Human Carcinoembryonic Antigen Functions As a General Inhibitor of Anoikis1

[CANCER RESEARCH 60, 3419–3424, July 1, 2000] Advances in Brief

Human Carcinoembryonic Antigen Functions as a General Inhibitor of Anoikis1

Cosme Ordon˜ez, Robert A. Screaton, Chris Ilantzis, and Clifford P. Stanners2 McGill Cancer Centre and Department of Biochemistry, McGill University, Montreal, Quebec, H3G1Y6 Canada

Abstract correct deployment of differentiation programs. It has been proposed that anoikis, a type of apoptotic program triggered when cells lose Human carcinoembryonic antigen (CEA), a widely used tumor marker, contact with the ECM (9–11), functions in vivo as a surveillance and CEACAM6 [formerly nonspecific cross-reacting antigen (NCA)] are mechanism that prevents dysplasia and preserves normal tissue archi- up-regulated in many types of human cancers, whereas family member tecture by destroying any cells that attempt to deviate from their CEACAM1 [formerly biliary glycoprotein (BGP)] is usually down- regulated. Deregulated overexpression of CEA/CEACAM6 but not normally operative spatial constraints (10). Our model (Fig. 4) would CEACAM1 can inhibit the differentiation and disrupt the polarization require that this process be inhibited, if aberrant multilayered tissue and tissue architecture of many different types of cells. In this report, we architecture initiated by CEA/CEACAM6 overexpression were to be show that CEA and CEACAM6, but not CEACAM1, markedly inhibit the maintained. We demonstrate here that forced overexpression of CEA/ apoptosis of cells when deprived of their anchorage to the extracellular CEACAM6 but not CEACAM1 on the surface of L6 rat myoblasts, matrix, a process known as anoikis. By blocking this tissue architecture MDCK epithelial cells, and human SW1222 and Caco-2 colorectal surveillance mechanism, the architectural perturbation initiated by CEA/ cancer cells inhibited their anoikis in vitro. CEACAM6 can thus be maintained. Materials and Methods Introduction Cell Lines. L6 rat myoblasts, SW1222 and Caco-2 human colorectal car- 3 CEA and highly related CEA family member CEACAM6, mem- cinoma cells, and MDCK epithelial cells were grown as monolayer cultures in bers of the immunoglobulin superfamily, show deregulated cell sur- DMEM (L6 and MDCK cells) or ␣-MEM (SW1222 and Caco-2 cells) con- face overexpression in about 50% of human cancers (1); CEACAM1, taining 10% fetal bovine serum (GM; Life Technologies, Inc., Gaithersburg, on the other hand, which differs from CEA/CEACAM6 chiefly by its MD) supplemented with 100 ␮g/ml streptomycin and 100 units/ml penicillin mode of cell membrane anchorage (transmembrane versus GPI), is (Life Technologies, Inc.) at 37°C in a humidified atmosphere with 5% CO2. usually down-regulated (2). These CEA family members function, in All cell lines were subcultured before confluence and seeded at a density of ϫ 4 2 vitro at least, as intercellular adhesion molecules (3–5). A common 1 10 cells/cm . cDNA Transfections and Infections. Transfection procedures and the view that CEA represents a “differentiation marker” that merely transfected cell lines used in this study have been described previously (6, reflects the differentiation status of the tumors that express it has been 7).4, 5 Briefly, stable transfectants of rat L6 myoblasts were obtained by the challenged recently by evidence that CEA and CEACAM6, but not calcium phosphate precipitation method using p91023B expression vector CEACAM1, could play an instrumental role in tumorigenesis by the (courtesy of R. Kaufman; Genetics Institute, Boston, MA) containing full- disruption of cell polarity and tissue architecture and the inhibition of length cDNAs encoding cell adhesion proteins CEA, CEACAM6, cell differentiation of many different types of cells4 (6–8). If dereg- CEACAM1-4L (formerly splice variant BGPa), CEA deletion mutant ⌬NCEA ulated CEA/CEACAM6 overexpression in colonocytes at the base of [lacking the last 75 amino acids of the NH2-terminal domain (6, 12)], NCAM- colonic crypts can indeed cause the aberrations in tissue architecture 125 [GPI-linked NCAM splice variant with a muscle-specific domain (13)] and pSV2neo plasmid as a dominant selectable marker. SW1222 and Caco-2 observed in human colonic carcinomas, CEA/CEACAM6 overexpres- 2ϩ sion must also overcome the control mechanisms that normally pre- transfectants were obtained with Zn -inducible episomal expression vector pML1 containing the hygromycin gene plus full-length cDNAs encoding either serve the tissue architecture of the crypts. We have obtained evidence CEACAM1, CEA, or ⌬NCEA or a cDNA containing the entire coding region that the function of specific integrins is perturbed by CEA/ of CEACAM6.4 SW1222-Hygro and Caco-2-Hygro (vector alone) controls 5 CEACAM6 overexpression, thus affecting cell-ECM interactions were obtained with pML1 containing the hygromycin gene. Pooled clones of that are necessary for the establishment of tissue architecture and the stably transfected cells were selected with 400 ␮g/ml neomycin (G418; L6 cells) or 200 ␮g/ml hygromycin B (SW1222 and Caco-2 cells). L6 myoblasts 5 Received 10/22/99; accepted 5/12/00. expressing comparably high levels (FACS mean value, 150–225) of the The costs of publication of this article were defrayed in part by the payment of page proteins encoded by the transfected cDNAs were selected by FACS using charges. This article must therefore be hereby marked advertisement in accordance with specific mAbs. After promoter induction with Zn2ϩ, the SW1222-CEACAM6 18 U.S.C. Section 1734 solely to indicate this fact. transfectant and the Caco-2-CEA/CEACAM6 double transfectant cells ex- 1 Supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. C. O. was supported by a Studentship from the Cancer pressed about 9- and 20-fold higher cell surface levels of CEACAM6 or Research Society of Canada. CEA/CEACAM6, respectively, than control cells transfected with the vector 2 To whom requests for reprints should be addressed, at McGill Cancer Centre, 3655 alone.4 Although G418 (L6) and hygromycin B (SW1222 and Caco-2) were Drummond Street, Montreal, Quebec, H3G1Y6 Canada. Phone: (514) 398-3535; Fax: removed from the culture media during functional assays, no loss of cell (514) 398-6769; E-mail: [email protected]. 3 The abbreviations used are: CEA, carcinoembryonic antigen; GM, growth medium, surface expression of the transfected cDNAs was observed (data not shown). ECM, extracellular matrix; TUNEL, terminal deoxynucleotidyl transferase-mediated nick L6 and MDCK cells in the exponential phase of growth were infected with end labeling; DAPI, 4Ј,6-diamidino-2-phenylindole; MDCK, Madin-Darby canine kid- a replication-defective recombinant retrovirus containing either pBabe(human ney; GPI, glycosylphosphatidyl inositol; FACS, fluorescence-activated cell sorting; bcl-2)puro [L6 (7)] or pLXSN(CEA cDNA)neo (MDCK) or the vector alone NCAM, neural cell adhesion molecule; PolyHEMA, poly(2-hydroxyethylmethacrylate); ␮ mAb, monoclonal antibody. as a control. Stably transfected MDCK cells were selected with 400 g/ml 4 C. Ilantzis, L. DeMarte, R. A. Screaton, and C. P. Stanners. Deregulated expression G418, and cells expressing high levels of CEA on the cell surface were of the human tumor marker CEA, and CEA family member NCA disrupts tissue archi- selected by FACS using specific anti-CEA mAbs. tecture and blocks colonocyte differentiation, submitted for publication. To avoid phenotypic perturbations due to clonal variation, all transfected 5 C. Ordon˜ez, R. A. Screaton, C. Ilantzis, M. Fan, and C. P. Stanners. Carcinoembry- onic antigen, a human tumor marker, inhibits cell differentiation and apoptosis by cell lines used in this study were prepared as pooled (total) populations ␣ ␤ perturbing the function of the 5 1 integrin receptor, submitted for publication. consisting of multiple clones selected with G418 or hygromycin. The poly- 3419

Fig. 1. AϪF, DAPI staining of L6 parental and transfected myoblasts suspended in GM for 48 h on PolyHEMA-coated surfaces. Apoptotic cells showed characteristic fragmented nuclei, whereas survivors showed an intact nuclear morphology. A, L6 parental cells. L6 myoblasts expressing human CEACAM1 (B), human Bcl-2 protein (C), the human GPI-linked isoform of NCAM (NCAM-125) (D), CEA (E), and CEACAM6 (F). G, the apoptotic index (see the graph) was calculated by scoring the percentage of apoptotic cells in three independent samples of 500–1000 cells each. The statistical average and SD of three independent experiments are indicated. Both CEA and CEACAM6 expression on the surface of L6 myoblasts significantly inhibited anoikis (P ϭ 0.005 and P ϭ 0.004, respectively). A second independent pooled population of stably transfected L6 myoblasts (L6- CEA-2) showed results similar (P ϭ 0.001) to those seen in L6-CEA transfected cells. These results were confirmed using the TUNEL assay in two separate experiments (data not shown).

clonality of the CEA-expressing L6 cell population was demonstrated by cells with DAPI, the cells were fixed on ProbeOnPlus microscope slides Southern blot (7). (Fisher Biotech) with 4% paraformaldehyde for 20 min, washed, permeabi- Apoptotic Assays. To induce overexpression of CEA/CEACAM6, lized for 5 min with 0.1% Triton X-100 (T-8787; Sigma, St. Louis, MO), and SW1222 and Caco-2 transfected cells were cultured in GM supplemented with stained with 10 ␮g/ml DAPI in PBS. Cells with fragmented (DAPI) or stained

0.1 mM ZnSO4 for 24 h before the experiment. To measure the anoikis of L6, (TUNEL) nuclei were scored as apoptotic cells. The apoptotic index was MDCK, and Zn2ϩ-induced SW1222 and Caco-2 cells (control and transfected calculated by scoring no less than 1000 cells. All observations were reproduced cells), 0.2 ϫ 106 cells/ml of each cell line were suspended in PolyHEMA at least twice by independent experiments. (Aldrich Chemicals, Milwaukee, WI)-coated 6-well tissue culture plates for a All cell lines used in the anoikis assays formed cellular aggregates in period of 12–72 h in the presence (GM) or absence (serum-free DMEM) of suspension over PolyHEMA-treated surfaces. The average size of these ag- growth factors. The percentage of apoptotic cells was estimated by staining the gregates was determined by dividing the total number of cells per sample by nuclei with DAPI (Boehringer Mannheim, Roche Diagnostics, Laval, Quebec, the total number of aggregates, which was determined in triplicate. To deter- Canada) or by using the TUNEL assay (Oncor; S7100-KIT, Intergen, Boston, mine the total number of cells per sample, the aggregates were dissociated at MA) following the instructions from the manufacturer. Briefly, to stain the 37°C for 30 min with a collagenase enzyme mixture that contained 690 units 3420

Fig. 2. Apoptotic cells detected by positive staining using the TUNEL assay. A, control attached L6 parental myoblasts (L6-att) were cultured attached to the tissue culture plastic surface and did not undergo apoptosis. BϪD, parental and transfected L6 myoblasts suspended in serum- free DMEM on PolyHEMA-coated surfaces for 24 h (B,L6 parental myoblasts; C, CEACAM1-expressing L6 myoblasts; D, CEACAM6-expressing L6 myoblasts). E, the apoptotic index was calculated as described in the Fig. 1 legend. These results were corroborated by DAPI staining in four independent experiments. F, quantitation of anoikis of transfected MDCK cells suspended in GM on PolyHEMA- coated surfaces for 24 h. The average and SDs of two independent experiments are presented.

crude collagenase/ml (code CLS-1; Worthington Biochemical Corp., Freehold, P Ͻ 0.001, respectively; Fig. 2, D and E); control L6 myoblasts NJ) in PBS. The cell concentration in the resulting single cell suspension was expressing CEACAM1 (Figs. 1B and 2, C and E) or cells transfected measured using a particle counter (Coulter Electronics Inc., Hialeah, FL). The with the vector alone (data not shown) showed about the same level average aggregate size of the parental L6 cells was 30% less than that of the of anoikis as the parental cells. transfectant populations, which had average aggregate sizes that were within To test for nonspecific effects due to intercellular adhesion, L6 5% of each other. Statistical Analysis. Differences between groups (CEA/CEACAM6- transfected myoblasts expressing high levels of the NCAM isoform expressing cells versus control cells) were analyzed by using Student’s t test. NCAM-125, another GPI-anchored intercellular adhesion molecule of P Ͻ 0.05 was considered significant. the immunoglobulin superfamily, were used as a control. NCAM-125, despite being expressed at higher levels than CEA [FACS mean value, Results 225 (NCAM-125) versus 190 (CEA)],5 did not inhibit anoikis (Fig. 1, Effects of CEA and CEACAM6 Expression on Anoikis of Rat D and G; Fig. 2E). This result indicates that the inhibitory effects of L6 Myoblasts. To test whether CEA and CEACAM6 overexpression CEA and CEACAM6 are not due to indirect effects of their intercel- on the cell surface inhibits anoikis in vitro, we used pooled stably lular adhesion function or GPI anchorage per se. As a further test of ⌬ transfected L6 rat myoblast clones as an ectopic model. These trans- specificity, L6 myoblasts expressing NCEA, a deletion mutant lack- fectants, together with appropriate controls, were cultured in suspen- ing the last two-thirds of the NH2-terminal domain (6, 12), were tested sion in serum-free DMEM or GM in tissue culture dishes coated with for anoikis. These transfectants were as prone to undergo anoikis as PolyHEMA to prevent cell attachment to the substratum. Under these L6 parental cells (Fig. 2E). The deleted portion of the NH2-terminal conditions, parental L6 myoblasts underwent anoikis due to the lack domain has been shown to be required for both intercellular adhesion of survival signals provided by the ECM or substratum, showing (12) and the differentiation inhibitory effects of CEA (6). characteristic fragmentation of their nuclei (Fig. 1A) and positive Parental L6 myoblasts suspended in the absence of growth factors staining by the TUNEL assay (Fig. 2B). L6 myoblasts expressing (serum-free DMEM) showed apoptotic features as early as 12 h (data CEA or CEACAM6 on their surfaces, on the other hand, were much not shown). However, in the presence of growth factors (GM), apo- less prone to undergo anoikis in GM (P ϭ 0.001 and P ϭ 0.004, ptosis of suspended L6 myoblasts was only 5% after 24 h (data not respectively; Fig. 1, EϪG) or serum-free DMEM (P ϭ 0.003 and shown) and increased to 40% after 48 h (Fig. 1G). As mentioned 3421

Fig. 3. AϪD, DAPI staining of SW1222 parental and transfected cells. A, control SW1222 parental cells attached to uncoated substratum. SW1222 parental cells (B), SW1222-CEACAM6 cells (C), and SW1222-Hygro (vector alone) (D) control cells cultured on PolyHEMA-coated dishes for 24 h in GM. E, quantitation of anoikis of transfected SW1222 cells (24 h). F, quantitation of anoikis of Caco-2 human colorectal cancer cells suspended in GM on Poly- HEMA-coated dishes for 48 h (left) and 72 h (right). The data represent the average of two typical experiments of six independent experiments with similar results.

above, CEA and CEACAM6 expression inhibited anoikis of L6 SW1222 and Caco-2 cell lines, both of which are capable of colonic myoblasts in both the presence and absence of growth factors. Sus- epithelial differentiation4 (14, 15), was measured. Parental SW1222 pended L6 myoblasts expressing human Bcl-2, a known inhibitor of and Caco-2 cells express relatively low levels of endogenous CEA anoikis (9, 10), were less prone to undergo cell death (P Ͻ 0.001) than and CEACAM6 (16). After promoter induction with Zn2ϩ, the L6 parental myoblasts (Fig. 1, C and G; Fig. 2E). Interestingly, SW1222-CEACAM6 transfectant and the Caco-2-CEA/CEACAM6 Bcl-2-mediated inhibition of anoikis was more prominent in the double transfectant cells from subconfluent growing cultures ex- presence of growth factors (Fig. 1G) than in the absence of growth pressed about 9- and 20-fold higher cell surface levels of CEACAM6 factors (Fig. 2E). The ectopic expression of CEA on the surface of and CEA/CEACAM6, respectively, than control cells transfected with MDCK cells inhibited anoikis (P ϭ 0.001) when these cells were the vector alone.4 These levels of CEA/CEACAM6 expression cor- suspended in PolyHEMA-coated dishes (Fig. 2F), thus confirming the respond to those observed in vivo in human colonic tumors (17). effect. SW1222 cells overexpressing CEACAM6 were found to be unable to Effect of CEA and CEACAM6 Overexpression on Anoikis of form glandular-like spheroids of polarized cells in collagen gels;4 Human Colonocytes. CEA and CEACAM6 are not endogenously Caco-2 cells overexpressing CEA/CEACAM6 lost their ability to expressed by either skeletal muscle cells or MDCK cells. To test the form monolayers of polarized cells and instead formed stratified inhibitory effects of CEA/CEACAM6 in a medically relevant model layers of disorganized cells, closely resembling dysplastic colorectal system, anoikis of stably transfected human colorectal carcinoma carcinomas.4 3422

Fig. 4. Tissue architecture model. AϪC, anoikis functions as a surveillance mechanism preserving the normal architecture of human colonic crypts. DϪG, CEA/CEACAM6-mediated inhibition of anoikis disrupts tissue architecture. A, CEA (denoted as thick dark lines) and CEACAM6 are apically expressed in the top third of normal human colonic crypts (17). To simplify the model, only CEA is indicated. B, in normal crypts, colonocytes that have lost contact with the basement membrane (ECM) undergo anoikis and are unable to survive out of the epithelial monolayer plane, thus (C) tissue architecture is preserved. D, unlike the expression pattern in normal colonic crypts, CEA and CEACAM6 are overexpressed over the entire surface of malignant colonocytes (circles). E, a CEA/CEACAM6-overexpressing cell that has lost cell-ECM adhesion has a longer survival capacity and prolif- erates (F) out of the plane of the single columnar epithelium, disrupt- ing normal tissue architecture and inhibiting cellular differentiation. The latter event, together with other genetic lesions, will contribute to tumor formation and progression (G).

CEACAM6-overexpressing SW1222 cells and CEA/CEACAM6- the number of cells in the aggregates. This is not the case, however, overexpressing Caco2 cells were 2- and 4-fold less prone to undergo because CEACAM1 and NCAM-125 are also intercellular adhesion anoikis, respectively, than transfectants expressing CEACAM1 or molecules, and cells expressing them showed the same number and ⌬NCEA or cells transfected with the vector alone (P Ͻ 0.01; Fig. 3). size of cellular aggregates as CEA/CEACAM6-expressing cells (see CEACAM6 alone also inhibited anoikis (P ϭ 0.01) of transfected “Materials and Methods”) but no inhibition of anoikis. We suggest Caco-2 cells (Fig. 3F). Transfected Caco-2 cells overexpressing only that intercellular interactions could nevertheless play a role in inhib- CEA could not be tested because of a pronounced tendency to lose iting anoikis by fostering clustering of CEA/CEACAM6 molecules by cell surface CEA expression during culture (data not shown). These both antiparallel and parallel binding on the cell surface, thus ampli- results show that deregulated overexpression of CEA and CEACAM6, fying a CEA/CEACAM6-mediated antiapoptotic signal. In support of but not CEACAM1, can not only disrupt cellular polarization and this suggestion, the deletion mutant ⌬NCEA, which is defective in 4 tissue architecture of human epithelial colonocytes but can also mediating both intercellular adhesion (12) and the myogenic differ- inhibit their architectural quality control mechanism, anoikis. entiation block (6), is also defective in inhibiting anoikis. CEACAM1 overexpression in L6 and Caco-2 transfected cells did Discussion not inhibit anoikis. These results are consonant with the observation The observed inhibition of a quality control mechanism preserving that CEA and CEACAM6 cell surface expression is up-regulated in tissue architecture by CEA/CEACAM6 cell surface overexpression many cancers, whereas CEACAM1 expression is down-regulated (see has important implications for the role of these molecules in human “Introduction”). L6 myoblasts expressing GPI-anchored NCAM-125 cancer, especially in light of the fact that so many cancers show also underwent anoikis as readily as parental and CEACAM1- overexpression of these CEA family members. The question of mech- expressing L6 myoblasts. This result suggests that the CEA/ anism arises. L6, MDCK, SW1222, and Caco-2 cells formed aggre- CEACAM6 inhibitory function is specific and is not an effect of GPI gates when cultured in suspension on PolyHEMA-coated surfaces anchorage per se. CEA/CEACAM6 inhibition of anoikis seems to be (data not shown). Apoptotic cells were detected in these cellular specific for this type of apoptosis because CEA expression on the aggregates, but most of the apoptotic cells detached from them. In surface of L6 myoblasts does not inhibit v-myc-induced apoptosis (7). ␣ ␤ contrast, most of the cells that survived anoikis were found in the The 5 1 integrin receptor, when bound to fibronectin, triggers a aggregates, suggesting that cell-cell interactions could provide a sur- survival signal in many tissue culture cell lines (10). In the absence of ␣ ␤ vival signal. This phenomenon has been observed before in 3T3 cells the 5 1 integrin/fibronectin interaction, many cell types undergo that become resistant to anoikis in the presence of insulin-like growth anoikis. We have recently demonstrated that CEA/CEACAM6 ex- factor I (18). This raises the question of whether the intercellular pression on the surface of L6 myoblasts modifies cell adhesion to ␣ ␤ adhesion activity of molecules such as CEA and CEACAM6 could fibronectin due to a change in the functional status of the 5 1 indirectly induce resistance to anoikis as a consequence of increasing integrin receptor.5 The antiapoptotic intracellular signals generated by 3423

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2000 American Association for Cancer Research. CEA INHIBITS ANOIKIS ␣ ␤ CEA/CEACAM6-mediated activation of the 5 1 integrin receptor CEA Family: Basic and Clinical Perspectives, Vol. 5, pp. 57–71. Amsterdam: are currently under investigation. Harwood Academic Publishers, 1998. 6. Eidelman, F. J., Fuks, A., DeMarte, L., Taheri, M., and Stanners, C. P. Human We hypothesize that the CEA/CEACAM6-mediated inhibition of carcinoembryonic antigen, an intercellular adhesion molecule, blocks fusion and anoikis could contribute to the disruption of normal tissue architecture differentiation of rat myoblasts. J. Cell Biol., 123: 467–475, 1993. that accompanies malignant transformation (Fig. 4). We propose that 7. Screaton, R. A., Penn, L. Z., and Stanners, C. P. Carcinoembryonic antigen, a human tumor marker, cooperates with Myc and Bcl-2 in cellular transformation. J. 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Cosme Ordoñez, Robert A. Screaton, Chris Ilantzis, et al.

Cancer Res 2000;60:3419-3424.

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