11 Mechanism of perineural invasion in head and neck cancer

e30

Abstracts / Oral Oncology 51 (2015) e27–e55

we found that miR-542-3p over-expression reduced cell viability and improved chemotherapy-induced apoptosis in HNSCC tumor cell lines. Upon miR-542-3p over-expression, we observed a dose-dependent down-regulation of Survivin levels, which could be rescued by enforced ectopic Survivin expression but not by a Survivin mutant currently isolated from primary tumor cells. Bioinformatic prediction as well as reporter assays confirmed direct targeting of Survivin by miR-542-3p. Targeting Survivin by siRNA or with miR-542-3ploaded nanoparticles synergistically sensitized HNSCC tumor cells to irradiation- or chemotherapeutic-induced cell death. Conclusion: Collectively, miR-542-3p seems to exerts its tumor suppressive function in HNSCC by targeting Survivin. Exploiting miR-542-3p re-expression by chemical or genetic decoys could be a promising therapeutic strategy for treating HNSCC. doi:10.1016/j.oraloncology.2015.02.011

9 Matrix remodeling in head and neck squamous cell carcinomais J. Dudas a, A. Fullar b, M. Bitsche a, A. Romani a, I. Kovalszky b, H. Riechelmann a a b

Innsbruck, Austria Budapest, Hungary

Introduction and Background: Extracellular matrix (ECM) remodeling is an inherent characteristic of invasive head and neck squamous cell carcinoma (HNSCC). We investigated the regulation of gene expression, synthesis and activation of ECM components and matrix remodeling enzymes in a co-culture model of head and neck squamous cell carcinoma (HNSCC) and in HNSCC tissue. Materials and methods: Periodontal ligament fibroblasts (PDL) and SCC-25 HNSCC cells were co-cultured for seven days, followed by mRNA and protein expression analysis of ECM components, ECM receptors, matrix metalloproteinases (MMPs) and proteinase inhibitors. The activity of MMPs was analyzed using gelatinase zymography. MMP-9, fibronectin and ‘‘antiadhesion proteins’’ were investigated by immunohistochemistry in HNSCC tissue. Results: In co-cultured fibroblasts, in addition to fibronectin (FN), the ‘‘antiadhesion proteins’’ tenascin C (TSC), thrombospondin 1 (TSP1) and latent TGF-beta binding protein 2 (LTBP2) were upregulated. In lower levels: FN, TSC and TSP1 were also produced by the tumor cells. In co-cultured fibroblasts, alpha 1 and 5 and beta 1 integrins, in co-cultured tumor cells alpha 3 and 5 integrins were upregulated. MMP-1 was upregulated in both co-cultured tumor cells and fibroblasts, MMP-2 in co-cultured fibroblasts, and MMP-9 in co-cultured tumor cells. MMP inhibitors were mainly produced in fibroblasts. The activation of MMP-2 required the co-culture with tumor cells. MMP-9 was exclusively produced in tumor cells. In sections of formalin-fixed paraffin embedded HNSCC tissue, FN and LTBP2 were detected in fibroblastic stroma surrounding tumor cell nests; TSP1 was detected both in tumor cell nests and in the surrounding fibroblastic stroma. Extended MMP-9 immunohistochemical staining was found in 27 of 280 HNSCC tissue samples, in a subpopulation of cells in tumor cell nests. Conclusion: Fibroblast – tumor cell communication is a major pathway of matrix remodeling in HNSCC tumor tissue. Carcinomaassociated fibroblasts produce new matrix elements as TSP1, TSC and LTBP2, both tumor cells and fibroblasts upregulate integrin receptors, and they also share tasks in synthesis and activation of matrix remodeling enzymes. Intensive matrix re-organization takes place at the borders of tumor cell nests. doi:10.1016/j.oraloncology.2015.02.012

10 Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients C. Schrader, M. Kolb, K. Zaoui, C. Flechtenmacher, N. Grabe, K.-J. Weber, P.K. Plinkert, J. Heß Heidelberg, Germany Introduction: The Kallikrein-related peptidase 6 (KLK6) belongs to a family of 15 secreted serine proteases with trypsin or chymotrypsin-like activity. Deregulation of KLK6 expression occurs in neurodegenerative and skin disorders, and is a common event in cancer. Most cancers were characterized by a strong increase of KLK6 transcript and protein levels as compared to normal tissues. Consequently, KLK6 represents a promising biomarker of early diagnosis and/or unfavorable prognosis in several human malignancies. However, the expression of KLK6 head and neck squamous cell carcinoma (HNSCC), and its molecular function has not been addressed so far. Materials and methods: We conducted loss-of-function and gainof-function approaches in mucosal tumor cell lines to investigate the contribution of KLK6 in the regulation of tumor development and malignant progression. We demonstrate that silencing of KLK6 expression promotes tumor cell proliferation, migration and invasion in vitro. This phenotype is accompanied by the induction of epithelial-to-mesenchymal transition (EMT) and augments resistance against irradiation. Tissue microarrays with primary HNSCC samples from a retrospective patient cohort (n = 162) were stained by immunohistochemistry and the correlation between KLK6 staining and survival was addressed by univariate Kaplan–Meier and multivariate Cox proportional hazard model analysis. Results: KLK6 expression was detected in HNSCC cell lines (FaDu, Cal27 and SCC25), but not in HeLa cervix carcinoma cells. ShRNAmediated silencing in FaDu cells and ectopic expression in HeLa cells unraveled an inhibitory function of extracellular functionary KLK6 on tumor cell proliferation and mobility. FaDu clones with silenced KLK6 expression displayed molecular features resembling EMT and exhibited higher resistance against irradiation. Low KLK6 protein expression in primary tumors from oropharyngeal and laryngeal SCC patients was significantly correlated with poor progression-free (p = 0.001) and overall survival (p < 0.0005), and served as an independent risk factor for unfavorable clinical outcome. Conclusions: This study demonstrates that low KLK6 expression serves as unfavorable risk factor for progression-free and overall survival of HNSCC patients, suggesting a context-dependent role of KLK6 in different human tumor entities. The presented data further provide experimental evidence that KLK6 is a key regulator of tumor cell proliferation, motility, and response to irradiation by modulating epithelial-to-mesenchymal transition. doi:10.1016/j.oraloncology.2015.02.013

11 Mechanism of perineural invasion in head and neck cancer S.W. Kim Pittsburgh, PA, United States Introduction: Perineural invasion (PNI) is one of the most prominent adverse prognostic factors in head and neck squamous cell carcinoma (HNC). However, the factors that regulate the interaction between nerves and HNC cells are poorly understood. In this study, we examined the hypothesis that neuronally produced neuregulin 1 (NRG1) and its receptor, HER3, expressed by HNC cells mediate the process of PNI. Materials and methods: The expression of HER3 in HNC cell lines were examined using Western blotting. The effects of HER3 activa-

Abstracts / Oral Oncology 51 (2015) e27–e55

tion on HNC cell lines were studied using migration and invasion assays. The effects of HER3 inhibition on HNC cell lines were studied with the use of lentiviral HER3 shRNA as well as therapeutic antiHER3 antibodies. We utilized novel co-culture assays to determine the effects of neurons on the migration and invasion of HNC cells. Lastly, a novel, dorsal root ganglion (DRG) based neurite-tumor interaction (NTI) assay was utilized to model and study the effects of HER3 downregulation on the movement of HNC cells along culture neurites. Results: HNC cell lines were found to express HER3. The activation of HER3 resulted in the activation Akt, MAPK, and focal adhesion kinase (Fak). The activation of HER3 also resulted in increased migration and invasion of HNC cells. Co-culture of HNC cells with neurons resulted in increased migration of HNC cells in a HER3 dependent fashion. Furthermore, co-culture of HNC cells with DRG in Matrigel also resulted in chemotactic invasion of the HNC cells towards the DRG in a HER3 dependent fashion. Lastly, the perineural migration of HNC cell along cultured neurites in NTI assay was inhibited by HER3 downregulation in HNC cells. Discussion: We have produced data using novel assays that suggest that the process of PNI in HNC may be mediated, at least in part, by NRG1 and HER3. Given that other members of the ErbB family have been proven to be effective targets in HNC and that therapeutic antibodies against HER3 are already in clinical development, the NRG1:HER3 axis represents an ideal target that can be explored as novel targeted therapy for HNC patients with PNI. doi:10.1016/j.oraloncology.2015.02.014

Session 3 – Use of high-throughput and omics technology to understand tumor–host interaction 12 Invited lecture: High-throughput functional genomics – An emerging field in head and neck cancer research R.H. Brakenhoff Amsterdam, Netherlands Introduction: Head and neck squamous cell carcinomas (HNSCC) belong to the six most frequent tumors worldwide, and the incidence is rising. HNSCCs develop in the mucosal linings of the upper aerodigestive tract, and are preceded by prenoplastic changes, a phenomenon coined as ‘‘field cancerization’’. Risk factors for HNSCC are smoking and excessive consumption of alcohol. Moreover, there are genetic predisposition syndromes, most particularly Fanconi anemia, associated with a very high risk for HNSCC. In recent years it became evident that also infection with the human papillomavirus (HPV) causes HNSCC, particularly tumors that arise in the oropharynx. HNSCC caused by HPV is a distinct class of tumors, both at the clinical and molecular level. Despite the application of combined treatment modalities that may severely impact quality of life with disfigurement, problems with speech and swallowing, the prognosis of HNSCC remains disappointing. New effective and less toxic treatments are urgently awaited. As a first step we tried to identify novel druggable gene targets. Material and methods: Two tumor cell lines were transfected with a genome-wide siRNA library, and cell survival assayed. Sublibraries of lethal siRNAs were rescreened on a larger panel of HPV-positive and HPV-negative cell lines as well as primary fibroblasts. Data were normalized and compared by bioinformatics. Druggable gene targets were mined. Results: We identified 362 genes that cause cell death in cancer cell lines, while only 20% of these cause cell death in primary fibroblasts. There were large differences between cell lines. One of the

e31

gene hits for which a small molecule inhibitor was available was analyzed in more detail. The inhibitor showed high efficacy in xenografted mouse models. Conclusion: In head and neck cancer only few druggable oncogenic mutations occur. Nonetheless using functional genetic screens and genome editing methods multiple gene targets can be identified that might be exploited for improved treatment protocols. doi:10.1016/j.oraloncology.2015.02.015

13 No molecular evidence for the role of the genetic background of the host in explaining lymph node metastasis in head and neck cancer S. Mes, B.J.M. Braakhuis, E. Bloemena, W.N. van Wieringen, C.R. Leemans, R.H. Brakenhoff Amsterdam, Netherlands Introduction: It has been shown for head and neck squamous cell carcinomas (HNSCC), that a specific gene expression profile in the primary tumor has predictive value for the presence of a lymph node metastasis. It is possible that these expression profiles are a result of acquired cancer-associated genetic aberrations during tumorigenesis. The alternative hypothesis, based on genetic studies in the mouse, is that the variation in the genome of the host determines the risk for metastasis. Aim of this study was to measure the global gene expression of the normal mucosa and the tumor as control of HNSCC patients to establish whether there is a difference between patients with and without lymph node metastasis. In addition, the paired analysis allowed the assessment of differential gene expression of HNSCC versus corresponding normal mucosa. Material and methods: We examined the global gene expression of normal oral mucosa and the paired tumors from 22 HNSCC patients of whom eight patients were with and fourteen without lymph node metastasis. Five HNSCC were in the oropharynx and 17 in the oral cavity; only HPV-negative tumors were selected. Cross-validation was used to establish the predictive value of a gene expression profile. Results: We showed in this case-control comparison that a gene expression profile in tumors had significant predictive value, as previously reported. When using expression profiles of the corresponding normal mucosa, however, the predictive value was lower and can be considered of little or no value for the prediction of lymph node status. Approximately 1600 unique and mapped transcripts were significantly and differentially expressed between tumor and mucosa, based on an p-value <0.05 and a fold-change 60.5 or P2.0. Canonical pathways related to immune cell adhesion and extravasation and extra-cellular matrix remodeling were altered in tumor cells. Conclusions: This study provides evidence that the metastatic potential in HNSCC is likely the result of the cancer-associated genetic changes, and not related to the genetic variation in the host. Second, we obtained a comprehensive and valuable view on the gene expression difference between HNSCC and paired normal mucosal tissue. doi:10.1016/j.oraloncology.2015.02.016

14 Next generation sequencing (NGS) approach to identify genetic alterations in head and neck squamous cell carcinoma F. Niehr a, R. Konschak a, S. Liebs b, M. Munz a, V. Budach a, U. Keilholz a, I. Tinhofer a a b

Berlin, Germany Heidelberg, Germany