Background
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 [
4] 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) [
8]. 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" [
9] that is involved cancer and stem cells' signalling [
12].
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 [
8]. 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 [
8]. 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.
Methods
Antibodies, cell lines, and plasmids
α-EpEX antibody HO.3 [
20], 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 × 10
5 or 3 × 10
6 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 × 10
5 cells/well in 6-well plates which represented 0.05 × 10
5 cells/cm
2; D2: 3 × 10
5 cells/well representing 0.31 × 10
5 cells/cm
2; D3: 20 × 10
5 cells/well representing 2.18 × 10
5 cells/cm
2 and treated similarly. Doubling times were calculated as described [
2]. In order to achieve different cell densities with fix cell numbers, 4 × 10
5 and 2 × 10
6 cells were plated in culture dishes with increasing areas. D1
4 × 105 = 0.07 × 10
5 cells/cm
2 and D1
2 × 106 = 0.14 × 10
5 cells/cm
2; D2
4 × 105 = 0.42 × 10
5cells/cm
2 and D2
2 × 106 = 0.35 × 10
5 cells/cm
2; D3
4 × 105 = 2 × 10
5 cells/cm
2 and D3
2 × 106 = 2.08 × 10
5 cells/cm
2.
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 [
20]. 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. [
21] 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.
Discussion
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 [
26] 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 [
16]. 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 [
34], 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 [
8].
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.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SD carried out cell and molecular biology studies; BM carried out cell imaging experiments; DM carried out cell and molecular biology studies, and participated in the coordination of the study; CE and GB participated in cell and molecular biology experiments; OG conceived the study, participated in its design and coordination, and wrote the manuscript.