- Email: [email protected]
Deletion of exon 8 increases cisplatin-induced E-cadherin cleavage
E XP E RI ME N TA L CE LL RE S EA RCH 3 14 ( 20 0 8 ) 1 5 3 –16 3
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Deletion of exon 8 increases cisplatin-induced E-cadherin cleavage Margit Fuchs a , Christine Hermannstädter a , Peter Hutzler b , Georg Häcker c , Ferdinand Haller a , Heinz Höfler a,b , Birgit Luber a,⁎ a
Technische Universität München, Klinikum rechts der Isar, Institut für Allgemeine Pathologie und Pathologische Anatomie, D-81675 München, Germany b GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Pathologie, D-85764 Neuherberg, Germany c Technische Universität München, Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, D-81675 München, Germany
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
E-Cadherin-mediated cell–cell adhesion plays a key role in epithelial cell survival and loss of
Received 26 May 2007
E-cadherin or β-catenin expression is associated with invasive tumor growth. Somatic
Revised version received
E-cadherin mutations have been identified in sporadic diffuse-type gastric carcinoma. Here,
27 August 2007
we analysed the fate of E-cadherin with an in frame deletion of exon 8 compared to wild-type
Accepted 6 September 2007
E-cadherin and the involved signalling events during cisplatin-induced apoptosis. We report
Available online 14 September 2007
that mutant E-cadherin was more readily cleaved during apoptosis than the wild-type form. Also β-catenin, an important binding partner of E-cadherin, was processed. E-cadherin
Keywords:
cleavage resulted in disconnection of the actin cytoskeleton and accumulation of E-cadherin
E-cadherin
and β-catenin in the cytoplasm. Inhibitor studies demonstrated that E-cadherin cleavage
Cleavage
was caused by a caspase-3-mediated mechanism. We identified the Akt/PKB and the ERK1/2
Cisplatin
signalling pathways as important regulators since inhibition resulted in increased
Akt/protein kinase B
E-cadherin cleavage and apoptosis. In summary, we clearly demonstrate that somatic
E-cadherin mutations
E-cadherin mutations affect apoptosis regulation in that way that they can facilitate the disruption of adherens junctions thereby possibly influencing the response to cisplatinbased chemotherapy. Elucidating the mechanisms that regulate the apoptotic program of tumor cells can contribute to a better understanding of tumor development and potentially be relevant for therapeutic drug design. © 2007 Elsevier Inc. All rights reserved.
Introduction In epithelial cells, E-cadherin is an important component of adherens junctions [1] and via its cytoplasmic domain it is
associated with β- or γ-catenin which in turn bind to α-catenin [2]. Recently, α-catenin has been demonstrated to act as a molecular switch that associates with E-cadherin–β-catenin and regulates the assembly of actin filaments [3,4]. E-cadherin
⁎ Corresponding author. Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität München, Trogerstr. 18, 81675 München, Germany. Fax: +49 89 4140 4915. E-mail address: [email protected] (B. Luber). Abbreviations: AEC, anti-E-cadherin antibody; Akt/PKB, Akt/protein kinase B; cl, cleaved; CTL, control; DAPI, 4,6-diamidino-2phenylindole; DMSO, dimethyl sulfoxide; del 8 E-cadherin, d8, E-cadherin with deletion of exon 8; fl, full length; MDA, untransfected, parental MDA-MB-435S cells; PI3-kinase, phosphatidylinositol 3-kinase; SD, standard deviation; RT, room temperature; wt, wild-type 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.09.004
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functions as a tumor and invasion suppressor [5] and in several tumors, including gastric [6] and breast carcinomas [7], loss of E-cadherin and/or β-catenin expression have been associated with invasive tumor growth [5,8,9]. In diffuse-type gastric carcinoma and their lymph node metastases, somatic E-cadherin mutations have been found in the mutational hotspot region [10] whereby exons 8 or 9 were frequently affected by in frame deletions of the whole exons [11]. Recent studies on these mutations revealed that they affect cell adhesion, motility, proliferation, proteolysis and also tumorigenicity in an animal model [10–19]. Today, cisplatin-based chemotherapy is worldwide used for the treatment of cancer patients including those suffering from gastric cancer. In response to cisplatin, a variety of cellular signalling pathways is modulated, among which are the Akt/PKB, ERK1/2, SAPK/JNK and p38 cascades [20]. These pathways regulate cell proliferation, DNA repair and apoptosis. The balance between pro- and anti-apoptotic factors is of critical importance since dysregulation can lead to inhibition of apoptosis and tumor development. Susceptibility to apoptosis is controlled by a variety of different regulatory proteins including E-cadherin that plays an important role in protecting cells from cell death [21–25]. Several mechanisms which control the apoptosis-regulating function of E-cadherin have been proposed [26], including activation of anti-apoptotic Akt/PKB by formation of Ecadherin-mediated cell–cell junctions [27,28] and association of E-cadherin with the epidermal growth factor receptor [23] and β-catenin [29]. Cleavage of cell adhesion components during apoptosis was therefore suggested to be an effective mechanism to eliminate the anti-apoptotic function of Ecadherin [30] and also to facilitate the separation of tumor cells which have started the apoptotic program from intact cells. Therefore, in the present study we addressed the question if cleavage of E-cadherin is affected by deletion of exon 8 during cisplatin-induced apoptosis and which are the consequences on cell survival. Further, we investigated signalling events including caspase cascades that are involved in the apoptotic scenario.
Materials and methods Cell cultivation and transfection The human E-cadherin-negative cell line MDA-MB-435S (ATCC, Rockeville, MD) and the wild-type (wt) and mutant E-cadherin-cDNA transfected derivatives that were established previously [11] were grown in Dulbecco's modified Eagle medium (Life Technologies, Karlsruhe, Germany) supplemented with 10% fetal calf serum (PAN Biotech, Aidenbach, Germany) and penicillin–streptomycin (50 IU/ ml and 50 mg/ml; Life Technologies) at 37 °C and 5% CO2. MDA-MB-435S is a strain which evolved from the parent line MDA-MB-435, isolated from the pleural effusion of a female with metastatic, ductal adenocarcinoma of the breast [31]. Two different clones were investigated for each E-cadherin expression construct in this study to exclude clonal variations.
Reagents Cisplatin (Sigma, Deisenhofen, Germany) was prepared as a 2.5-mM stock solution in H2O and used at a final concentration of 75 μM. 4,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma, prepared as a 1 mg/ml stock solution in H2O and used at a concentration of 500 ng/ml. PI3-kinase inhibitor LY294002 (Calbiochem, Schwalbach, Germany) was dissolved in DMSO as a 65-mM stock solution and used at a concentration of 40 μM. MEK1 inhibitor PD98059 (Sigma) was prepared as a 50mM stock solution in dimethyl sulfoxide (DMSO) and used at a final concentration of 50 μM. Caspase-3 inhibitor Z-DEVD-FMK (Calbiochem) was dissolved in DMSO at a concentration of 50 mM and used at 20 μM. DMSO was included in the negative control samples at appropriate concentrations when the effect of Z-DEVD-FMK was tested to exclude effects of the solvent. Rhodamine-coupled phalloidin was obtained from Sigma.
Counting of apoptotic cells Cells were plated on glass coverslips in 6-well plates at a density of 8 × 104 cells per well. After 1 day, cells were treated with 75 μM cisplatin for 8, 16, 24 and 32 h. Then cells were fixed with 3.7% formaldehyde and treated for 1 h at 37 °C with 3% (w/v) BSA/0.1% (w/v) saponin. Subsequently, cells were incubated with DAPI at a final concentration of 500 ng/ml for 45 min at 37 °C. The number of living or apoptotic cells (showing nuclear condensation and apoptotic figures) was counted in nine randomly chosen high power microscopic fields (magnification: 32×, Axiovert 135, Zeiss, Jena, Germany) and the percentage values were calculated setting the total number of cells to 100%. A total of 500–1200 cells were counted for each time point in two independent experiments.
Antibodies Mouse monoclonal antibodies against E-cadherin (clone 36, AEC: anti-E-cadherin antibody), β-catenin and cytochrome c (6H2.B4) were purchased from BD Biosciences (Heidelberg, Germany). Anti-E-cadherin antibody SHE78-7 was purchased from Jackson Immuno Research Laboratories (West Grove). Antibodies against caspase-3, cleaved caspase-3 as well as polyclonal antibodies against total or activated proteins were purchased from Cell Signalling Technology (Beverly, MA): Akt, phosphorylated Akt (Ser473), Erk1/2, phosphorylated Erk1/2 (Thr202/Tyr204). Monoclonal anti-α-tubulin antibody (Sigma) was used to stain α-tubulin as a loading control. The horseradish peroxidase-conjugated anti-mouse IgG was purchased from Amersham Pharmacia Biotech (Braunschweig, Germany) and anti-rabbit IgG was from BioSource (purchased from Invitrogen, Karlsruhe, Germany).
Western blot analysis Two independent cell lines were analysed for each construct by Western blot analysis, and the results of one representative clone are shown in all figures. Cells were seeded at a density of 3.5 × 105 cells per 5-cm tissue culture dish or at 9.5 × 105 cells per 10-cm tissue culture dish. After treatment, cells were harvested at the indicated time points with 200 μl or 300 μl
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L-CAM lysis buffer as described before [16]. Proteins were separated by SDS–polyacrylamide gel electrophoresis followed by transfer to nitrocellulose (Schleicher & Schuell, Dassel, Germany) membranes. Antibody dilutions were as follows: E-cadherin clone 36 1:5000, β-catenin 1:2000, caspase-3 1:2000, cleaved caspase-3 1:1000, Akt 1:1000, phosphorylated Akt (Ser473) 1:1000, Erk1/2 1:1000, phosphorylated Erk1/2 (Thr202/Tyr204) 1:2000 and α-tubulin 1:10000. For signal detection, the enhanced chemoluminescence system (Amersham Pharmacia Biotech) was used. For signal quantification, blots were scanned and densitometric analysis was performed with Scion Image Software from Scion Corporation (Version Beta 4.0.2, Frederick, USA). In all Western blot analyses, α-tubulin staining was performed to demonstrate loading of equal amounts of cellular lysates.
Immunofluorescence analysis, nuclear and actin filament staining Cells were plated on glass coverslips in 6-well plates at a density of 8 × 104 cells per well. After 1 day, cells were treated with 75 μM cisplatin for the times indicated in the figure legends. Then, cells were either fixed with 3.7% formaldehyde (β-catenin, p53, cytochrome c) or methanol (E-cadherin) and treated for 1 h at 37 °C with 3% (w/v) BSA/0.1% (w/v) saponin in PBS for permeabilisation and blocking. Cells were subsequently incubated for 1 h with primary antibody diluted in blocking solution (anti-β-catenin antibody 1:100, anti-E-cadherin antibody SHE78-7 1:100, anti-cytochrome c antibody 1:100). Then, cells were incubated for 1 h at RT with an FITCcoupled secondary antibody (goat-anti-mouse IgG; Dianova, Hamburg, Germany) diluted 1:500 in blocking solution. Hoechst (Sigma) was used for nuclear staining at a concentration of 1:200 for 7 min at RT, protected from light. Staining of actin filaments was performed as described previously [11]. Coverslips were mounted on slides using antifading reagent (Molecular Probes, Leiden, Netherlands) and analysed with a Zeiss Axiovert laser scanning microscope LSM 510 meta.
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tivation of the adherens junction components E-cadherin and β-catenin by caspase cleavage on specific motifs [34,35]. To investigate the impact of cisplatin on tumor-derived mutant E-cadherin with a deletion of exon 8 (del 8), MDA-MB-435S cells expressing either wt or mutant E-cadherin were treated with 75 μM cisplatin for 28 h and cell extracts were analysed by Western blot analysis. At this time point, approximately 30% of the cells were apoptotic which was similar in both tested cell lines (data not shown). It is of importance to note that we observed by flow cytometric analysis the absence of Ecadherin in the non-transfected parental MDA-MB-435S cell line and expression of either wt or mutant E-cadherin in transfected cell lines in N95% of the cells [18]. As shown in Fig. 1A, both the full-length protein and a 25-kDa cleavage product were detectable after cisplatin application using an antibody directed against the C-terminus of E-cadherin (clone 36). The molecular weight of the small fragment was consistent with cleavage at the caspase-3 consensus cleavage site 747-DTRD-750 [34], which is highly conserved in different species and represents the only caspase-3 consensus sequence in the cytoplasmic tail. While cisplatin treatment caused only a minor cleavage of wt E-cadherin, cleavage of del 8 E-cadherin was strongly enhanced at 28 h, resulting in a 7.4-fold increase of the intensity of the 25-kDa fragment compared to wt E-cadherin (Fig. 1B). In contrast, a significant decrease in full-length E-cadherin was observed in cells expressing del 8 E-cadherin. Taken together,
Assay for caspase-3 activity Cells were seeded at a density of 3 × 105 cells per 6-cm tissue culture dish. After 1 day, caspase-3 inhibitor Z-DEVD-FMK was added at a concentration of 20 μM. After 1 h, cisplatin was added at a concentration of 75 μM and cells were incubated for 28 h. Then, cells were lysed in NP-40 lysis buffer [32,33]. Triplicates of aliquots were incubated with a peptide containing 10 μM of a caspase-3 recognition sequence (DEVD-AMC) in buffer consisting of bovine serum albumine, HEPES and fluorometric substrate. Free AMC was measured after 1 h incubation at 37 °C. Values are presented as relative fluorescence units (mean ± SD of triplicate reactions).
Results Increased cisplatin-mediated cleavage of mutant E-cadherin Programmed cells death is characterised by a variety of morphological and biochemical features like functional inac-
Fig. 1 – Cisplatin-induced cleavage of E-cadherin. (A) Expression and protein cleavage of E-cadherin was investigated by immunoblot analysis after treatment with 75 μM cisplatin for 28 h. Extracts were derived from non-transfected (M) or E-cadherin (wt or del 8) expressing MDA-MB-435S cells. A representative of at least three independent experiments is shown. (B) Immunoblot analyses of (A) were quantified by densitometric analysis using Scion Image Software. Shown are means of wt or mutant E-cadherin cleavage products. Each bar represents the mean±SD of three independent experiments.
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E-cadherin with a deletion of exon 8 was more susceptible to cisplatin-induced cleavage compared to the wt form.
Cisplatin-mediated β-catenin cleavage At cell–cell contacts, E-cadherin is associated via β- and α-catenin with the actin cytoskeleton. β-Catenin is not only a binding partner of E-cadherin, it plays also an important role in the Wnt signalling pathway [36]. To examine the effect of cisplatin on β-catenin, we measured its cleavage after cisplatin treatment. A decrease in full-length β-catenin was observed in cells expressing del 8 E-cadherin. Further, we detected a variety of fragments (Fig. 2), which had previously been described to appear during apoptosis [35]. β-Catenin cleavage patterns were similar in both tested cell lines expressing E-cadherin independent of the mutational status (Fig. 2). Longer cisplatin treatment resulted in an increase of smaller β-catenin fragments. However, co-immunoprecipitation analysis revealed that binding of β-catenin to E-cadherin was still detectable at 28 h of cisplatin treatment (data not shown), underlining the observation that only a fraction of adherens junctions components was cleaved at this time point.
Cisplatin-induced relocalisation of E-cadherin and β-catenin To further analyse the impact of cisplatin on the cell adhesion complex, we next investigated the localisation of E-cadherin and β-catenin after cisplatin treatment by immunofluorescence staining. In untreated wt E-cadherin expressing cells, staining of E-cadherin with an anti-SHE78-7 E-cadherin antibody directed against the extracellular domain (Fig. 3A) and β-catenin (Fig. 3B) was mainly found at cell–cell contacts. In cells expressing the mutant form of E-cadherin, E-cadherin and β-catenin were detectable at cell–cell contact sites, but also additionally in lamellipodia and the perinuclear region as described previously [11,19]. After onset of apoptosis by cisplatin, a relocalisation of E-cadherin and β-catenin from the cell periphery to the cytoplasm was found in apoptotic wt and del 8 E-cadherin expressing cells which have already lost their cell–cell contacts (Fig. 3A). Interestingly, redistribution of del 8 E-cadherin from the cellular membrane to the cytoplasm seemed to precede apoptosis because it was already visible in cisplatin-treated cells without nuclear fragmentation. Further, to visualise the reorganisation of the actin cytoskeleton during cisplatin-induced apoptosis, co-staining of actin filaments
Fig. 2 – Cisplatin-induced cleavage of β-catenin. Expression and protein cleavage of β-catenin was investigated by immunoblot analysis after treatment with 75 μM cisplatin for 24 and 48 h. Extracts were derived from non-transfected (M) or E-cadherin (wt or del 8) expressing MDA-MB-435S cells. A representative of at least three independent experiments is shown.
with phalloidin was performed. As shown in Fig. 3C, cisplatin treatment caused disruption of actin filaments in shrinked apoptotic cells with condensed nuclei. Taken together, our immunofluorescence analysis clearly demonstrates a cisplatin-mediated loss of E-cadherin from cell–cell contacts which occurred earlier in del 8 E-cadherin expressing cells. β-Catenin seemed to follow the distribution of E-cadherin.
Caspase activation and cytochrome c release in response to cisplatin Caspases are major players in apoptosis regulation that convey the apoptotic signal in a proteolytic cascade, including cleavage and activation of downstream acting caspases. Subsequently, degradation of downstream cellular targets occurs, finally leading to apoptosis. MDA-MB-435S cells expressing wt or del 8 E-cadherin were exposed to cisplatin for 28 h and activation of caspase 8, 9 and 3 was examined by Western blot analysis as well as by a caspase-3 activation assay. Additionally, cytochrome c release from mitochondria was determined indicating the involvement of the intrinsic pathway in apoptosis regulation. Independent of the Ecadherin status, cytochrome c was released into the cytoplasm after cisplatin application as detected by immunofluorescence analysis (Fig. 4A) and cell fractionation assay (data not shown). While full-length caspase-9 was cleaved in a similar manner in all tested cell lines (data not shown), cleavage of caspase 8 and 3 was strongly enhanced in cells expressing del 8-E-cadherin reflecting the result obtained for E-cadherin cleavage (Fig. 4B and data not shown). In order to determine whether Ecadherin cleavage was induced by caspase-3, cells were treated with the commonly used caspase-3 inhibitor Z-DEVD-FMK and a caspase activity assay as well as Western blot analysis were performed. The activity of caspase-3 measured in cell extracts was significantly reduced by Z-DEVD-FMK (Fig. 4C). Further, Ecadherin cleavage was completely blocked in the presence of Z-DEVD-FMK, suggesting that caspase-3 was the protease which mediated the processing (Fig. 4D).
Involvement of PI3-kinase-Akt/PKB pathway in E-cadherin processing As we have recently described, anti-apoptotic Akt/PKB was twofold stronger activated in wt E-cadherin expressing MDAMB-435S cells than in the parental cell line or cells expressing mutant E-cadherin under normal cultivation conditions [16]. Similar in all cell lines, application of cisplatin for 24 h caused a transient increase of Akt/PKB phosphorylation on Ser 473 which was not due to increased expression (Fig. 5). In contrast, the total kinase content was reduced. After 48 h of cisplatin treatment, phosphorylation of Akt/PKB was strongly diminished which seemed to be a consequence of the decreased amount of total kinase. Interestingly, we determined by DAPI staining analysis more apoptotic cells in the presence of wt Ecadherin compared to del 8 E-cadherin after 24 and 32 h of cisplatin treatment (Fig. 5B). These data suggest that in the early phase of cisplatin treatment, the Akt/PKB survival pathway was strongly activated. Later on, survival signalling was diminished, leading to a change of balance of pro- and anti-apoptotic signalling pathways in advantage of cell death.
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Fig. 3 – Localisation of E-cadherin and β-catenin upon cisplatin-exposure. Localisation of E-cadherin and β-catenin as well as distribution of the actin cytoskeleton were visualised after 16 h of 75 μM cisplatin treatment. wt or del 8 E-cadherin expressing MDA-MB-435S cells were stained with anti-E-cadherin antibody SHE-78-7 (A) or anti-β-catenin antibody (B) and an FITC-conjugated secondary antibody. Nuclei were stained with Hoechst. A sequence of confocal images was taken at 0.7-μm intervals and one optical section is shown. Arrowheads indicate cells with redistribution of E-cadherin or β-catenin from the cellular membrane to the cytoplasm. (C) wt or del 8 E-cadherin expressing MDA-MB-435S cells were treated with 75 μM cisplatin for 32 h and actin filaments were stained with rhodamine-coupled phalloidin. Nuclei were stained with Hoechst. A sequence of confocal images was taken at 0.7-μm intervals and one optical section is shown. Arrowheads mark cells with nuclear fragmentation. Bars represent 20 μm.
Interestingly, processing of E-cadherin and β-catenin increased over time (Figs. 2 and 5A). However, this effect was more predominant in the presence of del 8 E-cadherin. To examine the role of the PI3-kinase-Akt/PKB pathway in more detail, cells were pre-treated for 1 h with the specific PI3-kinase
inhibitor, LY294002. The following cisplatin application for 28 h resulted in a marked reduction of cell viability of approximately 60% detected by DAPI staining (data not shown). While activation of Akt/PKB increased during cisplatin treatment, the LY294002/cisplatin regime abrogated completely Akt/PKB
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Fig. 4 – Cytochrome c release and caspase-3 cleavage during cisplatin-induced apoptosis. (A) wt and del 8 E-cadherin expressing MDA-MB-435S cells were treated for 16 h with 75 μM cisplatin and stained with anti-cytochrome c primary and an FITC-conjugated secondary antibody. Nuclei were stained with Hoechst. A sequence of confocal images was taken with a laser scanning microscope at 0.7 μm intervals and one optical section is shown. Arrowheads indicate cytochrome c release and nuclear fragmentation. Scale bars represent 20 μm. (B) Western blot analysis demonstrating processing of caspase-3 in cells that were either left untreated or incubated with 75 μM cisplatin for 28 h. A representative of at least three independent experiments is shown. (C) Non-transfected (M), wt and del 8 E-cadherin expressing MDA-MB-435S cells were treated for 28 h with 75 μM cisplatin and caspase-3 activity was measured in cell extracts. DMSO served as control. (D) MDA-MB-435S cells expressing del 8 E-cadherin were treated with 75 μM cisplatin for 28 h in the presence of 20 μM caspase-3 inhibitor Z-DEVD-FMK. The E-cadherin cleavage product was shown by immunoblot analysis. α-Tubulin was used as a loading control.
phosphorylation (Fig. 5C). As already shown before, the total Akt/PKB level was decreased during cisplatin treatment relative to untreated cells. Strikingly, the PI3-kinase inhibitor provoked a significant increase of cisplatin-induced E-cadherin cleavage (Fig. 5C).
levels were slightly decreased relative to untreated cells. In wt E-cadherin expressing cells, application of PD98059 resulted in enhanced nuclear fragmentation, significant inhibition of ERK activation as well as increased E-cadherin cleavage (Fig. 6B and data not shown).
Cisplatin-mediated activation of MAPK ERK1/2
Activation of stress-regulated kinases during cisplatin-induced apoptosis
Beside the PI3-kinase-Akt/PKB pathway, Erk1/2 plays also an important role in cell survival. To clarify the role of Erk1/2, cells were treated with cisplatin for 28 h and phosphorylation as well as expression of Erk1/2 was examined. For further evaluation, cells were pre-treated with a selective MEK1 inhibitor, PD98059, for 1 h prior to cisplatin application. The results indicate that Erk1/2 was strongly phosphorylated during cisplatin treatment in all cell lines to a similar extent (Fig. 6A). The increase in phosphorylated Erk1/2 (pErk1/2) did not result from increased expression of Erk1/2, as total Erk1/2
Among many biological, physical and chemical stimuli, cisplatin induces activation of SAPK/JNK and p38 MAPK [20]. Therefore, we finally investigated the impact of cisplatin on these kinases dependent on the mutational status of Ecadherin. As shown in Fig. 7, SAPK/JNK was strongly activated upon cisplatin application in all tested cell lines and this effect was not due to a difference in the expression level. However, after cisplatin treatment the phosphorylation in del 8 Ecadherin expressing cells was not as strong as compared to
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tion similar in all investigated cell lines. Activation of SAPK/ JNK and p38 occurred in a time-dependent manner, consistent with an increased number of apoptotic cells (data not shown). Taken together, cisplatin leads to a similar activation of stressregulated kinases independent of the E-cadherin status.
Discussion Cell–cell and cell–matrix contacts have important functions in epithelial cells and their disruption can lead to apoptosis. The E-cadherin–catenin complex plays a key role in the maintenance of epithelial cell adhesion and in protecting cells from cell death. In diffuse-type gastric cancer, somatic E-cadherin mutations were frequently found in a mutational hotspot region comprising exons 8 and 9. These mutations cause a loss of function (reduction of cell–cell adhesion and impairment of the tumor-suppressive function of E-cadherin) on one side and a gain of function (increase of cellular motility and upregulation of the proteolytic functions of tumor cells) on the other side [11,15–18]. In the present study, we investigated E-cadherin with a deletion of exon 8 (del 8) which affects a putative calcium binding motif located within the extracellular domain of E-cadherin [11, 13]. Since calcium binding is required for E-cadherin function and protease resistance [37], E-cadherin mutations might impair the overall structure and stability of the protein as a consequence of reduced calcium binding.
Fig. 5 – Impact of Akt/PKB on cisplatin-induced E-cadherin cleavage and apoptosis induction. (A) Western blot analysis demonstrating expression and phosphorylation of Akt/PKB and processing of E-cadherin. Cells were either left untreated or incubated with 75 μM cisplatin for 24 or 48 h. One representative of at least two independent experiments is shown. (B) Non-transfected (MDA) or E-cadherin expressing (wt or d8: del 8) MDA-MB-435S cells were treated with 75 μM cisplatin for the indicated times. Nuclei were stained with DAPI and apoptotic cells (showing nuclear condensation and apoptotic figures) were counted in nine randomly chosen high power microscopic fields. The mean percentage of apoptotic cells ± SD from two independent experiments is shown. (C) In the presence of the PI3-kinase inhibitor LY294002 (40 μM) or DMSO as a control, expression and phosphorylation of Akt/PKB as well as protein cleavage of del 8 E-cadherin was investigated by immunoblot analysis after application of 0 μM (CTL) or 75 μM cisplatin. Cells were treated with the inhibitor 1 h before cisplatin application. One representative of at least two independent experiments is shown.
wt E-cadherin expressing cells. Phosphorylation of the MAP kinase p38 was hardly detectable in untreated cells. In contrast, cisplatin caused a significant increase in p38 activa-
Fig. 6 – Role of ERK1/2 during cisplatin-induced E-cadherin cleavage. (A) Expression and phosphorylation of ERK1/2 was investigated by immunoblot analysis after application of 0 μM (CTL) or 75 μM cisplatin. One representative of at least three independent experiments is shown. (B) MDA-MB-435S cells transfected with wt E-cadherin were incubated with the MEK inhibitor PD98059 (50 μM) 1 h before cisplatin application (75 μM) for 28 h. Expression and phosphorylation of ERK1/2 as well as E-cadherin processing was examined. One representative of two independent experiments is shown. fl: full length; cl: cleaved.
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Fig. 7 – Activation of stress-activated kinases upon cisplatin application. Western blot analysis demonstrating expression and phosphorylation of JNK/SAPK and p38 MAPK in non-transfected (M) or E-cadherin (wt or del 8) transfected MDA-MB-435S cells which were treated with 75 μM cisplatin for 28 h. One representative of at least three independent experiments is shown.
Here, we analysed the fate of del 8 E-cadherin compared to wt E-cadherin during cisplatin-induced apoptosis in MDAMB-435S cells and the involved signalling events. To our knowledge, this is the first study of cisplatin-induced apoptosis in tumor cells harbouring a gastric carcinoma-derived somatic E-cadherin mutation. In transfected MDA-MB-435S cells, we observed during cisplatin-induced apoptosis cleavage of E-cadherin and β-catenin. Cleavage product of E-cadherin with apparent molecular mass of 25 kDa increased with prolonged chemotherapeutic treatment and the full-length protein was decreased, concomitantly. These results were consistent with immunofluorescence analysis, showing a decrease of E-cadherin and β-catenin at cell–cell contacts and an accumulation in the cytoplasm after apoptosis induction. Additionally, N-cadherin cleavage occurred (data not shown) which is in accordance with the literature [38]. Tumorderived mutant E-cadherin was cleaved by caspase-3 more readily than the wt protein. This can be explained by the fact that larger fractions of mutant E-cadherin are retained within the cytoplasm rather than inserted in the plasma membrane [19], where it may be more accessible to proteolytic degradation. From inhibitor studies, we assume that caspase-3 was responsible for E-cadherin processing. The effect of cisplatin on members of the cadherin family was investigated previously by Schmeiser and Grand [39]. They demonstrated that cisplatin treatment of human embryo retinoblasts resulted in 48 and 104 kDa fragments of both E- and P-cadherin which were produced by caspases. Complexes between E-cadherin and catenins were sustained in apoptotic cells as long as the protein components retained intact. Our data clearly show that deletion of exon 8 in E-cadherin altered the ability to influence cisplatin-induced apoptotic tumor cell response compared to wt E-cadherin. While the contribution of wt E-cadherin to apoptosis was investigated in several studies [21–25], there is only one report on the role of gastric carcinoma-associated E-cadherin variants during programmed cell death [40]. In this report, wt E-cadherin expressed in Chinese hamster ovary cells rendered cells more sensitive to Taxol-induced apoptosis than mutant E-cadherin variants with germline mutations T340A
and V832M which had been identified in patients with hereditary diffuse gastric cancer. This result is consistent with our observations where the number of apoptotic cells was increased in the presence of wt-E-cadherin compared to del 8 E-cadherin after 24 h and 32 h of cisplatin application. Taken together, our data not only confirm previous reports on E-cadherin cleavage during apoptosis [30,39] but in addition clearly demonstrate that somatic E-cadherin mutation affect apoptosis regulation in that way that they can facilitate the disruption of adherens junction thereby possibly influencing the response of cisplatin-based chemotherapy. Interestingly, distinct proteolytic activities can act on E-cadherin cleavage during apoptosis. Steinhusen and colleagues [30] demonstrated that in addition to caspase-3-mediated E-cadherin cleavage, a matrix metalloproteinase (MMP) sheds the extracellular domain from the cell surface during staurosporin-induced apoptosis. Furthermore, a connection between nuclear MMP3 and induction of apoptosis via its catalytic activity was described in the literature [41]. Recently, we observed in transfected MDA-MB-435S cells that MMP3 was significantly expressed in the presence of del 8 E-cadherin and thereby released into the medium compared to cells transfected with wt E-cadherin. Enhanced MMP3 expression was involved in increased motility that could be blocked by NNGH, an MMP inhibitor [15]. As shown here and also described before [11], the deletion of exon 8 altered the migration pattern of E-cadherin in that way that the mutant E-cadherin showed lower molecular mass compared to wt E-cadherin already in the control situation. Beside the full-length protein, also a smaller fragment of apparently 80 kDa was detectable for del 8 E-cadherin that increased after cisplatin application. To examine the possibility that this 80-kDa fragment was caused by MMP proteolysis, we applied NNGH to the cells and analysed E-cadherin by Western blot. Our results clearly demonstrate that application of NNGH significantly decreased the 80-kDa cleavage product, indicating that MMP3 proteolysis was involved. This is of interest because MMP3 could be responsible for destabilizing mutant E-cadherin by shedding the extracellular domain that renders the remaining fragment of the cell adhesion molecule more accessible to further proteolysis. This in turn might result in the increased 25-kDa cleavage fragment we detected after cisplatin-induced apoptosis. However, additional studies are necessary to determine the precise mechanism by which MMP3 might be involved in regulating the apoptosis response upon cisplatin application in the presence of mutant E-cadherin. The PI3-kinase-Akt/PKB pathway is a central regulator of cell growth and survival [42]. Akt/PKB has been implicated in modulating the sensitivity of cancer cells towards standard chemotherapy by virtue of its activation by cisplatin [43]. Furthermore, overexpression of Akt/PKB can lead to cisplatin resistance [44]. In the present study, we observed a transient activation of Akt/PKB caused by cisplatin which is in accordance with a previous report where Akt/PKB activation was described in response to apoptotic stimuli and suggested to act as a “brake” on the process [45]. The loss of Akt/PKB expression that we detected after prolonged cisplatin treatment (48 h) might be explained by cleavage of Akt/PKB during apoptosis [46,47]. Akt/PKB is a client of Hsp90 [48,49] and upon cisplatin application, the chemotherapeutic agent associates with
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Hsp90 and reduces its chaperone activity [50]. In turn, Akt/PKB might not be longer stabilised and is therefore susceptible to degradation. It is notable that Hsp90 was similarly expressed in all investigated cell lines (data not shown). Our data further demonstrate an important role of PI3-kinase-Akt/PKB in determining the fate of E-cadherin during apoptosis, since inhibition of this pathway led to increased E-cadherin cleavage after cisplatin treatment. Although in wt E-cadherin expressing cells Akt/PKB was stronger activated without treatment suggesting an advantage against apoptotic stimuli, they underwent more apoptosis during cisplatin application. At first glance this seems to be contrary but recently we observed an increased activation of the epidermal growth factor receptor in del 8 E-cadherin expressing cells (submitted for publication) which acts also anti-apoptotic. Several components of the PI3-kinase-Akt/PKB pathway are deregulated in cancers of the digestive tract [51]. For instance, amplification of the Akt-1 locus [52], frequent monoallelic deletion of PTEN [53] as well as promoter methylation and silencing of PTEN [54] have been reported in gastric carcinoma and may play an important role in apoptosis regulation in this type of cancer. The relationship between the presence of Ecadherin mutations and alterations of the PI3-kinase-Akt/PKB pathway in gastric carcinoma remains to be investigated. Members of the MAPK family play important roles in signal transduction processes of DNA-damage signals [20]. We observed activation of ERK1/2 during induction of apoptosis by cisplatin. Inhibition of the ERK1/2 pathway enhanced E-cadherin cleavage, demonstrating its important pro-survival role. The strength and duration of ERK1/2 activation during cisplatin treatment might be among the crucial factors deciding between cell proliferation and apoptosis. Elucidating the mechanisms that regulate the apoptotic program of tumor cells can contribute to a better understanding of tumor development and potentially be relevant for therapeutic drug design. In summary, our data demonstrate that during cisplatininduced apoptosis E-cadherin with a deletion of exon 8 was more readily cleaved than wt E-cadherin. We presume that this effect was caused by caspase-3 and possibly synergistically enhanced by a matrix metalloproteinase. E-cadherin cleavage resulted in disconnection of the actin cytoskeleton and accumulation of E-cadherin and β-catenin in the cytoplasm. We identified the PI3K and MAPK pathways as important signalling events during the balance of cell survival versus cell death. Our data suggest that E-cadherin mutations can influence the therapeutic response and further investigations are necessary to clarify the precise underlying molecular mechanism.
Acknowledgments The authors thank J. Vier for technical assistance and D. Poirier for helpful discussions. Our study was supported by a fellowship awarded to Dr. M. Fuchs by the Technische Universität München (HWP II), a grant to Drs. B. Luber and I. Becker from Wilhelm-Sander-Stiftung (Nr. 1999.118.2) and a grant to Drs. B. Luber and H. Höfler from the Deutsche Forschungsgemeinschaft (SFB 456).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2007.09.004.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Deletion of exon 8 increases cisplatin-induced E-cadherin cleavage Margit Fuchs a , Christine Hermannstädter a , Peter Hutzler b , Georg Häcker c , Ferdinand Haller a , Heinz Höfler a,b , Birgit Luber a,⁎ a
Technische Universität München, Klinikum rechts der Isar, Institut für Allgemeine Pathologie und Pathologische Anatomie, D-81675 München, Germany b GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Pathologie, D-85764 Neuherberg, Germany c Technische Universität München, Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, D-81675 München, Germany
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
E-Cadherin-mediated cell–cell adhesion plays a key role in epithelial cell survival and loss of
Received 26 May 2007
E-cadherin or β-catenin expression is associated with invasive tumor growth. Somatic
Revised version received
E-cadherin mutations have been identified in sporadic diffuse-type gastric carcinoma. Here,
27 August 2007
we analysed the fate of E-cadherin with an in frame deletion of exon 8 compared to wild-type
Accepted 6 September 2007
E-cadherin and the involved signalling events during cisplatin-induced apoptosis. We report
Available online 14 September 2007
that mutant E-cadherin was more readily cleaved during apoptosis than the wild-type form. Also β-catenin, an important binding partner of E-cadherin, was processed. E-cadherin
Keywords:
cleavage resulted in disconnection of the actin cytoskeleton and accumulation of E-cadherin
E-cadherin
and β-catenin in the cytoplasm. Inhibitor studies demonstrated that E-cadherin cleavage
Cleavage
was caused by a caspase-3-mediated mechanism. We identified the Akt/PKB and the ERK1/2
Cisplatin
signalling pathways as important regulators since inhibition resulted in increased
Akt/protein kinase B
E-cadherin cleavage and apoptosis. In summary, we clearly demonstrate that somatic
E-cadherin mutations
E-cadherin mutations affect apoptosis regulation in that way that they can facilitate the disruption of adherens junctions thereby possibly influencing the response to cisplatinbased chemotherapy. Elucidating the mechanisms that regulate the apoptotic program of tumor cells can contribute to a better understanding of tumor development and potentially be relevant for therapeutic drug design. © 2007 Elsevier Inc. All rights reserved.
Introduction In epithelial cells, E-cadherin is an important component of adherens junctions [1] and via its cytoplasmic domain it is
associated with β- or γ-catenin which in turn bind to α-catenin [2]. Recently, α-catenin has been demonstrated to act as a molecular switch that associates with E-cadherin–β-catenin and regulates the assembly of actin filaments [3,4]. E-cadherin
⁎ Corresponding author. Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität München, Trogerstr. 18, 81675 München, Germany. Fax: +49 89 4140 4915. E-mail address: [email protected] (B. Luber). Abbreviations: AEC, anti-E-cadherin antibody; Akt/PKB, Akt/protein kinase B; cl, cleaved; CTL, control; DAPI, 4,6-diamidino-2phenylindole; DMSO, dimethyl sulfoxide; del 8 E-cadherin, d8, E-cadherin with deletion of exon 8; fl, full length; MDA, untransfected, parental MDA-MB-435S cells; PI3-kinase, phosphatidylinositol 3-kinase; SD, standard deviation; RT, room temperature; wt, wild-type 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.09.004
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functions as a tumor and invasion suppressor [5] and in several tumors, including gastric [6] and breast carcinomas [7], loss of E-cadherin and/or β-catenin expression have been associated with invasive tumor growth [5,8,9]. In diffuse-type gastric carcinoma and their lymph node metastases, somatic E-cadherin mutations have been found in the mutational hotspot region [10] whereby exons 8 or 9 were frequently affected by in frame deletions of the whole exons [11]. Recent studies on these mutations revealed that they affect cell adhesion, motility, proliferation, proteolysis and also tumorigenicity in an animal model [10–19]. Today, cisplatin-based chemotherapy is worldwide used for the treatment of cancer patients including those suffering from gastric cancer. In response to cisplatin, a variety of cellular signalling pathways is modulated, among which are the Akt/PKB, ERK1/2, SAPK/JNK and p38 cascades [20]. These pathways regulate cell proliferation, DNA repair and apoptosis. The balance between pro- and anti-apoptotic factors is of critical importance since dysregulation can lead to inhibition of apoptosis and tumor development. Susceptibility to apoptosis is controlled by a variety of different regulatory proteins including E-cadherin that plays an important role in protecting cells from cell death [21–25]. Several mechanisms which control the apoptosis-regulating function of E-cadherin have been proposed [26], including activation of anti-apoptotic Akt/PKB by formation of Ecadherin-mediated cell–cell junctions [27,28] and association of E-cadherin with the epidermal growth factor receptor [23] and β-catenin [29]. Cleavage of cell adhesion components during apoptosis was therefore suggested to be an effective mechanism to eliminate the anti-apoptotic function of Ecadherin [30] and also to facilitate the separation of tumor cells which have started the apoptotic program from intact cells. Therefore, in the present study we addressed the question if cleavage of E-cadherin is affected by deletion of exon 8 during cisplatin-induced apoptosis and which are the consequences on cell survival. Further, we investigated signalling events including caspase cascades that are involved in the apoptotic scenario.
Materials and methods Cell cultivation and transfection The human E-cadherin-negative cell line MDA-MB-435S (ATCC, Rockeville, MD) and the wild-type (wt) and mutant E-cadherin-cDNA transfected derivatives that were established previously [11] were grown in Dulbecco's modified Eagle medium (Life Technologies, Karlsruhe, Germany) supplemented with 10% fetal calf serum (PAN Biotech, Aidenbach, Germany) and penicillin–streptomycin (50 IU/ ml and 50 mg/ml; Life Technologies) at 37 °C and 5% CO2. MDA-MB-435S is a strain which evolved from the parent line MDA-MB-435, isolated from the pleural effusion of a female with metastatic, ductal adenocarcinoma of the breast [31]. Two different clones were investigated for each E-cadherin expression construct in this study to exclude clonal variations.
Reagents Cisplatin (Sigma, Deisenhofen, Germany) was prepared as a 2.5-mM stock solution in H2O and used at a final concentration of 75 μM. 4,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma, prepared as a 1 mg/ml stock solution in H2O and used at a concentration of 500 ng/ml. PI3-kinase inhibitor LY294002 (Calbiochem, Schwalbach, Germany) was dissolved in DMSO as a 65-mM stock solution and used at a concentration of 40 μM. MEK1 inhibitor PD98059 (Sigma) was prepared as a 50mM stock solution in dimethyl sulfoxide (DMSO) and used at a final concentration of 50 μM. Caspase-3 inhibitor Z-DEVD-FMK (Calbiochem) was dissolved in DMSO at a concentration of 50 mM and used at 20 μM. DMSO was included in the negative control samples at appropriate concentrations when the effect of Z-DEVD-FMK was tested to exclude effects of the solvent. Rhodamine-coupled phalloidin was obtained from Sigma.
Counting of apoptotic cells Cells were plated on glass coverslips in 6-well plates at a density of 8 × 104 cells per well. After 1 day, cells were treated with 75 μM cisplatin for 8, 16, 24 and 32 h. Then cells were fixed with 3.7% formaldehyde and treated for 1 h at 37 °C with 3% (w/v) BSA/0.1% (w/v) saponin. Subsequently, cells were incubated with DAPI at a final concentration of 500 ng/ml for 45 min at 37 °C. The number of living or apoptotic cells (showing nuclear condensation and apoptotic figures) was counted in nine randomly chosen high power microscopic fields (magnification: 32×, Axiovert 135, Zeiss, Jena, Germany) and the percentage values were calculated setting the total number of cells to 100%. A total of 500–1200 cells were counted for each time point in two independent experiments.
Antibodies Mouse monoclonal antibodies against E-cadherin (clone 36, AEC: anti-E-cadherin antibody), β-catenin and cytochrome c (6H2.B4) were purchased from BD Biosciences (Heidelberg, Germany). Anti-E-cadherin antibody SHE78-7 was purchased from Jackson Immuno Research Laboratories (West Grove). Antibodies against caspase-3, cleaved caspase-3 as well as polyclonal antibodies against total or activated proteins were purchased from Cell Signalling Technology (Beverly, MA): Akt, phosphorylated Akt (Ser473), Erk1/2, phosphorylated Erk1/2 (Thr202/Tyr204). Monoclonal anti-α-tubulin antibody (Sigma) was used to stain α-tubulin as a loading control. The horseradish peroxidase-conjugated anti-mouse IgG was purchased from Amersham Pharmacia Biotech (Braunschweig, Germany) and anti-rabbit IgG was from BioSource (purchased from Invitrogen, Karlsruhe, Germany).
Western blot analysis Two independent cell lines were analysed for each construct by Western blot analysis, and the results of one representative clone are shown in all figures. Cells were seeded at a density of 3.5 × 105 cells per 5-cm tissue culture dish or at 9.5 × 105 cells per 10-cm tissue culture dish. After treatment, cells were harvested at the indicated time points with 200 μl or 300 μl
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L-CAM lysis buffer as described before [16]. Proteins were separated by SDS–polyacrylamide gel electrophoresis followed by transfer to nitrocellulose (Schleicher & Schuell, Dassel, Germany) membranes. Antibody dilutions were as follows: E-cadherin clone 36 1:5000, β-catenin 1:2000, caspase-3 1:2000, cleaved caspase-3 1:1000, Akt 1:1000, phosphorylated Akt (Ser473) 1:1000, Erk1/2 1:1000, phosphorylated Erk1/2 (Thr202/Tyr204) 1:2000 and α-tubulin 1:10000. For signal detection, the enhanced chemoluminescence system (Amersham Pharmacia Biotech) was used. For signal quantification, blots were scanned and densitometric analysis was performed with Scion Image Software from Scion Corporation (Version Beta 4.0.2, Frederick, USA). In all Western blot analyses, α-tubulin staining was performed to demonstrate loading of equal amounts of cellular lysates.
Immunofluorescence analysis, nuclear and actin filament staining Cells were plated on glass coverslips in 6-well plates at a density of 8 × 104 cells per well. After 1 day, cells were treated with 75 μM cisplatin for the times indicated in the figure legends. Then, cells were either fixed with 3.7% formaldehyde (β-catenin, p53, cytochrome c) or methanol (E-cadherin) and treated for 1 h at 37 °C with 3% (w/v) BSA/0.1% (w/v) saponin in PBS for permeabilisation and blocking. Cells were subsequently incubated for 1 h with primary antibody diluted in blocking solution (anti-β-catenin antibody 1:100, anti-E-cadherin antibody SHE78-7 1:100, anti-cytochrome c antibody 1:100). Then, cells were incubated for 1 h at RT with an FITCcoupled secondary antibody (goat-anti-mouse IgG; Dianova, Hamburg, Germany) diluted 1:500 in blocking solution. Hoechst (Sigma) was used for nuclear staining at a concentration of 1:200 for 7 min at RT, protected from light. Staining of actin filaments was performed as described previously [11]. Coverslips were mounted on slides using antifading reagent (Molecular Probes, Leiden, Netherlands) and analysed with a Zeiss Axiovert laser scanning microscope LSM 510 meta.
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tivation of the adherens junction components E-cadherin and β-catenin by caspase cleavage on specific motifs [34,35]. To investigate the impact of cisplatin on tumor-derived mutant E-cadherin with a deletion of exon 8 (del 8), MDA-MB-435S cells expressing either wt or mutant E-cadherin were treated with 75 μM cisplatin for 28 h and cell extracts were analysed by Western blot analysis. At this time point, approximately 30% of the cells were apoptotic which was similar in both tested cell lines (data not shown). It is of importance to note that we observed by flow cytometric analysis the absence of Ecadherin in the non-transfected parental MDA-MB-435S cell line and expression of either wt or mutant E-cadherin in transfected cell lines in N95% of the cells [18]. As shown in Fig. 1A, both the full-length protein and a 25-kDa cleavage product were detectable after cisplatin application using an antibody directed against the C-terminus of E-cadherin (clone 36). The molecular weight of the small fragment was consistent with cleavage at the caspase-3 consensus cleavage site 747-DTRD-750 [34], which is highly conserved in different species and represents the only caspase-3 consensus sequence in the cytoplasmic tail. While cisplatin treatment caused only a minor cleavage of wt E-cadherin, cleavage of del 8 E-cadherin was strongly enhanced at 28 h, resulting in a 7.4-fold increase of the intensity of the 25-kDa fragment compared to wt E-cadherin (Fig. 1B). In contrast, a significant decrease in full-length E-cadherin was observed in cells expressing del 8 E-cadherin. Taken together,
Assay for caspase-3 activity Cells were seeded at a density of 3 × 105 cells per 6-cm tissue culture dish. After 1 day, caspase-3 inhibitor Z-DEVD-FMK was added at a concentration of 20 μM. After 1 h, cisplatin was added at a concentration of 75 μM and cells were incubated for 28 h. Then, cells were lysed in NP-40 lysis buffer [32,33]. Triplicates of aliquots were incubated with a peptide containing 10 μM of a caspase-3 recognition sequence (DEVD-AMC) in buffer consisting of bovine serum albumine, HEPES and fluorometric substrate. Free AMC was measured after 1 h incubation at 37 °C. Values are presented as relative fluorescence units (mean ± SD of triplicate reactions).
Results Increased cisplatin-mediated cleavage of mutant E-cadherin Programmed cells death is characterised by a variety of morphological and biochemical features like functional inac-
Fig. 1 – Cisplatin-induced cleavage of E-cadherin. (A) Expression and protein cleavage of E-cadherin was investigated by immunoblot analysis after treatment with 75 μM cisplatin for 28 h. Extracts were derived from non-transfected (M) or E-cadherin (wt or del 8) expressing MDA-MB-435S cells. A representative of at least three independent experiments is shown. (B) Immunoblot analyses of (A) were quantified by densitometric analysis using Scion Image Software. Shown are means of wt or mutant E-cadherin cleavage products. Each bar represents the mean±SD of three independent experiments.
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E-cadherin with a deletion of exon 8 was more susceptible to cisplatin-induced cleavage compared to the wt form.
Cisplatin-mediated β-catenin cleavage At cell–cell contacts, E-cadherin is associated via β- and α-catenin with the actin cytoskeleton. β-Catenin is not only a binding partner of E-cadherin, it plays also an important role in the Wnt signalling pathway [36]. To examine the effect of cisplatin on β-catenin, we measured its cleavage after cisplatin treatment. A decrease in full-length β-catenin was observed in cells expressing del 8 E-cadherin. Further, we detected a variety of fragments (Fig. 2), which had previously been described to appear during apoptosis [35]. β-Catenin cleavage patterns were similar in both tested cell lines expressing E-cadherin independent of the mutational status (Fig. 2). Longer cisplatin treatment resulted in an increase of smaller β-catenin fragments. However, co-immunoprecipitation analysis revealed that binding of β-catenin to E-cadherin was still detectable at 28 h of cisplatin treatment (data not shown), underlining the observation that only a fraction of adherens junctions components was cleaved at this time point.
Cisplatin-induced relocalisation of E-cadherin and β-catenin To further analyse the impact of cisplatin on the cell adhesion complex, we next investigated the localisation of E-cadherin and β-catenin after cisplatin treatment by immunofluorescence staining. In untreated wt E-cadherin expressing cells, staining of E-cadherin with an anti-SHE78-7 E-cadherin antibody directed against the extracellular domain (Fig. 3A) and β-catenin (Fig. 3B) was mainly found at cell–cell contacts. In cells expressing the mutant form of E-cadherin, E-cadherin and β-catenin were detectable at cell–cell contact sites, but also additionally in lamellipodia and the perinuclear region as described previously [11,19]. After onset of apoptosis by cisplatin, a relocalisation of E-cadherin and β-catenin from the cell periphery to the cytoplasm was found in apoptotic wt and del 8 E-cadherin expressing cells which have already lost their cell–cell contacts (Fig. 3A). Interestingly, redistribution of del 8 E-cadherin from the cellular membrane to the cytoplasm seemed to precede apoptosis because it was already visible in cisplatin-treated cells without nuclear fragmentation. Further, to visualise the reorganisation of the actin cytoskeleton during cisplatin-induced apoptosis, co-staining of actin filaments
Fig. 2 – Cisplatin-induced cleavage of β-catenin. Expression and protein cleavage of β-catenin was investigated by immunoblot analysis after treatment with 75 μM cisplatin for 24 and 48 h. Extracts were derived from non-transfected (M) or E-cadherin (wt or del 8) expressing MDA-MB-435S cells. A representative of at least three independent experiments is shown.
with phalloidin was performed. As shown in Fig. 3C, cisplatin treatment caused disruption of actin filaments in shrinked apoptotic cells with condensed nuclei. Taken together, our immunofluorescence analysis clearly demonstrates a cisplatin-mediated loss of E-cadherin from cell–cell contacts which occurred earlier in del 8 E-cadherin expressing cells. β-Catenin seemed to follow the distribution of E-cadherin.
Caspase activation and cytochrome c release in response to cisplatin Caspases are major players in apoptosis regulation that convey the apoptotic signal in a proteolytic cascade, including cleavage and activation of downstream acting caspases. Subsequently, degradation of downstream cellular targets occurs, finally leading to apoptosis. MDA-MB-435S cells expressing wt or del 8 E-cadherin were exposed to cisplatin for 28 h and activation of caspase 8, 9 and 3 was examined by Western blot analysis as well as by a caspase-3 activation assay. Additionally, cytochrome c release from mitochondria was determined indicating the involvement of the intrinsic pathway in apoptosis regulation. Independent of the Ecadherin status, cytochrome c was released into the cytoplasm after cisplatin application as detected by immunofluorescence analysis (Fig. 4A) and cell fractionation assay (data not shown). While full-length caspase-9 was cleaved in a similar manner in all tested cell lines (data not shown), cleavage of caspase 8 and 3 was strongly enhanced in cells expressing del 8-E-cadherin reflecting the result obtained for E-cadherin cleavage (Fig. 4B and data not shown). In order to determine whether Ecadherin cleavage was induced by caspase-3, cells were treated with the commonly used caspase-3 inhibitor Z-DEVD-FMK and a caspase activity assay as well as Western blot analysis were performed. The activity of caspase-3 measured in cell extracts was significantly reduced by Z-DEVD-FMK (Fig. 4C). Further, Ecadherin cleavage was completely blocked in the presence of Z-DEVD-FMK, suggesting that caspase-3 was the protease which mediated the processing (Fig. 4D).
Involvement of PI3-kinase-Akt/PKB pathway in E-cadherin processing As we have recently described, anti-apoptotic Akt/PKB was twofold stronger activated in wt E-cadherin expressing MDAMB-435S cells than in the parental cell line or cells expressing mutant E-cadherin under normal cultivation conditions [16]. Similar in all cell lines, application of cisplatin for 24 h caused a transient increase of Akt/PKB phosphorylation on Ser 473 which was not due to increased expression (Fig. 5). In contrast, the total kinase content was reduced. After 48 h of cisplatin treatment, phosphorylation of Akt/PKB was strongly diminished which seemed to be a consequence of the decreased amount of total kinase. Interestingly, we determined by DAPI staining analysis more apoptotic cells in the presence of wt Ecadherin compared to del 8 E-cadherin after 24 and 32 h of cisplatin treatment (Fig. 5B). These data suggest that in the early phase of cisplatin treatment, the Akt/PKB survival pathway was strongly activated. Later on, survival signalling was diminished, leading to a change of balance of pro- and anti-apoptotic signalling pathways in advantage of cell death.
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Fig. 3 – Localisation of E-cadherin and β-catenin upon cisplatin-exposure. Localisation of E-cadherin and β-catenin as well as distribution of the actin cytoskeleton were visualised after 16 h of 75 μM cisplatin treatment. wt or del 8 E-cadherin expressing MDA-MB-435S cells were stained with anti-E-cadherin antibody SHE-78-7 (A) or anti-β-catenin antibody (B) and an FITC-conjugated secondary antibody. Nuclei were stained with Hoechst. A sequence of confocal images was taken at 0.7-μm intervals and one optical section is shown. Arrowheads indicate cells with redistribution of E-cadherin or β-catenin from the cellular membrane to the cytoplasm. (C) wt or del 8 E-cadherin expressing MDA-MB-435S cells were treated with 75 μM cisplatin for 32 h and actin filaments were stained with rhodamine-coupled phalloidin. Nuclei were stained with Hoechst. A sequence of confocal images was taken at 0.7-μm intervals and one optical section is shown. Arrowheads mark cells with nuclear fragmentation. Bars represent 20 μm.
Interestingly, processing of E-cadherin and β-catenin increased over time (Figs. 2 and 5A). However, this effect was more predominant in the presence of del 8 E-cadherin. To examine the role of the PI3-kinase-Akt/PKB pathway in more detail, cells were pre-treated for 1 h with the specific PI3-kinase
inhibitor, LY294002. The following cisplatin application for 28 h resulted in a marked reduction of cell viability of approximately 60% detected by DAPI staining (data not shown). While activation of Akt/PKB increased during cisplatin treatment, the LY294002/cisplatin regime abrogated completely Akt/PKB
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Fig. 4 – Cytochrome c release and caspase-3 cleavage during cisplatin-induced apoptosis. (A) wt and del 8 E-cadherin expressing MDA-MB-435S cells were treated for 16 h with 75 μM cisplatin and stained with anti-cytochrome c primary and an FITC-conjugated secondary antibody. Nuclei were stained with Hoechst. A sequence of confocal images was taken with a laser scanning microscope at 0.7 μm intervals and one optical section is shown. Arrowheads indicate cytochrome c release and nuclear fragmentation. Scale bars represent 20 μm. (B) Western blot analysis demonstrating processing of caspase-3 in cells that were either left untreated or incubated with 75 μM cisplatin for 28 h. A representative of at least three independent experiments is shown. (C) Non-transfected (M), wt and del 8 E-cadherin expressing MDA-MB-435S cells were treated for 28 h with 75 μM cisplatin and caspase-3 activity was measured in cell extracts. DMSO served as control. (D) MDA-MB-435S cells expressing del 8 E-cadherin were treated with 75 μM cisplatin for 28 h in the presence of 20 μM caspase-3 inhibitor Z-DEVD-FMK. The E-cadherin cleavage product was shown by immunoblot analysis. α-Tubulin was used as a loading control.
phosphorylation (Fig. 5C). As already shown before, the total Akt/PKB level was decreased during cisplatin treatment relative to untreated cells. Strikingly, the PI3-kinase inhibitor provoked a significant increase of cisplatin-induced E-cadherin cleavage (Fig. 5C).
levels were slightly decreased relative to untreated cells. In wt E-cadherin expressing cells, application of PD98059 resulted in enhanced nuclear fragmentation, significant inhibition of ERK activation as well as increased E-cadherin cleavage (Fig. 6B and data not shown).
Cisplatin-mediated activation of MAPK ERK1/2
Activation of stress-regulated kinases during cisplatin-induced apoptosis
Beside the PI3-kinase-Akt/PKB pathway, Erk1/2 plays also an important role in cell survival. To clarify the role of Erk1/2, cells were treated with cisplatin for 28 h and phosphorylation as well as expression of Erk1/2 was examined. For further evaluation, cells were pre-treated with a selective MEK1 inhibitor, PD98059, for 1 h prior to cisplatin application. The results indicate that Erk1/2 was strongly phosphorylated during cisplatin treatment in all cell lines to a similar extent (Fig. 6A). The increase in phosphorylated Erk1/2 (pErk1/2) did not result from increased expression of Erk1/2, as total Erk1/2
Among many biological, physical and chemical stimuli, cisplatin induces activation of SAPK/JNK and p38 MAPK [20]. Therefore, we finally investigated the impact of cisplatin on these kinases dependent on the mutational status of Ecadherin. As shown in Fig. 7, SAPK/JNK was strongly activated upon cisplatin application in all tested cell lines and this effect was not due to a difference in the expression level. However, after cisplatin treatment the phosphorylation in del 8 Ecadherin expressing cells was not as strong as compared to
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tion similar in all investigated cell lines. Activation of SAPK/ JNK and p38 occurred in a time-dependent manner, consistent with an increased number of apoptotic cells (data not shown). Taken together, cisplatin leads to a similar activation of stressregulated kinases independent of the E-cadherin status.
Discussion Cell–cell and cell–matrix contacts have important functions in epithelial cells and their disruption can lead to apoptosis. The E-cadherin–catenin complex plays a key role in the maintenance of epithelial cell adhesion and in protecting cells from cell death. In diffuse-type gastric cancer, somatic E-cadherin mutations were frequently found in a mutational hotspot region comprising exons 8 and 9. These mutations cause a loss of function (reduction of cell–cell adhesion and impairment of the tumor-suppressive function of E-cadherin) on one side and a gain of function (increase of cellular motility and upregulation of the proteolytic functions of tumor cells) on the other side [11,15–18]. In the present study, we investigated E-cadherin with a deletion of exon 8 (del 8) which affects a putative calcium binding motif located within the extracellular domain of E-cadherin [11, 13]. Since calcium binding is required for E-cadherin function and protease resistance [37], E-cadherin mutations might impair the overall structure and stability of the protein as a consequence of reduced calcium binding.
Fig. 5 – Impact of Akt/PKB on cisplatin-induced E-cadherin cleavage and apoptosis induction. (A) Western blot analysis demonstrating expression and phosphorylation of Akt/PKB and processing of E-cadherin. Cells were either left untreated or incubated with 75 μM cisplatin for 24 or 48 h. One representative of at least two independent experiments is shown. (B) Non-transfected (MDA) or E-cadherin expressing (wt or d8: del 8) MDA-MB-435S cells were treated with 75 μM cisplatin for the indicated times. Nuclei were stained with DAPI and apoptotic cells (showing nuclear condensation and apoptotic figures) were counted in nine randomly chosen high power microscopic fields. The mean percentage of apoptotic cells ± SD from two independent experiments is shown. (C) In the presence of the PI3-kinase inhibitor LY294002 (40 μM) or DMSO as a control, expression and phosphorylation of Akt/PKB as well as protein cleavage of del 8 E-cadherin was investigated by immunoblot analysis after application of 0 μM (CTL) or 75 μM cisplatin. Cells were treated with the inhibitor 1 h before cisplatin application. One representative of at least two independent experiments is shown.
wt E-cadherin expressing cells. Phosphorylation of the MAP kinase p38 was hardly detectable in untreated cells. In contrast, cisplatin caused a significant increase in p38 activa-
Fig. 6 – Role of ERK1/2 during cisplatin-induced E-cadherin cleavage. (A) Expression and phosphorylation of ERK1/2 was investigated by immunoblot analysis after application of 0 μM (CTL) or 75 μM cisplatin. One representative of at least three independent experiments is shown. (B) MDA-MB-435S cells transfected with wt E-cadherin were incubated with the MEK inhibitor PD98059 (50 μM) 1 h before cisplatin application (75 μM) for 28 h. Expression and phosphorylation of ERK1/2 as well as E-cadherin processing was examined. One representative of two independent experiments is shown. fl: full length; cl: cleaved.
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Fig. 7 – Activation of stress-activated kinases upon cisplatin application. Western blot analysis demonstrating expression and phosphorylation of JNK/SAPK and p38 MAPK in non-transfected (M) or E-cadherin (wt or del 8) transfected MDA-MB-435S cells which were treated with 75 μM cisplatin for 28 h. One representative of at least three independent experiments is shown.
Here, we analysed the fate of del 8 E-cadherin compared to wt E-cadherin during cisplatin-induced apoptosis in MDAMB-435S cells and the involved signalling events. To our knowledge, this is the first study of cisplatin-induced apoptosis in tumor cells harbouring a gastric carcinoma-derived somatic E-cadherin mutation. In transfected MDA-MB-435S cells, we observed during cisplatin-induced apoptosis cleavage of E-cadherin and β-catenin. Cleavage product of E-cadherin with apparent molecular mass of 25 kDa increased with prolonged chemotherapeutic treatment and the full-length protein was decreased, concomitantly. These results were consistent with immunofluorescence analysis, showing a decrease of E-cadherin and β-catenin at cell–cell contacts and an accumulation in the cytoplasm after apoptosis induction. Additionally, N-cadherin cleavage occurred (data not shown) which is in accordance with the literature [38]. Tumorderived mutant E-cadherin was cleaved by caspase-3 more readily than the wt protein. This can be explained by the fact that larger fractions of mutant E-cadherin are retained within the cytoplasm rather than inserted in the plasma membrane [19], where it may be more accessible to proteolytic degradation. From inhibitor studies, we assume that caspase-3 was responsible for E-cadherin processing. The effect of cisplatin on members of the cadherin family was investigated previously by Schmeiser and Grand [39]. They demonstrated that cisplatin treatment of human embryo retinoblasts resulted in 48 and 104 kDa fragments of both E- and P-cadherin which were produced by caspases. Complexes between E-cadherin and catenins were sustained in apoptotic cells as long as the protein components retained intact. Our data clearly show that deletion of exon 8 in E-cadherin altered the ability to influence cisplatin-induced apoptotic tumor cell response compared to wt E-cadherin. While the contribution of wt E-cadherin to apoptosis was investigated in several studies [21–25], there is only one report on the role of gastric carcinoma-associated E-cadherin variants during programmed cell death [40]. In this report, wt E-cadherin expressed in Chinese hamster ovary cells rendered cells more sensitive to Taxol-induced apoptosis than mutant E-cadherin variants with germline mutations T340A
and V832M which had been identified in patients with hereditary diffuse gastric cancer. This result is consistent with our observations where the number of apoptotic cells was increased in the presence of wt-E-cadherin compared to del 8 E-cadherin after 24 h and 32 h of cisplatin application. Taken together, our data not only confirm previous reports on E-cadherin cleavage during apoptosis [30,39] but in addition clearly demonstrate that somatic E-cadherin mutation affect apoptosis regulation in that way that they can facilitate the disruption of adherens junction thereby possibly influencing the response of cisplatin-based chemotherapy. Interestingly, distinct proteolytic activities can act on E-cadherin cleavage during apoptosis. Steinhusen and colleagues [30] demonstrated that in addition to caspase-3-mediated E-cadherin cleavage, a matrix metalloproteinase (MMP) sheds the extracellular domain from the cell surface during staurosporin-induced apoptosis. Furthermore, a connection between nuclear MMP3 and induction of apoptosis via its catalytic activity was described in the literature [41]. Recently, we observed in transfected MDA-MB-435S cells that MMP3 was significantly expressed in the presence of del 8 E-cadherin and thereby released into the medium compared to cells transfected with wt E-cadherin. Enhanced MMP3 expression was involved in increased motility that could be blocked by NNGH, an MMP inhibitor [15]. As shown here and also described before [11], the deletion of exon 8 altered the migration pattern of E-cadherin in that way that the mutant E-cadherin showed lower molecular mass compared to wt E-cadherin already in the control situation. Beside the full-length protein, also a smaller fragment of apparently 80 kDa was detectable for del 8 E-cadherin that increased after cisplatin application. To examine the possibility that this 80-kDa fragment was caused by MMP proteolysis, we applied NNGH to the cells and analysed E-cadherin by Western blot. Our results clearly demonstrate that application of NNGH significantly decreased the 80-kDa cleavage product, indicating that MMP3 proteolysis was involved. This is of interest because MMP3 could be responsible for destabilizing mutant E-cadherin by shedding the extracellular domain that renders the remaining fragment of the cell adhesion molecule more accessible to further proteolysis. This in turn might result in the increased 25-kDa cleavage fragment we detected after cisplatin-induced apoptosis. However, additional studies are necessary to determine the precise mechanism by which MMP3 might be involved in regulating the apoptosis response upon cisplatin application in the presence of mutant E-cadherin. The PI3-kinase-Akt/PKB pathway is a central regulator of cell growth and survival [42]. Akt/PKB has been implicated in modulating the sensitivity of cancer cells towards standard chemotherapy by virtue of its activation by cisplatin [43]. Furthermore, overexpression of Akt/PKB can lead to cisplatin resistance [44]. In the present study, we observed a transient activation of Akt/PKB caused by cisplatin which is in accordance with a previous report where Akt/PKB activation was described in response to apoptotic stimuli and suggested to act as a “brake” on the process [45]. The loss of Akt/PKB expression that we detected after prolonged cisplatin treatment (48 h) might be explained by cleavage of Akt/PKB during apoptosis [46,47]. Akt/PKB is a client of Hsp90 [48,49] and upon cisplatin application, the chemotherapeutic agent associates with
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Hsp90 and reduces its chaperone activity [50]. In turn, Akt/PKB might not be longer stabilised and is therefore susceptible to degradation. It is notable that Hsp90 was similarly expressed in all investigated cell lines (data not shown). Our data further demonstrate an important role of PI3-kinase-Akt/PKB in determining the fate of E-cadherin during apoptosis, since inhibition of this pathway led to increased E-cadherin cleavage after cisplatin treatment. Although in wt E-cadherin expressing cells Akt/PKB was stronger activated without treatment suggesting an advantage against apoptotic stimuli, they underwent more apoptosis during cisplatin application. At first glance this seems to be contrary but recently we observed an increased activation of the epidermal growth factor receptor in del 8 E-cadherin expressing cells (submitted for publication) which acts also anti-apoptotic. Several components of the PI3-kinase-Akt/PKB pathway are deregulated in cancers of the digestive tract [51]. For instance, amplification of the Akt-1 locus [52], frequent monoallelic deletion of PTEN [53] as well as promoter methylation and silencing of PTEN [54] have been reported in gastric carcinoma and may play an important role in apoptosis regulation in this type of cancer. The relationship between the presence of Ecadherin mutations and alterations of the PI3-kinase-Akt/PKB pathway in gastric carcinoma remains to be investigated. Members of the MAPK family play important roles in signal transduction processes of DNA-damage signals [20]. We observed activation of ERK1/2 during induction of apoptosis by cisplatin. Inhibition of the ERK1/2 pathway enhanced E-cadherin cleavage, demonstrating its important pro-survival role. The strength and duration of ERK1/2 activation during cisplatin treatment might be among the crucial factors deciding between cell proliferation and apoptosis. Elucidating the mechanisms that regulate the apoptotic program of tumor cells can contribute to a better understanding of tumor development and potentially be relevant for therapeutic drug design. In summary, our data demonstrate that during cisplatininduced apoptosis E-cadherin with a deletion of exon 8 was more readily cleaved than wt E-cadherin. We presume that this effect was caused by caspase-3 and possibly synergistically enhanced by a matrix metalloproteinase. E-cadherin cleavage resulted in disconnection of the actin cytoskeleton and accumulation of E-cadherin and β-catenin in the cytoplasm. We identified the PI3K and MAPK pathways as important signalling events during the balance of cell survival versus cell death. Our data suggest that E-cadherin mutations can influence the therapeutic response and further investigations are necessary to clarify the precise underlying molecular mechanism.
Acknowledgments The authors thank J. Vier for technical assistance and D. Poirier for helpful discussions. Our study was supported by a fellowship awarded to Dr. M. Fuchs by the Technische Universität München (HWP II), a grant to Drs. B. Luber and I. Becker from Wilhelm-Sander-Stiftung (Nr. 1999.118.2) and a grant to Drs. B. Luber and H. Höfler from the Deutsche Forschungsgemeinschaft (SFB 456).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2007.09.004.
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