From diabetes to heart disease : studies on dyslipidemia-induced B-cell dysfunction, immunomodulation in heart transplantation, and cardiac stem cell therapy

Diabetes is a growing epidemic with devastating human, social and economic impact. It is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent LDL and HDL particles in insulin-secreting β-cells. Purified human VLDL and LDL particles reduced insulin mRNA levels and β-cell proliferation, and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of β-cells involved caspase-3 cleavage and reduction in levels of the c-Jun N-terminal (JNK) Interacting Protein-1 (IB1/JIP-1). In contrast, the pro-apoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of JNK. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of the protein kinase Akt/PKB. Heart disease is a major cause of morbidity and mortality among patients with diabetes. When heart failure is refractory to medical therapy and cannot be improved by electrical resynchronization, percutaneous angioplasty or coronary graft bypass surgery, heart transplantation remains a "last resort" therapy. Nevertheless, it is limited by the side effects of immunosuppressive drugs and chronic rejection. Localized expression of immunomodulatory genes in the donor organ can create a state of immune privilege within the graft, and was performed in rodent hearts by infecting cells with an adenovirus encoding indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in the catabolism of tryptophane. Other strategies are based on genetic manipulation of dendritic cells (DCs) with immunosuppressive genes and in vitro exposure of DCs to agents that prevent their maturation by inflammatory cytokines. Finally, we used 5-bromo-2'-deoxyuridine, which is incorporated into DNA and diluted with cell division, to identify long-term label retaining cells in the adult rodent heart. The majority of these cells were positive for the stem cell antigen-1 (Sca-1) and negative for the endothelial precursor marker CD31. They formed cardiospheres in vitro and showed differentiation potential into mesenchymal cell lineages. When cultured in cardiomyogenic differentiation medium, they expressed cardiac-specific genes. Taken together, these data provide evidence of slow-cycling stem cells in the rodent heart. Chronic shortage of donor organs opens the way to cardiac stem cell therapy in humans, although the long way from animal experimentation to routine therapy in patients may still take several years. - Du diabète de type 2 à la maladie coronarienne : trois études sur les dysfonctions de la cellule sécrétrice d'insuline induites par les dyslipidémies, l'immunomodulation dans la transplantation cardiaque, et la thérapie par des cellules souches myocardiques. Le diabète de type 2 a pris les dimensions d'une épidémie, avec des conséquences sociales et économiques dont nous n'avons pas encore pris toute la mesure. La maladie s'accompagne souvent d'une dyslipidémie caractérisée par une hypertriglycéridémie, des taux abaissés de cholestérol HDL, et des concentrations de cholestérol LDL à la limite supérieure de ce qui est considéré comme acceptable. L'hypothèse à la base de cette étude est qu'une modification des taux plasmatiques de lipoprotéines pourrait avoir une influence directe sur la cellule β sécrétrice d'insuline en modifiant sa fonction, sa durée de vie et son taux de régénération. Dans un premier temps, nous avons mis en évidence, sur la cellule β, la présence de plusieurs récepteurs impliqués dans la captation des lipoprotéines. Nous avons confirmé la fonctionnalité de ces récepteurs en suivant l'internalisation de LDL et de HDL marqués. En présence de VLDL ou de LDL humains, nous avons observé une diminution de la transcription du gène de l'insuline, une prolifération cellulaire réduite, et une augmentation de l'apoptose, toutes fonctions de la dose et du temps d'exposition. L'apoptose induite par les VLDL passe par une activation de la caspase-3 et une réduction du taux de la protéine IB1/JIP-1 (Islet Brain1/JNK Interacting Protein 1), dont une mutation est associée à une forme monogénique de diabète de type 2. Par opposition, les HDL, ainsi que des peptides inhibiteurs de JNK, sont capables de contrer la cascade pro-apoptotique déclenchée, respectivement, par les LDL et les VLDL. Ces effets protecteurs comprennent l'inhibition du clivage de la caspase-3 et l'activation de la protéine kinase Akt/PKB. En conclusion, les lipoprotéines sont des éléments clés de la survie de la cellule β, et pourraient contribuer au dysfonctionnement observé dans le pancréas endocrine au cours du développement du diabète. La maladie cardiaque, et plus particulièrement la maladie coronarienne, est une cause majeure de morbidité et de mortalité chez les patients atteints de diabète. Plusieurs stratégies sont utilisées quotidiennement pour pallier les atteintes cardiaques: traitements médicamenteux, électromécaniques par resynchronisation électrique, ou communément appelés « interventionnels » lorsqu'ils font appel à l'angioplastie percutanée. La revascularisation du myocarde par des pontages coronariens donne également de très bons résultats dans certaines situations. Il existe toutefois des cas où plus aucune de ces approches n'est suffisante. La transplantation cardiaque est alors la thérapie de choix pour un nombre restreint de patients. La thérapie génique, en permettant l'expression locale de gènes immunomodulateurs dans l'organe greffé, permet de diminuer les réactions de rejet inhérentes à toute transplantation (à l'exception de celles réalisées entre deux jumeaux homozygotes). Nous avons appliqué chez des rongeurs cette stratégie en infectant le coeur greffé avec un adénovirus codant pour l'enzyme indoleamine 2,3-dioxygénase (IDO), une enzyme clé dans le catabolisme du tryptophane. Nous avons procédé de manière identique in vitro en surexprimant IDO dans les cellules dendritiques, dont le rôle est de présenter les antigènes aux lymphocytes Τ du receveur. Des expériences similaires ont été réalisées en traitant les cellules dendritiques avec des substances capables de prévenir, en partie du moins, leur maturation par des agents pro-inflammatoires. Finalement, nous avons exploré une stratégie utilisée couramment en hématologie, mais qui n'en est encore qu'à ses débuts au niveau cardiaque : la thérapie par des cellules souches. En traitant des rongeurs avec un marqueur qui s'incorpore dans l'ADN nucléaire, le 5-bromo- 2'-deoxyuridine, nous avons identifié une population cellulaire se divisant rarement, positive en grande partie pour l'antigène embryonnaire Sca-1 et négative pour le marqueur endothélial CD31. En culture, ces cellules forment des cardiosphères et sont capables de se différencier dans les principaux types tissulaires mésenchymateux. Dans un milieu de differentiation adéquat, ces cellules expriment des gènes cardiomyocytaires. En résumé, ces données confirment la présence chez le rongeur d'une population résidente de précurseurs myocardiques. En addenda, on trouvera deux publications relatives à la cellule β productrice d'insuline. Le premier article démontre le rôle essentiel joué par la complexine dans l'insulino-sécrétion, tandis que le second souligne l'importance de la protéine IB1/JIP-1 dans la protection contre l'apoptose de la cellule β induite par certaines cytokines.

ESCMID* guideline for the diagnosis and management of Candida diseases 2012: adults with haematological malignancies and after haematopoietic stem cell transplantation (HCT).

Fungal diseases still play a major role in morbidity and mortality in patients with haematological malignancies, including those undergoing haematopoietic stem cell transplantation. Although Aspergillus and other filamentous fungal diseases remain a major concern, Candida infections are still a major cause of mortality. This part of the ESCMID guidelines focuses on this patient population and reviews pertaining to prophylaxis, empirical/pre-emptive and targeted therapy of Candida diseases. Anti-Candida prophylaxis is only recommended for patients receiving allogeneic stem cell transplantation. The authors recognize that the recommendations would have most likely been different if the purpose would have been prevention of all fungal infections (e.g. aspergillosis). In targeted treatment of candidaemia, recommendations for treatment are available for all echinocandins, that is anidulafungin (AI), caspofungin (AI) and micafungin (AI), although a warning for resistance is expressed. Liposomal amphotericin B received a BI recommendation due to higher number of reported adverse events in the trials. Amphotericin B deoxycholate should not be used (DII); and fluconazole was rated CI because of a change in epidemiology in some areas in Europe. Removal of central venous catheters is recommended during candidaemia but if catheter retention is a clinical necessity, treatment with an echinocandin is an option (CII(t) ). In chronic disseminated candidiasis therapy, recommendations are liposomal amphotericin B for 8 weeks (AIII), fluconazole for >3 months or other azoles (BIII). Granulocyte transfusions are only an option in desperate cases of patients with Candida disease and neutropenia (CIII).

Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation.

Using quantitative fluorescence in situ hybridization and flow cytometry, the telomere length of telomere repeat sequences after stem cell transplantation (SCT) were measured. The study included the telomeres of peripheral blood monocytes that should reflect the length of telomeres in stem cells and the telomeres of T lymphocytes that could shorten as a result of peripheral expansion. The loss of telomeres in monocytes and in memory T cells, although accelerated initially, became comparable to the loss of telomeres in healthy controls from the second year after transplantation. In addition, the telomere length in the naive T cells that were produced by the thymus was comparable to the telomere length in the naive T cells of the donor. Compared to the total length of telomeres available, the loss of telomere repeats in leukocytes after SCT resembles the accelerated shortening seen in early childhood and remains, therefore, relatively insignificant.

Disseminated Rhizopus microsporus infection cured by salvage allogeneic hematopoietic stem cell transplantation, antifungal combination therapy, and surgical resection.

O. Lebeau, C. Van Delden, J. Garbino, J. Robert, F. Lamoth, J. Passweg, Y. Chalandon. Disseminated Rhizopus microsporus infection cured by salvage allogeneic hematopoietic stem cell transplantation, antifungal combination therapy, and surgical resection. Transpl Infect Dis 2010. All rights reserved Abstract: Invasive Zygomycetes infection complicating prolonged neutropenia is associated with high mortality in the absence of immune recovery. We report a patient who developed disseminated zygomycosis due to Rhizopus microsporus during induction chemotherapy for acute myeloid leukemia. Rescue allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed as her only chance of cure of this infection and to treat refractory leukemia. Posaconazole combined with liposomal amphotericin B contained the zygomycosis during prolonged neutropenia due to allo-HSCT followed by intense immunosuppression for grade IV acute graft-versus-host disease. Surgical removal of all infected sites after immune recovery, with prolonged posaconazole treatment, ultimately cured the infection. New combination antifungal therapies might sufficiently control disseminated zygomycosis to allow allo-HSCT to be performed, assuring life-saving immune recovery. Surgery appears to be necessary for definite cure of these infections.

Salvage Regimen With Autologous Stem Cell Transplantation With Or Without Rituximab Maintenance For Relapsed Diffuse Large B-Cell Lymphoma (Dlbcl): Coral Final Report

Times Cited: 0 References: 0 Citation MapAbstract : Background: Chemotherapy followed by autologous stem cell transplantation (ASCT) is the standard treatment for relapsed DLBCL. No study has compared salvage therapies and evaluated maintenance post ASCT.Methods: DLBCL CD 20+ in first relapse or pts refractory after first therapy were randomized between R ICE (rituximab, ifosfamide, etoposide, carboplatinum) or R DHAP (rituximab dexamethasone cytarabine cisplatinum). Responding patients received BEAM and ASCT then randomized between observation or maintenance with rituximab every 2 m for 1 yr (Gisselbrecht J Clin Oncol; 2010).Results: Analysis was made on 477 pts (R ICE: 243 pts; R DHAP: 234 pts): 255 relapses >12m, 213 refractory/early relapses; 306 pts had prior rituximab; secondary(s) IPI 0-1: 281 pts; s IPI 2-3:181pts. There was no difference in response rate between R ICE 63.6% and R DHAP 64.3%. There was no difference between R ICE and R DHAP at 4 yrs for EFS (26% vs 37% p=0.2) and OS (43% vs 51%, p=0.3). Factors affecting 4 yrs EFS, PFS and OS were: prior treatment with rituximab; early relapse< 12 m; s IPI 2-3. ASCT was performed in 255 pts and 242 randomized for maintenance: 122 pts rituximab (R), 120 pts observation (O). Distribution between R/O arms were respectively: median age 54 /53 yrs, Male 76/83; female 46/37; secondary IPI 0-1: 84/81; sIPI 2-3: 36/36. 89/76 relapses >12m., 33/41 refractory/early relapses. Median follow up was 44 m with 111 events. 4 yrs EFS was 52.8 % (CI 46-59) with 63% (CI 56-69) OS. There was no difference in EFS, PFS and OS between R and O arms. In multivariate analysis, sIPI2-3 significantly affected EFS, PFS, OS (p=0.0004). Women (83pts) had a better 4 yrs EFS 63% than male (159pts) 37% (p=0.01). The difference was only in the R arm (p=0.004). Gender was an independent prognostic factor in the R arm. Toxicity was mild with 12% SAE versus 4% for R /O respectively.Conclusions: There was no difference between R ICE and R DHAP and between post ASCT maintenance with R or O. Women did significantly better after ASCT with rituximab. Early relapses to upfront rituximab-based chemotherapy have a poor prognosis.

Functional analysis of different haematopoietic stem cell subsets

RÉSUMÉ : Elucider les bases moléculaires et cellulaires du fonctionnement des cellules souches s'avère crucial dans la compréhension de l'organisation cellulaire au sein des tissus et des organes ainsi que pour le développement de nouvelles stratégies thérapeutiques en médecine régénérative et en oncologie. Les cellules souches adultes les mieux connues sont celles responsables de l'hématopoïèse, les cellules souches hématopoïétiques (CSH). Durant ces dernières années, la recherche a porté une attention particulière à l'isolation prospective de CSH dérivées de la moelle osseuse de souris en utilisant des marqueurs de surface cellulaire ainsi que des propriétés fonctionnelles alléguées. Par la suite, la capacité fonctionnelle des CSH a été vérifiée classiquement par leur transplantation intraveineuse dans des souris réceptrices conditionnées et par l'analyse de leur aptitude à reconstituer le système hématopoïétique à long terme. Des études récentes suggèrent que la transplantation des cellules directement dans la moelle osseuse pourrait non seulement aboutir à une prise de greffe plus rapide et plus efficace, mais pourrait même aider à l'identification de cellules qui ont certes des propriétés intrinsèques de CSH, mais qui n'ont pas la capacité de trouver leur niche au sein de la moelle osseuse et ont donc échoué dans les analyses classiques de reconstitution. Dans cette étude, nous comparons à deux niveaux la fonction de différents sous-groupes de cellules souches de la moelle osseuse, définis par leur phénotype de surface cellulaire. Premièrement, nous étudions leur capacité à reconstituer des souris létalement irradiées après injection soit intraveineuse soit intrafémorale. Deuxièmement, par analyse cytométrique de flux à 8 couleurs, nous comparons leur activité relative de « side population » (SP) par exclusion du colorant fluorescent Hoechst 33342. Nos résultats préliminaires renforcent en effet l'idée que la transplantation intrafémorale aboutit à une greffe plus rapide et plus efficace. Par contre, en utilisant cette approche, nous n'arrivons pas à identifier des cellules capables de prendre greffe spécifiquement quand elles sont injectées en intrafémorale. Finalement, bien qu'une confirmation in vivo soit encore nécessaire, nous suggérons sur la base de nos analyses cytométriques de flux, que les cellules SP Sca1t~és éie~~ CD48t~és bas sont très enrichies en CSH. Ceci permettrait l'isolation ex vivo de CSH de la moelle osseuse de souris par une stratégie à la fois nouvelle et simple. SUMMARY : Elucidating the molecular and cellular bases of stem cell function is crucial for the understanding of cellular organisation within tissues and organs as well as for the development of new therapeutic strategies in regenerative medicine and oncology. The best-known adult stem cells are those responsible for haematopoiesis, the haematopoietic stem cells (HSCs). In recent years, much effort has been put into the prospective isolation of mouse bone marrow (BM)-derived HSCs using cell-surface markers and alleged functional properties. Upon isolation, the functional capacity of putative HSCs has been classically assessed by intravenous transplantation into conditioned recipient mice and analysis of their ability to reconstitute the haematopoietic system at long-term. It has recently been suggested that transplanting the cells directly into the BM might not only result in more rapid and more effective engraftment, but even help to identify cells that have intrinsic HSC properties but lack the ability to home to their BM niche and have thus failed to succeed in classical reconstitution assays. In this study, we compare the function of different BM cell subsets, as defined by their cell surface phenotype, on two levels. Firstly, we assess their ability to reconstitute lethally irradiated mice, when injected either intravenously or intrafemorally. Secondly, using 8-colour flow cytometric analysis, we compare their relative side population (SP) activity by exclusion of the fluorescent dye Hoechst 33342. Our preliminary results indeed reinforce the idea that intrafemoral transplantation results in faster and more effective engraftment, however, using this approach, we are unable to identify cells that are capable of engrafting specifically when injected intrafemorally. Finally, although in vivo confirmation is still required, we propose, based on the results of our flow cytometric analyses, that SP Scat Very h'9h CD48Very'°W cells should be highly enriched for HSCs. This would allow for a simple new strategy for the isolation of mouse BM HSCs ex vivo.

A TARBP2-dependent miRNA expression profile underlies cancer stem cell properties and provides candidate therapeutic reagents in Ewing sarcoma.

We have recently demonstrated that human pediatric mesenchymal stem cells can be reprogrammed toward a Ewing sarcoma family tumor (ESFT) cancer stem cell (CSC) phenotype by mechanisms that implicate microRNAs (miRNAs). Here, we show that the miRNA profile of ESFT CSCs is shared by embryonic stem cells and CSCs from divergent tumor types. We also provide evidence that the miRNA profile of ESFT CSCs is the result of reversible disruption of TARBP2-dependent miRNA maturation. Restoration of TARBP2 activity and systemic delivery of synthetic forms of either of two of its targets, miRNA-143 or miRNA-145, inhibited ESFT CSC clonogenicity and tumor growth in vivo. Our observations suggest that CSC self-renewal and tumor maintenance may depend on deregulation of TARBP2-dependent miRNA expression.

Autologous stem cell transplantation for enteropathy-associated T-cell lymphoma: a retrospective study by the EBMT.

Enteropathy-associated T-cell lymphoma (EATL) is a rare subtype of peripheral T-cell lymphomas with a poor prognosis. Autologous stem cell transplantation (ASCT) was retrospectively evaluated as a consolidation or salvage strategy for EATL. The analysis included 44 patients who received ASCT for EATL between 2000 and 2010. Thirty-one patients (70%) were in first complete or partial remission at the time of the ASCT. With a median follow-up of 46 months, relapse incidence, progression-free survival, and overall survival were 39%, 54%, and 59% at 4 years, respectively, with only one relapse occurring beyond 18 months posttransplant. There was a trend for better survival in patients transplanted in first complete or partial remission at 4 years (66% vs 36%; P = .062). ASCT is feasible in selected patients with EATL and can yield durable disease control in a significant proportion of the patients.

Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes.

Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling. Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks. These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

Toll-like receptor 4 polymorphisms and aspergillosis in stem-cell transplantation.

BACKGROUND: Toll-like receptors (TLRs) are essential components of the immune response to fungal pathogens. We examined the role of TLR polymorphisms in conferring a risk of invasive aspergillosis among recipients of allogeneic hematopoietic-cell transplants. METHODS: We analyzed 20 single-nucleotide polymorphisms (SNPs) in the toll-like receptor 2 gene (TLR2), the toll-like receptor 3 gene (TLR3), the toll-like receptor 4 gene (TLR4), and the toll-like receptor 9 gene (TLR9) in a cohort of 336 recipients of hematopoietic-cell transplants and their unrelated donors. The risk of invasive aspergillosis was assessed with the use of multivariate Cox regression analysis. The analysis was replicated in a validation study involving 103 case patients and 263 matched controls who received hematopoietic-cell transplants from related and unrelated donors. RESULTS: In the discovery study, two donor TLR4 haplotypes (S3 and S4) increased the risk of invasive aspergillosis (adjusted hazard ratio for S3, 2.20; 95% confidence interval [CI], 1.14 to 4.25; P=0.02; adjusted hazard ratio for S4, 6.16; 95% CI, 1.97 to 19.26; P=0.002). The haplotype S4 was present in carriers of two SNPs in strong linkage disequilibrium (1063 A/G [D299G] and 1363 C/T [T399I]) that influence TLR4 function. In the validation study, donor haplotype S4 also increased the risk of invasive aspergillosis (adjusted odds ratio, 2.49; 95% CI, 1.15 to 5.41; P=0.02); the association was present in unrelated recipients of hematopoietic-cell transplants (odds ratio, 5.00; 95% CI, 1.04 to 24.01; P=0.04) but not in related recipients (odds ratio, 2.29; 95% CI, 0.93 to 5.68; P=0.07). In the discovery study, seropositivity for cytomegalovirus (CMV) in donors or recipients, donor positivity for S4, or both, as compared with negative results for CMV and S4, were associated with an increase in the 3-year probability of invasive aspergillosis (12% vs. 1%, P=0.02) and death that was not related to relapse (35% vs. 22%, P=0.02). CONCLUSIONS: This study suggests an association between the donor TLR4 haplotype S4 and the risk of invasive aspergillosis among recipients of hematopoietic-cell transplants from unrelated donors.

Fourth European Conference on Infections in Leukaemia (ECIL-4): guidelines for diagnosis, prevention, and treatment of invasive fungal diseases in paediatric patients with cancer or allogeneic haemopoietic stem-cell transplantation.

Invasive opportunistic fungal diseases (IFDs) are important causes of morbidity and mortality in paediatric patients with cancer and those who have had an allogeneic haemopoietic stem-cell transplantation (HSCT). Apart from differences in underlying disorders and comorbidities relative to those of adults, IFDs in infants, children, and adolescents are unique with respect to their epidemiology, the usefulness of diagnostic methods, the pharmacology and dosing of antifungal agents, and the absence of interventional phase 3 clinical trials for guidance of evidence-based decisions. To better define the state of knowledge on IFDs in paediatric patients with cancer and allogeneic HSCT and to improve IFD diagnosis, prevention, and management, the Fourth European Conference on Infections in Leukaemia (ECIL-4) in 2011 convened a group that reviewed the scientific literature on IFDs and graded the available quality of evidence according to the Infectious Diseases Society of America grading system. The final considerations and recommendations of the group are summarised in this manuscript.

Calcineurin signaling as a negative determinant of keratinocyte cancer stem cell potential and carcinogenesis.

Calcineurin is the only known serine-threonine phosphatase under calcium-calmodulin control and key regulator of the immune system. Treatment of patients with calcineurin-inhibitory drugs like cyclosporin A and FK506 to prevent graft rejection dramatically increases the risk of cutaneous squamous cell carcinoma, which is a major cause of death after organ transplants. Recent evidence indicates that suppression of calcineurin signaling, together with its impact on the immune system, exerts direct tumor-promoting effects in keratinocytes, enhancing cancer stem cell potential. The underlying mechanism involves interruption of a double negative regulatory axis, whereby calcineurin and nuclear factors of activated T-cell signaling inhibits expression of ATF3, a negative regulator of p53. The resulting suppression of keratinocyte cancer cell senescence is of likely clinical significance for the many patients under treatment with calcineurin inhibitors and may be of relevance for other cancer types in which altered calcium-calcineurin signaling plays a role.

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells.

Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype.