FUS-DDIT3 Prevents the Development of Adipocytic Precursors in Liposarcoma by Repressing PPARγ and C/EBPα and Activating eIF4E

FUS-DDIT3 is a chimeric protein generated by the most common chromosomal translocation t(12;16)(q13;p11) linked to liposarcomas, which are characterized by the accumulation of early adipocytic precursors. Current studies indicate that FUS-DDIT3- liposarcoma develops from uncommitted progenitors. However, the precise mechanism whereby FUS-DDIT3 contributes to the differentiation arrest remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here we have characterized the adipocyte regulatory protein network in liposarcomas of FUS-DITT3 transgenic mice and showed that PPARgamma2 and C/EBPalpha expression was altered. Consistent with in vivo data, FUS-DDIT3 MEFs and human liposarcoma cell lines showed a similar downregulation of both PPARgamma2 and C/EBPalpha expression. Complementation studies with PPARgamma but not C/EBPalpha rescued the differentiation block in committed adipocytic precursors expressing FUS-DDIT3. Our results further show that FUS-DDIT3 interferes with the control of initiation of translation by upregulation of the eukaryotic translation initiation factors eIF2 and eIF4E both in FUS-DDIT3 mice and human liposarcomas cell lines, explaining the shift towards the truncated p30 isoform of C/EBPalpha in liposarcomas. Suppression of the FUS-DDIT3 transgene did rescue this adipocyte differentiation block. Moreover, eIF4E was also strongly upregulated in normal adipose tissue of FUS-DDIT3 transgenic mice, suggesting that overexpression of eIF4E may be a primary event in the initiation of liposarcomas. Reporter assays showed FUS-DDIT3 is involved in the upregulation of eIF4E in liposarcomas and that both domains of the fusion protein are required for affecting eIF4E expression. CONCLUSIONS/SIGNIFICANCE: Taken together, this study provides evidence of the molecular mechanisms involve in the disruption of normal adipocyte differentiation program in liposarcoma harbouring the chimeric gene FUS-DDIT3.Research in ISG group is supported partially by FEDER and by MEC (SAF2006-03726), Junta de Castilla y León (CSI03A05), FIS (PI050087, PI050116), Fundación de Investigación MMA, Federación de Cajas de Ahorro Castilla y León (I Convocatoria de Ayudas para Proyectos de Investigación Biosanitaria con Células Madre), CDTEAM project (CENIT-Ingenio 2010) and MEC Consolider-Ingenio 2010 (Ref. CSD2007-0017).Research in ISG group is supported partially by FEDER and by MEC (SAF2006-03726 and PETRI N° 95-0913.OP), Junta de Castilla y León (CSI03A05), FIS (PI050087, PI050116), Fundación de Investigación MMA, Federación de Cajas de Ahorro Castilla y León (I Convocatoria de Ayudas para Proyectos de Investigación Biosanitaria con Células Madre), CDTEAM project (CENIT-Ingenio 2010) and MEC Consolider-Ingenio 2010 (Ref. CSD2007-0017). MSM is supported by the Ramon y Cajal Scientific Spanish Program, Fondo Investigacion Sanitaria (FIS PI04-1271), Junta de Castilla y León (SA085A06) and Fundación Manuel Solorzano, University of Salamanca.Peer reviewe

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Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p < 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Targeted Cancer Cell Killing by Highly Selective miRNA-Triggered Activation of a Prokaryotic Toxin-Antitoxin System.

Although not evolved to function in eukaryotes, prokaryotic toxin Kid induces apoptosis in human cells, and this is avoided by coexpression of its neutralizing antitoxin, Kis. Inspired by the way Kid becomes active in bacterial cells we had previously engineered a synthetic toxin-antitoxin system bearing a Kis protein variant that is selectively degraded in cells expressing viral oncoprotein E6, thus achieving highly selective killing of cancer cells transformed by human papillomavirus. Here we aimed to broaden the type of oncogenic insults, and therefore of cancer cells, that can be targeted using this approach. We show that appropriate linkage of the kis gene to a single, fully complementary, target site for an oncogenic human microRNA enables the construction of a synthetic toxin-antitoxin pair that selectively kills cancer cells overexpressing that particular microRNA. Importantly, the resulting system spares nontargeted cells from collateral damage, even when they overexpress highly homologous, though nontargeted, microRNAs

Retroviral-mediated expression of PPARγ2 rescues the impaired adipogenesis of FUS-DDIT3 MEFs.

<p>A) FUS-DDIT3 MEFs were infected with either control retroviral vector or one expressing PPARγ2 (pQCXIP-PPARγ2) and selected for 3 days with 2 μg/ml puromycin. Then, wild-type MEF, FUS-DDIT3 MEF and PPARγ2 expressing FUS-DDIT3-MEF were cultered up to confluence and grown in the presence of standard adipose differentiation induction medium. At day 8 after induction of adipocyte differentiation, cells were fixed and stained for neutral lipids with Oil-Red-O and the morphological differentiation is shown (the original magnification is ×20). This experiment was repeated three times using cells prepared from all lines and from different embryos and similar results were obtained. B) Analysis of the PPARγ2 protein by western-blot in FUS-DDIT3 MEFs infected with either a control retroviral vector (pQCXIP) or one expressing PPARγ2 (pQCXIP- PPARγ2) 4 days after infection.</p

Adipogenic gene expression in liposarcomas of FUS-DDIT3 transgenic mice and in human liposarcoma cell lines carrying the translocation t(12;16)(q13;p11).

<p>(A) Hematoxylin/eosin stained sections showing the presence of lipoblasts with round nuclei and accumulation of intracellular lipid in a liposarcoma arisen in the chest region of FUS-DDIT3 mouse (10× and 40× magnifications are shown). (B) Western blot analyses of regulators of adipocyte function in white adipose tissue (WAT), liposarcoma arisen in FUS-DDIT3 transgenic mice and human liposarcomas cell lines expressing FUS-DDIT3 (LIS-3 and LIS-4). Cell and tissue extracts (10 μg) were resolved in SDS-PAGE gel (10% acrylamide), followed by immunoblotting analysis with anti-C/EBPβ, anti-C/EBPδ, anti-PPARγ, anti-C/EBPα and anti-actin antibodies. These data are representative of three independent experiments. (C) Western blot analysis of fat cell markers such as aP2 and adiponectin in liposarcomas of FUS-DDIT3 transgenic mice and in human liposarcoma cell lines carrying the translocation t(12;16)(q13;p11). These data are representative of three independent experiments. (D) Expression of the human <i>FUS-DDIT3</i> oncogene by RT-PCR both in liposarcomas of FUS-DDIT3 transgenic mice and in human liposarcoma cell lines carrying the translocation t(12;16)(q13;p11).</p

Model for the adipocyte differentiation arrest produced by <i>FUS-DDIT3</i> in liposarcoma development.

<p>(A) Scheme of the normal differentiation program in mesenchymal progenitor cells. (B) FUS-DDIT3 blocks the adipocyte differentiation program in mesenchymal cell progenitors by interfering with the PPARγ and C/EBPα activities at the transcriptional level. In addition, FUS-DDIT3 induces the expression of eIF4E, that in turns, is able to inactivate the C/EBPα pathway by shifting the normal isoform ratio towards the truncated p30- C/EBPα isoform, which has a negative effect on adipogenesis.</p

C/EBPα does not bypass adipogenesis blockade in FUS-DDIT3 expressing-MEF.

<p>(A) FUS-DDIT3 represses the C/EBPα transactivation induced by C/EBPβ. U2OS cells were cotransfected with 1 μg of pRL-SV40 (Renilla basal control (PROMEGA), lines 1–6) along with: 5 μg of pCEBP1171 (luciferase reporter vector containing 1171 of the rat C/EBPα promoter, samples 2–6); 3 μg ratC/EBPβwtpSG5 (C/EBPβ expressing vector, lines 3–6); 3, 5 and 7 μg pcDNA3-hFUS-DDIT3 (hFUS-DDIT3 expression vector, lines 4–6); 5 μg of the hFUS-DDIT3 expression vector (line 7). These data are representative of three independent experiments. (B) Retroviral expression of C/EBPα does not rescue the adipocyte differentiation blockade in FUS-DDIT3 MEFs. FUS-DDIT3 MEFs were infected with a retroviral vector expressing C/EBPα (pQCXIP-C/EBPα) and selected for 3 days with 2 μg/ml puromycin. Then, wild-type MEF, FUS-DDIT3 MEF and C/EBPα expressing FUS-DDIT3-MEFS were cultered up to confluence and grown in the presence of standard adipose differentiation induction medium. At day 8 after induction of adipocyte differentiation, cells were fixed and stained for neutral lipids with Oil-Red-O and the morphological differentiation is shown (the original magnification is ×20). This experiment was repeated three times using cells prepared from all lines and from different embryos and similar results were obtained. (C) Analysis of C/EBPα (p42-C/EBPα and p30-C/EBPα isoforms) protein expression by western-blot in FUS-DDIT3 MEFs infected with either a control retroviral vector (pQCXIP) or one expressing C/EBPα (pQCXIP- C/EBPα) 4 days after infection.</p

CombitTA-FUS-DDIT3 expression and effect of FUS-DDIT3 on adipocyte differentiaton.

<p>A) Analysis of the tetracycline (Doxycycline) dependent CombitTA-FUS-DDIT3 expression by RT-PCR in the presence (+tet) or in the absence (-tet) of doxycycline in MEF (the time of treatment with doxycycline was 48 hours). Actin was used to check the RNA integrity and loading. B) Adipocyte differentiation in CombitTA-FUS-DDIT3 MEFs after suppression of FUS-DDIT3 expression by tetracycline treatment. CombitTA-FUS-DDIT3 MEFs in the presence (+tet) or in the absence (-tet) of doxycycline were cultered up to confluence and grown in the presence of standard adipose differentiation induction medium. At day 8 after induction of adipocyte differentiation, cells were fixed and stained for neutral lipids with Oil-Red-O and the morphological differentiation is shown (the original magnification is ×20). This experiment was repeated three times using cells prepared from different embryos and similar results were obtained. C) Western blot analyses of PPARγ, and C/EBPα in liposarcoma arisen in CombitTA-FUS-DDIT3 mice in the presence (+tet) or in the absence (−tet) of doxycycline. Doxycycline was given at 4 mg/mL for 4 weeks.</p