According to the American Cancer Society, it has been estimated that in 2019 alone, there will be approximately 53,000 new cases of oropharyngeal cancers. Oropharyngeal cancers are the largest subset of head and neck squamous cell carcinomas (HNSCCs), which are the sixth most common cancer across worldwide populations. They, along with other HNSCCs, fall under a category of cancers known as Human papillomavirus (HPV)-associated cancers, and it has been found that upwards of 70% of these cancers can be attributed to high-risk HPV infections. Specifically, the high-risk HPV gene, E7, plays a key role in relieving cell cycle repression by disrupting the DREAM complex via competitive binding with p130, driving the cell cycle and cell proliferation. In order to combat this interaction, a LIN52-S20C mutation was developed, in hopes of reducing E7 binding of p130 and stabilizing the DREAM complex. We utilized human cervical cell lines, immortalized keratinocytes, and mouse fibroblasts, all of which contained the HPV16 genome, as models to observe the effects of the LIN52-S20C mutation on HPV-mediated hijacking of the cell cycle. Not only were we able to replicate the increased proliferation and upregulated DREAM gene expression in infected cells, but we were also able to observe some reversal of these effects in many of our cell models through the expression of the LIN52-S20C variant. The findings of these studies have been promising and provide a basis for future works, and we hope that the effects of the LIN52-S20C mutation can be translated into studies in in vivo models.

AIM & OBJECTIVES: Human papilloma virus (HPV) has been related to cervical infection, however, its part in Head and Neck Squamous Cell Carcinoma (HNSCC) is still debatable and is easy to refute. Suspicion of HPV causation is heightened when carcinomas arise in patients that are young and have never smoked. The present UK based study undertaken at Northampton NHS Trust endeavoured to determine the extent to which HPV is an entity in HNSCC in the UK. Furthermore, the study investigated whether sexual behaviour (as measured by sexual health clinic (SHC) attendance) is linked the acquisition of HPV associated HNSCC in young age groups. HNSCC incidences and sexual trends in the UK were collected from publicly available databases to identify if there were any changes at a national level in sexual behaviours and their influence on HNSCC in young age groups. MATERIALS & METHODS: PCR was used to evaluate the presence of HPV in biopsy samples from of 99 patients diagnosed with HNSCC at Northampton Hospital from 2006 to 2014. Patient demographics on age, sex, smoking, alcohol use and SHC attendance were also collected. All HPV PCR positive biopsies were further genotyped using an ABI 3130xl genetic analyser. Databases in the UK; including GLOBOCAN, NATSAL and PHE were searched for data on HNSCC prevalence, sexual behaviour trends and vaccine uptake. Multinomial regression explored the relationship between HPV positivity and sex, age, smoking, drinking, race and SHC attendance. RESULTS: PCR showed that 25.2% (25/99) of biopsies tested were positive for HPV and were all obtained from white participants. Most specimens (23, 92%) were high-risk (HR) HPV 16 positive with a mean age of 56 for HPV positivity and 72% of the cases 50-60 years old. Smokers were 11% in total (11/99) with most 88.9% participants (88/99) being non-smokers. HPV positivity was strongly linked with non-smoking history (p < 0.001); no alcohol abuse (p < 0.001); male gender (p < 0.001); young age less than 60 years (p < 0.001) and SHC attendance (p < 0.001). A Kruskal-Wallis post hoc test affirmed the impact of age on HPV positivity (p= < 0.05). GLOBOCAN and Cancer Research demonstrated a rising UK HNSCC pattern of over 200% for both sexes from 1975 to 2011. The three NATSAL surveys undertaken in 1990-1991, 1999-2001 and 2010-2012 demonstrated an overall increase in opposite and same sex partners. The UK average of individuals engaging in oral sex was in the younger age groups of between 16 and 54 with at least 70% of males and 63% females of that age engaging in oral sex. Finally, NASTAL 1, 2 and 3 surveys reported 20 vs 15; 25 vs 55; 55 vs 65 of males and females respectively with more than 10 sexual partners to have attended the SHC. The UK immunization take-up was over 90% countrywide. CONCLUSION: Few research studies have been conducted to date on HPV as a cause of HNSCC in the UK. The present research showed 25.2% of HNSCC to be caused by HPV, with the high risk (HR) genotype 16 (the leading cause of cervical cancer) accounting for 92% (23/25) of the cases. These outcomes affirmed the high prevalence of HR-HPV in HNSCC, with a rate of 25.2% similar to those reported previously. Routine HPV testing in those aged below 60 is therefore warranted. Smoking and drinking showed negative correlation; the young age of below 60 and attendance of the SHC for both sexes showed a positive correlation with HPV positive HNSCC. NATSAL data showed increased sexually risky behaviour coupled with attending the SHC in younger ages for both sexes. Increased sexually risky behaviour as shown in NASTAL surveys may be the reason why young age and SHC attendance is positively correlated with HPV HNSCC. The study highlights a conceivable relationship between HPV positive HNSCC in those under 60 years with no smoking history who attended the SHC. Smoking and drinking are known risks for HNSCC in those past 65 years of age; the negative association with HPV HNSCC in the young in the present research revealed smoking and drinking to have reduced association with HPV HNSCC. The reported HR-HPV positive HNSCC in young age groups inform future vaccination strategies and consequently decrease the quantity of HPV HNSCC's.

A ribonucleoproteína heterogênea nuclear K (hnRNP K) é uma proteína envolvida em processos de expressão gênica e tem sido proposta como ligante de RNAs mensageiros mitocondriais. Apesar de ser considerada um marcador de pior prognóstico no câncer de cabeça e pescoço, o papel da hnRNP K nesta doença ainda é pouco conhecido. O objetivo deste trabalho foi estudar o envolvimento da hnRNP K na mitocôndria com ênfase na bioenergética e na identificação de novos potenciais ligantes de hnRNP K. As linhagens celulares utilizadas foram de carcinoma de cabeça e pescoço (HN13 e CAL 27) com silenciamento de RNA para hnRNP K e células HEK293 com super-expressão de hnRNP K. O efeito do acúmulo celular da hnRNP K na cadeia transportadora de elétrons mitocondrial foi avaliado por meio da atividade dos complexos mitocondriais I, II e V em células HN13. A redução do nível de hnRNP K usando RNA de interferência promoveu uma diminuição da atividade dos complexos nas células HN13, indicando o envolvimento da proteína na eficiência do transporte de elétrons na cadeia respiratória mitocondrial. Células HEK293 com super-expressão da hnRNP K (HEK293/hnRNP K) e as linhagens HN13 e CAL 27 com silenciamento e redução estável de hnRNP K foram utilizadas para determinar o papel de hnRNP K no potencial de membrana mitocondrial, níveis de ATP, produção de lactato e consumo de oxigênio. Células HEK293/hnRNP K comparadas ao controle apresentaram maior nível de ATP, menor potencial de membrana mitocondrial, menor consumo de oxigênio e maior produção de lactato. As células HN13 com redução da hnRNP K apresentaram níveis mais baixos de ATP, com menor liberação de lactato para o meio extracelular e maior consumo de oxigênio. Esses resultados sugerem que o acúmulo da proteína hnRNP K tem ação importante na mitocôndria por alterar o metabolismo bioenergético celular de fosforilação oxidativa para glicólise anaeróbica. A estratégia de co-imunoprecipitação usando anticorpos para hnRNP K, digestão de proteínas com tripsina e cromatografia líquida acoplada a espectrômetria de massa foi usada para encontrar novos potenciais ligantes de hnRNP K. A análise dos dados com o software SEPro identificou 57 proteínas candidatas a ligantes da hnRNP K. Três proteínas foram validadas por co-IP e Western blotting: o fator de transcrição mitocondrial PTCD3, YB1 e PSF. Propomos que a hnRNP K apresenta função na energética mitocondrial, e provavelmente, a sua interação com PTCD3 participa desta função. / Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a protein involved in gene expression processes, which has been proposed to bind mitochondrial mRNAs. Despite it to be considered a prognostic marker in cancer, the hnRNPK role in this disease is unknown. We addressed the involvement of hnRNP K in mitochondria with emphasis on bioenergetics and identification of new potential ligands of hnRNP K. The cell lines used were from head and neck squamous cell carcinoma (HN13 and CAL 27) with RNA silencing for hnRNP K , and HEK293 cells with overexpression of hnRNP K. The effects of cellular accumulation of hnRNP K in mitochondrial electron chain carriers were assessed by the activity of mitochondrial complexes I, II and V in HN13 cells. Reduced levels of hnRNP K using RNA interference promoted a decrease in the activity of the complexes in HN13 cells, indicating the involvement of the protein in the efficiency of the electron transport in mitochondrial respiratory chain. HEK293 cells with overexpression of hnRNP K (HEK293/hnRNP K) and HN13 and CAL 27 cells with silencing and stable reduction of hnRNP K were used to determine the role of hnRNP K in mitochondrial membrane potential, ATP levels, lactate production and oxygen consumption. HEK293/hnRNP K, compared to control cells, showed higher levels of ATP, reduced mitochondrial membrane potential, lower oxygen consumption and higher production of lactate. HN13 cells with reduced hnRNP K had lower ATP levels, with lower release of lactate to the extracellular medium and higher oxygen consumption. These results suggest that accumulation of hnRNP K protein plays a role in mitochondria by changing the cellular energetic metabolism from oxidative phosphorylation to glycolysis. The strategy of co-immunoprecipitation using antibodies for hnRNP K, protein digestion with trypsin, and liquid chromatography, coupled to mass spectrometer, were used to search for new potential ligands of hnRNP K. Data analysis with software SEPro identified 57 candidate proteins binding to hnRNP K. Three proteins were validated by co-IP and Western blotting: the mitochondrial transcription factor PTCD3, YB1, and PSF. We propose that hnRNP K plays a role in the mitochondrial energetics, and probably its interaction with PTCD3 participates in this function.

Einleitung: Die zielgerichtete Therapie von Plattenepithelkarzinomen der Kopf-Hals-Region (HNSCC) durch simultanes Targeting von EGFR (epidermal growth factor-receptor) mit Cetuximab (E) und αVβ3 und αVβ5-Integrinen mit Cilengitide (Cil) könnte wegen dessen geringen Nebenwirkungen attraktiv sein. Wir analysierten die Koloniebildung epithelialer Zellen (CFec) und die Produktion pro-angiogener and pro-inflammatorischer Zytokine im Kurzzeit-Chemoresponsetest (FLAVINO). Methoden: Kollagenase-IV-verdaute Proben von 43 histopathologisch gesicherter HNSCC wurden in Laminin-beschichtete 96-well-Platten eingesät, die in Triplikaten E, Cil oder CilE in Endkonzentrationen von 66,7 µg/ml, 10 µM, und 66,7 µg/ml+10 µM enthielten. Kulturüberstände (KÜS) wurden nach 3 Tagen entnommen und adhärente Zellen Ethanol-fixiert. 39 HNSCC hatten CFec≥4/well. Interleukin 6 (IL-6), MCP-1 (monocyte chemoattractant protein 1) und VEGF (vascular endothelial growth factor A) in KÜS wurden mit ELISA quantifiziert. Ergebnisse: CFec auf Laminin wurde durch Cil, E und CilE signifikant unterdrückt. Die Produktion von MCP-1, IL-6 und VEGF wurde ebenfalls vermindert. CilE bewirkte die stärkste Suppression von CFec, MCP-1 und VEGF. Die Wirksamkeit von CilE überstieg dabei diejenige von E oder Cil allein. Der überwiegend additiv gesteigerte aber bei manchen HNSCC ausbleibende Effekt zeigt starke Heterogenität in der Response verschiedener HNSCC auf. Die IL-6-Freisetzung wurde durch E und verstärkt durch CilE, aber nicht durch Cil allein signifikant supprimiert. Schlussfolgerung: Kombiniertes Targeting von EGFR und Integrinen mit CilE erhöht die suppressiven Effekte auf CFec und pro-angiogene und pro-inflammatorische Zytokine, welche potentielle Bedeutung als Biomarker für Response erlangen.:1 Einleitung 3 1.1 Integrine – multifunktionale Transmembranproteine 4 1.1.1 Erkennungsmerkmal RGD-Sequenz 5 1.1.2 Integrine αVβ3 und αVβ5 sowie deren Rolle in Tumorangiogenese und Metastasierung 5 1.2 Cilengitide – ein Integrin-Inhibitor 6 1.3 Epidermaler Wachstumsfaktorrezeptor 8 1.3.1 Die Rolle des EGFR bei Tumoren 9 1.3.2 Cetuximab - ein EGFR-Inhibitor 10 1.4 Tumore der Kopf-Hals-Region und deren Biomarker 11 1.4.1 Therapie von Kopf-Hals-Tumoren 12 1.4.2 Klinische Erprobungen von Cilengitide 12 1.4.3 Targeted Therapie mit Cetuximab bei Kopf-Hals-Tumoren 13 1.4.4 Biomarker bei HNSCC 15 1.4.5 Interleukin 6 16 1.4.6 Vaskulärer endothelialer Wachstumsfaktor A – VEGF-A 17 1.4.7 Monozyten-Chemoattraktor Protein 1 – MCP-1 19 1.5 Chemoresponsetestung ex vivo 21 1.5.1 Der koloniebildende Kurzzeit-Chemoresponsetest FLAVINO 22 1.5.2 Flavin-induzierte photodynamische Degeneration 22 1.5.3 Ablauf des FLAVINO-Chemoresponsetests 23 1.6 Aufgabenstellung und neue Erkenntnisse 24 2 Publikationen 26 2.1 Abstract zum Vortrag zur 84. Jahrestagung der Deutschen Gesellschaft für Hals-, Nasen-, Ohrenheilkunde und Kopf- und Hals-Chirurgie 27 2.2 Publikation bei Anticancer Research 28 2.3 Abstract zum Beitrag zur 88. Jahrestagung der deutschen Gesellschaft für Hals-, Nasen-, Ohrenheilkunde und Kopf- und Hals-Chirurgie 36 3 Zusammenfassung 37 4 Literaturverzeichnis 41 5 Anlagen 49 5.1 Erklärung über die eigenständige Verfassung der Arbeit 49 5.2 Darstellung des Eigenanteils 50 5.3 Lebenslauf 52 5.4 Danksagung 53

Squamous cell carcinoma of the head and neck region (HNSCC) is an aggressive malignancy with generally poor prognosis and high mortality. The Special AT-rich binding protein 1 (SATB1) is a genome organizer protein that participates in regulating gene expression by acting as a trans-acting element as well as by recruiting chromatin remodeling complexes and enzymes. SATB1 is often overexpressed in cancer, and its possible role in tumour progression has been explored in several types of cancers and also suggested in HNSCC. However, its influence on molecular and cellular processes in HNSCC has not been examined, and, using primary cell lines, provided the basis of this thesis. This is a comprehensive study of molecular and cellular processes being affected upon siRNA-mediated SATB1 knockdown in vitro and in vivo. 15 HNSCC primary cell lines were obtained from the University of Turku and screened for SATB1 mRNA levels. The comparison of SATB1 mRNA levels with location, lymph node metastasis, disease staging (TNM) or SATB2 mRNA levels revealed no association. Hence, for deeper analysis 7 primary cell lines were selected based on growth inhibitory effects upon transient SATB1 knockdown, rather than their initial SATB1 mRNA levels. Growth inhibition upon SATB1 depletion was shown in monolayer (viable cell quantitation and colony forming ability) as well as non-adherent (spheroid assay) culture conditions. In some cell lines, cell death induced by apoptosis or retardation of cell cycle progression was observed as well. Parallel to this, using the FLAVINO assay, colony forming abilities of tumour cells from patient biopsies obtained from the University Hospital of Leipzig (Department of Otorhinolaryngology, Head and Neck Surgery) were tested post SATB1 knockdown. For molecular analysis, effects of SATB1 knockdown on transcription rates of selected oncogenes were analyzed. Among EMT markers, N-cadherin and beta catenin levels were found reduced upon SATB1 knockdown. The transcription of HER3 and its ligands Heregulin α & β was attenuated in all the seven primary cell lines, irrespectively of the growth inhibitory effects of SATB1 knockdown. These results demonstrated the role of SATB1 in the process of EMT and in autocrine signalling. Effects of HER3 inhibition on transcription rates of SATB1 were tested as well. HER3 inhibition was achieved by Patritumab, a novel monoclonal antibody against HER3. While SATB1 transcription rates remained unchanged upon HER3 inhibition, growth inhibition assays (2D and 3D) revealed that the combined use of HER1 and HER3 inhibitory antibodies provides better tumour cell inhibition over the single treatment. Finally, antitumor effects of SATB1 knockdown were monitored in vivo in two xenograft models (UT-SCC-14 and UT-SCC-42B). Treatment of tumor xenograft-bearing mice with siRNAs formulated in polymeric nanoparticles revealed reduced tumour growth, based on the knockdown of SATB1 as demonstrated on the protein level. Taken together, in this work SATB1 knockdown is demonstrated to mediate growth inhibition, induction of apoptosis, cell cycle retardation, negative impact on EMT and autocrine signaling and in vivo anti-tumour effects, thus highlighting the relevance of SATB1 in HNSCC.:156 pages

No description available.

No description available.

No description available.

Nach Daten des Robert-Koch-Instituts (RKI, Krebs in Deutschland 2015/2016) sind Tumore des Kopf- Hals-Bereichs (ICD-10 C00-C14 und C30-C32) in Deutschland bei Männern das siebthäufigste, bei Frauen das fünfzehnthäufigste Malignom. In den letzten Jahren sind dabei insgesamt leicht sinkende Neuerkrankungs- und Sterblichkeitsraten zu beobachten, wobei es regionale Unterschiede gibt. Während die Sterblichkeit unter männlichen Patienten in den alten Bundesländern gesunken ist, stieg sie in den neuen Bundesländern in den letzten 30 Jahren an. Nachdem die EXTREME-Studie (Vermorken et al., 2008) durch Hinzunahme von Cetuximab zum Duplet aus Cisplatin und 5-Fluoruracil (PFE) eine Verlängerung des Überlebens zeigen konnte, galt dieses Triplet als Standard in der Erstlinientherapie von Plattenepithelkarzinomen der Kopf-Hals-Region. Im Jahr 2020 wurden die NCCN-Leitlinien (Nationale Comprehensive Cancer Network) nach Veröffentlichung der KEYNOTE-048 Studienergebnisse (Burtness et al., 2019) angepasst, wonach sich die Erstlinientherapie nun nach dem CPS-Score richtet, der auf immunhistologischen Untersuchungen des Tumorgewebes beruht. Die Rationale unserer Analysen war die Frage nach dem bleibenden Stellenwert von PFE in der Erstlinientherapie rezidivierter, metastasierter Plattenepithelkarzinome der Kopf-Hals-Region (R/M HNSCC). In unsere retrospektive Studie konnten 124 Patienten des Universitätsklinikums Leipzig mit einem R/M HNSCC, die eine palliative Chemotherapie erhalten hatten, eingeschlossen werden. Die Auswertung der Überlebensdaten erfolgte mithilfe von Kaplan-Meier-Schätzern, Log-Rank-Tests und multivariaten Cox-Regressionen. Wir führten Subgruppenanalysen durch, um eine im klinischen Alltag möglichst handhabbare Beschreibung der Patientengruppen herauszuarbeiten, die in besonderem Maße von PFE profitieren. Ganz allgemein konnten wir feststellen, dass PFE im Gegensatz zu anderen Erstlinientherapien den in Studien beobachteten Überlebensvorteil auch in der klinischen Routine zeigte. Der Überlebensvorteil durch PFE war in unseren Analysen altersabhängig. Über alle Altersschichten hinweg sahen wir ein verlängertes Überleben durch PFE, allerdings zeigte sich dieser Gewinn an Überlebenszeit vor allem bei den jüngeren Patienten. Auch bei einer Teilung des Datensatzes bei 65 Jahren (vgl. Vermorken et al., 2008) konnten wir in beiden Gruppen einen Überlebensvorteil durch PFE sehen. Eine weitere Subgruppenanalyse zeigte, dass die Anzahl der Vorbehandlungen ein prädiktiver Faktor für den Erfolg einer palliativen Chemotherapie ist. Eine Systemtherapie nach genau einer Vorbehandlung weist hierbei im Vergleich zur palliativen Chemotherapie bei Erstdiagnose oder mehr als einer Vorbehandlung den größten prädiktiven Wert für ein verlängertes Überleben auf. Wir konnten weiterhin keinen Hinweis negativer Effekte einer vorangegangenen cisplatin-basierten Chemotherapie feststellen. Dies stellt im Kontext der Diskussion um die Toxizität kumulativer Cisplatin-Dosen einen Erkenntnisgewinn dar. Es zeigte sich, dass durch Hinzunahme weiterer Chemotherapeutika (zum Beispiel Taxane im CeFCiD- Schema PFTE, vgl. Klinghammer et al., 2019) keine weitere Verlängerung des Überlebens erreicht werden konnte. Um Verzerrungen in den Analyseergebnissen zu vermeiden, führten wir Sensitivitätsanalysen, Datenimputationen und Subgruppenanalysen durch. Trotzdem verbleiben einige Limitationen unserer retrospektiven Studie, da eine zufällige Beeinflussung der Ergebnisse durch eine möglicherweise nicht repräsentative Stichprobe nicht endgültig ausgeschlossen werden können. Die Bedeutung unserer Ergebnisse ergibt sich aus den durchgeführten Subgruppenanalysen und der daraus abgeleiteten Beschreibung der Patientengruppen, die einen besonders deutlichen Überlebensvorteil durch PFE zeigen. Wir leiten aus unseren Erkenntnissen einen weiterhin bestehenden, hohen Stellenwert von PFE in der Behandlung rezidivierter, metastasierter Plattenepithelkarzinome der Kopf-Hals-Region ab.:1 Einführung 1.1 Epidemiologie 1.2 Ätiologie und Risikofaktoren 1.3 Therapie der rezidivierten, metastasierten Tumoren der Kopf-Hals-Region 1.4 Rationale für die durchgeführte Studie 1.5 Vorstellung der Ergebnisse unserer Studie und weiterführender Subgruppenanalysen 1.5.1 Der Einfluss des Alters auf die palliative Chemotherapie 1.5.2 Zum Vergleich von Studienpatienten und Patienten aus der klinischen Routine 1.5.3 Der Einfluss von Vorbehandlungen auf die palliative Chemotherapie 1.6 Diskussion unserer retrospektiven Studie 2 Publikation 3 Zusammenfassung der Arbeit 4 Literatur 5 Anlagen Posterbeitrag zur 91. DGHNO-Jahrestagung Spezifizierung des eigenen wissenschaftlichen Beitrags Erklärung über die eigenständige Abfassung der Arbeit Verzeichnis der wissenschaftlichen Veröffentlichungen und Vorträge Lebenslauf Danksagung

No description available.