Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of immature nonfunctional highly proliferative hematopoietic cells in the blood, due to a blockage in myeloid differentiation at various stages. Since the success of the differentiation agent, All-trans retinoic acid (ATRA), in the treatment of acute promyelocytic leukemia (APL), much effort has gone into trying to find agents that are able to differentiate AML cells and specifically the leukemic stem cell (LSC). CD44 is a cell surface receptor that is over-expressed on AML cells. When bound to anti-CD44 monoclonal antibodies (mAbs), this differentiation block is relieved in AML cells and their proliferation is reduced. The molecular mechanisms that AML cells undergo to achieve this reversal of their apparent phenotype is not fully understood. To this end, we designed a study using quantitative phosphoproteomics approaches aimed at identifying differences in phosphorylation found on proteins involved in signaling pathways post-treatment with CD44-mAbs. The Rho family of GTPases emerged as one of the most transformed pathways following the treatment with CD44-mAbs. The P21 activated kinase 2(PAK2), a target of the Rho family of GTPases, was found to be differentially phosphorylated in AML cells post-treatment with CD44-mAbs. This protein has been found to possess a role similar to that of a switch that determines whether the cell survives or undergoes apoptosis. Beyond confirming these results by various biochemical approaches, our study aimed to determine the effect of knock down of PAK2 on AML cell proliferation and differentiation. In addition, over-expression of PAK2 mutants using plasmid cloning was also explored to fully understand how levels of PAK2 as well as the alteration of specific phospohorylation sites could alter AML cell responses to CD44-mAbs. Results from this study will be important in determining whether PAK2 could be used as a potential therapeutic target for AML once its levels are altered.

Le laboratoire INSERM U908 a montré que le Nerve Growth Factor (NGF) et son précurseur, le proNGF, sont impliqués dans l’agressivité des tumeurs mammaires via des effets sur la croissance, l’angiogenèse ou encore la migration/invasion. Pour autant, aucunes thérapies ciblées n’a à ce jour été approuvée suite à des essais cliniques de traitements visant à inhiber les effets du (pro)NGF et de leurs récepteurs dans les cancers. Ces résultats indiquent que la phosphorylation de TrkA est nécessaire mais pas suffisante pour expliquer le(s) mécanisme(s) par lequel(s) le NGF ou son précurseur participe(nt) au développement tumoral. L’obtention de lignées tumorales résistantes au lestaurtinib est un phénomène rapide, nous avons donc émis l’hypothèse que la signalisation de TrkA indépendante de sa phosphorylation existe de façon « innée » dans les cellules tumorales. Par l’analyse protéomique, nous avons ainsi découvert que le NGF induit le recrutement de CD44 et d’une cascade de signalisation p115RhoGEF/RhoA/ROCK1 (Aubert L*, Guilbert M* et al, Oncotarget, 2015 ; *co-premier auteur). Nous avons ainsi montré que le NGF via le complexe TrkA/CD44 induit la migration et l’invasion des cellules cancéreuses de sein in vivo, et augmente la tumorigénicité in vivo. Quant au proNGF, j’ai pu observer qu’il induit l’internalisation de l’EGFR par la phosphorylation du résidu Y1068. Il en résulte une diminution des effets prolifératif et pro-invasif de l’EGF dans les cellules tumorales. Ces résultats fondamentaux sont tout à fait intéressants même s’ils nécessitent leurs consolidations et doivent permettre de démontrer le caractère pronostic de la détection de TrkA et ses corécepteurs dans le cancer. Ainsi nos études permettront le développement de thérapie ciblée par des firmes pharmaceutiques. / The INSERM U908 unit has showed that Nerve Growth factor (NGF) and its precursor (ProNGF) are implicated in tumor agressivness via their effects on growth, angiogenesis or migration/invasion and metastasis. Nevertheless, (pro)NGF and their receptors targeted therapies failed to demonstrate efficiency and clinical trial are disappointing. These results indicate that TrkA phosphorylation is not sufficient to explain molecular mechanisms of (pro)NGF effects on tumors. Indeed, we obtained lestaurtinib resistant cell lines within 3 weeks of treatment which indicated that resistant mechanisms are innate. So, by functional proteomics analyses, we described that NGF induced the formation of TrkA/CD44 complex and then the recruitment of p115RhoGEF/RhoA/ROCK1 signalling cascade (Aubert L*, Guilbert M* et al, Oncotarget, 2015 ; *equally contributed to this work). We showed that CD44 mediated effects participate to invasive effects of NGF in vitro, and in vivo, we demontrated that CD44 increases NGF induced tumoriginicity. In a second part, I observed that the proNGF regulated EGFR turn over through its phosphorylation on Y1068. This effect on EGFR decreased proliferative and pro-invasive effects of EGF in cancer cells. These fundamental results are interesting and need to be consolidated to ensure development of prognosis or targeted therapies.

Progression to metastatic disease is the leading cause of deaths resulting from breast cancer. Understanding the mechanisms underlying a cell's ability to move away from its site of origin and populate a distant site is important for the future development of therapies. The interactions between a tumor cell and the microenvironment can modulate a cell's ability to invade through tissues and access distant organs. In this study we present evidence indicating the differential modulation of invasive and proliferative phenotypes by hyaluronan present in the cellular microenvironment.We establish the role of CD44, the primary receptor for hyaluronan, in breast cancer progression and metastasis through the use of transgenic mouse models of breast cancer. While no differences were seen in the onset of primary breast tumors, mice expressing CD44 had a reduced rate of pulmonary metastasis compared to mice that lacked CD44. This establishes an anti-invasive role for CD44 in breast tumor progression. We also identify a decreased population of alveolar macrophages in CD44 negative mice that could affect metastatic breast cancer cell colonization of the lungs.We then focused our study in vitro, where we assessed the invasive properties of breast cancer cells as they move through three dimensional (3D) matrices containing or lacking hyaluronan. We show that in 3D type I collagen gels, breast cancer cells invade more readily in the absence of hyaluronan compared to when hyaluronan (HA) is embedded within the gel. HA mediated inhibition of invasion is dependent on CD44 binding as demonstrated through the use of a CD44 functional blocking antibody.We also show that HA promotes differential phenotypes of breast cancer cell. HA promotes filopodia formation and invasion when soluble in the cell microenvironment. Alternatively, matrix-embedded HA inhibits invasion and promotes migration through the formation of lamellipodia. The differential HA invasive and proliferative phenotypes are mediated by differential activation of ERK or γPAK. Activation of γPAK is mediated by CD44 while ERK activation by HA occurs by CD44 independent mechanisms.We also demonstrate an inhibition of MMP9 mediated invasion by HA when embedded within a type IV collagen matrix, but not a type I collagen matrix. This differential activity indicates that it is not only the immobilization of HA in a matrix that determines its activity, but also the context in which it is present within the matrix.These data underscore the importance of studying matrix components in an environment that closely resembles in vivo conditions. HA is a prime example as it has the capability of both promoting and inhibiting invasion depending on how it is presented to a cell. Differential HA activity also underlies the importance of understanding extracelluar matrix degradation and the release of matrix components as these can adversely affect disease progression.

The objective of this study was to get a better knowledge of lymphocyte apoptosis, surface expression of CD44 on lymphocytes and infammatory cytokine production (TNF-alfa, IL-4, IL-10, and TGFbeta 1) throughout inflammatory response of bovine mammary gland caused by bacterial toxin lipopolysacharide (LPS) and muramyl dipeptide as a structural unit of peptidoglycan (MDP). In the experiments, there were used LPS and MDP at the specified concentrations (5 microg and 500 microg, respectively). The inflammatory response was analysed at selected time points (24, 48, 72, and 168 h) following stimulation of the mammmary gland. As a control, the same volumes of physiological saline solution was used. The lymphocyte apoptosis and the surface expression of the CD44 on the lymphocytes were detected in the mammary gland lavages collected from the 8 clinically healthy virgin Holstein x Czech Pied heifers by flow cytometry. Further, it was analysed the concentrations of the cytokines at the same selected time points by an ELISA method. The itramammary instillation using LPS and MDP resulted in the highest lymphocyte apoptosis 48 hours after the inductor challenge. The receptor CD44 was highly correlated with the lymphocyte apoptosis occurrence. It is likely that CD44 receptor is present throughout lymphocyte apoptosis process. The cytokine concentrations obtained indicate that the cytokines may play a role in apoptosis during mammary gland infammation.

Administration of Concanavalin-A (Con A) induces severe injury to the hepatocytes in mice and is considered to be a model for human hepatitis. In the current study, we investigated the role of CD44 in Con A induced hepatitis. Although immune cells have been identified as the causative agent of Con A-induced hepatitis, the exact mechanism of pathogenesis remains unclear. When Con A was injected into CD44 wild type (WT) mice, it induced hepatitis as evident from increased plasma aspartate aminotransferase (AST) levels accompanied by active infiltration of mononuclear cells in the liver and significant induction of apoptosis. Interestingly, Con A injected C57BL/6 CD44-knockout (KO) mice exhibited increased hepatitis with higher levels of apoptosis in the liver and increased plasma AST levels when compared to the CD44 WT mice. Also, transfer of T cells from Con A injected CD44-KO mice into CD44 WT mice induced higher levels of hepatitis when compared to transfer of similar cells from CD44 WT mice into CD44 WT mice. The increased hepatitis seen in CD44-KO mice was partially due to increased production of cytokines such as TNF-a, IL-2 and IFN-g, but not Fas or FasL. Also, it was not caused by altered presence of T cell subsets. The increased susceptibility of CD44 KO mice to hepatitis correlated with increased resistance of T cells from CD44 KO mice to undergo apoptosis when compared to the CD44 WT mice. Together, these data demonstrate that activated T cells use CD44 to undergo apoptosis, and dysregulation in this pathway could lead to increased pathogenesis in a number of diseases, including hepatitis. / Master of Science

Hyaluronan, an unbranched glycosaminoglycan of the extracellular matrix, has an amazingly simple structure. Initially thought to fulfil only hydrating and space-filling functions in tissues, evidence generated during the past decades shows that hyaluronan is involved in intriguingly complex signalling events in health and disease. In cancer, increased hyaluronan levels have been correlated with poor patient survival. The research underlying this thesis sheds light on the interplay between hyaluronan, its producing and degrading enzymes as well as the triggered intracellular signalling in the metastatic cascade. Utilising breast cancer and normal mammary cells, paper I and II investigate the initial steps of tumour progression: proliferation, invasion and epithelial-mesenchymal transition. Hyaluronan synthase 2 plays a central role in all these processes. In paper III, the focus is shifted toward growth factor-induced hyaluronan production. Stimulation with PDGF-BB, which can be secreted by tumour cells, increased hyaluronan production via upregulation of HAS2 in fibroblast cultures. Finally, paper IV discusses the involvement of hyaluronidases and CD44 in angiogenesis and intravasation – events that are associated with advanced cancer stages.

Reproduction de : Thèse doctorat : Biochimie : Reims : 2009. / Titre provenant de l'écran titre. Bibliogr.p.137-157 annexes.

In addition to the antigen-specific receptors, the T and B cells also express a variety of adhesion molecules, which are known to participate in cell-cell interaction, migration, homing and signal transduction. CD44 is a widely distributed cell surface glycoprotein whose principal ligand has been identified as hyaluronic acid (HA), a major component of the extracellular matrix. In the current study, we investigated whether HA or mAbs against CD44 would induce a proliferative response in mouse lymphocytes. Spleen cells from normal and nude but not severe combined immunodeficient mice, exhibited strong proliferative responsiveness to stimulation with soluble HA or anti-CD44 mAbs. Furthermore, purified B cells but not T cells were found to respond to HA. These data demonstrated that interaction between HA and CD44 can regulate murine B cell effector functions and that such interactions may play a critical role during normal or autoimmune responsiveness of B cells. Endothelial cell injury resulting in vascular leak syndrome (VLS) is one of the most widely noted phenomenons in a variety of clinical diseases, however, the underlying reason for which remains unclear. We used interleukin-2 induced VLS as a model to investigate the role of cytolytic lymphocytes in the direct cytotoxicity of endothelial cells. BL/6 wild-type mice developed significant VLS in the lungs, liver and spleen following IL-2 administration. Interestingly, perforin-knockout mice exhibited marked decrease in VLS in all three organs tested. Also, FasL-defective (gld) mice and Fas-deficient (lpr) mice exhibited decreased VLS in the liver and spleen, but not in the lungs. These results demonstrated for the first time that perforin and FasL may actively participate in endothelial cell injury and induction of VLS in a variety of organs. Inasmuch as, CD44 also plays a major role in the lymphocyte adhesion to the endothelial cells, we used CD44-knockout mice and observed that such mice exhibited markedly diminished VLS following IL-2-treatment. Our data also suggested that blocking CD44 helps in reducing the IL-2-induced VLS and therefore such an approach may serve as a useful tool to prevent endothelial cell damage seen in a variety of clinical disorders. / Ph. D.

Immunotherapy with IL-2 is accompanied by severe toxicity leading to development of vascular leak syndrome (VLS). Previous studies from our laboratory demonstrated that CD44 knockout mice exhibit marked decrease in IL-2 induced VLS, thereby suggesting a role for CD44 in VLS. In the current study we tested whether use of mAbs against CD44 or hyaluronic acid (HA), the ligand for CD44, can abrogate IL-2 induced VLS. Administration of IL-2 (75,000 U/mouse, three times a day for 4 days) into C57BL/6 mice triggered significant VLS in the lungs and liver. Interestingly, HA caused a marked increase in IL-2-induced VLS in the lungs and liver. In contrast, use of anti-CD44 mAbs reduced IL-2-induced VLS in the lungs and liver. The change in VLS seen following HA or anti-CD44 mAbs treatment was not due to any defect in lymphocyte migration or homing to various organs because histopathological studies in these mice demonstrated significant and often greater perivascular infiltration of lymphocytes when compared to mice treated with IL-2 alone. However, HA treatment exhibited a marked increase in IL-2-induced lymphokine-activated killer (LAK) cell activity while anti-CD44 mAbs treatment led to a significant decrease in IL-2-induced LAK cell activity. These studies demonstrate that HA or anti CD44 mAbs may serve as a useful tool to selectively alter the LAK activity as well as to prevent the toxicity induced by IL-2. Altering CD44-HA interactions in vivo may offer a novel therapeutic approach to prevent endothelial cell injury by cytotoxic lymphocytes in a variety of clinical diseases. / Master of Science

Metastasis is the leading cause of death in patients with cancer, and the extracellular matrix is critical to cancer dissemination. The adhesion receptor, CD44, mediates cellular communication with the extracellular matrix by binding to the glycosaminoglycan, hyaluronan (HA). CD44 and HA play critical roles in cancer progression and development. HA is deposited in extracellular and pericellular matrices where it directs intracellular signaling through interactions with cell-surface CD44. CD44-HA interactions, in turn, direct signaling that is relevant to cancer progression. Importantly, these molecules can both promote and inhibit the oncogenic cascade, although the mechanism by which they promote dual and contrasting functions is unknown.Here we show that HA can both activate and suppress EGFR, a critical regulator of oncogenic signaling, in a context-dependent fashion. Using a 3D collagen system in which HA is either polymerized in collagen matrix or provided soluble in the media (sHA) we report that collagen-embedded HA (eHA) inhibits EGFR activation, filopodia formation, and cell spreading on a collagen matrix. Additionally, we show that CD44 is subject to cell-type changes during cancer progression. We have found that CD44 is expressed in the myoepithelium of the developing mammary gland and regulates the normal function of this cell type. The myoepithelial function of CD44 is also relevant to its role in cancer progression as CD44 is expressed in the basal cells of early-stage breast and prostate cancer but undergoes a basal to luminal epithelial switch with increasing tumorigenicity and is strongly expressed by tumor epithelium. These findings demonstrate a novel role for eHA as a protective molecule when encountered in the collagen matrix during cancer progression and highlight the importance of understanding cell-type specific contributions during cancer progression. Taken together, the findings reported in this dissertation point to a mechanism by which CD44 and HA can function in tumor suppression and promotion, depending on cell-type specific expression and modulation of the extracellular matrix.