Reduced expression of EphrinA1 (EFNA1) inhibits three-dimensional growth of HT29 colon carcinoma cells

Cancer Letters 175 (2002) 187–195 www.elsevier.com/locate/canlet

Reduced expression of EphrinA1 (EFNA1) inhibits threedimensional growth of HT29 colon carcinoma cells Lyka Potla, Erwin R. Boghaert, Douglas Armellino, Philip Frost, Nitin K. Damle* Oncology/Immunology Division, Wyeth-Ayerst Research, 200/4604, 401 North Middletown Road, Pearl River, NY 10965-1299, USA Received 22 March 2001; received in revised form 22 May 2001; accepted 25 May 2001

Abstract Ephrin A1 (EFNA1) is a GPI-anchored ligand that preferentially binds to the receptor tyrosine kinase, EphA2. EphA2 is over-expressed in malignant melanocytes and in prostate carcinoma cells. Whether activation of EphA2 by EFNA1 is involved in aberrant growth or differentiation of cancer cells is currently not known. We studied the effect of reducing EFNA1 on the growth of a colon carcinoma cell line (HT29). HT29 cells were transfected with EFNA1 antisense yielding clones that expressed less than 25% of EFNA1 found in vector controls. EFNA1-antisense transfectants grew slower than controls when cultured as three-dimensional spheroids. When grown as monolayers, the transfectants had a similar doubling time of the vector controls. These results indicated that autocrine stimulation of EphA2 by EFNA1 could trigger an indirect growth signal by overcoming ‘contact inhibition’. Following addition of EFNA1-Fc to HT29 cells, tyrosine hyperphosphorylation of EphA2, E-cadherin, and b-catenin were observed. Because the function of E-cadherin is associated with contact inhibition of HT29 cells, phosphorylation of E-cadherin and b-catenin by activation of EphA1 is one possible mechanism by which HT29 cells alleviate contact inhibition. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: EphA2; EFNA1; E-cadherin; b-Catenin; Contact inhibition; Colon cancer

1. Introduction The uncontrolled proliferation of cancer cells is due to aberrant execution of growth and differentiation signals that are strictly controlled in differentiated tissues. In a differentiated epithelium, the intercellular contact of the epithelial cells inhibits their capacity to proliferate. This contact-dependent growth inhibition (‘contact inhibition’ [1]) needs to be at least partly neutralized by neoplastic cells to allow tumor growth. Surprisingly, many carcinoma cells retain the * Corresponding author. Tel.: 11-845-602-3984; fax: 11-845602-5557. E-mail address: [email protected] (N.K. Damle).

expression and function of adhesion molecules (e.g. E-cadherin [2]) that are responsible for contact inhibition. The molecular mechanisms used by a carcinoma cell to overcome this inhibition are largely unknown. Studies on EGF-induced [3] and HGF-induced [4] activation of their respective receptors showed that this activation resulted in a concomitant phosphorylation of b-catenin. This phosphorylation undermines the integrity of the cadherin complex (E-cadherin, a-, b-, and g-catenin) and is inversely proportional to the adhesive strength of E-cadherin [5]. The Ecadherin complex is responsible for the formation of intermediate junctions [6]. In addition, experimental evidence suggests that E-cadherin mediated cell–cell adhesion can inhibit cell proliferation [7,8]. Consequently, by disrupting the cadherin complex, activa-

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00613-9

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tion of certain growth factor receptors is a potential mechanism by which a cancer cell reduces contact inhibition. EphA2 (ECK), a member of the family of Eph receptor protein kinases, is over-expressed in various tumors [9–11]. Zantek et al. [12] reported that this receptor tyrosine kinase was located close to intermediate cell junctions following transfection of Ecadherin in MDA-MB-231 cells. Moreover, the EphA2 in the proximity of intermediate junctions is phosphorylated. In cells that lack the function of Ecadherin, EphA2 becomes distributed over the cell membrane and is largely unphosphorylated [11]. These data indicate that EphA2 could serve a role in tumor growth. The sole direct evidence for this hypothesis is that the stimulation of EphA2 by its ligand Ephrin A1 (EFNA1) enhances the growth of malignant melanocytes [9]. However, stimulation of proliferation by EphA2 activation in carcinoma cells has not been demonstrated. In view of these data, we questioned if activation of EphA2 serves a role in reducing contact inhibition. The present study shows two lines of evidence that support this possibility. A first series of experiments demonstrates that decreasing the amount of EFNA1 in HT29 colon carcinoma cells reduces their growth in three-dimensional cultures. These cultures were used because they reveal contact inhibition to a greater degree than by comparing confluent with subconfluent monolayer cultures [13]. A second series of experiments shows that exposure of HT29 cells to EFNA1 caused not only tyrosine hyperphosphorylation of EphA2 but also of E-cadherin and b-catenin.

2. Materials and methods 2.1. Cell lines and culture conditions HT29 was purchased from ATCC (Manassas, VA, USA). The cells were maintained in minimum essential medium (MEM) supplemented with 1 mM sodium pyruvate, non-essential amino acids, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml Gentamycin, and 10% fetal bovine serum (FBS). This mixture is hereafter called complete culture medium (CCM). All culture media and supple-

ments were purchased from Gibco (Grand Island, NY, USA). 2.2. Construction of an EFNA1 antisense expression vector The mammalian expression vector pCEP4, which encodes Epstein–Barr virus (EBV) episomal replication signals, a constitutive cytomegalovirus (CMV) promoter, and a hygromycin-resistance gene, was obtained from Invitrogen (Carlsbad, CA, USA). RNA was extracted (RNAzol B, TEL-TEST, Inc., Friendswood, TX, USA) from human umbilical vein endothelial cells (Clonetics, San Diego, CA, USA) that were stimulated with TNFa (10 ng/ml; R&D Systems, Minneapolis, MN, USA) for 4 h prior to extraction. Using a kit (Stratagene, La Jolla, CA, USA), the extracted RNA was transcribed into single-stranded cDNA. Ten micrograms of RNA was mixed with an oligo dT primer. This reaction mixture was heated to 658C for 5 min and slowly cooled to 228C. First-strand cDNA was synthesized in a mixture containing 5 ml of ten-fold concentrated first-strand buffer, 5 ml DTT, 1 ml of Rnase block, 2 ml dNTPs (1.25 mM) and 1 ml of MMLV reverse transcriptase (20 U/ml) in a total volume of 50 ml. The components were gently mixed and incubated at 378C for 1 h. This cDNA was used to amplify full-length, double-stranded EFNA1 and EFNA1 antisense (EFNA1aS) DNA. The following primers were used for the polymerase chain reaction (PCR): 5 0 -ACA GGT ACC CTC GAG ACC ACC ATG GAG TTC CTC TGG GCC CCT CTC TTG GGT CT-3 0 (EFNA1 sense) and 5 0 -ACA GGA TCC AAG CTT TCA TCA CGG GGT CAG CAG CAG AAG TGG AA-3 0 (EFNA1aS). The PCR took place in a mixture of 5 ml of first-strand cDNA, 100 ng of the sense and the antisense primer, 5 ml 10 £ PFU polymerase buffer, 500 mM MgCl2, 1.25 mM dNTPs, and 1 ml PFU enzyme (2 U/ml) in a total volume of 50 ml. The reaction consisted of 35 cycles of denaturation (958C, 1 min) and synthesis (728C, 4 min) and one termination cycle (728C, 7 min). The reaction product was analyzed by electrophoresis in 1% agarose. The EFNA1aS PCR product was purified, digested with HindIII and XhoI, and ligated into HindIII/XhoIdigested pCEP4 expression vector to create the EFNA1aS construct.

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2.3. Detection of EPHA2, EFNA1, E-cadherin, and b catenin in cell extracts and conditioned medium 2.3.1. Antibodies Rabbit antibodies against human EphA2 and EFNA1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal antibodies against E-cadherin, b-catenin, and phosphotyrosine were purchased from Transduction Laboratories (Lexington, KY, USA). Fluorescein-conjugated and phycoerythrin-conjugated F(ab 0 )2 fractions of affinity-purified goat anti-human or anti-rabbit antibodies were obtained from Zymed Corp. (South San Francisco, CA, USA). 2.3.2. Immunoprecipitation and immunoblotting Monolayers (75% confluence) were washed with PBS and lysed in 1 ml extraction buffer (1% NP-40, 150 mM NaCl, 50 mM Tris (pH 8.0), 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mg/ml aprotinin, 0.5 mg/ml leupeptin, 1 mg/ml pepstatin, and 170 mg/ ml PMSF). The protein concentration of each sample of the tumor cell lysates was determined by the BIORAD protein assay (BIO-RAD, Richmond, CA, USA). Protein extracts were preincubated with antibodies (1 mg/ml) for 2 h, 50 ml of proteinA-agarose (Calbiochem, La Jolla, CA, USA) was added and the mixture was shaken overnight at 48C. The proteinAagarose beads were washed three times with PBS containing 0.1% Tween 20, suspended in 25 ml of Laemmli sample buffer (BIO-RAD), and boiled for 5 min after addition of 5 mM 2-mercaptoethanol. The proteinA-agarose beads were pelleted by centrifugation and 25 ml of each sample was electrophoresed through 8% (for Eph family members) or 14% (for EFNA1) precast polyacrylamide gels (NOVEX, San Diego, CA, USA). Protein was then transferred to nitrocellulose membranes (Hybond ECL, Amersham, Arlington Heights, IL, USA). The membranes were blocked with 1% non-fat dry milk in Tris-buffered saline (50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 8). They were probed with the primary immunoprecipitating antibody (1 mg/ml), washed, and probed again with horseradishperoxidase-conjugated antirabbit antibody (dilution ¼ 1/10 4, Amersham). Immunoreactive bands were revealed by an Amersham ECL immunoblot detection system. To reveal tyrosine phosphorylation of E-cadherin, b-catenin, and

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EphA2, HT29 monolayer cultures were treated with EFNA1-Fc for various periods (see Fig. 4) and immunoprecipitated. Following electrophoresis and blotting, the membranes were probed with antibodies against E-cadherin, b-catenin, and EphA2, respectively. The membranes were then stripped and subsequently probed with an anti-phosphotyrosine antibody. 2.3.3. Flow cytometry Exponentially growing monolayer cultures were trypsinized to single cell suspensions. One hundred thousand cells from each suspension were centrifuged down in round-bottom microtiter culture wells and treated with 1 mg/ml of EFNA1-Fc in PBS supplemented with 2% bovine serum albumin (PBS–BSA). Human IgG1 was used as a negative control. EFNA1Fc is a chimera consisting of 200 amino acids of the N-terminal part of EFNA1 linked to the constant region of human IgG1 (ligand-body). The expression vector for the chimeric protein (generously provided by Dr D. Ceretti, Immunex, Seattle, WA, USA) was transfected into 293 cells (ATCC). EFNA1-Fc was purified from conditioned medium of these transfectants and affinity-purified using a protein A-Sepharose column (Pharmacia Biotech AB, Uppsala, Sweden). After 30 min of incubation at 48C, the cells were washed twice with PBS–BSA and counter-stained with a 1:100 dilution of fluorescein-labeled F(ab 0 )2 fragments of affinity-purified anti-human Fcg antibodies (Zymed). The stained cells were washed with PBS–BSA and suspended in 4% formaldehyde in PBS. Forward scatter, 908 light scatter, and fluorescence emission at 535 nm was determined with a FACSort flow cytometer (Becton Dickinson, San Jose, CA, USA). 2.4. Spheroid cultures Spheroids were made in static cultures according to the method of Yuhas et al. [14]. Briefly, 4 £ 10 5 cells suspended in 5 ml CCM were seeded in a Petri dish on a 5 ml semi-solid agar (0.66% in CCM) base. Spheroid formation was allowed during a 4-day period after which spheroids with a diameter of 0.15 mm were selected and placed in a 24-multiwell dish. Each well contained one spheroid on a 0.5 ml semi-solid agar base with 1 ml CCM overlay. The two diameters

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of each spheroid were measured at regular intervals during a period of 3 weeks. The volume of a spheroid was calculated according to the formula of Attia and Weiss [15]: a2 £ b £ 0:4, where a is the smaller diameter of the spheroid and b the larger one. The volume doubling time was determined by log 2/a, where a represents the slope of the log of the spheroid volume as a function of time. The statistical significance of the differences in spheroid volume doubling time was determined by Student’s t-test. 3. Results 3.1. Reduction of expression of EFNA1 in HT29 cells HT29 colon carcinoma cells expressed EphA2, EFNA1, E-cadherin, and b-catenin. This cell line was therefore used for all further described experiments. To evaluate if the interaction between EFNA1 and EphA2 had an influence on cell growth, we reduced the endogenous amount of EFNA1 available to bind EphA2. Transfection of HT29 cells with an EFNA1aS construct directly prevented the production of EFNA1 at the translational level. Fig. 1 illustrates that the expression of EFNA1 by the HT29/ EFNA1aS clones was decreased in comparison to HT29pCEP4. Two repeats of this experiment yielded identical results. The Western blots in Fig. 1A show this decrease in the cell lysates derived from these transfectants. The EphA2 levels (Fig. 1B) were also slightly decreased in the HT29/EFNA1aS clones. Nonetheless, FACS analysis demonstrated that a similar amount of EFNA1-Fc was bound to HT29/pCEP4 and to each HT29/EFNA1aS clone. The mean channel fluorescence (MCF) values were 22.75 for HT29/ pCEP4 and 17.69, 17.43, and 21.54 for three HT29/ EFNA1aS clones. None of the differences in MCF values was significant, demonstrating that the transfection did not influence the cell’s potential to bind exogenous EFNA1. 3.2. Influence of reduced EFNA1 expression on growth of HT29 cells In monolayers (Fig. 2), we did not observe significant differences in doubling times among four HT29/ EFNA1aS clones, HT29/pCEP4 and HT29. The average doubling times of the cultures during their expo-

nential growth phase were 29, 27, 26, 31, 27, and 26 h for HT29/EFNA1aS clones 10, 12, 13, and 14, HT29/ pCEP4, and HT29, respectively. After reaching confluence (day 7, Fig. 2), HT29/pCEP4 and HT29 continued to grow. In 4 days the cell numbers increased by 11 and 3% for these cell lines. In contrast, the maximum cell density of the HT29/ EFNA1aS coincided with the confluence. After reaching this maximum density, cultures of HT29/ EFNA1aS started to die. This finding virtually excluded that EphA2 stimulation by autocrine EFNA1 was an essential mitogenic signal for HT29. However, some growth inhibition of EFNA1aS clones was noticed in confluent cultures, indicating

Fig. 1. Influence of EFNA1 antisense transfection on expression of EFNA1 (A) and EphA2 (B) by HT29. (A) Immunoprecipitates of HT29, HT29pCEP4 and HT29/EFNA1aS, probed for EFNA1 after Western blotting. One million cells were grown as monolayers and protein extracts were made after 16 h. Five hundred microliters of each extract (0.1 mg protein/ml) was immunoprecipitated with an antibody against EFNA1. Each precipitate was suspended in 25 ml Laemmli buffer. The suspensions were electrophoresed through a 14% polyacrylamide gel, transferred to a nitrocellulose membrane and probed with anti-EFNA1. Notice the reduced intensity of protein bands at 27 and 23 kDa (compared to HT29). This reduction was not seen following transfection with pCEP4. (B) Samples from the cell extracts used in (A) were immunoprecipitated with an antibody against EphA2. The precipitates were electrophoresed through an 8% polyacrylamide gel and probed for EphA2 after Western blotting. The clonal variation of the EphA2 expression did not relate to the expression of EFNA1.

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clones formed spheroids on semi-solid agar, indicating that the level of EFNA1 expression did not influence the capacity of HT29 to form cell aggregates. Fig. 3 illustrates a comparison of the growth curves of HT29pCEP4 and various clones of HT29/ EFNA1aS in three independent experiments. The graphs show a slower growth of HT29/EFNA1aS clones as compared to HT29/pCEP4. Slight numerical differences in the doubling times were noted depending on the identity of the clone. Nonetheless, HT29/ EFNA1aS clones consistently (40% on average) had longer doubling times than HT29/pCEP4 (see Table 1). The observation that five clones demonstrated a consistent growth reduction in independent experiments renders it less likely that the growth reduction is merely a result of selecting a slowly growing subpopulation of HT29.

3.3. EFNA1 stimulation increases tyrosine phosphorylation of EphA2, E-cadherin, and b catenin in HT29

Fig. 2. Influence of reduced EFNA1 expression on growth of HT29 in monolayer cultures. The growth curves of HT29 and HT29/ pCEP4 (closed symbols) are compared to those of four EFNA1aS-transfectants of HT29 (open symbols). Each point represents the average of four samples. The error bars indicate the standard deviation. Notice that after reaching confluence (day 7), HT29/ pCEP4 and HT29 continued to grow contrary to the EFNA1aStransfectants.

that EFNA1 induction of EphA2 could alleviate contact inhibition. To verify this hypothesis, we compared growth of HT29 with HT29/EFNA1aS in spheroid cultures. These three-dimensional cultures (for recent review, see Ref. [16]) allow the study of tumor cell growth when the cells are in contact with one another at any given time during the experiment. Therefore, stimulation or inhibition of contact-inhibited growth would be easier visualized in such cultures. HT29/pCEP4 as well as HT29/EFNA1aS

The spheroid data showed that the absence of EFNA1 was growth inhibitory for HT29 but only at maximum cell density. This finding further confirms that stimulation of EPHA2 by EFNA1 could be involved in alleviating contact inhibition. Because the E-cadherin complex plays a role in maintaining this function in HT29 [12], we questioned whether stimulation of EPHA2 by EFNA1 could influence Ecadherin and associated catenins. To determine if EFNA1 could change tyrosine phosphorylation of Ecadherin and b-catenin, we treated monolayer cultures of HT29 cells with EFNA1-Fc (100 ng/ml). Fig. 4 shows the levels of tyrosine phosphorylation of EphA2, E-cadherin, and b-catenin at 10, 20, 40, and 80 min following exposure to EFNA1-Fc. Increased tyrosine phosphorylation of EphA2 was observed 10 min after the addition of EFNA1-Fc and phosphorylation levels remained elevated for at least 80 min. In contrast, increased tyrosine phosphorylation of Ecadherin was seen 20 min after the addition of EFNA1-Fc. Tyrosine phosphorylation of b-catenin started after 40 min. Despite the elevation of EphA2-phosphorylation for 80 min, increased tyrosine phosphorylation of E-cadherin and b-catenin was only observed before the 40-min time point.

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Fig. 3. Inhibition of HT29 spheroid growth following transfection with EFNA1 antisense. Three independent experiments are presented. Spheroid growth of HT29 cells that were transfected with the pCEP4 vector (closed symbols) was compared to the growth of various clones of EFNA1 antisense transfectants (open symbols). The ordinate represents the spheroid volume while the abscissa stands for the period of spheroid culture. Data points represent the average volume of at least four spheroids. Error bars represent the standard deviation. The initial diameter of each spheroid was between 0.1 and 0.2 mm.

Table 1 Increase of spheroid volume doubling time of EFNA1aS-transfectants Cell line

2 a T (h) (average ^ SD)

Number of spheroids

2 T increase (fold control)

Statistical significance (P-value Student’s t-test)

Experiment 1 HT29/pCEP4 HT29/EFNA1aS-c14

53 ^ 6 92 ^ 14

6 6

1.0 1.7

– 0.000096

Experiment 2 HT29/pCEP4 HT29/EFNA1aS-c10 HT29/EFNA1aS-c12 HT29/EFNA1aS-c13

65 ^ 16 86 ^ 9 88 ^ 18 83 ^ 10

5 4 5 5

1.0 1.3 1.4 1.3

– 0.047 0.06 0.06

Experiment 3 HT29/pCEP4 HT29/EFNA1aS-c9 HT29/EFNA1aS-c10

66 ^ 4 81 ^ 14 115 ^ 18

6 5 5

1.0 1.2 1.7

– 0.027 0.015

a 2

T, volume doubling time.

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Fig. 4. EFNA1 stimulates tyrosine phosphorylation of EphA2, E-cadherin, and b-catenin in HT29 cells. HT29 was treated for various periods with 100 ng/ml EFNA1-Fc prior to protein extraction. The extracts were immunoprecipitated with antibodies against EphA2 (A), E-cadherin (B), or b-catenin (C). The immunoprecipitates were subjected to Western blotting, probed with antibodies against phosphorylated tyrosine (A1, B1, C1) and reprobed with an antibody against EphA2 (A2), E-cadherin (B2), or b-catenin (C2).

4. Discussion The present study demonstrates that decreasing the EFNA1 expression in HT29 delayed the growth of these tumor cells in vitro. This delay only occurred when the cells were cultured in conditions that allow maximum intercellular contact (spheroid and confluent monolayer cultures). We also showed that stimulation of EphA2-phosphorylation by EFNA1 coincided with increased tyrosine-phosphorylation of E-cadherin and b-catenin. Taken together, these findings suggest that EphA2-activation by EFNA1 can downregulate E-cadherin mediated contact inhibition of carcinoma cells. Some studies indicate an increased expression of EphA2 in human cancers [9–11]. We also found this receptor and its ligand in a majority (nine out of 11) of carcinoma cell lines from human origin (data not shown). Various functions have been ascribed to EphA2 stimulation. Easty et al. [9] reported that EFNA1 can induce a mitogenic signal in malignant melanocytes. In intestinal epithelial cells, EphA2 and EFNA1 are part of an autocrine loop that regulates cell migration and barrier function [17]. More recently, Zantek et al. [12] linked the function of EphA2 to that of E-cadherin, a homotypic cell adhesion molecule responsible for the formation of adherence junctions in epithelia. The authors concluded that the loss of E-cadherin function could alter

neoplastic cell growth and adhesion via effects of EphA2. This linkage can help explain why the slower growth of EFNA1-deficient HT29 cells exclusively occurs in confluent monolayers and in spheroid cultures. Three-dimensional cultures of tumor cells (spheroids) have growth kinetics resembling those of tumor growth in vivo [18]. Indeed, the difficulty for cells in the spheroid core to obtain nutrients, growth factors, ions, and oxygen [16] can in part account for the slower growth of a spheroid as compared to that of a monolayer. Additionally, spheroid growth is perpetually subject to contact inhibition. This type of inhibition was originally described for monolayer cultures and it entails the reduction of growth when cells reach confluence. Cultures of transformed cells are usually less susceptible to this inhibition than cultures of normal cells [1]. Conceivably, this mechanism needs to be at least partly neutralized for tumor growth. Because EFNA1-deficient HT29 cells grew only slower than the vector controls when cultured as spheroids, the interaction between EFNA1 and EphA2 may be one way to neutralize this inhibition. The identical doubling times of EFNA1-deficient cells and their controls during the log-phase of monolayer growth exclude that EFNA1 would induce in HT29 cells a simple mitogenic signal as was found in malignant melanocytes. Homotypic cell adhesion molecules such as NCAM

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[19,20] and E-cadherin [8,13] can inhibit growth following cell to cell contact. Homotypically bound E-cadherin could reduce the cytoplasmic pool of bcatenin and therefore inhibit growth signals which rely on b-catenin for their signal transduction [5,6]. St. Croix et al. [13] evidenced that functional Ecadherin could upregulate the cyclin-dependent kinase inhibitor p27, hence causing inhibition of cell proliferation in three-dimensional cultures. Furthermore, the phosphorylation-status of the cadherin complex (E-cadherin, a-, b-, and g-catenin) is associated with its adhesion function in a way that tyrosine phosphorylation of predominantly b- and g-catenin, or to a lesser extent E-cadherin and a-catenin [5] is inversely proportional to adhesive strength of Ecadherin. In this regard, tyrosine phosphorylation of b-catenin can increase following activation of epidermal growth factor receptor [3] and hepatocyte growth factor receptor [4]. Hence, these growth factors could alleviate growth inhibition by destroying the functional integrity of the catenin/cadherin complex. Our finding of increased tyrosine phosphorylation of Ecadherin and b-catenin indicates that EphA2-activation can facilitate HT29 cell growth by alleviating contact inhibition. However, decreased E-cadherin mediated cell adhesion through increased phosphorylation of the E-cadherin complex has been deduced from associative evidence. Direct evidence linking the two phenomena is yet to be obtained [21]. The phosphorylation of E-cadherin and b-catenin induced by EFNA1-Fc was only demonstrable during 40 min of exposure of the cell culture. After this period, tyrosine-phosphorylation of E-cadherin and b-catenin was reduced to initial levels regardless of the presence of EPNA1-Fc. This finding implies that contact inhibition needed only to be alleviated for a short time period to provide a growth advantage for a tumor population. This idea is also consistent with the fact that HT29 cells can grow in three dimensions and remain organized as a spheroid. If the adherence junctions were continuously destabilized, one would expect the cells to form loose aggregates as observed in tumor cells void of E-cadherin or a-catenin (unpublished observation). Moreover, the existence of a mechanism that can neutralize contact inhibition for short time intervals could be an additional reason why E-cadherin function can be maintained in certain tumors. It has been postulated that E-cadherin expres-

sion can be advantageous to a tumor cell by preventing apoptosis (see Ref. [13] for discussion). Nonetheless, it remains an enigma how this advantage would outweigh the disadvantage of growth inhibition. Provision of a molecular mechanism to switch off this inhibition would neutralize this selective growth disadvantage of an E-cadherin expressing tumor cell. Addition of EFNA1-Fc cannot entirely compensate for the loss of endogenous EFNA1. EFNA1-Fc (1 or 2 mg/ml), did not stimulate the growth of EFNA1deficient spheroids (data not shown). These amounts of EFNA1-Fc are nevertheless capable of inducing phosphorylation of EphA2 in monolayer cultures. Several reasons can account for this discrepancy. The simplest explanation is that EFNA1-Fc has easier access to adherence junctions when the cells are cultured as monolayers as opposed to multilayered spheroid cultures. Additionally, the absence of the GPI-anchor in EFNA1-Fc may present another reason why the ligand body cannot substitute for the natural ligand. A GPI membrane anchor can sustain the apposition and binding of EphA2 and EFNA1. This sustained binding could then prevent internalization and cause oligomerization of EphA2. This notion is analogous to the ligand-induced oligomerization of EphB that is necessary for complete biological activity [22]. By conjecture, EFNA1-Fc may fail to cause oligomerization of the receptor and would therefore not substitute for the loss of the natural ligand. Further experimentation will be needed to distinguish the molecular consequences of EphA2-activation by EFNA1-Fc from the activation by the natural ligand. In conclusion, our data underscore that mechanisms distinct from growth stimuli of single cells may be operational to allow the growth of tumor cells in a tissue configuration. We provided evidence that at least in HT29 cells the autocrine production of EFNA1 can contribute to cell growth in three dimensions. The results also substantiate the hypothesis that “effects of E-cadherin on tumor cell behavior may occur via effects of Eph-A2” [12]. More specifically, the activation of Eph-A2 by EFNA-1 can alleviate E-cadherin mediated contact inhibition in tumor cells. The potential importance of this mechanism requires further studies of its molecular details and its role in carcinoma growth in general.

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Acknowledgements We thank Dr D. Cerretti for his gift of the EFNA1Fc expression plasmid and Dr T. Hunter for providing the EphA2 plasmid construct.

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