ANTICANCER RESEARCH 36: 2379-2384 (2016)

SOX18 Expression in Non-melanoma Skin Cancer

MACIEJ ORNAT1, CHRISTOPHER KOBIERZYCKI1, JEDRZEJ GRZEGRZOLKA1, BARTOSZ PULA1, ALEKSANDRA ZAMIRSKA2, ANDRZEJ BIENIEK2, JACEK C. SZEPIETOWSKI2, PIOTR DZIEGIEL1,3 and MARZENNA PODHORSKA OKOLOW1

Departments of 1Histology and Embryology, and 2Dermatology, Venereology and Allergology, Wroclaw Medical University, Wroclaw, Poland; 3Department of Physiotherapy, University School of Physical Education, Wroclaw, Poland

Abstract. Background/aim: SRY-related HMG box Classification of Tumours, the most common malignant skin 18 (SOX18) is a involved in a range of tumours are: basal cell carcinoma (BCC), squamous cell physiological processes, including differentiation of carcinoma (SCC) and melanoma malignum (1). BCC and endothelial cells during new vessel formation. Numerous SCC are jointly referred to as non-melanoma skin cancers studies are being conducted to determine its role in (NMSCs) and originating from stratified squamous carcinogenesis. Materials and Methods: Thirty-five cases of epithelium cells, as in contrast to melanoma, which is of squamous cell carcinoma (SCC), 61 cases of basal cell neuroectodermal origin (1, 2). In 2012, there were 5.4 million carcinoma (BCC), 15 cases of actinic keratosis (AK) and 15 NMSC cases, including 3.3 million of BCC and SCC (3). normal skin (NS) cases were examined in the study. Actinic keratosis (AK) is a common intraepithelial Expression of SOX18 was investigated with immuno - proliferative lesion classified as a preinvasive cancer (4). Up histochemistry and light optic microscopy. The obtained to 20% of all AK have a tendency towards transformation results were subjected to statistical analysis including into SCC and for patients diagnosed with at least 10 AK available clinicopathological data. Results: Nuclear lesions, the 5-year risk of development of SCC is expression of SOX18 was shown in vascular endothelial approximately 14% (5-7). The development of AK is induced cells, basal layer cells of NS epidermis, as well as in AK, by frequent skin exposure to intense ultraviolet radiation BCC and SCC cancer cells. Expression of SOX18 in SCC, (UV) (8). An increased number of NMSCs was also shown BCC and AK cells was significantly higher than in NS in patients with an excessive exposure to UV radiation, both (p<0.01, p<0.001 and p<0.01, respectively). Additionally, in UV-B and UV-A range (9, 10). This was probably higher expression of SOX18 in BCC than in SCC cells associated with an increased risk of DNA mutations in (p<0.001) was observed. Conclusion: SOX18 may play a keratinocytes, particularly in TP53 , as well as with role in the development of BCC and SCC. Further studies induction of local immunosuppressive state within the skin with the use of additional markers tested at the mRNA and as a result of such radiation. In turn, UV reduces the body's protein level are necessary for better explanation of SOX18 ability for an effective immune response against cancer cells function in cancer transformation. (11, 12). It was also shown that long-term exposure to UV radiation is associated primarily with an increase in the Nowadays, due to rapidly increasing incidence, malignant incidence of SCC (7). skin neoplasms are an important medical, economical and BCC originates from epidermal basal layer cells. social problem. Based on the World Health Organization Uncovered skin exposed long-term to UV radiation, mainly in the head and neck area, is a typical localization for BCC (85% of cases), with about 30% of cases located within the skin in the nasal area (6). Since BCC rarely metastasises, it Correspondence to: Professor Marzenna Podhorska-Okolow, MD, belongs to the so-called 'locally malignant lesions'. Growing, Ph.D., Department of Histology and Embryology, Wroclaw Medical it infiltrates local tissues therefore causing their destruction University, Chalubinskiego 6a, 50-368 Wroclaw, Poland. Tel: +48 and leading to the deformation of deeper structures, 717841670, Fax: +48 717840082, e-mail: marzenna.podhorska- including cartilage and bone tissue (13, 14). In turn, SCC [email protected] (also called carcinoma spinocellulare) is derived from the Key Words: BCC, SCC, SOX18, non-melanoma skin cancer, stratum spinosum and is much more aggressive in immunohistochemistry. comparison to BCC (7). Under microscopy, areas of

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Table I. Clinical characteristic of patients with squamous cell carcinoma (SCC), basal cell carcinoma (BCC), actinic keratosis (AK) and normal skin (NS).

Parameter SCC BCC AK NS

Number of patients 35 61 15 15 Male 21 24 5 8 Female 14 37 10 7 Mean, median and range of age (years) 66, 68 (37-97) 70, 74 (50-87) 70, 75 (53-85) 62, 57 (21-82) Time of diagnosis and treatment; mean and range (months) 30 (1-84) 27 (6-360) 30 (6-84) N/A

hyperkeratosis and keratin pearls (keratodes type) are often date, no expression of SOX18 in BCC and SCC has been present, especially in cancer classified as highly described and such research, as well as their results, may differentiated (1). SCC morbidity increases with age, mostly advance the knowledge on the role of SOX18 in NMSCs. after the sixth decade of life, and predominantly occurs in the areas of the skin exposed long-term to intense sunlight Materials and Methods (backs of hands, head and face skin, lips and auricles). Clinical presentation of SCC is highly diversified, ranging Patients. Material for the research was obtained between 2005 and from asymptomatic to moderately severe lesions, and up to 2007 from the Department of Dermatology, Venereology and Allergology of Wroclaw Medical University, Poland. A total of 126 ulcerative and bleeding exophytic tumours (14). archival paraffin blocks were used: 96 cases of invasive skin cancer In the all abovementioned dysplastic lesions, i.e. AK, BCC (35 of SCC and 61 of BCC), 15 cases of preinvasive skin cancer and SCC, treatment of choice is their complete surgical (AK) and 15 cases of normal skin (NS) collected during removal. Further therapeutic decisions are based on the dermatosurgical procedures. Available clinicopathological data results of histopathological examination, which, especially included: patient's age and sex, time between diagnosis and in the case of SCC, may be associated with the need for treatment, histological type and clinical stage, localization of adjuvant therapy with the use of radio- and phototherapy (7). primary lesion and presence of inflammation (Tables I-III). SRY-related HMG box protein 18 (SOX18) encoded by Immunohistochemistry (IHC). The reactions were conducted on 4 μm- SOX18 gene is responsible for the interaction with specific thick paraffin sections mounted on SuperFrost Plus microscope slides A A A DNA sequences (5'- /T /T CAA /TG-3'), thus functioning as (Menzel Gläser, Braunschweig, Germany). Deparaffinization, re- a transcription factor (15). It is a part of a family of SOX hydration and exposure of the epitopes were performed with use of factors consisting of eight groups (A–H), which are classified PreTreatment Link Rinse Station and Target Retrieval Solution (pH based on high homology of structure and function (16). 9, 97˚C, 20 min). Activity of endogenous peroxidase was blocked by SOX18 belongs to group F and is involved in maturation and 5 min exposure to Peroxidase-Blocking Reagent. All sections were rinsed with wash buffer and incubated for 20 min at room temperature differentiation of endothelial cells in the prenatal period and with the primary monoclonal antibodies directed against SOX18 postnatally in angiogenesis. The analogy of SOX18 function (clone D-8, 1:25; Santa Cruz Biotechnology, Santa Cruz, CA, USA). with widely known inductors of angiogenesis i.e. Secondary goat anti-mouse antibodies coupled to a dextran core, from the vascular endothelial growth factor family (VEGF), linked to horseradish peroxidase were applied. Next, visualization was has been shown (17). Expression of SOX18 was observed in performed using the EnVision™ FLEX+ system according to the cardiomyocytes (particularly in the walls of ventricles and manufacturer's instructions. The IHC reactions were performed in an interventricular septum), cells of gastric and jejune mucosa, Autostainer Link48 (DakoCytomation) automated staining platform. The reactions were visualized using 3,3’-diaminobenzidine as well in pneumocytes. SOX18 is probably not only tetrachlorohydrate (DAB+ chromogen). All slides were counterstained engaged in wingless-type MMTV integration site family with Mayer’s hematoxylin and subsequently closed with SUB-X (WNT) signalling pathways, but also in β-catenin/T-cell Mounting Medium in Cover Slipper (DakoCytomation). All reagents, factor complex-dependent transcriptional regulation (16, 18). except primary antibody against SOX18 were obtained from Taking into account that silencing of SOX18 gene may DakoCytomation, Glostrup, Denmark. cause inhibition of proliferation, migration and invasion of, e.g. hepatocellular carcinoma cells, and that expression of Analysis of IHC reactions. The intensity of IHC reactions was evaluated with the use of an Olympus BX41 microscope (Olympus, SOX18 is increased in numerous cancer types e.g. gastric Tokyo, Japan). Nuclear expression of the protein was assessed in stromal tumours, pancreatic, liver, ovary and breast cancer, the cells that exhibited colour reaction in three regions with the it seems that this protein may play an important role in the highest intensity of expression (hot spots), by calculating the processes of cancer promotion and progression (18-24). To percentage of cells with positive IHC reaction.

2380 Ornat et al: SOX18 Expression in Non-melanoma Skin Cancer

Statistical analysis. Prism 5.0 programme (GraphPad, La Jolla, CA, Table II. Pathological characteristics of the patients with squamous cell USA) was used for statistical calculations. Analysis of normal carcinoma (1). distribution of SOX18 expression was performed with the use of Shapiro–Wilk test. Non-parametric Kruskal–Wallis test was used for Parameter N (%) the evaluation of the average differences in SOX18 expression in groups and then multiple comparisons of mean ranks for all samples Histological type were performed (Dunn's test). In all evaluated cases, statistically Keratodes 27 (77.1) significant p-values were accepted at less than 0.05. Akeratodes 8 (22.9) Broder’s scale 1 4 (11.43) Results 2 10 (28.57) 3 11 (31.44) Nuclear expression of SOX18 was shown in vascular 4 8 (22.86) endothelial cells, basal layer cells of NS epidermis, as well Depth of infiltration as in AK, BCC and SCC cancer cells (Figure 1). Intensity of Epidermis 8 (22.9) Subcutaneous tissue 27 (77.1) observed SOX18 expression varied between cancer cells in Inflammation AK (mean±standard deviation: 53.07%±30.27), SCC Mild 6 (17.14) (47.76%±29.08) and BCC cases (73.50%±19.80), while in Moderate 22 (62.86) NS it was found rarely (4.86%±2.74). Statistically significant Extensive 7 (20.0) difference was shown between the levels of expression of Sun exposure Exposed 32 (91.43) SOX18 in each of the groups (AK, BCC, SCC) vs. NS Not exposed 3 (8.57) (p<0.01, p<0.001, p<0.01, respectively), wherein the level of expression was higher in cancerous lesions. Additionally, higher expression of SOX18 in BCC than in SCC cells (p<0.05) was found. The comparisons of BCC vs. AK and Table III. Pathological characteristics of the patients with basal cell SCC vs. AK were not statistically significant. carcinoma (1). According to the available clinicopathological data shown in the tables, no statistically significant differences were Parameter N (%) found in SOX18 expression. Histological type Superficial 19 (31.15) Discussion Nodular 36 (59.02) Adenoid 2 (3.27) There exist numerous studies regarding the role of SOX18 in Infiltrative 3 (4.92) carcinogenesis and its possible usage in diagnostic and Micronodular 1 (1.64) therapeutic process. Expression of this protein was shown in Sun exposure Exposed 42 (68.85) many malignant neoplasms e.g. ovarian, breast, gastric, lung Not exposed 16 (26.23) and liver cancer (18-24). Not defined 3 (4.92) In ovarian cancer, SOX18 expression was found in the nuclei of vascular and lymphatic endothelial cells, as well as in the nuclei and cytoplasm of cancer cells. In tumor cells, the expression was significantly higher in more advanced clinical stages of the disease (stage III and IV according to nodes. Furthermore, the recurrence-free survival time and the the International Federation of Gynaecology and Obstetrics). 5-year survival rate were significantly decreased in those Moreover, SOX18 expression was associated with shorter cases (21). Similar correlation was discovered as a result of patient survival (22). Similarly, in invasive ductal breast the research performed with the use of hepatocellular carcinoma (IDC), SOX18 expression was observed in carcinoma cell line, where overexpression of SOX18 was endothelial and cancer cells. Increased nuclear expression of positively correlated with shorter patient survival. Moreover, SOX18 in IDC cells was positively correlated with more its lowered expression was associated with decreased tumor advanced clinical stage of the disease and positive status of invasiveness and aggressiveness (18). The above observations human epidermal growth factor 2 (23). In the case were confirmed by research on cases of non-small cell lung of gastric cancer, the expression of SOX18 was significantly cancer. The level of SOX18 in such tumours was higher in cancer cells in comparison to surrounding significantly higher in cancer cells in comparison to non- noncancerous mucosa. Gastric tumors with SOX18 cancerous surrounding tissue. Additionally, survival was overexpression were characterized by cancer cells invading significantly shorter in patients with higher expression of lymphatic vessels and the presence of metastasis in lymph SOX18 (24).

2381 ANTICANCER RESEARCH 36: 2379-2384 (2016)

Figure 1. Immunohistochemical expression of SRY-related HMG box protein 18 (SOX18) in normal skin (A, B), actinic keratosis (C, D), squamous cell carcinoma (E, F) and basal cell carcinoma (G, H). Magnification ×200.

The above facts may indicate that SOX18 plays a role in the suggest that SOX18 is involved in the progression of specific progression of these cancer types. Our NMSC observations types of skin cancer. also confirm those findings. Higher expression of SOX18 in Studies on new markers involved in skin carcinogenesis the cases of preinvasive cancer in the form of AK, as well as may provide valuable information regarding the mechanisms invasive SCC and BCC in comparison to normal skin may of cancer formation, thus contributing to a better

2382 Ornat et al: SOX18 Expression in Non-melanoma Skin Cancer understanding of this process, and in the future, to more 13 Peter NC: Plastic Surgery, Third Edition. New York, Elsevier, precise diagnosis and more effective therapy. Our results may pp. 707-742, 2013. facilitate distinguishing of NMSCs (BCC and SCC) and AK 14 Miller SJ, Alam M and Andersen J: Basal cell and squamous cell from other noncancerous skin lesions, which may prove very skin cancers . J Natl Compr Canc Netw 8: 836-864, 2010. 15 Harley VR, Lovell-Badge R and Goodfellow PN: Definition of a useful at early stages of formation of dysplastic skin lesions. consensus DNA binding site for SRY. Nucleic Acids Res 22: 1500-1501, 1994. Acknowledgements 16 Dunn TL, Mynett-Johnson L, Wright EM, Hosking BM and Koopman PA: Sequence and expression of Sox-18 encoding a This work was funded by scientific grant no. ST 810 of the new HMG-box transcription factor. Muscat GE – Gene 161: Wroclaw Medical University. 223-225, 1995. 17 Downes M and Koopman P: SOX18 and the transcriptional References regulation of blood vessel development. Trends Cardiovasc Med 11: 318-324, 2001. 1 LeBoit PE, Burg G, Weedon D and Sarasin A: Pathology and 18 Saitoh T and Katoh M: Expression of human SOX18 in normal Genetics of Skin Tumours. World Health Organization tissues and tumors. Int J Mol Med 10: 339-344, 2002. Classification of Tumours. IARC Press, Lyon, 2006. 19 Wang G, Wei Z, Jia H, Zhao W, Yang G and Zhao H: 2 Erb P, Ji J, Kump E, Mielgo A and Wernli M: Apoptosis and Knockdown of SOX18 inhibits the proliferation, migration and pathogenesis of melanoma and nonmelanoma skin cancer. Adv invasion of hepatocellular carcinoma cells. Oncol Rep 34: 1121- Exp Med Biol 624: 283-295, 2008. 1128, 2015. 3 Rogers HW, Weinstock MA, Feldman SR and Coldiron BM: 20 Young N, Hahn CN, Poh A, Dong C, Wilhelm D, Olsson J, Incidence estimate of nonmelanoma skin cancer (keratinocyte Muscat GE, Parsons P, Gamble JR and Koopman P: Effect of carcinomas) in the U.S. population, 2012. JAMA Dermatol 151: disrupted SOX18 transcription factor function on tumor growth, 1081-1086, 2015. vascularization, and endothelial development. J Natl Cancer Inst 4 Cohen JL: Actinic keratosis treatment as a key component of 98: 1060-1067, 2006. preventive strategies for nonmelanoma skin cancer. J Clin 21 Eom BW, Jo MJ, Kook MC, Ryu KW, Choi IJ, Nam BH, Kim Aesthet Dermatol 3: 39-44, 2010. YW and Lee JH: The lymphangiogenic factor SOX18: A key 5 Patterson JW: Weedon's Skin Pathology, Fourth Edition. London, indicator to stage gastric tumor progression. Int J Cancer 131: Elsevier, 2015. 41-48, 2011. 6 Ferri FF: Actinic keratosis. In: Ferri's Clinical Advisor 2015. 22 Pula B, Kobierzycki C, Solinski D, Olbromski M, Nowak- New York, Elsevier, pp. 39-40, 2014. Markwitz E, Spaczynski M, Kedzia W, Zabel M and Dziegiel P: 7 Bolognia J, Jorizzo J and Schaffer J: Dermatology, Third SOX18 expression predicts response to platinum-based 34 Edition. New York, Elsevier, pp. 1773-1793, 2012. chemotherapy in ovarian cancer. Anticancer Res : 4029-4037, 2014. 8 Habif TB: Clinical Dermatology: A Color Guide to Diagnosis and Therapy, Fifth Edition. New York, Elsevier, pp. 801-846, 23 Pula B, Olbromski M, Wojnar A, Gomulkiewicz A, Witkiewicz 2010. W, Ugorski M, Dziegiel P and Podhorska-Okolow M: Impact of SOX18 expression in cancer cells and vessels on the outcome of 9 Cakir B, Adamson P and Cingi C: Epidemiology and Economic invasive ductal breast carcinoma. Cell Oncol 36: 469-483, 2013. Burden of Nonmelanoma Skin Cancer. Facial Plastic Surg Clinics N Am 20: 419-422, 2012. 24 Jethon A, Pula B, Olbromski M, Werynska B, Muszczynska- Bernhard B, Witkiewicz W, Dziegiel P and Podhorska-Okolow 10 Ley RD: Dose response for ultraviolet radiation A-induced focal M: Prognostic significance of SOX18 expression in non-small melanocytic hyperplasia and nonmelanoma skin tumors. cell lung cancer. Int J Oncol 46: 123-32, 2015. Photochem Photobiol 73: 20-23, 2001. 11 Brash DE: Roles of the transcription factor in keratinocyte carcinomas. Br J Dermatol 154: 8-10, 2006. 12 Ullrich SE: Modulation of immunity by ultraviolet radiation: key Received February 8, 2016 effects on antigen presentation. J Invest Dermatol 105: 30-36, Revised March 23, 2016 1995. Accepted March 28, 2016

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