EXPRESSION OF THE SONIC HEDGEHOG PATHWAY IN SQUAMOUS CELL CARCINOMA OF THE SKIN AND THE MUCOSA OF THE HEAD AND NECK

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1 ORIGINAL ARTICLE EXPRESSION OF THE SONIC HEDGEHOG PATHWAY IN SQUAMOUS CELL CARCINOMA OF THE SKIN AND THE MUCOSA OF THE HEAD AND NECK Sven Schneider, 1 Dietmar Thurnher, MD, 1 Philipp Kloimstein, 1 Verena Leitner, MD, 2 Peter Petzelbauer, MD, 2 Johannes Pammer, MD, 3 Markus Brunner, MD, 1 Boban M. Erovic, MD 1 1 Department of Otorhinolaryngology Head and Neck Surgery, Medical University of Vienna, Vienna, Austria. boban.erovic@meduniwien.ac.at 2 Department of Dermatology, Medical University of Vienna, Vienna, Austria 3 Department of Clinical Pathology, Medical University of Vienna, Vienna, Austria Accepted 19 February 2010 Published online 19 May 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: /hed Abstract: Background. Activation of the hedgehog pathway may contribute to carcinogenesis. This study characterizes the expression pattern in squamous cell carcinoma of the skin and the head and neck. Methods. Tissue microarrays were constructed with samples of squamous cell carcinoma of the skin and the head and neck. All tissue samples were immunohistochemically stained for 7 Hedgehog pathway molecules. Results. Significant (p <.0001) overexpression of all evaluated molecules could be observed in the tumor samples compared with healthy control tissues. Expression of Gli-2 showed significant upregulation and that of Smoothened and Patched significant downregulation in head and neck compared with skin carcinoma. High expression of Sonic hedgehog correlates significantly (p ¼.001) with poor overall survival in patients with head and neck cancer. Conclusions. Hedgehog signaling is differentially regulated in squamous cell carcinomas of the skin and the head and neck. Sonic hedgehog expression may serve as a prognostic factor in patients with head and neck cancer. VC 2010 Wiley Periodicals, Inc. Head Neck 33: , 2011 Keywords: Sonic hedgehog; squamous cell carcinoma; head and neck; skin; tissue microarray The hedgehog signaling pathway plays an essential role in decision making of left right asymmetry and differentiation of limbs, the nervous system, and digestive tract. 1,2 Furthermore, after embryogenesis the hedgehog signaling pathway is involved in the regulation of adult tissue stem cells during regeneration of adult tissue damage. 3 In the mammalian family of genes, 3 hedgehog homologs, ie, Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh), are known. These proteins activate a membrane receptor complex con- Correspondence to: B. M. Erovic VC 2010 Wiley Periodicals, Inc. sisting of Patched (Ptch) and Smoothened (Smo). Ptch, a 12-transmembrane protein, functions as a receptor, without transducing the intracellular signals himself. Smo, a 7-transmembrane protein, executes the signals, 4 but in the absence of Hedgehog, Smo is blocked by Ptch. Binding of the hedgehog protein to Ptch releases the repression of Smo and subsequently Gli zinkfinger transcription factors are activated. 5 The Gli protein family consists of 3 proteins with different cellular functions. Gli-1 and Gli-2 mediate Hedgehog signals and have been implicated in tumorigenesis as oncogenes within the activated signal transduction. 6 Gli-1 is transcriptionally regulated through Gli-3. Recent studies have shown a deregulation of the hedgehog pathway in several malignant tumors such as basal cell carcinoma of the skin, 7 medulloblastoma, 8 tumors of the lung, 9 digestive tract, 10 and prostate. 11 There is evidence that inappropriate activation of hedgehog signaling is involved in the occurrence of metastasis by increasing Snail protein expression, which is induced rapidly by Gli-1. Snail protein represses E-cadherin and thereby the loss of cellular adhesion. 12 Increased Hedgehog signal transduction also leads to a higher activation of anti-apoptotic genes, angiogenic factors such as angiopoietin-1 and angiopoietin-2, 13 cyclins D1 and B1, 14 and to a decrease of apoptotic gene activation. 15 Squamous cell carcinomas of the skin are known to be the second most common form of malignant skin tumors. 16 Interestingly, in most cases when identified early and treated promptly by surgical excision, patients 5-year overall survival is 95%, with approximately 4% risk of metastasis. 17,18 The most interesting fact is the significant discrepancy in patients prognosis with squamous cell carcinomas of the skin and head and neck mucosa. In particular, although in both patient groups the tumor cells are 244 Sonic Hedgehog Expression in Skin and Head and Neck Mucosa HEAD & NECK DOI /hed February 2011

2 squamous carcinoma cells, the typical 5-year combined survival rates for patients with head and neck tumors are still in the range of 62% to 67%. 19 Although the importance of the hedgehog signaling in tumor development is recognized in basal cell carcinomas, 20 there has been no study regarding the expression in squamous cell carcinoma of the head and neck mucosa. The aim of this pilot study was to determine the immunohistochemical-staining pattern of the hedgehog pathway in healthy skin and head and neck mucosa as well as in squamous cell carcinomas of the skin and mucosa of the head and neck. Furthermore, we correlated the expression of the hedgehog pathway in squamous cell carcinomas of the skin and mucosa of the head and neck to patients clinical data. MATERIALS AND METHODS Patients. In this study, samples from 60 patients (19 women, 41 men; mean age, 77 years; range, years) with squamous cell carcinoma of the skin were included. All patients were treated at the Department of Dermatology, Medical University Vienna in the year A pathologist with a high expertise in skin tumors (P.P.) reviewed all cases. The tumor sites were located in the head and neck region (78%) and on the extremities (22%). Clinical data of the follow-up were available for all of the patients, with a mean follow-up period of 11 months including overall survival, disease-free interval, and treatment information. The overall survival and disease-free interval for all patients was 2 to 28 months. Tumor biopsies of 56 untreated patients (11 women, 45 men; mean age, 60 years; range, years) with locally advanced head and neck cancer were obtained during diagnostic panendoscopy at the Department of Otorhinolaryngology, Head and Neck Surgery between the years 1999 and The sites of the primary carcinomas were hypopharynx in 23% of patients (T1: 1 patient; T2: 3 patients; T4: 9 patients), larynx in 13% (T2: 1 patient; T3: 2 patients; T4: 4 patients), and oral cavity and oropharynx in 64% of patients (T2: 3 patients; T3: 7 patients; T4: 26 patients). Ten patients were treated by surgery and consecutive chemotherapy, and 2 patients underwent surgery followed by radiotherapy; 22 patients were treated by primary chemotherapy, whereas 20 patients underwent primary radiotherapy; and 4 patients obtained combined radiochemotherapy. Healthy skin (n ¼ 5) and oral mucosa (n ¼ 6) were obtained during routine mastectomy and tonsillectomy, respectively. Construction and Slide Preparation of Tissue Microarrays. Tissue microarrays were assembled from formalin-fixed, paraffin-embedded tissues using a punch (Beecher Instruments, Silver Spring, MD) with a diameter of 0.6 mm. Before constructing a tissue microarray block, serial 5-lm sections were cut from all donor blocks and were stained with hematoxylin and eosin for targeting morphologically representative areas of the tumor. Afterward slices were cut and serially sectioned slides were prepared. This allowed a minimum variation during the staining process. The first tissue microarray encompasses 324 tissue cores of 60 patients with squamous cell carcinoma of the skin. The second tissue microarray contains 168 tissue cores of 56 patients with head and neck tumors. All tumor samples were arrayed in a rectilinear pattern, with 0.8 mm between the centers of each sample. Immunohistochemistry. Dewaxed and rehydrated tumor sections (of 2 3 lm thickness) were subjected to antigen retrieval in a microwave oven (600 W) using EDTA buffer. Blocking of unspecific binding was achieved with 5% Tris-buffered saline/bovine serum albumin (TBS/BSA; Sigma Aldrich, Seelze, Germany) for 1 hour at room temperature. Primary antibodies directed against Gli-1, Gli-3, Shh, Ihh, Ptch, and Smo (Santa Cruz Biotechnology, Santa Cruz, CA) and Gli-2 (Aviva Systems Biology, San Diego, CA) were obtained and tested for ideal dilution. Application of the primary antibody (Shh, Ihh, Gli-1, Gli-2, Gli-3, Ptch, Smo) in a dilution of 1:100 and appropriate negative and positive control was done at 4 C overnight. As a secondary antibody, multi-link antibody (1:200, biotinylated-polyclonal swine anti-goat, -mouse, -rabbit; Dako, Glostrup, Denmark) was used in 1% TBS/BSA for 1 hour at room temperature, followed by alkaline phosphatase conjugated Streptavidin-AP/TBS/BSA (1:250; Dako) for 1 hour at room temperature. Visualization was performed by fast red (Sigma, St. Louis, MO) and counterstained by hemalaun. Samples were analyzed using an Olympus BH-2 microscope (Olympus America, Center Valley, PA). Specimen Classification Based on Immunohistochemical Results and Morphological Features. All slides were assigned to 1 of 4 categories of marker expression: 0 ¼ <5%; 1 ¼ 5% to 30%; 2 ¼ 30% to 60%; 3 ¼ 60% to 100%. The average of the core stains was taken to determine the staining intensity, which was entitled as follows: 0 as negative, 1 as weak, 2 as moderate, and 3 as strong expression. The expression pattern of each marker was determined independently by 3 investigators in the complete tumor samples (S.S., P.K., and B.M.E.). Observer bias was avoided, as much as possible, by repeating the evaluation of protein expression at 2 different time points and without knowledge of patients clinical data. Statistical Analysis. The overall survival was defined as the period from the patient s first treatment until the end of the study (May 1, 2009). The disease-free interval was defined as the period from Sonic Hedgehog Expression in Skin and Head and Neck Mucosa HEAD & NECK DOI /hed February

3 the patient s first treatment to the final date of the study, or the period from the patient s first treatment to the date of recurrent disease being diagnosed. The overall survival and disease-free interval curves of all patients were estimated using the Kaplan Meier method and the log-rank test. Cox regression analysis was performed to correlate protein expression to each patient s data. A value of p <.05 was considered statistically significant. The Statistical Package for the Social Sciences Software, version 15.0 for Windows (SPSS UK Ltd, Surrey, UK), was used for analysis. RESULTS Immunohistochemistry. Tissue microarrays, containing core biopsies of patients with squamous cell carcinomas of the skin and mucosa of the head and neck, were stained immunohistochemically with antibodies directed against the Hedgehog signaling molecules including Shh, Ihh, Gli-1, Gli-2, Gli-3, Ptch, Smo (Figures 1 and 2). The expression pattern of these antigens was correlated to histopathological diagnosis and patients clinical data. None of the patients with squamous cell carcinoma of the skin died as a result of the tumor disease. Therefore, no statistical analyses including overall survival and disease-free interval were performed. Expression of the Hedgehog Pathway in Normal Skin and Head and Neck Mucosa. No expressions of Gli-1, Gli-2, Gli-3, Shh, Ihh, Ptch, and Smo were found in normal skin and normal oral mucosa. Expression of the Hedgehog Pathway in Squamous Cell Carcinomas of the Skin and Mucosa of the Head and Neck. All proteins of the hedgehog-signaling pathway were expressed in the cytoplasm of squamous cell carcinoma cells. Gli-1 was negative in 32 of 56 skin specimens (57%), whereas in 21 of the 56 specimens (37%) and in 3 of the 56 specimens (5%) weak and moderate expressions were observed, respectively. In squamous cell carcinomas of the head and neck 23 of 43 biopsies (47%) showed no expression of Gli-1, but in 18 and 2 of the 43 samples (42% and 5%, respectively) weak to moderate expressions could be observed, respectively. Gli-2 was weakly and highly expressed in 5 and 3 of 56 samples (9% and 5%, respectively) of skin tumors, whereas 48 of 56 biopsies (85%) stayed completely negative; 32, 20, and 2 of 54 samples (59%, 37%, and 4%, respectively) of patients with head and neck tumor showed no, weak, and moderate expression, respectively. Comparison of Gli-2 expression in skin and mucosal tumors showed a high statistical difference (p ¼.004), with higher expression in head and neck cancer. Gli-3 expression was weakly and moderately expressed in 21 and 1 of 54 patients (39% and 2%, respectively) with skin tumor, whereas in 32 biopsies (59%) no Gli-3 could be detected. Samples of mucosal tumors were negative in 21 of 44 patients (47%), weakly positive in 19 of 44 patients (43%), and moderately positive 4 of 44 patients (9%) for Gli-3. Shh was weakly and moderately detectable in 33 and 5 of 57 skin tumor samples (58% and 9%, respectively); 19 of the 57 samples (33%) stayed negative for Shh. In mucosal tumors 23 of 53 biopsies (43%) were negative, whereas 23 of the 53 patients (43%) showed weak expression, 6 of the 53 patients showed moderate expression (11%), and 1 of the 53 patients showed strong expression (2%) of Shh. Ihh expression in squamous cell carcinoma of the skin was observed in only 6 of 57 samples (11%). In 51 cases (89%) no expression was detectable. Ihh in squamous cell carcinoma of head and neck mucosa was weakly and moderately expressed in 9 and 2 of 52 biopsies (17% and 4%, respectively). No Ihh synthesis could be found in 41 samples (79%). In skin tumors, weak, moderate, and strong expression of Smo was observed in 32, 13, and 4 of 59 patients biopsies (54%, 22%, 7%, respectively); 10 samples (17%) stayed completely negative for Smo. In mucosal tumors, Smo was weakly, moderately, and strongly detectable in 24, 6, and 1 of 56 samples (43%, 11%, 2%, respectively); 25 biopsies (45%) showed no immunostaining. Expression of Smo in skin tumors was significantly higher (p <.001) than that in mucosal carcinomas. Ptch expression in squamous cell carcinoma of the skin was weak, moderate, and strong in 27, 20, and 4 of 57 patients (47%, 35%, and 7%, respectively). Six of 57 cases (10%) lacked any Ptch expression. From 53 patients with squamous cell carcinomas of the head and neck, 20 showed no expression (38%), 28 showed weak expression (53%), 4 showed moderate expression (8%), and 1 showed strong expression (2%). There was a strong difference between skin and mucosa with significantly higher expression (p <.0001) in carcinomas of the skin. Patients Survival Data. With respect to patients survival data, 7% of patients with head and neck carcinoma were alive without and 2% with evidence of disease, whereas 76% had died of cancer before the end of the observation period (May 1, 2009). Recurrences occurred in 12 patients (21%) and 15% did not appear for scheduled follow-up sessions. Overall survival and disease-free interval ranged from 3 to 94 and 7 to 94 months, respectively. Median survival time was 21 months. Univariate analysis showed that moderate overexpression of Shh significantly correlates (p ¼.001) with decreased patients overall survival. Kaplan Meier curves of overall survival and Shh expression are shown in Figure 3. Multivariate analysis did not reveal any variables significantly associated with overall survival. 246 Sonic Hedgehog Expression in Skin and Head and Neck Mucosa HEAD & NECK DOI /hed February 2011

4 FIGURE 1. Schematic representation of the staining intensity of 2 tissue-microarrays. The left panel shows results for the squamous cell carcinomas (SCCs) of the head and neck mucosa, whereas the right panel shows the results for SCCs of the skin, respectively. SHH, sonic hedgehog; IHH, Indian hedgehog; SMO, Smoothened; PATCH, Patched. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] DISCUSSION Although head and neck as well as skin squamous cell carcinoma originate from the same histologic background, proliferation, incidence of metastasis, and patients survival differ significantly. The clinical outcome of patients suffering from head and neck squamous cell carcinoma is significantly worse than that in patients with squamous cell carcinoma of the skin. The aim of this study was to determine and characterize the expression pattern of hedgehog-signaling molecules in head and neck squamous cell carcinoma and squamous cell carcinoma of the skin. Outcomes of immunohistochemical analysis of the 2 tumor entities were compared and statistically Sonic Hedgehog Expression in Skin and Head and Neck Mucosa HEAD & NECK DOI /hed February

5 FIGURE 2. Expression of hedgehog signaling molecules in squamous cell carcinoma (SCC) of oral mucosa is shown in pictures A G; expression in SCC of the skin is shown in pictures a g. Negative expression of hedgehog signaling molecules in SCC of oral mucosa is shown in pictures H N; negative expression in SCC of the skin is shown in pictures h n. All pictures taken at 200 magnification. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] correlated. To our knowledge, this is the first evidence of elevated hedgehog signal transduction in human tissue samples of squamous cell carcinomas of the skin and head and neck mucosa. The connection between hedgehog signaling and carcinogenesis was first detected in patients with basal cell naevus syndrome (Gorlin s syndrome). In patients suffering from this syndrome mutations in the Patched gene could be found. 21 The enhanced expression of hedgehog-signaling molecules has also been demonstrated in tumor tissues of the breast, 22 stomach, 23 endometrium, 24 cervix, 25 pancreas, 26 and colon. 27 The importance of the hedgehog pathway could also be shown in squamous cell carcinoma cell lines by growth suppression through introduction of wild-type Patched gene. 28 In oral squamous carcinoma cell lines, inhibition of G1/S transition and apoptotic cell death by treatment with cyclopamine, a 248 Sonic Hedgehog Expression in Skin and Head and Neck Mucosa HEAD & NECK DOI /hed February 2011

6 FIGURE 3. Kaplan Meier curves of patients overall survival and Sonic hedgehog. Moderate overexpression of Sonic hedgehog correlates significantly (p ¼.001) to poor overall survival in patients with oral cancer. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] steroidal alkaloid that blocks the intracellular Shh signaling, could be demonstrated. 29 It is also known that in eyelid epithelial neoplastic lesions in mice cyclopamine inhibits proliferation and induces apoptosis in epithelial tumor cells in vivo. 30 Also, a combination of cyclopamine with epidermal growth factor receptor inhibitor gefitinib showed synergistic effects in inhibiting the growth of prostate cancer cells in vitro. 31 Besides cyclopamine, there are other promising modulators (ie, agonists and antagonists) of hedgehog signaling. One such is CUR61414, a selective inhibitor that was identified as a lead compound in a screen of 100,000 synthetic organic molecules. 32 It binds to and antagonizes the activity of Smo. CUR61414 was found to suppress proliferation and induce apoptosis in organotypic cultures of basal cell carcinoma-like lesions without affecting proliferation of normal skin cells. Still, in head and neck neoplastic lesions and squamous cell neoplastic lesions of the skin the functional relevance of the hedgehog-signaling pathway is still not completely investigated. Our analysis showed elevated pathway activation in the examined malignant epithelial tissue compared with the control samples and are in line with other recently published reports In particular, neoplastic lesions show a considerably different expression pattern of the sonic hedgehog pathway. The expression of Gli-1, Ptch, Smo, and Shh is elevated to a great extent in both head and neck and skin neoplasia, and strongly contrasts the expression profile observed in normal tissue. Ptch and Smo proteins showed stronger expression in squamous cell carcinomas of the skin compared with lesions of the head and neck mucosa. On the other hand, transcription factors Gli-1 and Gli- 3 and the Ihh protein were raised in mucosal carcinomas, although the differences were not significant. Only the expression of Gli-2 transcription factor arose significantly higher in mucosal tumors. Expression patterns of hedgehog signaling proteins in both types of cancer are summarized in Figure 4. We also examined the prognostic value of the members of hedgehog-signaling transduction by correlating expression patterns to overall survival. Interestingly, it could be shown that high expression of the Shh ligand is significantly correlated with poor prognosis in patients with squamous cell carcinoma of the head and neck mucosa (see Figure 3). The Kaplan Meier curves show evidence for reduced overall survival in patients with moderate and strong expression of sonic hedgehog protein. In particular, reduced overall survival is significantly correlated (p <.001) with moderate Sonic hedgehog expression. The worse outcome in patients with moderate compared with strong expression may be explained by the small sample size. Probably, a study in a bigger cohort is needed to show correlation even more clearly. As Shh ligand is inducing growth stimulation, this finding indicates that the hedgehog-signaling pathway becomes important for the poor prognosis of patients with head and neck neoplastic lesions. Another underlying mechanism for poor prognosis in patients with highly expressed hedgehog proteins may be the cross-linking of hedgehog signaling to chemoresistance via multidrug resistance protein 1 and breast cancer resistance protein. 33 This phenomenon hardly occurs in treatment of squamous cell carcinoma of the skin in which chemotherapeutics are used less frequently, but possibly has a more relevant function in head and neck squamous cell carcinoma. This aspect potentially leads to the assumption that hedgehog signaling might influence the outcome of patients with head and neck carcinoma in multiple modes, whereas in squamous cell carcinoma of the FIGURE 4. Histogram showing the differential expression of hedgehog proteins in mucosal and skin squamous cell carcinomas of tumor specimen in percent: significant different expressions of Gli-2, Smo, and Ptch are marked by an asterisk. Shh, sonic hedgehog; Ihh, Indian hedgehog; Smo, Smoothened; Ptch, Patched. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Sonic Hedgehog Expression in Skin and Head and Neck Mucosa HEAD & NECK DOI /hed February

7 skin the relevance might be greater for tumor growth than that for treatment response. Increased expression of Sonic hedgehog ligand and Gli-1 protein, as a major transcription factor in hedgehog signaling, is a strong indication for activation of hedgehog signaling in squamous cell carcinomas of the head and neck and squamous cell carcinomas of the skin, respectively. Therefore, it is possible that there is an impact of pathway activation on the tumorigenesis and proliferation of both types of neoplasias. Activation of the hedgehog-signaling molecules may occur as a widespread phenomenon in neoplastic transformation, rather than a specific step in tumor development. Besides, the increased expression may play a prominent role in tumorigenesis. Although the functional significance of these findings remains to be determined, these evidences indicate that the hedgehog-signaling pathway is involved in the proliferation of malignant tissues and may represent a possible molecular target for both squamous cell carcinomas of the skin and especially head and neck mucosa. REFERENCES 1. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 2001;15: Lum L, Beachy PA. The hedgehog response network: sensors, switches, and routers. Science 2004;304: Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in carcinogenesis. Nature 2004; 432: Zhao Y, Tong C, Jiang J. Hedgehog regulates smoothened activity by inducing a conformational switch. Nature 2007;450: Altaba Ari, Palma V, Dahamane N. Hedgehog-Gli signaling and the growth of the brain. Nat Neurosci 2002;3: Matise P, Joyner AL. Gli genes in development and cancer. Oncogene 1999;18: Daya-Grosjean L, Couve-Privat S. 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