CD44 variant isoforms associate with tetraspanins and EpCAM

Experimental Cell Research 297 (2004) 329 – 347 www.elsevier.com/locate/yexcr

CD44 variant isoforms associate with tetraspanins and EpCAM Dirk-Steffen Schmidt, a Pamela Klingbeil, a Martina Schno¨lzer, b and Margot Zo¨ller a,c,* a

Department of Tumor Progression and Tumor Defense, German Cancer Research Center, Heidelberg, Germany b Department of Central Protein Analytic, German Cancer Research Center, Heidelberg, Germany c Department of Applied Genetics, University of Karlsruhe, Karlsruhe, Germany Received 29 December 2003 Available online 20 April 2004

Abstract The metastasizing subline of the rat pancreatic adenocarcinoma BSp73 expresses a set of membrane molecules, the combination of which has not been detected on non-metastasizing tumor lines. Hence, it became of interest whether these molecules function independently or may associate and exert specialized functions as membrane complexes. Separation of CD44v4-v7 containing membrane complexes in mild detergent revealed an association with the alpha3 integrin, annexin I, EpCAM, and the tetraspanins D6.1A and CD9. EpCAM and the tetraspanins associate selectively with CD44 variant (CD44v), but not with the CD44 standard (CD44s) isoform. The complexes are found in glycolipid-enriched membrane (GEM) microdomains, which are dissolved by stringent detergents, but the complexes are not destroyed by methyl-h-cyclodextrin (MhCD) treatment, which implies that complex formation does not depend on a lipid-rich microenvironment. However, a complex-associated impact on cell – matrix and cell – cell adhesion as well as on resistance towards apoptosis essentially depended on the location in GEMs. Thus, CD44v-specific functions may well be brought about by complex formation of CD44v with EpCAM, the tetraspanins, and the alpha3 integrin. Because CD44v4-v7 – EpCAM complex-specific functions strictly depended on the GEM localization, linker or signal-transducing molecules associating with the complex are likely located in GEMs. D 2004 Elsevier Inc. All rights reserved. Keywords: Adhesion molecules; Membrane complexes; Rat; Metastasis

Introduction It is well established that the large array of functions, which a tumor cell has to fulfill to settle as a metastasis in a distant organ, requires cooperative activities between the tumor and the surrounding tissue and that several classes of molecules are involved, like cell – cell and cell – matrix adhesion molecules and matrix-degrading enzymes, to name

Abbreviations: CD44s, CD44 standard isoform; CD44v, CD44 variant isoforms; CHO, Chinese hamster ovary; ECL, enhanced chemiluminescence; FCS, fetal calf serum; GEM, glycolipid-enriched membrane microdomain; HA, hyaluronan; HRPO, horseradish peroxidase; IP, immunoprecipitation; mAB, monoclonal antibody; MhCD, methyl-hcyclodextrin; PI, propidium iodine; PMA, phorbolmyristate acetate. * Corresponding author. Department of Tumor Progression and Tumor Defense, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Fax: +49-6221-424760. E-mail address: [email protected] (M. Zo¨ller). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.02.023

only a few. Furthermore, metastasis formation requires concerted activities between tumor cells, surrounding cells, and matrix elements and between individual molecules of the tumor cell itself [1,2]. One of the molecules frequently discussed to be associated with tumor progression are CD44 variant isoforms (CD44v) (review in Refs. [3,4]). Originally, CD44v4-v7, particularly CD44v6, has been described to suffice for the induction of the metastatic phenotype in the non-metastasizing subline BSp73AS of a rat pancreatic adenocarcinoma. Yet, the transfer of CD44v4-v7 did not suffice to induce the miliary type of metastases as observed in the parental BSp73ASML subline [5]. The potential involvement of CD44v6 in tumor progression was confirmed for many tumor types in numerous clinical studies (review in Refs. [3,6 – 8]). Interestingly, this metastasis-associated molecule is also known to be involved in tissue formation and patterning [9 – 11] and in T cell activation, frequently in the context of autoimmune disease [12 –19]. Despite

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this wide range of interest in the CD44v6 molecule, its function(s) remains elusive. As CD44 isoforms do not differ in the cytoplasmic tail, CD44v6-specific functions could be due to different folding of CD44 molecules containing variant exon products, for example, it has been shown that CD44v6 has a higher binding affinity for hyaluronate than CD44 standard isoform (CD44s) [20 – 22]. Alternatively, CD44v6-specific functions could be due to differences in associating molecules, which either bind selectively to the CD44v6 exon product or bind to CD44s exon products that are differently exposed in the presence of the CD44v6 exon product. In an elegant study [23], it was shown that CD44v6 is required for c-Met phosphorylation via the formation of a CD44v6 – HFG – c-Met multiprotein complex. Furthermore, it has been suggested that CD44v6 associates differently with the cytoskeleton, in particular with ankyrin [24,25] and members of the ERM family including Merlin [26 –29], where the latter could promote tumor growth or inhibition depending on cell density [30]. Also, c-Met-induced activation of the MEK or Erk pathway requires not only the CD44v6– HFG – c-Met complex, but, in addition, an association of the cytoplasmic tail of CD44 with ERM proteins [23]. We recently described that in metastasizing, but not in non-metastasizing rat tumor lines, additional membrane molecules are selectively up-regulated [31,32]. These are the tetraspanin D6.1A [33], the rat EpCAM homologue, D5.7A [34], the uPAR-related molecule C4.4A [35], and the a6h4 integrin [36]. Overexpression of these molecules in a non-metastasizing subline supported tumor progression, though the a6h4 integrin does so only when coexpressed with the tetraspanin D6.1A [36]. However, as stated above for CD44v4-v7, overexpression of none of these molecules induced miliary metastasis formation. Furthermore, all of these molecules, though not in concert, are expressed on nontransformed cells [31]. Taking these two observations, we speculated that it may be an interaction between these or additional molecules that promote tumor progression. Because of the strong impact of CD44v6 on tumor progression, we concentrated in the first instance on whether any of the additional metastasis-associated membrane molecules or others may associate with CD44v4-v7, which is the major CD44 isoform on BSp73ASML cells [5], and whether such an association has functional consequences. We show that the tetraspanins D6.1A and CD9 associate with CD44v4-v7, tetraspanins being known as molecular facilitators [37], which besides of their association with integrins [37 – 40] have also been described to weakly associate with CD44 [41]. CD44v4-v7 also associates with EpCAM. Notably, EpCAM only binds to CD44v4-v7 and not to CD44s. The formation of CD44v4-v7 – EpCAM containing complexes can influence cell– cell and cell – matrix adhesion and apoptosis resistance. Thus, complex formation between these two metastasis-associated molecules could well be an important parameter of tumor progression.

Materials and methods Tumor lines and treatments The following tumor lines were used: BSp73ASML (metastasizing pancreatic adenocarcinoma), BSp73AS (non- or low-metastasizing pancreatic adenocarcinoma) [42], BSp73AS-14 (BSp73AS transfected with the CD44v4-v7 cDNA) [5], BSp73AS-D5.7A (BSp73AS transfected with the EpCAM cDNA) [34], and BSp73AS-D6.1A (BSp73AS transfected with the D6.1A cDNA) [33]. These tumor lines were derived from the BDX rat strain. The tumor line Progressor, a metastasizing colon carcinoma of the BDIX strain, was kindly provided by F. Martin [43]. The tumor line 804G, a metastasizing bladder carcinoma line, was kindly provided by J.C. Jones [44]. The adherently growing tumor lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS). Chinese hamster ovary (CHO)-K1 cells, obtained from the American Type Culture Collection, were cultured in DMEM/F12 medium supplemented with 10% FCS. CHO-meta-1, CHO-mut-1, CHO-mut-2, and CHO-mut-1/2 were stably transfected with CD44v4-v7 cDNA (meta-1), where the membrane proximal cysteines C286 (mut-1) or C295 (mut-2) or both (mut-1/2) were exchanged to alanine (see below). Confluent cultures were trypsinized and split. Where indicated, cells were subjected to the following treatments: For PKC activation, cells were starved by culture in serum-free medium and were incubated for 1 – 2 h with 10 7 – 10 8 M phorbolmyristate acetate (PMA). Raft domains were destroyed by washing cells with HEPES buffer (25 mM HEPES, 150 mM NaCl, 5 mM MgCl2, pH 7.2) and cultured in the presence of methyl-h-cyclodextrin (MhCD) (10 – 50 mM for 30 min at 37jC). For the destruction of microfilaments, cells were incubated for 30 –60 min at 37jC in serum-free medium containing 1 AM latrunculin B or 30 AM cytochalasin D. Antibodies, flow cytometry, and immunofluorescence The monoclonal antibody (mAB) A2.6 (CD44v6 specific), D5.7 (EpCAM specific), C4.4 (C4.4A specific) [all mouse IgG1 (mIgG1)], and an isotype-matched control antibody, 3– 9, have been described [45,46]. A rat CD44sspecific mAB (5G8) recognizing an epitope that is lost upon insertion of variant exons was kindly provided by J. Sleeman, Institute of Genetics, University of Karlsruhe, Germany [22]. A rat CD71-specific hybridoma (Ox26, mIgG2a) and a rat panCD44-specific hybridoma (Ox50, mIgG1) were obtained from the European Association of Animal Cell Cultures. Anti-ezrin and anti-moesin (polyclonal rabbit sera) were obtained from Upstate Biotechnology. Two rat CD9-specific hybridoma (clones B2C11 and RPM.7, mIgG), Phalloidin-FITC, unlabeled and dyeor enzyme-labeled secondary antibodies were obtained from Southern Biotechnology (Birmingham, AL) or Phar-

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mingen (Hamburg, Germany) or DIANOVA (Hamburg, Germany). For FACS analysis, cells were washed and resuspended at 5  105 cells/50 Al PBS containing 2% FCS. Cells were incubated with the first antibody (10 Ag/ml) for 30 min at 4jC and after washing with the second, dye-labeled antibody (30 min at 4jC) at appropriate dilutions. Fluorescence staining was evaluated using a FACSCalibur (Becton-Dickinson, Heidelberg, Germany). Colocalization was analyzed by immunofluorescence. Cells were seeded overnight on hyaluronan (HA)-coated glass slides and were incubated for 15 min at 37jC with Ox50 or A2.6 or D5.7. After washing, cells were incubated for 30 min at 37jC with an excess of Cy2-, Texas-Red (TxR)-, or rhodamine (Rho)-labeled anti-mIgG. The same staining procedure was used when analyzing co-capping. However, co-capping was analyzed on freshly harvested cells, which were seeded in round-bottom microtiter plates and did not adhere to a substrate. After first antibody staining and cross-linking with a dye-labeled secondary antibody, cells were centrifuged onto cover slides. Cells were washed with ice-cold PBS, fixed for 30 min with 4% paraformaldehyde (w/v in PBS), and free binding sites of the dye-labeled anti-mIgG were blocked by incubation with an excess of mIgG. Thereafter, cells were incubated for 1 h at 4jC with a second, directly dye-labeled antibody. When evaluating colocalization with cytoskeletal proteins, cells were permeabilized for 4 min with 0.1% Triton X-100 (v/v) after fixation. Fixed and permeabilized cells were incubated for 1 h at 4jC with Phalloidin-FITC or with anti-ezrin or anti-moesin and dye-labeled anti-rabbit IgG. After washing, slides were mounted in Elvanol. Digitized images were generated using a Leica DMRBE Microscope. In two-color experiments, digital images were overlayed electronically. Generation of mutated CD44v4-v7 and transfections of BSp73AS and CHO-K1 cells Three mutants of the CD44v4-v7 cDNA were generated by PCR-based site-directed mutagenesis using as template the pcDNA3 vector containing the wild-type CD44v4-v7 cDNA. For exchanging Cys 286 to alanine, the primer 5VCTTGCCGTCGCCATTGCTGTC-3V was used, and for exchanging Cys 295 to alanine, the primer 5V-TAGGAGAAGGGCTGGGCAGAA-3V and the reverse primer 5VTAGAAGGCACAGTCGAGG-3V were used. The PCR reaction was performed for 30 cycles (15 s 94jC, 20 s 50jC, 3 min 72jC). Using the same PCR conditions, the PCR products were used as primers in a second PCR reaction together with the T7 primer 5V-TAATACGACTCACTATAGGG-3V. PCR products were cloned into the pcDNA3 vector using the restriction endonucleases HindIII and XbaI. The double mutated isoform was generated by using the Cys295Ala mutant as the template for site-directed mutagenesis. Mutations were verified by sequencing (SeqLab, Goettingen).

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Mutated isoforms were transfected into CHO-K1 cells using Polyfect (QIAGEN) according to the manufacturer’s recommendation. Transfected CHO-K1 cells were selected by growth in DMEM/F12 medium, 10% FCS, 1 mg/ml G418. Transfected CHO-K1 cells with comparable expression rates for the wild-type and the different mutants were used. Mass spectrometry BSp73ASML cells (5  109) were subjected to a largescale immunoprecipitation (IP) with the mAb Ox50 under Lubrol lysis conditions. After precipitation, the ProteinG Sepharose beads were eluted with 2 ml RIPA buffer and mixed with 3 vol methanol, 1 vol chloroform and 3 vol ddH2O, vortexed and centrifuged for 1 min. The upper phase was taken off and 3 vol methanol was added. After vortexing and centrifugation, the protein pellet was air dried, dissolved in 200 Al reducing Laemmli buffer and boiled for 5 min. An aliquot of 100 Al was separated in an SDS-PAGE gradient (4– 20%) and the gel was stained with colloidal coomassie (Sigma) according to the manufacturer’s recommendation. Eleven bands were cut out, proteins were eluted and digested with trypsin. The resulting peptide mixture was subjected to MALDI mass spectometry. Single charged monoisotypic peptide masses were used as inputs for database searching. Searches were performed against the NCBInr database using the ProFound search algorithm and the Protein prospector software developed at the University of California, San Francisco. Isoelectric points were allowed to range from 0 to 14, and the oxidation of methionine was included as possible modification. Up to one missed tryptic cleavage was considered, and the mass tolerance for the monoisotopic peptide mass was set to F100 ppm. Searches with fragment masses from PDS experiments were performed against the NCBInr databases using the MS-Tag search algorithm provided by the Protein prospector software package. Parent mass tolerance was set to F0.1 Da and fragment ion tolerance was set to F0.8 Da. Immunoprecipitation of biotinylated membrane proteins and reprecipitation Where indicated, surface proteins were labeled with water-soluble NHS-X-Biotin (Calbiochem) (200 Ag biotin/ ml lysis buffer without detergent, 30 min, room temperature) under mild agitation. Biotinylation was stopped by washing with ice-cold PBS containing 200 mM glycine. For immunoprecipitation, the cells were washed three times with detergent-free lysis buffer (25 mM HEPES, 150 mM NaCl, 5 mM MgCl2, pH 7.2) in cell culture dishes and then scraped in ice-cold lysis buffer containing 1% of the indicated detergent and appropriate amounts of proteinase inhibitors (2 mM PMSF, 1  proteinase inhibitor cocktail without EDTA) (Roche-Diagnostics, Mannheim, Germany). Cells were lysed for 1 h at 4jC on a rocking platform. After

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centrifugation (13,000  g, 20 min, 4jC), the lysates were precleared by overnight incubation at 4jC with ProteinG Sepharose (1:1 in PBS, 20 Al/ml lysate) and 1 ml lysate was immunoprecipitated with 2 Ag/ml antibody (1 h at 4jC and addition of 40 Al ProteinG Sepharose for 2 h at 4jC). ProteinG Sepharose beads were pelleted for 15 s at 13,000  g. Pellets were washed four times in 1% lysis buffer. Proteins were eluted with 1 nonreducing Laemmli buffer. For reprecipitation, the proteins were eluted for 15 min, 37jC, under vigorous shaking and using a more stringent detergent. Western blotting Whole cell lysates (1% detergent in 25 mM HEPES pH7.2, 150 mM NaCl, 5 mM MgCL2), immunoprecipitated proteins, or sucrose density gradient fractions were mixed at a 1:6 ratio with 6 nonreducing Laemmli buffer and boiled for 5 min at 95jC. Proteins were separated in 6%, 12%, or 4 –20% continuous gradient SDS-PAGE, were transferred on PVDF membranes at 30 vol overnight, and membranes were blocked for 1 h at room temperature with 10% fat-free milk in PBS/0.1% Tween 20. For protein detection, the indicated primary antibodies were applied as hybridoma supernatant or at 1 Ag/ml purified antibody in PBS/0.1% Tween 20 (1 h, room temperature). Membranes were washed (three times) in PBS/0.1% Tween 20, incubated with the secondary HRP-conjugated antibodies (Amersham, Braunschweig, Germany) (1 h, room temperature), washed again, and developed with the enhanced chemiluminescence (ECL) detection system (Amersham) according to the manufacturer’s recommendations. Palmitoylation assay CHO-K1 transfectants were grown to 80% confluency, washed, and starved for 4 h in serum-free RPMI. Starved cells were incubated for 2 h with 0.25 mCi [3H]-palmitic acid in RPMI/5% FCS. Labeled cells were lysed (1% TX100) and tetraspanins and CD44 were immunoprecipitated. After SDS PAGE and Western blotting, the PVDF membranes were washed in detergent-free lysis buffer, air-dried, submerged in scintillation fluid, and air-dried again. Membranes were exposed for 21 days at 70jC to a Kodak film.

(GEM) fractions (fractions 2 – 4) and soluble fractions (fractions 10– 12) were analyzed by immunoprecipitation or Western blotting. Adhesion, migration, and agglutination In adhesion studies, [3H]thymidine-labeled cells were seeded on a monolayer of adherent cells or BSA- or hyaluronic acid (HA) (100 Ag/ml)-coated plates. Where indicated, mAb D5.7 or Ox50 or A2.6 (10 Ag/ml) were added to the culture medium. Cells were incubated for 2 h. Nonadherent cells were vigorously washed off, adherent cells were lysed (2% SDS), harvested with an automatic harvester, and counted in a h-counter. The percentage of adherent cells is shown. Migratory activity was evaluated in an in vitro wound healing assay. Tumor cells (5  105) were grown in Petri dishes until a subconfluent stage was reached. The monolayer was scratched with a blunt-edged needle. Cells were incubated for another 48 h, were fixed and stained with hematoxilin-eosin, and ‘‘wound healing’’ was microscopically evaluated. Cell agglutination was evaluated by incubating equal amounts of unlabeled and FITC-labeled cells, which had or had not been treated with MhCD in culture chambers on a rocking platform (2 h, 37jC). Medium was sucked off, cells were dried and embedded in Evanol. Aggregate formation was evaluated using a Leica DMRBE Microscope. Digital images were overlayed electronically. Proliferation and apoptosis Proliferation was tested by [3H]thymidine incorporation. Cells were seeded in flat-bottom microtiter plates, [3H]thymidine was added, and cells were incubated at 37jC for 8 h. Cells were trypsinized, harvested, and transferred to a hcounter to evaluate [3H]thymidine incorporation. Apoptosis was determined after tumor cells had been seeded on flat bottom microtiter plates in medium containing 1% FCS and, where indicated, 10 8 M PMA. Apoptotic cell death was determined after 24 h by flow cytometry [annexin V-FITC staining and propidium iodine (PI) uptake]. Statistics

Sucrose density gradient centrifugation Cells (5  106) were lysed in 2 ml lysis buffer and 800 Al of the cleared lysate were mixed with 800 Al 80% sucrose solution. The 40% sucrose solution was overlayed with 1.6 ml 30% sucrose and 800 Al 5% sucrose in lysis buffer without detergent. Centrifugation was performed for 16 h at 200,000  g and 4jC. Fractions (14) of 300 Al (starting from the top) were carefully pipetted off. Fractions were analyzed by dot blot using biotinylated Cholera Toxin B subunit as a GM1 marker. Glycolipid-enriched membrane

Significance of differences was evaluated by the two tailed Student’s t test.

Results The metastasis-associated molecules, CD44v4-v7, D6.1A (homologue of CO-029) EpCAM, and C4.4A, are strongly overexpressed on the metastasizing subline of a rat pancreatic adenocarcinoma, BSp73ASML (Fig. 1). Notably,

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BSp73ASML cells are equally stained by Ox50, a pan CD44 antibody that recognizes an epitope in the standard part of CD44, and A2.6, which recognizes an epitope of the v6 exon product. As was demonstrated before [45], this is because BSp73ASML cells rather exclusively express CD44 variant isoforms including the v6 exon product. Expression of CD44v4-v7, D6.1A, C4.4A, and EpCAM has not been seen in the non-metastasizing subline of this pancreatic adenocarcinoma, BSp73AS [31]. The non-metastasizing subline expresses CD44s at a high level. Both lines, BSp73ASML and BSp73AS, express the tetraspanin CD9 and the transferrin receptor (CD71) at a comparable level. Because of the repeatedly observed coexpression of EpCAM and D6.1A with CD44v4-v7 on metastasizing tumor lines, it became of interest whether the molecules may be physically linked and/or may exert concerted functional activities. Co-immunoprecipitation of CD44v4-v7, EpCAM, and D6.1A in BSp73ASML cells

Fig. 1. Relative expression level of metastasis-associated surface molecules on BSp73ASML cells: BSp73ASML cells and for comparison the nonmetastasizing subline BSp73AS were stained with Ox50 (anti-panCD44), A2.6 (anti-CD44v6), D5.7 (anti-EpCAM), D6.1 (anti-D6.1A), C4.4 (antiC4.4A), Ox26 (anti-CD71), B2C11 (anti-CD9), or mIgG (negative control) and PE-labeled anti-mIgG. The mean fluorescence intensity is indicated.

BSp73ASML cells were biotinylated and lysed with the strong detergents Triton X-100 and Brij96, with the ionic detergent CHAPS and with the mild detergents Lubrol and Brij98. Lysates were immunoprecipitated with A2.6, which recognizes an epitope on CD44v6. Only after lysis in the mild detergents, the precipitates contained several proteins besides of CD44v. A prominent band at 35 kDa was suspected to be EpCAM and a smear between 25 and 30 kDa was suspected to be D6.1A (data not shown). To verify the assumption, cells were lysed in Lubrol and lysates were precipitated with Ox50, D5.7, and D6.1 and reprecipitated in a crisscross fashion (Fig. 2A). In fact, the three molecules co-immunoprecipitated. Also, when CD44v4-v7 was cross-linked on BSp73ASML cells via A2.6 to induce capping, the three molecules, CD44v4-v7, EpCAM, and D6.1A, were found to co-cap (Fig. 2C). Co-immunoprecipitation and co-capping of the three molecules were also found in colorectal and bladder carcinoma rat lines that metastasize via the lymphatic system (data not shown). To confirm the existence of CD44v4-v7 – EpCAM – D6.1A complexes on metastasizing rat tumor lines and to see whether additional molecules may be contained in CD44v complexes, BSp73ASML cells were cultured on large scale, lysed in Lubrol, precipitated with A2.6, and precipitated proteins were separated by SDS-PAGE. Prominent bands were cut out, digested with trypsin, and peptides were subjected to mass spectrometry. A 35-kDa band was identified as EpCAM and a 20-kDa band as the tetraspanin CD9, a tetraspanin with some similarity to D6.1A. The D6.1A molecule was not detected by mass spectrometry, which is likely due to its very variable degree of glycosylation [33], such that no sharp protein bands are seen after SDS-PAGE. Mass spectrometry revealed, in addition, coimmunoprecipitation of the a3 integrin chain and of annexin

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I with CD44v. Additional bands were nonidentified keratins, a DNA-type K chaperon, h actin, the mouse IgG heavy, and light chains and fragments thereof. These proteins are frequently found as contaminants due to the preparation regimen. Bands of low molecular weight have not yet been identified (Fig. 2C). Thus, mass spectrometry confirmed the association between the metastasis-associated molecules CD44v4-v7 and EpCAM and revealed, in addition, an association with CD9, a3, and annexin I. Although the latter associations could well be of interest for tumor progression, for the sake of clarity, we concentrated in the following experiments on the ‘‘metastasis-associated’’ molecules CD44v, EpCAM, and the tetraspanin D6.1A. The tetraspanin CD9 was, in addition, included because it has some similarity to D6.1A and is expressed by both the metastasizing and the non-metastasizing subline of the BSp73 tumor. Localization of the CD44v – EpCAM – D6.1A membrane complexes

Fig. 2. Coprecipitation and colocalization of the metastasis-associated molecules EpCAM and D6.1A with CD44v: (A) Biotinylated BSp73ASML cells were lysed in 1% Lubrol. Lysates were precipitated with Ox50 or D5.7 or D6.1. Precipitates were dissolved in 1% TX-100 and were reprecipitated in a crisscross fashion. Proteins were separated in a 12% SDS-PAGE and were blotted and detected by extravidin – horseradish peroxidase (HRPO) staining. (B) BSp73ASML cells were incubated with A2.6 (10 Ag/ml, 15 min, 37jC) and, after washing, with an excess of Cy2-labeled anti-mIgG (30 min, 37jC). Cells were washed in ice-cooled PBS and counterstained with rhodamine-labeled D5.7 or D6.1. Individual staining and the digital overlays are shown, scale bar: 5 Am. (C) A membrane preparation from 109 BSp73ASML cells was lysed in 1% Lubrol and precipitated with Ox50. Proteins were eluted in RIPA buffer at room temperature and thereafter at 37jC. Proteins were separated under reducing conditions on a 4 – 20% linear SDS-PAGE. The marked bands were cut out, digested, analyzed by mass spectrometry, and identified as indicated.

As mentioned above, the complexes were dissolved by TX-100, but not by Lubrol, which does not disrupt cholesterol-rich rafts. Therefore, it was of interest whether the complexes containing CD44v would be recovered in the light fractions after sucrose density gradient centrifugation. Caveolin and the GPI-anchored C4.4A, which are considered as classical raft markers, and CD71, a classical nonraft marker, served as controls. Lubrol lysates were subjected to sucrose density gradient centrifugation, light and dense fractions were collected, separated by gel electrophoresis and blotted with anti-caveolin, C4.4, Ox26 (anti-CD71), A2.6, D5.7, D6.1, and RPM.7 (antiCD9). CD71 was exclusively recovered in the heavy fractions. Caveolin and C4.4A were strongly enriched in the light fractions. CD44v, EpCAM, D6.1A, and CD9 were recovered in the light and the dense fractions. As revealed by sucrose density gradient separation of lysed BSp73AS cells that expressed, after transfection, either CD44v4-v7 (BSp73AS-14) or EpCAM (BSp73ASD5.7A), the noncomplexed molecules CD44v4-v7 and EpCAM were also recovered in the light and the dense fractions (Fig. 3A). MhCD is known to remove about 50% of the cholesterol from membrane preparations, which does not suffice to remove caveolin from the light fraction. Yet, after MhCD treatment, CD44v4-v7 was exclusively recovered from the heavy fraction and EpCAM as well as D6.1A and CD9 were strongly enriched in the heavy fractions. Also, CD44v4-v7 was only detected in the heavy fractions of MhCD-treated BSp73AS-14 cells and hardly any EpCAM was seen in the light fractions of BSp73AS-D5.7A cells (Fig. 3B). After 100,000  g centrifugation, incompletely lysed membrane fractions, that is, rafts, are recovered in the pellet. Thus, 100,000  g centrifugation provided another

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Fig. 3. GEM localization of CD44v complexes: (A) BSp73ASML, BSp73AS-14, and BSp73AS-D5.7A cells were lysed in 1% Lubrol. After sucrose gradient centrifugation and collection of 300 Al fractions (starting at the top), aliquots were separated by SDS-PAGE. After blotting, proteins were detected with the indicated antibodies. (B) Cells were treated for 30 min at 37jC with 10 mM MhCD, biotinylated, lysed, and treated as in A. (C) BSp73ASML cells were biotinylated and lysed in 1% Lubrol. Lysates were freed of nuclei and large membrane fractions (17,000  g centrifugation) and were fractioned by sucrose density gradient centrifugation. (D) The supernatant of lysates after 17,000  g centrifugation (lane 2), the pellet of the lysate after 100,000  g centrifugation (lane 1), and the pellet after 100,000  g centrifugation of the fractions 2 – 4 from C (lane 3) and of the fractions 9 – 11 from C (lane 4) were separated by SDSPAGE. Gels were blotted and proteins were detected by extravidin – HRPO staining. Several repetitions of the experiments shown in A or B and C or D revealed corresponding results.

means to control for the location of CD44v4-v7, EpCAM, D6.1A, and CD9 in GEM fractions. Lubrol lysates were freed of incompletely lysed large membrane fractions and micels by 17,000  g centrifugation. Thereafter, the supernatant was fractionated by sucrose density gradient centrifugation. Pooled light and dense sucrose density gradient fractions were centrifuged at 100,000  g. Significantly, more CD44v, EpCAM, D6.1A, and CD9 were recovered in the pellet of the light than of the dense fractions (Figs. 3C and D). We conclude that the vast majority of the complex molecules are located in GEM microdomains, but not in classical (TX-100 insoluble) rafts, because even a partial depletion of cholesterol sufficed to interfere with the raft localization. EpCAM, CD9, and D6.1A co-immunoprecipitate with CD44v4-v7 as opposed to CD44s Because CD44 variant isoforms, but not CD44s, have been associated with metastasis formation in the BSp73 tumor model [5], it was of interest whether EpCAM and the tetraspanin D6.1A selectively co-immunoprecipitate with CD44v, but not with CD44s. The tetraspanin CD9 was included because BSp73AS cells express CD9, but not

D6.1A. Lubrol lysates were precipitated with Ox50 (panCD44), RPM.7 (CD9), D5.7 (EpCAM), and Ox26 (CD71). Precipitates were separated by gel electrophoresis and were blotted in a crisscross fashion (Fig. 4). Ox50 precipitates of BSp73ASML cells contained CD44v, D6.1A, EpCAM, and CD9. Accordingly, RPM.7 precipitates contained CD9, D6.1A, CD44v, and EpCAM. Because BSp73ASML cells hardly express CD44s molecules, the experiment was repeated with BSp73AS cells that constitutively express CD9 and CD44s and that were transfected with CD44v4-v7 cDNA (BSp73AS-14) or EpCAM cDNA (BSp73AS-D5.7A). Ox50 precipitates of BSp73AS-14, but not of BSp73AS-D5.7A, contained CD9. CD9 precipitates of BSp73AS-14, but not of BSp73ASD5.7A, contained CD44 (CD44v4-v7). Importantly, Ox50 precipitates of BSp73AS-D5.7A did not contain EpCAM and D5.7 precipitates did not contain CD44. Also, D6.1 did not precipitate CD44s in BSp73AS-D6.1A cells (data not shown). The experiment has been repeated using 5G8, a CD44s-specific antibody, for precipitation. CD44s precipitates did contain neither EpCAM, D6.1A, nor CD9 (data not shown). Thus, at least in the tumor lines tested, EpCAM, CD9, and D6.1A selectively co-immunoprecipitate with CD44v, but not with CD44s.

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Fig. 4. EpCAM and the tetraspanin CD9 associate with CD44v4-v7, but not with CD44s: BSp73ASML, BSp73AS-14, and BSp73AS-D5.7A cells were biotinylated and lysed in 1% Lubrol. Lysates were precipitated with Ox50, RPM.7, D5.7, and Ox26 (negative control). Precipitated proteins were eluted with TX-100 and were reprecipitated in a crisscross fashion. Reprecipitated proteins were separated by SDS-PAGE. After blotting, proteins were detected by extravidin – HRPO staining. Ox50 as well as RPM.7 precipitates of BSp73ASML lysates contained CD44v, D6.1, EpCAM, and CD9. Ox50 and RPM.7 precipitates of BSp73AS-14 lysates also contained CD44v and CD9. However, Ox50 and RPM.7 precipitates of BSp73AS-D5.7A lysates did not contain EpCAM, and D5.7 precipitates did not contain CD9. Thus, EpCAM and CD9 selectively coprecipitated with CD44v. The experiment has been repeated five times providing consistent results.

The localization of the CD44v4-v7—EpCAM complex in glycolipid-enriched membrane microdomains does not depend on palmitoylation of CD44v4-v7 but might be facilitated by the association of CD44v4-v7 with ezrin and moesin We have shown above that CD44v – EpCAM– D6.1A complexes are recovered mainly in the light fractions after sucrose gradient centrifugation (Fig. 3D); but after partial cholesterol depletion, the molecules were recovered in the heavy fractions. Thus, the question arose whether MhCD treatment may have destroyed the complexes or whether the complexes remained intact after destruction of GEMs. As shown in Figs. 5A and B, the complexes remained stable after partial cholesterol depletion. After treatment with up to

50 mM MhCD, Ox50 precipitates contained EpCAM and CD9, and D5.7 precipitates contained CD44v and CD9 (Fig. 5A). Also, EpCAM colocalized with antibody-induced clusters of CD44v on untreated as well as on MhCD-treated BSp73ASML cells (Fig. 5B). Thus, we speculated that the complexes may become actively recruited to or retained in the rafts. Palmitoylation supports anchoring of membrane proteins to cholesterol and CD44 has two potential palmitoylation sites. Hence, palmitoylation of CD44 could drive the complexes into GEMs. To test this hypothesis, both palmitoylation sites of CD44 were separately or jointly mutated and CHO-K1 cells were transfected with the three mutant cDNA of CD44v4-v7. Three cultures with mutations at the potential palmitoylation sites Cy 286 or Cy

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Fig. 5. CD44v4-v7 – EpCAM complexes remain stable after partial cholesterol depletion and do not require CD44v palmitoylation for GEM localization: (A) BSp73ASML cells, untreated or MhCD-treated (30 min, 37jC, 10 or 50 mMol MhCD), were lysed in 1% Lubrol. Lysates were immunoprecipitated with Ox50 and D5.7. Immunoprecipitates were separated on SDS-PAGE and were blotted on PVDF membranes. Proteins were detected with an antibody mix containing Ox50, D5.7, and RPM.7. (B) CD44v4-v7 was cross-linked on untreated and MhCD-treated (10 mMol, 30 min, 37jC) BSp73ASML cells via A2.6 (15 min, 37jC) and an excess of anti-mIgG-Cy2 (30 min, 37jC). Cells were washed, fixed with 4% paraformaldehyde, and free binding sites of the anti-mIgG were blocked by incubation with mIgG. Cells were counterstained with rhodamine-labeled D5.7. The individual staining and the digital overlay are shown, scale bar: 7.5 Am. (C) Cysteines 286 and 295 in the cytoplasmic tail of CD44 were exchanged by alanine as described in Material and methods and CHO cells were transfected with the empty pCDNA3 vector or the vector containing either CD44v4-v7 or CD44v4-v7 with alanine at position 286 or 295 or both 286 and 295. CHO cells expressing CD44v4-v7 at a comparable intensity were selected by FACS analysis. (D) Transfected CHO cells, as shown in C were lysed in 1% Lubrol and the lysate was fractioned by sucrose density gradient centrifugation. Fractions 2 – 4 and 9 – 11 were separated by SDS-PAGE and were blotted on a PVDF membrane. CD44v was detected by staining with A2.6. One representative experiment out of three experiments is shown.

295 or both Cy286 and Cy 295, which expressed CD44v at a high and comparable level (Fig. 5C), were lysed in Lubrol and blotted with A2.6 after sucrose density gradient centrifugation. The location of CD44v4-v7 in the light fractions was unaltered after destroying either one or both palmitoylation sites (Fig. 5D). Hence, palmitoylation of CD44v apparently does not influence the Lubrol raft association of the CD44v complexes. However, A2.6 recognized a higher molecular weight protein when both palmitoylation sites were destroyed. Whether this is due to altered glycosylation and/or transport of mutated CD44v4v7 remains to be explored. CD44 can be anchored to the actin cytoskeleton via members of the ERM family [28] that have been described to account for the redistribution of membrane molecules during formation of the immunological synapse on T cells, that is, guide CD43 out of the central area [47]. Hence, a selective association of CD44v4-v7 or of the complex with ERM proteins could also contribute to the recruitment of the complex into GEMs. CD44, CD44v6, EpCAM, and D6.1A were cross-linked on BSp73ASML, BSp73AS,

BSp73AS-14, BSp73AS-D5.7A, and BSp73AS-D6.1A cells, and cells were counterstained with anti-ezrin (Fig. 6A). On BSp73ASML cells, a considerably part of ezrin colocalized with CD44, EpCAM, and D6.1A. Partial colocalization of ezrin with CD44v was also seen in BSp73AS-14 cells. However, ezrin hardly colocalized with CD44s, EpCAM, and D6.1A in BSp73AS, BSp73ASD5.7A, and BSp73AS-D6.1A cells. The same observations accounted for moesin (data not shown). Since ezrin links transmembrane molecules to the actin cytoskeleton, colocalization of CD44v4-v7, EpCAM, and D6.1A with the ERM proteins was controlled by counterstaining with phalloidin-FITC (Fig. 6B). In BSp73ASML cells, most clusters of CD44v, EpCAM, and D6.1A were stained by phalloidin-FITC. In BSp73AS-14 cells too, most CD44v4-v7 clusters were stained by phalloidin-FITC. In BSp73AS cells, hardly any colocalization of CD44s with actin bundles was seen; and in transfected BSp73AS cells, only a minor portion of EpCAM and D6.1A colocalizes with actin fibers. Thus, in transfected BSp73AS cells, ezrin and actin fibers readily colocalized with CD44v4-v7, but

338 D.-S. Schmidt et al. / Experimental Cell Research 297 (2004) 329–347 Fig. 6. Colocalization of ezrin and actin bundles with CD44v: BSp73AS, BSp73AS-14, BSp73AS-D5.7A, BSp73AS-D6.1A, and BSp73ASML cells were seeded on HA-coated plates. (A) Cells were stained with Ox50, A2.6, D5.7, and D6.1 and were counterstained with (A) Cy2-labeled anti-mIgG or (B) anti-mIgG-TxR as described in Fig. 5B. After washing, fixing, permeabilization, and blocking, cells were stained with (A) anti-ezrin and were counterstained with anti-rabbit IgG-TxR or (B) Phalloidin-FITC. (A and B) Single staining and the digital overlay are shown, scale bar: 10 Am.

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Fig. 6 (continued).

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far less efficient with EpCAM and D6.1A; whereas in BSp73ASML cells, ezrin and actin bundles readily colocalized with all three molecules, CD44v4-v7, EpCAM, and D6.1A. This finding suggests that the association of CD44v4-v7 and of CD44v4-v7 containing complexes with ezrin could, at least partly, account for the localization of the complexes in GEM microdomains. Taken together, EpCAM, D6.1A, and CD9 associate selectively with CD44 variant isoforms, but not with CD44s. The complexes are located in cholesterol-depletion-sensitive GEM microdomains but remain stable after partial cholesterol depletion. Recruitment of the complexes in GEMs is independent of CD44v4-v7 palmitoylation but may be supported by the association of CD44v with ezrin or moesin and the actin cytoskeleton. The association of CD44v4-v7 with EpCAM influences cell – cell and cell –matrix adhesion Cell – cell and cell – matrix adhesion as well as cell motility are important parameters for tumor progression and have been disputed to be influenced by CD44 and EpCAM. CD44 is a cell –matrix adhesion molecule and the principle receptor for hyaluronan. EpCAM is a homotypic cell – cell adhesion molecule. To obtain a hint, whether the association of the two molecules modulates cell – matrix or cell –cell adhesion, we evaluated adhesiveness of BSp73ASML and Progressor cells that both express CD44v4-v7, EpCAM, and D6.1A and of BSp73AS cells in comparison to BSp73AS cells transfected with the CD44v4-v7 or the EpCAM cDNA. We first evaluated adhesion of CD44 to HA. As could have been expected, BSp73ASML, Progressor, BSp73AS, BSp73AS-14, and BSp73AS-D5.7A cells, which all express CD44 at a high level, adhered better to HA- than to BSAcoated plates (Fig. 7), although we could not define signif-

icant differences between CD44s (BSp73AS) versus CD44v (BSp73AS-14) expression [22]. Because CD44v4-v7 strongly associates with ezrin and moesin (Fig. 6 and Ref. [48]), it became of interest to explore the adhesion to HA after destruction of the microfilament network. When cells were treated with latrunculin, adhesion to plastic and HA was significantly reduced. The same observations accounted for cells treated with cytochalasin D, albeit inhibition of adhesion was weaker (data not shown). Both anti-panCD44 and anti-CD44v6 partly inhibited adhesion of BSp73AS, BSp73AS-14, BSp73AS-D5.7A, as well as of BSp73ASML and Progressor cells to HA (data not shown). These findings confirmed the contribution of CD44 to HA adhesion but did not provide evidence for a contribution of CD44v4-v7 – EpCAM complexes to HA adhesion. However, a selective contribution of CD44v4-v7 – EpCAM complexes became apparent when cells were treated with MhCD. Adhesion of MhCD-treated BSp73AS, BSp73AS-14, and BSp73ASD5.7A cells to plastic and HA was unaltered, but adhesion of BSp73ASML and Progressor cells was significantly reduced. We interpret the finding in the sense that the CD44v – EpCAM complex formation does not influence adhesion to HA when located in GEM microdomains, but it might hamper adhesion when located outside of rafts. To evaluate the contribution of EpCAM versus CD44v – EpCAM-containing complexes to cell – cell adhesion, BSp73AS, BSp73AS-14, BSp73AS-D5.7A, BSp73ASML, and Progressor cells were seeded on the respective monolayers. EpCAM-positive cells (BSp73ASML, Progressor, and BSp73AS-D5.7A) adhered better to EpCAM-positive than EpCAM-negative cells, whereas BSp73AS and BSp73AS-14 cells adhered less efficiently but equally well to EpCAM-positive and EpCAM-negative lines (Fig. 8A). Thus, the CD44v4-v7 – EpCAM complex (BSp73ASML and Progressor cells) apparently did not interfere with homotypic

Fig. 7. Influence of CD44v4-v7 – EpCAM complex formation on hyaluronan adhesion: [3H]thymidine-labeled BSp73ASML, Progressor, BSp73AS, BSp73AS-14, and BSp73AS-D5.7A cells (2  105) were untreated or treated with 1 AM latrunculin or with 20 mM MhCD (30 min, 37jC) and were seeded in triplicates on BSA- or HA-coated plates. Cells were incubated for 2 h at 37jC and were washed vigorously. Adherent cells were detached by trypsin treatment (30 min, 37jC). Plates were harvested and the percentage of adherent cells was evaluated in a h-counter. Mean values + SD are shown. Significance of differences ( P < 0.01) is indicated by an asterisk.

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Fig. 8. Influence of CD44v4-v7 – EpCAM complex formation on cell – cell adhesion and aggregation: (A and B) Cells were seeded on flat bottom microtiter plates and grown to confluency or were labeled with [3H]thymidine. [3H]thymidine-labeled cells (2  105) were added in triplicates to the cell monolayer and were incubated for 2 h at 37jC. (B) Where indicated, [3H]thymidine-labeled cells were treated with 20 mM MhCD (30 min, 37jC). (A and B) Plates were washed vigorously and adherent cells were detached by incubation in 1% trypsin. The percentage of adherent cells was evaluated in a h counter. Mean values + SD are shown. Significance of differences ( P < 0.01) is indicated by an asterisk. (C) Unlabeled and FITC-labeled cells were mixed at a 1:1 ratio and were incubated for 2 h on a rocking platform at 37jC. Where indicated, the FITC-labeled cells were MhCD-treated (as in B). Cells were allowed to settle, medium was sucked off, and after drying cells were embedded in Mowiol. Cluster formation was evaluated at a fluorescence microscope. Digital overlays of the green fluorescence image and the direct light appearance are shown, scale bar: 100 Am.

EpCAM adhesion. However, and similar to HA adhesion, cell – cell adhesion of BSp73ASML and Progressor cells was significantly impaired after MhCD treatment, whereas weak adhesion of BSp73AS and BSp73AS-14 (data not shown) and strong adhesion of BSp73AS-D5.7A cells remained unaltered (Fig. 8B). The analysis of cluster formation provided similar results. In the aggregation assay, one cell partner was unlabeled while the other was FITC labeled. The FITClabeled cells were either untreated or MhCD treated. Cells were incubated on a rocking platform for 2 h. After settling down, medium was sucked off and cluster formation was evaluated microscopically (Fig. 8C). Cluster formation was most readily observed with Progressor (data not shown), BSp73ASML, and BSp73AS-D5.7A cells. Few and small clusters were seen with BSp73AS cells and BSp73AS-14 cells. Cluster formation was strongly reduced in MhCDtreated FITC-labeled BSp73ASML cells, whereas cluster formation of the unlabeled and untreated BSp73ASML cells was unimpaired. However, FITC-labeled BSp73ASD5.7A cells clustered equally well after partial cholesterol depletion. This is shown only for the mixtures of unlabeled and FITC-labeled cells of the individual lines.

Corresponding findings were seen when evaluating cluster formation between unlabeled and FITC-labeled cells in a crisscross fashion (data not shown). Cell migration was evaluated by an in vitro wound healing assay. We did not observe differences in wound healing between the BSp73AS lines expressing or not expressing CD44v or EpCAM. We also did not obtain any evidence that A2.6 or D5.7 would inhibit cell migration. Instead, wound healing of all lines was significantly inhibited in the presence of anti-CD44s (data not shown). Thus, there is no evidence that the CD44v4-v7 – EpCAM complexes influence the migratory capacity of the cells. Also, the complexes apparently are not essential for cell – cell and cell – matrix adhesion. However, cells expressing the CD44v4-v7 – EpCAM complex show strongly reduced HA and cell – cell adhesion after destruction of GEM microdomains. The impact of CD44v4-v7 –EpCAM complexes on apoptosis resistance Besides altered adhesiveness and migratory capacity, apoptosis resistance is yet another feature frequently ob-

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served in metastasizing tumor cells. In fact, BSp73ASML cells are more apoptosis-resistant than BSp73AS cells. BSp73ASML cells were also shown to grow in vivo and in vitro more slowly than BSp73AS cells [49]. We first evaluated whether expression of CD44v4-v7 or of EpCAM may have bearing on the proliferative activity of BSp73AS cells, as this could have indirectly influenced the apparent rate of apoptosis. Transfection of BSp73AS cells with either CD44v4-v7 or EpCAM had no impact on the proliferation rate of BSp73AS cells. Also, the proliferative activity of BSp73AS, BSp73AS-14, and BSp73AS-D5.7A cells was not significantly influenced by cross-linking of CD44s, CD44v6, or EpCAM or by PMA treatment. Instead, proliferative activity of BSp73ASML and Progressor cells was slightly improved by antibody cross-linking of CD44v and EpCAM and was significantly inhibited by PMA treatment. All five lines hardly proliferated after MhCD treatment (data not shown). To support apoptosis induction, cells were grown for 24 h in the absence of FCS. Where indicated, cells were stimulated by PMA and/or GEMs were destroyed by MhCD treatment. Apoptosis was determined by Annexin-V-FITC and PI staining. BSp73ASML and Progressor cells were significantly more apoptosis resistant than BSp73AS cells. Transfection of the latter with the CD44v4-v7 cDNA had no influence on apoptosis susceptibility. However, a reduction in apoptosis susceptibility was seen consistently in BSp73AS-D5.7A cells. PMA treatment as well as antibody cross-linking of CD44v and EpCAM exerted no effect on apoptosis resistance of untransfected and transfected BSp73AS cells. Apoptosis resistance of BSp73ASML and Progressor cells was slightly increased by PMA treatment and antibody crosslinking. Instead, MhCD treatment increased apoptosis susceptibility. Although all five lines became more apoptosis susceptible by MhCD treatment, the effect was

much stronger on BSp73ASML and Progressor cells, which became equally apoptosis susceptible to BSp73AS cells. PKC activation did not prevent the high apoptosis susceptibility of MhCD-treated BSp73ASML and Progressor cells (Fig. 9). The fact that PMA treatment and antibody cross-linking (though only to a minor degree) affected apoptotic cell death only in BSp73ASML and Progressor cells argues for an initiation of signal transduction that, at least partly, depends on CD44v4-v7 – EpCAM complex formation. Because MhCD treatment increased the rate of apoptosis in BSp73ASML and Progressor cells by a factor of 6.9 and 3.9, respectively, in BSp73AS-D5.7A cells by a factor of 2.3, but in BSp73AS and BSp73AS-14 cells only by a factor of 1.4 and 1.2, respectively, we hypothesize that EpCAM and the CD44v4-v7 – EpCAM complex likely are associated with apoptosis-preventing signal-transducing molecules only when located in GEMs.

Discussion It is well accepted that metastasizing tumor cells require altered expression of a multitude of molecules to proceed through all steps of the metastatic cascade. Adhesion molecules are involved [50 – 53]. We and other groups provided evidence that CD44v6 may be one of the central players (Ref. [5] and reviewed in Refs. [3,4,8]). EpCAM too is known to be strongly up-regulated in several epithelial tumors [54 – 56]. Furthermore, several tetraspanins have been reported either to promote [33,57] or to suppress the process of tumor progression [58 – 60]. We report here for the first time that the metastasisassociated molecules CD44v and EpCAM form a complex that also contains the tetraspanins CD9 and D6.1A. Under selective conditions, functional activity of the CD44v –

Fig. 9. Influence of CD44v4-v7 – EpCAM complex formation on apoptosis susceptibility: Cells (5  105) were cultured for 24 h in serum-free medium in flatbottomed 96-well plates that had been coated with mIgG or Ox50 or A2.6 or D5.7. Where indicated, cells were treated with MhCD (as described above) or medium contained 10 8 M PMA. Thereafter, cells were stained with FITC-labeled annexin V and PI to evaluate the percentage of dead cells by flow cytometry. Mean values + SD of triplicates are shown. Significance of differences ( P < 0.01) is indicated by an asterisk.

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EpCAM complex differs from those of the individual molecules.

Membrane subdomain localization of CD44v4-v7 –EpCAM– tetraspanin complexes

Proteins associating with CD44v4-v7

Because of its relevance for functional activity (see below), we would first like to comment on the possible nature of the association. The CD44v –EpCAM– tetraspanin complexes were preserved in the mild detergent Lubrol but were disintegrated in strong detergents like TX-100 and were recovered from the light fractions after sucrose density gradient centrifugation. One could argue that this is evidence for an indirect association of the molecules, for example, via lipid interactions [73 – 75]. However, the fact that the complexes were not destroyed by partial cholesterol depletion provides an argument for protein –protein interactions. Because the complexes remained stable after partial cholesterol depletion, that is, irrespective of the GEM microenvironment, and because the individual components were also located in GEMs, it is difficult to argue whether the complexes are actively recruited towards GEMs. Nonetheless, the complexes display several features that support their localization in GEMs: (i) Tetraspanins are retained in GEM microdomains via palmitoylation and lipid anchoring [39,76]. CD44 also has two palmitoylation sites. However, our finding that mutations at both potential palmitoylation sites of CD44 did not interfere with GEM localization argues against palmitoylation of CD44v4-v7 as a potential means to recruit the complex. (ii) CD44 has been described to be associated with lck and lyn [78 – 80], members of the src family of protein tyrosine kinases that are GEM associated [40]. This could provide another means for the recruitment of CD44s and CD44v into GEMs. (iii) CD44—preferentially CD44 variant isoforms—associate with members of the ERM family [27,48]. Our data provided evidence that EpCAM and the tetraspanins CD9 and D6.1A also come into proximity to ezrin and moesin via their association with CD44v4-v7. As the ERM proteins are involved in membrane protein sorting [47], the preferential association of ERM proteins with CD44v could also account for the localization of the CD44v4-v7 –EpCAM– tetraspanin complex in GEMs. (iv) Finally, annexins are phospholipid binding proteins and are known to recruit associating proteins into lipid-rich membrane domains [77]. Annexin II has been shown to colocalize with CD44 in GEMs, where CD44 interacts with the underlying cytoskeleton. Notably too, annexin II and CD44 are released from GEMs by sequestration of plasma membrane cholesterol [70]. Furthermore, annexin I, which is part of the CD44v4-v7 –EpCAM– tetraspanin complex and can associate with actin and tubulin, is involved in signal transduction and apoptosis resistance [71,72]. (v) According to our findings, EpCAM does not associate with ERM proteins and it has no potential palmitoylation site. Thus, the question of the mechanism that anchors EpCAM itself in GEMs remains to be answered. Taken together, the CD44v4-v7 – EpCAM – tetraspanin complex formation appears to be based on low affinity protein – protein interactions. Its preferential localization in

CD44 variant isoforms exert a variety of functions, not ascribed to CD44s [3,4,7,8]. As the CD44s and CD44v isoforms share the cytoplasmic tail, it has been suggested that ligands exist, like bFGF, that binds CD44v3 [61,62] and osteopontin [63,64] that also selectively binds to CD44 variant isoforms [65]. Alternatively, transmembrane molecules may associate with CD44v, but not with CD44s, as described for the association of CD44v6 with scatter factor and c-Met [23]. In view of our observation that CD44v4-v7 and a set of four additional molecules is coexpressed exclusively on metastasizing tumor lines [66], we speculated that functions mediated by CD44v, but not by CD44s, may depend on an association of CD44v with these molecules. We could demonstrate that in the metastasizing rat tumor line BSp73ASML as well as in two additional metastasizing rat tumor lines, Progressor and 804G (data not shown), CD44v co-immunoprecipitates with EpCAM and D6.1A. The complex contains, in addition, the tetraspanin CD9, the a3 integrin, and annexin I. In the tested tumor lines, EpCAM, CD9, and D6.1A co-immunoprecipitate only with CD44v4-v7 and not with CD44s. We are currently generating deletion mutants of D6.1A, CD9, and EpCAM to define the domains associating with CD44v. We are also generating additional CD44 variant isoforms and chimeric CD44 molecules that will allow to define whether EpCAM, D6.1A, and CD9 bind directly to the variant exon product or to regions in the CD44s molecule that are folded or exposed differently due to the insertion of variant exon products. We have not yet analyzed in detail the nature of the co-immunoprecipitation of a3 and annexin I with CD44. There are reports describing CD44 expression influencing a3 integrin expression [67], but to our knowledge coimmunoprecipitation of CD44 and a3 has only been described in complexes containing CD9 [41]. In fact, the a3 integrin is known to bind directly to CD9 [68,69], and thus it is most likely that a3 will be recruited into the CD44v – EpCAM –D6.1A – CD9 complex via CD9. Also, the association of annexin I with the complex has not been explored. Notably, the closely related annexin II has also been observed to colocalize with CD44 in epithelial cells [70]. Irrespective of the contribution of a3 and annexin I as well as of the precise definition of the binding sites, we could demonstrate that complex formation between CD44, EpCAM, D6.1A, and CD9 essentially depends on the expression of CD44 variant isoforms. Besides the CD44v6 –HGF – c-Met complex [23], co-immunoprecipitation of CD44v4-v7 with EpCAM, D6.1A, and CD9 provides a second example for a CD44v-specific complex. As the set of these molecules is only expressed in metastasizing tumor cells, we speculated that complex formation may support steps of the metastatic cascade.

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GEMs could be due to the palmitoylation of the tetraspanins or the ERM association of CD44v. We consider annexin I to be the most promising candidate for the localization of the CD44v4-v7 – EpCAM –tetraspanin complex in GEMs, as the association with annexin I can also account for the release of the complex from GEMs by cholesterol depletion. Also, the functional activity ascribed to annexin I is well in line with our findings on functional activity of the complex. CD44v4-v7 –EpCAM complex-specific functions essentially depend on the GEM localization As shown for c-Met [23], functional activities claimed to be specific for CD44 variant isoforms can actually be a consequence of CD44v-selective binding partners, such that CD44v or the associating molecules gain access to adapter and signal-transducing molecules not associating with CD44s. We tried to experimentally support this hypothesis particularly for the complex of CD44v with EpCAM. We restricted our studies to this aspect because tetraspanins, known to form multiprotein complexes [38,81,82], are argued to exert functional activities mainly via complex formation [37], whereas several functional activities are directly described to CD44 and EpCAM. CD44, apart from other functions, is known as a major receptor for hyaluronan [83,84] and EpCAM was described as a homotypic cell –cell adhesion molecule [54,85]. These ‘‘complex-independent’’ functions facilitated the evaluation of loss or gain of function by complex formation. CD44 binds to hyaluronan [83,84,86,87]. Although antibody blocking studies confirmed the contribution of CD44 to HA binding (data not shown), we did not see significant differences in HA adhesion between cells expressing CD44s, CD44v, or the complex in the unperturbed state. However, after MhCD treatment that sufficed to destroy the GEM localization of the complex as well as of the individual molecules, adhesiveness of cells expressing the complex was strongly reduced, whereas adhesiveness of cells expressing only CD44v4-v7 remained unaltered. It has already been described in 1993 that CD44 can be expressed in three states, HA binding, inducible for HA binding, and uninducible [88]. We now would argue that the active state can also depend on the membrane microenvironment and that inducibility can depend on associated molecules such that associating molecules prevent binding of CD44v4-v7 to HA when the complex is located outside of GEMs. MhCD treatment also affected cell – cell adhesion and cluster formation in cells expressing the complex, but not in cells expressing only EpCAM. EpCAM has two EGF-like repeats, which are required for homophilic adhesion. The first repeat supports the interaction with the adjacent cell, while the second repeat mediates the lateral interaction between EpCAM molecules to form tetramers [54,89]. Tetramer formation depends on the association with F-actin via a-actinin and may be the first step for establishing the

intercellular contacts [90]. We interpret the finding that MhCD treatment interfered with cell –cell adhesion only in cells expressing the CD44v –EpCAM complex in the sense that depending on the membrane microdomain environment CD44v4-v7 may compete for EpCAM, such that only by the enrichment of the molecules in GEMs sufficient EpCAM is available for tetramer formation; whereas after destruction of GEMs, CD44v4-v7 efficiently interferes with the lateral EpCAM– EpCAM association, thus inhibiting tetramer formation and weakening EpCAM-mediated homotypic cell – cell adhesion. Work is in progress to control the hypothesis. We did not see changes in the migratory capacity of cells expressing CD44v4-v7 or EpCAM or both, and accordingly, migration was neither inhibited nor strengthened by the respective antibodies. This is worth mentioning because migration was inhibited by an antibody, Ox50, binding to an undefined region of the CD44 standard molecule that is not occupied by either the variant exon products or by the association of CD44v4-v7 with EpCAM. BSp73AS cells are rather apoptosis-susceptible and transfection of the cells with CD44v4-v7 or with EpCAM cDNA had no impact (CD44v4-v7) or only a minor impact (EpCAM) on apoptosis susceptibility. BSp73ASML cells are apoptosis resistant [49]. Yet, when BSp73ASML or Progressor cells were treated with MhCD, both lines became highly apoptosis susceptible. First to mention, annexin I, which has been found in association with the complex, is known to be involved in apoptosis resistance [71,72], and annexin II and CD44 are released from GEMs by sequestration of plasma membrane cholesterol [70]. These findings could well explain our observations of the loss of complexmediated apoptosis resistance by cholesterol depletion. Furthermore, the strong linkage between apoptosis resistance and GEM localization implicates, as has been suggested repeatedly [91,92], a strict structural organization of the inner side of the plasma membrane and could well be explained by the fact that several small GTPases as well as members of the src family of protein tyrosine kinases have been described to be located in GEMs [75,93,94]. Although the signal-transducing molecule(s) associating selectively with the complex is not yet defined, we know that a serine or threonine kinase, which phosphorylates a 23-kDa protein, becomes activated upon antibody crosslinking or via PMA stimulation only in cells expressing the CD44v4-v7 – EpCAM complex [95], that is, different signaling cascades are likely initiated by the complex as compared to the individual molecules. As (i) none of the associating molecules has intrinsic phosphatase or phosphokinase activity and (ii) co-immunoprecipitation, antibody blocking and antibody cross-linking studies provided evidence for an association of the extracellular domains of the molecules, it is obviously the joint recruitment of cytoplasmic and/or membrane-anchored adaptor and signal-transducing molecules by the individual partners that accounts for complex-mediated functions. It is well known that tetraspanins recruit PKC into proximity with specific

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integrins [39,69], and signal transduction initiated by transmembrane molecules associating with CD44 has repeatedly been described, for example, association of the cytoplasmic region of the TGFh-receptor I with that of CD44 [96], the association of scatter factor and c-met with CD44v6 [23], or the association of MMP-7 and the heparin binding epidermal growth factor precursor with extracellular domains of CD44 [97]. Notably, all these partner molecules of CD44 have intrinsic catalytic activity. This does not account for EpCAM. Thus, the cooperative activity of the CD44v4-v7 – EpCAM complex provides a first example that these two molecules in their complexed form activate a signal transduction cascade that cannot be triggered by the individual components. This is the first report to show that selectively CD44v4-v7, but not CD44s, associates with EpCAM, the complex containing, in addition, the tetraspanins CD9 and D6.1A, the a3 integrin, and annexin I. Furthermore, the complexed form of CD44v4-v7 fulfills different functions than CD44v4-v7 by itself. Thus, it is the complex that accounts, at least in part, for functions selectively attributed to CD44 variant isoforms. Notably too, CD44v4-v7 –EpCAM complex-mediated functions strictly depend on the membrane subdomain localization of the complex, that is, the complex strongly supports apoptosis resistance only when located in GEMs.

Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (Zo40-8/1) (MZ).

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