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Expression of CYLD, NF-κB and NF-κB-related Factors in Salivary Gland Tumors

MASAKATSU FUKUDA1, FUMIE FUKUDA1, YUKO HORIUCHI1, YUKA OKU1, SEIJI SUZUKI1, KAORU KUSAMA2 and HIDEAKI SAKASHITA1

1Second Division of Oral and Maxillofacial Surgery and 2Division of Pathology, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Sakado, Saitama, Japan

Abstract. The cylindromatosis (CYLD) was originally phosphorylated by the IκB kinase (IKK) complex and identified as a tumor suppressor that is mutated in familial degraded by the proteasome, allowing NF-κB to translocate cylindromatosis, an autosomal dominant condition that confers to the nucleus and activate its target gene (11, 13). Loss of a predisposition to multiple tumors of the skin appendages. the deubiquitinating activity of CYLD is correlated with CYLD has activity and inhibits the tumorigenesis. It has been reported that CYLD expression is activation of transcription factor NF-κB. Therefore, loss of detectable in the brain, testis, skeletal muscle, spleen, liver, CYLD function correlates with tumorigenesis. Expression of heart, lung and leukocytes (5). However, it is still unknown CYLD has been detected in various organs, but its expression whether CYLD is expressed in salivary glands. Moreover, it is in salivary gland tumor (SGT) is still unknown. Adenoid cystic also unclear whether loss of CYLD is associated with the carcinoma (ACC) is a well known and typical malignant SGT. development of salivary gland tumors. ACC was previously known as cylindroma in view of its marked Adenoid cystic carcinoma (ACC) is a well known and histological resemblance to dermal cylindroma. In this study, typical malignant salivary gland tumor. ACCs are the expressions of CYLD and NF-κB mRNA in HSG, a human biologically aggressive and can give rise to metastases many SGT cell line, were found to be increased by TNF-· years after excision of the primary tumor. This can lead to stimulation. Immunohistochemistry clearly demonstrated the facial palsy due to perineural invasion. ACC was previously expression of CYLD and NF-κB-related factors in ACC tissue. called cylindroma because of its marked histological resemblance to dermal cylindroma. Molecular studies of familial and sporadic cylindromas, which In the present study, the expression of CYLD and NF-κB arise from the eccrine or apocrine cells of the skin (1, 2), have in HSG, a human salivary gland tumor cell line, was analyzed. shown frequent alterations at 16q12-13 that Furthermore, the expression and distribution of CYLD, NF- have recently been found to house the cylindromatosis κB and NF-κB-related factors in ACC tissue were also (CYLD) gene (3-6). CYLD, a tumor suppressor gene, has investigated. This is the first reported study of CYLD deubiquitinating enzyme activity and inhibits the activation expression in salivary gland tumors in vitro and in vivo. of transcription factor NF-κB (5, 7-9), which plays key roles in inflammation, immune responses, tumorigenesis and Materials and methods protection against apoptosis (10-12). In most cells, NF-κB is kept inactive in the cytoplasm through association with IκB Reagents. Rabbit anti-human CYLD polyclonal antibody (PAb CYLD), mouse anti-human NF-κB p65 monoclonal antibody inhibitors. Upon stimulation by various reagents, IκB is (MAb NF-κB), rabbit anti-human IκB-· polyclonal antibody (PAb IκB-·, mouse anti-human IKK· monoclonal antibody (MAb IKK·) and goat anti-human IKK‚ polyclonal antibody (PAb Correspondence to: Assistant Professor Masakatsu Fukuda, Second IKK‚) were purchased from Santa Cruz Biotechnology (CA, Division of Oral and Maxillofacial Surgery, Department of USA). Biotinylated horse anti-mouse IgG (H+L) antibody and Diagnostic and Therapeutic Sciences, Meikai University School of streptavidin-peroxidase were obtained from Vector Laboratories Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283, Japan. Tel: (Burlingame, CA, USA) and Gibco-BRL, Life Technologies (MD, (+81)-49-285-5511, Fax: (+81)-49-279-1193, e-mail: fukudam@ USA), respectively. dent.meikai.ac.jp Cell culture. The HSG cell line, derived from a human Key Words: Cylindromatosis (CYLD) gene, nuclear factor-kappa B submandibular salivary gland, was established by Shirasuna et al. (NF-κB), inhibitory kappa B (IκB), IκB kinase-· (IKK·), IκB (14). The HSG cells were maintained in RPMI-1640 medium kinase-‚ (IKK‚), adenoid cystic carcinomas (ACCs). supplemented with 10% heat-inactivated fetal bovine serum (FBS),

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100 IU/mL penicillin and 100 Ìg/mL streptomycin, and grown to Table I. The primer sequences used. confluence in 25-cm2 culture flasks at 37ÆC in a humidified 5% CO2 incubator until use. As a positive control, human uterine Oligonucleotides: cervical adenocarcinoma cells (HeLa) obtained from the American Type Culture Collection (ATCC; VA, USA) were used. CYLD primer pairs 1. forward primer: 5’-TCGCTGGACTGGAACTGGAAG-3’ 2. reverse primer: 5’-GGATGAAGGTGGACTTGAGTGTTG-3’ RNA extraction and RT-PCR. Total RNA was extracted from the HSG cells by the acid-guanidinium-phenol-chloroform (AGPC) NF-κB primer pairs method (15). Briefly, the monolayers were scraped off and 1. forward primer: 5’-CACTTATGGACAACTATGAGGTCTC dissolved in solution D (4 M guanidinium thiocyanate solution TGG-3’ containing 25 mM sodium citrate, 0.5% sarcosyl and 0.1 M 2. reverse primer: 5’-CTGTCTTGTGGACAACGCAGTGGAAT 2-mercaptoethanol), then extracted with phenol-chloroform and TTTAGG-3’ chloroform. The RNA was alcohol-precipitated and the pellet was dissolved in sterile water. Random hexamer-primed single- ‚-actin primer pairs stranded cDNA was synthesized with an RNA LA PCRì Kit 1. forward primer: 5’-GTGGGGCGCCCCAGGCACCA-3’ (Takara Shuzo, Shiga, Japan) from 1.0 Ìg of total RNA in a final 2. reverse primer: 5’-CTCCTTAATGTCACGCACGATTTC-3’ volume of 20 Ìl with 0.25 units of avian myeloblastosis virus reverse transcriptase at 42ÆC for 60 min. An aliquot (1.0 Ìl) of the reaction mixture was diluted with 10 Ìl of PCR buffer containing 4 pmol each of 5' and 3' CYLD, NF-κB and ‚-actin primer sets (Table I). The PCR reaction was then performed with 2.5 units of (T1N0M0; stage I) arising from the hard palate. The pathological TaKaRa LA Taqì (Takara). The samples were subjected to diagnosis was based on histological examination of hematoxylin and denaturation at 94ÆC for 2 min. This was followed by 35 cycles of eosin-stained slides according to the WHO classification (16). The denaturation at 94ÆC for 30 sec, annealing at 56ÆC for CYLD patient had not undergone preoperative chemotherapy or mRNA and 65ÆC for NF-κB mRNA detection for 1 min each, and radiotherapy. extension at 72ÆC for 1 min. For the last cycle, the extension period was 10 min. The amplified DNA was electrophoresed on a Immunostaining of adenoid cystic carcinoma. The sections were 2.0% agarose gel, stained with ethidium bromide and visualized immersed in absolute methanol containing 0.3% H2O2 for 20 min on a UV illuminator, then photographed. at room temperature to block endogenous peroxidase activity. After washing, each section for staining of CYLD, NF-κB, IκB, Western blot analysis. The monolayer cells (1x106 cells/ml) were IKK· and IKK‚ was immersed in 0.01 M citrate buffer (pH 6.0) lysed in cell lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 and heated in a microwave oven for 15 min, as described by Shi et mM NaCl, 0.02% sodium azide, 1% Triton X-100, 1 Ìg/ml al. (17). After washing with PBS (pH 7.4), the sections were aprotinin and 100 Ìg/ml phenylmethylsulfonyl fluoride (PMSF), incubated in 2% BSA-PBS for 15 min at room temperature to and scraped from the dishes with a cell scraper (Nalge Nunc Int., block non-specific reactions. Diluted PAb CYLD (1:200), MAb Naperville, IL, USA). After 20 min on ice, the lysate was NF-κB (1:100), PAb IκB-· (1:100), MAb IKK· (1:50) and PAb centrifuged for 5 min at 15,000 rpm at 4ÆC, and the soluble IKK‚ (1:50) were respectively applied to the sections for 60 min at supernatant fraction was used for Western blot analysis. Protein room temperature. After washing with PBS, the slides were concentrations were measured by Bio-Rad protein assay. For gel incubated with secondary antibodies diluted 1:1000, including horse electrophoresis, 30-Ìg protein samples were mixed with an equal anti-mouse IgG (H+L) antibody for NF-κB and IKK·, goat anti- volume of SDS-PAGE sample buffer and boiled for 5 min. The rabbit IgG antibody for IκB-· and CYLD or rabbit anti-goat IgG samples were loaded in each lane and separated on a 5% gel, then antibody for IKK‚, for 30 min at room temperature. Diluted the proteins were electroblotted onto nitrocellulose membranes. streptavidin-peroxidase (1:1000) was applied to the sections for 30 The membranes were blocked at non-specific binding sites with min. The sections were immersed for 10 min in 0.05% 3,3'- 2% bovine serum albumin in phosphate-buffered saline (BSA- diaminobenzidine tetrahydrochloride in 0.05 M Tris-HCl buffer PBS) for 30 min at room temperature. Appropriately diluted (pH 7.6) containing 0.01% H2O2 and then counterstained with antibodies were applied to the sections for 60 min at room Mayer’s hematoxylin. temperature. The filters were individually washed three times for 10 min in PBS containing 0.05% Tween-20 (PBT). After Ethical considerations. The study was approved by the Research incubation with secondary biotinylated horse anti-mouse IgG Ethics Committee of Meikai University School of Dentistry, (H+L) antibody (1:1000) for 30 min at room temperature, the Saitama, Japan. filters were again washed three times for 10 min in PBT. Streptavidin-peroxidase, diluted 1:1000, was then applied to the Results strips for 30 min. Finally, the membranes were immersed for 2 min in 0.05% 3,3'-diaminobenzidine tetrahydrochloride in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% H O . 2 2 Detection of CYLD mRNAs in HSG cells. To examine Primary tumor sample. A formalin-fixed, paraffin-embedded whether the HSG cells expressed CYLD mRNA, RT-PCR specimen was obtained by surgical biopsy at Meikai University was performed. The expression of CYLD was detected in Hospital, Japan, from a 57-year-old male patient with ACC the total RNA from HSG and HeLa cells (Figure 1). As an

468 Fukuda et al: Expression of CYLD and NF-κB-related Factors in Salivary Gland Tumors

Figure 2. Western blot analysis. The CYLD peptide induced in the HSG cells was expressed spontaneously at 119 kDa.

Detection of CYLD and NF-κB mRNAs in HSG cells stimulated with TNF-·. To examine how CYLD and NF-κB Figure 1. RT-PCR. CYLD mRNA was clearly detected from the total expression is regulated in HSG cells upon stimulation with RNA of the HSG cells. As an internal control, ‚-actin mRNA detection revealed that the total RNA obtained was intact. TNF-·, semi-quantitative RT-PCR was carried out. This showed that the expressions of both CYLD and NF-κB mRNA were increased in HSG cells stimulated with 10 ng/ml TNF-·, in comparison with non-stimulated HSG cells (Figure internal control, ‚-actin mRNA detection was performed to 4A and B). assess the integrity of the cDNA obtained from the total RNA extracted from the HSG cells, and the total RNA Discussion obtained was confirmed to be intact. The CYLD gene, a tumor suppressor gene showing ubiquitous Expression of CYLD protein in HSG cells. SDS-solubilized expression, has been localized to chromosome 16q12-13 (3, 5, cell extracts of each tumor cell type were subjected to 18, 19). The CYLD gene is composed of 20 exons and the Western blot analysis to identify the presence of CYLD protein comprises 956 amino acids (5). CYLD is a newly- protein and to determine the quantity of this protein identified member of the deubiquitinating enzyme family and expressed in salivary gland tumor cells. Western blot inhibits the activation of NF-κB (5, 7-9), which plays key roles analysis revealed that CYLD induced in the salivary gland in inflammation, immune responses, tumorigenesis and tumor cells was the 119-kDa peptide (Figure 2). We, protection against apoptosis (10-12). In most cells, NF-κB is therefore, had demonstrated that CYLD peptides were kept inactive in the cytoplasm through association with IκB spontaneously expressed in the salivary gland tumor cells. inhibitors. After stimulation by various reagents, such as TNF-·, IκB is phosphorylated by the IκB kinase (IKK) Immunohistochemical detection of CYLD, NF-κB and NF-κB- complex and degraded by the proteasome, allowing NF-κB to related factors in ACC tissue. A case of ACC was subjected translocate to the nucleus and activate its target gene (11, 13). to immunohistochemistry. The histological type of the As shown in Figure 5, CYLD binds to the NF-κB essential studied ACC was cribriform. Immunoreactivity for CYLD, modulator (NEMO), also known as IKKÁ, component of the NF-κB and NF-κB-related factors was found sporadically IKK complex, and appears to regulate its activity through and/or in an aggregated pattern in the cribriform and duct- deubiquitination of TRAF2, since TRAF2 deubiquitination like structures of the ACC tissue. A positive reaction for can be modulated by CYLD (7-9), thus preventing the CYLD was weakly evident on the membrane of the cancer activation of the IKK complex and consequently inhibiting the cells (Figure 3A). CYLD was also detected in the activation of NF-κB. Therefore, loss of the deubiquitinating histologically-normal salivary gland tissue adjacent to sites activity of CYLD is correlated with tumorigenesis. Germline of malignancy (data not shown). NF-κB positivity was CYLD mutations have been detected in families affected by observed in the cytoplasm of the cancer cells (Figure 3B). cylindromatosis, and somatic mutations have been seen in both However, a slight positive reaction for NF-κB was also sporadic and familial cylindromas (3, 5, 18). Although CYLD detected in the nucleus. Interestingly, positivity for IκB-· was detected in the brain, testis, skeletal muscle, spleen, liver, was observed not only in the cytoplasm, but also the heart, lung and leukocytes, CYLD expression in the salivary nucleus of the cancer cells (Figure 3C). The cytoplasm glands had not been confirmed. Furthermore, it is also unclear showed strong reactivity for IKK· (Figure 3D), but weak whether loss of CYLD is associated with the development of positivity for IKK‚ (Figure 3E). salivary gland tumors.

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Figure 3. Immunohistochemistry. Immunoreactivity for CYLD, NF-κB and NF-κB-related factors was noted sporadically and/or in an aggregated manner in the cribriform and duct-like structures of ACC tissue. A weak positive reaction for CYLD was observed on the membrane of the cancer cells (A) (x66). A positive reaction for NF-κB was observed in the cytoplasm of the cancer cells (B) (x66). A slight positive reaction for NF-κB was also detected in the nucleus of the cancer cells (indicated by arrows). A positive reaction for IκB-· was observed in the nucleus (indicated by arrows) and cytoplasm of the cancer cells (C) (x66). A strong positive reaction for IKK· was observed in the cytoplasm of the cancer cells (D) (x66). A weak positive reaction for IKK‚ was observed in the cytoplasm of the cancer cells (E) (x66).

470 Fukuda et al: Expression of CYLD and NF-κB-related Factors in Salivary Gland Tumors

Figure 4. Semi-quantitative RT-PCR. The expression of both CYLD (A) and NF-κB (B) mRNA was increased in HSG cells stimulated with 10 ng/ml TNF-·, in comparison with non-stimulated HSG cells.

In the present study, CYLD expression and its correlations with NF-κB and NF-κB-related factors were investigated in salivary gland tumor, in vitro and in vivo. Our data indicated that both CYLD mRNA and protein are expressed spontaneously in the salivary gland tumor cell line, HSG. Using immunohistochemistry, it was also demonstrated that ACC arising from the hard palate was distinctly positive for CYLD, NF-κB, IκB, IKK· and IKK‚. Although CYLD showed weak immunoreactivity on the membrane of the cancer cells, NF-κB was detected in the cancer cell cytoplasm in ACC tissue, suggesting that NF-κB activity is inhibited. However, a slight positive reaction for NF-κB was detected in the nucleus of cancer cells in ACC tissue. Interestingly, IκB was expressed in the nucleus of the ACC cells. To our knowledge, this is a novel finding, as Figure 5. Schematic representation of the autoregulatory pathway through previously it was believed that IκB was localized in the which TRAF-2 is deubiquitinated by TNF-·-induced CYLD, which in cytoplasm (7-9, 20). The reason why IκB is localized in the turn leads to inhibition of NF-κB signaling. nucleus, and its possible function, remain to be determined. Furthermore, the fact that the IKK· expression was stronger than the IKK‚ expression in ACC tissue suggests that IKK· has a dominant role in comparison to IKK‚ in ACC. functions normally in salivary gland tumors and whether it Based on these observations, it can be hypothesized that shows gene alterations. loss of CYLD function is associated with development of This is the first report of CYLD expression in ACC and salivary gland tumors such as ACC. We, therefore, the human salivary gland tumor cell line HSG, a neoplastic attempted to examine the state of CYLD and NF-κB transformed cell line established by Shirasuna et al. (14) expression when salivary gland tumor cells were stimulated from intercalated duct cells of a human submandibular with TNF-·. Semi-quantitative PCR revealed that the salivary gland after exposure to radiation. expressions of both CYLD and NF-κB mRNA in HSG The current findings suggest that the HSG cell line may cells were increased upon stimulation of TNF-· for 24 h. be a useful model for studies on CYLD regulation in This indicated that CYLD did not inhibit the activity of malignant tumors of the salivary gland. Further investigation NF-κB, suggesting that loss of CYLD function might be of the role of CYLD, including its interaction with NF-κB- associated with human salivary gland tumorigenesis, related factors and analysis of CYLD gene alterations in including growth, development and perineural invasion. It salivary gland tumor cells, will be required to establish a will be necessary further to examine whether CYLD strategy for CYLD-based therapy of salivary gland tumors.

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References 12 Orlowski RZ and Baldwin AS Jr: NF-kappaB as a therapeutic target in cancer. Trends Mol Med 8(8): 385-389, 2002. 1 Lever W and Schaumburg-Lever G: Histopathology of the Skin. 13 Smahi A, Courtois G, Rabia SH et al: The NF-kappaB 7th ed. Lippincott, JB, Philadelphia, pp. 848-851, 1990. signalling pathway in human diseases: from incontinentia 2 Tellechea O, Reis J, Ilheu O et al: Dermal cylindroma. An pigmenti to ectodermal dysplasias and immune-deficiency immunohistochemical study of thirteen cases. Am J syndromes. Hum Mol Genet 11(20): 2371-2375, 2002. Dermatopathol 17: 260-265, 1995. 14 Shirasuna K, Sato M and Miyazaki T: A neoplastic epithelial 3 Biggs PJ, Wooster R, Smith N et al: Familial cylindromatosis duct cell line established from an irradiated human salivary (turban tumour syndrome) gene localised to chromosome gland. Cancer 48: 745-752, 1981. 16q12-q13: evidence for its role as a tumour suppressor gene. 15 Chomczynski P and Sacchi N: Single-step method of RNA Nat Genet 11: 441-443, 1995. isolation by acid guanidinium thiocyanate-phenol-chloroform 4 Biggs PJ, Chapman P, Lakhani SR et al: The cylindromatosis extraction. Anal Biochem 162: 156-159, 1987. gene (cyld1) on chromosome 16q may be the only tumour 16 Seifert G (ed.): Histological Typing of Salivary Gland Tumours. suppressor gene involved in the development of cylindromas. (2nd Ed.), World Health Organization International Oncogene 12: 1375-1377, 1996. Histological Classification of Tumors. Springer-Verlag, Berlin, 5 Bignell GR, Warren W, Seal S et al: Identification of the Heidelberg, New York, pp. 21-47, 1991. familial cylindromatosis tumour-suppressor gene. Nat Genet 25: 17 Shi SR, Key ME and Kalra KL: Antigen retrieval in formalin- 160-165, 2000. fixed and paraffin-embedded tissues: an enhancement method 6 Takahashi M, Rapley E, Biggs P et al: Linkage and LOH studies for immunohistochemical staining based on microwave oven in 19 cylindromatosis families show no evidence of genetic heating of tissue sections. J Histochem Cytochem 39: 741-748, heterogeneity and refine the CYLD locus on chromosome 1991. 16q12-q13. Hum Genet 106: 58-65, 2000. 18 Thomson SA, Rasmussen S, Zhang J et al: A new hereditary 7 Brummelkamp TR, Nijman SMB, Dirac AMG et al: Loss of the cylindromatosis family associated with CYLD1 on chromosome cylindromatosis tumour suppressor inhibits apoptosis by 16. Hum Genet 105(1-2): 171-173, 1999. activating NF-kappaB. Nature 424(6950): 797-801, 2003. 19 Leonard N, Chaggar R, Jones C et al: Loss of heterozygosity at 8 Kovalenko A, Chable-Bessia C, Cantarella A et al: The tumour cylindromatosis gene locus, CYLD, in sporadic skin adnexal suppressor CYLD negatively regulates NF-kappaB signalling by tumours. J Clin Pathol 54(9): 689-692, 2001. deubiquitination. Nature 424(6950): 801-805, 2003. 20 Frelin C, Imbert V, Griessinger E et al: Targeting NF-kappaB 9 Trompouki E, Hatzivassilliou E, Tsichritzis T et al: CYLD is a activation via pharmacologic inhibition of IKK2-induced deubiquitinating enzyme that negatively regulates NF-kappaB apoptosis of human acute myeloid leukemia cells. Blood 105(2): activation by TNFR family members. Nature 424(6950): 793- 804-811, 2005. 796, 2003. 10 Li Q and Verma IM: NF-kappaB regulation in the immune system. Nat Rev Immunol 2(10): 725-734, 2002. 11 Karin M, Cao Y, Greten FR et al: NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2(4): 301- Received March 28, 2006 310, 2002. Accepted April 18, 2006

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