No description available.

No description available.

Die strahleninduzierte Mucositis enoralis ist eine der bedeutendsten und häufig dosislimitierenden frühen Nebenwirkungen der Strahlentherapie fortgeschrittener Kopf Hals Tumoren. Bis heute hat sich noch kein allgemein gültiges Konzept zur Therapie und Prophylaxe der Mundschleimhautentzündung durchsetzen können. Ein Ansatz zur selektiven, auf der Tumorbiologie beruhenden Beeinflussung der Strahlenempfindlichkeit von Tumoren ist die Blockade des epidermalen Wachstumsfaktor-Rezeptors (EGFR). In Kombination mit Strahlentherapie sollen so die lokale Tumorkontrolle und die Heilungschancen verbessert werden. Die Wirkung der Tyrosinkinase-Inhibitoren BIBX1382BF und Erlotinib auf histomorphologische Parameter in der Mundschleimhaut sowie auf die Expression der als Stammzellmarker diskutierten Proteine p63, Integrin β1 und CD44 wurde in der vorliegenden Arbeit im Vergleich zur alleinigen fraktionierten Bestrahlung untersucht. Für die histologischen Studien erfolgte die zweiwöchige fraktionierte Bestrahlung der Schnauzen von Mäusen des Inzuchtstammes C3H/Neu mit zehn Fraktionen zu je 3 Gy (Tag 0-4, Tag 7-11). Die Versuche gliederten sich in vier Gruppen: • I/A (54 Tiere) und II/A (40 Tiere): fraktionierte Bestrahlung, keine weitere Behandlung • I/B (51 Tiere): fraktionierte Bestrahlung, zusätzlich orale Gabe von BIBX1382BF, 50 mg/kg KG per os, von Tag 0-14 je 30 min nach der Bestrahlung • II/B (35 Tiere): fraktionierte Bestrahlung, zusätzlich orale Gabe von Erlotinib, 50 mg/kg KG per os, von Tag 0-11 je 30 min nach der Bestrahlung. Die Entnahme der Zungen erfolgte im Versuch I bei jeweils drei Tieren pro Tag von Tag 0 bis Tag 17. Im Versuch II wurden an den Tagen 0, 2, 4, 6, 8, 10, 12 und 14 jeweils die Zungen von fünf Tieren entnommen. Anschließend folgten die Fixierung der Zungen in Formalin, die Einbettung in Paraffin und die Anfertigung 3 µm dicker Gewebeschnitte. Die Zungenpräparate wurden für die histologischen Untersuchungen mit Hämatoxylin-Eosin gefärbt. Für die immunhistochemischen Färbungen wurde die ABC-Methode eingesetzt. Das Epithel der Zungenunterseite wurde lichtmikroskopisch hinsichtlich Zellzahl, Schichtdicke und Expression der potentiellen Stammzellmarker p63, Integrin β1 und CD44 ausgewertet. Aufgrund der geringen Gruppengröße (Versuch I: drei Tiere pro Datenpunkt; Versuch II: fünf Tiere pro Datenpunkt) wurde auf eine eingehende statistische Testung verzichtet. Die vorliegende Arbeit beschränkt sich auf eine beschreibende Darstellung des Verlaufs der Einzelparameter über den Gesamtzeitraum. Die Zellzahlen verringerten sich während der ersten Bestrahlungswoche auf 60-70 % der Ausgangswerte, stagnierten in der zweiten Woche und stiegen schließlich bis zum Ende der Nachbeobachtung wieder an. Zwischen den nur bestrahlten und den zusätzlich mit BIBX1382BF behandelten Tieren war kein Unterschied feststellbar. Ein gleichsinniger Verlauf war auch in Versuch II zu beobachten, wobei die Zellzahlen der mit Erlotinib behandelten Tiere in der Funktionsschicht durchgängig höher ausfielen als in Versuchsreihe A. Die Dicke des Gesamtepithels bzw. der einzelnen Epithelschichten zeigte im Versuch I unter Bestrahlung große individuelle Schwankungen. Unter zusätzlicher BIBX1382BF-Gabe wurden oft niedrigere Werte gemessen. Im Versuch II blieb die Dicke des Gesamtepithels unter Fraktionierung konstant. Von Tag 0-12 wurden bei zusätzlicher Erlotinib-Applikation geringere Werte der Gesamtdicke gemessen als unter alleiniger Bestrahlung, ansonsten fielen die Veränderungen der Epitheldicke unabhängig von der Erlotinib-Gabe gering aus. Der kurzzeitigen, mit dem allgemeinen Zellverlust einhergehenden Verringerung der p63-Expression zu Beginn der Bestrahlung folgt bis zum Ende des Beobachtungszeitraumes die Normalisierung der p63-positiven Zellen. Mit EGFR-Blockade sind gegenüber der alleinigen Bestrahlung keine Unterschiede in der p63-Expression festzustellen. Die Integrin β1-Expression nahm im Verlauf der Bestrahlung ab. Unter EGFR-Blockade mit BIBX1382BF zeigte sich an den Tagen 2-9 und 12-16 ein schwächeres Färbesignal als im fraktioniert bestrahlten Epithel, was für eine mögliche Interaktion des EGF-Rezeptors mit Integrin β1 spricht. Im Versuch II waren unabhängig von der Erlotinib-Gabe keine Unterschiede in der Expression von Integrin β1 feststellbar. Die CD44-Expression im Epithel wurde durch Bestrahlung gefördert. Übereinstimmend konnte in der vorliegenden Arbeit in beiden Versuchen eine Steigerung der CD44-Färbeintensität über den jeweiligen Referenzbereich festgestellt werden. Eine Blockade der EGFR-Aktivität durch Erlotinib reduzierte die Expression von CD44, wie in Versuch II/B im initialen Abfall der CD44-Färbeintensität deutlich wurde. Doch schon ab Tag 4 wurden im Versuch II/A und II/B gleich starke Färbesignale für CD44 erfasst. Insgesamt ergaben sich unter EGFR-Inhibition mittels BIBX1382BF oder Erlotinib keine Hinweise auf Veränderungen der untersuchten Parameter während einer zweiwöchigen fraktionierten Bestrahlung. Ob diese Ergebnisse auch auf andere Tyrosinkinase-Inhibitoren bzw. unterschiedliche Wirkstoffklassen (z. B. Anti-EGFR-Antikörper) übertragbar sind, muss in weiteren Studien untersucht werden. / Radiation-induced oral mucositis is one of the most important and often dose limiting early side effects of radiotherapy of advanced tumours in the head-and-neck region. To this day, no general concept for therapy and prophylaxis of the oral mucositis has been established. The inhibition of the epidermal growth factor receptor (EGFR) is one approach to a selective increase of the radiosensitivity of tumours based on the tumour biology. In combination with radiotherapy, application of EGFR-inhibitors is supposed to increase the local tumour control and the chances of cure. The aim of the present study was to investigate the effect of the tyrosine kinase inhibitors BIBX1382BF and Erlotinib on the radiation response of the oral mucosa and on the expression of different proteins that are discussed to be markers of epithelial stem cells. For the histological studies, the snouts of C3H/Neu mice were irradiated with ten daily fractions of 3 Gy over two weeks (on days 0-4, 7-11). The experiments comprised four treatment groups: • I/A (54 animals) and II/A (40 animals): fractionated irradiation, no further treatment • I/B (51 animals): fractionated irradiation, administration of BIBX1382BF, 50 mg/kg per os, once daily (days 0-14) 30 min after the radiation treatment • II/B (35 animals): fractionated irradiation, administration of Erlotinib, 50 mg/kg per os, once daily (days 0-11) 30 min after the radiation treatment. Between day 0 and 17, three animals of the groups I/A and I/B were euthanised per day. In the experimental arms II/A and II/B five mice were killed on day 0, 2, 4, 6, 8, 10, 12 and 14, respectively. The tongues were excised, fixed in formalin, embedded in paraffin and 3 µm thick sections were prepared. Subsequently, the tongue sections were stained with haematoxylin and eosin or with an ABC kit to visualise proteins of interest. The epithelium of the lower tongue was examined by light microscopy regarding the following parameters: cell numbers, thickness of epithelial layers and expression of the potential stem cell markers p63, integrin β1 and CD44. Due to the limited number of animals per data point (experiment I: three mice per data point; experiment II: five mice per data point), a detailed statistical analysis was not performed. The present study is determined to describe the parameter variations over the observation period. Cell numbers decreased to 60-70 % of the pre-treatment control values within the first week of irradiation alone, remained constant in the second week, and then slowly increased until the end of the observation period. There was no difference between radiotherapy alone or combined treatment with BIBX1382BF. In experiment II similar observations were made with higher cell numbers in the functional layer of the epithelium of the Erlotinib treated animals than in the irradiated group. The thickness of the epithelium and its individual layers showed high inter individual differences in experiment I. In treatment group I/B, lower values of thickness were often detected in comparison to group I/A. In experiment II the thickness of the epithelium remained constant under fractionated irradiation. Between day 0 and 12 the Erlotinib treatment slightly decreased the thickness of the whole epithelium in comparison to the irradiated group. Besides, there were only minor changes in the thickness of the different layers. Associated with the general loss of cells, radiation treatment led to a transient decrease in the expression of p63. The number of p63-positive cells recovered until the end of the observation period. A similar expression pattern of p63-positivity was found independent of EGFR inhibition. The expression of integrin β1 decreased during fractionated irradiation. On days 2-9 and 12-16, the changes were more pronounced in combination with BIBX1382BF treatment which indicates a potential interaction of the EGF receptor with integrin β1. In experiment II, no differences between the exclusively irradiated group and the combined treatment with Erlotinib were found for the expression patterns of integrin β1. Irradiation alone resulted in a higher epithelial expression of CD44. Accordingly, a general increase of CD44 staining intensity was observed in both experiments exceeding control values. Due to the EGFR inhibition with Erlotinib, the expression of CD44 initially decreased. However, by day 4 no persisting differences in staining intensity could be observed independent of EGFR inhibition. In summary, EGFR inhibition via BIBX1382BF or Erlotinib did not result in alterations of the analysed parameters during two weeks of fractionated irradiation. Further studies are required to demonstrate if the present findings are transferable to other tyrosine kinase inhibitors or different substance classes (e.g. inhibiting receptor antibodies).

No description available.

Introdução: O Erlotinibe é um inibidor oral da tirosina quinase localizada no domínio intracelular do receptor do fator de crescimento epidérmico (EGFR). É uma droga ativa contra o carcinoma epidermóide de cabeça e pescoço (CECCP) que apresenta alta expressão deste receptor, demonstrando desta forma possível sinergismo com a quimioterapia e a radioterapia. Objetivo: Avaliar a expressão do EGFR e da proteína Akt fosforilada por imuno-histoquímica como fator preditivo a resposta terapêutica ao Erlotinibe em um estudo fase II que incluiu 32 pacientes com CECCP localmente avançado; também foram analisados mutações do gene EGFR nos éxons 18,19,20 e 21. Pacientes e métodos: Neste estudo pacientes portadores de CECCP localmente avançado foram tratados com uma combinação de Cisplatina 100mg/m2 intravenoso, administrada nos dias 8, 29 e 50 do tratamento; e radioterapia na dose de 70 Gy administrada em 39 frações a partir do dia 8. O Erlotinibe foi iniciado uma semana antes da radioterapia e mantido até o último dia da radioterapia. Biópsias pré-tratamento, extraídas dos blocos de parafina, foram analisadas por imunohistoquímica para avaliar a expressão do EGFR e da Akt fosforilada. O resultado dessas amostras foi quantificado por um programa de análise digital de imagem. O status mutacional do gene EGFR (nos éxons 18, 19,20 e 21) foi analisado utilizando PCR convencional e sequenciamento. Resultados: A resposta completa ao tratamento ocorreu em vinte pacientes (62,5%), sendo que dois foram tratados com Laringectomia de resgate e ficaram sem evidência de doença. A análise de sobrevida com relação ao estadiamento e com o sítio anatômico evidenciou diferença estatisticamente significativa (p= 0.05). A análise das proteínas EGFR e p-Akt por imuno-histoquímica, quando os sítios estavam agrupados não apresentou valor preditivo de resposta ao tratamento; no entanto ao avaliarmos os sítios anatômicos separadamente, apenas a quantificação de EGFR em hipofaringe foi uma variável preditiva de resposta ao tratamento com erlotinibe (p=0.05). Em relação às análises moleculares nenhuma mutação foi detectada no seqüenciamento dos éxons estudados da proteína EGFR. Conclusão: A expressão do EGFR parece ser um fator preditivo à reposta terapêutica, no entanto outros estudos com identificação de outros biomarcadores e amostras maiores são necessários para elucidar quais pacientes com CECCP podem ser beneficiados com este tratamento / Purpose: Erlotinib, an oral tyrosine-kinase inhibitor, is active against squamous cell carcinoma of the head and neck (HNSCC) and possibly has a synergistic interaction with chemotherapy and radiotherapy. We investigated the expression of EGFR and phosphorylated AKT by immunohistochemistry as predictors of response to Erlotinib in a cohort of 32 locally advanced HNSCC, enrolled in a Phase II trial. In addition, we assessed mutation on hotspots of EGFR gene (exons18,19,20,21). Patients and Methods: This study was conducted in a Phase I/II trial of cisplatin 100 mg/m2 on days 8, 29 and 50; and radiotherapy 70 Gy starting on day 8. Erlotinib was started orally 1 week before chemo radiation and continued daily just to the last day of chemo radiation. Pretreatment archival tumor specimens were evaluated for EGFR and phosphorylated-Akt (p-Akt) by immunohistochemistry. These immunostains were quantified by digital image analysis. EGFR gene mutational status was also assessed using conventional PCR and sequencing. Results: Complete response to treatment occurred in twenty patients (62.5%), and two were treated with salvage laryngectomy and were without evidence of disease. Survival analysis in relation to the staging and the tumor site showed a statistically significant difference (p = 0.05). Analysis of EGFR protein and p-Akt by immunohistochemistry, when sites were grouped showed no predictive value for treatment response, however when evaluating the anatomical sites separately, only the quantification of EGFR in the hypopharynx was a significant predictor of response to treatment with erlotinib (p = 0.05). Regarding the molecular analysis no mutations were detected in the sequencing of the exons studied EGFR protein. Conclusion: The expression of EGFR seems to be a predictive factor for response to therapy, although other studies with identification of other biomarkers and larger samples are needed to elucidate which patients may benefit HNSCC with this treatment

This thesis reports the investigation of spontaneous Raman Spectroscopy (RS) for its applicability in early drug discovery. A key focus has been to develop an understanding of the applicability of RS for the quantification and localisation of compound concentration inside mammalian cells. Further investigation into the use of Surface Enhanced Raman Spectroscopy (SERS) for research on Visceral Leishmaniasis (VL) and Leishmania donovani as well as investigating applicability for cancer research are decisive parts of this work. The key work described in this thesis is the investigation of whole cell concentration of compounds inside THP-1 and Madin Darby Canine Kidney (MDCK) cells. For true quantification the Cell Silent Region (CSR) is used to measure without interference from cellular background signal. The model compound is erlotinib, an anti-cancer drug with an alkyne group expressing a peak in the CSR. The developed RS system is calibrated using the current gold standard technique Ultra Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS). However, because of the single cell nature of the RS information on inter cell variability can be extracted. The RS measurements suggest that there is a large variation of concentration within single cell populations. The RS measurements can therefore give insight in single cell behaviour within a large cell population. Findings shows that washing cycles, before fixation, alter the intra-cellular concentrations significantly. This is hypothesised to be caused by the sudden change in concentration on the outside of the cell that applies an osmotic pressure, leading to loss of substance from inside the cell wall. Localisation of erlotinib is shown within THP-1 cells and points towards an accumulation inside the cell nucleus. Later, internalised Au nano-particles in the range of 30 nm to 80 nm have been investigated for their enhancement effects and localisation inside THP-1 cells. Au nano-particles are found to be internalised easily by differentiated THP-1 cells and accumulate in lysosomes. This allows for a high local enhancement of the spontaneous Raman signal. However, no advantage for the detection of lysosomally trapped compounds (chloroquine, chlorpromazine) was achieved. The detection of substances without a signal in the CSR was achieved without enhancement. Nonetheless, compounds with intrinsic peaks in the CSR could benefit from this enhancement. Lastly the RS system is explored for alternative uses in early drug discovery. This includes the detection of toxicity as well as the discrimination of cell types. Toxicity has been detected using optically trapped THP-1 cells and doxorubicin. Utilising Principal Component Analysis (PCA) combined with Linear Discriminant Analysis (LDA) on these measured spectra, allowed for a clear discrimination of toxically influenced from healthy cells. Differences mainly show up in DNA content caused by the mode of action of doxorubicin and caused by the trapping, which generates most of the signal within the nucleus of the cell. Discriminating cancerogenic (DU145) from healthy prostate cells (PNT2) has been achieved by probing fixed cells and evaluating the acquired Raman spectra with a PCA/LDA combination. The accuracy of separation of these cells when tested with a 10-fold cross-validation technique, is above 98 %, allowing a good discrimination.

Introdução: O Erlotinibe é um inibidor oral da tirosina quinase localizada no domínio intracelular do receptor do fator de crescimento epidérmico (EGFR). É uma droga ativa contra o carcinoma epidermóide de cabeça e pescoço (CECCP) que apresenta alta expressão deste receptor, demonstrando desta forma possível sinergismo com a quimioterapia e a radioterapia. Objetivo: Avaliar a expressão do EGFR e da proteína Akt fosforilada por imuno-histoquímica como fator preditivo a resposta terapêutica ao Erlotinibe em um estudo fase II que incluiu 32 pacientes com CECCP localmente avançado; também foram analisados mutações do gene EGFR nos éxons 18,19,20 e 21. Pacientes e métodos: Neste estudo pacientes portadores de CECCP localmente avançado foram tratados com uma combinação de Cisplatina 100mg/m2 intravenoso, administrada nos dias 8, 29 e 50 do tratamento; e radioterapia na dose de 70 Gy administrada em 39 frações a partir do dia 8. O Erlotinibe foi iniciado uma semana antes da radioterapia e mantido até o último dia da radioterapia. Biópsias pré-tratamento, extraídas dos blocos de parafina, foram analisadas por imunohistoquímica para avaliar a expressão do EGFR e da Akt fosforilada. O resultado dessas amostras foi quantificado por um programa de análise digital de imagem. O status mutacional do gene EGFR (nos éxons 18, 19,20 e 21) foi analisado utilizando PCR convencional e sequenciamento. Resultados: A resposta completa ao tratamento ocorreu em vinte pacientes (62,5%), sendo que dois foram tratados com Laringectomia de resgate e ficaram sem evidência de doença. A análise de sobrevida com relação ao estadiamento e com o sítio anatômico evidenciou diferença estatisticamente significativa (p= 0.05). A análise das proteínas EGFR e p-Akt por imuno-histoquímica, quando os sítios estavam agrupados não apresentou valor preditivo de resposta ao tratamento; no entanto ao avaliarmos os sítios anatômicos separadamente, apenas a quantificação de EGFR em hipofaringe foi uma variável preditiva de resposta ao tratamento com erlotinibe (p=0.05). Em relação às análises moleculares nenhuma mutação foi detectada no seqüenciamento dos éxons estudados da proteína EGFR. Conclusão: A expressão do EGFR parece ser um fator preditivo à reposta terapêutica, no entanto outros estudos com identificação de outros biomarcadores e amostras maiores são necessários para elucidar quais pacientes com CECCP podem ser beneficiados com este tratamento / Purpose: Erlotinib, an oral tyrosine-kinase inhibitor, is active against squamous cell carcinoma of the head and neck (HNSCC) and possibly has a synergistic interaction with chemotherapy and radiotherapy. We investigated the expression of EGFR and phosphorylated AKT by immunohistochemistry as predictors of response to Erlotinib in a cohort of 32 locally advanced HNSCC, enrolled in a Phase II trial. In addition, we assessed mutation on hotspots of EGFR gene (exons18,19,20,21). Patients and Methods: This study was conducted in a Phase I/II trial of cisplatin 100 mg/m2 on days 8, 29 and 50; and radiotherapy 70 Gy starting on day 8. Erlotinib was started orally 1 week before chemo radiation and continued daily just to the last day of chemo radiation. Pretreatment archival tumor specimens were evaluated for EGFR and phosphorylated-Akt (p-Akt) by immunohistochemistry. These immunostains were quantified by digital image analysis. EGFR gene mutational status was also assessed using conventional PCR and sequencing. Results: Complete response to treatment occurred in twenty patients (62.5%), and two were treated with salvage laryngectomy and were without evidence of disease. Survival analysis in relation to the staging and the tumor site showed a statistically significant difference (p = 0.05). Analysis of EGFR protein and p-Akt by immunohistochemistry, when sites were grouped showed no predictive value for treatment response, however when evaluating the anatomical sites separately, only the quantification of EGFR in the hypopharynx was a significant predictor of response to treatment with erlotinib (p = 0.05). Regarding the molecular analysis no mutations were detected in the sequencing of the exons studied EGFR protein. Conclusion: The expression of EGFR seems to be a predictive factor for response to therapy, although other studies with identification of other biomarkers and larger samples are needed to elucidate which patients may benefit HNSCC with this treatment

No description available.

La sclérodermie systémique (ScS) est une maladie auto-immune caractérisée par une fibrose cutanée et viscérale ainsi que par des anomalies microcirculatoires. Son origine multifactorielle et ses manifestations cliniques variées en font une maladie à la physiopathologie complexe. Cette maladie rare demeure une affection dont l’étiologie est encore inconnue et pour laquelle il n’existe aucun traitement curatif. Notre laboratoire a mis en évidence le rôle des formes réactives de l’oxygène en mettant au point un modèle animal induit par l’acide hypochloreux. Ce modèle nous a permis d’explorer différentes voies de signalisation intracellulaire, impliquées dans la formation de FRO, favorisant la fibrose. Dans ce travail, nous avons choisi d’explorer dans la sclérodermie systémique différentes voies de signalisation impliquées dans l’inflammation à l’aide d’un modèle de réaction du greffon contre l’hôte sclérodermiforme (GVH-Scl) et d’un modèle de sclérodermie induit par les formes réactives de l’oxygène. De plus, nous avons étudié l’impact de molécules ciblant ces voies afin de fournir de nouvelles données permettent d’enrichir l’arsenal thérapeutique de cette maladie, actuellement pauvre. Les souris obtenues à partir du premier modèle présentent une fibrose cutanée et pulmonaire, une alopécie, une diarrhée et une inflammation hépatique. Une quantité importante d’auto-anticorps ainsi qu’une activation du système immunitaire sont retrouvées. Nous avons observés une activation de la voie de l’EGFR, de STAT3, de Wnt/β-caténine, d’AKT, d’ERK1/2 et de Notch dans la GVH-Scl. L’inhibition de la voie de l’EGFR par l’erlotinib a montré une amélioration clinique associée à une réduction de la fibrose cutanée et de l’inflammation cutanée et hépatique. De plus, l’erlotinib agit sur le système immunitaire et restaure la proportion de LT CD4+ naïfs chez les souris malades ainsi que le taux d’auto-anticorps anti-topoisomérase 1. La co-inhibition des voies de signalisation STAT3, Wnt/β-caténine, AKT, ERK1/2 et Notch par le niclosamide améliore les symptômes de la GVH-Scl chez la souris. Nous avons noté une diminution de la fibrose cutanée et pulmonaire, une diminution de l’inflammation cutanée, hépatique et gastro-intestinale et une diminution de la production d’auto-anticorps. La proportion de cellules naïves parmi les LT CD4+ et CD8+ est plus élevée chez les souris malades traitées que chez les malades non traitées. Les souris du second modèle présentent une fibrose cutanée et pulmonaire et une activation du système immunitaire accompagnée d’une production d’autoanticorps anti-topoisomérase. Nous avons retrouvé une activation des voies de signalisation de STAT3, de Wnt/β-caténine et d’AKT chez les souris sclérodermiques. Nous avons également observés une activation de la voie de signalisation de STAT6 et une surexpression de KLF4 chez ces souris malades. La co-inhibition des voies de signalisation STAT3, Wnt/β-caténine et AKT par le niclosamide améliore la fibrose cutanée et pulmonaire chez les souris sclérodermiques. Cette molécule diminue le nombre et l’activation des LB et des LT CD4+ et CD8+ ainsi que la production des auto-anticorps chez les souris malades. Le traitement de souris sclérodermique par le léflunomide, inhibiteur de STAT6, a aussi montré une amélioration de la fibrose cutanée et pulmonaire et des anomalies immunitaires présentées par les souris sclérodermiques. De plus l’inhibition de STAT6 et de KLF4 par le léflunomide inhibe la polarisation des macrophages en macrophages M2. Ainsi nous avons mis en évidence le rôle des voies de signalisation de l’EGFR, de STAT3, de Wnt/β-caténine, d’AKT, de STAT6 et de KLF4 dans la physiopathologie de la sclérodermie systémique. (...) / Systemic sclerosis (SSc) is a connective tissue disorder that results in skin and inner organs fibrosis, microvascular injuries and auto-immunity. This rare disease has a complex physiopathology which is due to its multifactorial origin and its various clinical manifestations. Its etiology remains unknown and no curative treatment exists at present. Our team highlighted the role of reactive oxygen species (ROS) by developing a hypochlorous acid (HOCl)-induced mouse model of SSc. This model allowed the exploration of several intracellular signalizing pathways which were involved in ROS production and promote fibrosis. In this work, we choose to investigate inflammatory signalizing pathways in SSc with a sclerodermatous graft versus host disease (Scl-GVHD) mouse model and a ROS-induced mouse model. Moreover, we studied the effects of drugs targeting these pathways in order to reinforce data providing new therapeutics. Mice from Scl-GVHD model developed a diffuse cutaneous SSc with pulmonary fibrosis, alopecia, diarrhea and liver inflammation. Production of anti-DNA topoisomerase 1 auto-antibodies and immunological activation was also found. We observed an activation of EGFR, STAT3, Wnt/β-catenin, AKT, ERK1/2 and Notch signaling pathways in Scl-GVHD. Inhibition of EGFR by Erlotinib showed clinical amelioration with a decreased skin fibrosis and decreased skin and liver inflammation. Moreover, Erlotinib decreased production of activated/memory CD4+ T cells and of auto-antibody anti-topoisomerase1. Co-inhibition of STAT3, Wnt/β-catenin, AKT, ERK1/2 and Notch pathways by Niclosamide reversed clinical symptoms of Scl-GVHD in mice. We observed an improvement of skin and lung fibrosis, of cutaneous, hepatic and intestinal inflammation, and a reduced production of auto-antibodies. The ratio of CD4 and CD8 naive T cells was higher in Niclosamide-treated GVHD mice than in untreated GVHD mice. Mice from HOCl-model present skin and lung fibrosis and an immune activation along with the production of auto-antibodies anti-topoisomerase. We found an activation of STAT3, Wnt/β-catenin and AKT signaling pathways in HOCl-mice. We also observed an activation of STAT6 signaling pathway and an overexpression of KLF4 in these sicked mice. Co-inhibition of STAT3, Wnt/β-catenin and AKT pathways by Niclosamide improves skin and lung fibrosis in HOCl-mice. This drug decreased number and activation of B cells and CD4+ and CD8+ T cells and auto-antibodies production in mice. Treatment of HOCl-mice with Leflunomide, STAT6 inhibitor, also showed an improvement of skin and lung fibrosis and of immunological abnormalities in HOCl-mice. Moreover, inhibition of STAT6 and KLF4 by Leflunomide inhibits M2 polarization of macrophages. Thus, we highlighted the role of EGFR, STAT3, Wnt/β-catenin, AKT, STAT6 and KLF4 signaling pathways in physiopathology of SSc. Use of inhibitors of these pathways such as Etrlotinib, Niclosamide and Leflunomide, indicate a clinical and biological efficacity in mouse. These drugs allow the control of the 3 characteristic features of SSc reproduced in these animal models: fibrosis, inflammation and autoimmunity and could thus be effective in fighting the development of clinical-biological abnormalities of SSc.

<p>Lung cancer is the third most prevalent cancer in the world; however it is the leading cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for ~85% of the lung cancer cases. The current strategies to treat NSCLC patients with frequent causal genetic mutations is through targeted therapeutics. Approximately 10-35% of NSCLC patient tumors have activated mutations in the Epidermal Growth Factor Receptor (EGFR) resulting in uncontrolled cellular proliferation. The standard-of care for such patients is EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs), a class of targeted therapeutics that specifically inhibit EGFR activity. One such EGFR-TKI used in this study is erlotinib. Following erlotinib treatment, tumors rapidly regress at first; however, over 50% of patients develop erlotinib resistance within a year post treatment. Development of resistance remains to be the major challenge in treatment of NSCLC using EGFR-TKIs such as erlotinib. </p> <p>In approximately 60% of cases, acquired erlotinib resistance in patients is attributed to a secondary mutation in EGFR, whereas in about 20% of cases, activation of alternative signaling pathways is the reported mechanism. For the remaining 15-20% of <a>cases</a> the mechanism of resistance remains unknown. Therefore, it can be speculated that the common methods used to identify genetic mutations in tumors post erlotinib treatment, such as histologic analysis and genetic screening may fail to identify alterations in epigenetic mediators of erlotinib resistance, also including microRNAs (miRNAs). MiRNAs are short non-coding RNAs that post-transcriptionally negatively regulate their target transcripts. Hence, in this study two comprehensive screens were simultaneously conducted in erlotinib sensitive cells: 1) a genome-wide knock-out screen, conducted with the hypothesis that loss of function of certain genes drive erlotinib resistance, 2) a miRNA overexpression screen, conducted with the hypothesis that certain miRNAs drive the development of erlotinib resistance when overexpressed. The overreaching goal of the study was to identify novel drivers of erlotinib resistance such as microRNAs or other epigenetic factors in NSCLC.</p><p>The findings of this study led to the identification of a tumor suppressive protein and an epigenetic regulator, SUV420H2 (KMT5C) that has never been reported to be involved in erlotinib resistance. On the other hand, the miRNA overexpression screen identified five miRNAs that contribute to erlotinib resistance that were extensively analyzed using multiple bioinformatic tools. It was predicted that the miRNAs mediate erlotinib resistance via multiple pathways, owing to the ability of each miRNA to target multiple transcripts via partial complementarity. Importantly, a correlation between the two screens was identified clearly supporting the use of two simultaneous screens as a reliable technique to determine highly significant miRNA-target interactions. Overall, the findings from this study suggest that epigenetic factors, such as histone modifiers and miRNAs function as critical mediators of erlotinib resistance, possibly belonging to the 15-20% of NSCLC cases with unidentified mechanisms involved in erlotinib resistance.</p><p></p>