Ectopie Expression of Carcinoembryonic Antigen by a Melanoma Cell Leads to Changes in the Transcription of Two Additional Cell Adhesion Molecules1

[CANCER RESEARCH 55, 3254-3257, August 1, 1995] Advances in Brief

Thomas Grimm and Judith P. Johnson2

Institute of Immunology, University of Munich, Goelhestrasse 31, D-80336 Munich, Germans

Abstract Materials and Methods

Ectopie expression of Carcinoembryonic antigen (CEA) by the mela Cells, Probes, and Antibodies. Two independent stable CEA transfectant noma cell line Mel Wei led to alterations in cell morphology and to cell lines, CEA-Mel Wei 1 and II, were generated from the melanoma cell line changes in the expression of two melanoma-associated cell adhesion mol Mel Wei. Lipofectin (GIBCO BRL, Gaithersburg, MD) was used to transfect ecules, neural cell adhesion molecule and MUC18. The normally flat, Mel Wei cells with the full-length CEA cDNA clone 641 in the expression triangular cells developed neurite-like extensions and exhibited a less vector pcDNA-1, together with the neo resistance plasmid pGM-1 (5). The organized growth pattern. When compared to untransfected Mel Wei cells stable transfectant line MWAS was generated by transfecting Mel Wei with an or to those transfected with an irrelevant cDNA, two independent CEA irrelevant antisense cDNA clone (dropSAS). Hybridoma BBM.1 directed transfectants showed a decrease in the expression of neural cell adhesion against ÃŸ-2microglobulin was obtained from the American Type Culture molecule and an increase in the expression of MUC1S. These changes, Collection (Rockville, MD). Anti-MUC18 mAbs MUC18 and MUCBA18.4 which are characteristic of the metastatic phenotype in melanomas, were (6) and anti-intracellular adhesion molecule-1 antibody P3.58BA3 (7) were observed at the cell surface and at the level of mRNA and were independ produced in our laboratory. The anti-NCAM mAbs Leul9 and T199 were ent of adherent growth. Steady-state levels of neural cell adhesion mole obtained from Becton and Dickinson (Cockeysville, MD) and Dianova cule mRNA were reduced in CEA-expressing cells by approximately (Hamburg, Germany), respectively. Mouse myeloma protein UpclO was ob 5-fold, while MUC18 mRNA showed an 8-fold increase. No significant tained from Sigma Chemical Co. (St. Louis, MO). Peroxidase-conjugated differences in the expression of intercellular adhesion molecule-1 or ÃŸ-2 rabbit anti-mouse immunoglobulin was obtained from Dianova. microglobulin were observed between Mel Wei and CEA-Mel Wei. These Fluorescein-conjugated goat anti-mouse immunoglobulin was obtained data indicate that changes in the expression of a single cell adhesion from Dakopatts (Hamburg, Germany). molecule such as CEA can lead to alterations in the expression of unre Immunoperoxidase. Cells were grown on microscope slides (Dunn, lated cell adhesion molecules and may contribute to the general derange Asbach, Germany), fixed in acetone (5 min at room temperature), and stained ment of adhesive interactions observed frequently in tumor cells. using standard immunoperoxidase methods (6). RNA Extraction and Northern Blot Analysis. Total RNA was extracted Introduction from 2 X IO7 cells using RNazol (WAK-Chemie, Bad Homburg, Germany) according to the principles of the acid guanidine-chloroform-phenol procedure Changes in the expression of cell adhesion molecules are frequently (8). Fifteen iig RNA/lane were size separated, transferred to Hybond N observed during malignant transformation and are postulated to con (Amersham), and hybridized with previously radiolabeled DNA probes as tribute to the development of the metastatic phenotype. Normally described (9). The Northern blots were hybridized with the full-length MUC18 constitutive cell adhesion molecules such as E-cadherin are often cDNA (10), with NCAM oligonucleotide E7, which hybridizes to all NCAM down-regulated in carcinomas (1), whereas other cell adhesion mol transcripts (11) and which corresponds to bp 1177-1224 of the published cDNA sequence, and with an oligonucleotide (5'-CCCTGGTGAC CAGGCG- ecules, such as the carbohydrate ligands of endothelial selectins, are GCCA ATACGGCCAA TCCGTTGACT CCGACTTTCC AC-3') from the newly expressed (2). Although the mechanisms responsible for these first exon of the GAPDH gene (12). The roentgen films were evaluated using changes remain mostly unknown, they do not appear to generally reflect mutations or other genetic alterations. CEA3 is an epithelial an elscript 400 Scanner (ATH, Taufkirchen, Germany). cell surface glycoprotein that is frequently increased in expression and Results altered in cellular localization following malignant transformation (3). CEA has been shown to function as a homotypic intercellular adhe Expression of CEA Leads to Morphological Changes in Mel sion molecule (4), and it has been speculated that alterations in its Wei Cells. Transfection of the melanoma cell line Mel Wei with the expression in carcinoma cells may lead to a general derangement in CEA cDNA clone 641 led to high levels of cell surface CEA expres cell adhesion and consequent disruption of normal cell-cell interac sion (Fig. \A) in two independent transfectants (CEA-Mel Wei I and CEA-Mel Wei II), whereas untransfected cells or cells transfected tions. Here we report that the de novo expression of CEA by a melanoma cell line leads to selective changes in the expression of two with an irrelevant cDNA clone (MWAS cells; data not shown) are melanoma-associated cell adhesion molecules. These results indicate CEA negative. The expression of CEA in the melanoma cells was that alterations in the expression of one cell adhesion molecule can associated with a change in the growth characteristics of the mela lead to changes in the expression of additional, unrelated cell adhesion noma cells. Untransfected Mel Wei cells growing in tissue culture molecules. flasks or Petri dishes have a flat triangular shape (Fig. 2B). In contrast, both CEA-Mel Wei I and II produce long, neurite-like extensions (Fig. 2D). In confluent cultures, newly produced Mel Wei cells are Received 3/31/95; accepted 6/8/95. The costs of publication of this article were defrayed in part by the payment of page shed into the medium, whereas CEA-Mel Wei cells remain adherent charges. This article must therefore be hereby marked advertisement in accordance with and pile on top of each other, suggesting changes in contact inhibition. 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by a grant from the Mildred-Scheel-Stiftung, Deutsche Since the independent transfectants CEA-Mel Wei I and II showed Krebshilfe, Bonn, Germany. identical changes in morphology that were not seen in cells trans 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: CEA, Carcinoembryonic antigen; GAPDH, glyceralde- fected with an irrelevant cDNA, it appears to be the expression of the hydephosphate dehydrogenase; ICAM, intercellular adhesion molecule; NCAM, neural CEA molecule itself that is responsible for the changes in the growth cell adhesion molecule. characteristics of the cells. 3254

B melanoma cells. No significant differences between Mel Wei and CEA-Mel Wei cells were observed in the surface expression of intracellular adhesion molecule-1 (Fig. 1Â£)and ÃŸ-2microglobulin (Fig. ID); the differences of the S mean channels were 6 and 10, respectively (Fig. IF). In contrast, major changes were seen in the expression of NCAM (Fig. IÃŸ)and MUC18 (Fig. 1C). The anti- NCAM mAb Leul9 reacted with Mel Wei (Figs. IB, thin lines) but only weakly with CEA-Mel Wei I and II (Fig. IÃŸ,heavy and dotted lines, respectively), indicating that surface expression of NCAM is down-regulated on CEA-expressing cells. Identical results were ob tained with a second mAb (T199; data not shown). Analyses with mAbs directed against two different epitopes on the MUC18 molecule 400 - indicated that expression of this antigen is increased on CEA-express 300 - ing cells. Reactivity of Mel Wei (Fig. 1C, thin line) and CEA-Mel

200 - Wei I and II (Fig. 1C, heavy and dotted lines, respectively) with mAb MUCBA18.4 is shown. Identical results were obtained with 100 - mAb MUC18. The 8 mean channel values for antibody binding on 0 - I Mel Wei (Fig. IF, â€¢) and CEA-Mel Wei I (Fig. IF, D) and II FJ95 LeulS TI 99 MUCBA18.4 MUC18 P3.58BA3 BBM.l (Fig. IF, H) are shown. Fig. 1. Expression of cell surface molecules by Mel Wei and CEA-Mel Wei. A-E, Immunoperoxidase staining of cells grown on microscope slides FACs profiles. A, mAb FJ95 (anli-CEA); B. mAb Leul9 (anti-NCAM); C. mAb MUCBA18.4 (ami-MUC18); D. mAb BBM.l (anti-/3-2 microglobulin); Â£. mAb yielded the same results. As shown in Fig. 3, NCAM expression P3.58BA3 (anti-ICAM-1). Thin line, Mel Wei; heavy line, CEA-Mel Wei I; dolled line. (assessed with mAb Leu 19) is reduced in CEA-Mel Wei (Fig. 3, CEA-Mel Wei II. Hori~onial axis, relative logarithmic fluorescence intensity. Vertical C versus A), whereas MUC18 (assessed with mAb MUCBA18.4; axis, relative cell number. For each cell, the isotype control histograms have been Fig. 3, D versus B) is enhanced. subtracted. F, mean fluorescence intensity. The height of the columns represents the mean logarithmic fluorescence intensity after subtraction of the mean logarithmic fluorescence Changes in Antigen Expression Are Also Observed in Cells intensity of the corresponding UpclO negative control (ÃŸmeanchannel). â€¢,Mel Wei; D, Grown in Suspension. To determine whether the observed changes CEA-Mel Wei I; D, CEA-Mel Wei II. in NCAM and MUC18 expression were dependent on cell morphol ogy, the normally adherent cells were cultured in bacterial Petri dishes Changes in the Cell Surface Expression of MUC18 and NCAM where the cells do not adhere. After 24 h, cells cultured in the tissue Are Observed in Mel Wei Cells following Transfection with CEA. culture Petri dishes were adherent (Fig. 2, B and D) and demonstrated The changes observed in the morphology of the Mel Wei cells the CEA-associated changes in cell shape, while those in bacterial following the expression of CEA prompted us to look for alterations Petri dishes remained in suspension (Fig. 2, A and C). Examination of in the expression of cell surface molecules normally found on these the Mel Wei (Fig. 2, E and C) and CEA-Mel Wei I (Fig. 2, F and H)

Fig. 2. MUC18 and NCAM expression by adherent and nonadherent Mel Wei and CEA-Mel Wei. Mel Wei and CEA-Mel Wei I were grown for 24 h in suspension cultures (uncoated bacterial culture dishes) or as adherent cells (coated tissue culture dishes). A-D. micrographs of Mel Wei (A and ÃŸ)and CEA-Mel Wei I (C and D) in suspension culture (A and (7) and adherent (B and D). E-H, surface reactivity of Mel Wei (E and G) and CEA-Mel Wei I (F and H) determined by immunofluorescence. E and F, Leu 19 (anti-NCAM); G and H, MUCBA18.4 (anti-MUC18). Thin lines, cells in suspension culture; heavy lines, adherent cells. Horizontal axis, relative logarithmic fluorescence intensity. Vertical axis, relative cell number. For each cell, the isotype control histograms have been subtracted. 3255

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Fig. 3. Immunoperoxidase detection of MUC18 b "**â€¢*.- and NCAM Expression. Mel Wei (A and B) and 7s CEA-Mel Wei I (C and D) grown on coated micro scope slides. A and C, mAb Leu 19 (anti-NCAM); B 1'.v.. and D, mAb MUCBA18.4 (anti-MUC18). rfeiv'iÃ¤ kw

cells for NCAM (Fig. 2, E and F) and MUC18 (Fig. 2, G and H) in changes in mRNA levels, Northern blot analysis of Mel Wei and expression indicated that the CEA-related changes in expression were CEA-Mel Wei transfectants were performed. CEA-Mel Wei I found under both adhesion-permissive (Fig. 2, heavy lines) and non- (Fig. 4A, Lane 3) and II (Fig. 4A, Lane 4) showed a decrease in permissive (Fig. 2, thin lines) culture conditions, indicating that they expression of the 6.7-kb NCAM mRNA as compared to Mel Wei (Fig. were not a result of the observed changes in the cell shape but rather 4A, Lane 1) and MWAS (Fig. 4/4, Lane 2). Densitometric comparison a result of CEA expression. Similar results were obtained when the of NCAM and GAPDH expression in each cell line (Fig. 4Â£)revealed incubation period under these different conditions was prolonged to a decrease of the NCAM:GAPDH ratio from 1.00 (set as standard) in 48 h. Mel Wei and 0.72 in MWAS, to 0.28 and 0.09 in the CEA-Mel Wei Changes in the Expression of MUC18 and NCAM in the CEA transfectants. Conversely, expression of the 3.3-kb MUC18 mRNA Transfectants Occur at the Transcriptional Level. To investigate (Fig. 4B) was increased in the CEA-transfected Mel Wei cells (Fig. whether the changes observed in antigen expression are also reflected 4B, Lanes 3 and 4) compared to untransfected Mel Wei and MWAS (Fig. 4ÃŸ,Lanes 1 and 2). The MUC18/GAPDH ratios were 0.07 (Mel Wei), 0.15 (MWAS), 1.00 (CEA-Mel Wei I, set as standard) and 0.74 (CEA-Mel Wei II). 1.0-, Discussion 0.8

0.6 In the study reported here, de novo expression of CEA by the melanoma cell line Mel Wei has been shown to induce changes in cell 0.4 morphology and in the expression of two melanoma-associated cell adhesion molecules, NCAM and MUC18. The changes in cell adhe 0.2 sion molecule expression observed were found in two independently B E 0 generated CEA transfectants and were not seen in Mel Wei cells transfected with an irrelevant cDNA. They were also independent of cell adherence and the formation of neurite-like extensions and thus 1.0 appear to be directly related to the expression of CEA. 0.8 That ectopie expression of CEA can dramatically alter the function of a cell has been shown in studies with rat myoblasts (3). Expression 0.6 of CEA by these cells prevented cell fusion and blocked their entire 0.4- differentiation program. This required the presence of the adhesion- mediating domains of CEA, suggesting that it was dependent on intra- 0.2- or intercellular adhesive interactions. The down-regulation of NCAM F 0- CZl and up-regulation of MUC18 occurred at the transcriptional level, 1 indicating that the expression of CEA leads to the transduction of a signal into the nucleus. This could involve either signaling via CEA Fig. 4. MUC18 and NCAM mRNA expression in Mel Wei and CEA-Mel Wei. Northern blot analysis of Mel Wei (Lane 1), MWAS (Lane 2), CEA-Mel Wei I (Lane 3), itself or signaling via a CEA ligand expressed on Mel Wei. Recent CEA-Mel Wei II (Lane 4). RNA Blots were hybridized with an NCAM probe (A) or a studies on neuronal cell adhesion molecules have begun to shed light MUC18 probe (C) and subsequently with a GAPDH probe (B and D). Densitometric on how cell adhesion molecules can generate intracellular signals analysis of Northern blots. E, NCAM:GAPDH RNA ratios (with the highest value set to (13). NCAM, LI, and N-cadherin share short-sequence homologies 1). F, MUC18:GAPDH RNA ratios (with the highest value set to 1). D, Mel Wei; â€¢ MWAS; D, CEA-Mel Wei I; m, CEA-Mel Wei II. with fibroblast growth factor receptor, and these mediate binding 3256

between the cell adhesion molecules and the growth factor receptor. antigen correlates with poor survival in patients with colorectal carcinoma: clinico- pathological and Â¡mmunohislochemical study. Cancer Res., 53: 3632-3637, 1993. This binding leads to activation of the receptor and is required for cell 3. Eidelman, F. J., Fuks, A., DeMarte, L., Tahery, M., and Stanners, C. P. Human adhesion molecule-directed neurite outgrowth. Although melanoma carcinoembryonic antigen, an intercellular adhesion molecule, blocks fusion and cells generally express fibroblast growth factor receptor, such homol differentiation of rat myoblasls. J. Cell Biol., 123: 467-475, 1993. ogous sequences could not be identified in CEA. It seems likely that 4. Benchimol, S., Fuks, A., Jothy, S., Beauchemin, N., Shirota, K., and Stanners, C. P. Carcinoembryonic antigen, a human tumor marker, functions as an intercellular this molecule interacts with an as yet unidentified melanoma cell adhesion molecule. Cell, 57: 327-334, 1989. surface molecule to generate an intracellular signal leading to changes 5. Grimm, T., RiethmÃ¼ller,G., and Johnson. J. P. Characteristics of carcinoembryonic in the transcription of NCAM and MUC18. Although CEA is perhaps antigen (CEA) expressed in different cell types: evidence that CEA can function as an adhesion molecule and as a repulsion molecule. Biochem. Biophys. Res. Commun., unlikely to be the molecule that triggers these changes in melanoma 204: 1225-1234, 1994. cells in vivo, it should share a binding specificity (and perhaps 6. Lehmann, J. M., Holzmann, B., Breitbari, E. W., Schmiegelow, P., RiethmÃ¼ller,G., sequence homology) with the molecule involved. Not all melanoma and Johnson, J. P. Discrimination between benign and malignant cells of melanocytic lineage by two novel antigens, a glycoprotein with a molecular weight of 113,000 and cell lines appear to be sensitive to this regulatory effect of CEA a protein with a molecular weight of 76,000. 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MUC18, a melanoma-progression that have been associated with the acquisition of metastatic capacity. associated molecule, and its potential role in tumor vascularization and hcmatogenous The cell surface glycoproteins NCAM and MUC18, both members of spread. Cancer Res., 54: 5689-5694, 1994. 10. Lehmann, J. M., RiethmÃ¼ller,G., and Johnson, J. P. MUC18, a marker of tumor the immunoglobulin superfamily, are expressed on a variety of ma progression in human melanoma, shows sequence similarity to the neural cell adhe lignant tumors, where their presence has, in some cases, been asso sion molecules of the immunoglobulin superfamily. Proc. Nati. Acad. Sci. USA, 86: ciated with prognosis (14, 15). NCAM has been shown to function as 9891-9895, 1989. 11. Small, S. J., Shull, G. E., Santoni. M. J., and Akeson, R. Identification of a cDNA a homophilic cell adhesion molecule (16), while recent evidence clone that contains the complete coding sequence for a 140-kD rat NCAM polypep- suggests that MUC18 may interact with a heterophilic ligand (17). In tide. J. Cell Biol., 105: 2335-2345, 1987. models of metastasis formation using immune-deficient mice, meta 12. Dugaiczyk, A., HarÃ³n, J. A., Stone, E. M., Dennison. O. E., Rothblum, K. N., and Schwartz, R. J. Cloning and sequencing of a deoxyribonucleic acid copy of glycer- static variants of melanoma cells are NCAM low and MUC18 high, aldehyde-3-phosphate dehydrogenase messenger ribonucleic acid isolated from whereas nonmetastatic variants can be NCAM high and MUC18 chicken muscle. Biochemistry, 22: 1605-1613, 1983. negative (18, 19). Furthermore, transfection of a glioma cell line with 13. Williams, E. J., Furness, J., Walsh, F. S., and Doherty, P. Activation of the FGF receptor underlies neurite outgrowth stimulated by LI, N-CAM and N-cadherin. NCAM encoding cDNA reduced its invasive properties in vivo (20), Neuron, 13: 583-594, 1994. and an examination of tumors in situ has shown an increase in 14. Holzmann, B., BrÃ¶cker.E. B., Lehmann, J. M.. Ruiter, D. J., Sorg, C., RiethmÃ¼ller, MUC18 expression in advanced as opposed to early lesions (6). G., and Johnson, J. P. Tumor progression in human malignant melanoma: five stages defined by their antigenic phenotypes. Int. J. Cancer, 39: 466-471, 1987. The expression of CEA can induce metastatic potential in previ 15. Roth, J., Zuber, C., Wagner, P., Taatjes, D. J., Weisgerber, C., Heitz, P. U., Goridis, ously nonmetastatic cells, an effect that has been attributed to CEA C., and Bitter-Suermann, D. Reexpression of poly(sialic acid) units of the neural cell adhesion molecule in Wilms' tumor. Proc. Nati. Acad. Sci. USA, 85: 2999-3003, binding to a protein expressed on the surface of Kupffer cells (21). However, the expression of CEA may affect more than CEA-mediated 1988. 16. Rao, Y., Wu, X. F., Gariepy, J., Rutishauser, U., and Siu, C. H. Identification of a adhesion. In two different systems, the expression of CEA has now peptide sequence involved in homophilic binding in the neural cell adhesion molecule been shown to result in changes in the transcription of other genes. NCAM. J. Cell Biol., 118: 937-949, 1992. 17. Shih, I-M., Elder, D. E., Speicher, D., Johnson, J. P., and Herlyn, M. Isolation and Since the changes reported here concern cell adhesion molecules, both functional characterization of Ihe A32 melanoma-associated antigen. Cancer Res., 54: of which have been implicated in metastasis, it is intriguing to 2514-2520, 1994. postulate that changes in CEA expression, as are observed in many 18. I inun n,mu. D., Raz, A., and Bock, E. Differential expression of cell adhesion molecules in variants of K1735 melanoma cells differing in metastatic capacity. Int. carcinomas, may contribute in several different ways to tumor J. Cancer, 43: 709-712, 1989. progression and to the development of the metastatic phenotype. 19. Luca, M., Hunt, B., Bucana, C. D., Johnson, J. P., Fidler, I. J., and Bar-Eli, M. Direct correlation between MUC18 expression and metastatic potential of human melanoma References cells. Melanoma Res., 3: 35-41, 1993. 20. Edvardsen, K., Pedersen, P. H., Bjerkvig, R., Hermann, G. G., Zeuthen, J., Laerum, 1. Mayer, B., Johnson, J. P., Leill, F., Jauch, K. W., Heiss, M. M., Schildberg, F. G., 0. D., Walsh, F. S., and Bock, E. Transfection of glioma cells with the neural-cell Birchmeier, W., and Funke, I. E-cadherin expression in primary and metastatic gastric adhesion molecule NCAM: effect on glioma-cell invasion and growth in vivo. Int. J. cancer: down-regulation correlates with cellular dedifferentiation and glandular Cancer, 58: 116-122, 1994. disintegralion. Cancer Res., 53: 1690-1695, 1993. 21. Thomas, P., Petrick, A., Toth, C. A., Fox, E. S., Elting, J. J., and Steele, G., Jr. Peptide 2. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. Ectopic Expression of Carcinoembryonic Antigen by a Melanoma Cell Leads to Changes in the Transcription of Two Additional Cell Adhesion Molecules

Thomas Grimm and Judith P. Johnson

Cancer Res 1995;55:3254-3257.

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