Published October 15, 1991 A Role for the E-Cadherin Cell-Cell Adhesion Molecule during Tumor Progression of Mouse Epidermal Carcinogenesis Pilar Navarro,* Manuel Gómez,* Angel Pizarro,t Carlos Gamallo,§ Miguel Quintanilla,* and Amparo Cano* * Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientfficas, Departamento de Bioqufmica, Facultad Medicina, Universidad Autónoma de Madrid 28029 Madrid, Spain; t Servicio de Dermatologfa, Hospital La Paz, Madrid, Spain; and § Servicio de Anatomfa Patol6gica, Hospital La Paz, Facultad Medicina, Universidad Autónoma de Madrid, Madrid, Spain Abstract. The expression of the cell-cell adhesion observed in spindle cell carcinomas induced by CarB molecules E- and P-cadherin has been analyzed in or CarC cells. P-cadherin protein was detected in all seven mouse epidermal keratinocyte cell lines repre- cell lines that exhibit an epithelial (MCA3D, AT5, PDV, sentative of different stages of epidermal carcinogene- and PDVC57) or epithelioid (HaCa4) morphology, as Downloaded from sis. An inverse correlation between the amount of well as in nude mouse tumors, independent of their E-cadherin protein and tumorigenicity of the cell lines tumorigenic capabilities. However, complete absence has been found, together with a complete absence of of P-cadherin was observed in the fibroblast-like cells E-cadherin protein and mRNA expression in three car- (CarB and CarC) and in spindle cell carcinomas. The cinoma cell lines (the epithelioid HaCa4 and the introduction of an exogenous E-cadherin cDNA into fibroblastoid CarB and CarC cells) . A similar result HaCa4 cells, or reactivation of the endogenous jcb.rupress.org has been detected in tumors induced in nude mice by E-cadherin gene, leads to a partial suppression of the the cell lines, where induction of E-cadherin expres- tumorigenicity of this highly malignant cell line. These sion takes place in moderately differentiated squamous results suggest a role for E-cadherin in the progression cell carcinomas induced by HaCa4 cells, although at to malignancy of mouse epidermal carcinogenesis. much lower levels than in well-differentiated tumors They also suggest that the loss of both E- and P-cad- induced by the epithelial PDV or PDVC57 cell lines. herin could be associated to the final stage of carcino- on May 4, 2010 Complete absence of E-cadherin expression has been genesis, the development of spindle cell carcinomas. ELL adhesion mechanisms play a major role in vital level (Nose et al., 1987; Hatta et al., 1988; Ringwald et al., processes such as embryogenesis, tissue and organ 1987), but distinct immunological properties and binding pattern formation and maintenance of specific tissue specificities (Nose et al., 1988; Miyatani et al., 1989). The Carchitecture (Edelman, 1985 ; Thiery et al., 1982) . Calcium- pattern ofexpression and binding specificities ofthe different dependent cell-cell adhesion molecules are one ofthe major subfamilies are consistent with the proposed role of these components involved in the various processes of intercellular molecules in the segregation of differentiating tissues during adhesion (Takeichi et al., 1985) . In vertebrates, calcium- embryogenesis and in the morphogenetic processes involved dependent cell-cell adhesion molecules identified so far con- in the generation of an organism (Takeichi, 1988, 1990, stitute a family of transmembrane glycoproteins, of 119-135 1991). kD, for which the general term cadherins has been proposed During tumorigenesis, profound alterations in intercellu- (see Takeichi, 1988, 1990 for reviews) . lar and cell-to-substratum interactions take place (for re- The different members of the family, identified in various views, see Hynes, 1989 ; Parish et al ., 1987) . Such altera- tissues and species, can be grouped in three distinct catego- tions are particularly evident in tumorigenesis of epithelial ries, exemplified in the mouse by (a) N-cadherin (Hatta and tissues, where the malignant cells have to pass through the Takeichi, 1986; Hatta et al., 1985), present mostly in neural basement membrane barrier in order to invade and metasta- tissues ; (b) E-cadherin (identical to uvomorulin) (Yoshida- size other tissues. Even before invading adjacent tissues, the Noro et al., 1984; Hyafil et al., 1980; Peyrieras et al., 1985), malignant cells loosen their adhesion to their original neigh- expressed in epithelial tissues; and (c) P-cadherin, present bors. The molecular mechanisms underlying such altera- both in epithelial and nonepithelial tissues with major preva- tions are largely unknown and only recently have begun to lence at the placenta (Nose and Takeichi, 1986) . All mem- be investigated. Among them, changes in cell-cell interac- bers of the family present strong homology at the amino acid tions mediated by the cadherin family of molecules in rela- © The Rockefeller University Press, 0021-9525/91/10/517/17 $2 .00 TheJournal ofCell Biology, Volume 115, Number2, October 1991517-533 517 Published October 15, 1991 tion to malignancy have received increasing interest in the role in the further progression towards poorly differentiated last few years. Studies on tissue sections of various human spindle cell carcinomas. carcinomas have shown that E-cadherin is expressed in both differentiated and dedifferentiated tumors (Eidelman et al., Materials and Methods 1989; Shimoyama et al., 1989), although lower levels appear to be present in poorly differentiated carcinomas (Shimo- CellLines and Growth Conditions yama et al., 1989) . A recent study on E-cadherin expression in several human carcinoma cell lines indicates that acquisi- Theorigin of the mouseepidermal keratinocyte cell lines used in the present study (MCA3D, AT5, PDV, PDVC57, HaCa4, CarB andCarQ is described tion ofthe invasion properties ofdedifferentiated fibroblastoid elsewhere (Dfaz-Guerra, M., C. Bauluz, S. Haddow, J. L. Jorcano, A. carcinoma cells is associated with the loss of E-cadherin ex- Cano, A. Balmain, and M. Quintanilla, manuscript submitted for publica- pression (Frixen et al., 1991). These observations, together tion) and is briefly summarized in Thble I. All of the epidermal cell lines with previous similar results with H-ras-transformed MDCK were grown in HAM F-12 medium supplemented with a complete set of amino acids (Gibco Ltd ., Paisley, Scotland), 5 or 10% FCS and the anti- cells (Behrens et al., 1989), support a role for E-cadherin biotics amphotericin B (2.5 /ig/ml), ampicillin (100 kg/ml), and gentamicin as an antiinvasive molecule, and as a marker ofthe differenti- (32 pg/ml) (Sigma Chemical Co., St. Louis, MO). Mouse 10TV2 fibro- ation grade of epithelial carcinomas (Frixen et al., 1991) . blasts were cultured in Dulbeccds MEM supplemented with 10% FCS The expression of P-cadherin has so far only been reported and antibiotics. Cells were grown at 37°C in a humidified 5% COZ at- in one study ofhuman epithelial carcinomas (Shimoyama et mosphere . al., 1989). Our laboratory is investigating the role ofE- and P-cadherin 7Mnsfection Procedure during the tumorigenic process ofmouse epidermal carcino- HaCa4cells werecotransfected with the pBATEM-2 (Noseet al., 1988) and genesis. Both cadherins are the only members ofthe calcium the pSV2neo vectors (Southern and Berg, 1982) by the calcium-phosphate by Nose . (1988) . dependent cell-cell adhesion molecules expressed in normal coprecipitation method essentially as described et al Transfection with 1 jug of pBATEM-2 and 0.1 ug of pSV2neo was carried mouse skin with P-cadherin restricted to the proliferative out in Dulbeccds MEM supplemented with 10% FCS. After 18 h, theDNA- Downloaded from basal layer and E-cadherin expressed in the basal and supra- containing medium was replaced by fresh HAM F-12 containing 10% FCS, basal layers (Nose and Takeichi, 1986). The model of two- and 24 h later selection with 0.4 mg/ml G418 (Gibco Ltd .) was begun. stage mouse skin carcinogenesis is based in the progressive Resistant colonies were isolated and tested for E-cadherin expression by im- munofluorescence staining and Western immunoblotting. Positive cells induction of premalignant papillomas and invasive carci- were subcloned and used for further studies. The pBATEM-2 vector was nomas by treatment with initiating and promoting chemical kindly provided by M Takeichi (University of Kyoto, Kyoto, Japan), and the agents (Boutwell, 1974; Hecker, 1978; Hennings et al., pSV2neo vector by J. L. Jorcano (CIEMAT, Madrid, Spain) . 1985). A variety ofcell lines can be derived from tumors and by in vitro transformation of primary epidermal cells, 7lumorigenicity Assays in Nude Mice jcb.rupress.org providing the opportunity of an in vitro approach to the For the tumorigenicity test, cells were collected and washed in PBS andsub- mouse skin carcinogenesis system (for review, see Balmain cutaneously injected in male nu/nu mice, 8-10 wk old . 1 x 106 cells were and Brown, 1988). used for a single injection. In different experiments, mice were either indi- In this study, we have analyzed the expression of P- and vidually injected with the same cell line at three different sites (the two flanks and the neck) or double injected with distinct cells on the left and E-cadherin in seven mouse epidermal keratinocyte cell lines, right flanks. Animals were observed for tumor formation and sacrificed on May 4, 2010 ranging from immortalized nontumorigenic to highly malig- when tumors reached a size of ti 2 cm . nant carcinoma-derived cells, which express morphological phenotypes in culture