BTG1 inactivation drives lymphomagenesis and promotes lymphoma dissemination through activation of BCAR1.

Understanding the functional role of mutated genes in cancer is required to translate the findings of cancer genomics into therapeutic improvement. BTG1 is recurrently mutated in the MCD/C5 subtype of diffuse large B-cell lymphoma (DLBCL), which is associated with extranodal dissemination. Here, we provide evidence that Btg1 knock out accelerates the development of a lethal lymphoproliferative disease driven by Bcl2 overexpression. Furthermore, we show that the scaffolding protein BCAR1 is a BTG1 partner. Moreover, after BTG1 deletion or expression of BTG1 mutations observed in patients with DLBCL, the overactivation of the BCAR1-RAC1 pathway confers increased migration ability in vitro and in vivo. These modifications are targetable with the SRC inhibitor dasatinib, which opens novel therapeutic opportunities in BTG1 mutated DLBCL.

Chronic T cell receptor stimulation unmasks NK receptor signaling in peripheral T cell lymphomas via epigenetic reprogramming.

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

Physical exercise rescues defective neural stem cells and neurogenesis in the adult subventricular zone of Btg1 knockout mice.

Adult neurogenesis occurs throughout life in the dentate gyrus (DG) and the subventricular zone (SVZ), where glia-like stem cells generate new neurons. Voluntary running is a powerful neurogenic stimulus triggering the proliferation of progenitor cells in the DG but, apparently, not in the SVZ. The antiproliferative gene Btg1 maintains the quiescence of DG and SVZ stem cells. Its ablation causes intense proliferation of DG and SVZ stem/progenitor cells in young mice, followed, during adulthood, by progressive decrease of the proliferative capacity. We have previously observed that running can rescue the deficit of DG Btg1-null neurogenesis. Here, we show that in adult Btg1-null SVZ stem and neuroblast cells, the reduction of proliferation is associated with a longer cell cycle and a more frequent entry into quiescence. Notably, running increases proliferation in Btg1-null SVZ stem cells highly above the levels of sedentary wild-type mice and restores normal values of cell cycle length and quiescence in stem and neuroblast cells, without affecting wild-type cells. Btg1-null SVZ neuroblasts show also increased migration throughout the rostral migratory stream and a deficiency of differentiated neurons in the olfactory bulb, possibly a consequence of premature exit from the cycle; running, however, normalizes migration and differentiation, increasing newborn neurons recruited to the olfactory circuitry. Furthermore, running increases the self-renewal of Btg1-null SVZ-derived neurospheres and, remarkably, in aged Btg1-null mice almost doubles the proliferating SVZ stem cells. Altogether, this reveals that SVZ stem cells are endowed with a hidden supply of self-renewal capacity, coupled to cell cycle acceleration and emerging after ablation of the quiescence-maintaining Btg1 gene and following exercise.

The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway.

The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells. In addition, both the formation and the integrity of the blood-testis barrier are compromised owing to mislocalization of N-cadherin and connexin 43 at the surface of Sertoli cells. We further establish that Mex3b acts to regulate the cortical level of activated Rap1, a small G protein controlling phagocytosis and cell-cell interaction, through the activation and transport of Rap1GAP. The active form of Rap1 (Rap1-GTP) is abnormally increased at the membrane cortex and chemically restoring Rap1-GTP to physiological levels rescues the phagocytic and adhesion abilities of Sertoli cells. Overall, these findings implicate Mex3b in the spatial organization of the Rap1 pathway that orchestrates Sertoli cell functions.

Running rescues defective adult neurogenesis by shortening the length of the cell cycle of neural stem and progenitor cells.

Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild-type and Btg1-null mice, using a new method to determine the cell cycle length. Our data show that in wild-type mice running leads to a cell cycle shortening only of NeuroD1-positive progenitor cells. In contrast, in Btg1-null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S-phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein(+) and Sox2(+)) and progenitor (NeuroD1(+)) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1-null early-postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1-null mice after running provides a long-lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1-null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long-term modulation of neurogenesis.

CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness.

The homeobox transcription factor CDX2 plays a crucial role in intestinal cell fate specification, both during normal development and in tumorigenic processes involving intestinal reprogramming. The CDX2 regulatory network is intricate, but it has not yet been fully uncovered. Through genome-wide screening of a 3D culture system, the RNA-binding protein MEX3A was identified as putatively involved in CDX2 regulation; therefore, its biological relevance was addressed by setting up cell-based assays together with expression studies in murine intestine. We demonstrate here that MEX3A has a repressive function by controlling CDX2 levels in gastric and colorectal cellular models. This is dependent on the interaction with a specific binding determinant present in CDX2 mRNA 3'untranslated region. We have further determined that MEX3A impairs intestinal differentiation and cellular polarization, affects cell cycle progression and promotes increased expression of intestinal stem cell markers, namely LGR5, BMI1 and MSI1. Finally, we show that MEX3A is expressed in mouse intestine, supporting an in vivo context for interaction with CDX2 and modulation of stem cell properties. Therefore, we describe a novel CDX2 post-transcriptional regulatory mechanism, through the RNA-binding protein MEX3A, with a major impact in intestinal differentiation, polarity and stemness, likely contributing to intestinal homeostasis and carcinogenesis.

Btg1 is Required to Maintain the Pool of Stem and Progenitor Cells of the Dentate Gyrus and Subventricular Zone.

Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ). Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons. Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at 2 months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit the cell cycle after completing the S phase, express p53 and p21 at high levels and undergo apoptosis within 5 days. In the SVZ of adult (two-month-old) Btg1-null mice we observed an equivalent decrease, associated to apoptosis, of stem cells, neuroblasts, and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity. We conclude that ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in the dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, probably reflecting impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Btg1 appears, therefore, to be required for maintaining adult stem and progenitor cells quiescence and self-renewal.

Role of miR-34c microRNA in the late steps of spermatogenesis.

Spermatogenesis is a cyclic process in which diploid spermatogonia differentiate into haploid spermatozoa. This process is highly regulated, notably at the post-transcriptional level. MicroRNAs (miRNAs), single-stranded noncoding RNA molecules of about 20-25 nucleotides, are implicated in the regulation of many important biological pathways such as proliferation, apoptosis, and differentiation. We wondered whether miRNAs could play a role during spermatogenesis. The miRNA expression repertoire was tested in germ cells, and we present data showing that miR-34c was highly expressed only in these cells. Furthermore, our findings indicate that in male gonads, miR-34c expression is largely p53 independent in contrast to previous results showing a direct link in somatic cells between the miR-34 family and this tumor suppressor protein. In order to identify target genes involved in germinal lineage differentiation, we overexpressed miR-34c in HeLa cells, analyzed the transcriptome of these modified cells, and noticed a shift of the expression profile toward the germinal lineage. Recently, it has been shown that exogenous expression of Ddx4/Vasa in embryonic chicken stem cells (cESC) induces cESC reprogramming toward a germ cell fate. When we simultaneously expressed miR-34c in such cells, we could detect an up-regulation of germ cell-specific genes whereas the expression of other lineage specific markers remained unchanged. These data suggest that miR-34c could play a role by enhancing the germinal phenotype of cells already committed to this lineage.

Transcriptional activation of hTERT, the human telomerase reverse transcriptase, by nuclear factor of activated T cells.

Telomerase is essential for telomere maintenance, and its activation is thought to be a critical step in cellular immortalization and tumorigenesis. Human telomerase reverse transcriptase (hTERT) is a major component of telomerase activity. We show here that hTERT is expressed soon after lymphocyte activation and that its expression is inhibited by rapamycin, wortmannin, and FK506, which was the most potent inhibitor. These results suggest a potential role for the transcription factor nuclear factor of activated T cells (NFAT) in the regulation of hTERT expression. Five putative NFAT-binding sites were identified in the hTERT promoter. In luciferase assays, the hTERT promoter was activated by overexpressed NFAT1. Moreover, serial deletions revealed that the promoter activation was mainly due to a -40 NFAT1-binding site flanked by two SP1-binding sites. Mutation of the -40 NFAT-binding site caused a 53% reduction in the transcriptional activity of hTERT promoter. Simultaneous mutations of the -40 NFAT-responsive element together with one or both SP1-binding sites led to a more dramatic decrease in luciferase activity than single mutations, suggesting a functional synergy between NFAT1 and SP1 in hTERT transcriptional regulation. NFAT1 overexpression in MCF7 and Jurkat cell lines induced an increase in endogenous hTERT mRNA expression. Inversely, its down-regulation was induced by NFAT1 silencing. Furthermore, chromatin immunoprecipitation assay demonstrated that NFAT1 directly binds to two sites (-40 and -775) in the endogenous hTERT promoter. Thus, we show for the first time the direct involvement of NFAT1 in the transcriptional regulation of hTERT.

Vinorelbine induces beta3-tubulin gene expression through an AP-1 Site.

Although the correlation between beta3-tubulin expression level in tumors has been well correlated with clinical outcome in patients receiving microtubule-targeted agents, the regulation of this protein remains poorly understood. Recently, new insight of regulatory processes was offered with the cloning of the gene promoter. In this study beta3-tubulin gene expression was induced in response to exposure to various antimicrotubule agents such as vinorelbine and paclitaxel. The exploration of the beta3-tubulin gene promoter by successive deletions followed by site-directed mutagenesis led to the localization of a vinorelbine-responsive element containing an activator-protein 1 (AP-1) site. Among the various antimicrotubule agents tested, it appeared that the implicated AP-1 site was activated only after exposure to vinorelbine. This study confirms the inducible nature of the beta3-tubulin gene promoter.

Two new miR-16 targets: caprin-1 and HMGA1, proteins implicated in cell proliferation.

BACKGROUND INFORMATION: miRNAs (microRNAs) are a class of non-coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3' UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR-16 (miRNA-16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR-16. RESULTS: In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR-16, caprin-1 (cytoplasmic activation/proliferation-associated protein-1) and HMGA1 (high-mobility group A1), and we also studied cyclin E which had been previously recognized as an miR-16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR-16 interacts with the 3' UTR of the three target mRNAs. We showed that miR-16, in MCF-7 and HeLa cell lines, down-regulates the expression of caprin-1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. CONCLUSIONS: Taken together, our data demonstrated that miR-16 can negatively regulate two new targets, HMGA1 and caprin-1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.

Coactivation of nuclear receptors and myogenic factors induces the major BTG1 influence on muscle differentiation.

The btg1 (B-cell translocation gene 1) gene coding sequence was isolated from a translocation break point in a case of B-cell chronic lymphocytic leukaemia. We have already shown that BTG1, considered as an antiproliferative protein, strongly stimulates myoblast differentiation. However, the mechanisms involved in this influence remained unknown. In cultured myoblasts, we found that BTG1 stimulates the transcriptional activity of nuclear receptors (T3 and all-trans retinoic acid receptors but not RXRalpha and PPARgamma), c-Jun and myogenic factors (CMD1, Myf5, myogenin). Immunoprecipitation experiments performed in cells or using in vitro-synthesized proteins and GST pull-down assays established that BTG1 directly interacts with T3 and all-trans retinoic acid receptors and with avian MyoD (CMD1). These interactions are mediated by the transactivation domain of each transcription factor and the A box and C-terminal part of BTG1. NCoR presence induces the ligand dependency of the interaction with nuclear receptors. Lastly, deletion of BTG1 interacting domains abrogates its ability to stimulate nuclear receptors and CMD1 activity, and its myogenic influence. In conclusion, BTG1 is a novel important coactivator involved in the regulation of myoblast differentiation. It not only stimulates the activity of myogenic factors, but also of nuclear receptors already known as positive myogenic regulators.

Efficient somatic gene targeting in the lymphoid human cell line DG75.

Among the different approaches used to define the function of a protein of interest, alteration and/or deletion of its encoding gene is the most direct strategy. Homologous recombination between the chromosomal gene locus and an appropriately designed targeting vector results in an alteration or knockout of the gene of interest. Homologous recombination is easily performed in yeast or in murine embryonic stem cells, but is cumbersome in more differentiated and diploid somatic cell lines. Here we describe an efficient method for targeting both alleles of a complex human gene locus in DG75 cells, a cell line of lymphoid origin. The experimental approach included a conditional knockout strategy with three genotypic markers, which greatly facilitated the generation and phenotypic identification of targeted recombinant cells. The vector was designed such that it could be reused for two consecutive rounds of recombination to target both alleles. The human DG75 cell line appears similar to the chicken DT40 pre B-cell line, which supports efficient homologous recombination. Therefore, the DG75 cell line is a favorable addition to the limited number of cell lines amenable to gene targeting and should prove useful for studying gene function through targeted gene alteration or deletion in human somatic cells.

CCR4-associated factor CAF1 is an essential factor for spermatogenesis.

The CCR4-associated protein CAF1 has been demonstrated to play several roles in the control of transcription and of mRNA decay. To gain further insight into its physiological function, we generated CAF1-deficient mice. They are viable, healthy, and normal in appearance; however, mCAF1(-/-) male mice are sterile. The crossing of mCAF1(+/-) mice gave a Mendelian ratio of mCAF1(+/+), mCAF1(+/-), and mCAF1(-/-) pups, indicating that haploid mCAF1-deficient germ cells differentiate normally. The onset of the defect occurs during the first wave of spermatogenesis at 19 to 20 days after birth, during progression of pachytene spermatocytes to haploid spermatids and spermatozoa. Early disruption of spermatogenesis was evidenced by Sertoli cell vacuolization and tubular disorganization. The most mature germ cells were the most severely depleted, but progressively all germ cells were affected, giving Sertoli cell-only tubes, large interstitial spaces, and small testes. This phenotype could be linked to a defect(s) in germ cells and/or to inadequate Sertoli cell function, leading to seminiferous tubule disorganization and finally to a total disappearance of germ cells. The mCAF1-deficient mouse provides a new model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

Characterization of an excreted/secreted antigen form of 14-3-3 protein in Toxoplasma gondii tachyzoites.

The 14-3-3 protein was shown to be present into the parasitophorous vacuole of Toxoplasma gondii-infected human monocyte cells and in the excreted/secreted antigens (ESA). The ESA 14-3-3 protein migrates electrophoretically as the cytosol and the main membranous 14-3-3 isoforms. The excretion/secretion of 14-3-3 was not sensitive to cycloheximide, a protein synthesis inhibitor, even at a concentration which inhibited the production of 14-3-3 inside the tachyzoites. Recombinant 14-3-3/GST protein was used to test the presence of 14-3-3 antibodies in different human sera. A positive immunoreactivity was observed with sera corresponding to acute toxoplasmosis and a possible involvement of 14-3-3 in host immunity is discussed.

Stability of the Peutz-Jeghers syndrome kinase LKB1 requires its binding to the molecular chaperones Hsp90/Cdc37.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder characterized by the presence of multiple gastrointestinal polyps and an increased risk for various types of cancers. Inactivating germline mutations of the LKB1 gene, which encodes a serine/threonine kinase, are responsible for the majority of PJS cases. Here, we show that the heteromeric complex containing the molecular chaperones Hsp90 and Cdc37/p50 interacts with the kinase domain of LKB1. Treatment of cells with either geldanamycin or novobiocin, two pharmacological inhibitors of Hsp90 causes the destabilization of LKB1. Furthermore, geldanamycin treatment leads to the ubiquitination and the rapid degradation of LKB1 by the proteasome-dependent pathway. In addition, we found that a LKB1 point mutation identified in a sporadic testicular cancer, weakens the interaction of LKB1 with both Hsp90 and Cdc37/p50 and enhances its sensitivity to the destabilizing effect of geldanamycin. Collectively, our results demonstrate that the Hsp90/Cdc37 complex is a major regulator of the stability of the LKB1 tumor suppressor. Furthermore, these data draw attention to the possible adverse consequences of antitumor drugs that target Hsp90, such as antibiotics related to geldanamycin, which could disrupt LKB1 function and promote the development of polyps and carcinomatous lesions.

Subcellular localization of 14-3-3 proteins in Toxoplasma gondii tachyzoites and evidence for a lipid raft-associated form.

A polyclonal antibody was raised against a Toxoplasma gondii 14-3-3-gluthatione S-transferase fusion protein obtained by cloning a 14-3-3 cDNA sequence determined from the T. gondii database. This antibody specifically recognized T. gondii 14-3-3 without any cross-reaction with mammalian proteins. Immunofluorescence microscopy studies of the tachyzoites or the T. gondii-infected cells suggested cytosolic and membranous localizations of 14-3-3 protein. Different subcellular fractions were prepared for electrophoresis analysis and immunodetection. 14-3-3 proteins were found in the cytosol, the membrane fraction and Triton X-100-resistant membranes. Two 14-3-3 isoforms were detected. The major one was mainly cytoplasmic and to a lesser extent membrane-associated, whereas the minor isoform was associated with the detergent-resistant lipid rafts.

Interaction of PRMT1 with BTG/TOB proteins in cell signalling: molecular analysis and functional aspects.

BACKGROUND: Several recent reports have connected protein methylation with differentiation. Furthermore, the BTG/TOB proteins have also been implicated in such control. BTG1 and 2 have been shown to interact with PRMT1 (predominant cellular arginine N-methyltransferase of type I). RESULTS: First, we have studied the interaction between PRMT1 and the proteins of the BTG/TOB family. We show that boxC, a sequence present only in BTG1 and BTG2, is essential for this association. Using boxC peptide, we have investigated the importance of PRMT1/BTG protein association during type I protein methylation reactions. Finally, we show that the addition of boxC fused to penetratin interferes with the neuronal differentiation of PC12 cells and ES cell-derived neurones. CONCLUSIONS: Taken together, these results indicate that PRMT1/BTG proteins could play a key role in the arginine methylation-mediated signalling pathway as well as in neuronal differentiation.