Modulating expression of LAMPs and ABH histo-blood group antigens in normal and neoplastic human skin

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1 DOI: /s Research article CEJMed 1(2) Modulating expression of LAMPs and ABH histo-blood group antigens in normal and neoplastic human skin Victoria S. Sarafian 1,DorianI.Dikov 2, Milen P. Karaivanov 3 1 Department of Medical Biology, Medical University of Plovdiv, Plovdiv-4000, Bulgaria 2 Service d Anatomie et de Cytologie Pathologiques, Centre Hospitalier de Lagny-Marne-La-Vallée, Lagny-sur-Marne-77495, France 3 Department of General and Clinical Pathology, Medical University of Pleven, Pleven-6500, Bulgaria Received 22 January 2006; accepted 13 April 2006 Abstract: Although the precise biological role of lysosomal membrane-associated glycoproteins (LAMPs) and ABH histo-blood group antigens (HBGAs) remains somewhat unclear, they are thought to be related to cell differentiation, cellular adhesion, and tumorigenesis. Here, we present the first comparative immunohistochemical study of both LAMPs and HBGAs in normal and neoplastic skin. Their localization is compared to that of high molecular weight cytokeratin and cytokeratin MNF 116. LAMPs and HBGA were differentially expressed in the normal stratified squamous epithelium, suggesting that they are involved in the initial steps of the differentiation process, whereas HBGAs are characteristic of terminal keratinocyte differentiation. No change in the reactivity for HBGA was detected in the stratified epithelium overlying squamous cell or basal cell carcinomas, whereas a considerable loss of LAMPs was detected. LAMPs were overexpressed in tumor cells, whereas HBGAs were lost in tumor zones of basocellular carcinomas. In spinocellular carcinomas, HBGAs were detected in tumor keratinocytes and in keratin pearls. These results provide new evidence for the differential expression of LAMPs and HBGAs in the normal stratified squamous epithelium, as well as the presence of a modulating reactivity in basocellular and spinocellular carcinomas, suggesting that these glycoproteins are involved in differentiation and tumorigenesis of human skin. c Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved. Keywords: Lysosomal membrane-associated glycoproteins (LAMPs), ABH histo-blood group antigens (HBGAs), skin, basal cell carcinoma, squamous cell carcinoma sarafian@abv.bg, v sarafian@yahoo.com

2 120 V.S. Sarafian et al. / Central European Journal of Medicine 1(2) Introduction Although human skin is well known to act as a barrier and sensory organ, increasing evidence suggests that it has additional, nonclassical functions, including the synthesis, processing, and metabolism of a range of structural proteins, glycans, lipids, and signaling molecules [1]. The ABH histo-blood group antigens (HBGAs) and the lysosomal membrane-associated glycoproteins (LAMPs) (LAMP-1 and LAMP-2) are glycoconjugates with a wide cellular distribution. These glycoproteins are thought to have diverse biological and pathological functions, but their precise roles have not yet been defined. LAMPs are transmembrane glycoproteins found predominantly in lysosomal membranes. LAMP-1 is a marker of differentiation and development [2]. Also, LAMPs are thought to participate in neoplastic progression [3, 4] and cellular adhesion [5]. In addition, HBGAs appear to participate in epithelial cell differentiation [6], intercellular communications [7], tumorigenesis [8], histogenesis, and evolution [9]. Abnormal epidermal proliferation is characteristic of a number of disorders, including the two most common epithelial cell-derived neoplasms basal cell and squamous cell carcinomas. Whereas both clinicopathological entities arise from epidermal keratinocytes, the natural history of their development, their molecular pathogenesis, and their clinical courses seem to be significantly different [10]. In the current study, we hypothesized that LAMPs and HBGAs participate in the differentiation of normal human epidermis, as well as in the pathological changes associated with neoplastic processes in the skin. We present novel evidence that the expression of both glycoproteins differs between normal keratinocytes and basal and squamous cell carcinomas. 2 Materials and methods 2.1 Specimens Biopsy specimens were obtained by excision from patients (age, 53 to 82 years) with squamous (spinocellular; n=18) or basal cell (basocellular; n=21) carcinomas. The tissue samples were examined by the Service d Anatomie et Cytologie Pathologiques, Centre Hospitalier, Lagny-Marne-La-Vallée, France and by the Department of General and Clinical Pathology, Medical University of Pleven, Bulgaria. None of the patients had received chemotherapy or radiotherapy. Normal skin from age-matched individuals was obtained from discarded pathological specimens. Informed consent for use of the tissue samples was obtained in accordance with the Helsinki Declaration. Prior to surgery, the blood group phenotypes of all patients were assessed serologically.

3 V.S. Sarafian et al. / Central European Journal of Medicine 1(2) Immunohistochemistry Tissue specimens were taken immediately after surgical resection, fixed in 10% buffered formalin, and embedded in paraffin. Routine microscopy with hematoxylin-eosin was performed prior to the immunohistochemical study. Paraffin sections were stained by the indirect immunoperoxidase technique using a universal LSAB-2 kit (DAKO) and the chromogen aminoethylcarbazole as previously described [9]. Monoclonal antibodies to the following proteins were used as the primary antibodies: human HBGAs A and B (BulBio); human LAMP-1 and LAMP-2 (clones H4A3 and H4A4, Developmental Studies Hybridoma Bank, University of Iowa); human high molecular weight cytokeratin (CK HMW; DAKO); human cytokeratin MNF 116 (CK MNF 116; DAKO). Positive and negative controls were examined in parallel. The specificity of staining was assessed using negative controls in which [1] the primary antibody was absent; [2] the primary and secondary antibodies were replaced with 3% bovine serum albumin; and [3] the primary antibody was replaced with preimmune mouse serum. Nuclei were counterstained with Mayer s hematoxylin. Immunoreactivity was calculated using the following semiquantitative scale: 3+, 80% to 100% positive epithelial cells; 2+, 40% to 80% positive epithelial cells; 1+, 20% to 40% positive epithelial cells; and -, no reaction. 3 Results The expression of HBGAs and LAMPs was examined in spinocellular and basocellular carcinomas and in normal age-matched skin, and cytokeratin staining was used to identify epithelial cells. The blood group phenotypes as determined by serological and immunohistochemical analysis of HGBA expression were identical. Loss of HGBA reactivity was observed in some tumor keratinocytes. In normal skin, HBGAs were present in the stratum spinosum and stratum granulosum of the stratified squamous epithelium (Fig. 1a). The basal layer did not contain immunoreactivity for HGBAs. Interestingly, LAMPs showed an opposite pattern of expression; they were detected in the less differentiated keratinocytes in the stratum basale. In addition, LAMP-2 was also present in the squamous layer. The overlying epidermal layers were deficient in LAMPs (Fig. 1b). The epithelial cells of hair follicles and of eccrine glands were positive for both HBGAs and LAMPs. These two types of glycoproteins were found in epithelial cells of the sebaceous glands but not in the adipocytes of the subcutaneous fat tissue (Figs. 1c and d). Endothelial cells and erythrocytes were intensely stained for HBGAs only. We next examined the stratified squamous epithelium above the tumor as well as the tumor zones for HBGA and LAMP expression in basal and squamous cell carcinomas and we compared their localization with that of cytokeratins. We found no difference in the expression of either glycoprotein in epithelial skin appendages compared to normal skin. Staining for the glycoproteins showed the same pattern in the squamous epithelium of spinocellular carcinomas as in normal skin. HBGAs were found in the cytoplasm of

4 122 V.S. Sarafian et al. / Central European Journal of Medicine 1(2) Fig. 1 Expression of HBGAs and LAMPs in normal human skin (Biotin-streptavidinperoxidase reaction). a) Focal expression of HBGA A in the stratum spinosum and stratum granulosum of the stratified squamous epithelium. The arrowhead indicates positively stained endothelial cells in dermal capillaries. Original magnification, 280. b) Expression of LAMP-2 in stratum basale and stratum spinosum of the stratified squamous epithelium. Original magnification 280. c) Expression of HBGA B in epithelial cells of hair follicles (arrow) and sebaceous glands (arrowhead). Original magnification, 200. d) Expression of LAMP-1 in epithelial cells of a sebaceous gland. Original magnification, 280. tumor keratinocytes in the center of some tumor lobules, as well as in keratin pearls, the endothelium of capillaries, red blood cells, and heterogeneously in some inflammatory cells within the perivascular infiltrates (Fig. 2a). Also, HBGAs were detected in the epithelium of basal cell carcinomas. Interestingly, there was a complete loss of HBGAs in tumor keratinocytes (Fig. 2b). Another unexpected observation was that the expression of LAMPs was lost in the epithelium above the tumor in 76% of basocellular and in 61% of spinocellular carcinomas. Tumor keratinocytes in both carcinomas, however, were positive for LAMPs. Staining for LAMP-2 was considerably more intense and granular than staining for LAMP-1 (Figs. 2c and d).

5 V.S. Sarafian et al. / Central European Journal of Medicine 1(2) Fig. 2 Expression of HBGAs and LAMPs in skin carcinoma (biotin-streptavidinperoxidase reaction). a) Squamous cell carcinoma. Intensive cytoplasmic reactivity for HBGA A in tumor keratinocytes (arrow), with initial structuring of keratin pearls in the center of tumor follicles. Heterogeneous expression in some inflammatory cells of perivascular infiltrates (outline arrow). Positive endothelial cells and erythrocytes (arrowhead). Original magnification, 200. b) Basal cell carcinoma. Expression of HBGA B in the normal overlying epidermis (arrow) and in endothelial cells and erythrocytes (arrowhead). Loss of HNGA B expression in the tumor zone (the unstained region on the right). Original magnification, 400. c) Squamous cell carcinoma. Intense cytoplasmic staining for LAMP-2 in tumor keratinocytes. Original magnification, 400. d) Basal cell carcinoma. Intense granular cytoplasmic staining for LAMP-2 in tumor keratinocytes. Original magnification, 280. We also compared the expression of HBGAs and LAMPs with that of CK HMW and CK MNF 116. CK HMW was expressed homogenously throughout the normal squamous epithelium, as well as focally in some regions overlying the tumor. Except for the stratum corneum of spinocellular carcinomas, the epithelium was not stained for CK MNF 116. Tumor cells and keratin pearls in squamous cell carcinomas were positive for both cytokeratins. In tumor keratinocytes, the staining for CHK HMW was more intense and homogenous than that for CK MNF 116. In contrast, in keratin pearls, staining for CK

6 124 V.S. Sarafian et al. / Central European Journal of Medicine 1(2) Fig. 2 (continued) Expression of HBGAs and LAMPs in skin carcinoma (biotinstreptavidin-peroxidase reaction). e) Squamous cell carcinoma. Expression of CK MNF 116 in a keratin pearl. Original magnification, 200. f) Basal cell carcinoma. Intense cytoplasmic staining for CK HMW in tumor keratinocytes (arrow) as well in the normal overlying epidermis (outline arrow) and in basal and parabasal cells of a sebaceous gland (arrowhead). Original magnification, 100. MNF 116 was more intense than staining for CK HMW but noticeably weaker than that for HBGAs (Fig. 2e). Also, expression of CK HMW was heterogeneous in tumor keratinocytes within basal cell carcinomas. In this case, the immunoreactivity was highest in the central part of tumor lobules. Overall, the cytoplasm of tumor cells showed diffuse staining for CK HMW (Fig. 2f). 4 Discussion The difference in antigenic profiles of keratinocytes in normal and pathological conditions is of current interest in cutaneous biology. However, parallel investigations of LAMPs and HBGAs in human skin have not yet been carried out. In this report, we present the first comparative immunohistochemical study of both glycoproteins in normal and neoplastic skin in relation to two types of cytokeratins, CK HMW and CK MNF 116. Interestingly, we observed the opposite pattern of expression of LAMPs and HBGAs in the stratified squamous epithelium of normal skin: LAMPs and HGBAs were present in the basal and upper layers of the epidermis, respectively. The carbohydrate ABH- HBGAs are known to be differentially expressed in epithelia, and their expression has been shown to closely correlate with cellular differentiation [11]. Mandel et al. [12] reported the presence of A antigen and its corresponding glycosyltransferase only in the spinous layer of stratified nonkeratinized squamous epithelium. In the keratinized epithelium, we discovered ABH antigens both in the spinous and granular layers, suggesting that their presence is related to the stage of terminal keratinocyte differentiation. Expression of the

7 V.S. Sarafian et al. / Central European Journal of Medicine 1(2) ABH antigens could provide immunomorphological evidence for the sequential migration of proliferative cells towards the surface of the epidermis during the acquisition of a mature keratinocyte phenotype. In an in vitro model of spontaneous migration, the ability of HaCaT keratinocytes to migrate was reduced by inhibition of the lysosomal enzyme cathepsin B, which contributes to remodeling of the extracellular matrix [13]. Another lysosomal protease, cathepsin L, has been confirmed to be an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling [14]. Because LAMPs are integral elements of lysosomal membranes, it is very possible that they participate in the process of keratinocyte differentiation. The expression of LAMPs is restricted to the basal layers, suggesting that, in contrast to HBGAs, they are involved in the initial steps of the differentiation process. Blood group carbohydrates have been proposed as markers not only for epithelial but also for endothelial differentiation [15]. Our findings indicate that both glycoproteins are differentially expressed in the normal stratified squamous epithelium. The parallel analysis of LAMP, HBGA, and cytokeratin expression confirmed that expression of LAMPs and HBGAs correlate with epithelial differentiation. Both of the cytokeratins examined, CK HMW and CK MNF116, showed characteristic staining patterns, allowing them to be used as controls for the detection of LAMP- and ABH-positive cells. To determine how the expression of the two types of glycoproteins is altered in neoplastic skin, we examined the localization and intensity of their staining in tumor cells from spinocellular and basocellular carcinomas and compared it to that of normal skin. We examined not only tumor keratinocytes but also the stratified epithelium overlying the tumor. We did not detect a change in the reactivity for HBGAs in the epithelium, but there was a considerable loss of LAMPs in the overlying epithelium of both carcinomas. Assuming that LAMPs in normal stratified epithelium are associated with the initial differentiation of keratinocytes, their loss in the epithelium above the tumor may reflect a commitment to dedifferentiation during the neoplastic process. Tumor progression is often associated with altered glycosylation of the cell-surface proteins and lipids. Our analysis revealed that the expression of LAMPs and HBGAs is modified in skin carcinomas: LAMPs were overexpressed and HBGAs were lost in basal cell carcinomas. In a previous study, total and patchy loss of A/B antigen expression was reported in 24 of 32 oral squamous cell carcinomas [16]. Recently, altered expression of HBGAs in malignant oral tissues was suggested to result in increased cell migration because normally migrating oral epithelial cells like malignant cells showed lack of A/B antigens. These molecules may bind key receptors that control adhesion and motility, such as integrins, cadherins, and CD44 [17]. Our study revealed a complete loss of HBGAs in some tumor zones of basal cell carcinomas, whereas, in squamous cell carcinomas, the antigens were detected in the cytoplasm of tumor keratinocytes and in keratin pearls. Keratin pearls in well-differentiated carcinomas simulate the differentiation pathway of the normal oral squamous epithelium. Expression of involucrin and HBGAs has been shown to correspond with terminally keratinized cells in keratin pearls [18]. Because the

8 126 V.S. Sarafian et al. / Central European Journal of Medicine 1(2) HBGAs were detected in terminally differentiated keratinocytes in the normal stratified epithelium, it may be normal to find these antigens in keratin pearls. Tumor keratinocytes in both types of carcinomas were intensely stained for LAMPs. Furuta et al. [3] reported a similar finding in the epithelium of colorectal neoplasms, suggesting that the expression of LAMPs is related to malignant progression. Our previous investigations proved that LAMP expression is enhanced on the surface of various tumor cell lines. Such enhanced expression of LAMPs may facilitate tumor progression by binding as ligands to galectin-3 [5]. Local tumor progression of pancreatic carcinoma is also influenced by LAMP-1 [4]. In conclusion, we describe a comparative immunohistochemical study on LAMPs and HBGAs in normal and neoplastic skin. The results suggest that these glycoconjugates are differentially expressed in the normal stratified squamous epithelium and that there is a modulating reactivity in basal cell and squamous cell carcinomas. Therefore, these glycoconjugates may participate in differentiation and tumorigenesis in human skin. Acknowledgment The study was supported in part by Grant N:01/2005 from the Medical University of Plovdiv, Bulgaria. References [1] C.M. Chuong, B.J. Nickoloff, P.M. Elias et al.: What is the true function of skin?, Exp. Derm., Vol. 11, (2002), pp [2] R.D. Moreno: Differential expression of lysosomal associated membrane protein (LAMP-1) during mammalian spermiogenesis, Mol. Reprod. Dev., Vol. 66, (2003), pp [3] K. Furuta, M. Ikeda, Y. Nakayama, K. Nakamura, M. Tanaka, N. Hamasaki, M. Himeno, S.R. Hamilton and J.T. August: Expression of lysosome-associated membrane proteins in human colorectal neoplasms and inflammatory diseases, Am. J. Pathol., Vol. 159, (2001), pp [4] B.M. Kunzli, P.O. Berberat, Z.W. Zhu, M. Martignoni, J. Kleeff, A.A. Tempia- Caliera, M. Fukuda, A. Zimmermann, H. Friess and M.W. Buchler: Influences of the lysosomal associated membrane proteins (Lamp-1, Lamp-2) and Mac-2 binding protein (Mac-2-BP) on the prognosis of pancreatic carcinoma, Cancer, Vol. 94, (2002), pp [5] V.S. Sarafian, M. Jadot, J.M. Foidart, J.J. Letesson, F. van den Brule, V. Castronovo, R. Wattiaux, S. Wattiaux-De Coninck: Expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in human tumor cells, Int. J. Cancer, Vol. 75, (1998), pp [6] V. Ravn and E. Dabelsteen: Tissue distribution of histo-blood group antigens, Acta Pathol., Microbiol. Immunol. Scand., Vol. 108, (2000), pp

9 V.S. Sarafian et al. / Central European Journal of Medicine 1(2) [7] K.H. Nomura, R. Kobayashi, Y. Hirabayashi, M. Fujisue-Sakai, S. Mizuguchi and K. Nomura: Involvement of blood-group-b-active trisaccharides in Ca2+-dependent cell-cell adhesion in the Xenopus blastula, Dev. Genes and Evolution, Vol. 208, (1998), pp [8] J. Le Pendu, S. Marionneau, A. Cailleau-Thomas, J. Rocher, B. Le Moullac-Vaidye and M. Clement: ABH and Lewis histo-blood group antigens in cancer, Acta Pathol., Microbiol. Immunol. Scand., Vol. 109, (2001), pp [9] V.S. Sarafian, E. Tomova and S. Kalaydjiev: Stomach expression of histo-blood group antigens A and B in some vertebrates, Acta Zool., Vol. 85, (2004), pp [10] B.J. Nickoloff, J.Z. Qin, V. Chaturvedi, P. Bacon, J. Panella and M.F. Denning: Life and death signaling pathways contributing to skin cancer, J. Invest. Dermatol., Symposium Proceedings, Vol. 7, (2002), pp [11] V. Ravn and E. Dabelsteen : Tissue distribution of histo-blood group antigens, APMIS, Vol. 108, (2000), pp [12] U. Mandel, T. White, J. Karkov, S. Hakomori, H. Clausen and E. Dabelsteen: Expression of the histo-blood group ABO gene defined glycosyltransferases in epithelial tissues, J. Oral Pathol. Med., Vol. 19, (1990), pp [13] H. Buth, B. Wolters, B. Hartwig, R. Meier-Bornheim, H. Veith, M. Hansen, C.P. Sommerhoff, N. Schaschke, W. Machleidt, N.E. Fusenig, P. Boukamp, K. Brix: Ha- CaT keratinocytes secrete lysosomal cysteine proteinases during migration, Eur. J. Cell Biol., Vol. 83, (2004), pp [14] D.J. Tobin, K. Foitzik, T. Reinheckel, L. Mecklenburg, V.A. Botchkarev, C. Peters and R. Paus: The lysosomal protease cathepsin L is an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling, Am. J. Pathol., Vol. 160, (2002), pp [15] V.S. Sarafian, P. Dimova, I. Georgiev and H. Taskov: ABH blood group antigen significance as markers of endothelial differentiation of mesenchymal cells, Folia Med., Vol. 2, (1997), pp [16] S. Gao, E.P. Bennett, J. Reibel, X. Chen, M.E. Christensen, A. Krogdahl and E. Dabelsteen: Histo-blood group ABO antigen in oral potentially malignant lesions and squamous cell carcinoma-genotypic and phenotypic characterization, Acta Pathol., Microbiol. Immunol. Scand., Vol. 112, (2004), pp [17] E. Dabelsteen and S. Gao : ABO blood-group antigens in oral cancer, J. Dent. Res., Vol. 84, (2005), pp [18] P.D. Toto and H. Nadimi: Coexpression of cytokeratins, involucrin, and blood group antigens in oral squamous cell carcinomas, Oral Surgery, Oral Med., Oral Pathol., Vol. 90, (1990), pp