Histone deacetylase inhibitors inhibit metastasis by restoring a tumor suppressive microRNA-150 in advanced cutaneous T-cell lymphoma.

Tumor suppressive microRNA (miR)-150 inhibits metastasis by combining with the C-C chemokine receptor 6 (CCR6) "seed sequence" mRNA of the 3'-untranslated region (3'-UTR) in advanced cutaneous T-cell lymphoma (CTCL). Because the histone deacetylase inhibitor (HDACI) vorinostat showed excellent outcomes for treating advanced CTCL, HDACIs may reduce the metastasis of CTCL by targeting miR-150 and/ or CCR6. To examine whether these candidate molecules are essential HDACI targets in advanced CTCL, we used the My-La, HH, and HUT78 CTCL cell lines for functional analysis because we previously demonstrated that their xenografts in NOD/Shi-scid IL-2γnul mice (CTCL mice) induced multiple metastases. We found that pan- HDACIs (vorinostat and panobinostat) inhibited the migration of CTCL cells and downregulated CCR6. The miRNA microarray analysis against CTCL cell lines demonstrated that these pan-HDACIs commonly upregulated 161 miRNAs, including 34 known tumor suppressive miRNAs such as miR-150. Although 35 miRNAs possessing the CCR6 "seed sequence" were included in these 161 miRNAs, miR-150 and miR-185-5p were downregulated in CTCL cells compared to in normal CD4+ T-cells. The transduction of 12 candidate miRNAs against CTCL cells revealed that miR-150 most efficiently inhibited their migration capabilities and downregulated CCR6. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that miR-150 was downregulated in advanced but not early CTCL primary cases. Finally, we injected miR-150 or siCCR6 into CTCL mice and found that mouse survival was significantly prolonged. These results indicate that miR-150 and its target, CCR6, are essential therapeutic targets of pan-HDACIs in advanced CTCL with metastatic potential.

Erythroblast enucleation is a dynein-dependent process.

Mammalian erythroblasts undergo enucleation through a process thought to be similar to cytokinesis. Microtubule-organizing centers (MTOCs) mediate organization of the mitotic spindle apparatus that separates the chromosomes during mitosis and are known to be crucial for proper cytokinesis. However, the role of MTOCs in erythroblast enucleation remains unknown. We therefore investigated the effect of various MTOC inhibitors on cytokinesis and enucleation using human colony-forming units-erythroid (CFU-Es) and mature erythroblasts generated from purified CD34(+) cells. We found that erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), a dynein inhibitor, and monastrol, a kinesin Eg5 inhibitor, as well as various inhibitors of MTOC regulators, including ON-01910 (Plk-1), MLN8237 (aurora A), hesperadin (aurora B), and LY294002 (PI3K), all inhibited CFU-E cytokinesis. Among these inhibitors, however, only EHNA blocked enucleation. Moreover, terminally differentiated erythroblasts expressed only dynein; little or none of the other tested proteins was detected. Over the course of the terminal differentiation of human erythroblasts, the fraction of cells with nuclei at the cell center declined, whereas the fraction of polarized cells, with nuclei shifted to a position near the plasma membrane, increased. Dynein inhibition impaired nuclear polarization, thereby blocking enucleation. These data indicate that dynein plays an essential role not only in cytokinesis but also in enucleation. We therefore conclude that human erythroblast enucleation is a process largely independent of MTOCs, but dependent on dynein.

MicroRNA-150 inhibits tumor invasion and metastasis by targeting the chemokine receptor CCR6, in advanced cutaneous T-cell lymphoma.

In this study, we show that microRNA-150 (miR-150) is significantly downregulated in advanced cutaneous T-cell lymphoma (CTCL), and that this downregulation is strongly associated with tumor invasion/metastasis. Inoculation of CTCL cell lines into nonobese diabetic/Shi-scid interleukin 2γ (IL-2γ) null mice led to CTCL cell migration to multiple organs; however, prior transfection of the cells with miR-150 substantially reduced the invasion/metastasis by directly downregulating CCR6, a specific receptor for the chemokine CCL20. We also found that IL-22 and its specific receptor subunit, IL22RA1, were aberrantly overexpressed in advanced CTCL, and that production of IL-22 and CCL20 was increased in cultured CTCL cells. IL22RA1 knockdown specifically reduced CCL20 production in CTCL cells, suggesting that IL-22 upregulation may activate the production of CCL20 and its binding to CCR6, thereby enhancing the multidirectional migration potential of CTCL cells. CTCL cells also exhibited nutrition- and CCL20-dependent chemotaxis, which were inhibited by miR-150 transfection or CCR6 knockdown. From these findings, we conclude that, in the presence of continuous CCR6 upregulation accompanied by miR-150 downregulation, IL-22 activation leads to continuous CCL20-CCR6 interaction in CTCL cells and, in turn, autocrine metastasis to distal organs. This suggests miR-150, CCL20, and CCR6 could be key targets for the treatment of advanced CTCL.

A synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34(+) cells.

Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type-dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)-mediated very rapid apoptosis occurs within 1 hour in CD34(+) cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34(+) cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34(+) cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34(+) cells, neither exogenous IFN-α nor IFN-ß induces rapid apoptosis in CD34(+) cells and neutralization or blocking of type-I IFN receptor does not rescue CD34(+) cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TLR3, signaling triggers poly I:C-induced rapid apoptosis of human CD34(+) cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders.

CpG-ODN 2006 and human parvovirus B19 genome consensus sequences selectively inhibit growth and development of erythroid progenitor cells.

Recent studies have shown that anemia is commonly observed after exposure to pathogens or pathogen-derived products, which are recognized via Toll-like receptor 9 (TLR9). In the current study, we demonstrate that CpG oligodeoxynucleotide-2006, a TLR9 ligand with phosphodiester (PO; 2006-PO) but not with the phosphorothioate backbone, selectively inhibits the erythroid growth derived from human CD34(+) cells. The 2006-PO was internalized by the erythroid progenitors within 30 minutes; however, expression of TLR9 mRNA was not detected in these cells. The 2006-PO directly inhibited burst-forming unit-erythroid growth, resulted in the accumulation of cells in S and G(2)/M phases, and increased cell size and frequency of apoptotic cells. These features were similar to those observed in erythroid progenitors infected with human parvovirus B19 that causes pure red cell aplasia. The consensus sequence of 2006-PO was defined as 5'-GTTTTGT-3', which was located in the P6-promoter region of B19 and inhibited erythroid growth in a sequence-specific manner and down-regulated expression of erythropoietin receptor (EPOR) mRNA and EPOR. B19 genome extracted from serum also inhibited erythroid growth and down-regulated expression of EPOR on glycophorin A(+) cells. These results provide a possible insight into our understanding of the mechanisms of human parvovirus B19-mediated inhibition of erythropoiesis.

Aberrant overexpression of microRNAs activate AKT signaling via down-regulation of tumor suppressors in natural killer-cell lymphoma/leukemia.

The gene(s) responsible for natural killer (NK)-cell lymphoma/leukemia have not been identified. In the present study, we found that in NK-cell lymphoma lines (n = 10) and specimens of primary lymphoma (n = 10), levels of miR-21 and miR-155 expression were inversely related and were significantly greater than those found in normal natural killer (CD3(-)CD56(+)) cells (n = 8). To determine the functions of these microRNAs in lymphomagenesis, we examined the effects of antisense oligonucleotides (ASOs) targeting miR-21 (ASO-21) and/or miR-155 (ASO-155) in NK-cell lymphoma lines overexpressing one or both of these miRNAs. Conversely, cells showing little endogenous expression of miR-21 or miR-155 were transduced by the use of lentiviral vectors, leading to their overexpression. Reducing expression of miR-21 or miR-155 led to up-regulation of phosphatase and tensin homologue (PTEN), programmed cell death 4 (PDCD4), or Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1). ASO-21- and ASO-155-treated cell lines all showed down-regulation of phosphorylated AKT(ser473). Moreover, transduction with either miR-21 or miR-155 led to down-regulation of PTEN and PDCD4 or SHIP1 with up-regulation of phosphorylated AKT(ser473). Collectively, these results provide important new insight into the pathogenesis of NK-cell lymphoma/leukemia and suggest targeting miR-21 and/or miR-155 may represent a useful approach to treating NK-cell lymphoma/leukemia.

Phagocytosis of co-developing neutrophil progenitors by dendritic cells in a culture of human CD34(+) cells with granulocyte colony-stimulating factor and tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce the differentiation of CD34(+) cells toward dendritic cells (DCs). We have previously shown that DCs are co-generated from human CD34(+) cells during erythroid or megakaryocytic differentiation in the presence of TNF-alpha, and those DCs are able to stimulate autologous T cell proliferation. The aim of this study was to learn whether the co-stimulation of granulocyte colony-stimulating factor (G-CSF) and TNF-alpha would generate neutrophil progenitors and DCs together from human CD34(+) cells, and if this was the case, to clarify the phenotypic and functional characteristics of these DCs. When highly purified human CD34(+) cells were cultured for 7 days with G-CSF alone, the generated cells predominantly expressed a granulocyte marker, CD15, and then differentiated into neutrophils after 14 days of culture. The addition of TNF-alpha with G-CSF markedly decreased the number of CD15(+) cells without affecting the total number of cells during 7 days of culture. Almost one third of the generated cells were positive for CD11c and CD123. Furthermore, CD11c(+) cells were found to phagocytose CD15(+) cells and were able to induce allogeneic, but not autologous, T cell proliferation in the mixed lymphocyte reaction (MLR). On the other hand, the CD11c(+) cells generated by TNF-alpha and cytokines capable of inducing erythroid differentiation were able to stimulate autologous T cells. There was a difference in the expression of CD80, CD83 and CD86 among CD11c(+) cells induced by G-CSF plus TNF-alpha and those generated by interleukin-3, stem cell factor, and erythropoietin plus TNF-alpha. These results indicate that the co-stimulation of human CD34(+) cells with G-CSF and TNF-alpha induces the phagocytosis of co-developing neutrophil progenitors by DCs, and the stimulatory effects of these DCs on autologous T cells is different from that of DCs generated from CD34(+) cells during erythroid differentiation.

Phagocytosis of codeveloping megakaryocytic progenitors by dendritic cells in culture with thrombopoietin and tumor necrosis factor-alpha and its possible role in hemophagocytic syndrome.

Tumor necrosis factor-alpha (TNF-alpha) and thrombopoietin (TPO) have been shown to induce the differentiation and proliferation of CD34+ cells toward dendritic cells (DCs) in the presence of multiacting cytokines. We hypothesized that the costimulation of TPO and TNF-alpha generates megakaryocytic progenitors and DCs together from human CD34+ cells and that the interaction of these cells may indicate a physiologic and/or a pathologic role of DCs in megakaryopoiesis. When highly purified human CD34+ cells were cultured for 7 days with TPO alone, the generated cells expressed megakaryocytic markers, such as CD41, CD42b, and CD61. The addition of TNF-alpha with TPO remarkably decreased the number of megakaryocytic progenitor cells without affecting the cell yield. Almost half of the cells thus generated expressed CD11c, and most of them were positive for CD4 and CD123. Furthermore, CD11c+ cells were found to capture damaged CD61+ cells and to induce autologous T-cell proliferation, although the cytokine productions were low. We also confirmed an engulfment of CD61+ cells and their fragment by CD11c+ cells in bone marrow cells from patients with hemophagocytic syndrome. These findings suggest that DCs generated under megakaryocytic and inflammatory stimuli are involved in megakaryopoiesis and the subsequent immune responses to self-antigens.