EpCAM Is Involved in Maintenance of the ... - Wiley Online Library

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS EpCAM Is Involved in Maintenance of the Murine Embryonic Stem Cell Phenotype BA´RBARA GONZA´LEZ,a SABINE DENZEL,b BRIGITTE MACK,b MARCUS CONRAD,c OLIVIER GIRESa,b a

Clinical Cooperation Group Molecular Oncology, Helmholtz-Zentrum Mu¨nchen, German Research Center for Environmental Health, and Head and Neck Research Department, Ludwig-Maximilians-University of Munich, Munich, Germany; bDepartment of Otorhinolaryngology, Head and Neck Surgery, Grosshadern Medical Center, Ludwig-Maximilians-University of Munich, Munich, Germany; cHelmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Munich, Germany Key Words. Cell adhesion molecules • Cell surface markers • Embryonic stem cells • Self-renewal

ABSTRACT Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is expressed on subsets of normal epithelia, numerous stem- and progenitor-type cells, and most carcinomas and highly overexpressed on cancer-initiating cells. The role of EpCAM in early development, particularly in stem-like cells, has remained unclear. Here, we show that the maintenance of self-renewal in murine embryonic stem (ES) cells depends on the high-level expression of EpCAM. Cultivation of ES cells under differentiation conditions in the absence of leukemia inhibitory factor (LIF) caused down-regulation of EpCAM along with decreased expression of cellular myelocytomatosis oncogene (c-Myc),

Sex-determining region Y-Box 2, Octamer 3/4 (Oct3/4), and Stat3. As a consequence ES cells were morphologically differentiated and ceased to proliferate. RNA interference-mediated inhibition of EpCAM expression under self-renewal conditions resulted in quantitatively decreased proliferation, decreased Oct3/4, SSEA-1, and c-Myc expression, and diminished alkaline phosphatase activity. Conversely, exogenous expression of EpCAM partially compensated for the requirement of ES cells for LIF to retain a stem cell phenotype. Thus, murine EpCAM is a transmembrane protein, which is essential but by itself is not sufficient for maintenance of the ES cell phenotype. STEM CELLS 2009;27:1782–1791

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Murine embryonic stem (ES) cells were first described in 1981 and derive from the inner cell mass [1]. Pluripotency of stem cells is defined as the capacity to self-renew and differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) with adequate triggers and is controlled by diverse signaling molecules and transcription factors [2–8]. In the murine system, leukemia inhibitory factor (LIF) signaling, signal transducer and activator of transcription (Stat) 3, Nanog, Octamer 3/4 (Oct3/4), Kru¨ppel-like factor 4, Sex-determining region Y-Box 2 (Sox2), and cellular myelocytomatosis oncogene (c-Myc), and Wnt signals are decisive factors that contribute to the phenotype of ES cells [3–5, 7, 9–12]. Notably, the expression of some of these proteins, although still defining valuable markers for pluripotency and self-renewal, is not restricted to ES cells. Although these key members of stem cell renewal regulators have been identified, the orchestrated activity of major factors in ES cells is only partially understood. The interleukin (IL)-6 cytokine family member LIF, whose activity affects Stat3 and other transcription factors, is

a master regulator of stem cellness in mice [6]. Understanding regulatory events that impinge on the fate of ES cells is of both academic and clinical value. Epithelial cell adhesion molecule EpCAM (CD326) [13] is a transmembrane glycoprotein with accepted relevance in human hepatoblasts [14, 15] and malignant derivatives thereof [16], during renal regeneration [17], in human carcinomas [18–20], and during morphogenesis [21]. In 2004 investigations on human EpCAM revealed for the first time a role in signal transduction and cell cycle regulation [22, 23]. EpCAM was reckoned to be a promoter of proliferation, migration, and invasion and a potential antagonist of differentiation [22– 25]. So far, a large part of this phenotype has been attributed to its role in the regulation of transcription of the oncogenic factor c-Myc and cyclins A and E [22]. Recently, the signaling mode of EpCAM was uncovered and revealed to rely on regulated intramembrane proteolysis and nuclear translocation of the intracellular domain of EpCAM in conjunction with bcatenin and lef-1 [26]. Notably, transcription of the epcam gene itself depends on transcription factors that are regulators of ES cell fate, that is, members of the Wnt signaling pathway such as T-Cell factor 4 [27].

Author contributions: B.G.: Collection and assembly of data; Data analysis and interpretation; Manuscript writing support; S.D. and B.M.: Collection and assembly of data; M.C.: Conception and design; Manuscript writing support; O.G.: Conception and design; Data analysis and interpretation; Manuscript writing. Correspondence: Olivier Gires, Ph.D., Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Marchioninistr. 15, D-81377, Munich, Germany. Telephone: 49-89-70953895; Fax: 49-89-70956896; e-mail: [email protected]. de Received December 16, 2008; accepted for publication April 15, 2009; first published online in STEM CELLS EXPRESS April 23, C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.97 2009. V

STEM CELLS 2009;27:1782–1791 www.StemCells.com

Gonza´lez, Denzel, Mack et al.

1783

We show here that EpCAM is expressed to higher levels in murine ES cells under self-renewal conditions than during morphological differentiation of these cells. After withdrawal of LIF signals, ES cells downregulate EpCAM expression at their cell surface along with classic markers of stem cells. Exogenous inhibition of EpCAM expression induced features of differentiating ES cells such as loss of proliferation and of classic ES cell markers. Sustained expression of EpCAM was able to complement partly for the loss of LIF-mediated signals. Hence, expression of EpCAM is relevant in the maintenance of an ES-like phenotype in murine ES cells.

MATERIALS

AND

METHODS

scope; Leica, Solms, Germany, http://www.leica.com). The average depth of stapled sections during recording was 100-180 nm. For immunocytochemistry, cells were stained as mentioned above and detected with the avidin-biotin-peroxidase complex method (Vectastain; Vector Laboratories, Burlingame, CA, http:// www.vectorlabs.com) according to the manufacturer’s protocol. Images were recorded on a Zeiss Standard 25 bright-field microscope (Carl Zeiss, Inc., Jena, Germany, http://www.zeiss.com). Negative controls represented staining with a suitable immunoglobulin isotype control instead of the specific primary antibody. In addition, primary antibodies were substituted for phosphatebuffered saline (PBS) only. The specificity of EpCAM antibodies was further assessed with EpCAM-negative and -positive HEK293 transfectants and murine fibroblast transfectants [22] (data not shown).

Cell Lines and Cell Numbers

Flow Cytometry

Murine mF9 teratoma cells and human head and neck carcinoma cells were cultured in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 15% fetal calf serum (FCS) (Biochrom AG, Heidelberg, Germany, http://www.biochrom.de), except for FaDu cells (10% FCS). Murine E14TG2a and mPfes 2 cells were cultured in Stempan Gmem (PAN-Biotech, Aidenbach, Germany, http://www.pan-biotech.com). E14TG2a and mPfes 2 cells were additionally supplemented with LIF (1,000 U/ml; GibcoBRL, Gaithersburg, MD, http://www.gibcobrl.com or SigmaAldrich, Munich, Germany, http://www.sigmaaldrich.com) and grown on 0.1% gelatin-coated culture dishes. mPfes 2 were supplemented with 50 mM mercaptoethanol. For assessment of cell numbers, E14TG2a cells were plated at densities of 3 105 cells in the presence or absence of LIF. At the indicated time points, living cells were assessed by trypan blue exclusion count.

Cells were stained with an anti-EpCAM antibody (CD326; BD Biosciences; 1:50 dilution in PBS-3% FCS) or an anti-stage-specific mouse embryonic antigen (SSEA)-1 antibody (mouse polyclonal antibody MC480; Abcam) for 15 minutes on ice, washed three times in PBS-3% FCS, and stained with fluorescein isothiocyanate-conjugated secondary antibody. Measurement of cell surface expression of EpCAM was performed in a FACSCalibur device (BD Pharmingen, Heidelberg, Germany, http://www. bdbiosciences.com). Negative controls represented staining of cells in the absence of primary antibody. Given are ratios of mean fluorescence intensities after antibody staining and controls.

Immunocytochemistry and Immunofluorescence, Confocal Laser Scanning, and Bright-Field Microscopy Cells were fixed according to Brock et al. [28] and stained with specific antibodies (CD326 rat anti-mouse EpCAM; BD Biosciences, Heidelberg, Germany, http://www.bdbiosciences.com), followed by Hoechst 33342 labeling of nuclear DNA (SigmaAldrich). Oct3/4 was stained with a specific rabbit polyclonal antibody (no. 19857; Abcam, Cambridge, U.K., http://www. abcam.com). Cells were analyzed in a fluorescence laser scanning system (TCS-SP2 scanning system and DM IRB inverted micro-

Immunoblotting E14TG2a cells were lysed in buffer containing 1% Triton and protease inhibitors (Complete; Roche, Mannheim, Germany, http://www.roche-applied-science.com) for 10 minutes on ice, and debris was pelleted by centrifugation at 13,000 rpm (10 minutes, 4 C). Equal amounts of proteins in the supernatants were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred on polyvinylidene difluoride membranes. EpCAM, c-Myc, Oct3/4, and actin were detected with specific antibodies (BD Biosciences Abcam, and Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com, respectively) and horseradish peroxidase-conjugated secondary antibodies with ECL reagent (Amersham, Freiburg, Germany, http://www.gelifesciences.com). Chemoluminescence was visualized on an x-ray film.

> Figure 1. Phenotype of E14TG2a embryonic stem (ES) cells. (A): E14TG2a murine ES cells were stained with antibodies specific for the extracellular domain of murine EpCAM in conjunction with fluorochrome-labeled secondary antibodies (green) and analyzed in a laser scanning microscope. Nuclear DNA was visualized with Hoechst 33342 dye (blue). (B): Expression of murine EpCAM was assessed by immunocytochemistry in E14TG2a ES cells. Shown is one representative E14TG2a cell colony. The innermost cells display the highest EpCAM expression. (C): E14TG2a and mPfes 2 murine ES cells, murine teratoma mF9 cells, and human head and neck carcinoma cells were stained with antibodies specific for the extracellular domain of murine or human EpCAM in conjunction with fluorescein isothiocyanate-labeled secondary antibodies and analyzed by flow cytometry. Shown are control stainings in the absence of primary antibody (open diagram) and EpCAM stainings (gray diagram) of representative experiments. (D): E14TG2a murine ES cells were cultured under self-renewal conditions (þLIF) or under differentiation conditions (LIF) for the indicated time periods. After 6 days cells were supplemented with LIF or kept further under differentiation conditions. Shown are representative microscopic images at 200 magnification from three independent experiments. (E): Cells were treated as in (D) and stained with eosin (upper panel). Enzymatic activity of AP in E14TG2a ES cells was determined under self-renewal conditions or after LIF withdrawal for the indicated time period. Shown are representative stainings of three independent experiments (lower panel). (F): mRNA expression of c-myc, Sox2, LIF-R, Oct3/4, Stat3, and GAPDH as a control was analyzed by reverse transcriptase-polymerase chain reaction in E14TG2a cells under self-renewal (þLIF) or differentiation conditions (LIF). Shown are the representative results of three independent experiments. (G): Oct3/4 protein expression was analyzed by immunoblotting with specific antibodies in E14TG2a cells under self-renewal (þLIF) or differentiation conditions (LIF) at day 6. Shown are the representative results of three independent experiments. GAPDH levels served as loading controls. (H): SSEA-1 and EpCAM protein expressions was analyzed in parallel by flow cytometry with specific antibodies in E14TG2a cells under self-renewal (þLIF) or differentiation conditions (LIF) at day 6. Shown are the mean and SD of mean fluorescence intensity ratios of three independent experiments. Self-renewal conditions were set to 100% for comparability. (I): E14TG2a cells were cultured under self-renewal or differentiation conditions, and cell numbers were assessed at the indicated time points. At day 6 after LIF withdrawal cells were supplemented with LIF where indicated. Data are presented as the mean SD of three independent experiments. Abbreviations: AP, alkaline phosphatase; c-myc, cellular myelocytomatosis oncogene; d, day; DAPI, 4,6-diamidino2-phenylindole; E, embryonic day; Ep, EpCAM; FSC, forward light scatter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LIF, leukemia inhibitory factor; LIF-R, leukemia inhibitory factor receptor; MFI, mean fluorescence intensity; Oct3/4, Octamer 3/4; siRNA, small interference RNA; Sox2, Sex-determining region Y-Box 2; SSEA-1, stage-specific mouse embryonic antigen-1; Stat3, signal transducer and activator of transcription.

www.StemCells.com

EpCAM in Maintenance of Murine ES Cell Phenotype

1784

Figure 1.


1785

RNA Interference Expression of EpCAM was inhibited by transient transfection of murine EpCAM-specific small interference (si) RNA (50 -UAUU CAUUCAGAGAGGAACGGG-30 and 50 -GUUGCUCUGUGAAU GAAUAGG-30 ). As a control, an siRNA-negative control duplex (OR-0030-NEG; Eurogentec, Seraing, Belgium, http://www.eurogentec.be) was transfected similarly. Cells (2 106) were transfected with 5 lg of siRNA oligonucleotides using an Amaxa transfection system (Amaxa, Cologne, Germany, http://www.amaxa. com) and thereafter were plated in 10-cm culture plates for further cultivation and analysis. Comparison of electroporation (1.5% on average), magnet-assisted transfection (19.5% on average), and Amaxa transfections with peGFP-C1 and YFP plasmids revealed the highest transfection efficiencies with the Amaxa system (57% on average). Percentages refer to green fluorescent protein- or yellow fluorescent protein-positive cells as assessed by fluorescenceactivated cell sorting 24 hours after transfection.

Alkaline Phosphatase Activity Alkaline phosphatase activity was assessed as described previously [29].

Reverse-Transcription-Polymerase Chain Reaction Total RNA from cell lines was isolated using an RNeasy Mini Kit (Qiagen, Du¨sseldorf, Germany, http://www1.qiagen.com), and cDNA was generated using a reverse transcription system (Promega, Madison, WI, http://www.promega.com) according to the manufacturer’s instructions. The following primer pairs were used for the amplification of target mRNAs: mouse (m) Oct3/4, forward 50 -ATCGGACCAGGCTCAGAGGTATTG-30 and reverse 50 -GTTCTCATTGTTGTCGGCTTCC-30 ; mSSEA-1, forward 50 CGTGGACGATTTCCCTAATGC-30 and reverse 50 -CAGTCTGC CAAGTTGTGGATGC-30 ; mgp130, forward 50 -AAGCTATCA TGGGTCAGTTCAGGGC-30 and reverse 50 -ATTGGTGAGATT CACGGTCAGCTCT-30 ; mLIF receptor, forward 50 -TCAGGAA AATCGGCAGTAT-30 and reverse 50 -GTCTACAGCAACATGG TAAG-30 ; mNanog, forward 50 -CTGGGAACGCCTCATCAA TG-3 and reverse 50 -GGTTTTTCTGCCACCGCTTG-30 ; mSox2, forward 50 -GGAGTGGAAACTTTTGTCCGAGAC-3 and reverse 50 -TGGAGTGGGAGGAAGAGGTAACC-30 ; mc-Myc, forward 50 -TCTGTGGAGAAGAGGCAAACCC-30 and reverse 50 -TGTG CTCGTCTGCTTGAATGG-30 ; mStat3, forward 50 -AGTTCC TGGCACCTTGGATTG-30 and reverse 50 -TCTGCTGCTTCTC TGTCACTACGG-30 ; mb-catenin, forward 50 -TTTGACGCTGCT CATCCCAC-3 and reverse 50 -GCTTGCTCTCTTGATTGCCA TAAG-30 ; and mouse glyceraldehyde-3-phosphate dehydrogenase, forward 50 -TGTCGCTGTTGAAGTCAGAGGAGA-30 and reverse 50 -AGAACATCATCCCTGCCTCTACTG-3.

RESULTS EpCAM Expression on Murine ES Cells Murine E14TG2a ES cells were stained with antibodies specific for the extracellular domain of EpCAM in combination with fluorescence-labeled secondary antibodies, and confocal laser-scanning microscope images were recorded. E14TG2a cells displayed strong expression of EpCAM, which was restricted to the plasma membrane (Fig. 1A). Comparable results were obtained after immunocytochemical detection of EpCAM and for flow cytometry (Fig. 1B, 1C). Note that E14TG2a cells in aggregates displayed variances in EpCAM expression levels. Cells at the inner part of aggregates expressed EpCAM more strongly than cells at the periphery, which were partially characterized by protrusions and incipient morphological differentiation (see Fig. 1B for a detailed view). Strong expression of EpCAM was not only characteristic of E14TG2a but also was common to other ES cell lines www.StemCells.com

Figure 2. Effects of LIF withdrawal on EpCAM expression in E14TG2a embryonic stem (ES) cells. (A): E14TG2a murine ES cells were cultured under self-renewal conditions (þLIF) or under differentiation conditions (LIF). After 6 days EpCAM expression was monitored by immunocytochemistry. Shown are representative microscopic images at 400 magnification from three independent experiments. (B): E14TG2a murine ES cells were treated as in (A) and EpCAM expression was assessed by flow cytometry. Shown are mean fluorescence intensity ratios and SDs of cells grown under differentiation versus self-renewal conditions (LIF or þLIF) from three independent experiments. (C): Protein expression of EpCAM, c-Myc, and actin as a control was visualized by immunoblotting with specific antibodies in combination with horseradish peroxidase-conjugated secondary antibodies and the ECL detection system. Cells were grown as indicated (þLIF or LIF). Shown are representative results of three independent experiments. Abbreviations: c-myc, cellular myelocytomatosis oncogene; IB, immunoblot; LIF, leukemia inhibitory factor.

(mPfes 2) and malignant murine teratoma lines (mF9) (Fig. 1C). Human EpCAM mean fluorescence intensity was also assessed on the head and neck squamous cell carcinoma line FaDu, revealing expression levels comparable to those of teratoma cells (Fig. 1C).

EpCAM Expression Is Diminished during Early Differentiation For maintenance of self-renewal, murine ES cells rely on the supplementation of culture media with the IL6 cytokine family member LIF [6]. First, the effects of LIF withdrawal on ES cells were recapitulated with E14TG2a cells. Six days after withdrawal a substantial proportion of ES cells had adopted a spindle-shaped morphology and appeared as single cells rather than cells growing in colonies (Fig. 1D). After 12 days this phenotype was even more pronounced, and almost all cells displayed long protrusions and were spindle-shaped (Fig. 1D). With readdition of LIF after 6 days, remaining undifferentiated cells within the differentiation conditions took over the culture and generated colonies of densely packed, round-shaped ES cells (Fig. 1D). Morphological differentiation of ES cells after LIF withdrawal was further visualized when cells were stained with eosin (Fig. 1E, upper panel). In these cells, enzymatic activity of alkaline phosphatase (AP) was assessed as a surrogate marker for the self-renewal capacity of ES cells [8]. High AP activity was a characteristic of undifferentiated E14TG2a cells grown in the presence of LIF (Fig. 1E, lower panel). As expected, under differentiation conditions the enzymatic activity of AP decreased substantially (Fig. 1E), as did classic markers of self-renewing ES cells such as c-Myc, Sox2, LIF receptor, Oct3/4, and Stat3 (Fig. 1F–1H). In accordance with a loss of ES cell marker expression, E14TG2a cell numbers

1786


Figure 3. EpCAM and Oct3/4 expression in E14TG2a embryonic stem (ES) cells. (A): E14TG2a murine ES cells were cultured in the presence (left and right panels) or absence of LIF (middle panel). EpCAM (brown) and Oct3/4 (blue) were visualized in double stainings. Shown are representative cells from two independent experiments. Under ES cell conditions (þLIF), cells occasionally morphologically differentiated. Such spindle-shaped cells commonly decreased EpCAM and Oct3/4 expression. (B): E14TG2a murine ES cells were treated as in (A) and stained for EpCAM (green), Oct3/4 (red), and cellular DNA (blue). Stainings were visualized upon laser scanning confocal microscopy and digitally merged (lower right image in every quadrant). Cellular morphology is depicted below as transmission images. Shown are representative cells from two independent experiments. Right panels show morphologically undifferentiated and differentiated ES cells. Undifferentiated cells were characterized as EpCAMþ/Oct3/4þ. Abbreviation: LIF, leukemia inhibitory factor; Oct3/4, Octamer 3/4.

decreased by 10-fold on average with LIF withdrawal. Supplementation of cells with LIF 6 days after withdrawal reinduced proliferation of undifferentiated cells, and cell numbers were restored within 6 days (Fig. 1I). Next, we assessed whether EpCAM expression on E14TG2a cells was sensitive to culture conditions. As shown with immunocytochemical staining, LIF withdrawal resulted in substantial loss of EpCAM expression (Fig. 2A). Mean fluorescence intensity ratios of EpCAM expression at the cell surface, as determined with flow cytometry, were diminished by 60% under differentiation conditions and were restored to 70% of initial levels with LIF readdition for 6 days (Fig. 2B). Concomitant with the decrease in EpCAM cell surface expression, an overall reduction in EpCAM protein levels and in its major downstream target, c-Myc, was observed (Fig. 2C). Hence, EpCAM is expressed in murine ES cells in the presence of LIF, and levels parallel those of c-Myc as was already shown in human carcinomas, in HEK293 cells, and in NIH3T3 fibroblast model systems of de novo EpCAM expression [22]. Expressions of EpCAM and of the ES cell marker Oct3/4 were assessed simultaneously in double stainings. In the presence of LIF, EpCAM and Oct3/4 were co-expressed in ES cells and expression intensities were correlated. E14TG2a cells at the inner part of colonies expressed the highest levels of

EpCAM and Oct3/4, whereas ES cells at the outer part of colonies expressed less of both proteins and showed signs of incipient morphological differentiation (Fig. 3A, left and right panels). In sharp contrast, with LIF withdrawal cells decreased expression of EpCAM and Oct3/4 (Fig. 3A, center panel). These findings were corroborated by immunofluorescence imaging of Oct3/4 and EpCAM and confocal laser scanning microscopy assessment (Fig. 3B). Strong expression of EpCAM correlated with Oct3/4 expression at the single cell level. Occasionally, ES cells showed features of morphologically differentiated cells even in the presence of LIF in culture media. These cells were strongly increased in size and characterized by protrusions (Fig. 3B, transmission pictures, right panel). In such cases, however, differentiated cells showed low to no EpCAM expression along with a substantial decrease of Oct3/4 levels (Fig. 3B, right panels). Undifferentiated ES cells in the same field of view were EpCAMþ/Oct3/4þ.

Suppression of EpCAM Perturbed ES Cell Phenotype RNA interference was used to suppress the expression of EpCAM in E14TG2a cells under self-renewal conditions. After 48 hours cells treated with EpCAM-specific siRNA


1787

Figure 4. Effects of EpCAM inhibition on E14TG2a self-renewal. (A): E14TG2a murine embryonic stem (ES) cells were transiently transfected with control (csi) or EpCAM-specific siRNA oligonucleotides (siRNA) under self-renewal conditions. EpCAM cell surface expression was monitored after 2 days by flow cytometry. RNA interference yielded a 40% reduction of EpCAM expression in average. (B): E14TG2a cells were treated as in (A), and cell numbers were determined after 2 days. Data are presented as mean total cell numbers SD from three independent experiments. (C, D): Enzymatic activity of AP in E14TG2a ES cells was assessed under self-renewal conditions and after inhibition of EpCAM expression via RNA interference with a control siRNA (csi) or an EpCAM-specific siRNA (siRNA). Shown are mean percentages of AP (h)- and EpCAM (n)-positive cells from three independent experiments (C) and one representative example (D). (E): E14TG2a cells were transiently transfected with siRNAs under self-renewal conditions as in (A), and cell surface expression of EpCAM (n) and the ES cell marker SSEA-1 (h) was assessed after 2 days. Shown are mean percentages with SDs from three independent experiments. (F): E14TG2a cells were transiently transfected with siRNAs under self-renewal conditions as in (A) and mRNA expression of c-myc, Sox2, LIF-R, Stat3, and GAPDH as a control was assessed after 2 days. Shown are representative results from three independent experiments. (G): E14TG2a cells were transiently transfected with siRNAs under self-renewal conditions as in (A) and Oct3/4 and EpCAM protein expressions were assessed upon immunoblotting with specific antibodies in combination with horseradish peroxidase-conjugated secondary antibodies. Actin levels served as a loading control. Abbreviations: AP, alkaline phosphatase; c-myc, cellular myelocytomatosis oncogene; csi, control; E, embryonic day; GAPDH, glyceraldehyde-3phosphate dehydrogenase; FSC, forward light scatter; LIF, leukemia inhibitory factor; LIF-R, leukemia inhibitory factor receptor; mEp, mouse EpCAM; MFI, mean fluorescence intensity; Oct3/4, Octamer 3/4; siRNA, small interference RNA; Sox2, Sex-determining region Y-Box 2; SSEA-1, stage-specific mouse embryonic antigen-1; Stat3, signal transducer and activator of transcription; wt, wild-type.

displayed a 40% reduction of EpCAM surface expression compared with control siRNA (Fig. 4A, left panel). From density plots and transfection controls it was obvious that a rather large proportion of cells were transfected with siRNA. This led to an intermediate downregulation of approximately 40www.StemCells.com

50% in all cells (Fig. 4A, right panels), which fits to the halflife of its human homolog [30]. Concomitantly, cell numbers of EpCAM knockdown cells were quantitatively decreased by 40% 2 days after culturing in the presence of LIF (Fig. 4B). In addition, both the enzymatic activity of alkaline

1788


EpCAM Partly Compensates for LIF Withdrawal

Figure 5. EpCAM knockdown does not induce apoptosis. (A): E14TG2a murine ES cells were transiently transfected with control (csi) or EpCAM-specific siRNA oligonucleotides (siRNA) under selfrenewal conditions. EpCAM cell surface expression was monitored after 2 days by flow cytometry. (B, C): After transient knockdown of EpCAM, the percentage of E14TG2a murine embryonic stem cells that displayed sub-G1 fragmented DNA was assessed with propidium iodide. Shown are representative results in (B) and the mean with SD from three independent experiments in (C). Abbreviations: csi, control; E, embryonic day; siRNA, small interference RNA.

phosphatase and the expression of an additional ES cell marker, SSEA-1 [31, 32], were diminished from 82 4 to 60 5% and down to 47 12%, respectively (Fig. 4C–4E). EpCAM downregulation further resulted in a decrease of cmyc transcripts, but, unlike for LIF withdrawal (compare Fig. 2C), the mRNA levels of Sox2 and LIF receptor remained unaffected (Fig. 4F). Stat3 mRNA levels were only marginally affected after EpCAM downregulation (Fig. 4F). Downregulation of EpCAM expression also resulted in a substantial loss of Oct3/4 protein expression in E14TG2a cells (Fig. 4G). Hence, suppression of EpCAM expression specifically interfered with the expression of distinct stem cells markers and consequently with the self-renewal potential of E14TG2a cells. Next, we analyzed whether the observed effects were due to incipient apoptosis after the decreased expression of EpCAM. E14TG2a cells were transiently transfected with control or EpCAM-specific siRNA, and the expression of EpCAM was monitored by flow cytometry. As expected, EpCAM expression was diminished in the specific samples (Fig. 5A). The formation of sub-G1 DNA was assessed in those samples and was low (3%), independent of the transfected siRNA (Fig. 5B, 5C). Hence, apoptosis apparently did not account for the observed phenotype of loss of proliferation after EpCAM knockdown in ES cells.

Murine EpCAM was cloned from E14TG2a cells and expressed from a human cytomegalovirus immediate early enhancer (HCMVIEE) coupled to the chicken b-actin promoter and first intron in the pCAG eukaryotic expression vector. Bulk cultures (n ¼ 3) and single cell clones (n ¼ 3) of pCAG control (pCAG-ø) or EpCAM (pCAG-mEp) E14TG2a cells were analyzed. Expressions of endogenous and hemagglutinin (HA)-tagged transgenic EpCAM were assessed with EpCAM- and HA-specific antibodies by immunoblotting (Fig. 6A). Exogenous EpCAM was expressed to levels slightly greater than those of endogenous EpCAM and independent of the LIF supply. Stable cell clones were cultured in the presence or absence of LIF. In the presence of LIF both cell transfectants did not differ significantly in their proliferation rates and yielded similar cell numbers after 6 days (Fig. 6B). Under differentiation conditions, in which endogenous EpCAM expression was decreased (Figs. 3A, 6C), exogenously added EpCAM provided cells with a proliferation advantage. Cells that constitutively expressed EpCAM grew to an average total cell number of 1.9 107 at day 6, whereas control transfectants yielded 1.1 107 cells when starting from equal cell numbers (3 105) (Fig. 6B). This number represented a significant 1.7-fold enhanced proliferation rate upon exogenous EpCAM expression and under differentiation conditions (p ¼ .009). Effects of exogenous expression of EpCAM on the differentiation status of ES cells were monitored in stable E14TG2a transfectants. To this end, both cell lines (i.e., pCAG-ø and pCAG-mEp) were cultured under self-renewal or differentiating conditions, and alkaline phosphatase enzymatic activity and Oct3/4 protein expression were determined. AP enzymatic activity and EpCAM cell surface expression were comparable for both cell lines in the presence of LIF in cell culture media (Fig. 6C). Under differentiation conditions, however, control transfectants revealed sharp drops of both AP enzymatic activity and EpCAM expression, from approximately 80-85% positive cells down to 10% positive cells for both markers. In contrast, enforced EpCAM expression was detectable in 70% of pCAG-mEp cells, and EpCAM-expressing cells showed a threefold higher number of AP enzymatic activity-positive cells compared with pCAG-ø cells (p ¼ .03). Expression of the ES cell marker Oct3/4 was assessed via immunoblot in pCAG-ø and pCAG-mEp clones in the presence and absence of LIF in culture media. Withdrawal of LIF in pCAG-ø cells led to strong downregulation of Oct3/4 expression (Fig. 6D). In contrast, constitutive expression of EpCAM in pCAG-mEp cells resulted in a steady expression of Oct3/4 (Fig. 6D). Thus, EpCAM expression alone is able to partially maintain the phenotype of ES cells in the absence of otherwise essential signals triggered by LIF.

DISCUSSION Embryonic stem cells derive from the inner cell mass [1] and are competent to generate every cell type and organ in the developing embryo. Stem cell self-renewal and differentiation are driven by extracellular and cell-autonomous triggers. Extracellular input is provided in the form of secreted factors and cell surface-associated molecules by cells of the microenvironment, which constitute the stem cell niche [33]. Because of this pluripotency, there was great hope for use of embryonic and tissue-specific stem cells in regenerative medicine


1789

Figure 6. Effects of exogenous EpCAM expression in E14TG2a cells. (A): E14TG2a murine embryonic stem (ES) cells were stably transfected with a pCAG-ø or pCAG-mEp expression vector. Lysates of stable transfectants were separated by SDS-polyacrylamide gel electrophoresis and EpCAM, HA-tagged EpCAM, and actin were visualized with specific antibodies. (B): E14TG2a murine ES cells were stably transfected with a pCAG-ø) or pCAG-mEp expression vector. Stable transfectants were cultured under self-renewal (þLIF) or differentiation conditions (LIF), and total cell numbers were determined after 6 days. Shown are mean cell numbers with SDs from three independent experiments. (C): E14TG2a stable transfectants were cultured under self-renewal (þLIF) or differentiation conditions (LIF), and EpCAM expression (n) and alkaline phosphatase enzymatic activity (h) were assessed. Shown are mean percentages with SDs from three independent experiments. (D): E14TG2a stable transfectants were cultured under self-renewal (þLIF) or differentiation conditions (LIF), and the expression of Oct3/4 was assessed with immunoblotting with specific antibodies. As a control, actin levels were assessed on the same membrane. Shown are representative results of three independent experiments. Abbreviations: E, embryonic day; HA, hemagglutinin; LIF, leukemia inhibitory factor; mEp, mouse EpCAM; Oct3/4, Octamer 3/4; pCAG-ø, pCAG control; pCAG-mEp, pCAG EpCAM.

and cancer treatment [34–38]. Engraftment of stem cells in degenerating tissue constitutes an outstanding task, which was fulfilled already for some diseases such as muscular dystrophy [39]. However, precise knowledge of the regulation of stem cellness is mandatory in this regard. Consequently, strong emphasis is placed on the elucidation of molecular mechanisms of stem cellness. Here, we identified epithelial cell adhesion molecule EpCAM as a receptor and cell adhesion molecule involved in the fate of murine ES cells. EpCAM was described before as being present on the surface of cells of the germ line. Anderson et al. [40] already proposed a role for this receptor ‘‘in the development of the germ line and the behavior of totipotent cells’’; however, to date no function could be assigned to EpCAM in ES cells. We show that EpCAMhigh expression was characteristic for ES cells under self-renewal and pluripotency conditions. In contrast, EpCAM was rapidly downregulated with incipient differentiation along with established ES cell marker genes such as Sox2, c-Myc, Oct3/4, and Stat3. This overall downregulation was reversed with LIF resupplementation, presumably because of cells that remained undifferentiated even after LIF withdrawal and that grew again as pluripotent cells with readdition of LIF. By using RNA interwww.StemCells.com

ference and overexpression of EpCAM under self-renewal and differentiation conditions, respectively, we now provide evidence that EpCAM is essential but by itself is not sufficient to sustain a comprehensive stem cell program in vitro. Inhibition of EpCAM expression in the presence of LIF induced features of differentiating ES cells, with decreased proliferation, diminished AP activity, and diminished Oct3/4, c-Myc, and SSEA-1 protein expression. Conversely, enforced expression of EpCAM in the absence of LIF partly counteracted differentiation, including the expression of Oct3/4 and increased proliferation of cells, pointing to an active role of EpCAM in ES cell fate regulation. It is conceivable that EpCAM actively participates in the regulation of stem cellness via its ability to increase c-myc expression [22], Oct3/4, and other target genes involved in cell cycle regulation [41]. The most recent insights in EpCAM signaling via regulated intramembrane proteolysis and in conjunction with b-catenin/Lef in carcinoma cells [26, 42] will need attention in ES cells, too and should be subject of future experimental work. Expression levels of Sox2 and LIF receptor mRNAs remained unaffected by EpCAM knockdown. Hence, EpCAM selectively targets distinct subsets of genes involved in the phenotype of ES cells, that is, c-myc and Oct3/4, while leaving the


1790

expression of other genes unaffected. This finding speaks in favor of a specific effect rather than a nonspecific or even an off-target effect of EpCAM knockdown in ES cells. The observed expression pattern of EpCAM in murine ES cells was reminiscent of tissue-specific human hepatic progenitor cells. EpCAMhigh was a feature of undifferentiated precursors, whereas terminally differentiated liver cells in contrast were negative for EpCAM [15, 43]. It is noteworthy that repopulation of the liver, after injury or neoplastic transformation, was highly successful when cells expressed high levels of EpCAM [44]. Dysregulation of EpCAM expression in liver precursor cells, conversely, is highly detrimental as shown for the case of hepatoblastoma, in which EpCAMhigh is characteristic [16]. Comparable findings were reported for regenerating kidney after ischemia/reperfusion injury [17]. In line with these findings, EpCAM, which is a long-known tumor-associated antigen [18], displayed its highest expression in cancer stem cells of various malignancies [45–49]. It is therefore legitimate to speculate that EpCAM plays a roll in signaling processes that might govern cell proliferation and dedifferentiation in general and particularly in rapidly growing ES or precursor cells, as was anticipated early on [22, 40, 50, 51]. Further investigations on signaling pathways upstream and downstream of EpCAM in ES cells should elucidate this issue.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13

14 15 16 17 18

Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156. Boyer LA, Lee TI, Cole MF et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005;122:947–956. Cartwright P, McLean C, Sheppard A et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 2005;132:885–896. Liu N, Lu M, Tian X et al. Molecular mechanisms involved in selfrenewal and pluripotency of embryonic stem cells. J Cell Physiol 2007;211:279–286. Matsuda T, Nakamura T, Nakao K et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999;18:4261–4269. Okita K, Yamanaka S. Intracellular signaling pathways regulating pluripotency of embryonic stem cells. Curr Stem Cell Res Ther 2006;1: 103–111. Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res 2007;17:42–49. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature 2005;434:843–850. Reya T, Duncan AW, Ailles L et al. A role for Wnt signalling in selfrenewal of haematopoietic stem cells. Nature 2003;423:409–414. Tada T. Toti-/pluripotential stem cells and epigenetic modifications. Neurodegener Dis 2006;3:32–37. Wernig M, Meissner A, Foreman R et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007;448: 318–324. Gires O. TACSTD1 (tumor-associated calcium signal transducer 1). Atlas of Genetics and Cytogenetics in Oncology and Haematology. Available at http://atlasgeneticsoncology.org/Genes/TACSTD1ID42459ch2p21. html. Accessed. Schmelzer E, Reid LM. EpCAM expression in normal, non-pathological tissues. Front Biosci 2008;13:3096–3100. Schmelzer E, Zhang L, Bruce A et al. Human hepatic stem cells from fetal and postnatal donors. J Exp Med 2007;204:1973–1987. Cairo S, Armengol C, De Reynies A et al. Hepatic stem-like phenotype and interplay of Wnt/b-catenin and Myc signaling in aggressive childhood liver cancer. Cancer Cell 2008;14:471–484. Trzpis M, McLaughlin P, van Goor H et al. Expression of EpCAM is up-regulated during regeneration of renal epithelia. J Pathol 2008;216: 201–208. Baeuerle PA, Gires O. EpCAM (CD326) finding its role in cancer. Br J Cancer 2007;96:417–423.

In light of the present findings, enrichment of pluripotent cells based on high-level expression of EpCAM can be envisaged, for example, with an adapted microchip based on EpCAM-specific antibodies as was reported for circulating tumor cells in the blood [52]. Taken together, the results indicate that EpCAM is a novel cell surface receptor involved in the fate of ES/progenitor cells in health and disease that contributes to the intricate program governing stem cellness.

ACKNOWLEDGMENTS We cordially thank Dr. Alexander Seiler for the mPfes 2 ES cells and Marie Bouquet for the F9 cell line. Work by O.G. is supported by the Helmholtz Center Munich, Deutsche Krebshilfe, and Deutsche Krebshilfe.

DISCLOSURE

OF OF

POTENTIAL CONFLICTS INTEREST

The authors indicate no potential conflicts of interest. 19 Balzar M, Winter MJ, de Boer CJ et al. The biology of the 17–1A antigen (Ep-CAM). J Mol Med 1999;77:699–712. 20 Armstrong A, Eck SL. EpCAM: A new therapeutic target for an old cancer antigen. Cancer Biol Ther 2003;2:320–326. 21 Trzpis M, McLaughlin PM, de Leij LM et al. Epithelial cell adhesion molecule. more than a carcinoma marker and adhesion molecule. Am J Pathol 2007;171:386–395. 22 Mu¨nz M, Kieu C, Mack B et al. The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene 2004;23:5748–5758. 23 Osta WA, Chen Y, Mikhitarian K et al. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res 2004;64:5818–5824. 24 Mu¨nz M, Hofmann T, Scheibe B et al. The carcinoma-associated antigen EpCAM induces glyoxalase 1 resulting in enhanced methylglyoxal turnover. Cancer Genomics Proteomics 2004;1:241–247. 25 Mu¨nz M, Zeidler R, Gires O. The tumor-associated antigen EpCAM up-regulates the fatty acid binding protein E-FABP. Cancer Lett 2005; 225:151–157. 26 Maetzel D, Denzel S, Mack B et al. Nuclear signalling by tumourassociated antigen EpCAM. Nat Cell Biol 2009;11:162–171. 27 Yamashita T, Budhu A, Forgues M et al. Activation of hepatic stem cell marker EpCAM by Wnt-b-catenin signaling in hepatocellular carcinoma. Cancer Res 2007;67:10831–10839. 28 Brock R, Hamelers IH, Jovin TM. Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry 1999;35:353–362. 29 Berrill A, Tan HL, Wuang SC et al. Assessment of stem cell markers during long-term culture of mouse embryonic stem cells. Cytotechnology 2004;44:77–91. 30 Munz M, Fellinger K, Hofmann T et al. Glycosylation is crucial for stability of tumour and cancer stem cell antigen EpCAM. Front Biosci 2008;13:5195–5201. 31 Solter D, Knowles BB. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U|S|A 1978; 75:5565–5569. 32 Solter D. From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research. Nat Rev Genet 2006;7: 319–327. 33 Jones DL, Wagers AJ. No place like home: anatomy and function of the stem cell niche. Nat Rev Mol Cell Biol 2008;9:11–21. 34 Gordon MY. Stem cells for regenerative medicine—biological attributes and clinical application. Exp Hematol 2008;36:726–732. 35 Corsten MF, Shah K. Therapeutic stem-cells for cancer treatment: hopes and hurdles in tactical warfare. Lancet Oncol 2008;9:376–384. 36 Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 2008;132:661–680. 37 Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 2005;19:1129–1155.


38 Pera MF, Trounson AO. Human embryonic stem cells: prospects for development. Development 2004;131:5515–5525. 39 Cerletti M, Jurga S, Witczak CA et al. Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles. Cell 2008;134:37–47. 40 Anderson R, Schaible K, Heasman J et al. Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line. J Reprod Fertil 1999;116:379–384. 41 Maaser K, Borlak J. A genome-wide expression analysis identifies a network of EpCAM-induced cell cycle regulators. Br J Cancer 2008; 99:1635–1643. 42 Carpenter G, Red Brewer M. EpCAM: another surface-to-nucleus missile. Cancer Cell 2009;15:165–166. 43 Schmelzer E, Wauthier E, Reid LM. The phenotypes of pluripotent human hepatic progenitors. Stem Cells 2006;24:1852–1858. 44 de Boer CJ, van Krieken JH, Janssen-van Rhijn CM et al. Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J Pathol 1999;188:201–206. 45 Ricci-Vitiani L, Lombardi DG, Pilozzi E et al. Identification and expansion of human colon-cancer-initiating cells. Nature 2007;445: 111–115.

www.StemCells.com

1791

46 Dalerba P, Dylla SJ, Park IK et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA 2007; 104:10158–10163. 47 Al-Hajj M, Wicha MS, Benito-Hernandez A et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003;100:3983–3988. 48 Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008;8: 755–768. 49 Gires O, Klein CA, Baeuerle PA. On the abundance of EpCAM on cancer stem cells. Nat Rev Cancer 9:143, 2009; author reply 143. 50 Litvinov SV, van Driel W, van Rhijn CM et al. Expression of EpCAM in cervical squamous epithelia correlates with an increased proliferation and the disappearance of markers for terminal differentiation. Am J Pathol 1996;148:865–875. 51 Scho¨n MP, Schon M, Klein CE et al. Carcinoma-associated 38-kD membrane glycoprotein MH 99/KS 1/4 is related to proliferation and age of transformed epithelial cell lines. J Invest Dermatol 1994;102:987–991. 52 Nagrath S, Sequist LV, Maheswaran S et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 2007;450:1235–1239.