J Oral Pathol Med (2015) 44: 444–448 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

doi: 10.1111/jop.12253

wileyonlinelibrary.com/journal/jop

Aberrant expression of caspase 14 in salivary gland carcinomas Juliana Lemound1, Angelika Stucki-Koch2, Marcus Stoetzer1, Horst Kokem€ uller1, Nils-Claudius Gellrich1, 2 2 Hans Kreipe , Kais Hussein 1

Department of Craniomaxillofacial Surgery, Hannover Medical School, Hannover, Germany; 2Institute of Pathology, Hannover Medical School, Hannover, Germany

OBJECTIVES: Caspase 14 is reduced in adenocarcinomas of the stomach and colon. In contrast, breast and lung adenocarcinomas frequently show an overexpression of caspase 14. Salivary gland adenocarcinomas have not been evaluated for potential aberrant caspase 14 expression. MATERIALS AND METHODS: Samples from salivary gland carcinomas (n = 43) were analysed by immunohistochemistry (caspase 14, filaggrin, GATA3 and Ki67) and fluorescence in situ hybridization. RESULTS: Caspase 14 is not expressed in normal salivary glands, while in a subfraction of carcinomas (32%) an aberrant expression was found. Filaggrin could not be detected. Caspase 14 staining was not associated with tumour dedifferentiation, GATA3 expression or amplification of gene locus 19p13. CONCLUSION: In summary, aberrant expression of caspase 14 can be found in a subfraction of salivary gland carcinomas but could not be used as a biomarker for a specific carcinoma subtype of the salivary gland. J Oral Pathol Med (2015) 44: 444–448 Keywords: caspase 14; filaggrin; GATA3; salivary gland carcinoma

Introduction Caspases are the central molecules of the apoptotic process. One exception is caspase 14 which is not involved in apoptosis but in cell differentiation (1–4). Caspase 14 is primarily involved in epidermal cornification and skin barrier formation, particularly hydration and UVB protection (5–7). Although protein expression is prominently

Correspondence: Juliana Lemound, Department of Cranio-Maxillofacial Surgery, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Tel: + 49 511 532 4878, Fax: + 49 511 532 4740, E-mail: [email protected] Accepted for publication July 28, 2014

present in differentiating and cornifying layers of the epidermis and hair follicles, caspase 14 has been identified in complex and simple epithelia like human cervical and oral epithelium, the Hassall’s bodies of the thymus, choroid plexus, retinal pigment epithelium, placental trophoblast cells and deep glands of the stomach (6–12). In keratinocytes of the skin, caspase 14 is involved in differentiation, not in proliferation and apoptosis (13). Accordingly, the basal cell layer displays weak or absent expression of caspase 14 and, paralleling keratinocytic differentiation, the expression of the protein increases in the stratum spinosum and corneum (6, 13–15). Caspase 14 is involved in the cleavage of its cofactor profilaggrin to filaggrin, and filaggrin is expressed and accumulates in the stratum corneum (7, 8, 16–20). Caspase 14 is encoded on chromosome segment 19p13.1. The transcriptional regulation of the gene is not fully understood. An increased keratinocyte differentiation by growth stimulation in vitro or by addition of vitamin D3 leads to up-regulation of caspase 14. Ca2+ addition, which usually increases the cell differentiation, does not increase the expression of caspase 14 (6, 21–24). Several caspase 14-associated transcription factors have been found, for example GATA binding protein 3 (GATA3) (13, 24–27). A deregulation of caspase 14 expression has been described in neoplastic human tissues as well as in cancer cell lines. In cases of cervical, stomach, colon and ovarian carcinoma, decreased expression of caspase 14 seems to be associated with higher grades of dedifferentiation. In contrast, in advanced tumour stages of ductal infiltration and invasive breast carcinoma as well as lung adenocarcinoma, overexpression of caspase 14 was observed (10, 28, 29). The described effects could be mostly confirmed in corresponding cancer cell lines (13, 14, 22, 28–30). Salivary gland cancer tissue samples from patients have not yet been systematically investigated. Therefore, in this current investigation we evaluated whether aberrant caspase 14 expression is involved in different types of salivary gland carcinomas, which could be useful as new tumour markers.

Caspase 14 in salivary gland cancer Lemound et al.

Material and methods Study group Patient cohort and tumour samples

Salivary gland neoplasms were as follows: adenoid cystic carcinomas (n = 16), adenocarcinomas, not otherwise specified (NOS) (n = 13), mucoepidermoid carcinomas (n = 12), carcinomas ex pleomorphic adenoma (n = 2) and pleomorphic adenomas (n = 4). Non-neoplastic controls comprised 26 normal glands from unrelated cases. Two tissue microarrays (each with 60 samples) were used. All samples had been taken as part of standard clinical care. Samples were formalin-fixed and paraffin-embedded (FFPE) and were retrieved from the tissue archive of the Institute of Pathology, Hannover Medical School. The retrospective analysis of these clinical samples, after the diagnosis had been established, was approved by the local Ethics Committee, Hannover Medical School. Detection of caspase 14, filaggrin and GATA3 protein expression

For protein expression analysis, we used ~2–3 lm FFPE sections. Xylol-deparaffinized sections were pretreated in an autoclave prior to incubation with antisera: anticaspase 14 antibody (1:200, monoclonal mouse, clone D10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and antifilaggrin antibody (1:100, monoclonal mouse, clone SPM181, GeneTEx, Inc., Irvine, CA, USA), anti-GATA3 antibody (1:200, monoclonal mouse, clone L50-823, Biocare Medical, Concord, CA, USA) and Ki67 (ready to use, monoclonal rabbit, clone 30-9, Ventana, Basle, CH) (31, 32). Anticaspase 14 and antifilaggrin antibodies detect the inactive precursor proteins of caspase 14 and filaggrin as well as the active proteins. Both antibodies have been validated in FFPE sections from skin tissue. A ZytoChem-Plus HRP Polymer kit (Zytomed Systems, Berlin, Germany) and DAB (Dako, Glostrup, Denmark) were used for visualization of immunostaining. Sections were counterstained with haematoxylin. Immunohistochemistry for caspase 14, filaggrin and GATA3 was scored semi-quantitatively as described previously (31): 0 (no apparent reaction), 1+ (positivity in

Aberrant expression of caspase 14 in salivary gland carcinomas.

Caspase 14 is reduced in adenocarcinomas of the stomach and colon. In contrast, breast and lung adenocarcinomas frequently show an overexpression of c...
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