Fawzia Louache

The chemokine receptor CXCR4 strongly promotes neuroblastoma primary tumour and metastatic growth, but not invasion.

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.

Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures.

In contrast to myeloid and B‐lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+CD38+ and CD34+CD38low cells from both cord blood and adult bone marrow reproducibly develop into CD4+CD8+T cells when introduced into NOD‐SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC (fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T‐cell potential of human primitive progenitor cells. The frequency of T‐cell progenitors among cord‐blood‐derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD‐SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD4lowCD8−sCD3−TCRαβ–CD5+CD1a+T cells were mixed with CD4+CD8+ cells and more mature CD4+CD8−TCRαβ+cells. However, in FTOC initiated with bone marrow T progenitors, <10% double‐positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T‐cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine‐stimulated CD34+CD38low cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.

Novel Anti-Metastatic Action of Cidofovir Mediated by Inhibition of E6/E7, CXCR4 and Rho/ROCK Signaling in HPV+ Tumor Cells

Cervical cancer is frequently associated with HPV infection. The expression of E6 and E7 HPV oncoproteins is a key factor in its carcinogenicity and might also influence its virulence, including metastatic conversion. The cellular mechanisms involved in metastatic spread remain elusive, but pro-adhesive receptors and their ligands, such as SDF-1α and CXCR4 are implicated. In the present study, we assessed the possible relationship between SDF-1α/CXCR4 signaling, E6/E7 status and the metastatic process. We found that SDF-1α stimulated the invasion of E6/E7-positive cancer cell lines (HeLa and TC-1) in Matrigel though CXCR4 and subsequent Rho/ROCK activation. In pulmonary metastatic foci generated by TC-1 cells IV injection a high proportion of cells expressed membrane-associated CXCR4. In both cases models (in vitro and in vivo) cell adhesion and invasion was abrogated by CXCR4 immunological blockade supporting a contribution of SDF-1α/CXCR4 to the metastatic process. E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1α-induced cell invasion. In addition, Cidofovir inhibited lung metastasis (both adhesion and invasion) supporting contribution of E6 and E7 oncoproteins to the metastatic process. Finally, potential signals activated downstream SDF-1α/CXCR4 and involved in lung homing of E6/E7-expressing tumor cells were investigated. The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor). These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition.

Reversing resistance to vascular disrupting agents by blocking late mobilization of circulating endothelial progenitor cells

UNLABELLEDnThe prevailing concept is that immediate mobilization of bone marrow-derived circulating endothelial progenitor cells (CEP) is a key mechanism mediating tumor resistance to vascular-disrupting agents (VDA). Here, we show that administration of VDA to tumor-bearing mice induces 2 distinct peaks in CEPs: an early, unspecific CEP efflux followed by a late yet more dramatic tumor-specific CEP burst that infiltrates tumors and is recruited to vessels. Combination with antiangiogenic drugs could not disrupt the early peak but completely abrogated the late VDA-induced CEP burst, blunted bone marrow-derived cell recruitment to tumors, and resulted in striking antitumor efficacy, indicating that the late CEP burst might be crucial to tumor recovery after VDA therapy. CEP and circulating endothelial cell kinetics in VDA-treated patients with cancer were remarkably consistent with our preclinical data. These findings expand the current understanding of vasculogenic rebounds that may be targeted to improve VDA-based strategies.nnnSIGNIFICANCEnOur findings suggest that resistance to VDA therapy may be strongly mediated by late, rather than early, tumor-specific recruitment of CEPs, the suppression of which resulted in increased VDA-mediated antitumor efficacy. VDA-based therapy might thus be significantly enhanced by combination strategies targeting late CEP mobilization.

Identification of CXCR4 as a new nitric oxide-regulated gene in human CD34+ cells.

As an intracellular second messenger, nitric oxide (NO) is increasingly implicated in the control of transcriptional machinery and gene expression. Here, we show that cell surface expression of CXCR4 on CD34+ cells was increased in a dose‐ and time‐dependent manner in response to NO donors. Augmented surface expression was correlated with an increase in CXCR4 mRNA level. A specific NO scavenger prevented the elevation in CXCR4 mRNA caused by NO donors, suggesting a direct signaling action mediated by NO on CXCR4 transcription. NO treatment had no significant effect on CXCR4 mRNA stability. However, induction of CXCR4 mRNA by NO was still observed in conditions in which initiation of translation was inhibited, suggesting that the NO effect must be mediated by a pre‐existing protein. CXCR4 mRNA induction did not involve cGMP (guanosine 3′, 5′‐cyclic monophosphate) generation but was most likely mediated via oxidation of intracellular protein thiols. Finally, CD34+ cells pretreated with NO donors exhibited an increased chemotactic response. This study demonstrates that the NO pathway can modulate CXCR4 expression in human CD34+ cells and suggests that NO may play a critical role in the trafficking of hematopoietic progenitors.

Generation and characterisation of Rhd and Rhag null mice

Mouse Rhd* and Rhag* genes were targeted using insertional vectors; the resulting knockout mice, and double‐knockout descendants, were analysed. Rhag glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM‐4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM‐4, and only a moderate reduction of Rhag expression. Double knockout phenotype was similar to that of Rhag targeted mice. Rhd and Rhag deficient mice exhibited neither the equivalent of human Rhnull haemolytic anaemia nor any clinical or cellular abnormalities. Rhd−/− and Rhag−/− erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM‐4 membrane expression. There was no difference in recovery from phenylhydrazine‐induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM‐4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in Rhag−/− but only slightly in Rhd−/− animals that significantly expressed Rhag, supporting the view that RhAG and Rhag, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and Rhag proteins in the red cell membrane.

Lymphoid differentiation of hematopoietic stem cells requires efficient Cxcr4 desensitization

The CXCL12/CXCR4 signaling exerts a dominant role in promoting hematopoietic stem and progenitor cell (HSPC) retention and quiescence in bone marrow. Gain-of-function CXCR4 mutations that affect homologous desensitization of the receptor have been reported in the WHIM Syndrome (WS), a rare immunodeficiency characterized by lymphopenia. The mechanisms underpinning this remain obscure. Using a mouse model with a naturally occurring WS-linked gain-of-function Cxcr4 mutation, we explored the possibility that the lymphopenia in WS arises from defects at the HSPC level. We reported that Cxcr4 desensitization is required for quiescence/cycling balance of murine short-term hematopoietic stem cells and their differentiation into multipotent and downstream lymphoid-biased progenitors. Alteration in Cxcr4 desensitization resulted in decrease of circulating HSPCs in five patients with WS. This was also evidenced in WS mice and mirrored by accumulation of HSPCs in the spleen, where we observed enhanced extramedullary hematopoiesis. Therefore, efficient Cxcr4 desensitization is critical for lymphoid differentiation of HSPCs, and its impairment is a key mechanism underpinning the lymphopenia observed in mice and likely in WS patients.

Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines.

Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.

Wiskott-Aldrich syndrome protein-deficient hematopoietic cells can be efficiently mobilized by granulocyte colony-stimulating factor

The Wiskott-Aldrich syndrome protein is an essential cytoskeleton regulator found in cells of the hematopoietic lineage and controls the motility of leukocytes. The impact of WAS gene deficiency on the mobilization of hematopoietic progenitor/stem cells in circulation has remained unexplored but information would be pertinent in the context of autologous gene therapy of Wiskott-Aldrich syndrome. The response to granulocyte-colony stimulating factor mobilization was investigated in a murine WAS knock-out model of the disease, by measuring hematologic parameters, circulation and engraftment of hematopoietic progenitor/stem cells. In the steady-state, adult WAS knock-out mice have B-cell lymphopenia, marked neutrophilia, increased counts of circulating hematopoietic progenitor cells and splenomegaly, presumably caused by the retention of hematopoietic progenitor cells due to high levels of splenic CXCL12. In spite of these anomalies, the administration of granulocyte-colony-stimulating factor mobilizes progenitor/stem cells in WAS knock-out mice to the same level and with the same kinetics as in wild-type control mice. Mobilized peripheral blood cells from WAS knock-out mice can be transduced and are able to engraft into lethally-irradiated hosts reconstituting multiple lineages of cells and providing more effective radio-protection than mobilized cells from wild-type control mice. Surprisingly, the homing and the peripheral blood recovery of B lymphocytes was influenced by the background of the host. Thus, in the absence of Wiskott-Aldrich syndrome protein, effective mobilization is achieved but partial correction may occur as a result of an abnormal hematopoietic environment.

Cutting Edge: NANOG Activates Autophagy under Hypoxic Stress by Binding to BNIP3L Promoter

Hypoxia upregulates the core pluripotency factors NANOG, SOX2, and OCT4, associated with tumor aggressiveness and resistance to conventional anticancer treatments. We have previously reported that hypoxia-induced NANOG contributed in vitro to tumor cell resistance to autologous-specific CTL and in vivo to the in situ recruitment of immune-suppressive cells. In this study, we investigated the mechanisms underlying NANOG-mediated tumor cell resistance to specific lysis under hypoxia. We demonstrated the tumor-promoting effect of hypoxia on tumor initiation into immunodeficient mice using human non–small lung carcinoma cells. We next showed a link between NANOG and autophagy activation under hypoxia because inhibition of NANOG decreased autophagy in tumor cells. Chromatin immunoprecipitation and luciferase reporter assays revealed a direct binding of NANOG to a transcriptionally active site in a BNIP3L enhancer sequence. These data establish a new link between the pluripotency factor NANOG and autophagy involved in resistance to CTL under hypoxia.