (Figure 2A, lanes 2 and 4). To test whether E-cadherin-dependent EGFR activation provides the survival signal in aggregates, we incubated cells with tyrphostin AG1478. EGFR blockade with 1 μmol/L AG1478 induced apoptosis in HSC-3 cell aggregates cultured for 48 hours, indicated by DNA laddering (Figure 2A, lane 3). The DNA laddering induced by AG1478 was similar to that of HSC-3 single cells cultured in suspension (Figure 2A, lane 4). By contrast, AG1478-treated HSC-3 monolayer cells showed no evidence of apoptosis (Figure 2A, lane 5). Next, we analyzed DNA fragmentation to identify apoptotic cells by TUNEL assay. Again, AG1478 treatment dramatically increased the percentage of TUNEL-positive cells in HSC-3 MCAs at 48 hours (Figure 2B). Control monolayer HSC-3 cells showed low levels of TUNEL-positive cells, whereas, after suspension as single cells for the same time period, more than 40% of the cells were apoptotic. To further test the idea that E-cadherin engagement can induce EGFR activation and rescue cells from apoptosis, we exogenously expressed mouse E-cadherin in E-cadherin-negative C8 cells, a SCC cell line that fails to forms poor intercellular adhesions and undergoes apoptosis in suspension.10Kantak SS Kramer RH E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells.J Biol Chem. 1998; 273: 16953-16961Crossref PubMed Scopus (209) Google Scholar The E-cadherin-transfected C8 (C8/E-cad) cells expressed high levels of the receptor (Figure 3A). To determine whether the exogenously transfected E-cadherin co-localizes with EGFR at cell-cell boundaries, aggregates of C8/E-cad cells were processed for immunofluorescence analysis. Similar to what was observed with HSC-3 MCAs (Figure 1A), we found that in C8/E-cad MCAs the EGFR was also co-localized with E-cadherin and that significant levels of phosphorylated EGFR was detected by immunolabeling at cell-cell junctions (Figure 3B). In parental C8 cells that poorly aggregate, the distribution of EGFR remained uniformly distributed when cells are in suspension (data not shown). Next, EGFR activation was assessed by immunoblotting for EGFR tyrosine phosphorylation in the C8/E-cad cells after they were plated as MCAs for 24 hours. No EGFR activation was detected in MCAs from E-cadherin–negative C8 cells, but strong phosphorylation of EGFR was induced in MCAs from C8/E-cad cells (Figure 3C). The level of EGFR activation was nearly as intense as that generated in high-E-cadherin-expressing D1 cells, a positive control. Subsequently we analyzed the effect of ectopically expressed E-cadherin on survival in suspension by TUNEL assay in which the C8/E-cad cells showed enhanced resistance to anoikis (Figure 3D). Whereas ∼33% of C8 cells cultured in suspension were TUNEL-positive, only 9.6% of the C8/E-cad cells were apoptotic. Again, this enhanced anoikis resistance of C8/E-cad cells was reversed by EGFR kinase blockade with tyrphostin AG1478 (22.7%). Therefore, ectopic expression of E-cadherin is able to induce EGFR activation in C8 MCAs and partially rescue the C8 cells from anoikis. Among downstream pathways of EGFR, MEK/MAPK (ERK1/2) and PI3K/Akt pathways are believed to be the main pathways involved in cell survival.19Datta SR Brunet A Greenberg ME Cellular survival: a play in three Akts.Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3755) Google Scholar We tested whether E-cadherin-dependent phosphorylation of EGFR activates these two pathways. After induction of cell-cell adhesion, there was a progressive and sustained elevation of phosphorylated ERK1/2 that was evident as early as 6 hours and peaked at ∼24 hours (Figure 4A). This time-course activation of ERK1/2 closely paralleled EGFR activation after aggregate formation. Moreover, inhibition of EGFR kinase activity with 1 μmol/L tyrphostin AG1478 abolished ERK1/2 phosphorylation, indicating that ERK1/2 activation in MCAs was induced by EGFR transactivation (Figure 4B). To determine whether downstream MAPK phosphorylation is required for the survival of MCAs in suspension, we used U0126, a specific MEK inhibitor, and measured DNA fragmentation by TUNEL assay to quantify apoptotic cells. As shown in Figure 4B, 10 μmol/L U0126 blocked ERK1/2 phosphorylation in HSC-3 cell aggregates. At this concentration, U0126 also induced apoptosis in HSC-3 aggregates at 48 hours (Figure 4C). In contrast to the robust ERK1/2 activation after intercellular adhesion, phosphorylated Akt was not detected at significant levels in MCAs (Figure 4D). However, Akt activation was readily observed in substrate-adherent HSC-3 monolayer cells, and the level of activated Akt was strongly elevated after EGF treatment. The apparent lack of a role for Akt in cell-cell adhesion-induced survival is also suggested by the effect of the specific PI3K inhibitor LY294002, which had no detectable effect on MCA cell survival measured by both TUNEL assay (Figure 4C) and DNA laddering assay (Figure 4E). In contrast, treatment of MCAs with AG1478 to block EGFR, or with U0126 to inhibit MAPK, induced apoptosis as evidenced by extensive DNA laddering. These findings imply that MCA cell survival depends primarily on the E-cadherin/EGFR/MAPK signaling pathway. Previously we found that the level of anti-apoptotic Bcl-2 protein is elevated in response to E-cadherin-mediated adhesion.10Kantak SS Kramer RH E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells.J Biol Chem. 1998; 273: 16953-16961Crossref PubMed Scopus (209) Google Scholar These results indicated that the susceptibility to apoptosis in suspended single cells is correlated with the loss of Bcl-2 expression. To test whether E-cadherin-mediated adhesion regulates Bcl-2 expression via the EGFR/MAPK pathway, we treated HSC-3 aggregates with either EGFR inhibitor AG1478, MAPK inhibitor U0126, or PI3K inhibitor LY294002 (Figure 5A). As expected, both monolayers and MCAs exhibited high Bcl-2 levels. But after treatment with either AG1478 or U0126, the level of Bcl-2 decreased drastically, whereas the level of Bax remained relatively constant. The Bcl-2/Bax protein ratio decreased by more than threefold in MCAs after blockade of EGFR or MAPK. Consistent with what we observed above, treatment with LY294002 did not affect the Bcl-2/Bax protein ratio in MCAs, further suggesting that PI3K/Akt pathway was not essential for E-cadherin-mediated cell survival. We also examined the levels of the related Bcl-2 family member, Bcl-xL (Figure 5B). Bcl-xL also showed high levels of expression after MCA formation but was down-regulated in suspended single cells paralleling the response seen for Bcl-2. To establish whether elevated Bcl-2 is linked to HSC-3 cell survival, we stably transfected HSC-3 cells with Bcl-2. Overexpression of Bcl-2 in HSC-3 cells was then confirmed by Western blotting (Figure 5C). Compared with control cells, Bcl-2 protein levels were much higher in both monolayer and single-cell suspension culture of Bcl-2-transfected cells. Importantly, in viability assays, when both cell lines were subjected to single-cell suspension for 48 hours and then replated onto culture dishes, no visible colonies of control cells survived (Figure 5D). However, in the Bcl-2-transfected cells, a significant number of cells were able to survive and proliferate to form colonies after replating. Similarly, mock-transfected HSC-3 cells were unable to survive (data not shown). Next, we used a TUNEL assay to analyze the effect of Bcl-2 overexpression on anoikis. As show
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