Involvement of miRNAs in the differentiation of human glioblastoma multiforme stem-like cells

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process

A cyclin-D1 interaction with BAX underlies its oncogenic role and potential as a therapeutic target in mantle cell lymphoma

The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 overexpression plays an essential role in the development of mantle cell lymphoma (MCL), an aggressive tumor that remains incurable with current treatment strategies. Cyclin-D1 has been postulated as an effective therapeutic target, but the evaluation of this target has been hampered by our incomplete understanding of its oncogenic functions and by the lack of valid MCL murine models. To address these issues, we generated a cyclin-D1-driven mouse model in which cyclin-D1 expression can be regulated externally. These mice developed cyclin-D1-expressing lymphomas capable of recapitulating features of human MCL. We found that cyclin-D1 inactivation was not sufficient to induce lymphoma regression in vivo; however, using a combination of in vitro and in vivo assays, we identified a novel prosurvival cyclin-D1 function in MCL cells. Specifically, we found that cyclin-D1, besides increasing cell proliferation through deregulation of the cell cycle at the G(1)-S transition, sequestrates the proapoptotic protein BAX in the cytoplasm, thereby favoring BCL2's antiapoptotic function. Accordingly, cyclin-D1 inhibition sensitized the lymphoma cells to apoptosis through BAX release. Thus, genetic or pharmacologic targeting of cyclin-D1 combined with a proapoptotic BH3 mimetic synergistically killed the cyclin-D1-expressing murine lymphomas, human MCL cell lines, and primary lymphoma cells. Our study identifies a role of cyclin-D1 in deregulating apoptosis in MCL cells, and highlights the potential benefit of simultaneously targeting cyclin-D1 and survival pathways in patients with MCL. This effective combination therapy also might be exploited in other cyclin-D1-expressing tumors

Expression of MALT1 oncogene in hematopoietic stem/progenitor cells recapitulates the pathogenesis of human lymphoma in mice

Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1(+)Lin(-) hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas

LITAF, a BCL6 Target Gene, Regulates Autophagia in B Cells and Is Essential for T-Cell Dependent Humoral Responses

Abstract Abstract 1391 LITAF was discovered as a p53-induced transcript that promoted TNFa secretion in monocytes in response to LPS. We previously reported that LITAF is inactivated by deletion or promoter hypermethylation in germinal center-derived B-cell lymphomas. However, the function of LITAF in B lymphocytes is unknown. Using gene expression analysis of isolated B-cell subpopulation and immunohistochemical studies of tonsil lymphoid follicles we found that LITAF is expressed in naïve B lymphocytes and is repressed within the germinal centers (GCs). Thus, LITAF showed an opposite expression to BCL6, an essential regulator of GC development and function. Likewise, expression of LITAF and BCL6 were inversely correlated in cell lines and biopsies from patients with B-cell lymphoma, further suggesting a link between LITAF and BCL6. ChIP-on-chip and ChIP-sequencing analyses of B cells coupled with luciferase reporter assays revealed that BCL6 repressed LITAF expression by binding to its promoter. Accordingly, BCL6 silencing with siRNAs or after exposure to a BCL6-inhibitor peptide increased LITAF expression, indicating that LITAF is transcriptionally repressed by BCL6 in GC B lymphocytes and in B-cell lymphoma cells. To initially elucidate the function of LITAF in B cells, gain-and-loss of function experiments were performed in different cellular models. LITAF expression was not related to TNFa secretion after LPS exposure, nor modulated cell proliferation or apoptosis in B cells. However, sustained expression of LITAF in B-cell lymphoma cells increased cell size, lysosome content and mitochondrial mass. Gene expression microarray studies defined a LITAF-related transcriptional signature containing genes involved in the regulation of endomembranes, vesicle trafficking and protein transport. Accordingly, immunofluorescence analysis co-localized LITAF with lysosomes and with autophagosomes expressing LC3, the mammalian homolog of yeast autophagy-related protein (Atg8), as well as with the lysosomal sorting-associated proteins NEDD4 and TSG101, both in normal CD19+ B lymphocytes and in B-cell lymphoma cells. In addition, LITAF expression induced autophagic activity in B cells, shown by an increase in the FL1/FL3 ratio after acridine orange staining and by converting LC3-I to LC3-II, which were more evident upon cell starvation. Together, these data suggest that LITAF may play a role in the processing of proteins in autophagosomes through regulating autophagy. To investigate LITAF function in vivo, we generated mice with targeted deletion of the Litaf gene in B lymphocytes by using the Cre-loxP system. Litaf -mb1-Cre (Litaf−/− ) mice developed healthy and showed normal distribution of hematopoietic cell subpopulations. However, Litaf−/− mice were unable to develop full T-cell dependent immune responses, presenting PNA-stained, Litaf-negative GCs that were absent or had marked reduction in size and number. Accordingly, reduced amounts of IgM, IgG1 and IgG3 antibodies as a consequence of abnormal class switch recombination (CSR) were detected in immunized mice. However, in experiments testing CSR in vitro, in which B cells are artificially activated in the absence of T cells, the amounts of IgM/IgG1/IgG3 did not differ between knock-out and control groups. Similarly, mouse immunization with a T-cell independent antigen did not induce differences in immunoglobulin production. Further studies of GCs in T-cell immunized Litaf−/− mice using an antibody for the Class II-associated invariant chain peptide (CLIP) revealed that the atrophic GCs in Litaf−/− mice showed strong CLIP expression in comparison to wild-type littermates. In normal immune responses, CLIP peptides bind to MHC class II molecules in endolysosomes, until they are displaced by the antigen, then releasing CLIP and allowing MHC II-antigen complexes to be transported to the cell membrane for T-cell presentation. The failure to develop appropriate immune responses together with the accumulation of CLIP peptides in Litaf -deficient mice indicate that Litaf is essential for adequate T-cell dependent immune responses in GC B lymphocytes, possibly through facilitating the presentation of the antigens to MHC II molecules in the endolysosomes. Once this process is assembled and the T-cell activated B lymphocytes enter the GCs, BCL6 represses LITAF to prevent additional interactions between B and T cells during BCR editing. Disclosures: No relevant conflicts of interest to declare

Involvement of miRNAs in the differentiation of human glioblastoma multiforme stem-like cells.

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process

The NS cultures are enriched in GICs when compared to the glioblastoma cell line U87MG.

<p><i>In </i><i>vitro</i> self-renewal limiting dilution assays, performed in 12 wells per dilution in triplicate experiments (A), and clonogenic assays, performed in quadruplicate 96-well plates (B) for three NS cell lines: G63, G52 and GN1C; as well as for the U87MG cell line, used as a negative control, are depicted. * indicates statistical p value <0.05 using unpaired t-test with the Holm-Bonferroni correction for multiple comparisons. Tumors obtained from <i>in </i><i>vivo</i> xenografts (1x10⁶ cells injection) in the brain striatum of BALB/c-Rag2^-/--IL2γc^-/- mice (n=6 per cell line) were detected using microPET and are shown circled by a dotted line (C). Tumor images corresponding to G63, G52 and G97C xenografts, as well as a negative control brain, are displayed, along with the corresponding quantification of maximum value of standardized caption (SUV_max). A coronal section (200 mm thick) of a tumor originated by G97C was observed by means of a stereoscopic microscope (D) for tumor localization (black arrowhead) and a semi-thin section of the tumor was stained with hematoxylin-eosin (E).</p

miR-21 over-expression in GICs induces GFAP expression, decreases Nestin levels and targets <i>SPRY1</i>.

<p>miR-21 over-expression was analyzed by q-RT-PCR 7 days after transfection and growth in NS medium (A). 2^-ΔCt was calculated as miR-21 expression relative to RNU6B expression for GN1C cells transfected with pre-miR negative control (pre-C-) or miR-21 precursor (pre-21). mRNA expression levels of Nestin (NES) as well as astrocytic (GFAP) and neuronal (TUJ1) differentiation markers were measured by q-RT-PCR (B). 2^-ΔΔCt was calculated relative to GAPDH expression and GN1C cells transfected with pre-miR negative control (dotted lines). Transfections were carried out in triplicate. Protein expression levels of Nestin, GFAP and TUJ1 were visualized by immunofluorescence (C), 7 days after transfection of GN1C cells with miR-21 precursor (pre-21) or pre-miR negative control (pre-C-). Images were acquired at 20X magnification with a 739CCD camera coupled to an Axio Imager Z1 microscope (Carl Zeiss Inc.) using the Isis Imaging System software. Quantification of mean intensity for Nestin, GFAP and TUJ1 fluorescence is displayed (D). SPRY1 and β-Actin (loading control) protein levels were measured by Western blot in the GN1C and G63 cell lines at the NS state (0h) and after 96 hours (96h) of induction of their differentiation (E), as well as 96 hours after transfection with pre-miR-21 (21) or a pre-miR negative control (C-) (F). A time-course study of SPRY protein expression by Western blot was also performed at 48, 72 and 96 hours after transfection with pre-miR-21 (21) or a pre-miR negative control (C-) (G). Quantification of at least three independent replicates was performed using ImageJ software and is shown as SPRY1/β-Actin expression relative to pre-miR negative control transfected cells (100%). Specific binding of miR-21 to the 3´-UTR binding sites of <i>SPRY1</i> was assessed in the GN1C cell line using luciferase assays and site-directed mutagenesis (H) “Mut 1” corresponds to mutation of the site in position chr4:124324121-124324128 and “Mut 2” to chr4:124323893-124323900. Transfections were carried out in triplicate. * indicates p value <0.05 in unpaired t test statistical analysis using the Holm-Bonferroni correction for multiple comparisons. </p

Inhibition of miR-221/222 in differentiating GICs increases Nestin expression and decreases GFAP and TUJ1 levels.

<p>miR-221 and miR-222 inhibition in GN1C cells growing in differentiation medium was carried out with specific anti-miRs and was confirmed by q-RT-PCR 14 days after transfection, compared to cells transfected with anti-miR negative control (anti-C-) (A, B). The expression of miR-221 and miR-222 was normalized with respect to RNU6B as 2^-ΔCt. The expression of the progenitor marker Nestin (NES) as well as of astrocytic (GFAP) and neuronal (TUJ1) differentiation markers was also measured by q-RT-PCR and normalized with respect to GAPDH and to the cells transfected in parallel with the anti-miR negative control as 2^-ΔΔCt (C, D). Transfections were carried out in triplicate. Dotted lines indicate the expression level of GN1C cells transfected with anti-miR negative control. The corresponding protein expression levels of Nestin, GFAP and TUJ1 were visualized by immunofluorescence (E) using antibodies conjugated to FITC (green) or Texas Red (red) counterstained with DAPI (blue). Images were acquired at 20X magnification with a 739CCD camera coupled to an Axio Imager Z1 microscope (Carl Zeiss Inc.) using the Isis Imaging System software. Quantification of mean intensity for Nestin, GFAP and TUJ1 fluorescence is displayed (F). * indicates p value <0.05 using unpaired t-test or Mann-Whitney U test for statistical analysis and the Holm-Bonferroni correction for multiple comparisons. </p

Over-expression of miR-29a/29b targets <i>MCL1</i> and promotes apoptosis in GICs.

<p>miR-29a/b over-expression in GN1C cells transfected with pre-miR-29a (pre-29a) or pre-miR-29b (pre-29b) compared to pre-miR negative control (pre-C-) was confirmed by q-RT-PCR 4 days after transfection (A, B). Cell viability assays using MTS (C) and apoptosis assessment by Cell Death Detection kit (D) were carried out 4 days after transfection. MCL1 protein levels were also measured by Western blot at the same time point, using β-Actin as loading control (E). Quantification of Western blots was performed with ImageJ (F) and is displayed as the MCL1/β-actin ratio relative to the negative control (100%). Regulation of the 3´-UTR of <i>MCL1</i> by miR-29a and miR-29b was analyzed by luciferase assays (G). All experiments were carried out at least in triplicate. * indicates p value <0.05 in unpaired t test or Mann-Whitney U test, using the Holm-Bonferroni correction for multiple comparisons.</p

Microarray profiling of miRNA expression during NS differentiation.

<p>A heat map of a hierarchical clustering analysis of the microRNAs with differential expression between the NS cell lines at their basal state (NS) and after 4 (4d) and 14 (14d) days of induction of differentiation is displayed (A). Expression data are represented as log2Ratio and were mean centered for each miRNA. Validation of the differential expression of miR-29a (B), miR-29b (C), miR-221 (D), miR-222 (E), miR-21 (F), miR-93 (G) and miR-106a (H) was carried out by q-RT-PCR using specific TaqMan microRNA assays, normalizing their expression values with respect to RNU6B levels and to the NS state by calculating 2^-ΔΔCt. * indicates statistical p value <0.05 using unpaired t-test with the Holm-Bonferroni correction for multiple comparisons; dotted lines indicate the basal expression level at the NS state.</p