Hedgehog Targeting by Cyclopamine Suppresses Head and Neck Squamous Cell Carcinoma and Enhances Chemotherapeutic Effects

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Hedgehog Targeting by Cyclopamine Suppresses Head and Neck Squamous Cell Carcinoma and Enhances Chemotherapeutic Effects ANTICANCER RESEARCH 33: 2415-2424 (2013) Hedgehog Targeting by Cyclopamine Suppresses Head and Neck Squamous Cell Carcinoma and Enhances Chemotherapeutic Effects CHRISTIAN MOZET*, MATTHAEUS STOEHR*, KAMELIA DIMITROVA, ANDREAS DIETZ and GUNNAR WICHMANN Department of Otolaryngology, Head and Neck Surgery, University of Leipzig, Leipzig, Germany Abstract. Background: The hedgehog signaling pathway prognosis is worse for those with metastatic disease (2). (HH) is involved in tumorigenesis in a variety of human Besides known risk factors (e.g. alcohol and smoking) and malignancies. In head and neck squamous cell carcinomas occupational exposure to other poorly understood factors, the (HNSCC), Hh overexpression was associated with poor entirety of the relevant tumor biology is still unclear. prognosis. Therefore, we analyzed the effect of Hh signaling Mortality rates of HNSCC have remained almost unchanged blockade with cyclopamine on colony formation of cells from for decades, even though the detection of tumor-specific HNSCC samples. Patients and Methods: HNSCC biopsies pathways has developed rapidly and offers a number of new were cultured alone for reference or with serial dilutions of targets in tumor therapy regimes (e.g. the epidermal growth cyclopamine (5-5,000 nM), docetaxel (137.5-550 nM), or factor-receptor, EGFR, and its tyrosine kinases). Proven cisplatin (1,667-6,667 nM) and their binary combinations. standardized concepts of tumor therapy in first- or second- Cytokeratin-positive colonies were counted after fluorescent line protocols are combinations of well-established cytostatic staining. Results: Cyclopamine concentration-dependently drugs such as platinum-derivates like cisplatin, taxanes like inhibited HNSCC ex vivo [(IC50) at about 500 nM]. In binary docetaxel or others (e.g. 5-fluorouracil, 5-FU), and their combinations, cyclopamine additively enhanced the binary or tertiary combinations. These are also recommended suppressive effects of cisplatin and docetaxel on HNSCC as treatment options together with targeted therapy. Agents colony formation. Conclusion: Our findings define SMO – a successfully used in targeted therapy studies in HNSCC, so Hh component- as a potential target in HNSCC and suggest far, are mainly those targeting EGFR or interfering with its the utility of Hh targeting in future multimodal treatment intrinsic tyrosine-kinase activity (by tyrosine-kinase regimens for HNSCC. inhibitors; TKI). To date, only cetuximab (Erbitux®), a monoclonal antibody acting as an EGFR inhibitor, enhancer Head and neck squamous cell carcinoma (HNSCC) is a of EGFR degradation and trigger of antibody-dependent cell- highly aggressive cancer with poor prognosis and low overall mediated cytotoxicity, has been approved for HNSCC survival (1). Worldwide incidence rates are at approximately treatment. Data regarding its value in combination with 650,000 new cases every year, primarily diagnosed in radiotherapy or single-agent treatment have been published advanced stages (Union for International Cancer Control (3, 4). This first monoclonal antibody for HNSCC therapy (UICC III/IVA/IVB) with about 50% five-year survival; the aroused the vision of more efficient targeted treatment in the near future because the EGFR pathway is only one of numerous molecular pathogenic factors which seem to be This article is freely accessible online. responsible for HNSCC development and spread. These factors include numerous pathways which are either *These Authors contributed equally to the writing of the manuscript. dysregulated due to the presence of oncogenic proteins of human papillomavirus (HPV) subtypes and involved in cell- Correspondence to: Christian Mozet, MD, Department of cycle dysregulation (e.g. CDKN2A [p16INK4A] or increased Otolaryngology, Head and Neck Surgery, University of Leipzig, p53 degradation), but also altered expression and signaling of Liebigstrasse 10-14, 04103 Leipzig, Germany. Tel: +49 3419721700, pathways involved in angiogenesis and vascularization Fax: +49 3419721709, e-mail: [email protected] leipzig.de, http://www.uni-leipzig.de/~hno/ [vascular endothelial growth factor (VEGF) and its receptors (VEGF -R-1,2, and 3)], degradation of the extracellular Key Words: Head and neck squamous cell carcinoma (HNSCC), matrix [via activated matrix metalloproteinases (MMP)], and hedgehog pathway, cyclopamine, cisplatin, docetaxel, synergism. are mostly caused by genomic instability and somatic 0250-7005/2013 $2.00+.40 2415 ANTICANCER RESEARCH 33: 2415-2424 (2013) mutations (5). Some of the aberrant pathways might offer Table I. Description of samples from patients with head and neck additional potential targets for tumor therapy in the near squamous cell carcinoma. future. In this context of potential biomarkers and key Number Gender Localization TNM UICC Grading molecules in tumor development, a growing number of studies have now also focused on the hedgehog signaling 1 M Paranasal sinus T4aN0M0 IVA 2 pathway (HH) and suggested its pivotal role in 2 M Larynx T3N3M0 IVB 2 carcinogenesis. It is one of the most interesting and 3 M Tonsil T2N1M0 III 3 fundamental pathways in embryo development, and stem cell 4 M Larynx T2N2bM0 IVA 3 5 M Tonsil T3N2cM0 IVA 3 regulation, and even carcinogenesis. In the recent past, 6 M Oral cavity T4aN2cM0 IVA 2 (re)activated Hh pathway and overexpression of its signaling 7 M Supraglottic T2N2bM0 IVA 3 components have been detected in a variety of solid human 8 F Metastasis cervical TxN3M0 IVB 3 malignancies, and it was held responsible for tumorigenesis, 9 M Tonsil T3N2bM0 IVA Not defined spread, and poor prognosis in tumors of the pancreas (6), 10 M Metastasis cervical T0NxM1 IVC 2 11 M Metastasis cervical TxNxM1 IVC 2 prostate (7), breast (8), and stomach (9). Promising anti- 12 M Nasopharynx T4aN2bM0 IVA Not defined tumoral results in animal xenograft models by blocking the 13 M Oral cavity T4aN0M0 IVA 2 Hh signaling pathway were published. In brain and skin 14 F Larynx T4aN1M0 IVA G2 tumors, phase II studies with Hh pathway inhibitors (e.g. 15 M Oral T4bN3M0 IVB 2 GDC-0449) have been successfully performed, and the 16 M Oral T2N0M0 II 3 17 M Metastasis cervical TxN2bM0 IVB 3 primary data have aroused the hope of clinical researchers 18 M Paranasal sinus T4aN0M0 IVA 3 (10). However, the relevance of the Hh pathway in HNSCC is still unclear. The first hints of the importance of Hh Localization, stage [TNM, Union of International Cancer Control expression in HNSCC were published by Chung et al. (11), (UICC)], and grading of biopsies analyzed in the FLAVINO assay who observed an association of high GLI1-expression, the according to the Cancer Staging Manual (Sixth Edition) published by the American Joint Committee on Cancer, 2009 (19). transcription factor of the Hh pathway, with poor survival of patients with head and neck cancer treated with radiation therapy. The first data about Hh protein (over)expression in HNSCC were published recently by Schneider et al. (12) and tumors or recurrent disease (Table I). After resection, probes were immediately placed into tubes containing tumor medium (TM; see Wang et al. (13), but they did not consider all components. below) and immediately transferred to the laboratory, weighed, and Our own quantitative analysis of Hh expression in HNSCC studied by means of a quality controlled colony formation assay underlines the meaning of SHH, GLI2 and in particular GLI1 (FLAVINO assay) under flavin-protecting conditions as described (14). Assuming that Hh overexpression in HNSCC influences previously (15-18). Since colony formation of the tumors ex vivo tumor growth and spread, Hh inhibitory agents might have was not always sufficient for analysis of dose-response curves, not beneficial effects, and may possibly add to the tumor- all of them could be investigated. Included in this study were 18 inhibiting effects of cytostatic drugs. The aim of this study HNSCC samples fulfiling the inclusion criteria of pathophysiologically confirmed HNSCC plus sufficient colony was to determine the effects of the steroid alkaloid formation of epithelial cells [≥4 colonies consisting of ≥3 vital cells, cyclopamine, a smoothened (SMO) and subsequent Hh (CFEC)] in untreated controls. The stages of the 18 further signaling inhibitor, on tumor growth and colony formation of investigated HNSCC following the criteria of the American Joint epithelial cells from HNSCC ex vivo. We initially investigated Committee on Cancer (AJCC) (19) are shown in Table I. The the impact of cyclopamine on growth of the cell-line KB, but, investigational study was approved by the Ethics Committee of the however, mainly focused on the impact of cyclopamine on Medical Faculty of the University Leipzig (no. 201-10-12072010 colony formation of primary epithelial cells derived from and no. 202-10-1207210). biopsy samples of HNSCC. We were interested in its ability Materials. TM was used as the cell culture medium for to affect the latter if applied alone or in combination with the chemoresponse testing of the HNSCC samples as well as of the KB guideline-conforming cytostatic drugs cisplatin and docetaxel cells for quality control. TM consisted of phenol red and flavin-free and analyzed the mode of action exerted by cyclopamine in RPMI-1640 (Bio&Sell, Feucht, Germany) supplemented with 10% binary mixtures. To the best of our knowledge, this is the first (v/v) fetal calf serum (FCS; Invitrogen®, Darmstadt, Germany), study on the effect of Hh blockade in HNSCC. amikacin, nystatin, penicillin,
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