Initial activation of EpCAM cleavage viacell-to-cell contact
- Sabine Denzel†1,
- Dorothea Maetzel†1,
- Brigitte Mack2,
- Carola Eggert2,
- Gabriele Bärr2 and
- Olivier Gires1, 2Email author
© Denzel et al; licensee BioMed Central Ltd. 2009
Received: 4 June 2009
Accepted: 19 November 2009
Published: 19 November 2009
Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and β-catenin, and drives cell proliferation.
EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems.
EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce c-myc and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects.
Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).
Epithelial cell adhesion molecule EpCAM is a membrane-bound glycoprotein involved in signalling that promotes gene transcription and cell proliferation [1–3]. The high-level over-expression of EpCAM in a plethora of carcinomas  led to the use of it as a marker with prognostic quality and as a target for therapeutic strategies [5–7]. Most-recent findings revealed the necessity for regulated intramembrane proteolysis (RIP) for the induction of EpCAM-related signal transduction, which initiates at the plasma membrane [8, 9]. EpCAM becomes proteolytically activated via cleavage by TACE (tumour necrosis-factor α converting enzyme) and a gamma-secretase complex comprising presenilin 2 (PS2) . After RIP, the intracellular domain of EpCAM (EpICD) is released in the cytoplasm and shuttles into the cell nucleus in a complex with the scaffold protein FHL2 (four and a half lim domain protein 2) and β-catenin. Thereupon, EpICD contacts members of the TCF/Lef family of transcription factors, binds DNA at Lef consensus sites, and induces transcription of target genes, including c-myc, cyclins, and genes related to proliferation [2, 3, 8]. Expression of EpCAM in murine and human embryonic stem (ES) cells revealed essential to the maintenance of the pluripotent and proliferative phenotype in vitro. SiRNA-mediated inhibition of mEpCAM expression in ES cells in the presence of factors necessary for a de-differentiated phenotype induced differentiation, reduced proliferation, and diminished expression levels of classical ES cell markers such as Oct3/4 and c-Myc [10, 11]. Owing to its mode of action and capacities, EpCAM was termed a "surface-to-nucleus missile"  that is involved cancer and stem cells' signalling .
Both, full-length EpCAM but also EpICD, which is composed of twenty-six amino acids only, rendered HEK293 cells tumourigenic in vivo and yielded large tumours with high efficiency after xenotransplantation in SCID mice. Likewise, EpICD alone sufficed to substitute for the deficiency to express EpCAM in vitro and supported proliferative signals in the absence of the remaining domains of EpCAM . It is further important to note that the over-expression of EpCAM is part of the signature of cancer-initiating cells at least in human colon, breast, and pancreas carcinomas [13–15]. Thus, the aptitude of EpCAM to regulate gene transcription alongside with the Wnt pathway and its strong oncogenic potential pinpoint an important role in cancer, eventually related to the origin of malignancies, i.e. cancer-initiating cells.
It is however still not entirely understood how EpCAM cleavage and the subsequent signalling cascades are triggered. First indication for a potential mechanism came from stainings of cell agglomerates, where EpCAM was essentially cleaved at areas of cell-to cell contact . Additionally, it was demonstrated that ectodomain shedding resulted in the formation of soluble EpEX, which is instrumental as a ligand in EpCAM signalling. Treatment of EpCAM-positive cells with a recombinant version of EpEX (rEpEX) induced EpCAM cleavage, suggesting that after an initial releasing trigger (i.e. in a juxtacrine fashion), soluble EpEX might provide cells with a paracrine signal, as was shown for L1, EGF-R, TNF-R, and others [16–19].
We assessed the dependency of EpCAM cleavage, signalling, and proliferation for cell-to-cell contacts. EpCAM cleavage and subsequent proliferative signals were observed only in cells grown at sufficient initial density to allow for cell-to-cell contact at the onset of the experiment. Oppositely, cells expressing the cleaved intracellular domain EpICD instead of full-length EpCAM were independent of contacts to neighbouring cells for proper proliferation. Thus, cell-to-cell contact is one initial trigger for RIP of EpCAM and nuclear translocation of the released signalling moiety is mandatory for the induction of gene transcription, and for cellular proliferation.
Antibodies, cell lines, and plasmids
α-EpEX antibody HO.3 , α-EpICD antibody (guinea pig antibody raised against the intracellular domain; PSL, Peptide Specialty Laboratories, Heidelberg, Germany), HA-tag (Roche, Heidelberg, Germany), c-Myc, eFABP, Cyclin A and E, ERα (F-10) (Santa Cruz, Santa Cruz, USA) were used. For laser-scanning fluorescence microscopy, dye-coupled Alexa antibodies (Alexa- 488, 594, and 647; Molecular Probes, Karlsruhe, Germany) were used as secondary antibodies.
EpCAM, EpICD, and EpICD-HA cDNAs were cloned into the eukaryotic expression vector pCAG-141 to achieve constitutive expression. Additionally, EpICD was fused to a mutated ligand-binding domain of the human estrogen receptor (ERT, kind gift of Prof. Dr. Georg Bornkamm) and cloned into pCAG141. All constructs were expressed in human embryonic kidney cells (HEK293). Induction of nuclear translocation of EpICD-ERT was accomplished with 100 nM 4-hydroxytamoxifen (Sigma, Munich, Germany). Stable cell clones were generated by transfection using MATra (IBA, Göttingen, Germany) and selection with 1 μg/ml puromycin (Sigma, Munich, Germany). HEK293 transfectants, HCT-8 and MCF-7 wild type cells were cultured in DMEM with 10% fetal calf serum.
Cell counting and doubling time
HEK293 transfectants were plated in 10 cm dishes at different densities (3 × 105 or 3 × 106 cells/dish). Cell numbers were assessed at different time points upon trypan blue exclusion assay as indicated. Colon carcinoma cells (HCT-8) and breast carcinoma cells (MCF-7) were plated at different densities as follows: D1: 0,5 × 105 cells/well in 6-well plates which represented 0.05 × 105 cells/cm2; D2: 3 × 105 cells/well representing 0.31 × 105 cells/cm2; D3: 20 × 105 cells/well representing 2.18 × 105 cells/cm2 and treated similarly. Doubling times were calculated as described . In order to achieve different cell densities with fix cell numbers, 4 × 105 and 2 × 106 cells were plated in culture dishes with increasing areas. D14 × 105 = 0.07 × 105 cells/cm2 and D12 × 106 = 0.14 × 105 cells/cm2; D24 × 105 = 0.42 × 105cells/cm2 and D22 × 106 = 0.35 × 105 cells/cm2; D34 × 105 = 2 × 105 cells/cm2 and D32 × 106 = 2.08 × 105 cells/cm2.
Immunoblot and immunoprecipitation
For immunoblot analysis, all cell lines were seeded as described for cell counting. Cells were lysed at the indicated time points in 50 μl lysis buffer (1% Triton X100 in TBS). Amounts of proteins were assessed with the BCA™ Protein Assay Kit (Pierce, Thermo Scientific, Rockford, IL, USA). 50 μg of protein lysate were mixed with SDS-PAGE loading buffer (25 mM TrisHCl pH7, 5% glycerin, 1% SDS, 2% beta-mercaptoethanol, bromphenol blue). Proteins were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore, Bedford, US), and detected using specific antibodies in combination with horseradish peroxidase (HRP)-conjugated secondary antibodies and the enhanced chemiluminescence (ECL) reagent (Amersham Biosciences, Freiburg, Germany).
For immunoprecipitation, cells were lysed in PBS/1% triton X100 and protease inhibitors (Roche, Mannheim, Germany). Precleared cell-free supernatants (100,000 g, 30 min) were incubated at 4°C overnight with protein G beads (30 μl, Amersham Biosciences, Freiburg, Germany) loaded with 1 μg of the EpEX-specific antibody HO.3 . Protein G beads were collected by centrifugation, and the pellets were washed five times in cold lysis buffer. Immunoprecipitates were eluted in SDS-PAGE loading buffer (25 mM TrisHCl pH7, 5% glycerin, 1% SDS, 2% beta-mercaptoethanol, bromphenol blue). Immunoprecipitates were analysed by immunoblotting with EpEX-specific antibodies.
Laser scanning fluorescence microscopy
HCT-8, and MCF-7 cells and HEK293 transfectants were analyzed with a fluorescence laser scanning system (TCS-SP2 scanning system and DM IRB inverted microscope, Leica, Solms, Germany). If stapled sections were recorded, depth of section was 100-180 nm in average. For EpEX and EpICD detection, cells were fixed according to Brock et al.  and stained with specific antibodies, followed by Hoechst 33342 labelling of nuclear DNA (Sigma, Munich, Germany). Profiling of EpEX, EpICD, and nuclear DNA localisation was conducted with the Leica LCS Lite software with a minimum of 900 measurement points per cell. Where indicated, HCT-8 cells were treated with with 1 μg rEpEX (recombinantly expressed in yeast, Dr. H. Lindhofer, Trion Pharma, Munich Germany) before confocal microscopy was performed.
EpCAM cleavage depends on cell-to-cell contact
Regulated intramembrane proteolysis (RIP) is mandatory for activation of EpCAM-signalling in carcinoma cells and may represent the basis for the observed decrease of intact EpCAM molecules at the cell surface owing to EpEX shedding. Cell-free supernatants of HCT8 and MCF7 cells (400 μg each) cultured under D1, D2, and D3 conditions were immunoprecipitated with antibodies specific for the shed ectodomain EpEX. Detectable amounts of soluble EpEX were observed in the culture supernatant of HCT8 and MCF7 cells at day 1 under D2 and D3 conditions but hardly under density condition 1 (Figure 1D, E). Amounts of soluble EpEX were further increased at day 2 (Figure 1D, E). Inductions of EpEX secretion were measured upon densitometry and compared amongst cells with differing initial seeding (D2/D1 and D3/D1). Ratios were 5.8- and 20-fold for HCT8, and 7- and 23-fold for MCF7 at day 1, respectively. Inductions were 1.8- and 3.5-fold for HCT8, and 9.5- and 11-fold for MCF7 at day 2 (see n-fold ratios in Figure 1D, E). In order to further control and validate these findings, two fix cell numbers (4 × 105 and 2 × 106) were each plated in culture dishes with increasing sizes resulting in starting densities comparable to D1-3 for both starting cell numbers (see Materials and Methods for details). At the time points of assessment, supernatants were equilibrated to the maximal volumes and served as a source to immunoprecipitate EpEX with specific antibodies. As for the case of differing cell numbers, EpEX shedding increased along with rising densities and resulted in similar n-fold inductions (Figure 1F and 1G). Hence, EpEX amounts shed per cell increased with enhanced densities.
Induction of EpCAM targets and proliferation
Doubling times of HCT8 colon carcinoma cells depending on initial seeding density.
Doubling time (td) in days
td 24 hrs
td 48 hrs
0.5 × 105
0,43 ± 0,06
0,49 ± 0,03
3 × 105
0,34 ± 0,02*
0,43 ± 0,012*
20 × 105
0,34 ± 0,0*
0,54 ± 0,006*
Cleaved EpICD is functionally independent of cell density
In these cell transfectants, the induction of c-Myc was monitored in dependency of the initial seeding density. A ten-fold difference in seeding density was chosen (3 × 105 and 3 × 106 in a 10 cm culture dish format). At low seeding density, EpCAM expression did not influence c-Myc expression as compared with control cells (ratio EpCAM/control = 1.1), while EpICD and EpICD-HA expression associated with a two-fold increase in c-Myc levels (Figure 5B left panel). In contrast, EpCAM expression induced enhanced levels of c-Myc when cells were seeded at higher density, which allowed for cell-to-cell contact. Under these conditions, induction of c-Myc by EpCAM, EpICD, and EpICD-HA was similar and superior to EpCAM-negative control cells (Figure 5B right panel; 1.9-, 2-, and 3.7-fold, respectively).
Next, we compared the effect of EpCAM and EpICD on cell proliferation depending on the initial cell density in the genetic background of EpCAM-negative HEK293 cells. As for the induction of c-Myc, EpCAM expression resulted in increased cell numbers as compared to control transfectants only under high-density conditions (Figure 5C, right panel). EpICD and EpICD-HA remained unaffected by cell densities in their capacity to increase cell proliferation. EpICD-positive cells steadily grew to numbers three-fold higher than control cells at day three, independently of the seeding density (Figure 5C, left panel). Hence, EpICD was independent of cell-to-cell contact with respect to its capacity to induce a proliferation phenotype in HEK293 cells, while EpCAM requires sufficient initial density.
Nuclear translocation is mandatory to develop EpICD effects
More and more, a dual role for EpCAM becomes evident as was long known for cadherins and proteins of the immunoglobulin superfamily [24, 25]. EpCAM is a transmembrane protein engaged in cell adhesion  and nuclear signalling [8, 9], which is instrumental in cell proliferation and morphoregulation [27, 28]. Generally speaking, high expression of EpCAM associated with a proliferative and regenerative phenotype in normal tissues [29–31] with active sites of cell division, and with cancer-initiating cells in tumours in vivo [32, 33]. Eventually, EpCAM is reckoned as a potent oncogenic factor, which is activated via regulated intramembrane proteolysis [8, 9] and which plays an important role in cancer and stem cell signalling [10–12]. In this respect, the remarkably short intracellular domain termed EpICD is necessary and sufficient to deploy oncogenic effects in vitro and in animal models of cancer [2, 8].
In the present work, we have assessed the actual need for cell-to-cell contact for the activation of EpCAM cleavage via regulated intramembrane proteolysis. Upon variation of cell densities, it became clear that cell-to-cell contact was involved in the initial activation of EpCAM signal transduction by cleavage. If cellular contact was allowed, then cleavage proceeded, was sufficient to generate soluble EpEX, to release EpICD from its membrane-associated localisation, and to induce target genes. Soluble EpEX conferred cleavage to single cells in the culture under these conditions. This type of initial juxtacrine activation following cell-to-cell contact may allow for a restricted radius of strong effects and may achieve fine patterns of cellular cross-talk . The ability to create a soluble ligand in the form of shed EpEX provides even more flexibility. Upon initial local contact, cells generate a means for long range paracrine signalling including a gradient of activation. Interestingly, EpCAM interacts with CD44 , itself a transmembrane protein that becomes cleaved, which hence appears as a common theme of receptor co-activation that is apparently governed by tetraspanins and associated proteins [35–37]. The assembly and disruption of such complexes is yet another level of regulation of signalling, which in vivo might be affected by cell-to-cell contacts. Certainly, the differential localisation of EpCAM in normal tissue (basolateral) versus carcinomas (homogenous distribution at the membrane) will further influence EpCAM interactions and activation.
Cleavage and nuclear translocation of EpCAM is associated with an induction of target genes [2, 23] and with reduced doubling times given the fact that cells were not contact inhibited. Thus, EpCAM interactions may allow a sensing of the presence of cognate cells to trigger proliferation via EpCAM cleavage and nuclear translocation of EpICD such as in condition D2. This would rather mimic the state of micrometastasis and of normal stem/progenitor cells, e.g. when repopulating injured organs. Notably, the regeneration of damaged liver and kidney were conducted by progenitor cells, which re-expressed EpCAM to high levels. Upon differentiation to hepatocytes and completion of organ regeneration, EpCAM expression is lost again [31, 38, 39]. In case neighbouring cells are missing, like it is e.g. the case for disseminated tumour cells, cells might decrease levels of EpCAM activation, receive less proliferation signals, and might rest in a state of quiescence (condition D1). For the case of contact inhibition of cell proliferation, although cleavage of EpCAM occurred to high extent, nuclear translocation was reduced and EpICD accumulated as peri-nuclear speckles besides nuclear speckles. Nonetheless, these remaining levels of nuclear EpICD were instrumental for the rapid induction of target genes, which reached a plateau at day two of assessment. For the case of MCF-7 cells, which are bigger than HCT-8, a reduction of the target gene c-Myc was observed under D3 conditions already at day 2 and might hence be anticipated for HCT-8 at later time points. These imaging results were paralleled by corroborative cells numbers. Cells grown under high density conditions only displayed low doubling-times at day one, with a sharp increase of doubling times at day two. Inhibitory effects impacting on EpICD nuclear translocation might further account for differences in localisation as observed in vivo in normal colonic mucosa and colon carcinomas .
The molecular basis for this partial reduction of EpICD nuclear translocation and its potential implication in the regulation of EpCAM effects are totally unclear and deserve further research. Most probably, effects of EpCAM on proliferation are overcome and dampened by mechanisms of contact inhibition, which might even actively impair on EpICD nuclear translocation, e.g. via cytoplasmic inhibitors which retain EpICD. This means of regulation of EpCAM effects at the level of subcellular localisation was further underscored by experiments using conditional systems in vitro. For the first time, targeted translocation of EpICD demonstrated the necessity of nuclear localisation in order to deploy regulatory effects on the expression of c-Myc and on cell proliferation.
In summary, EpCAM becomes cleaved upon cell-to-cell contact in a juxtacrine manner and additionally fosters signalling in a paracrine fashion using soluble EpEX.
Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).
epithelial cell adhesion molecule
EpCAM extreacellular domain
EpCAM intracellular domain
Part of the work was supported by grant #107105 of the Deutsche Krebshilfe to OG.
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