LRP1 est un récepteur d’endocytose multifonctionnel capable non seulement d’interagir avec de nombreux ligands et de réguler leur endocytose, mais également de moduler certaines voies de signalisation intracellulaire. Ce récepteur se révèle implique dans de nombreux mécanismes physiologiques et pathologiques, comme l’athérosclérose, la maladie d’Alzheimer ou encore le cancer. La multiplicité de ses partenaires fait émerger le concept selon lequel LRP1 est capable de réguler le protéome membranaire et ainsi d’influencer le comportement migratoire de nombreuses cellules. De plus, les résultats récents obtenus au sein de notre laboratoire indiquent que ce récepteur d’endocytose est capable de contrôler la dynamique d’adhérence des cellules tumorales. Nous avons donc cherche à identifier un nouveau partenaire membranaire de LRP1, capable de participer à la régulation de l’adhérence tumorale. Notre étude s’est portée sur le récepteur d’adhérence CD44. L’utilisation de RAP, un antagoniste de LRP1 révèle que ce récepteur d’endocytose module la présence de CD44 à la membrane plasmique. Nous avons également découvert que ces deux récepteurs colocalisent fortement dans la cellule tumorale, que ce soit à la surface cellulaire ou au niveau intracellulaire. De plus LRP1 et CD44 co-immunoprécipitent à partir d’extraits totaux et membranaires, indiquant que ces deux récepteurs sont fortement associés au sein d’un même complexe moléculaire. L’étude de la répartition membranaire du complexe LRP1/CD44 a montré qu’il était en grande partie situé dans les radeaux lipidiques et particulièrement les cavéoles. Toutefois ces structures n’apparaissent pas nécessaires à l’établissement du complexe et ne sont pas impliquées dans les processus d’endocytose relatifs à ces récepteurs. En effet, des expérimentations visant à quantifier l’endocytose de CD44 couplées à différents traitements (β-MCD, conditions hyperosmotiques) révèlent que l’endocytose de ce complexe implique principalement la voie des vésicules tapissées de clathrine. Nous avons également démontré que ce mécanisme dynamique permettait de réguler l’adhérence tumorale. Enfin, des travaux sur le shedding de CD44 ont permis d’identifier les protéases impliquées (MT1MMP, ADAMs 10 et 17) et mettent en évidence un effet protecteur de LRP1 sur le clivage de l’ectodomaine de CD44. / The low-density lipoprotein receptor-related protein-1(LRP-1) is a large endocytic recept or mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP1-metiated endocytosis was first associated to anti-properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1-silencing or RAP (receptor-associated protein) treatment led to tumor cell surface accumulation of CD44. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived mini-receptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. CD44 labeling with EEA1 and LAMP-1 showed that internalized CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44-silencing abolishes RAP-induced tumor cell attachment, revealing that cell-surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation for tumor cell adhesion. Altogether, our data shed light on the LRP-1 mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells.

Tumoren weisen oftmals einen veränderten Energiestoffwechsel auf, der durch den Begriff „Warburg-Effekt“ bzw. „aerobe Glykolyse“ charakterisiert ist. Hierunter wird die Stoffwechselsituation verstanden, auch in Gegenwart von Sauerstoff aus Glukose Laktat zu bilden. Benigne Zellen zeigen diesen Effekt in aller Regel nicht so stark wie Tumorzellen. In dieser Arbeit wurden neun Tumorzelllinien, die von unterschiedlichen humanen Tumoren etabliert wurden, hinsichtlich ihres Energiestoffwechsels untersucht. Da der Warburg-Effekt auf einem erhöhten Glukosemetabolismus beruht, wurde die Stärke der Glukoseaufnahme und Laktatbildung für die neun Tumorzelllinien bestimmt. Wesentliches Ziel dieser Arbeit war, einen Zusammenhang zwischen dem Warburg-Effekt und weiteren Eigenschaften der Tumorzellen wie ihrem Oberflächenprofil bzw. ihrer Malignität nachzuweisen. Zur Phänotypisierung wurden die Oberflächenmoleküle CD44 und CD24 ausgewählt, da sie Zellinteraktionen, Zelladhäsion und Zellmigration von Tumorzellen vermitteln. Die Malignität wurde im Xenograft-Modell überprüft. Hierzu wurden Tumorzellen unter die Haut immuninkompetenter Mäuse injiziert und anschließend die Wachstumsgeschwindigkeit der entstehenden, soliden Tumoren gemessen. Die Menge an produziertem Laktat durch Tumorzellen war eindeutig von der Anwesenheit von Glukose im Nährmedium abhängig. Insgesamt waren fünf der neun Tumorzelllinien starke Laktatbildner, was einer Laktatbildung von über 2,5 mmol/L innerhalb von 48 Stunden entspricht. Hierzu gehört u.a. die Mammakarzinomzelllinie MDAMB-231, wohingegen MCF-7, eine andere Mammakarzinomzelllinie, nur wenig Laktat bildete. Im Gegensatz zu MDAMB-231 wuchs MCF-7 zudem auch nicht im Xenograft-Modell. Dies scheint ein Hinweis darauf zu sein, dass starke Laktatbildner aggressiver, d.h. schneller wachsen, als schwache Laktatbildner. Darüber hinaus wurde in dieser Arbeit eine temperaturabhängige Aufnahme des Glukoseanalogons 2-NBDG beo-bachtet. Auch hier fiel die Tumorzelllinie MCF-7 im Vergleich zur Tumorzelllinie MDAMB-231 durch eine geringere Aufnahme von 2-NBDG auf. Dies wird als Hinweis darauf gedeutet, dass MCF-7 Glukose in erster Linie in den Mitochondrien veratmet, anstatt sie zu Laktat zu vergären. Die beiden Glukosetransporter GLUT-1 und GLUT-3 wurden hingegen auf den Zellen aller neun Tumorzelllinien gleichermaßen nachgewiesen. Lediglich auf den Zellen der Tumorzelllinien HT-29 und MDAMB-468 war GLUT-3 schwach exprimiert. Sechs der neun Tumorzelllinien weisen den so genannten „Phänotyp 1“ auf. Hierunter wird in dieser Arbeit die gemeinsame Expression von CD44 und CD24 auf der Zelloberfläche verstanden. Zwei Zelllinien waren ausschließlich positiv für CD44 („Phänotyp 2“), eine Zelllinie ausschließlich positiv für CD24 („Phänotyp 3“). Keiner dieser Phänotypen wies eine charakteristische Glukoseaufnahme bzw. Laktatbildung auf, auch wenn drei der fünf Zelllinien mit besonders starker Laktatbildung zum Phänotyp 2 bzw. 3 gehörten. Die im menschlichen Blut vorzufindende nicht-essentielle Aminosäure Glutamin ist auch in Kulturmedien unerlässlich. Deshalb wurde die Laktatbildung aus Glukose in neun Tumorzelllinien sowohl in An- als auch Abwesenheit von Glutamin gemessen. Dabei wurde festgestellt, dass sich die Menge an gebildetem Laktat nach Inkubation in Glukose-haltigem, aber Glutamin-freiem Medium (Glc+ Gln-) besonders stark verringerte. Dieses Phänomen wurde für die Tumorzelllinien HT-29, 23132/87, BXPC-3 und HRT-18 beobachtet und deutet darauf hin, dass Glutamin in diesen Tumorzelllinien in irgendeiner Form auf die Laktatbildung aus Glukose Einfluss nimmt. Denkbar wäre, dass Glutamin Enzyme der Glykolyse über einen allosterischen Effekt moduliert, wodurch die Laktatbildung ansteigt. Dass diese Zellen eine Glutamin-abhängige Laktatbildung in Form der Glutaminolyse aufweisen, konnte in dieser Arbeit nicht beobachtet werden. Um diese Beobachtung abschließend zu klären, sind jedoch weiterführende Untersuchungen notwendig. / Unlike normal cells, the energy metabolism in tumour cells is characterised by the Warburg effect, which means high aerobic glycolysis even in the presence of oxygen. Most of the glucose is metabolised to lactate, which is not observed to this extent in normal cells. The present thesis examines the energy metabolism of nine cancer cell lines which were all established from different human solid tumours. Because of the high glucose consumption of the Warburg effect, glucose uptake and lactate output were quantified. The objective was to find out whether there is a link between the Warburg effect and other properties of the tumour cells, e.g. the cell-surface phenotype or malignancy. The surface proteins CD44 and CD24, markers for cellular interactions, cellular adhesion and migration, were selected as phenotyping molecules. Tumour malignancy was validated in xenograft experiments in which tumour cells were injected subcutaneously into immunodeficient mice and the subsequent rate of tumour growth determined. The amount of lactate produced in tumours cells was dependent on the presence of glucose in the culture medium. Five out of nine tumour cell lines produced lactate in high concentrations (on average more than 2.5 mmol/L within 48 hours). One of these was MDAMB-231, a breast tumour cell line. In contrast, another breast tumour cell line, MCF-7, showed a low rate of lactate production and no growth in xenograft experiments. This implies that lactate production correlates with malignancy. Moreover, the uptake of the glucose analogon 2-NBDG was dependent on temperature. Again, MCF-7 showed a lower uptake of 2-NBDG than the cells of MDAMB-231, which suggests that MCF-7 metabolizes glucose in the mitochondria rather than via aerobic glycolysis. However, the expression of glucose carriers GLUT-1 and GLUT-3 was nearly equal on all tumour cell lines except for the cells of HT-29 and MDAMB-468 where GLUT-3 was seen only sparsely. Six out of nine tumour cell lines displayed the phenotype CD44+ / CD24+, meaning the concomitant expression of CD44 and CD24. The single expression of CD44 was seen on two tumour cell lines and one tumour cell line showed the single expression of CD24. A correlation between glucose uptake, lactate production and a particular phenotype respectively could not be shown unambiguously, albeit three out of five tumour cell lines with a high lactate production exhibited the single expression of CD44 or CD24. The non-essential amino-acid glutamine is a constituent of human blood and therefore, was added to the culture medium. The tumour cell lines’ lactate production was measured in the presence and absence of glutamine. The lactate production in some tumour cell lines decreased strongly in glutamine-free but glucose-containing culture medium (Glc+ Gln-). The tumour cell lines HT-29, 23132/87, BXPC-3 and HRT-18 showed this phenomenon, indicating an influence of glutamine on the metabolism of glucose to lactate. For example, glutamine may affect glycolysis by an allosteric impact on particular enzymes, resulting in a higher lactate production. Lactate production by glutaminolysis could not be proven within this thesis. Further investigations are required to elucidate this.

Rheumatoid Arthritis (RA) is an autoimmune disease characterized by inflammation and eventual destruction of the synovial joints. The role of platelets in the pathophysiology of arthritis has only recently been established. Because antibodies to CD44 can deplete platelets, we hypothesized that these antibodies might be effective in arthritis through a platelet-depletion mechanism. We examined the K/BxN passive transfer mouse model of arthritis and found that most antibodies against CD44 were capable of depleting platelets. However, anti-CD44 treatment is effective when administered during developing arthritis, while anti-platelet treatment was not. While CD44 antibodies may be therapeutic through platelet-dependant and independent mechanisms, the ability of CD44 antibodies to decrease platelet counts does not seem to be the critical factor in resolving arthritis in the K/BxN model.

CD44 es una familia de moléculas de adhesión que se expresa en distintos tejidos normales y neoplásicos. Esta familia de proteínas se ha relacionado a diversas funciones que incluyen la activación y el "homing"linfocitarios, hemopoyesis, la interacción intercelular y de las células con el medio extracelular y la migración. Para poder ejercer todas estas funciones CD44 recurre a un mecanismo de splicing alternativo. Algunas de las isoformas de CD44 parece que intervienen en la producción de las metástasis. Específicamente una línea no metastásica de adenocarcinoma pancreático se ha demostrado que adquiere potencial metastásico al ser transfectada con una variante de Cd44 que contiene el exón v6 (CD44v6) , un efecto que puede ser bloqueado con anticuerpos anti v6.En lo que se refiere al adenocarcinoma gástrico y colónico, la expresión de CD44 se ha relacionado con la progresión tumoral.En vista a lo antes expuesto, nosotros nos hemos propuesto investigar la participación de CD44 en la progresión tumoral de los procesos neoplásicos más frecuentes de esófago estómago y páncreas.Hemos llevado a cabo un estudio inmunohistoquímico con anticuerpos contra las diversas isoformas de CD44 en muestras obtenidas del Archivo del Departamento de Patología del Hospital Germans Trias i Pujol.La expresión de CCD44v3 en el carcinoma escamoso y en adenocarcinoma de esófago indica una posible participación de Cd44v3 en el desarrollo de ambos tipos de carcinoma, mediante el reclutamiento y presentación de factores de crecimiento a las células diana y la consiguiente activación de la proliferación celular.En cuanto a las neoplasias pancreáticas, nuestros resultados parecen indicar que la expresión de CD44v6 en el adenocarcinoma pancreático puede estar relacionada a un posible papel de esta isoforma variante en los mecanismos de invasión y metástasis de esta neoplasia.En lo que concierne al cáncer gástrico, hemos detectado con mayor frecuencia las isoformas que contienen el exón v6, en los adenocarcinomas metastatizantes de tipo intestinal que en los de tipo difuso.Además la expresión de Cd44v6 es mucho más frecuente en las metástasis ganglionares de los adenocarcinomas de tipo intestinal que en los de tipo difuso.Estos hallazgos sugieren una intervención relevante del exón v6 en el proceso metastásico, especialmente en la diseminación linfática de las células del adenocarcinoma de tipo intestinal. Por el contrario, la invasión de los ganglios linfáticos por las células del adenocarcinoma de tipo difuso parece seguir una vía independiente del exón v6. / CD44, a familiy of cell surface adhesion molecules, is expressed in a variety of normal and neoplastic tissues. This protein family has been linked to a number of functions including lymphocite homing and activation, hemopoiesis, cell-cell and cell-extracellular matrix interactions, and cell migration. To be able to exert such diversity of effects, CD44 resorts to the generation of numerous isoforms through a mechanism of alternative splicing. Some of these CD44 isoforms also seem to play a role in the production of tumour metastasis. Specifically, a non metastatic cell line of rat pancreatic adenocarcinoma has been seen to acquire metastatic potential when transfected with CD44 variants containing exon v6 (CD44v6), an effect that can be blocked by anti-v6 antibodies.In regard to gastric and colonic adenocarcinoma, CD44v6 expression has been related to tumour progression.In view of the aforesaid we have tried to asses CD44 participation in the tumour progression of many of the most usual neoplastic entities of esofagous, stomach and pancreas.Immunohistoquemical study with antibodies against many CD44 isoforms has been performed in samples obtaines from the archives of the Departement of Pathology, Hospital Germans Trias i Pujol.CD44v3 expression in squamous carcinoma and oesophageal adenocarcinoma promtsthe consideration of a putative role of CD44v3 in the developement of both types of carcinoma, by the grow factors recruitment and presentation to the target cells and further activation of cell proliferation.Refering to pancreatic neoplasms, our results seem to indicate that CD44v6 overexpression in human pancreatic adenocarcinoma may be related to a possible role of this variant isoform in the invasive and metastasic mechanism of this neoplasms.Concerning gastric cancer isoforms containing exon v6 have been more frequently detected in metastasizing intestinal type carcinomas than in metastasizing diffuse type carcinomas. Moreover, CD44v6 has been seen to be significantly more frequent in lymph node metastases of intestinal type carcinomas than in those of difuse type cases. These findings suggest an important role of exon v6 in the metastatic process, particularly in the lymph node spreading of the intestinal adenocarcinoma cells. On the contrary, invasion of lymph nodes by difuse type tumour cells seems to be independent of exon v6.

Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma (MM) cell lines. Due to the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the parent linear peptide. These efforts led to the development of a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as a single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment resulted in mechanistically similar hallmarks of HYD1 induced cell death, namely the generation of ROS, depletion of ATP levels, and failure to activate caspase-3. Moreover, MTI-101 was shown to be cross-resistant in the HYD1 acquired resistant H929-60 cell line that was previously developed in our laboratory. In the present study, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and LC-MS/MS analysis to identify binding partners of MTI-101. Using this approach, CD44 was identified as a predominant binding partner. Using an ELISA based assay, we showed that biotinylated peptide bound to full length recombinant human CD44 in a concentration dependent manner. Reducing the cell surface expression of CD44 was accompanied by the activation of caspase-3 and cell death was observed in the NCI-H929 and U266 MM cell lines, indicating that MM cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide in the 8226 MM cell line, and this was further corroborated using CD44 knockout mice which also showed less peptide binding compared to wild-type. However, ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101 induced cell death. Mechanistically, we show that MTI-101 induced a pro-survival signal through the activation of Erk1/2 and that CD44 formed a complex with Pyk2. These data corroborate with that of which was previously observed with the parental peptide being a partial agonist and inducing an autophagic survival signal. With respect to cell death, we showed that CD44 forms a complex with known death inducing proteins caspase-8, caspase-10, Rip1, Rip3, Drp1, TNFAIP8, and PGAM5. Furthermore, we demonstrated that MTI-101 induced mitochondrial fission which may be modulated by a Rip1, Rip3 or Drp1 dependent and independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma. Together these data continue to support the further development of this class of compounds as well as identify CD44 as a therapeutic target for the treatment of MM.

Rheumatoid Arthritis (RA) is an autoimmune disease characterized by inflammation and eventual destruction of the synovial joints. The role of platelets in the pathophysiology of arthritis has only recently been established. Because antibodies to CD44 can deplete platelets, we hypothesized that these antibodies might be effective in arthritis through a platelet-depletion mechanism. We examined the K/BxN passive transfer mouse model of arthritis and found that most antibodies against CD44 were capable of depleting platelets. However, anti-CD44 treatment is effective when administered during developing arthritis, while anti-platelet treatment was not. While CD44 antibodies may be therapeutic through platelet-dependant and independent mechanisms, the ability of CD44 antibodies to decrease platelet counts does not seem to be the critical factor in resolving arthritis in the K/BxN model.

Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2001. / Errata slip inserted at back. "August 2001." Includes bibliographical references (leaves 254-296).

Thesis (Ph.D.) -- Virginia Commonwealth University, 2006. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: p. 152-184. Also available online.