Dissertations / Theses on the topic 'EGFR-Inhibition'

Prognosis in advanced stage lung cancer is extremely poor with few effective therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations and are now an established part of therapy in selected patients. Such advances herald a previously unprecedented enthusiasm for the possibilities of targeted therapy. Acquired resistance however is widespread - the EGFR T790M mutation in particular represents approximately 50% of these. MET amplification is also an important route of resistance and preclinical data suggests synergy between therapies targeting these two receptors. We hypothesized that EGFR mutation status determines the EGFR-MET interaction and response to MET inhibition. We tested this hypothesis by using cells derived from NCI-H1975, which possess L858R and T790M EGFR mutations. This cell model and a derived murine xenograft experiment provided a platform with which to test these ideas by using assays of tumorigenicity in vitro; tumour growth/stroma formation in vivo and a selective MET kinase inhibitor, SGX523. EGFR-MET interaction was assessed by a Förster Resonance Energy Transfer (FRET) Fluorescence Lifetime Imaging Microscopy (FLIM) assay developed as part of this thesis that quantified EGFR-MET dimer formation. SGX523 significantly reduced cell proliferation, xenograft tumour growth and ERK phosphorylation in the presence of the EGFR L858R-T790M mutations but not with EGFR L858R alone where SGX523 reduced stroma formation but not growth. SGX523 reduced EGFR-MET dimerisation in the EGFR L858R-T790M mutant but increased EGFR-MET interaction in the presence of EGFR L858R alone. Little effect was seen with EGFR WT in response to SGX523 for any of these indices. This thesis provides novel data for the mechanistic understanding of EGFR-MET heterodimerisation and the accompanying discussion explores how this is relevant for EGFR and MET targeted therapies.

Le récepteur de facteur de croissance épidermique (EGFR) est une cible commune de thérapie anticancéreuse. Aujourd'hui, la recherche de nouvelles molécules inhibitrices de ce récepteur se tourne vers des substances naturelles. Un des composés naturels les plus prometteurs qui ont montré une activité anti-EGFR est la curcumine, un polyphénol présent dans les rhizomes de Curcuma longa. Il y a de nombreux rapports décrivant son effet sur l'activité kinase du récepteur, le rendement d'autophosphorylation, le niveau d'expression et les processus liés à la fonction EGFR comme la prolifération cellulaire. Néanmoins, l'ensemble des mécanismes d’intercation de la curcumine avec l'de l'EGFR n’est pas entièrement élucidée. Nous avons démontré que le mode d'action de la curcumine est double. La curcumine est capable d'inhiber partiellement, mais directement l'activité enzymatique du domaine intracellulaire de l'EGFR. Mais le travail présenté attire l'attention sur le rôle de l'environnement de la membrane de l'EGFR au niveau d'action de la curcumine. Nous avons montré que l'insertion de curcumine dans la membrane plasmique aboutit à sa rigidification et par conséquent la limitation de la diffusion du récepteur. Le suivi de particules à l'unité analyses a confirmé que le coefficient de diffusion de l'EGFR dans la membrane des cellules cancéreuses diminué de manière significative en présence de la curcumine, susceptibles d'influencer la dimérisation du récepteur et l'activation tour à tour
Epidermal Growth Factor Receptor (EGFR) is a common target of anticancer therapy. Nowadays the search for new molecules inhibiting this receptor is turning towards natural substances. One of the most promising natural compounds that have shown an anti-EGFR activity is curcumin, the polyphenol found in the rhizomes of Curcuma longa. There are numerous reports describing its effect on the receptor kinase activity, the autophosphorylation yield, the expression level and the processes related to EGFR function like cell proliferation. Nevertheless, the entire mechanism of how curcumin interact with the EGFR is not fully elucidated. We demonstrated that the mode of action of curcumin is dual. Curcumin is able to inhibit directly but partially the enzymatic activity of the EGFR intracellular domain. But the presented work brings attention to the role of the EGFR membrane environment at the curcumin action level. We showed that the curcumin insertion in plasma membrane leads to its rigidification and as a consequence to limitation of the receptor diffusion. Single particle tracking analyses confirmed that the diffusion coefficient of EGFR in the cancer cell membrane significantly decreased in the presence of the curcumin, which might influence the receptor dimerization and in turns its activation

Despite progress being made in our understanding of triple negative breast cancer (TNBC), the overall survival and disease-free survival for TNBC patients continues to be considerably poorer than their ER/PR/HER2+ counterparts. Metastasis and chemoresistance are the pivotal issues holding back the long-term success of TNBC treatments. In addition to the bulk tumor cells, cancer stem cells (CSCs) have emerged as important targets for alleviating TNBC progression and relapse. Cisplatin, a platinum based chemotherapeutic agent, has shown promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with tumor hypoxia that in turn promotes CSC enrichment and drug resistance. My work is to develop a combinational treatment to improve the long-term therapeutic potential of cisplatin that not only targeted the bulk TNBC population but also ALDHhigh and CD44+/CD24- CSC populations. Through clinical dataset analysis, I found that patient TNBC tumors expressed high levels of epidermal growth factor receptor (EGFR) and hypoxia genes. A similar expression pattern was demonstrated in cisplatin-resistant ovarian cancer. I therefore developed a combinational therapeutic to co-inhibit EGFR and hypoxia using metformin (an AMPK activator) and gefitinib (an EGFR inhibitor), which sensitized bulk TNBC cells to cisplatin and also led to the effective inhibition of both CD44+/CD24- and ALDHhigh CSCs. I obtained similar results by using clinically relevant TNBC patient samples ex vivo. Since these drugs are already frequently used in the clinic, this study illustrates a novel, clinically translatable therapeutic approach to improve the long-term therapeutic outcome of cisplatin for TNBC treatment.

Bei der Strahlentherapie fortgeschrittener Tumoren im Kopf-Hals-Bereich gilt die radiogene Mucositis enoralis als schwerwiegende und dosislimitierende frühe Nebenwirkung. Sehr häufig führt sie zu einer Unterbrechung der Behandlung, mit der Folge einer Reduktion der Tumorheilungschancen. Während einer fraktionierten Strahlenexposition kommt es in der Mundschleimhaut zu einer erhöhten Expression des Epidermalen Wachstumsfaktors (Epidermal Growth Factor, EGF) und dessen Rezeptors (EGFR). Durch eine Blockade des EGFR, als anerkannte Strategie zur Verbesserung der Tumorheilung, besteht deshalb die Gefahr, dass es zu einer Verschlimmerung der Schleimhaut-Nebenwirkungen kommt. Der Einsatz von Keratinozyten-Wachstumsfaktor (KGF) zeigt positive Ergebnisse bezüglich einer Reduktion der Schleimhautveränderungen. In dieser Arbeit wird deshalb im Tiermodell einerseits die Auswirkung einer Blockade des EGFR auf die Schleimhautreaktion, und andererseits eine mögliche Interaktion der Blockade mit der schleimhautschützenden Wirkung von KGF untersucht. Insgesamt kann keine signifikante Veränderung der Schleimhauttoleranz durch die EGFR-Inhibition mittels BIBX1382BF innerhalb der ersten beiden Wochen einer fraktionierten Bestrahlung festgestellt werden; lediglich das Auftreten ulzerativer Läsionen nach der zweiten Woche ist vorverlagert

PI3K/Akt is over-expressed in 50-70% of pancreatic ductal adenocarcinoma (PDAC). The hypothesis of this study is that PI3K and EGFR co-inhibition may be effective in PDAC with upregulated PI3K/Akt/mTOR (PAM) signaling. Five primary PDAC and two erlotinib acquired resistant (ER) cell lines with significantly over-expressed AKT2 gene, total Akt and pAkt, were used. Multiple inhibitors of the MAPK and PAM were tested alone or in combination by western blotting, cell proliferation, cell cycle, clonogenic, apoptosis, and migration assays. Erlotinib acted synergistically with PI3Kα inhibitor BYL in both ER cell lines (synergy index, SI=1.71 and 1.44 respectively). Treatment of ER cell lines by this dual blockade caused significant G1 cell cycle arrest (71%, P<0.001; 58%, P=0.003), inhibition of colony formation (69% and 72%, both P<0.001), and necrosis and apoptosis (75% and 53%, both P<0.001), more so compared to parent cell lines. In primary patient-derived tumor subrenal capsule (n=90) and subcutaneous (n=22) xenografts, Erlotinib plus BYL significantly reduced tumor volume (P=0.005). Strong pEGFR and pAkt immunostaining (2+/3+) was correlated with high response to erlotinib and low response to erlotinib plus BYL respectively. In conclusion, PDAC with increased expression of the PAM signaling were susceptible to PI3K/ EGFR co-inhibition suggesting oncogenic dependence. Erlotinib plus BYL should be considered for a clinical study in PDAC; further evaluation of pEGFR and pAkt expression as potential predictive biomarkers is warranted.

This thesis focuses on the evaluation of biomarkers for radio-immunodiagnostics and radio-immunotherapy and on radiosensitization strategies after HSP90 inhibition, as a step towards more personalized cancer medicine. There is a need to develop new tracers that target cancer-specific biomarkers to improve diagnostic imaging, as well as to combine treatment strategies to potentiate synergistic effects. Special focus has been on the cell surface molecule CD44 and its oncogenic variants, which were found to exhibit unique expression patterns in head and neck squamous cell carcinoma (HNSCC). The variant CD44v6 seems to be a promising target, because it is overexpressed in this cancer type and is associated with radioresistance. Two new radioconjugates that target CD44v6, namely, the Fab fragment AbD15179 and the bivalent fragment AbD19384, were investigated with regard to specificity, biodistribution and imaging performance. Both conjugates were able to efficiently target CD44v6-positive tumors in vitro and in vivo. PET imaging of CD44v6 with 124I-AbD19384 revealed many advantages compared with the clinical standard 18F-FDG. Furthermore, the efficacy of the novel HSP90 inhibitor AT13387 and its potential use in combination with radiation treatment were evaluated. AT13387 proved to be a potent new cancer drug with favorable pharmacokinetics. Synergistic combination effects at clinically relevant drug and radiation doses are promising for both radiation dose reduction and minimization of side effects, or for an improved therapeutic response. The AT13387 investigation indicated that CD44v6 is not dependent on the molecular chaperone HSP90, and therefore, radio-immunotargeting of CD44v6 in combination with the HSP90 inhibitor AT13387 might potentiate treatment outcomes. However, EGFR expression levels did correlate with HSP90 inhibition, and therefore, molecular imaging of EGFR-positive tumors may be used to assess the treatment response to HSP90 inhibitors. In conclusion, these results demonstrate how tumor targeting with radiolabeled vectors and chemotherapeutic compounds can provide more specific and sensitive diagnostic tools and treatment options, which can lead to customized treatment decisions and a functional diagnosis that provides more precise and safer drug prescribing, as well as a more effective treatment for each patient.

ZEB1 (Zinc finger E-box binding homeobox 1) est un facteur de transcription qui favorise l'invasion et les métastases tumorales en induisant une transition épithélio-mésenchymateuse (TEM) dans les cellules de carcinome. La TEM joue un rôle majeur dans l’embryogenèse et la progression maligne, mais aussi dans l'acquisition de propriétés des cellules souches cancéreuses (CSC) et la résistance aux traitements. L’objectif de ce travail était d’étudier le rôle de ZEB1 dans la progression et la chimiorésistance du CCA. Nos précédents travaux, ont montré une surexpression de ZEB1 dans les cellules résistantes au traitement anti-EGFR dans le CCA. Les données cliniques, ont révélé une expression de ZEB1 dans les échantillons humains de CCA et ont associé son expression à un mauvais pronostic. Pour les études in vitro, des modèles d’études cellulaires ont été réalisés dans lesquels l’expression de ZEB1 a été soit invalidée soit forcée. Les résultats ont montré l’implication de ZEB1 dans la plasticité cellulaire, par la mise en place d’un phénotype de TEM et de CSC dans les cellules exprimant ZEB1 comparées aux cellules contrôles. Les modèles d’invalidation de ZEB1 ont montré une inhibition de ces processus. Par ailleurs, les expériences ont permis d’identifier ZEB1 comme un facteur de résistance au traitement anti-EGFR dans les modèles de CCA. Les modèles d’invalidation de ZEB1 ont montré une sensibilisation des cellules à ce traitement. En conclusion, nous avons démontré dans cette étude que ZEB1 contribuerait à la plasticité cellulaire et à la chimiorésistance des cellules de CCA, faisant de lui une cible de choix dans le développement de nouvelles stratégies thérapeutiques
ZEB1 (Zinc Finger E-box Binding Homeobox 1) is a transcription factor that promotes tumor invasion and metastasis by inducing epithelial-mesenchymal transition (EMT) in carcinoma cells. EMT not only plays an important role in embryonic development and malignant progression, but is also implicated in acquisition of cancer stem cell properties and cancer therapy resistance. The aim of the study was to evaluate ZEB1 role in cholangiocarcinoma progression and chemoresistance. Our previous study in the first article showed ZEB1 overexpression in resistant cells to anti-EGFR treatment in CCA. Clinical data revealed ZEB1 expression in human tumor CCA samples and correlated its expression with a poor prognosis. To perform in vitro studies, different CCA cellular models were established, in which ZEB1 expression has been either invalidated or forced. We showed that ZEB1 regulates cellular plasticity characterized by an acquisition of TEM and CSC phenotypes in overexpressing ZEB1 cells. ZEB1 invalidation models showed a downregulation of these processes. In addition, cell viability analyzes identified ZEB1 as a resistance factor to anti-EGFR treatment in CCA models. ZEB1 inhibition models showed a cell re-sensitization to anti-EGFR treatment. In conclusion, we propose in this study that ZEB1 regulates CCA cells plasticity and chemoresistance, that could be a prime target in the development of new therapeutic strategies in CCA

In this thesis,the association of FASN (fatty acid synthase) expression with clinicopathological and anthropometric characteristics in breast cancer patients is studied to find out FASN role as a prognostic in early stage breast cancer. We also study the expression of FASN and its implication (alone or in combination with EGFR/HER/ErbB pathway inhibition) in cellular and animal models (xenografts and patient derived xenografts) or HER1+/FASN+ lung cancer and HER2+/FASN+ breast cancer. Finally, we have developed pre-clinical models of HER2+ breast cancer resistant to anti-HER2 therapies(trastuzumab and lapatinib), to study expression of FASN and other proteins involved in resistance, and, in vivo, the anti-tumoral efficacy of FASN inhibitors, alone or in combination. As a general conclusion, FASN is described as a new possible anti-tumoral target (alone or in combination) for future pre-clinical and clinical studies in FASN-positive tumor models
En aquesta tesi s’estudia la relació entre l’expressió de FASN (sintasa d'àcids grassos) i les característiques clinicopatològiques i antropomètriques en pacients amb càncer de mama, per esbrinar el paper de FASN com a pronòstic de càncer de mama d’estadis primerencs. També s'estudia l’expressió de FASN i la seva inhibició (sola o en combinació amb la inhibició de la ruta EGFR/HER/ErbB) en models cel·lulars i animals (xenografts i ortoxenopatients) de càncer de pulmó HER1+/FASN+, i en càncer de mama HER2+/FASN+. Finalment, s’han desenvolupat models pre-clínics de càncer de mama HER2+ resistents a teràpies anti-HER2 (trastuzumab i lapatinib) per estudiar l’expressió de FASN i altres proteïnes involucrades en la resistència i també, l’eficàcia anti-tumoral in vivo dels inhibidors de FASN, sols o en combinació. Com a conclusió, es descriu FASN com a possible nova diana anti-tumoral (sola o en combinació) per futurs estudis pre-clínics i clínics en models tumorals FASN+
Doctorat en Ciències Experimentals i Sostenibilitat

Dysregulation of protein activity, caused by alterations in protein sequence, expression, or localization, is associated with numerous diseases. In order to control the activity of harmful protein entities, affinity ligands such as proteins, oligonucleotides or small molecules can be engineered to specifically interact with them to modulate their function. In this thesis, non-immunoglobulin based affinity proteins known as affibody molecules are used to functionally inhibit proteins important for signaling through pathways that are overactive in different cancers.   In Paper I and Paper II, affibody molecules with high affinity for the receptor tyrosine kinases HER2 or EGFR are expressed in the secretory compartments of model cancer cell lines SKOV3 or A431 using a retrovirus-based gene delivery system. Equipping the affinity proteins with an ER retention tag, the affibody molecules together with their target protein are retained in the secretory compartments as shown by confocal fluorescence imaging. Flow cytometric analysis showed a 60 % or 80 % downregulation of surface located HER2 or EGFR in these cell lines, respectively. A significant decreased in proliferation rate of the cells was also observed, which for EGFR retention could be correlated with inhibition of phosphorylation in the kinase domain. In Paper III, novel affibody molecules interacting with the hormone binding site of the insulin growth factor-1 receptor were generated. One variant had high (1.2 nM) affinity for the receptor and could be used for immunofluorescence analysis and for receptor pull-out from cell lysates. Addition of this affibody molecule to MCF-7 cells had a dose dependent growth inhibitory effect on the cells. In Paper IV, novel affibody molecules against the intracellular oncoproteins H-Ras and Raf-1 were selected and characterized, and they proved to be specific for their target proteins. Mapping experiments showed that the affibody molecules selected against H-Ras interacted at over-lapping epitopes not affecting the interaction between Ras and Raf. In contrast, the predominant variant isolated during selection against Raf-1 could completely inhibit the Ras/Raf interaction in a real-time biospecific interaction analysis.   Taken together, the affibody molecules presented here and the strategies by which they are used to interfere with cancer related proteins and pathways may be valuable tools for further investigation of these systems and may possibly also be used to generate molecules suitable for cancer therapy.
QC 20100818

Le cancer colorectal (CCR) est la troisième maladie maligne la plus fréquente et la deuxième cause de décès par cancer. La famille des récepteurs tyrosine kinases transmembranaires ErbB a été identifiée comme l'un des principaux moteurs du développement et de la progression du CCR et l'un de ses membres les plus connus, le récepteur du facteur de croissance épidermique (EGFR / ERBB1 / Her1), considéré comme l'une des cibles les plus importantes en traitement CRC. Deux autres membres de la famille ErbB, les récepteurs Her2 et Her3, apparaissent également comme de nouvelles cibles importantes pour le CRC en raison de la mutation somatique, de l’amplification génique ou de la résistance aux traitements anti-EGFR. La protéine transmembranaire, Fas (TNFRSF6 / CD95), est un membre de la superfamille des récepteurs du facteur de nécrose tumorale (TNFRSF). Il peut transmettre des signaux multiples qui mènent à des destins de cellules complètement différents. Selon les contextes cellulaires, Fas initie la mort cellulaire par apoptose, essentielle pour arrêter les réponses immunitaires chroniques et prévenir l'auto-immunité et le cancer, ou pour stimuler la survie, la prolifération et la motilité des cellules, ce qui favorise l'auto-immunité, la croissance cancéreuse et les métastases. Avec des preuves de plus en plus nombreuses de la signalisation pro-survie médiée par Fas, les activités de promotion du cancer chez les patients atteints de cancer sont maintenant reconnues comme étant significatives et cliniquement pertinentes. Bien que cette polyvalence de signalisation ait été particulièrement bien démontrée dans le cancer du côlon, les mécanismes moléculaires qui sous-tendent les voies de survie sont encore largement inconnus. Dans ce contexte, l'objectif principal de mon doctorat Le projet visait à étudier l’importance du crosstalks entre les membres de la famille Fas et ErbB et, plus particulièrement, à déterminer si la signalisation Fas pouvait influencer la signalisation de l’EGFR favorisant le cancer.Plus précisément, je décris comment l’état de phosphorylation de la tyrosine Fas influence fortement la signalisation de la voie EGFR dans les cellules colorectales. Mes données démontrent que Fas dans son état prosurvival, phosphorylé à Y291 (pY291-Fas), interagit en effet avec EGFR et que cette interaction intensifie significativement la signalisation de l'EGFR dans les cellules cancéreuses colorectales anti-EGFR via la voie Yes-1 / STAT3. Le pY291-Fas s'accumule dans le noyau lors du traitement par EGF et favorise la localisation nucléaire du phospho-EGFR et du phospho-STAT3, l'expression de la cycline D1, l'activation des voies Akt et MAPK médiées par STAT3 et enfin la prolifération et la migration cellulaires. De plus, je découvre également le rôle potentiel que Her3 pourrait jouer avec Fas dans la libération des cellules cancéreuses colorectales de l'inhibition anti-EGFR.Tous ensemble mon doctorat des études permet de mieux comprendre le rôle des voies de survie de Fas dans la signalisation ErBb dans le CRC. Fait important, en démontrant un lien entre l'émergence d'une résistance aux traitements anti-ErbB et le signal de Fas pro-survie, mon travail justifie le développement d'une thérapie ciblée Fas / phospho-Fas comme nouvelle option thérapeutique pour surmonter les anti-EGFR, chez les patients présentant une résistance anti-EGFR secondaire
Colorectal cancer (CRC) is the third most common malignant disease and the second most frequent cause of cancer-related death. The ErbB family of transmembrane receptor tyrosine kinases has been identified as a major driver of the development and progression of CRC and one its best-known member, the epidermal growth factor receptor (EGFR /ERBB1/Her1), considered one of the most important targets in CRC treatment. Two others members of the ErbB family, the receptors Her2 and Her3, also emerge as important new targets for CRC due to the somatic mutation, gene amplification or resistance to the anti-EGFR therapies. The transmembrane protein, Fas (TNFRSF6/CD95), is a member of the tumor necrosis factor receptor superfamily (TNFRSF). It can transmit multiple signals that lead to completely different cell fates. Depending on cellular contexts, Fas either initiates cell death by apoptosis, which is essential for shutting down chronic immune responses and preventing autoimmunity and cancer, or stimulates cell survival, proliferation, and motility, which can promote autoimmunity, cancer growth, and metastasis. With increasing evidence of Fas-mediated pro-survival signaling, the cancer-promoting activities of Fas are now recognized as significant and clinically relevant. While this signaling versatility has been particularly well demonstrated in colon cancer, the molecular mechanisms underlying the survivals pathways are still largely unknown. In this context, the main aim of my Ph.D. project was to study the importance of the crosstalks between Fas and the ErbB family members and more specifically to determine whether the Fas signaling could influence the cancer-promoting signaling of EGFR.More precisely, I describe how the Fas tyrosine phosphorylation status strongly influences the signaling of the EGFR pathway in colorectal cells. My data demonstrate that Fas in its prosurvival state, phosphorylated at Y291 (pY291-Fas), indeed interacts with EGFR and that this interaction significantly intensifies EGFR signaling in anti-EGFR-resistant colorectal cancer cells via the Yes-1/STAT3-mediated pathway. The pY291-Fas accumulates in the nucleus upon EGF treatment and promotes the nuclear localization of phospho-EGFR and phospho-STAT3, the expression of cyclin D1, the activation of STAT3-mediated Akt and MAPK pathways, and finally the cell proliferation and migration. Additionally, I also uncover the potential role that Her3, may play along with Fas, in the colorectal cancer cell escape from anti-EGFR inhibition. All together my Ph.D. studies provide a better understanding of the role of the Fas survival pathways in the ErBb signaling in CRC. Importantly, by demonstrating a connection between the emergence of resistance to anti-ErbB therapies and the Fas pro-survival signal, my work provides a rationale for the development of Fas/phospho-Fas targeted therapy as a new therapeutic option for overcoming anti-EGFR, in patients with secondary anti-EGFR resistance

Le cancer colorectal (CCR) est la troisième maladie maligne la plus fréquente et la deuxième cause de décès par cancer. La famille des récepteurs tyrosine kinases transmembranaires ErbB a été identifiée comme l'un des principaux moteurs du développement et de la progression du CCR et l'un de ses membres les plus connus, le récepteur du facteur de croissance épidermique (EGFR / ERBB1 / Her1), considéré comme l'une des cibles les plus importantes en traitement CRC. Deux autres membres de la famille ErbB, les récepteurs Her2 et Her3, apparaissent également comme de nouvelles cibles importantes pour le CRC en raison de la mutation somatique, de l’amplification génique ou de la résistance aux traitements anti-EGFR. La protéine transmembranaire, Fas (TNFRSF6 / CD95), est un membre de la superfamille des récepteurs du facteur de nécrose tumorale (TNFRSF). Il peut transmettre des signaux multiples qui mènent à des destins de cellules complètement différents. Selon les contextes cellulaires, Fas initie la mort cellulaire par apoptose, essentielle pour arrêter les réponses immunitaires chroniques et prévenir l'auto-immunité et le cancer, ou pour stimuler la survie, la prolifération et la motilité des cellules, ce qui favorise l'auto-immunité, la croissance cancéreuse et les métastases. Avec des preuves de plus en plus nombreuses de la signalisation pro-survie médiée par Fas, les activités de promotion du cancer chez les patients atteints de cancer sont maintenant reconnues comme étant significatives et cliniquement pertinentes. Bien que cette polyvalence de signalisation ait été particulièrement bien démontrée dans le cancer du côlon, les mécanismes moléculaires qui sous-tendent les voies de survie sont encore largement inconnus. Dans ce contexte, l'objectif principal de mon doctorat Le projet visait à étudier l’importance du crosstalks entre les membres de la famille Fas et ErbB et, plus particulièrement, à déterminer si la signalisation Fas pouvait influencer la signalisation de l’EGFR favorisant le cancer.Plus précisément, je décris comment l’état de phosphorylation de la tyrosine Fas influence fortement la signalisation de la voie EGFR dans les cellules colorectales. Mes données démontrent que Fas dans son état prosurvival, phosphorylé à Y291 (pY291-Fas), interagit en effet avec EGFR et que cette interaction intensifie significativement la signalisation de l'EGFR dans les cellules cancéreuses colorectales anti-EGFR via la voie Yes-1 / STAT3. Le pY291-Fas s'accumule dans le noyau lors du traitement par EGF et favorise la localisation nucléaire du phospho-EGFR et du phospho-STAT3, l'expression de la cycline D1, l'activation des voies Akt et MAPK médiées par STAT3 et enfin la prolifération et la migration cellulaires. De plus, je découvre également le rôle potentiel que Her3 pourrait jouer avec Fas dans la libération des cellules cancéreuses colorectales de l'inhibition anti-EGFR.Tous ensemble mon doctorat des études permet de mieux comprendre le rôle des voies de survie de Fas dans la signalisation ErBb dans le CRC. Fait important, en démontrant un lien entre l'émergence d'une résistance aux traitements anti-ErbB et le signal de Fas pro-survie, mon travail justifie le développement d'une thérapie ciblée Fas / phospho-Fas comme nouvelle option thérapeutique pour surmonter les anti-EGFR, chez les patients présentant une résistance anti-EGFR secondaire
Colorectal cancer (CRC) is the third most common malignant disease and the second most frequent cause of cancer-related death. The ErbB family of transmembrane receptor tyrosine kinases has been identified as a major driver of the development and progression of CRC and one its best-known member, the epidermal growth factor receptor (EGFR /ERBB1/Her1), considered one of the most important targets in CRC treatment. Two others members of the ErbB family, the receptors Her2 and Her3, also emerge as important new targets for CRC due to the somatic mutation, gene amplification or resistance to the anti-EGFR therapies. The transmembrane protein, Fas (TNFRSF6/CD95), is a member of the tumor necrosis factor receptor superfamily (TNFRSF). It can transmit multiple signals that lead to completely different cell fates. Depending on cellular contexts, Fas either initiates cell death by apoptosis, which is essential for shutting down chronic immune responses and preventing autoimmunity and cancer, or stimulates cell survival, proliferation, and motility, which can promote autoimmunity, cancer growth, and metastasis. With increasing evidence of Fas-mediated pro-survival signaling, the cancer-promoting activities of Fas are now recognized as significant and clinically relevant. While this signaling versatility has been particularly well demonstrated in colon cancer, the molecular mechanisms underlying the survivals pathways are still largely unknown. In this context, the main aim of my Ph.D. project was to study the importance of the crosstalks between Fas and the ErbB family members and more specifically to determine whether the Fas signaling could influence the cancer-promoting signaling of EGFR.More precisely, I describe how the Fas tyrosine phosphorylation status strongly influences the signaling of the EGFR pathway in colorectal cells. My data demonstrate that Fas in its prosurvival state, phosphorylated at Y291 (pY291-Fas), indeed interacts with EGFR and that this interaction significantly intensifies EGFR signaling in anti-EGFR-resistant colorectal cancer cells via the Yes-1/STAT3-mediated pathway. The pY291-Fas accumulates in the nucleus upon EGF treatment and promotes the nuclear localization of phospho-EGFR and phospho-STAT3, the expression of cyclin D1, the activation of STAT3-mediated Akt and MAPK pathways, and finally the cell proliferation and migration. Additionally, I also uncover the potential role that Her3, may play along with Fas, in the colorectal cancer cell escape from anti-EGFR inhibition. All together my Ph.D. studies provide a better understanding of the role of the Fas survival pathways in the ErBb signaling in CRC. Importantly, by demonstrating a connection between the emergence of resistance to anti-ErbB therapies and the Fas pro-survival signal, my work provides a rationale for the development of Fas/phospho-Fas targeted therapy as a new therapeutic option for overcoming anti-EGFR, in patients with secondary anti-EGFR resistance

EGFR TKIs are standard therapy for advanced NSCLC. In order to define their role in early disease, we implemented a phase II trial of neoadjuvant gefitinib in clinical stage I NSCLC. Tumour shrinkage was seen in 43% of patients, with 11% achieving RECIST partial response (PR). Analysis of molecular markers showed EGFR TKD mutations in 17% of cases, being the only associated with PR. For the first time we defined the histopathological response of NSCLC to these agents, characterized by reduction in tumour cellularity and proliferative index as well as presence of non-mucinous BAC histology. Clinical PR tumours also presented large areas of stromal fibrosis with presence of focal residual tumour. In a characterization of intracellular signalling response, EGFR dephosphorylation in the residues Y1068 and Y1173 was not concordant and only the former was significantly reduced. pAkt Ser473/Akt and Thr308/Akt ratios were significantly reduced but observed among both, clinical responders and resistant patients. Interestingly, reduction in pEGFR Y1068 was significantly associated with increase in tumour cellularity (p=0.047), Ki-67 index (p=0.018) and tumour growth (p=0.019) with a residual perinuclear localization been detected, suggesting a novel mechanism of resistance involving receptor internalization. Finally, we determined that the EGFR protein remains stable up to one hour of post resection ischemia but two to three tumour samples are necessary for an adequate tumour representation. Furthermore, EGFR cytoplasmic compartment presented the best association with clinical response in our cohort. Taking all together, we were able to generate the first clinical trial exploring the use of an EGFR TKI in early NSCLC, characterizing for the first time the histopathological and signalling responses to these agents with an evidence of a potential novel mechanism of resistance. Finally, we observed that multiple samples collection for an adequate tumour representation, and assessment of the cytoplasmic compartment, are warrant.

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).

碩士
長庚科技大學
林口校區護理系碩士在職專班
107
High prevalence of dermatologic toxicities and it generated appearance change and negative impact on psychological and quality of life in metastatic colorectal cancer patients receiving epidermal growth factor receptor inhibition target therapy. The purposes of the study were (1) to determine the relationship of EGFR inhibitor-associated dermatologic toxicities, body image, self-esteem, and EGFR inhibitor health-related quality of life; and (2) to identify the predictors of EGFR inhibitor health-related quality of life in metastatic colorectal cancer patients receiving epidermal growth factor receptor inhibition target therapy. A cross-sectional, correlation study was conducted. Patients were assessed by using　Symptom Distress Scale (SDS), NCI-CTCAE4.03, Appearance Anxiety Inventory (AAI), Rosenberg Self-Esteem Scale, (RSE), Functional Assessment of Cancer Therapy-Epidermal Growth Factor Receptor Inhibitors (The FACT-EGFRI-18), and background information form. Patients were recruited using consecutive sampling from colorectal cancer and oncology wards in a medical center in northern Taiwan. Data were analyzed by descriptive analysis, Person’s Product-moment correlation, and multiple linear regressions. A total of 111 patients were included in the study. Patients’ EGFR-related quality of life was negatively related to age. Patients’ EGFR-related quality of life was positively related to target therapy dose, times of treatment, acne, paronychia, symptom distress, and body image. Multiple linear regression analysis indicated that age, target therapy dose, number of target therapy cycles, acne, paronychia, symptom distress, and body image accounted for 65.6% of variations in EGFR-related quality of life. The results can help clinical health providers to provide symptom management for metastatic colorectal cancer patients with skin toxicities and apply in clinical care.

Non-small cell lung cancer (NSCLC) is the most frequent cause of death from cancer worldwide. Despite the availability of active chemotherapy regimens and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, all advanced patients develop recurrent disease after first-line therapy. While heat shock protein 27 (Hsp27) is a stress-induced chaperone that promotes acquired resistance in several cancers, its relationship to treatment resistance in NSCLC has not been defined. Understanding adaptive responses of acquired resistance will help guide new strategies to control NSCLC. Hsp27 levels were evaluated in an HCC827 erlotinib-resistant derived cell line (HCC-827Resistant), and sensitivity to erlotinib was examined in Hsp27 over-expressing A549 cells. The role of Hsp27 in both erlotinib and cytotoxic treatment resistance was evaluated in HCC827 and A549 NSCLC cells using the Hsp27 antisense drug OGX-427 and VPC-27, a new functional small molecule inhibitor of Hsp27. The effect of Hsp27 inhibitors in combination with erlotinib was also assessed in mice bearing A549 or HCC827 xenografts. Hsp27 is induced by erlotinib and protects NSCLC cells from treatment-induced apoptosis, while Hsp27 inhibition sensitizes NSCLC cells to erlotinib. Interestingly, increased resistance to erlotinib was observed when Hsp27 was increased either in HCC827 ER or over-expressing A549 cells. Combining OGX-427 or VPC-27 with erlotinib significantly enhanced antitumor effects in vitro and delayed NSCLC cells growth in vivo. These data indicate that treatment-induced Hsp27 (by inhibition of EGFR) contributes to the development of resistance, and provides pre-clinical proof-of-principle that inhibition of stress adaptive pathways mediated by Hsp27 enhances the activity of erlotinib in NSCLC.

碩士
國立清華大學
生物資訊與結構生物研究所
105
Head and neck squamous cell carcinoma (HNSCC) is the sixth common cancer worldwide and the fifth most common cancer in Taiwan in 2014. Despite various therapeutic strategies were used, the 5-year overall survival rate of locally advanced HNSCC patients is still poor. Thus, investigation of novel therapeutic agents against HNSCC is in urgent need. It was well-characterized that the expression of epidermal growth factor receptor (EGFR) is highly associated with HNSCC pathogenesis and poor prognosis. We recently identified a novel EGFR tyrosine kinase inhibitor (EGFR-TKI), BPR3K007S0, with potent activity against HNSCC growth. In the present study, we aimed to investigate the anticancer functionality and mechanism of action of BPR3K007S0 in EGFR-overexpressing HNSCC cell line, FaDu. Based on cell proliferation assay, BPR3K007S0 is more effective than that of clinical used EGFR-TKI gefitinib (Iressa). BPR3K007S0 inhibited the expression of activated EGFR and its downstream pathway, such as ERK and AKT. Clonogenic assay demonstrated that BPR3K007S0 significantly reduces the colony size without affected colony formation. Cell cycle analysis showed that FaDu cells treated with BPR3K007S0 lead to increase cell cycle arrest in G1 phase without sub-G1 phase appearance. BPR3K007S0 also induced a senescence-associated secretory phenotype in FaDu cells. On the other hand, since EGFR activation is a major cause of metastasis in HNSCC, the anti-metastatic potential of BPR3K007S0 is investigated in FaDu cells. BPR3K007S0 induced mesenchymal-epithelial transition (MET) with upregulation of E-cadherin and downregulation of N-cadherin. The wound healing and transwell invasion assays revealed that cell migration and invasion ability were inhibited by BPR3K007S0. The in vivo study revealed that BPR3K007S0 significantly inhibited tumor and showed better anti-tumor efficacy than that of gefitinib.BPR3K007S0 also reduced the incidence of pulmonary metastasis in the in vivo experimental metastasis model. The number of metastatic pulmonary nodules was significantly reduced in the BPR3K007S0-treated group compared with the control and gefinitib treated group. Taken together, we proposed that BPR3K007S0 is a potent EGFR-TKI and may have a therapeutic benefit in treatment of HNSCC.

Oocyte maturation (OM) in teleosts is under precise hormonal control by estrogens and progestins. We show here that estrogens activate an epidermal growth factor receptor (EGFR) signaling pathway through the G protein-coupled estrogen receptor (GPER) to maintain meiotic arrest of full-grown zebrafish (Danio rerio) oocytes in an in vitro germinal vesicle breakdown (GVBD) bioassay. A GPER- specific agonist decreased OM and a GPER-specific antagonist increased spontaneous OM, whereas specific nuclear estrogen receptor (ERα and ERβ) agonists did not affect OM, which suggests the inhibitory action of estrogens on OM are solely mediated through GPER. Furthermore, a peptide-bound estrogen, which cannot enter the oocyte, decreased GVBD, showing that these estrogen actions are mediated through a membrane receptor. Treatment of oocytes with actinomycin D, a transcription inhibitor, did not block the inhibitory effects of estrogens on OM, indicating that estrogens act via a nongenomic mechanism to maintain oocyte meiotic arrest. EGFR mRNA was detected in denuded zebrafish oocytes by reverse transcription polymerase chain reaction (RT-PCR). Therefore, the potential role of transactivation of EGFR in estrogen inhibition of OM was investigated. The matrix metalloproteinase inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor (HB-EGF), increased spontaneous OM. Moreover, specific EGFR1 (ErbB1) inhibitors and inhibitors of extracellular-related kinase 1 and 2 (ERK1/2) increased spontaneous OM. Previously, estrogens have been shown to increase 3’-5’-cyclic adenosine mono phosphate (cAMP) levels through GPER in zebrafish oocytes during meiotic arrest. Taken together these present results suggest that estrogens also act through GPER to maintain meiotic arrest through a second signaling pathway involving transactivation of EGFR and activation of ERK 1 and 2.
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