Tesi sul tema "He53"

The medical treatment of colorectal cancer (CRC) has evolved greatly in the last years, involving combined chemotherapy protocols and, more recently, new biologic agents. Nevertheless, prognosis remains adverse for patients with metastatic disease and a significant portion of early-stage patients develop recurrence after chemotherapy. Clinical trials are now directed to evaluate new drug combinations and treatment schedules in order to overcome the mechanisms of chemoresistance. By the use of two patient-derived colon cancer cell lines, CC09 (BRAFV600E) and R511 (wt BRAF), and the established colon cancer cell line HT29 (BRAFV600E ; PIK3CAP449T), we found that the tyrosine kinase receptor HER3 is strongly involved in the mechanisms of resistance to 5FluoroUracil (5-FU) and Oxaliplatin drugs. By the use of a monoclonal antibody targeting HER3, named U3-1287, we found down-regulation of HER3 phosphorylation, HER3 internalization and degradation in all cell lines. Functionally, U3-1287 inhibits tumor cell proliferation inducing growth arrest in the G1 phase of the cell cycle, and reduces tumor mass in a CC09-derived xenograft model. Even though U3-1287 administration is higly efficient in abrogating the HER3-dependent activation of PI3K pathway in colon cancer cells, we also found that it induces a compensatory mechanism, involving the increase of HER2 receptor expression that in turn activates MAPK pathway. To overcome U3-1287-induced activation of MAPK, we used a combination therapy with U3-1287 antibody and the MEK-inhibitor Trametinib. We show that Trametinib alone induces the phosphorylation of HER3 receptor that in turn activates PI3K pathway; the combination therapy results in the complete abrogation of both PI3K and MAPK pathways, and in a significant reduction of cell survival in vitro in all three cancers cell lines. These data identify a new combination strategy that, independently of the genetic background of the cells, may overcome the mechanisms of resistance to chemotherapy in HER3-overexpressing colon cancers.

Descripción: Estrategias de Redacción es un curso de Humanidades, que brinda al participante un conjunto de herramientas lingüísticas para la redacción en el entorno laboral, tales como el uso apropiado de la normativa del español y la aplicación de estrategias de redacción como la enumerativa y la causal. Estas últimas se emplean en la elaboración de un tipo de documento: el informe de recomendación. Propósito: El curso desarrolla la competencia de comunicación escrita en el primer nivel de logro. La asignatura se orienta a la redacción de textos administrativos que ayuden a optimizar el desempeño profesional del participante, ya sea en el entorno universitario o en el ámbito laboral.

Creatividad y Liderazgo es un curso de formación general de carácter teórico y práctico que está dirigido a estudiantes de quinto ciclo a más de las Facultades de negocios e Ingeniería y que desarrolla una de las competencias generales de nuestro modelo educativo: Pensamiento Innovador.El curso Creatividad y Liderazgo surge como una respuesta a los vertiginosos cambios tecnológicos y sociales que ocurren en nuestros días los que a su vez producen profundas transformaciones en la cultura corporativa de empresas e instituciones. Esto enfrenta a los ejecutivos a cada vez mayores exigencias profesionales requeridas por las organizaciones que necesitan personas capaces de ejercer un liderazgo en equipos gestores de cambio e innovación. La formación por competencias que se brinda permite al estudiante ejecutivo un aprender haciendo. Las experiencias de aprendizaje se desarrollan en espacios de experimentación y simulación que facilitan al estudiante transferir lo aprendido de manera autónoma a los diferentes ámbitos de su vida y en particular al laboral. Así la formación que llevamos a cabo centra su atención en estrategias de indagación crítica y creativa de problemas reales influencia en los procesos de deliberación así como los modos de interacción. El objetivo central es preparar al ejecutivo para enfrentar de manera exitosa dos problemas: la resolución creativa de problemas y capacitarlos en el ejercicio de un liderazgo proactivo dentro de equipos de trabajo marcados cada vez más por una naturaleza interdisciplinaria.

Creatividad y Liderazgo es un curso de formación general de carácter teórico y práctico que está dirigido a estudiantes de quinto ciclo a más de las Facultades de negocios e Ingeniería y que desarrolla una de las competencias generales de nuestro modelo educativo: Pensamiento Innovador.El curso Creatividad y Liderazgo surge como una respuesta a los vertiginosos cambios tecnológicos y sociales que ocurren en nuestros días los que a su vez producen profundas transformaciones en la cultura corporativa de empresas e instituciones. Esto enfrenta a los ejecutivos a cada vez mayores exigencias profesionales requeridas por las organizaciones que necesitan personas capaces de ejercer un liderazgo en equipos gestores de cambio e innovación. La formación por competencias que se brinda permite al estudiante ejecutivo un aprender haciendo. Las experiencias de aprendizaje se desarrollan en espacios de experimentación y simulación que facilitan al estudiante transferir lo aprendido de manera autónoma a los diferentes ámbitos de su vida y en particular al laboral. Así la formación que llevamos a cabo centra su atención en estrategias de indagación crítica y creativa de problemas reales influencia en los procesos de deliberación así como los modos de interacción. El objetivo central es preparar al ejecutivo para enfrentar de manera exitosa dos problemas: la resolución creativa de problemas y capacitarlos en el ejercicio de un liderazgo proactivo dentro de equipos de trabajo marcados cada vez más por una naturaleza interdisciplinaria.

Creatividad y Liderazgo es un curso de formación general de carácter teórico y práctico que está dirigido a estudiantes de quinto ciclo a más de las Facultades de Negocios e Ingeniería y busca desarrollar una de las competencias generales de nuestro modelo educativo: Pensamiento Innovador.El curso Creatividad y Liderazgo surge como una respuesta a la necesidad de profesionales que cuenten con estrategias y metodologías para afrontar los vertiginosos cambios que ocurren en nuestros días los que a su vez producen profundas transformaciones en la cultura corporativa de empresas e instituciones. Esto enfrenta a los ejecutivos a cada vez mayores exigencias por parte de las organizaciones las cuales requieren personas capaces de involucrar en su práctica y ejercer cotidianamente un liderazgo en equipos gestores de cambio e innovación.La formación por competencias que se brinda permite al estudiante ejecutivo un aprender haciendo. Las experiencias de aprendizaje se desarrollan en espacios de experimentación y simulación que facilitan al estudiante transferir lo aprendido de manera autónoma a los diferentes ámbitos de su vida y en particular al laboral. Así la formación que llevamos a cabo centra su atención en estrategias de indagación crítica y creativa de problemas reales influencia en los procesos de deliberación así como los modos de interacción. El objetivo central es preparar al ejecutivo para enfrentar de manera exitosa dos situaciones: la resolución creativa de problemas y capacitarlos en el ejercicio de un liderazgo proactivo dentro de equipos de trabajo marcados cada vez más por una naturaleza interdisciplinaria.

The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3.

QC 20170330

The receptor tyrosine kinase HER3, a pseudokinase in the epidermal growth factor receptor (EGFR) family, is involved in responses to ligands and in the acquired resistance against HER2-directed targeted therapy in breast cancer. In this thesis I aim to further investigate the structure/function relationships at the basis of HER2- HER3 heterodimerisation, particularly in response to inhibitor treatment. The data presented here shows that in HER2+ breast cancer cells treated with the HER2 inhibitor lapatinib, stimulation with the HER3 ligand NRG is able to not only rescue cytotoxicity, but even promote proliferation in a drug-growth factor cooperative manner. We show that in response to lapatinib treatment, inactive heterodimers of HER2-HER3 can form, and that these heterodimers adopt a different conformation to the canonical, asymmetric active heterodimer. Instead, lapatinib promotes a head-to-head, symmetrical heterodimer, in which the α-C helices of both receptors form the interface. For the formation of the inactive heterodimer, as well as for promoting ligandinduced signalling, we show that HER3 requires ATP-binding capability. Although there are implications that HER3 retains a measure of catalytic activity, I show that the ATP-binding requirement for dimerisation and signalling is independent of activity. To highlight this, I have identified a Src/Abl kinase inhibitor, bosutinib, which has high affinity to HER3. SKBR3 cells treated with bosutinib in the absence of exogenously added ligand show an increase in proliferation in 2D and 3D culture models. This indicates an importance for conformational stabilisation of the HER3 kinase domain in the formation of active signalling heterodimers. As a pseudokinase, the role of HER3 may be conceived as being of lesser importance than that of its heterodimerisation partners such as HER2. The structure/function studies presented here show that HER3 remains of great importance in providing an allosteric platform for HER2, together forming the HER2-HER3 oncogenic heterodimer.

With the lighting speed revolution of technologies in chemistry and biology, increasing number of proteins which eluded scientists' efforts to block them before and were labeled as `undruggable', were successfully targeted with small molecule inhibitors. During the past five years, I have been working on figuring out the path to inhibit two elusive cancer targets: Her3 and KRas.
Chemistry and Chemical Biology

Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.

Background HER2-positive breast cancer is a poor prognostic subgroup, even if treated with anti-HER2 directed therapy. Trastuzumab is an important HER2-targeting antibody but only limited patients respond to this drug, and acquired resistance is a common problem. HER3 has been shown to be a key candidate in mediating resistance to trastuzumab and other ErbB inhibitors. The aims of the project are to investigate the resistance mechanisms and the relevant biomarkers in relation to trastuzumab treatment and resistance in HER2-positive breast cancer, in particular, HER3 subcellular localisation and HER3 phosphorylation. Methods Effects of trastuzumab on HER3 subcellular localisation and HER3 phosphorylation in relation to MET receptor were studied using western blots, nuclear fractionation, confocal microscopy, and immunoprecipitation in a panel of HER2-positive cell lines, including SKBr3 and BT474 breast cancer cells in which trastuzumab resistance was induced by long-term drug exposure. Effects of drug and knockdown experiments were tested by cell viability and proliferation assays. HER3 and MET expression was assessed by immunohistochemistry in xenograft tumours and human tissue samples, and clinical impact was assessed in different cohorts of HER2-positive breast cancer patients. Results Acquired trastuzumab resistant SKBr3 cells showed an increase of nuclear HER3_100kD, which was derived from C-terminus of HER3. Nuclear HER3_100kD could be due to the proteolytic cleavage of HER3 since it was reduced by ADAM17 or gamma-secretase inhibitor. In a panel of HER2-positive cell lines and xenograft samples, nuclear HER3 was observed only in the resistant cells. In addition, nuclear HER3 was associated with poor progression-free and overall survivals in HER2-positive breast cancer patients. It was also found that HER3 phosphorylation was maintained in acquired trastuzumab resistant cells, which was contributed by the ligand independent interaction of MET and HER3. Higher MET expression was associated with better overall survival in HER2-positive, breast cancer patients who were not treated with trastuzumab. Conclusions Nuclear HER3 was found in trastuzumab resistant cells and appeared to result from HER3 proteolytic cleavage mediated by ADAM17 and gamma-secretase. Further studies are required to investigate its mechanism and to identify the HER3 cleavage sites. MET was a key factor in maintaining HER3 phosphorylation during trastuzumab resistance. Lastly, nuclear HER3 and MET could be two potential biomarkers in HER2-positive breast cancer.

De part, leur implication dans la prolifération cellulaire, l'invasion et leur surexpression dans de nombreux cancers, les récepteurs à tyrosine kinase de la famille HER constituent des cibles de choix en oncologie. Parmi ces récepteurs, le récepteur HER3 semble pertinent car il est impliqué dans la tumorigenèse de nombreux cancers (sein, ovaire, pancréas, mélanome…) et il est associé à un mauvais pronostic. De plus, la surexpression du récepteur HER3 est souvent associée à l'apparition de résistance aux thérapies ciblées. Nous avons sélectionné plusieurs anticorps anti-HER3 humains et murins respectivement par « phage display » et fusion cellulaire. Ces derniers reconnaissent spécifiqument le récepteur HER3. Parmi les nombreux anticorps découvert, nous avons sélectionné un anticorps anti-HER3 humain H4B-121 et 2 anticorps anti-HER3 murins 9F7-F11 et 16D3-C1. Ces derniers ont la capacité à faire régresser des tumeurs épidermoide, pancréatique et triple négative chez la souris, en présence ou en abscence de neuréguline et indépendamment du status HER2 et P53/PTEN. Cette inhibition est possible grâce à un blocage du cycle cellulaire en phase G1, une inhibition de la prolifération ainsi qu'une induction de l'apoptose. Ces trois anticorps sont capables de bloquer l'hétérodimérisation des récepteurs HER2/HER3 ainsi que la phosphorylation du récepteur HER3. Ils sont aussi capables d'inhiber la phosphorylation de la protéine AKT ainsi que de ses cibles (MDM2, FOXO et XIAP). De plus, nous avons montré que nos anticorps sont capables de lyser les cellules tumorales par ADCC (Antibody-dependent cell-mediated cytotoxicity). Cette étude démontre que ces anticorps anti-HER3 représentent une nouvelle thérapie pour les cancers du pancréas et du sein triple négatif
Due to their implication in the cellular proliferation, the invasion and their surexpression in numerous cancers, the tyrosine kinase receptors of HER family constitute one of the best targets in oncology. Within this family, the human epidermal growth factor receptor 3 (HER3) plays a role in tumorigenesis of different cancers (Breast, melanoma, pancreas and ovary). This receptor is implicated in drug resistance and he is over expressed in cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells independently of NRG addiction, HER2 status and p53/PTEN mutations. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2(low) cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G1 cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. Anti-HER3 Abs can also induice antibody dependant cell-mediated cytotoxicity. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers

Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In the studies presented herein, MTK1 is shown to be required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced extracellular acidification and cell migration in MCF-7 breast cancer cells. Furthermore, it was shown that HRG stimulation leads to association of MTK1 with tyrosine phosphorylated HER3 in MCF-7 and T-47D breast cancer cells. The MTK1/HER3 association was dependent on HER2 activation and was decreased by pre-treatment with the HER2 inhibitor, lapatinib. Furthermore, HER2 does not directly associate with MTK1, but phosphorylates HER3 transiently. MTK1 also has a role in the ERK1/2 MAPK signaling pathway in response to heregulin (HRG) stimulation in T-47D and MCF-7 breast cancer cells. In addition to MTK1, Shc, Grb2 and GIT1 proteins are all involved in the ERK1/2 MAPK pathway in response to growth factor stimulation. MTK1 was also shown to associate with activated ERK1/2, GIT1, Shc, Grb2 and p85 of PI3K in response to heregulin stimulation. ERK1/2 kinase activity is involved in aberrant signaling that leads breast cancer progression. GIT1 is a scaffolding protein that is linked to growth factor mediated ERK1/2 signaling in cell migration. Moreover, we also identify the actin interacting region (AIR) on MTK1 and disruption of actin cytoskeletal polymerization with cytochalasin D inhibited the interaction between HER3 and MTK1, indicating that f-actin (which is needed for cell migration) is required for the MTK1/HER3 association. Additionally, HRG stimulation leads to extracellar acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.

Avec un taux de survie à 5 ans inférieur à 5%, l'adénocarcinome pancréatique (PDAC) est l'un des cancers les plus agressifs et pour lequel les thérapies existantes sont largement insuffisantes. Ce cancer est caractérisé par un tissu fibreux dense et un stroma très développé, en constante interaction avec la tumeur, favorisant le développement d'un environnement propice à la progression tumorale. Actuellement, la gemcitabine est la seule chimiothérapie approuvée capable de prolonger légèrement la survie des patients. Une thérapie ciblée dirigée contre l'EGFR, l'erlotinib, a prouvé que le ciblage de cette famille pourrait être une stratégie intéressante dans cette pathologie mais qu'une sélection des patients était indispensable pour augmenter les réponses thérapeutiques. Deux récepteurs de cette famille, HER2 et HER3, dimérisent pour former une entité particulièrement agressive et impliquée dans la croissance tumorale des PDACs. Diverses techniques récemment développées sont utilisées pour quantifier ces dimères, afin d'étudier leur rôle, mais également pour tenter de les cibler, et empêcher leur signalisation dans différents cancers. A l'heure actuelle, le pertuzumab est le seul anticorps monoclonal dirigé contre le récepteur HER2, capable de bloquer sa dimérisation, et utilisé en clinique. Dans un premier temps, nous nous sommes intéressés au rôle de HER3 en tant que cible et marqueur pronostique de l'effet du pertuzumab sur les PDAC exprimant faiblement HER2. Puis dans une deuxième partie, nous avons étudié et comparé les effets de différents anticorps monoclonaux dirigés contre HER2 et/ou HER3, sur la prolifération tumorale de tumeurs du pancréas. L'ensemble de ces résultats a permis d'établir que le ciblage du dimère HER2/HER3 s'avère être une stratégie prometteuse pour inhiber la croissance des tumeurs du pancréas exprimant faiblement HER2, et que le récepteur HER3 pourrait être un marqueur pronostique de l'effet du pertuzumab
With a 5-year survival lower than 5%, PDAC is one of the worst cancers in terms of mortality, and for which existing therapies are unsatisfying. This cancer is characterized by a dense fibrotic tissue and an over-developed stroma, in continual interaction with the tumor, promoting the development of an ideal environment for tumor progression.To date, gemcitabine is the only approved chemotherapy able to slightly increase patients' survival. The use of erlotinib, an EGFR targeting therapy, underlined that EGFR family targeting could be an interesting treatment strategy in this pathology, but that a better patients' selection is essential to increase therapeutic response. Two receptors of this family, HER2 and HER3, are able to dimerize to consititute an aggressive entity, involved in PDAC tumoral growth. Various recently developed techniques are used to quantify those dimers, in order to study their role, but also to target them and block their signaling in cancer cells. Pertuzumab is currently the only HER2-targeting monoclonal antibody able to block its dimerization and used in clinic. We first evaluated the role of HER3 as therapeutic target and prognostic marker of pertuzumab efficacy on HER2-low expressing PDACs. We then studied and compared therapeutic effects on pancreatic tumor proliferation of different antibodies targeting HER2 and/or HER3. Taken together, those results demonstrated that HER2/HER3 dimers targeting is a promising strategy to inhibit low-HER2 expressing pancreatic tumor growth, and that HER3 could be a pronostic marker for pertuzumab efficacy in those cancers

Elisidepsin es un nuevo depsipéptido sintético resultante del programa de investigación interna de PharmaMar para la obtención de productos sintéticos derivados de compuestos naturales de origen marino. Elisidepsin es un fármaco antiproliferativo que presenta actividad contra un amplio conjunto de tipos de tumores, entre ellos él de mama. El presente trabajo de investigación se centra en el estudio y caracterización de mecanismos de sensibilidad y resistencia a elisidepsin y en factores predictivos de respuesta al fármaco en un modelo “in vitro” con diversas líneas celulares tumorales. En éste hemos visto que este compuesto tiene una alta actividad citotóxica frente a diferentes líneas celulares, y aunque todavía se desconoce su mecanismo de acción, hemos visto que hay evidencias que relacionan la sensibilidad de esta sustancia con las vías de señalización dependientes de los receptores HER. Tras su tratamiento hemos observado una bajada de la expresión de los niveles de los receptores epidermoides, en especial de HER3 y con él una disminución de la actividad de las vías de supervivencia celular por AKT. La hipótesis de trabajo establecida fue que el receptor HER3 actúa como mediador de la señalización en respuesta al fármaco elisidepsin. Con la finalidad de evaluar esta relación, hemos visto que cuando se modula la expresión de HER3 en diferentes líneas celulares se modifica la sensibilidad de éstas frente el tratamiento de elisidepsin, haciéndolas más resistentes cuando disminuimos los niveles de expresión de HER3 mediante short hairpin específicos y más sensibles cuando se sobreexpresa dicho receptor. Todo esto apunta a un papel importante de este receptor en la mediación de la respuesta a éste fármaco. Además, con la generación de un mutante de HER3 sin dominio tirosina quinasa y tras la realización de diferentes ensayos de viabilidad celular y “cross-linking” se encontró una interacción del receptor HER3 en su porción intracitoplasmática con elisidepsin. Por otro lado, el estudio del receptor HER3 en tumores humanos es todavía muy preliminar, con un escaso número de publicaciones. Debido a que observamos una mayor respuesta de líneas tumorales de mama expresando HER3, continuamos el proyecto analizando con más detalle los niveles de este receptor en una amplia serie de carcinomas de mama pertenecientes a nuestro banco de tumores del Hospital Vall d’Hebron. La evaluación del HER3 en la serie clínica llevó a resultados interesantes, correlacionando la expresión del HER3 con los niveles de receptores estrogénicos, abriendo asimismo una nueva vía fisiopatológica para entender los mecanismos de sensibilidad y resistencia a fármacos antiestrógenos. Finalmente, tras constatar la asociación de la respuesta celular con la expresión de HER3, se planteó una posible asociación también de los niveles de HER3 con otros parámetros importantes en la carcinogénesis de cáncer de mama, como son los receptores hormonales y el estudio molecular de la posible interrelación de las vías bioquímicas relacionadas con la expresión del factor epidérmico HER3 y de los receptores estrogénicos. Para ello se hicieron estudios “in vitro” en el modelo celular MCF7 con el fármaco antiestrogénico (fulvestrant) donde se observó un incremento de los niveles de HER3 tras su administración y la implicación de este receptor en una mayor resistencia al tratamiento de fulvestrant.
Elisidepsin is a new synthetic depsipeptide, resultant from the PharmaMar Development Program to obtain synthetic products of marine origin-derived compounds. Elisidespin is a drug with antiproliferative activity against a wide range of tumors, including breast. The present research work focuses on the study and characterization of mechanisms of sensitivity and resistance to elisidepsin, and predictive factors of the drug response in an "in vitro" model of a broad panel of tumor cell lines. In this, we have seen that this compound has high cytotoxic activity in different cell lines, and although its mechanism of action is still unknown, we have seen evidence that relate the sensitivity of this chemical with signaling pathways dependent on HER receptors. After treatment we observed a decrease of the expression levels of the epidermal receptors, especially HER3 and with it a decreased activity of cell survival pathways by AKT. The working hypothesis is that HER3 receptor mediates signaling in response to elisidepsin. In order to assess this relationship, we saw that when HER3 expression is modulated in different cell lines their sensitivity to elisidepsin treatment is modified, making them more resistant when we down-regulated HER3 expression levels with a HER3-specific short hairpin and more sensitive when the receptor is overexpressed. All this points to an important role of HER3 receptor in mediating the response to this drug. Moreover, after the generation of a HER3 mutant tyrosine kinase free and cell viability and "cross-linking" assays a binding with HER3 receptor intracytoplasmic domain and elisidepsin was found. On the other hand, the study of HER3 receptor in human tumors is still not fully characterized, with a small number of publications. Since we saw a higher response of breast tumor cell lines expressing HER3, we decided to continue the project analyzing in more detail the levels of HER3 in a wide range of breast carcinoma samples from our database of Hospital Vall d'Hebron. The evaluation of HER3 in the clinical series led to interesting results, correlating the expression of HER3 with estrogen receptor levels, opening at the same time a new window for understanding the pathophysiological mechanisms of drug sensitivity and resistance to antiestrogen treatments. Finally, after confirming the association of the cellular response to elisidepsin with the expression of HER3, a possible association of HER3 levels with other important parameters in breast cancer carcinogenesis, such as hormone receptors, and the molecular study of the possible interrelationship of the biochemical pathways related to the expression of HER3 epidermal factor and estrogen receptors were also raised. For this, studies "in vitro" in a MCF-7 cellular model were made with an antiestrogen drug (fulvestrant), which showed an increase of HER3 expression levels after its administration and the involvement of this receptor in an increased resistance to fulvestrant treatment.

The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy. Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated. In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules. Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation. The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.

Affibody molecules are small protein scaffolds that have been engineered to bind to a variety of targets with diversetherapeutic and diagnostic applications. In this study, an array of affibody containing therapeutic constructs,targeting HER2 and HER3, and diagnostic anti-HER3 imaging agents have been purified in preparation for subsequentcancer cell assays and imaging studies in tumour-bearing mice respectively. Herein, the workflow for severalpurification techniques is delineated.

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Four members belong to the erbB family, erbB1, erbB2, erbB3, and erbB4. Several reports have investigated the expression of members from this family, however with only few regarding to the role of erbB3 on non-small cell lung cancer (NSCLC). The aim of this study was to describe the expression frequency of erbB3 protein in NSCLC and evaluate a possible relationship with clinical parameters such as: age, gender, histological subtype, stage, histological grade, and survival. A total of 66 samples were analyzed by immunohistochemistry. ErbB3 expression frequency was detected in 57 samples (86. 4%). No statistical significance was found between expression degree and age, histological subtype, stage, histological grade, and survival. However, when samples were classified into two expression categories (0-75% and 75-100%) a statistic significance was found (P=0. 04) related to grade II (72. 5%). Although we did not found a relation between erbB3 expression and clinical and/or pathological parameters, probably due to the low sample number, it was possible to define that 86. 4% of samples did express the erbB3 protein in at least 25% of the tumor cells. This result agrees with other described in the literature. Investigation of other members belonging to this family in the same sample here evaluated could improve the results obtained since they have been implicated with tumor malignancy and that their role in tumorigenesis is probably associated to a collaborative interaction.
A família erbB é composta por quatro membros: erbB1, erbB2, erbB3 e erbB4. Essa família tem aparecido amplamente em diversos trabalhos, porém, o estudo particular da proteína erbB3 no câncer de pulmão ainda permanece muito restrito. O objetivo desse estudo foi descrever a freqüência de expressão da proteína erbB3 em amostras de câncer de pulmão não-pequenas (NSCLC) células, assim como correlacionar os achados da freqüência de expressão da proteína com as seguintes características clinico-patológicas dos pacientes como: idade, gênero, tipo histológico, estadiamento patológico, grau histológico, óbito e sobrevida. Um total de 66 espécimes foram analisados por imuno-histoquímica. A freqüência de expressão da proteína erbB3 esteve presente em 57 casos (86,4%). Não foi encontrada relação estatisticamente significativa entre a freqüência de expressão da proteína e as variáveis: idade, gênero, tipo histológico, estadiamento, grau histológico, óbito e sobrevida. Entretanto, quando as amostras foram agrupadas em categorias de expressão entre 0 e 75% e 75 e 100% das células tumorais positivas para a proteína erbB3, houve significância estatística com relação ao grau histológico (P=0,04) sendo o de maior freqüência o grau histológico tipo II (72,5%). Embora não tenha sido possível correlacionar a presença da expressão da proteína erbB3 com o prognóstico e os outros parâmetros clínicos, provavelmente devido ao pequeno número de amostras investigadas neste estudo, foi possível observar a presença desta proteína em 86,4% das amostras estudadas, comprovando que, assim como o EGFR e o erbB2, o erbB3 pode ser encontrado no NSCLC de forma bastante pronunciada. É possível que informações relativas à expressão dos outros membros desta família nestas mesmas amostras, possam trazer novas interpretações, lembrando, novamente, que seus membros, já caracterizados como proteínas participantes na oncogênese, atuam de forma sinérgica.

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

Dans ce mémoire, nous étudions l'effet de la polarisation nucléaire sur l'he#3 liquide. La polarisation change les propriétés physiques de ce système par l'intermédiaire du principe de Pauli. Nous montrons expérimentalement qu'il est possible de produire de l'he#3 liquide avec une polarisation hors équilibre mais stationnaire, dans un réfrigérateur à dilution spécial (de type leiden). Le processus de dilution mis en jeu produit de l'he#3 à la fois froid (13 mk) et polarise (15%, 7 fois la valeur à l'équilibre sous un champ de 7 t). Le comportement du polariseur nous a permis de déterminer la pression à laquelle les susceptibilités molaires de l'he#3 concentre et dilue sont égalés, et de corriger les résultats antérieurs obtenus sur la susceptibilité de la phase diluée d'une solution saturée. Cette correction et une analyse des anciennes données sur la dépendance en température de la pression osmotique nous ont permis de déterminer les paramètres de landau f#o#a et m* de l'he#3 dilué saturé à quelques pourcents près. Grâce à notre dispositif, nous avons mesuré la dépendance en polarisation de la pression osmotique de la phase diluée d'une solution saturée. Ces mesures permettent de déduire une susceptibilité de la phase diluée en accord avec notre détermination de f#o#a et m*. Nous ouvrons également des perspectives pour élargir ces mesures vers des polarisations plus élevées.

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A fam?lia erbB ? composta por quatro membros: erbB1, erbB2, erbB3 e erbB4. Essa fam?lia tem aparecido amplamente em diversos trabalhos, por?m, o estudo particular da prote?na erbB3 no c?ncer de pulm?o ainda permanece muito restrito. O objetivo desse estudo foi descrever a freq??ncia de express?o da prote?na erbB3 em amostras de c?ncer de pulm?o n?o-pequenas (NSCLC) c?lulas, assim como correlacionar os achados da freq??ncia de express?o da prote?na com as seguintes caracter?sticas clinico-patol?gicas dos pacientes como: idade, g?nero, tipo histol?gico, estadiamento patol?gico, grau histol?gico, ?bito e sobrevida. Um total de 66 esp?cimes foram analisados por imuno-histoqu?mica. A freq??ncia de express?o da prote?na erbB3 esteve presente em 57 casos (86,4%). N?o foi encontrada rela??o estatisticamente significativa entre a freq??ncia de express?o da prote?na e as vari?veis: idade, g?nero, tipo histol?gico, estadiamento, grau histol?gico, ?bito e sobrevida. Entretanto, quando as amostras foram agrupadas em categorias de express?o entre 0 e 75% e 75 e 100% das c?lulas tumorais positivas para a prote?na erbB3, houve signific?ncia estat?stica com rela??o ao grau histol?gico (P=0,04) sendo o de maior freq??ncia o grau histol?gico tipo II (72,5%). Embora n?o tenha sido poss?vel correlacionar a presen?a da express?o da prote?na erbB3 com o progn?stico e os outros par?metros cl?nicos, provavelmente devido ao pequeno n?mero de amostras investigadas neste estudo, foi poss?vel observar a presen?a desta prote?na em 86,4% das amostras estudadas, comprovando que, assim como o EGFR e o erbB2, o erbB3 pode ser encontrado no NSCLC de forma bastante pronunciada. ? poss?vel que informa??es relativas ? express?o dos outros membros desta fam?lia nestas mesmas amostras, possam trazer novas interpreta??es, lembrando, novamente, que seus membros, j? caracterizados como prote?nas participantes na oncog?nese, atuam de forma sin?rgica

Abstract Cancer treatments have remarkably improved over the past years since targeted therapies and immunotherapy have been introduced to the field of oncology. The benefit of these new therapies is often limited, however, by de novo or acquired therapy resistances, which should be noticed when making clinical decisions. In this current work, we studied the prognostic and predictive values of several immunological markers in metastatic HER2-positive breast cancer treated with trastuzumab, because trastuzumab is still given to patients according to the HER2 status only, without certainty of tumor response. We also determined the role of HER2 and HER3 for cancer stem cells (CSC) in ALK translocated non-small cell lung cancer (NSCLC) cell lines since the CSCs are causing therapy resistance and cancer recurrence. The results demonstrated that a high number of cytotoxic T cells, together with a high number of M1-like macrophages in the center of the tumor (CT), are promising and independent prognostic factors in HER2-positive breast cancer. These markers together also can predict the progression of the disease and the length of trastuzumab discontinuation in tumor response. Expression of HER2 and HER3 increased the stem-like properties of ALK translocated NSCLC cells, which were decreased when the expressions were downregulated. HER2-HER3-dependent CSCs also mediated the ALK therapy resistance. In conclusion, this study suggests that patients with a favorable immunological tumor profile (high number of cytotoxic T cells and M1-like macrophages in the CT) could be treated in a less-intensive manner, that trastuzumab discontinuation could be feasible for these patients, and that targeting of HER2 and HER3 receptors can lead to more effective killing of cancer stem-like cells and should be further studied
Tiivistelmä Syöpähoidot ovat kehittyneet huomattavasti, kun kohdennetut hoidot ja immunologiset hoidot ovat tulleet perinteisten hoitojen rinnalle. Usein näiden hoitojen hyötyä kuitenkin rajoittaa jo olemassa oleva lääkeresistenssi tai sen kehittyminen, mikä tulisi ottaa huomioon hoitoja suunniteltaessa. Tässä työssä tutkittiin immunologisia merkkiaineita, joilla voitaisiin ennustaa trastutsumabi-hoidon vastetta sekä potilaiden ennustetta levinneessä HER2-positiivisessa rintasyövässä. Tällä hetkellä trastutsumabi-hoitopäätös tehdään pelkän HER2-geenimonistuman mukaan ilman varmuutta siitä, hyötyykö potilas oikeasti hoidosta. Lisäksi tutkimme HER2- ja HER3-reseptorien merkitystä syövän kantasoluille ALK-translokoituneessa ei-pienisoluisessa keuhkosyövässä (NSCLC), sillä syövän kantasolut ovat yksi merkittävimmistä tekijöistä lääkeresistenssin kehittymisessä ja syövän uusiutumisessa. Työssä havaittiin, että kasvaimen keskellä oleva suuri määrä sytotoksisia T-soluja sekä M1-tyypin makrofageja on yhteydessä potilaiden parempaan ennusteeseen ja että kyseiset merkkiaineet ovat toisistaan riippumattomia. Merkkiaineet pystyivät ennustamaan myös taudin etenemistä sekä trastutsumabi-hoitokeskeytyksen pituutta. HER2- ja HER3-proteiinien tuotto lisäsi ALK-translokoituneiden NSCLC-solujen kantasolumaisia ominaisuuksia, jotka puolestaan vähenivät, kun proteiinien tuotto estettiin. Lisäksi HER2-HER3 -riippuvaiset syövän kantasolut säätelivät lääkeresistenssiä kyseisessä taudissa. Työn tulokset viittaavat siihen, että potilaita, joilla on suotuisa kasvaimen immunoprofiili (suuri määrä sytotoksisia T-soluja ja M1-tyypin makrofageja kasvaimen keskellä) pystyttäisiin hoitamaan keveimmillä hoidoilla ja HER2-hoitokeskeytys voisi olla mahdollinen näillä potilailla. Lisäksi työ korostaa HER2- ja HER3-reseptorien kohdentamista syövän kantasolumaisten solujen tehokkaamman tuhoamisen saavuttamiseksi
Huomautus/Notice Painetussa virheelliset ISBN -tunnukset: ISBN (print) 978-952-42-2343-8 pitäisi olla 978-952-62-2343-8. ISBN (PDF) 978-952-42-2344-5 pitäisi olla 978-952-62-2344-5. Printed version has incorrect ISBNs: ISBN (print) 978-952-42-2343-8 it should be 978-952-62-2343-8. ISBN (PDF) 978-952-42-2344-5 it should be 978-952-62-2344-5

Le cancer est l'une des principales causes de décès dans le monde. D'après la société américaine contre le cancer; en 2015, un quart des décès aux Etats-Unis est du au cancer du poumon avant même les maladies cardiaques. Cette situation nous incite et bien d'autres scientifiques dans le monde à développer des moyens plus efficaces de traitement, le diagnostic et le dépistage de la maladie. Parce que près de 90% des décès par cancer sont dus à des métastases, de nombreuses études se sont concentrées sur le mécanisme de métastases et sur son impact clinique. Les cellules tumorales circulantes (CTC) sont les cellules s’échappent de tumeurs primaires ou métastatiques pour rejoindre le flux sanguin périphérique, ces cellules sont un élément de transition dans le processus métastatique et portent ainsi des informations cruciales sur ce mécanisme encore mal compris. Les CTCs ont déjà montré leur potentiel comme biomarqueur de pronostic de la progression de la maladie et de l'indicateur de l'efficacité du traitement en fonction l’augmentation ou de la diminution de leur nombre. Leur caractérisation moléculaire peut également donner des informations vis à vis de cibles thérapeutiques possibles et des mécanismes de progression de la maladie ou de la résistance aux médicaments. Leur comptage au cours du traitement combiné avec leur caractérisation moléculaire devrait améliorer la prise en charge des patients dans le cadre de la médecine personnalisée. Cependant CTCs sont extrêmement rares, 1 à 10 cellules / ml de sang parmi les 106 globules blancs et 109 globules rouges, leur capture à partir du sang reste donc un challenge analytique. Dans les dernières décennies, Une grande variété de techniques d'enrichissement et de capture a été mise au point et l'approche microfluidique est l'une des méthodes efficaces, flexibles et à haut débit. Au sein de notre équipe, un dispositif microfluidique (système Ephesia) puissant pour la capture et l'analyse des cellules tumorales circulantes a déjà été mis au point précédemment. Le principe de capture est basé sur l'auto-assemblage de billes magnétiques greffées par des anticorps, grâce aux quelles les cellules sont enrichies via l’interaction Ab- l'antigène de surface EpCAM que l'on trouve communément dans les cellules cancéreuses d'origine épithéliale. Ce système a déjà été validé avec des lignées cellulaires et des échantillons de patients. Cependant, le système n'a pas permis l'isolement / détection des sous-populations de CTCs ou d'effectuer une caractérisation moléculaire très poussée. Par conséquent, mon projet de thèse vise à améliorer encore les capacités du système sur les deux principaux aspects: le ciblage sous-populations de CTC et à l'étude des interactions des protéines à la surface des CTCs dans le Système Ephesia
Metastasis is the advanced stage of cancer progression and is the cause of 90% of deaths in cancer disease. During metastatic cascade, it is suggested that the successful metastatic initiation depends on the survival of circulating tumor cells (CTCs). CTCs are the cells that shed from the primary or secondary tumor sites into the blood circulation. it is now widely recognized as potential biomarker for companion diagnostics in which high number of CTCs in blood can indicate association with poor survival or high risk of disease progression. Besides, following the number of CTCs during the course of treatment can help to adapt the selected therapy and predict the treatment efficacy. On the other hand molecular characterization can provide patient stratification and identifying the therapeutic targets. However they are extremely rare in the bloodstream, estimated between 1-10 CTC among 6×106 leukocytes, 2×108 platelets and 4×109 erythrocytes per one mL of blood which makes their isolation very challenging. A very attractive way of isolation of CTCs is to integrate microfluidics. Microfluidics offers great advantages such as low volume of reagent consumption and short analysis times with automation as well as isolation and detection analysis can be integrated resulting in highly efficient biomedical devices for diagnostics. As parallel to state of the art, a powerful microfluidic device for circulating tumor cells capture and analysis had already been developed previously in our laboratory. The principle of capture is based on self-assembly of antibody-coated (EpCAM) magnetic beads in which the cells are enriched by EpCAM surface antigen which is found commonly in epithelial origin cancer cells. This system was already validated with cell lines and patients samples. However, the system did not allow isolation/detection of subpopulations of CTCs or performing high content molecular characterization. Therefore, my PhD project aimed at further improving the capabilities of the system on the main two aspects: targeting subpopulations of CTC and studying of protein interactions of CTCs in Ephesia System

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

Recently HER3, member of the epidermal growth factor receptor family (EGFR), has been found to play a crucial role in the development of resistance towards inhibitors that are given to patients with HER1- and HER2-driven cancers. As HER3 is up-regulated or over-activated in several types of human cancers, it is of outmost importance that new innovative drugs target its oncologic activity. The Affibody® Molecule Z08698 inhibits the heregulin induced signalling of HER3 with high affinity (KD~50 pM). As the Affibody® Molecule is small, has high solubility and outstanding folding kinetics, an effective penetration of tumour tissue is suggested together with a rationalized manufacturing process. Further coupling to an albumin binding domain (ABD) expands the plasma half-life of the molecule, hence increasing the molecule's potential of serving as a therapeutic. A process development for production of Z08698-VDGS-ABD094 has been established, where the molecule is efficiently produced in the E. coli host strain BL21(DE3), through a T7 based expression system. Cultivations were performed with a fed-batch fermentation process and the conditions were further optimized in order to obtain highest expression, while avoiding undesirable modifications like gluconoylations. By employing Design of experiments in combination with multivariate data analysis, a production process resulting in ~3.5 g product/ l culture could be verified. Moreover, thermolysis was evaluated as a suitable method for cell disruption, enabling an easy and cost-effective manufacturing process of the ABD fused Affibody® Molecule.

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

Introduction The adult brain exhibits low regenerative ability. Stem cell-based transplantation approaches have been largely unsuccessful, due to the difficulty to recapitulate the complex cytoarchitecture of the central nervous system (CNS). eNSCs are a new therapeutic option as pharmacological activation and increase of their number in vivo is accompanied by powerful neuroprotection in various disease models. Hes3 is expressed in both proliferating and quiescent NSCs, which makes it a useful biomarker for NSC identification. Direct injections of insulin in the adult brain increase the number of eNSCs and promote rescue of injured neurons via a novel molecular mechanism, the STAT3-Ser/Hes3 Signaling Axis. This molecular pathway with the STAT3-Ser phosphorylation at its core regulates Hes3 and together they form a merging point for several signals including insulin receptor activation. Main aim and Hypothesis Beyond the brain, STAT3-Ser/Hes3 signaling regulates various plastic cell populations in other organs of the endocrine/neuroendocrine system. In the pancreas, Hes3 is expressed in islets cells and regulates their growth, regeneration, and insulin release. Hes3 is also expressed in mouse hypothalamic tanycytes, which are diet responsive cells and play a very crucial role for the communication between the brain and the endocrine system. Also, Hes3 is expressed in the adrenal gland (both in the cortex and medulla); cultured adrenal progenitors express Hes3 and various treatments that induce Hes3 expression promote their growth. Therefore, STAT3-Ser/Hes3 Signaling may be involved in tissue problems that result from metabolic dysfunction. Metabolic syndrome often results in diabetes (Type I, Type II) and insulin resistance, suggesting that eNSCs may be affected by the condition. There is evidence that obesity induces inflammatory reactions in the hypothalamus, leading to NSC loss. However, it is not clear if damage to NSCs is also directly linked to insulin signaling disruption. Results Our results show that various parameters affect Hes3 levels in the brain. Aging decreased Hes3 mRNA expression. Type I diabetes increased Hes3 expression. Type II diabetes decreased Hes3 expression. Thus, we conclude that eNSCs are modulated by diabetes in an age-dependent manner. We also investigated whether common medication for metabolic related dysfunction also affects Hes3 expression in the adult brain. Indeed, our results show that metformin decreases Hes3 expression in the mouse hypothalamus. To address whether metformin has a direct effect on NSCs we treated primary mouse fNSCs with metformin. Metformin decreases cell number, proliferation and affects cell morphology, giving a more differentiated appearance (large, flat cell body with wider projections). Hes3 expression increases significantly at 72 hours of treatment. The metformin result opens the question if the increase in the Hes3 expression is a direct effect of the signal transduction pathways activated by metformin or due to a stress reaction. To address this we treated NSCs with exendin-4, another diabetes drug that we previously showed to both elevate Hes3 expression and cell number using a mouse insulinoma cell line (MIN6). Exendin-4 increases fNSC cell number but it did not affect the morphology. Similar to metformin proliferation was not affected. Hes3 expression increased significantly at 72 hours of treatment as well. This result indicates the distinctive action of the drugs on the STAT3-Ser/Hes3 signaling pathway. Specifically it dissociates Hes3 levels from other cellular parameters. Importantly it shows that two common diabetes medications have very different effects on NSCs. Because Hes3 is strongly regulated by metabolic parameters and medication we addressed potential roles of Hes3 using an established Hes3 null mouse line. Hes3 null mice exhibit no obvious phenotypes under normal conditions. However, we previously showed that when stressed by chemical induced damage, they exhibit low regenerative potential in the pancreas and brain. To identify additional phenotypes, we performed a phenotypic analysis of the Hes3 null mouse line under normal diet and HFD conditions (which induced type II diabetes). We found mild phenotypes that relate to the nervous system, the immune system and metabolism. At the molecular level, Hes3 deletion affects the expression of other genes within the Hes superfamily in the adult mouse brain. However, we did not observe these molecular differences in the HFD condition, suggesting an interplay between metabolic parameters (possibly, circulating insulin) and the regulation of Hes/Hey genes in the brain. Our data suggest a broad range of roles for Hes3, particularly under abnormal conditions. Conclusions Our work establishes that multiple parameters of metabolic state as well as diabetes medication affect Hes3 expression in the brain. Metabolic syndrome is a risk factor for many neurological disorders such as Alzheimer’s disease, Parkinson’s disease and Multiple Sclerosis. It is important to understand at the molecular and cellular level how metabolic dysfunction affects the brain. Here, we introduced a new cellular biomarker and signaling component that is greatly regulated in metabolic dysfunction.:1 Introduction 18 1.1 The ''plastic brain'': Neural Stem Cells, progenitors and precursors 19 1.2 Functional adult neurogenesis 19 1.3 NSCs in conventional and nonconventional regions of the adult brain 20 1.4 Neurodegenerative diseases, cell replacement and endogenous NSCs 21 1.5 The STAT3-Ser/Hes3 signaling axis in NSCs 24 1.6 Beyond the brain: The STAT3-Ser/Hes3 signaling axis operates in plastic cells 27 1.6.1 STAT3-Ser/Hes3 Signaling Axis in the pancreatic islet 27 1.6.2 STAT3-Ser/Hes3 Signaling Axis in the adrenal cortex and medulla 28 1.6.3 STAT3-Ser/Hes3 Signaling Axis in tanycytes of the hypothalamus? 28 1.6.4 STAT3-Ser/Hes3 Signaling: A new molecular component of the neuroendocrine system? 29 1.7 Metabolic syndrome and neurological disease 31 1.7.1 Metabolic dysfunction and Alzheimer's disease 31 1.7.2 Metabolic dysfunction and Parkinson's disease 31 1.7.3 Metabolic dysfunction and Multiple Sclerosis 32 1.7.4 Metabolism and neurodegenerative disease: Are they connected? 32 1.8 Main Aim – Hypothesis 33 2 Materials and Methods 34 2.1 Animal experiments 34 2.1.1 Animal use authorization 34 2.1.2 Genotyping 34 2.1.3 In vivo models 36 2.1.4 In vivo metabolic Analyses 36 2.1.5 Nociception 37 2.1.6 Histology 38 2.1.7 PCR and Real-Time quantitative PCR (qPCR) 39 2.1.8 Western Blot 41 2.2 Mouse phenotyping 42 2.3 Neural stem cell cultures 43 2.3.1 Preparation – Coatings 43 2.3.2 Cell Isolation and Cell Culture 43 2.3.3 Pharmacological Manipulation (Metformin – Exendin-4) 43 2.4 Heat maps 44 2.5 Statistical analyses 44 3 Results 45 3.1 Hes3 is expressed in the mouse brain 46 3.2 Aging and diabetes models alter Hes3 in the brain 48 3.2.1 Hes3 expression decreases with age 48 3.2.2 Pancreatic islet damage by streptozotocin increases Hes3 expression in the brain 48 3.2.3 High Fat Diet reduces Hes3 expression in the brain 49 3.3 Common diabetes medication affect neural stem cells (NSCs) in the brain 53 3.3.1 Metformin decreases Hes3 expression in the brain 53 3.3.2 Metformin opposes growth but increases Hes3 expression in cultured NSCs 54 3.3.3 Exendin-4 promotes growth and increases Hes3 expression in cultured NSCs 54 3.3.4 Metformin and Exendin-4 affect the STAT3-Ser/Hes3 signaling axis 59 3.4 Hes3 null mice exhibit a quasi-normal phenotype 60 3.4.1 Phenotypic Analysis - Normal Diet (ND) 60 3.4.2 Metabolism Relevant Phenotypes – HFD challenge 63 3.4.3 Phenotypic Analysis – Molecular 67 4 DISCUSSION 70 4.1 Diabetes affects the brain 71 4.2 STAT3-Ser/Hes3: a putative mediator 71 4.3 Hes3 is a special member of the Hes/Hey gene family 72 4.4 Patterns of Hes3 expression may be specific to cell type and microenvironment 72 4.5 Metabolic dysfunction and diabetes medication affect brain Hes3 73 4.5.1 Age regulates Hes3 73 4.5.2 Diabetes models regulate Hes3 expression in the brain 74 4.5.3 Metformin regulates Hes3 expression in the brain 74 4.6 Hes3 phenotyping provides clues to Hes3 functions 76 4.7 Hes3 and metabolic dysfunction: Are they connected? 77 5 Conclusions and Future Remarks 79 References 81

Cancer is the most fatal disease after cardiovascular disease with over 8.2 million deaths worldwide each year. Ever since the serendipitous discovery of mustard gas as an anti-cancer therapeutic in the 1940s, serious efforts have been put into discovering more chemotherapies. Chemotherapies can be categorized into different groups such as alkylating agents (cisplatin, cyclophosphamide), antimetabolites (5-fluorouracil, Ara-C) and mitotic inhibitors (taxanes, vinca alkoids) among others. While chemotherapies have proven to kill cancer cells by targeting cell division processes, over time, tumor cells can adapt and become resistant to these drugs. With a growing understanding of cell signaling networks, targeted therapies are being developed to overcome the issue of chemotherapy resistance. Targeted therapies are highly specific molecules that bind to a specific cellular protein or molecule and block signaling networks associated with biological processes. One of the most frequently dysregulated receptor systems in cancers is the receptor tyrosine kinase family with ErbB being one of the most studied receptors families. ErbB or HER receptors consists of four structurally related receptor tyrosine kinases namely, EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4. The ErbB family of receptors plays a major role in morphogenesis of the human body as well as various cellular responses such as cell growth, differentiation and proliferation. Overexpression and dysregulation of these receptors, particularly EGFR and HER2, have been linked to a number of cancers such as breast cancer, gastric cancer, ovarian cancer and non-small cell lung cancer, to name a few. One of the most successful therapies against ErbB related cancers have been targeted therapies. Targeted therapies for ErbB related cancers are of two kinds: (i) Small molecule tyrosine kinase inhibitors (such as erlotinib and gefitinib against EGFR) and, (ii) Monoclonal antibodies (such as trastuzumab against HER2 and cetuximab against EGFR). These drugs function either by inhibiting the kinase activity of the receptor and preventing phosophorylation of tyrosine residues, or binding to some other site on the extracellular domain of the receptor and preventing ligand binding and heterodimerization of ErbB monomers. These drugs have proven to have limited efficacy as monotherapy, but are more effective in combination with standard chemotherapies. However, tumor cells can adapt their signaling networks developing resistance to targeted therapies over the course of treatment and lead to cancer progression. While overexpression and dysfunction of EGFR and HER2 are implicated in most ErbB driven cancers, recent studies have found HER3 playing a pivotal role in inducing resistance to EGFR and HER2 targeted therapies in various cancers and has been found to be the most sensitive node in driving the PI3K pathway leading to tumorigenesis. Thus, there is an urgent need to develop drugs targeted against HER3 and bring them into the clinic. Since HER3 lacks kinase activity, only monoclonal antibodies can be developed against it. Currently, there are a number of molecules in clinical development that target HER3. For example, patritumab and MM-121 are humanized monoclonal antibodies that target the extracellular domain of HER3 receptor and leads to inhibition of HER3-PI3K signaling followed by rapid internalization of the receptor. MM-111 and MM-141, two different bispecific monoclonal antibodies that bind to HER2, HER3 and IGFR-1, HER3, respectively, are currently in clinical development. HER3 inhibitors provide hope to effectively overcome HER3 induced tumor resistance and successfully treat several ErbB driven cancers. However, further development of HER3 inhibitors is necessary by taking strategic approaches. One of these approaches it the utilization of systems biology, a branch of biology that involves computational and mathematical modeling of complex biological systems with the aim of discovering emergent properties of biological systems. Systems biology enables researchers to get a deeper understanding of biological networks such as that of ErbB and make predictive models and test outcomes. This approach was used by Merrimack Pharmaceuticals to develop novel monoclonal antibodies against HER3. Computational outcomes were successfully validated by in vitro and in vivo experiments. Thus, this suggests that systems biology might be the future of designing and developing HER3 inhibitors that would successfully overcome HER3 resistance and cancer progression.

Assessing hormone receptors (the estrogen and progesterone receptors) and the human epidermal growth factor receptor 2 (HER2) to guide clinical decision making revolutionized treatment for breast cancer patients. However, in the years since these biomarkers were first incorporated into routine clinical care, only a few others have been validated as clinically useful in guiding adjuvant chemotherapy decisions and are recommended by the American Society of Clinical Oncology (ASCO) for patients with hormone-positive breast cancer. For patients with triple-negative breast cancer (TNBC), which lacks hormone and HER2 receptors, not any of these biomarkers are recommended by ASCO due to insufficient evidence that they meaningfully improve clinical outcomes. Breast cancer is the second-leading cause of cancer-related death among women in the US, indicating an unmet need to improve treatments, which can be accomplished in part by identifying and validating novel predictive and prognostic biomarkers that yield actionable information about the clinical course of breast cancers, especially TNBCs. A major obstacle to improving outcomes for breast cancer patients is intratumor heterogeneity (ITH), which can be extensive in breast cancer and drives treatment resistance and relapse. I envision that assaying drivers of ITH can inform clinicians about which breast tumors may be intrinsically more aggressive and carry a greater risk of breast cancer-related morbidity and mortality. My research, presented here, primarily focuses on testing the impact of drivers of ITH (namely, centrosome amplification [CA], the clustering protein KIFC1, and mitotic propensity and its drivers) on clinical outcomes in breast cancer in multivariable models as well as the correlates of in vitro efficacy of centrosome declustering drugs (which can selectively eliminate cancer cells with CA). Collectively, these studies reveal gene signatures and immunohistochemical biomarkers that are independent predictors of aggressive breast cancer course and rational strategies to optimize targeted therapy to combat cancer cells exhibiting CA, thereby contributing to the literature on the development of precision medicine for breast cancer patients, including TNBC patients.

Ο καρκίνος του παχέος εντέρου αποτελεί ένα από τα συχνότερα κακοήθη νεοπλάσματα παγκοσμίως, προκαλώντας σημαντική νοσηρότητα και θνητότητα. Η συστηματική διερεύνηση της παθογένειας συντελεί σημαντικά στην πρόληψη, έγκαιρη διάγνωση, θεραπεία και πρόγνωση της νόσου. Στην εποχή της μοριακής ιατρικής, η κατανόηση των μοριακών μηχανισμών καρκινογένεσης έχει αναδείξει το ρόλο ενδοκυττάριων σηματοδοτικών μονοπατιών, τα οποία ρυθμίζουν κυτταρικές διαδικασίες, όπως πολλαπλασιασμός, διαφοροποίηση, απόπτωση, αγγειογένεση, διήθηση. Κομβικά συστατικά τέτοιων μοριακών δικτύων αποτελούν οι υποδοχείς αυξητικών παραγόντων, όπως ο Human Εpidermal Receptor-1 (ΗΕR-1/EGFR), ο ρόλος του οποίου έχει τεκμηριωθεί στο αδενοκαρκίνωμα παχέος εντέρου, επιτείνοντας το ενδιαφέρον στη διευκρίνηση του ρόλου γειτονικών υποδοχέων, όπως του Human Εpidermal Receptor-3 (HER-3). O HER-3 συνιστά μεμβρανικό υποδοχέα που συμμετέχει σε μοριακά μονοπάτια ενδοκυτταρικής σηματοδότησης. Επομένως, ποσοτικές μεταβολές στην έκφρασή του αλλά και ποιοτικές αλλαγές στη δομή του, συντελούν δυνητικά στην κυτταρική εξαλλαγή. Τα δεδομένα από τη μελέτη του HER-3 στο αδενοκαρκίνωμα παχέος εντέρου είναι περιορισμένα και ο ρόλος του στην παθογένεια, πρόγνωση και θεραπεία της νόσου παραμένει ασαφής. Αντίστοιχα με τους μεμβρανικούς υποδοχείς, πυρηνικοί υποδοχείς ενέχονται σε διαδικασίες ρύθμισης γονιδιακής έκφρασης. Οι οιστρογονικοί υποδοχείς (ER) α και β διαμεσολαβούν την επίδραση των οιστρογόνων σε κυτταρικό επίπεδο, μεταποιώντας τα επίπεδα των ορμονών σε μεταβολές γονιδιακής έκφρασης. Οι διαφορές στην επίπτωση του καρκίνου του παχέος εντέρου ανάμεσα στα δύο φύλα καθώς και πρόσφατα βιβλιογραφικά δεδομένα έχουν αναδείξει τη σημασία της έκφρασης αυτών των υποδοχέων στην καρκινογένεση του παχέος έντερου. Παράλληλα, η εξειδίκευση της οιστρογονικής δράσης σε επίπεδο ιστού και κυττάρου εξασφαλίζεται μέσω της δράσης συγκεκριμένων συμπαραγόντων (ενεργοποιητών/καταστολέων). Αυτές οι πρωτεΐνες είναι ειδικοί μεταγραφικοί παράγοντες, οι οποίοι συνδεόμενοι με τους οιστρογονικούς αλλά και άλλους υποδοχείς «εξατομικεύουν» την ορμονική επίδραση στο γονιδίωμα. Ο proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), γνωστός και ως modulator of non-genomic activity of ER (MNAR), ο amplified in breast cancer-1 (AIB1) και ο transcriptional intermediary factor-2 (TIF2) είναι σημαντικοί συμπαράγοντες οιστρογονικών υποδοχέων, με συνέπεια η μελέτη τους να αποτελεί αναπόσπαστο μέρος της διερεύνησης οιστρογονο-εξαρτώμενων μηχανισμών. Η συνεχής αλληλεπίδραση HER- και ER-εξαρτώμενων μονοπατιών έχει περιγραφεί σε ποικίλλους ιστούς. Σκοπός της παρούσας μελέτης είναι η διερεύνηση του ρόλου της (υπερ)έκφρασης του μεμβρανικού υποδοχέα HER-3, των οιστρογονικών υποδοχέων (ER) και των συμπαραγόντων PELP1/MNAR, AIB-1 και TIF-2 στον καρκίνο του παχέος εντέρου. Υλικά και μέθοδοι Στην παρούσα μελέτη χρησιμοποιήθηκαν ιστικά δείγματα, μονιμοποιημένα σε φορμόλη και εκλεισμένα σε παραφίνη από 140 ασθενείς με αδενοκαρκίνωμα παχέος εντέρου. Η έκφραση των επιπέδων HER-3 mRNA εκτιμήθηκε με τη μέθοδο της ποσοτικής αλυσιδωτής αντίδρασης πολυμεράσης (RT-PCR) σε 54 αδενοκαρκινώματα και 29 δείγματα φυσιολογικού βλεννογόνου. Η έκφραση των επιπέδων της φωσφορυλιωμένης (pHER-3) και μη φωσφορυλιωμένης HER-3 πρωτείνης εκτιμήθηκε με τη μέθοδο της ανοσοιστοχημείας σε 110 δείγματα φυσιολογικού βλεννογόνου, 24 αδενώματα και 140 αδενοκαρκινώματα. Η έκφραση των επιπέδων του οιστρογονικού υποδοχέα α και β και του PELP1/ΜΝΑR εκτιμήθηκε με τη μέθοδο της ανοσοιστοχημείας σε 113 αδενοκαρκινώματα, 30 αδενώματα και 88 δείγματα φυσιολογικού βλεννογόνου. Αντίστοιχα η έκφραση των επιπέδων του ΑΙΒ1 και TIF-2 εκτιμήθηκε με τη μέθοδο της ανοσοιστοχημείας σε 110 αδενοκαρκινώματα, 30 αδενώματα και 83 δείγματα φυσιολογικού βλεννογόνου. Αποτελέσματα Η πρωτεΐνη HER-3 εκφράζεται στο κυτταρόπλασμα και στον πυρήνα επιθηλιακών κυττάρων παχέος εντέρου σε φυσιολογικό βλεννογόνο, αδενώματα και αδενοκαρκινώματα και φαίνεται να μεταβάλλεται τοπογραφικά (από τον πυρήνα στο κυτταρόπλασμα) κατά τη διαδικασία της καρκινογένεσης. Ωστόσο, η έκφραση της πρωτεΐνης HER-3 σε αδενοκαρκινώματα δε φαίνεται να συσχετίζεται σημαντικά με τις κλινικοπαθολογικές παραμέτρους της νόσου. Η φωσφορυλιωμένη μορφή της πρωτεΐνης HER-3 (pHER-3) εκφράζεται στον πυρήνα και τη μεμβράνη επιθηλιακών και λείων μυικών κυττάρων παχέος εντέρου σε φυσιολογικό βλεννογόνο, αδενώματα και αδενοκαρκινώματα. Η πυρηνική έκφραση pHER-3 δε διαφέρει σημαντικά ανάμεσα σε φυσιολογικό βλεννογόνο, αδενώματα και αδενοκαρκινώματα. Ωστόσο, αυξημένα επίπεδα πυρηνικής έκφρασης pHER-3 συσχετίζονται σημαντικά με μεγαλύτερο στάδιο της νόσου, χωρίς να συσχετίζονται με τα επίπεδα έκφρασης της μη φωσφορυλιωμένης μορφής. Τα επίπεδα έκφρασης του γονιδίου ΗER-3 δε φαίνεται να αυξάνουν σημαντικά κατά την καρκινογένεση. Ωστόσο, αυξημένα επίπεδα HER-3 mRNA στα αδενοκαρκινώματα σχετίζονται με μεγαλύτερη ηλικία των ασθενών, εντόπιση του όγκου στο αριστερό κόλον και το ορθό και με λεμφαδενική διήθηση. Επίσης, συχετίστηκαν με αυξημένη πιθανότητα υποτροπής της νόσου και μειωμένο χρονικό διάστημα ως την υποτροπή. Ο ERα εκφράζεται σπάνια στο παχύ έντερο σε αντίθεση με τον ERβ, ο οποίος εκφράζεται συχνά στον πυρήνα επιθηλιακών αλλά και στρωματικών κυττάρων. Η έκφραση της πρωτεΐνης ERβ καθίσταται πιο έντονη κατά τη διάρκεια της καρκινογένεσης σε άνδρες, και στα αδενοκαρκινώματα συσχετίζεται με την πιθανότητα υποτροπής της νόσου. Ο συμπαράγοντας PELP1/MNAR ανιχνεύεται στον πυρήνα επιθηλιακών αλλά και στρωματικών κυττάρων του παχέος εντέρου και η έκφρασή του αυξάνει κατά την καρκινογένεση και στα αδενοκαρκινώματα συσχετίζεται με την έκφραση του ERβ. Ωστόσο, η υπερέκφραση του PELP1/MNAR στα ERβ θετικά αδενοκαρκινώματα συσχετίζεται με μεγαλύτερη συνολική επιβίωση των ασθενών. Ο AIB1 και ο TIF2 ανιχνεύονται στον πυρήνα επιθηλιακών αλλά και στρωματικών κυττάρων του παχέος εντέρου. Η έκφραση του ΑΙΒ1 αυξάνει κατά την καρκινογένεση και συσχετίζεται με τοπική ανάπτυξη του όγκου. Ωστόσο, στην πολυπαραγοντική ανάλυση ο ΑΙΒ1 αναδεικνύεται ως ανεξάρτητος ευνοϊκός προγνωστικός παράγοντας ως προς τη συνολική επιβίωση. Η έκφραση του ΤΙF2 αυξάνει κατά την καρκινογένεση και στα αδενοκαρκινώματα συσχετίζεται με την έκφραση του AIB1. Ωστόσο, η έκφρασή του στα αδενοκαρκινώματα δε σχετίζεται με κλινικοπαθολογικές παραμέτρους της νόσου. Το πρότυπο έκφρασης των συμπαραγόντων AIB1 και TIF2 δε σχετίζεται σημαντικά με εκείνο του ERβ. Συζήτηση-συμπεράσματα-προοπτικές Η παρούσα μελέτη αναδεικνύει τη σημασία της έκφρασης και φωσφορυλίωσης του ΗΕR3 στην καρκινογένεση του παχέος εντέρου, υποστηρίζοντας τον πιθανό ρόλο τους στην πρόγνωση της νόσου. Τα ευρήματα της μελέτης συμφωνούν με συγκεκριμένα βιβλιογραφικά δεδομένα ως προς τη συχνότητα ανίχνευσης του υποδοχέα στον παχύ έντερο, ενώ είναι η πρώτη η οποία αναδεικνύει τα επίπεδα HER-3 mRNA ως πιθανό προγνωστικό βιοδείκτη. Η διερεύνηση της έκφρασης του οιστρογονικού υποδοχέα β (ERβ1) ανέδειξε αύξηση των επιπέδων του κατά την καρκινογένεση, εύρημα που βρίσκεται σε συμφωνία με μερικά και σε αντιδιαστολή με άλλα βιβλιογραφικά δεδομένα. Η προγνωστική σημασία της έκφρασης των πυρηνικών υποδοχέων εξαρτάται από ποικίλους παράγοντες, όπως τα επίπεδα ειδικών συμπαραγόντων, ομοιοπολικές τροποποιήσεις, την τοπογραφία τους μέσα στο κύτταρο και τα επίπεδα συγκεκριμένων συγκεντρώσεων προσδέτη/ορμόνης. Η αύξηση των επιπέδων των συμπαραγόντων του οιστρογονικού υποδοχέα PELP1/MNAR, ΑΙΒ1 και ΤΙF2 κατά την καρκινογένεση υποδηλώνει πιθανή συμμετοχή τους σε οιστρογονοεξαρτώμενη ογκογόνο σηματοδότηση. Ωστόσο, η απουσία ισχυρής συσχέτισης ανάμεσα στα επίπεδα έκφρασής τους και στα επίπεδα έκφρασης του οιστρογονικού υποδοχέα (ERβ1) υπογραμμίζει την πλειοτροπική τους δράση και τον επιπρόσθετο ρόλο τους σε οιστρογονο-ανεξάρτητη ενδοκυττάρια σηματοδότηση. Περισσότερες μελέτες σε μεγάλο αριθμό ασθενών, κατάλληλα σχεδιασμένες και εκτελεσμένες, με τη χρήση ευαίσθητων και ειδικών πειραματικών τεχνικών καθώς και μεθόδων στατιστικής ανάλυσης απαιτούνται για την επιβεβαίωση των ευρημάτων της παρούσας μελέτης. Στην εποχή της μεταγονιδιωματικής ιατρικής, η αναζήτηση χρήσιμων μοριακών βιοδεικτών δύναται να συντελέσει στη βελτίωση της πρόγνωσης και της ποιότητας ζωής των ασθενών με καρκίνο του παχέος εντέρου.
Colorectal cancer is a major cause of cancer-related morbidity and mortality in the western world and has a significant impact on the health care systems. The deep understanding of molecular mechanisms that underline cellular transformation and tumor progression leads to the identification of key-molecules that are appropriate targets for sophisticated therapy in cancer. One such targeted approach exploits the presence of specific biomarkers that could be considered essential for tumor development. The role of such a biomarker, Human Εpidermal Receptor-1 (ΗΕR-1/EGFR), has been established in the development of colorectal cancer, suggesting the potential involvement of neighboring receptors, such as Human Εpidermal Receptor-3 (HER-3). HER-3 is a membranic receptor implicated in intracellular cell proliferation signaling. Thus, quantitative modifications in its expression and/or qualitative changes in its structure may contribute to cellular malignant transformation. The significance of HER3 expression, localization and phosphorylation remains elusive and data regarding its role in the pathogenesis, diagnosis, prognosis and management of colorectal cancer is limited. Apart from their mebranic counterparts, nuclear receptors are implicated in the regulation of gene transcription. Estrogen receptors (ER) α and β mediate the estrogen actions in the subcellular microenvironment. Differences in the incidence of colorectal cancer in the two genders have underlined the significance of ER expression in colorectal carcinogenesis. The specificity of estrogen activities in various cell types is mediated by the presence of tissue-specific coregulators (coactivators/corepressors). These proteins are specific transcription factors that bind to nuclear receptors, orchestrating their actions on the genome. Frequently, such coregulators are located in the cytoplasm, regulating the non genomic activity of the estrogens. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), also known as modulator of non-genomic activity of ER (MNAR), amplified in breast cancer-1 (AIB1) and transcriptional intermediary factor-2 (TIF2) are considered major ER-coregulators. Thus, the investigation of their expression is inherent to the evaluation of estrogen-mediated mechanisms. The dynamic cross-talk between HER- and ER-driven signaling pathways has been described. The aim of this study is the investigation of the role of HER3, ER and coregulators PELP1/MNAR, AIB-1, TIF-2 (over)expression in the pathogenesis and prognosis of colorectal cancer. Material and Methods Sections from formalin-fixed, paraffin-embedded colorectal tissue blocks, derived from 140 patients with colorectal cancer, were used. HER-3 mRNA levels of expression were assessed by quantitative RT-PCR in 54 colorectal adenocarcinomas and 29 normal mucosa specimens. The expression levels of both phosphorylated and unphosphorylated HER-3 protein were assessed by immunohistochemistry in 110 normal mucosa specimens, 24 adenomas and 140 adenocarcinomas. The expression levels of ER α and β, PELP1/ΜΝΑR were assessed by immunohistochemistry in 88 normal mucosa specimens, 30 adenomas and 113 adenocarcinomas. Additionally, the expression levels of ΑΙΒ1 and TIF-2 protein were assessed by immunohistochemistry in 83 normal mucosa specimens, 30 adenomas and 110 adenocarcinomas. Results HER-3 was detected both in the cytoplasm and nucleus, whereas pHER-3 was observed in the nucleus and membrane of cells. A possible switch in HER-3 topography from the nucleus to the cytoplasm during colorectal tumorigenesis is suggested. The expression of pHER-3 did not differ significantly in normal tissue, adenomas and carcinomas, but was related to disease stage. HER-3 mRNA overexpression was significantly associated with decreased time to disease progression. It was also correlated with higher median age, left colon and rectal tumour sites and lymph node involvement. ERα expression was extremely rare in colorectal tissue of our cohort and its expression did not appear to be associated with colorectal carcinogenesis. ERβ and PELP1/MNAR were detected in the nucleus of epithelial, endothelial, inflammatory, smooth muscle cells and myofibroblasts. When intensity of staining was taken into account, the expression of both proteins was significantly increased in epithelial cells of carcinomas compared to normal mucosa. ERβ expression in epithelial cells was correlated with decreased disease progression-free survival. PELP1/MNAR overexpression in epithelial cells was found to be an independent favorable prognostic factor. AIB1 and TIF2 were detected in the nucleus of epithelial, endothelial, inflammatory, smooth muscle cells and myofibroblasts. The expression of both proteins was significantly increased in epithelial cells of carcinomas compared to normal mucosa. In carcinomas, a significant correlation between the levels of expression of AIB1 and TIF2 was noted, but there was no correlation between the expression patterns of these two proteins and ERβ. Although AIB1 overexpression was associated with local tumor invasion, it was also found to correlate independently with prolonged overall survival. TIF2 overxpression did not appear to correlate with clinicopathological parameters. Conclusion/Discussion This study highlights the significance of ΗΕR3 expression and phosphorylation in colorectal carcinogenesis, supporting also its potential prognostic significance. This study supports literature data regarding HER3 expression in colorectal tissue, while is the first to imply a possible prognostic significance of HER-3 mRNA expression levels and to suggest a topographic switch of HER3 protein during colorectal carcinogenesis. ERβ1 protein levels were found to increase during colorectal carcinogenesis, a finding which corresponds only to a small portion of literature data. The prognostic role of nuclear receptors depends on a number of factors, such as coregulator expression levels, chemical modifications, subcellular localization and ligand/hormone levels. The concomitant increase in the expression levels of coregulators PELP1/MNAR, ΑΙΒ1 and ΤΙF2 during colorectal carcinogenesis might imply their potential participation in estrogen-mediated signaling. However, the characteristic absence of strong correlation between their expression pattern and ERβ1 expression pattern underlines their pluripotent role and their possible contribution to estrogen-independent signaling. Further studies with a large number of patients, appropriately designed and conducted using sensitive experimental and statistical methods, are required for the confirmation of our hypothesis generation results. In the post-genomic era, identification of useful molecular biomarkers might contribute to the improvement of the management and prognosis of patients with colorectal cancer.