Leukemia inhibitory factor: a newly identified metastatic factor in rhabdomyosarcomas.

Rhabdomyosarcoma frequently infiltrates bone marrow and this process involves the stromal-derived factor-1 (SDF-1)-CXCR4 axis. Because leukemia inhibitory factor (LIF), like SDF-1, is secreted by bone marrow stroma and directs the regeneration of skeletal muscles, we examined whether the LIF-LIF receptor (LIF-R) axis affects the biology of rhabdomyosarcoma cells. We found that in rhabdomyosarcoma cells, LIF stimulates the following: (a) phosphorylation of mitogen-activated protein kinase p42/44, AKT, and signal transducers and activators of transcription 3, (b) adhesion and chemotaxis, and (c) increased resistance to cytostatics. To compare the biological effects of LIF versus SDF-1, we examined the RH30 cell line, which is highly responsive to both ligands, and found that the chemotaxis of these cells is significantly reduced when the inhibitors of both receptors (T140 for CXCR4 and gp190 blocking antibody for LIF-R) are added simultaneously. Subsequently, by using repetitive chemotaxis to LIF or SDF-1, we selected from the RH30 line subpopulations of cells that respond to LIF but not SDF-1 (RH30-L) or to SDF-1 but not LIF (RH30-S). We found that (a) RH30-L cells seed better to the bone marrow, liver, and lymph nodes of immunodeficient mice than RH30-S cells and (b) mice inoculated i.m. with the RH30-L cells had more rhabdomyosarcoma cells in the bone marrow and lung after 6 weeks. Thus, we present the first evidence that the LIF-LIF-R axis may direct rhabdomyosarcoma metastasis. Further, because we showed that the in vivo metastasis of RH30 cells is inhibited by small interfering RNA against LIF-R, molecular targeting of this axis could become a new strategy to control the metastasis of rhabdomyosarcoma.

The effect of a novel recombination between the homeobox gene NKX2-5 and the TRD locus in T-cell acute lymphoblastic leukemia on activation of the NKX2-5 gene.

BACKGROUND AND OBJECTIVES: The NK-like homeobox gene (NKX2-5/CSX) plays a crucial role in cardiac development but is not normally expressed in hematopoietic cells. Here, we describe for the first time a fusion between NKX2-5 and the T-cell receptor delta locus (TRD) resulting in NKX2-5 activation in a case of T-cell acute lymphoblastic leukemia (T-ALL). DESIGN AND METHODS: Genomic DNA from a T-ALL patient with an atypical rearrangement, detected by Southern blotting, was analyzed by ligation-mediated polymerase chain reaction (PCR) with TRD-specific primers. Expression of NKX2-5 was analyzed by real-time quantitative PCR in the T-ALL case with the NKX2-5-TRD rearrangement, 18 other cases of T-ALL, three T-ALL derived cell lines, two non-hematopoietic cell lines, peripheral blood mononuclear cells from six healthy individuals and sorted thymocyte subsets. RESULTS: Sequence analysis of ligation-mediated PCR products revealed a novel rearrangement between the third diversity segment of the TRD locus (TRDD3) and a region on chromosome 5q35.1 located 32 kb downstream of the NKX2-5/CSX gene. As a result of this recombination NKX2-5 was placed under influence of the TRD enhancer, resulting in strong ectopic NKX2-5 expression. High NKX2-5 expression was also found in the T-cell lines PEER and CCRF-CEM, which harbor an NKX2-5-BCL11B rearrangement, and in the embryonic kidney cell line 293. NKX2-5 was not expressed in any of the major thymocyte subsets, in normal peripheral blood mononuclear cells, or in the majority (17/18) of the other cases of T-ALL. INTERPRETATION AND CONCLUSIONS: Our finding of overexpression of yet another homeobox gene in T-ALL further supports the hypothesis that homeobox genes play an important role in malignant transformation of particular types of T-ALL.

Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis.

The alpha-chemokine stromal-derived factor (SDF)-1 and the G-protein-coupled seven-span transmembrane receptor CXCR4 axis regulates the trafficking of various cell types. In this review, we present the concept that the SDF-1-CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells. Supporting this is growing evidence that SDF-1 plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells (HSCs) and their homing/retention in bone marrow. Moreover, functional CXCR4 is also expressed on nonhematopoietic tissue-committed stem/progenitor cells (TCSCs); hence, the SDF-1-CXCR4 axis emerges as a pivotal regulator of trafficking of various types of stem cells in the body. Furthermore, because most if not all malignancies originate in the stem/progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and, as a result, the SDF-1-CXCR4 axis is also involved in directing their trafficking/metastasis to organs that highly express SDF-1 (e.g., lymph nodes, lungs, liver, and bones). Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, and we discuss here the common molecular mechanisms involved in these processes. Finally, the responsiveness of CXCR4+ normal and malignant stem cells to an SDF-1 gradient may be regulated positively/primed by several small molecules related to inflammation which enhance incorporation of CXCR4 into membrane lipid rafts, or may be inhibited/blocked by small CXCR4 antagonist peptides. Consequently, strategies aimed at modulating the SDF-1-CXCR4 axis could have important clinical applications both in regenerative medicine to deliver normal stem cells to the tissues/organs and in clinical hematology/oncology to inhibit metastasis of cancer stem cells.

Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient.

We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte-colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)- and bone marrow (BM)-derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.

Identification of a new cluster of T-cell receptor delta recombining elements.

Within the human T-cell receptor delta (TCRD) gene we have identified a new cluster of seven delta recombining elements (deltaRec2.1-2.7), located 2.6-5.2 kilobases downstream of the Vdelta2 gene segment. The deltaRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Ddelta3 and Jdelta1 segments of the TCRD gene as well as with the psiJalpha of the TCRA gene. Rearrangements involving the deltaRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100-fold lower than that of classical deltaRec rearrangements. The total frequency of deltaRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T-cell development. The decrease of the frequency of the deltaRec2-Ddelta3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the deltaRec2-Jdelta1 rearrangements contained the Ddelta3 segment in the junctional region, we assume that they are derived from the deltaRec2-Ddelta3 rearrangements. In contrast, the majority of deltaRec2-psiJalpha rearrangements did not contain the Ddelta3 segment, indicating that they are single step rearrangements. The deltaRec2-Jdelta1 and deltaRec2-psiJalpha rearrangements seem to be T-lineage specific, but the deltaRec2-Ddelta3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that deltaRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Valpha-Jalpha rearrangements. We hypothesize, that in a similar manner to the classical deltaRec rearrangements, the deltaRec2 rearrangements might also contribute to T-cell differentiation towards the TCR-alphabeta lineage.