O carcinoma de cabeça e pescoço (HNSCC) é um dos tipos de câncer que mais acomete as pessoas ao redor do mundo, sendo responsável por 300 mil mortes anualmente. Essa alta taxa de mortalidade está diretamente associada ao diagnóstico tardio, ausência de biomarcadores e tratamento inespecífico. Diversos estudos mostraram que a expressão desregulada da classe de serino proteases do tipo II está intimamente relacionada com a etiologia de diversos tipos de carcinomas, como é o caso da via proteolítica da matriptase. Ainda, a matriptase, no contexto normal da descamação epitelial, ativa as KLKs 5 e 7, as quais são inibidas por LEKTI. A proteína LEKTI, codificada pelo gene SPINK5, possui 15 domínios diferentes, os quais são secretados de forma individual para a matriz extracelular, onde participa da inibição das KLKs 5 e 7. Resultados ainda não publicados do nosso laboratório mostraram que LEKTI é capaz de inibir o fenótipo pré-maligno causado pela superexpressão da matriptase na camada basal do epitélio de camundongos, quando superexpresso nessa mesma região. Ainda, marcações imunohistoquímicas de LEKTI, em amostras de carcinomas orais de humanos, mostraram que essa proteína está também presente nos carcinomas de cabeça e pescoço. Dessa forma, a hipótese levantada neste estudo é a de que LEKTI possui papel inibitório durante a carcinogênese de cabeça e pescoço. Sendo assim, para testar nossa hipótese, a presente dissertação buscou nocautear LEKTI, por meio da tecnologia de edição gênica CRISPR/Cas9, em linhagens de queratinócito imortalizado, através da dissecção por biologia celular, da função de LEKTI nestas células. Os resultados, obtidos por meio de Western Blot, imunofluorescência e sequenciamento das células potencialmente nocauteadas, mostram as dificuldades e desafios de nocautear células hipotetraplóides, como é o caso da linhagem celular utilizada neste estudo. / Head and neck squamous cell carcinoma (HNSCC) is one of the most common type of cancers around the world and it\'s responsible for 300.000 deaths every year. This high mortality rate is directly related with late diagnosis, lack of biomarkers and specific treatment. A fair amount of studies have shown that the deregulated expression of type II serine proteases, such as the matriptase proteolytic pathway, is intimately related with the etiology of a large number of carcinomas. Still, in a normal epithelial desquamation context, matriptase is able to activate KLKs 5 and 7, which are inhibited by LEKTI. The protein LEKTI, encoded by SPINK5 gene, has 15 different domains, which are secreted individually to the extracellular matrix, where acts inhibiting KLKs 5 and 7. Non published results from our laboratory has shown that LEKTI is able to inhibit the pre-malignant phenotype caused by the super-expression of matriptase in the epithelia\'s basal layer of mice, when super-expressed in the same region. Moreover, immunohistochemistry staining of LEKTI in human oral carcinomas showed that this protein is also present in head and neck carcinomas. In this way, the hypothesis of this study is that LEKTI has an inhibitory role during carcinogenesis of head and neck carcinomas. Therefore, to test our hypothesis, this dissertation aimed to knockout LEKTI in an immortalized keratinocyte cell line, using CRISPR/Cas9 editing technology, through cell biology dissection of LEKTI function in those cells. The results, obtained by Western Blot, immunofluorescence and sequencing of the cells, shows the difficulties and challenges to knockout cells that are hypo-tetraploids, just like HaCaT cell line.

An 100 Tumorproben mit HNSCC (head and neck squamous cell carcinoma) von 92 Patienten wurde mit Hilfe des FLAVINO-Assays erstmalig das Adhärenzverhalten von Plattenepithelzellen bei unterschiedlichen Beschichtungen untersucht und anschließend eine Ex-vivo-Chemoresponsetestung durchgeführt. Hierbei kamen als Beschichtungsproteine humanes Laminin [L], humanes Fibronektin [hFN], Kollagen I [K] aus Rattenschwänzen und eine vorteilhafte Mischung aus unterschiedlichen extrazellulären Proteinen [ECM] zur Anwendung. Zusätzlich wurde eine ECM-Platte noch mit 2,2 Gy einmalig bestrahlt. Ziel der Chemo-responsetestung war es, den Einfluss des Integrin-Inhibitor Cilengitide [Cil] auf HNSCC gegenüber den Standardtherapeutika Cisplatin [Cis], Docetaxel [DTX] und Cetuximab [Cetux] zu testen und letztendlich eine optimale Kombination zur Verbesserung der Prognose für HNSCC zu gewinnen. 41% der Tumorproben zeigten ein Koloniewachstum, wobei die höchste Koloniebildung auf der bestrahlten ECM-Beschichtung ausgezählt werde konnte, dicht gefolgt von der Kollagenbeschichtung. 10 µM Cil als Monotherapie konnten bei HNSCC eine Koloniereduktion von 23% erzielen, 66,6 µg/ml Cetux von 53% und beide in Kombination 55%. Cis als potente Monosubstanz in Verwendung 6,667 µM erzielten eine fast vollständige Koloniereduktion von 96%, wohingegen 550 nm DTX nur 66% Reduktion erreichten. In binärer, tertiärer und quartärer Kombination aller vier Chemotherapeutika ist nur eine komplette Koloniereduktion zu erreichen, wenn Cis in der Dosierung 6,667 µM verwendet wird. Cil in Kombination mit DTX, Cetux und Cis konnte nach zusätzlicher Bestrahlung eine 100%ige Koloniereduktion erreichen und kann somit als potenter Radiosensitizer angesehen werden.

In der vorliegenden Arbeit wurde die Expression des Hedgehog Signaltransduktionsweges (Hh) in Kopf-Hals-Tumoren (Head and Neck Squamous Cell Carcinoma, HNSCC) im Vergleich zu gesunder Mukosa immunhistochemisch untersucht und quantitativ analysiert. Dabei wurde die Expressionsstärke mit Hilfe einer eigens weiterentwickelten objektiven Auswertungsmethode analysiert, die sich auf die Rot-Grün-Blau(RGB)-Farblehre stützte. Untersucht wurden histologisch gesicherte Proben von Kopf-Hals-Tumoren und gesunde Mukosa der Mundhöhle. Nach Färbung mittels spezifischen Immunfluoreszenzantikörpern gegen die Hedgehog Komponenten Sonic Hedgehog (SHH), Patched-1 (PTCH1), Patched-2 (PTCH2), Smoothened (SMO), Glioma-Associated Oncogene Homolog-1, -2 und -3 (Gli-1, Gli-2 und Gli-3) erfolgte die bildanalytische Auswertung der Fluoreszenzsignale. Dabei zeigte sich eine deutliche Überexpression aller Hh-Komponenten in den Tumorproben gegenüber der gesunden Mukosa. Die Expression des Transkriptionsfaktors Gli-1 erreichte dabei etwa zehnfach höhere Werte als in der Mukosa und die des Liganden SHH etwa vierfach höhere Werte. Die nachgewiesene Überexpression des Hh-Signalwegs in den Tumorproben deuten wir als potentiellen Kofaktor in der Genese von Kopf-Hals-Tumoren. Eine mögliche Bedeutung für neue zielgerichtete Therapieansätze in der Zukunft wird diskutiert.

Despite the incorporation of the epidermal growth factor receptor (EGFR) inhibitor cetuximab into the clinical management of recurrent and metastatic (R/M) head and neck squamous cell carcinoma (HNSCC), only a small subset of patients responds to cetuximab, despite EGFR overexpression in virtually all of their tumors. At this time, there is a lack of validated predictive biomarkers to predict which patients will respond to cetuximab. Our previous work suggests that cetuximab activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the implications of activating this pathway are unclear. The IL-1 pathway plays a central role in immune response and displays both pro-tumor and anti-tumor activities. IL-1 may promote tumor growth by upregulating the secretion of pro-inflammatory mediators involved in angiogenesis and metastasis. On the other hand, IL-1 signaling may promote antitumor immunity via enhancement of natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell activity, which are important mechanisms of action of cetuximab. The goal of this work is to determine how modulation of the IL-1 pathway affects HNSCC tumor response to cetuximab and if IL-1 may serve as a predictive biomarker for patient response to cetuximab. Blockade of IL-1 signaling did not enhance the anti-tumor efficacy of cetuximab, while IL-1α overexpression and treatment with recombinant IL-1α and IL-1α nanoparticles increased HNSCC tumor response to cetuximab in immunodeficient and immunocompetent HNSCC mouse models. Mechanistically, these results appear to be due to activation of an anti-tumor NK and T cell-mediated immune response. Additionally, we found that both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and inducible nitric oxide synthase (iNOS) activity may be involved in the efficacy of IL-1-induced ADCC against cetuximab-coated HNSCC cells. Altogether, these results suggest that IL-1 signaling is necessary for HNSCC tumor response to cetuximab. Furthermore, we have shown that pre-treatment serum and tumor IL-1 ligands can predict progression-free survival of HNSCC patients treated with standard-of-care cetuximab and chemotherapy, cetuximab combined with other targeted therapies, and cetuximab monotherapy. Overall, we propose that IL-1α warrants further study as a novel therapeutic to enhance response to cetuximab and as a predictive biomarker for HNSCC response to cetuximab.

Bei dieser Arbeit handelt es sich um eine retrospektive Studie bezüglich Inzidenz und Therapie von Mundhöhlen- und Oropharynxkarzinomen, welche in dem Zeitraum von 1993 bis 2009 in der HNO-Klinik der Universität Leipzig als „High Volume Center“ behandelt wurden, mit besonderem Augenmerk auf die Stadien III-IV nach UICC/AJCC. In unserer Studie konnten wir eine Zunahme der an der Universität in Leipzig registrierten Patienten mit Mundhöhlen- und Oropharynxkarzinomen über die Jahre 1993 bis 2009 verzeichnen. Männer waren mehr als fünfmal so häufig betroffen wie Frauen. Diese Verteilung hielt sich über den Beobachtungszeitraum konstant. Bei den weiblichen Patientinnen wurden im Durchschnitt niedrigere Tumorstadien bei Erstdiagnose festgestellt. Frauen hatten insgesamt eine höhere 5-Jahresüberlebensrate. Die Stadienverteilung nach UICC/AJCC bei Erstdiagnose eines Mundhöhlen- und Oropharynxkarzinoms blieb über die Jahre hinweg annähernd gleich. Es wurden meist hohe Stadien festgestellt (62,7% Stadium IV). Das mittlere Erkrankungsalter von Mundhöhlen- und Oropharynxkarzinomen sank über den Beobachtungszeitraum. Das Alter bei Rezidivmanifestation blieb allerdings über die Jahre gleich und lag unter dem durchschnittlichen Alter bei Erstdiagnosestellung. In diesem Zeitraum wird in der Literatur eine zunehmende Infektionsrate mit HPV beschrieben, welche gemäß der Literaturdaten mit einem jüngeren Erkrankungsalter einhergeht. Über die Jahre fanden wir in dem von uns untersuchten Patientenkollektiv der Universitätsklinik Leipzig eine stetige Verbesserung der mittleren Überlebensraten. In zeitlicher Assoziation zu dieser Entwicklung fand eine Änderung der Therapiemodalitäten insbesondere mit Einführung der systemischen Chemotherapie ab ca. 2004 in Richtung einer zunehmenden multimodalen Therapie statt. Es konnte ein Vorteil im Gesamtüberleben nach einer kombiniert-operativen Therapie mit adjuvanter Radiochemotherapie gegenüber allen anderen Therapieformen gezeigt werden. Insbesondere im Stadium IV nach UICC/AJCC zeigte sich ebenfalls ein Vorteil der kombinierten Radiochemotherapie gegenüber einer alleinigen Radiotherapie.

Selective up-regulation of the mRNA of LOXL4, a member of the LOX matrix amine oxidase family, significantly correlated with lymph node metastases and higher tumour stages in head and neck squamous cell carcinomas (HNSCC). To evaluate the diagnostic and prognostic value of the protein we produced an antibody specific for LOXL4 and assessed the expression in 317 human HNSCC specimens. The LOXL4 protein was detected in 92.7% of primary tumours, in 97.8% of lymph node metastases and in affected oral mucosa with high-grade dysplasia, but was absent in various non-neoplastic tissues of the head and neck. TNM categories and overall survival did not link to grades of immunoreactivity. Studies in cultured primary hypopharyngeal HTB-43 carcinoma cells detected perinuclear and cell surface expression of LOXL4, but no nuclear localisation. Therefore, its interactive SRCR-domains and catalytic activity combined with tumour cell specific expression and cell surface associated location indicate multiple functions in tumour cell adhesion and interactions with the extracellular matrix. Our data suggest that LOXL4 is useful both as tumour marker and target in the treatment of HNSCC.

No description available.

Head and neck cancer accounts for approximately 3 percent of all cancers in the United States, and over 90% of them are head and neck squamous cell carcinoma (HNSCC). Chemotherapeutic drugs that treat HNSCC can activate BCL-2 family dependent apoptosis. Pro-apoptotic NOXA induced by adenovirus (Ad-NOXA) or fenretinide inactivates anti-apoptotic MCL-1, while ABT-263 can inactivate other anti-apoptotic BCL-2 family members such as BCL-2 and BCL-XL. We used p53 inactive HN8 and HN12, p53 wild-type UMSCC1, and HPV-positive UMSCC47 human HNSCC cell lines and five mouse HNSCC cell lines. Cells were treated with Ad-NOXA, ABT-263, and fenretinide alone or in combinations. Combinational treatment of ABT-263 with Ad-NOXA or fenretinide enhanced cell death among all cell lines we tested regardless of p53 status. These findings support the hypothesis that combinational treatment of Ad-NOXA or fenretinide with ABT-263 increases cell death by simultaneously inhibiting all anti-apoptotic BCL-2 family proteins in HNSCC cells.

Epidermal growth factor receptor (EGFR) is highly expressed in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) and is a key regulator of tumor cell growth and survival. Erlotinib, also known as tarceva, (a first-generation) and afatinib, also known as giotrif, (a second-generation) are tyrosine kinase inhibitors (TKIs) of EGFR. These TKIs are recognized therapeutic agents in these tumor types, as they inhibit EGFR signaling but show limited activity as single agents. Novel strategies will likely require EGFR-TKIs combination with an agent(s) that will enhance their therapeutic efficacy. Recently, we have demonstrated that combining statins, inhibitors of the mevalonate pathway, with erlotinib enhanced EGFR inhibition and induced synergistic cytotoxicity through the activation of cellular integrated stress response pathway (ISR) regulated by the induction of activating transcription factor 3 (ATF3). In our Phase I clinical trial, combining rosuvastatin with erlotinib, while demonstrating clinical activity, this treatment also showed statin-induced myopathies likely the result of diminished ubiquinone levels, which limited their utilization. Therefore, alternative strategies are warranted. Targeting geranylgeranyl diphosphate (GGPP) synthesis or its incorporation, a downstream mevalonate metabolite, represents such an approach with the potential to circumvent statin-associated toxicities but retain the efficacy in combination with EGFR inhibitors. In this project, we evaluated the effect of the combination of geranylgeranyl transferase-I inhibitor (GGTI-298) with the EGFR inhibitor, tarceva, (aim 1) and a GGPP synthase inhibitor, digeranyl bisphosphonate (DGBP), with the EGFR inhibitor, afatinib, (aim 2). For aim 1, we demonstrated that GGTI-298 treatment induced ATF3 expression in SCC9 and SCC25 cells and in a cohort of ex-vivo tumor tissues. Furthermore, GGTI-298 and tarceva induced synergistic cytotoxicity in SCC cells that was dependent on ATF3 expression, as ATF3 deficient murine embryonic fibroblasts (ATF3-/- MEFs) displayed attenuated cytotoxicity in response to GGTI-298 alone and in combination with tarceva. Similarly, SCC9 sub-lines that were selected as resistant to GGTI-298 through prolonged exposure to this agent also failed to demonstrate synergy with treatment of GGTI-298 in combination with tarceva. For aim 2, we demonstrated that the specific GGPP synthase inhibitor, DGBP, induced cytotoxicity in SCC cells. We further demonstrated this specificity as specific shRNA targeting of GGPP synthase as well as the inhibitor DGBP significantly enhanced the cytotoxic activity of the EGFR-TKI afatinib in SCC cells. DGBP as well as afatinib treatments induced ATF3 expression in SCC cells in vitro and in a cohort of ex-vivo tumor tissues. Co-administration of the downstream metabolite GGPP inhibits the induction of ATF3 and the cytotoxic and apoptotic effects associated with DGBP treatment. Furthermore, the synergistic cytotoxicity induced by the combination of DGBP and afatinib in SCC cells was also dependent on the expression of ATF3 through the induction of cellular stress response pathways. Taken together, these results suggest the potential clinical utility of combining downstream mevalonate inhibitors (GGTI-298 or DGBP) with EGFR inhibitors in HNSCC patients as a novel and more refined combination therapeutic approach.

No description available.