The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Giant extraskeletal myxoid chondrosarcoma of the thigh treated by wide extraarticular resection and reconstruction with a tumor prosthesis

Introduction: Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue tumour with a high risk for local recurrence and metastases. While this entity is resistant to radio- or chemo-therapy, wide resection remains the treatment of choice. Case report: A 60 year old man presented to our service with a large mass in his right thigh, slowly evolving over the past 7 years. His main complaint was the volume of his thigh. Imaging showed a 23x13x14 cm tumour in the quadriceps, eroding the cortical bone and with potential contamination of the knee joint. The risk of a pathological fracture was estimated considerable. A CT-guided core-needle biopsy revealed a FNCLCC grade 2 EMC. A thoraco-abdominal CT scan showed multiple pulmonary metastases. Due to the palliative situation with a very slow disease progression, a wide extraarticular resection of the distal femur and reconstruction with a megaprosthesis were performed. Extensive skin necrosis necessitated three revision procedures for débridement and confection of a pediculated lateral gastrocnemius muscle flap. No complementary treatment was possible for the pulmonary metastases. At 18 months follow-up, he walked without crutches, was able to do his activities of daily living. He was painfree and highly satisfied with the result. During the follow-up, slow progression of the pulmonary metastases was noted, which remained asymptomatic. Conclusion: Extraskeletal myxoid chondrosarcoma is a rare soft tissue tumour, and wide excision remains the treatment of choice. Whenever possible, limb salvage should be proposed to preserve function and quality of life.

Pseudoprogression after high-dose busulfan-thiotepa with autologous stem cell transplantation and radiation therapy in children with brain tumor: impact on survival

Objective: To demonstrate the incidence, time course, predisposing factor and reversibility of neurotoxicity in children with brain tumors treated with high dose busulfan-thiotepa with autologous stem cell transplantation (ASCT) and radiation therapy in our institutional experience.Materials and Methods: We performed a retrospective analysis of prospectively collected data. Between May 1988 and May 2007, 110 patients, median age 3.6 years (range, 1 months-15.3 years), with brain tumors were treated with surgical intervention and conventional chemotherapy. All patients received one course of high-dose busulfan-thiotepa with stem cell rescue, followed or preceded by radiotherapy.Results: Twenty-three patients (21%) developed neuroradiological abnormalities on follow-up imaging studies at a median time of 9.2 months (range, 5.6-17.3 months) after day 0 of ASCT. All MRI-lesions appeared in patients receiving radiotherapy after ASCT and were localized inside the 50-55 Gy isodoses. They disappeared in 14 of 23 patients with a median time of 8 months (range, 3-17 months). The presence of MRI-abnormalities was a favorable prognostic factor for overall survival on univariate analysis (hazard ratio: 0.12, 95% confidence interval [0.04, 0.33]), with a 5-year overall survival in patients with MRI-abnormalities of 84% (95% CI, 62-94), comparedto 27% (95% CI, 19-37) in those without lesions. On multivariate analysis, the presence of MRI-abnormalities was an independent prognostic factor for overall survival.Conclusion: MRI-detectable brain abnormalities are common early findings in children treated with high-dose busulfan-thiotepa followed by radiation therapy, and may mimic early tumor recurrence. They are correlated with a better outcome.

Dukes B colorectal cancer: distinct genetic categories and clinical outcome based on proximal or distal tumor location.

PURPOSE: The aim of this study was to determine whether tumor location proximal or distal to the splenic flexure is associated with distinct molecular patterns and can predict clinical outcome in a homogeneous group of patients with Dukes B (T3-T4, N0, M0) colorectal cancer. It has been hypothesized that proximal and distal colorectal cancer may arise through different pathogenetic mechanisms. Although p53 and Ki-ras gene mutations occur frequently in distal tumors, another form of genomic instability associated with defective DNA mismatch repair has been predominantly identified in the proximal colon. To date, however, the clinical usefulness of these molecular characteristics remains unproven. METHODS: A total of 126 patients with a lymph node-negative sporadic colon or rectum adenocarcinoma were prospectively assessed with the endpoint of death by cancer. No patient received either radiotherapy or chemotherapy. p53 protein was studied by immunohistochemistry using DO-7 monoclonal antibody, and p53 and Ki-ras gene mutations were detected by single strand conformation polymorphism assay. RESULTS: During a mean follow-up of 67 months, the overall five-year survival was 70 percent. Nuclear p53 staining was found in 57 tumors (47 percent), and was more frequent in distal than in proximal tumors (55 vs. 21 percent; chi-squared test, P < 0.001). For the whole group, p53 protein expression correlated with poor survival in univariate and multivariate analysis (log-rank test, P = 0.01; hazard ratio = 2.16; 95 percent confidence interval = 1.12-4.11, P = 0.02). Distal colon tumors and rectal tumors exhibited similar molecular patterns and showed no difference in clinical outcome. In comparison with distal colorectal cancer, proximal tumors were found to be statistically significantly different on the following factors: mucinous content (P = 0.008), degree of histologic differentiation (P = 0.012), p53 protein expression, and gene mutation (P = 0.001 and 0.01 respectively). Finally, patients with proximal tumors had a marginally better survival than those with distal colon or rectal cancers (log-rank test, P = 0.045). CONCLUSION: In this series of Dukes B colorectal cancers, p53 protein expression was an independent factor for survival, which also correlated with tumor location. Eighty-six percent of p53-positive tumors were located in the distal colon and rectum. Distal colon and rectum tumors had similar molecular and clinical characteristics. In contrast, proximal neoplasms seem to represent a distinct entity, with specific histopathologic characteristics, molecular patterns, and clinical outcome. Location of the neoplasm in reference to the splenic flexure should be considered before group stratification in future trials of adjuvant chemotherapy in patients with Dukes B tumors.

Notch1 functions as a tumor suppressor in mouse skin.

Notch proteins are important in binary cell-fate decisions and inhibiting differentiation in many developmental systems, and aberrant Notch signaling is associated with tumorigenesis. The role of Notch signaling in mammalian skin is less well characterized and is mainly based on in vitro studies, which suggest that Notch signaling induces differentiation in mammalian skin. Conventional gene targeting is not applicable to establishing the role of Notch receptors or ligands in the skin because Notch1-/- embryos die during gestation. Therefore, we used a tissue-specific inducible gene-targeting approach to study the physiological role of the Notch1 receptor in the mouse epidermis and the corneal epithelium of adult mice. Unexpectedly, ablation of Notch1 results in epidermal and corneal hyperplasia followed by the development of skin tumors and facilitated chemical-induced skin carcinogenesis. Notch1 deficiency in skin and in primary keratinocytes results in increased and sustained expression of Gli2, causing the development of basal-cell carcinoma-like tumors. Furthermore, Notch1 inactivation in the epidermis results in derepressed beta-catenin signaling in cells that should normally undergo differentiation. Enhanced beta-catenin signaling can be reversed by re-introduction of a dominant active form of the Notch1 receptor. This leads to a reduction in the signaling-competent pool of beta-catenin, indicating that Notch1 can inhibit beta-catenin-mediated signaling. Our results indicate that Notch1 functions as a tumor-suppressor gene in mammalian skin.

Low doses of ionizing radiation promote tumor growth and metastasis by enhancing angiogenesis.

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

Three Essays on the Formation and Impact of Economic Policy

Introduction In my thesis I argue that economic policy is all about economics and politics. Consequently, analysing and understanding economic policy ideally has at least two parts. The economics part, which is centered around the expected impact of a specific policy on the real economy both in terms of efficiency and equity. The insights of this part point into which direction the fine-tuning of economic policies should go. However, fine-tuning of economic policies will be most likely subject to political constraints. That is why, in the politics part, a much better understanding can be gained by taking into account how the incentives of politicians and special interest groups as well as the role played by different institutional features affect the formation of economic policies. The first part and chapter of my thesis concentrates on the efficiency-related impact of economic policies: how does corporate income taxation in general, and corporate income tax progressivity in specific, affect the creation of new firms? Reduced progressivity and flat-rate taxes are in vogue. By 2009, 22 countries are operating flat-rate income tax systems, as do 7 US states and 14 Swiss cantons (for corporate income only). Tax reform proposals in the spirit of the "flat tax" model typically aim to reduce three parameters: the average tax burden, the progressivity of the tax schedule, and the complexity of the tax code. In joint work, Marius Brülhart and I explore the implications of changes in these three parameters on entrepreneurial activity, measured by counts of firm births in a panel of Swiss municipalities. Our results show that lower average tax rates and reduced complexity of the tax code promote firm births. Controlling for these effects, reduced progressivity inhibits firm births. Our reading of these results is that tax progressivity has an insurance effect that facilitates entrepreneurial risk taking. The positive effects of lower tax levels and reduced complexity are estimated to be significantly stronger than the negative effect of reduced progressivity. To the extent that firm births reflect desirable entrepreneurial dynamism, it is not the flattening of tax schedules that is key to successful tax reforms, but the lowering of average tax burdens and the simplification of tax codes. Flatness per se is of secondary importance and even appears to be detrimental to firm births. The second part of my thesis, which corresponds to the second and third chapter, concentrates on how economic policies are formed. By the nature of the analysis, these two chapters draw on a broader literature than the first chapter. Both economists and political scientists have done extensive research on how economic policies are formed. Thereby, researchers in both disciplines have recognised the importance of special interest groups trying to influence policy-making through various channels. In general, economists base their analysis on a formal and microeconomically founded approach, while abstracting from institutional details. In contrast, political scientists' frameworks are generally richer in terms of institutional features but lack the theoretical rigour of economists' approaches. I start from the economist's point of view. However, I try to borrow as much as possible from the findings of political science to gain a better understanding of how economic policies are formed in reality. In the second chapter, I take a theoretical approach and focus on the institutional policy framework to explore how interactions between different political institutions affect the outcome of trade policy in presence of special interest groups' lobbying. Standard political economy theory treats the government as a single institutional actor which sets tariffs by trading off social welfare against contributions from special interest groups seeking industry-specific protection from imports. However, these models lack important (institutional) features of reality. That is why, in my model, I split up the government into a legislative and executive branch which can both be lobbied by special interest groups. Furthermore, the legislative has the option to delegate its trade policy authority to the executive. I allow the executive to compensate the legislative in exchange for delegation. Despite ample anecdotal evidence, bargaining over delegation of trade policy authority has not yet been formally modelled in the literature. I show that delegation has an impact on policy formation in that it leads to lower equilibrium tariffs compared to a standard model without delegation. I also show that delegation will only take place if the lobby is not strong enough to prevent it. Furthermore, the option to delegate increases the bargaining power of the legislative at the expense of the lobbies. Therefore, the findings of this model can shed a light on why the U.S. Congress often practices delegation to the executive. In the final chapter of my thesis, my coauthor, Antonio Fidalgo, and I take a narrower approach and focus on the individual politician level of policy-making to explore how connections to private firms and networks within parliament affect individual politicians' decision-making. Theories in the spirit of the model of the second chapter show how campaign contributions from lobbies to politicians can influence economic policies. There exists an abundant empirical literature that analyses ties between firms and politicians based on campaign contributions. However, the evidence on the impact of campaign contributions is mixed, at best. In our paper, we analyse an alternative channel of influence in the shape of personal connections between politicians and firms through board membership. We identify a direct effect of board membership on individual politicians' voting behaviour and an indirect leverage effect when politicians with board connections influence non-connected peers. We assess the importance of these two effects using a vote in the Swiss parliament on a government bailout of the national airline, Swissair, in 2001, which serves as a natural experiment. We find that both the direct effect of connections to firms and the indirect leverage effect had a strong and positive impact on the probability that a politician supported the government bailout.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.

FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1.

The mechanisms of blood vessel maturation into distinct parts of the blood vasculature such as arteries, veins, and capillaries have been the subject of intense investigation over recent years. In contrast, our knowledge of lymphatic vessel maturation is still fragmentary. In this study, we provide a molecular and morphological characterization of the major steps in the maturation of the primary lymphatic capillary plexus into collecting lymphatic vessels during development and show that forkhead transcription factor Foxc2 controls this process. We further identify transcription factor NFATc1 as a novel regulator of lymphatic development and describe a previously unsuspected link between NFATc1 and Foxc2 in the regulation of lymphatic maturation. We also provide a genome-wide map of FOXC2-binding sites in lymphatic endothelial cells, identify a novel consensus FOXC2 sequence, and show that NFATc1 physically interacts with FOXC2-binding enhancers. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.