Extracellular Matrix Receptors and Mouse Skin Carcinogenesis: Altered Expression Linked to Appearance of Early Markers of Tumor Progression1

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(CANCER RESEARCH 52. 2966-2976, May 15, 1992] Extracellular Matrix Receptors and Mouse Skin Carcinogenesis: Altered Expression Linked to Appearance of Early Markers of Tumor Progression1

Tamar Tennenbaum,2 Stuart H. Yuspa, Atul Grover, Vincent Castronovo, Mark E. Sobel, Yoshihiko Vainada, and Luigi M. De Luca Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology [T. T., S. H. Y., A. G., L. M. D.J and Laboratory of Tumor Pathology /K C, M. E. S.J, National Cancer Institute, and Laboratory of Developmental Biology, National Institute of Dental Research [Y. Y.], National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT Interaction of basal cells with components of the ECM3 is modified by the composition of the matrix and the expression Interaction of cells with the basement membrane is important for cell of several ECM cell surface receptors (7-9). Laminin is a major proliferation and differentiation. Disruption of the basement membrane glycoprotein of basement membranes, and in the epidermis is is an early event during progression of benign tumors to cancer. Using the techniques of immunohistochemistry and immunofluorescence, we found in the most proximal subepidermal region adjacent to show that cell-matrix interactions via the cell surface integrin receptors the basal cells (10). Laminin is composed of 3 major polypeptide a3/31, aS/Ã®l, o6/S4, the M, 67,000 laminili receptor (67LR) laminin- chains that contain multiple active sites (11-14). In vitro, many binding protein, and the secreted matrix protein laminin are strictly cell types are influenced by interaction with laminin, but the regulated during differentiation of mouse epidermis. While a6ÃŸ4and responses are complex and include stimulation of cell attach a5/31 are polarized to the basal surface of basal cells in contact with the ment, spreading, and migration (8, 14) as well as stimulation basement membrane, a3ÃŸl and the non-integrin 67LR are primarily of tumor cell invasion and metastasis (15, 16). Depending on detected in the cell periphery of suprabasal cells, where cell to cell the cell type, laminin can modify cell growth and differentiation contacts are found. Sequential changes in expression of matrix receptors (17, 18). occur following multistage Carcinogenesis of mouse skin. In an analysis of benign and malignant skin tumors induced by chemical carcinogens or The complex response to laminin by various cell types could oncogene transduction, we found that a3ÃŸl and aSÃŸlas well as the non- be due to specific interactions of active sites on laminin with integrin 67LR are sequentially down-regulated in the progression from different cell surface receptors (12, 13, 19). The integrins are a benign to malignant, while a6ÃŸ4is the predominant receptor expressed class of glycoprotein heterodimers that are thought to act as in the carcinomas. Tumor expression of a6ÃŸ4 is not polarized and is mediators of laminin function as well as mediators for the dissociated from its colocalized normal partner huilons pemphigoid an effects of other ECM molecules. Each integrin molecule is tigen, which remains restricted to the basement membrane. The changes composed of a- and 0-subunits that span the plasma membrane in matrix receptors are linked to appearance of keratin 13 in suprabasal and interact with the matrix extracellularly and the cytoskeletal regions, but always in a6ÃŸ4negative cells. The predominance of a6ÃŸ4in network intracellularly (12, 19, 20). For keratinocytes and the proliferating cells during progression is associated with decreased several other cell types, Â«3/31integrin serves as a promiscuous expression of keratin 13 in carcinomas. These results suggest that matrix receptor for laminin, fibronectin, and collagen type IV (21-23); interactions with its receptors are important determinants of ordered differentiation in normal skin and show characteristic alterations during u l .il and ii2.il integrins are active as laminin and collagen type Carcinogenesis that parallel changes in differentiation of the tumors. IV receptors (24, 25) and are also present in keratinocytes (4, 26). a6ÃŸland Â«6/34integrins have been identified as laminin receptors (27, 28), although only Â«6/34has been detected in INTRODUCTION keratinocytes (22, 23, 27, 29). Studies on integrin localization Cell interaction with the basement membrane is important in human skin reveal 2 distinct patterns of expression. The a6 to normal skin proliferation and differentiation. In normal skin, integrin subunit, which conjugates with the 04 polypeptide in proliferation occurs only in the basal cell layer that is in contact epithelial cells, is expressed polarized in the basal surface of basal cells in a classical cell-substrate distribution (4, 30-32). with the basement membrane (1, 2). Differentiation is linked to the loss of proliferation, detachment of the basal cells from However, in developing fetal skin (33), Â«6and ÃŸ4subunits the basement membrane, and migration into suprabasal layers. could be detected in lateral and apical surfaces of basal cells This regulation is altered in skin tumors where basal-like cells and suprabasal layers (33). The 01 integrins in epidermis, including Â«3/31and Â«2/31,are distributed in cell-cell contacts proliferate in several layers disconnected from the basement membrane (1,2). This change in the regulation of proliferation surrounding the entire plasma membrane on both basal and occurs early in Carcinogenesis since it is recognized in benign suprabasal layers (22,23,34). This nonpolarized and suprabasal tumors where the basement membrane is still intact (3). During distribution of Â«3/31andÂ«2/31integrins suggests a role for these receptors in cell-cell interactions in addition to cell-substrate the progression of benign tumors to a malignant phenotype, the basement membrane is disrupted, and many proliferating adhesion. Several non-integrin laminin binding proteins have also been cells invade into the stroma and migrate to the surrounding tissues (4-6). identified in various cell types (13, 35, 36). Among these is the 67LR, whose expression has been linked to neoplastic progres Received 12/4/91; accepted 3/10/92. sion and metastasis in certain epithelial tumors (37, 38). This The costs of publication of this article were defrayed in part by the payment receptor is linked to Ca2+-induced differentiation of keratino of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cytes in culture (39). ' This work was supported by a grant from Johnson & Johnson, Consumer In an effort to evaluate the interaction of laminin with epi Products corporation. T. T. is the recipient of a fellowship from the Israel Cancer dermal cells during Carcinogenesis, we have studied the distri- Research Fund and the European Organization for Research and Treatment of Cancer. 2To whom requests for reprints should be addressed, at National Cancer 3The abbreviations used are: ECM, extracellular matrix; BPA, bullous pem Institute. Laboratory of Cellular Carcinogenesis and Tumor Promotion, Building phigoid antigen; 67LR, M, 67,000 laminin receptor; GGT, -y-glutamyl transpep- 37, Room 3B25, Bethesda, MD 20892. tidase; PBS, phosphate-buffered saline. 2966

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1992 American Association for Cancer Research. ECM RECEPTORS, KERATIN 13, AND TUMOR PROGRESSION bution of laminili and several laminin receptors in normal skin kit (Vector, Burlingame, CA). Peroxidase staining was developed by incubation of sections with 3,3'-diaminobenzidine (Kodak, Rochester, and benign and malignant skin tumors. Using chemically in duced papillomas and carcinomas (40), as well as tumors pro NY). Sections were counterstained with contrast green (KPL, Bethesda, duced by introduction of specific oncogenes into normal kera- MD). Immunofluorescence. Five-^m serial frozen sections were obtained tinocytes (41), we show that the distribution of laminin and laminin receptors is strictly regulated during differentiation of from newborn mouse skin and tumor tissues. After rinsing with PBS (pH 7.6), the sections were incubated with mixtures of the primary normal skin. Furthermore, characteristic changes occur in the antibodies from different species in the appropriate dilutions overnight expression of laminin and laminin receptors during carcinogen- at 4Â°C.After incubation, the sections were washed twice for 10 min esis, and these changes coincide with the appearance of other with PBS and incubated with biotinylated secondary antibodies for 20 aberrant markers of malignant progression. These results indi min. Biotinylated secondary antibodies were removed with PBS washes, cate that early changes in the distribution of laminin receptors and sections were exposed to fluorescein isothiocyanate-conjugated may serve as predictive markers of malignant progression in secondary antibodies mixed together with streptavidin Texas red for 20 skin carcinogenesis. min to visualize 2 antigens simultaneously. The sections were rinsed thoroughly with PBS and examined with a Zeiss Axiophot fluorescence microscope equipped with filter for differential detection of fluorescein MATERIALS AND METHODS isothiocyanate and Texas red. Tissue Specimens. Newborn mouse skin was obtained from 2-day- â€¢¿y-GlutamyltranspeptidaseAnalysis.-y-Glutamyltranspeptidase analy old BALB/c mice. Tumor tissues were obtained from skin papillomas sis was performed according to the method of Ruttenberg et al. (50). and carcinomas induced in SENCAR mice by 7,12-dimethyl- The analysis was done on frozen tumor sections with ./V-(7-l-glutamyl)- benz(fl)anthracene initiation and promotion with 12-O-tetradecanoyl- 4-methoxy-2-naphthylamide (Polysciences Inc.) Fast Blue BB salt phorbol-13-acetate (40, 42) for up to 40 weeks. Forty tumors from (Sigma) and glycylglycine (Sigma). Frozen sections of mouse kidney several experiments were analyzed. Tumor tissues were also obtained tissue were used as positive controls. by harvesting papillomas and carcinomas evolving 4 weeks after grafting of oncogene-transduced cultured cells. Approximately 20 tumors were analyzed. Primary mouse keratinocytes were prepared from newborn RESULTS BALB/c skin as described (43) and cultured for 3 days in Eagle's minimal essential medium containing 0.05 mM Ca2+.Cells were infected For analysis of laminin and laminin receptors during tumor with a defective retroviral vector containing a neomycin resistance gene progression, we have used 2 comparable but distinct model (neor), a \-ras"' gene, or a \-fos gene as described previously (41, 44). systems. The introduction of the v-rasHa and \-fos oncogenes Five days postinfection, cells were trypsinized and combined with via retroviral transduction into cultured primary keratinocytes dermal fibroblasts for grafting onto the backs of nude mice, as described (41, 44). Four weeks after grafting, keratinocytes with a neo' gene was used to produce either benign or malignant tumors (41) in produced normal skin grafts, v-ras?1"keratinocyte grafts produced pap skin grafts. An alternative approach was the classical produc illomas, and keratinocytes infected with both v-ras"* and \-fos retrovi- tion of chemically induced papillomas and carcinomas using ruses developed carcinomas. Results were similar with cells derived topical 7,12-dimethylbenz(a)anthracene as the initiator and from either BALB/c or SENCAR mice in grafting studies or with multiple treatments with 12-0-tetradecanoylphorbol-13-acetate chemically induced tumors (45). as the promoter (42). Both systems resulted in tumors that Antibodies. Rabbit anti-human and rabbit anti-rat laminin antibody express an activated rasHa gene. Primary mouse keratinocytes (1:4000) were purchased from Telios, La Jolla, CA. Rabbit anti-human containing the transduced v-ras"" gene formed papillomas Â«s(1:200) antibody raised to a specific peptide corresponding to the carboxy terminus of the human Â«5subunit, and rabbit anti-chicken Â«3 within a few weeks of grafting. Both oncogene-induced papil antibody (1:200) raised against a peptide of the carboxy terminus of lomas and the chemically induced papillomas collected from chicken a3, were a gift of Dr. R. E. Hynes (46). The rat monoclonal mice 10 weeks after initiation and promotion were well orga anti-mouse a6 (GOH3) 1:500, which recognizes both a6ÃŸ4and a6/31 nized squamous lesions (Fig. IA) with an intact and clearly complexes (27), was purchased from Amac, Westbrook, ME. Rabbit defined basement membrane. Papillary projections were sur anti-human 01 antibody (1:500) raised to a synthetic peptide from the rounded by a vascular stroma, and a progressive pattern of carboxy terminus of the human Q\ integrin subunit was purchased from Chemicon, Temecula, CA (47). Goat anti-hamster Â«501(1:500), which suprabasal differentiation culminated in extensive cornified areas (Fig. IA). The introduction of both v-fos and \-ras"" genes recognizes both Â«5and ÃŸlheterodimers, was a gift from Dr. R. L. Juliano (48). Monospecific keratin antibodies to keratin 14 (1:1000), into primary mouse keratinocytes resulted in large malignant keratin 13 (1:500), and keratin 1 (1:500) were prepared in rabbits tumor grafts that were invasive and metastatic. Histologically, against synthetic peptides corresponding to unique sequences at the these tumors were disorganized, poorly differentiated carcino carboxy terminus of mouse keratins (49). Rabbit antibodies to the mas (41). Chemically induced squamous cell carcinomas were precursor of the 67LR ( 1:200) were prepared against synthetic peptides heterogeneous, demonstrating several stages of malignant pro derived from the cDNA (36, 37). Bullous pemphigoid serum (1:500) was provided by Dr. J. R. Stanley (3). All antibodies react with the gression. In the same tumor tissue, and often in the same tissue sections, areas with well-differentiated or dysplastic papillom- murine homologue of the antigen of interest. Peroxidase Antiperoxidase Staining. Five-^m frozen sections were atous morphology (Fig. IB) were adjacent to well-differentiated rehydrated for 15 min in PBS. Endogenous peroxidase activity was (Fig. 1C) or poorly differentiated carcinomatous regions (Fig. blocked with 0.3% hydrogen peroxide in methyl alcohol. For reducing ID). Mixed histology was also typical of chemically induced nonspecific binding, tissue sections were incubated with goat serum for papillomas collected after 40 weeks of promotion where well- 20 min or with rabbit serum (for goat anti-a501 antibody) followed by organized and severely dysplastic but noninvasive benign squa incubation with primary antibodies or the IgG control (for GOH3) overnight at 4"C in 12% bovine serum albumin in PBS. The immunol- mous areas were noted on the same section. Comparative ocalization was detected by using biotinylated goat anti-rabbit serum analysis of changes in the 2 model systems indicated that the or biotinylated goat anti-rat serum (for GOH3) or biotinylated rabbit markers under evaluation changed according to the histological anti-goat for a5ÃŸl antibody or biotinylated rabbit anti-human for progression and differentiation of the tumor rather than to the huilons pemphigoid antibody and the Vectastain avidin-biotin complex carcinogenic process by which the tumors were produced. 2967

Fig. 1. HistodifTerentiation of skin tumors derived by chemical induction. A, chemically induced papilloma, 10-week TPA promotion (x 400); B, chemically induced papilloma, 40-week TPA promotion (x 400); C, well differentiated carcinoma (x 200); D, poorly differentiated carcinomas (x 200).

Laminin Distribution component of the cell membrane localized laterally and apically between cells. In vitro studies indicate that 67LR is membrane- In newborn mouse skin, laminili staining was confined to the associated in mouse keratinocytes induced to differentiate by basement membrane of the epidermis and the outer root sheath calcium (39). In papillomas, the distribution of 67LR was of the hair follicles as well as the endothelial basement mem similar to that in newborn skin (Fig. 3/f), with intense im brane of blood vessels located in the dermis (Fig. 2A). In chemically induced and v-ras"" (Fig. 2B) papillomas, the lami- munostaining in the more differentiated layers of the tumor, corresponding to upper spinous and granular cells. Carcinomas nin pattern was very similar to normal skin. The laminin of well-differentiated carcinomas surrounded spheroids of invasive had lower amounts of immunodetectable 67LR, depending on tumor cells in a basement-membrane-like pattern and persisted the differentiation stage of the tissue. In poorly differentiated in the endothelium of developing capillaries (Fig. 1C). In poorly carcinomas, antigenic 67LR was not detected (Fig. 3C), whereas in well-differentiated carcinomas the antigen was con differentiated carcinomas, however, laminin was detected throughout the tumor tissue surrounding epithelial cell aggre fined to the differentiating regions. gates and developing capillaries (Fig. 2D). t*3/81.The distribution of integrin a3ÃŸ\was evaluated using an antibody against the a3 subunit. Primarily Â«3/31was ex Distribution of Laminin Receptors pressed in the differentiating cells of normal epidermis, similar 67LR. Very little 67LR was present in the basal surface of to that of 67LR (Fig. 3D). In addition, Â«3/31wasdetected along basal cells in normal epidermis, the region in contact with the basal side of cells in contact with the basement membrane laminin (Fig. .11), although it was detected in lateral borders of and surrounding normal basal cells. In well-differentiated pap occasional cells in the basal layer. However, the major expres illomas, n3.il was present in the basal surface of basal cells as sion of 67LR was suprabasal with the greatest intensity in the in normal skin, where it outlined the basement membrane zone spinous and granular cell layers (Fig. 3/4). The peripheral (Fig. 3E). There appeared to be a reduction in staining around location of the staining suggested that the protein might be a the remainder of the basal cells and in the lowest suprabasal 2968

Fig. 2. Immunohistochemical detection of laminin in normal epidermis and in benign and malignant skin tumors. A. normal epidermis; B, chemically induced papilloma; <". chemi cally induced well differentiated carcinoma; /), oncogene transformed poorly differentiated carcinoma. Note the basement membrane as sociated laminin staining indicated by E in B of the papilloma tissue, and surrounding the invading carcinoma tissue (E in ('). A-D, X 200. E, epithelial cells in tumors; S, stroma.

t*% â€¢¿Ã•%4,4>r-it-^:-% 3Â¡i

cell layers, but the staining intensity increased with advancing nant cells throughout the tissue, distributed in areas rich with differentiation state. In more dysplastic tumors, a3ÃŸlwas es laminin staining in doubly stained sections (Fig. 5F). sentially absent from most cell layers and only the most super BPA. BPA is a transmembrane protein that colocalizes with ficial layers expressed this antigen. Carcinoma tissue (Fig. 3F) Â«6/34in the hemidesmosomes of normal skin (30-32) and showed weak staining even in well differentiated areas, and appears as part of distinct attachment complexes of basal Â«3/ilwas absent in less differentiated regions. Similar results keratinocytes (4, 22, 26). Interestingly, in areas of papillomas were obtained using an antibody against ÃŸl,the 0-subunit of where Â«6/34extended to suprabasal layers (Fig. 5E), BPA the a3ÃŸl heterodimer that recognizes ÃŸlsubunits linked to remained confined to the basal surface of the basal layer. In heterogeneous a-heterodimers (data not shown). carcinomas, BPA was displayed diffusely throughout the tissue oc6ÃŸ4.Antibody to <>6integrin stained only the basal surface limited to areas of laminin-rich basement membrane surround of the basal layer in newborn skin (Fig. 3G). This highly ing epithelial cell aggregates (data not shown). BPA and Â«604 polarized distribution colocalized with laminin at the basement were not necessarily colocalized in undifferentiated carcinoma membrane but did not extend along the full length of the hair sections. follicles as did laminin (Fig. 2A). Concurrent with laminin a5/31. Similar to Â«6/34,the a5/31 integrin is polarized in the expression in papillomas, a6ÃŸ4integrin appeared specifically basal surface of the normal skin adjacent to the basement localized at the basal surface of the basal cells in well-differen membrane (data not shown). However, a5ÃŸl remained re tiated areas (Fig. 3//). However, areas of focal extensions stricted to basement membrane-associated cells with a polarized surrounding basal and suprabasal cells were noted and involved distribution in hyperplastic skin or papillomas (Fig. 6, C and progressive numbers of cell layers as tumors advanced from D), whereas Â«6/34extended to suprabasal layers (Fig. 6, A and well organized to dysplastic papillomas (Figs. 3H and 4, A and B). In progressing papillomas and in carcinomas, the expression B). As the number of layers expressing a6 increased, the char of a5ÃŸlwas low even in well-differentiated areas surrounded acteristic polarized staining coinciding with the basement mem by a basement membrane (data not shown). brane became less pronounced (Figs. 3H and 4B). When the Correlation of Changes in Surface Receptors to Other Markers expression of Â«6extended suprabasally from the basement of Neoplasia membrane, laminin remained confined to the basement mem brane regions as shown by double staining and immunofluores- To determine the significance of changes in cell surface cence (Fig. 5D). Invasive regions of carcinomas (Fig. 37) ex antigens to tumorigenesis, we studied a number of stage-specific pressed a6ÃŸ4integrin at the cell surface of most of the malig phenotypic markers identified during the evolution of skin 2969

Fig. 3. Immunohistochemical detection of surface receptors in normal skin (A, D, and G), papilloma (B, E, and //), and carcinoma (C, F, and /) tissue. Staining with polyclonal antibody to the 67LR (A-C) or a3 (D-F) or monoclonal antibody to Â«6(G-/). A, D, G, and /, x 400; A, Z.,Â£,F, and //, x 200. Dashed lines underline part of the tissue basement membrane. 2970

f B

â€¢¿ â€¢¿v

Fig. 4. Expression of a(s integrin subunit (.Ã•andli) and differential keratin distribution: keratin 13 (C, D, and C), keratin 14 (Â£),and keratin 1 (f) in papillomas. Note the differ ential expression of Â«6and K13 (A, C, and B, D) in corresponding fields in serial sections of the same papilloma including K13-negative and K13-positive areas. Note the differential expression of keratins (E-G) in corresponding fields of the same papilloma section including K1- and K13-positive or K1-positive and K13- negative. Numbers in each panel, same field in serial sections.

tumors through the benign and malignant stages. Well-differ However, these regions never contained K13-positive cells, even entiated benign tumors express keratins Kl (Fig. 4F), K10, as in a6/34-negative areas (data not shown). well as K5 and K14 (Fig. 4Â£),but the suprabasal markers Kl Tumor progression has been associated with an increase in and K10 are lost early in malignant progression (51). Charac GGT activity in carcinomas (52, 53). In the several tumors that teristically, the suprabasal keratin 13 is detected as papillomas were analyzed, low GGT activity was detected in focal areas of become dysplastic and progress to cancer (49, 52). This keratin carcinomas with no correlation to the dysplastic appearance or of internal stratified epithelium is not expressed in normal or stage of histological conversion of the tumors (data not shown). hyperplastic skin. In benign tumors, the regional distribution No association was evident in the appearance of GGT-positive of K13 was closely correlated to the extension of a6/J4 beyond lesions and <*6ÃŸ4expressionor the appearance of keratin 13. the basement membrane zone, but distinctly different cells expressed each marker within the tumor focus (Fig. 4, A, C). DISCUSSION When K13 appeared in a typical pattern in the differentiated suprabasal layers, a6ÃŸ4was present in the proliferating basal Previous studies have documented the strict regulation of the and suprabasal layers below K13-positive cells. Double staining expression of specific cytoplasmic markers during epidermal immunofluorescence detection of a6ÃŸ4and K13 revealed a differentiation (54, 55). For example, keratins K5 and K14 are reciprocal association: as the expression of suprabasal Â«6/34 expressed predominantly in basal cells, whereas keratins Kl ceased, subsequent strata contained K13-expressing cells (Fig. and K10 are products of cells committed to migrate suprabas- 5, A-C). As more a6j34-positive cells developed in progressing ally. Even suprabasal markers are differentially regulated since papillomas and carcinomas, the expression of keratin K13 Kl and K10 are transcribed in spinous cells, while only cells in actually decreased and was only focally expressed in regions the more superficial living layers express filaggrin, loricrin, and that were negative for a6ÃŸ4expression (Fig. 5C). In hyperpro- epidermal transglutaminase (55, 56). We now show that cell liferative skin above carcinomas, but not in the adjacent normal surface matrix receptors such as Â«3/31,a5/31, and a6ÃŸ4inte- skin, a6|34 expression extended into the suprabasal layers. grins, and 67LR, as well as the matrix protein laminin, are also 2971

Fig. 5. Localization of a6ÃŸ4related markers by double staining with monoclonal rat antibody against a6 (red) and with antibodies from rabbit (green) against K13 (A-C) or laminin (D and F) or human anti-BPA (E) in normal skin (A) papilloma (B, D, and E) and carcinoma (C and F). Coexpression of both antigens appears as yellow staining. Note the uneven distribution of a6 in adjacent regions of the same papilloma (B). White dashed lines underline areas of basement membrane. Note that the overexposure in A is done for clarity of Â«6staining (red). The cornified layer is staining nonspecifically in yellow. tightly regulated during differentiation of mouse epidermis. integrin is not exclusively mediating the function of laminin in Furthermore, the distribution of these markers is sequentially epidermis. In addition to laminin, a3ÃŸ\integrin has binding altered in benign and malignant skin tumors concordant with activity for several other ligands including fibronectin, collagen the expression of cytoplasmic markers of neoplastic progres type IV, and collagen type I (21, 57). This integrin was shown sion. The pattern of alterations seems predictable as the changes to be localized with other integrins as a component of the focal were observed in tumors produced by chemical carcinogens or adhesion plaques in human keratinocytes (22), suggesting mul by direct transduction with specific oncogenes. The coexpres- tiple functions for this receptor in keratinocytes and other cell sion of markers in the normal skin phenotype does not predict types. Alternatively, a3/31 and other ÃŸlintegrins that appear in coordinate alterations during neoplastic development. Each cell-cell contacts could recognize laminin-related proteins in component of the matrix/matrix-receptors analyzed demon the intercellular spaces that are not detected by antibodies strated specific patterns of changes in the tumors. generated to whole laminin (58, 59). The predominantly supra- In normal mouse skin, laminin is confined to the basement basal distribution of a3ÃŸlis shared by the non-integrin 67LR. membrane in contact with epidermal cells (Fig. 2A) and follows In studies with cultured keratinocytes (data not shown), 67LR virtually the same distribution as the integrin a6ÃŸ4(Fig. 3G) antibodies recognized a A/r 37,000 protein in the cytosol of and a5ÃŸ\(data not shown), which are localized to the basal basal cells that is translocated to the cell membrane during surface of basal cells. In contrast to the polarized a6ÃŸ4/a5ÃŸl terminal differentiation induced by Ca2+ (39). The M, 37,000 integrins expressed in a classical cell-matrix distribution, a3ÃŸl polypeptide has been identified as the precursor of the 67LR in and 67 LR localized in the cell periphery both in basal cells and pulse chase studies (60) and contains a laminin-binding site (61, suprabasal cells of mouse skin. a3ÃŸiwas also detected in the 62). These results, combined with the in vivo data, suggest that basal surface of basal cells, but the major site of detection was 67LR at the cell surface has a differentiation-related function, between the cells in the suprabasal differentiating layers. In perhaps in maintaining cell-cell contacts or interacting with an contrast, in human skin, Â«3/31is predominantly expressed in intercellular matrix component. Its absence from the basal the basal cell compartment (4, 22, 26, 34). The distribution of surface of basal cells suggests it is not required for keratinocytes ÃŸ\ integrins in newborn mouse skin resembles the pattern to interact with laminin. However, we cannot rule out the observed in earlier development of fetal and neonatal human possibility that interaction with basement membrane laminin skins (4, 26). The absence of detectable laminin in the regions causes a change in the epitope and loss of 67LR antibody of intense Â«301localization in mouse skin suggests that this recognition. 2972

Fig. 6. Â»6expression extends to suprabasal layers whereas o5 remains polarized in the basal surface of the basal layer. nS (C and /' ) or Â»6(Aand B) subunits in hyperplastic skin (A and C) and papilloma (B and />).

A surface receptor that localizes with the laminin basement plasma membrane of the basal layer. During progression of the membrane is Â«6/34integrin. Although Â«6/34shares a common papilloma, a6ÃŸ4is expressed in cells several layers above the itti subunit with Â«6/31,only Â«6/31has been shown to bind with basement membrane that have acquired the capacity to prolif high affinity to laminin (27). The function of Â«6/34asa laminin erate away from the basement membrane zone. However, the receptor, however, has been supported by studies showing that process is not restricted to proliferation of the basal cells above antibodies to either Â«6or /Â¡'4subunits blocked attachment to the basal layer, since nonproliferative suprabasal cells in hyper- laminin in epidermal cell cultures as well as other epithelial plastic (but not neoplastic) epidermis also express this integrin. cells (22, 23, 33). Recent reports linking Â«6/34to BPA suggest It is possible that Â«6/34expression could be related to changes that Â«6/34could associate with BPA in the hemidesmosome to in motility of cells under conditions of high cell turnover, where form a complex with intracellular cytoskeletal components via transit time into the suprabasal layers is shortened. The change the extended cytoplasmic domain of ÃŸ4similar to integrins in Â«6/34in benign tumors is not necessarily associated with interacting with talin and vinculin in the focal contact (30, 32, other changes in basal cell matrix-related proteins, since lami 63). nin and BPA do not change distribution at this stage. Further Characteristic changes in the patterns of laminin, integrin more, the fibronectin receptor, Â«5/91,does not change its nor expression, and 67LR are associated with epidermal carcino- mal epidermal basal surface localization in the basal layer in genesis (summarized in Table 1). The changes are stage-specific benign and progressive papillomas and is reduced in carcino and consistent in tumors induced by chemicals or by direct mas. It is possible that the change in Â«6/34distribution is related introduction of oncogenes. In the early papilloma stage, Â«6/34 to growth factors in the tumor or hyperplastic environment, integrin loses its polarized distribution and surrounds the cell and studies to test this possibility are in progress. 2973

Table 1 Expression and localization of laminin, matrix receptors, BPA keratins, and GGT in normal epidermis and during tumor development Papilloma EpidermisBasal CarcinomaBasal layer layerDifferentiating layersDifferentiating Laminin (+)"Differentiating

layers layers layersDifferentiating 67LR (+)Differentiating (++)Differentiating

layersÂ«301 layers(++)Basal layersBasal (+)Basal

layerBasal layers(++)Basal layers (+++)(++++)Basal

layer layerDysplastic layers BPA (+)EarlyBasal <+) (++)

GGT

Differentiating layers Differentiating layers Focal Kl (+) (+/-)

Focal Differentiating layers K13 " The intensity of expression of the different markers in skin tumors is described relative to the expression in normal epidermis (first column). * (+/â€”),focal staining of single cells. ' (++++), extensive distribution throughout the tissue. '(-), absent.

In tumors, the extension of a6ÃŸ4expression into suprabasal Clonal lines with reduced expression of a5/31 in Chinese ham cells is linked to altered differentiation. Regions where this ster ovary cell cultures demonstrated increased tumorigenicity integrin is abnormal are frequently the site of aberrant expres in vivo (68). The importance of 67LR protein appears to vary sion of keratin 13, a marker of neoplastic progression (49, 52), in different types of tumors. The expression of this laminin which is not expressed in normal or hyperplastic skin (64). receptor was increased in colon and breast adenocarcinomas Similarly in these sites, the expression of the normal keratino- when compared to their normal tissue homologues in vivo (37, cyte differentiation markers Kl, a3/31, and 67LR are frequently 38). The results of the current report suggest that the opposite lost or greatly diminished. However, the aberrant markers are may be the case in squamous skin carcinomas, and changes in not found in hyperplastic skin where Â«6(34can also be supra- this protein may be cell type-specific during the process of basal, suggesting that other changes in tumor cells are required carcinogenesis. for the aberrant expression of K13. In tumors, K13 is only The predominant integrin expressed in skin carcinomas is expressed in cells superficial to those expressing a6/34. There o6j34. Expression of Â«6/34integrin has been observed in other is no obligatory link between the expansion of <*6/34integrin epithelial tumorigenic cell lines (24), while a6ÃŸlwas highly expression and K13 expression. Both in the tongue and in the expressed in non-epithelial tumorigenic cell lines (66). High forestomach epithelium, where keratin 13 is the normal cyto- expression of a6ÃŸ4was also found in human skin squamous keratin in stratified layers suprabasally, <>ti,i4integrin is ex cell carcinomas (29). In the skin carcinogenesis model, the pressed strictly in a linear pattern in the basal surface of the predominance of a6ÃŸ4positive cells associated with malignant basal layer.4 progression correlates with the diminished expression of K13 During the process of malignant conversion, laminin is de in more advanced and poorly differentiated tumors (Fig. 5, A- tected at the invading front of the malignant cells that protrude C). Taken together with the exclusion of K13 from a6ÃŸ4 into the stroma and also is abundant throughout the tissue of positive cells in the benign lesions, changes in matrix receptors less differentiated tumors (Fig. 2, C and />). The diffuse expres and matrix components during carcinogenesis could contribute sion of laminin in carcinoma tissue could influence the pres to morphological variations and aberrant expression of markers entation and interaction of different functionally active sites on in tumor cells. Conversely, the tightly regulated expression of laminin to the surface receptors on the malignant cells (58, 59). these receptors and matrix components in skin could be impor Furthermore, malignant epidermal cells are deficient in detect tant in signaling the expression of normal markers of able a3ÃŸland 67LR, particularly in less differentiated regions. differentiation. The loss of ÃŸlintegrins is not a general phenomenon in carcinogenesis, but both a3ÃŸ\and a5ÃŸlalterations have been ACKNOWLEDGMENTS seen in progression of other tumor cells (65, 66) including We would like to thank Drs. Ulrike Lichti, Adam Click, and Tonja human skin carcinomas (4). The influence of integrins on tumor Kartasova for helpful discussions and critical review of the manuscript. cell behavior has yielded ambiguous diverse results, possibly We are grateful to Margaret Taylor for skillful technical assistance in due to the common assessment of integrins in tumor cell lines preparing the manuscript. Special thanks to the photography within where the adaptation to an in vitro environment may alter the NIH for professional artwork. response. In a study where a5ÃŸlintegrin was overexpressed in cells, tumorigenicity was reduced upon grafting in vivo (67). REFERENCES I. Fukuda, M., Okamura, K., Rohrbach, R., HÃ¶hm.N., and Fujita, S. Changes 4 T. Tennenbaum, S. H. Yuspa, A. Grover, V. Castronovo, M. E. Sobel, Y. in cell population kinetics during epidermal carcinogenesis. Cell Tissue Yamada, and L. M. De Luca, unpublished observations. Kinet., //: 611-621, 1978. 2974

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1992 American Association for Cancer Research. Extracellular Matrix Receptors and Mouse Skin Carcinogenesis: Altered Expression Linked to Appearance of Early Markers of Tumor Progression

Tamar Tennenbaum, Stuart H. Yuspa, Atul Grover, et al.

Cancer Res 1992;52:2966-2976.

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