Predominant role of Tax sumoylation in Tax-induced NF-kB activation in T cells

International audiencen.

In Vivo Detection of Succinate by Magnetic Resonance Spectroscopy as a Hallmark of SDHx Mutations in Paraganglioma

International audiencePurpose: Germline mutations in genes encoding mitochon-drial succinate dehydrogenase (SDH) are found in patients with paragangliomas, pheochromocytomas, gastrointestinal stromal tumors, and renal cancers. SDH inactivation leads to a massive accumulation of succinate, acting as an oncometabolite and which levels, assessed on surgically resected tissue are a highly specific biomarker of SDHx-mutated tumors. The aim of this study was to address the feasibility of detecting succinate in vivo by magnetic resonance spectroscopy. Experimental Design: A pulsed proton magnetic resonance spectroscopy (1 H-MRS) sequence was developed, optimized, and applied to image nude mice grafted with Sdhb À/À or wild-type chromaffin cells. The method was then applied to patients with paraganglioma carrying (n ¼ 5) or not (n ¼ 4) an SDHx gene mutation. Following surgery, succinate was measured using gas chromatography/mass spectrometry, and SDH protein expression was assessed by immunohistochemistry in resected tumors. Results: A succinate peak was observed at 2.44 ppm by 1 H-MRS in all Sdhb À/À-derived tumors in mice and in all paragangliomas of patients carrying an SDHx gene mutation, but neither in wild-type mouse tumors nor in patients exempt of SDHx mutation. In one patient, 1 H-MRS results led to the identification of an unsus-pected SDHA gene mutation. In another case, it helped define the pathogenicity of a variant of unknown significance in the SDHB gene. Conclusions: Detection of succinate by 1 H-MRS is a highly specific and sensitive hallmark of SDHx mutations. This non-invasive approach is a simple and robust method allowing in vivo detection of the major biomarker of SDHx-mutated tumors. Clin Cancer Res; 22(5); 1120–9. Ó2015 AACR

The Warburg Effect Is Genetically Determined in Inherited Pheochromocytomas

The Warburg effect describes how cancer cells down-regulate their aerobic respiration and preferentially use glycolysis to generate energy. To evaluate the link between hypoxia and Warburg effect, we studied mitochondrial electron transport, angiogenesis and glycolysis in pheochromocytomas induced by germ-line mutations in VHL, RET, NF1 and SDH genes. SDH and VHL gene mutations have been shown to lead to the activation of hypoxic response, even in normoxic conditions, a process now referred to as pseudohypoxia. We observed a decrease in electron transport protein expression and activity, associated with increased angiogenesis in SDH- and VHL-related, pseudohypoxic tumors, while stimulation of glycolysis was solely observed in VHL tumors. Moreover, microarray analyses revealed that expression of genes involved in these metabolic pathways is an efficient tool for classification of pheochromocytomas in accordance with the predisposition gene mutated. Our data suggest an unexpected association between pseudohypoxia and loss of p53, which leads to a distinct Warburg effect in VHL-related pheochromocytomas

Survey of Human Genes of Retroviral Origin: Identification and Transcriptome of the Genes with Coding Capacity for Complete Envelope Proteins

Sequences of retroviral origin occupy approximately 8% of the human genome. Most of these “retroviral” genes have lost their coding capacities since their entry into our ancestral genome millions of years ago, but some reading frames have remained open, suggesting positive selection. The complete sequencing of the human genome allowed a systematic search for retroviral envelope genes containing an open reading frame and resulted in the identification of 16 genes that we have characterized. We further showed, by quantitative reverse transcriptase PCR using specifically devised primers which discriminate between coding and noncoding elements, that all 16 genes are expressed in at least some healthy human tissues, albeit at highly different levels. All envelope genes disclose significant expression in the testis, three of them have a very high level of expression in the placenta, and a fourth is expressed in the thyroid. Besides their primary role as key molecules for viral entry, the envelope genes of retroviruses can induce cell-cell fusion, elicit immunosuppressive effects, and even protect against infection, and as such, endogenous retroviral envelope proteins have been tentatively identified in several reports as being involved in both normal and pathological processes. The present study provides a comprehensive survey of candidate genes and tools for a precise evaluation of their involvement in these processes

Succinate detection using in vivo 1H-MR spectroscopy identifies germline and somatic SDHx mutations in paragangliomas

International audiencePurpose: Germline mutations in genes encoding succinate dehydrogenase (SDH) are frequent in patients with pheochromocytoma and paraganglioma (PPGL). They lead to SDH inactivation, mediating a massive accumulation of succinate, which constitutes a highly specific biomarker of SDHx-mutated tumors when measured in vitro. In a recent pilot study, we showed that magnetic resonance spectroscopy (1H-MRS) optimized for succinate detection (SUCCES) could detect succinate in vivo in both allografted mouse models and PPGL patients. The objective of this study was to prospectively assess the diagnostic performances of 1H-MRS SUCCES sequence for the identification of SDH deficiency in PPGL patients.Methods: Forty-nine patients presenting with 50 PPGLs were prospectively enrolled in our referral center for 1H-MRS SUCCES. Two observers blinded to the clinical characteristics and genetic status analyzed the presence of a succinate peak and confronted the results to a composite gold standard combining PPGL genetic testing and/or in vitro protein analyses in the tumor.Results: A succinate peak was observed in 20 tumors, all of which had proven SDH deficiency using the gold standard (17 patients with germline SDHx mutations, 2 with a somatic SDHD mutation, and 1 with negative SDHB IHC and SDH loss of function). A false negative result was observed in 3 tumors. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 1H-MRS SUCCES were respectively 87%, 100%, 100%, 90%, and 94%.Conclusions: Detection of succinate using 1H-MRS is a highly specific and sensitive hallmark of SDH-deficiency in PPGLs

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription

<div><p>The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5’LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-<i>N</i>-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: <i>O</i>-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and <i>O</i>-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.</p></div

Effect of OGA inhibition on LTR activation and CREB O-GlcNAcylation in Tax expressing cells.

<p>(<b>A, B</b>) Effect of OGA inhibition on Tax-mediated LTR transactivation in (<b>A</b>) C8166 T cells or (<b>B</b>) HEK-293T cells. Left panels: Cells were transfected with the U3R-LTR-luciferase and pRL-TK plasmid and in the case of HEK-293T cells, the Tax plasmid, and were incubated or not with the OGA inhibitor Thiamet G for 24h. Luciferase production was then measured using the dual luciferase assay. Results are means ± SEM of 4 independent experiments performed in duplicates. Right panels: level of O-GlcNAcylation and Tax expression in each condition. (<b>C</b>) Effect of Tax on CREB O-GlcNAcylation in HEK-293T cells. HEK-293T cells were transfected with either the control or Tax plasmid and treated or not with the OGA inhibitor Thiamet G. Cell extracts were prepared two days post-transfection. Left panel: total proteins (lysates) or WGA-bound proteins (WGA) were separated by SDS-PAGE and blotted with either an anti-O-GlcNAc or an anti-CREB antibody. Tax expression in cell lysates is also shown. Middle and right panel: Total O-GlcNAc signal (middle panel) or WGA/lysate ratio for CREB signal (right panel) in two independents experiments. (<b>D</b>) Effect of Tax on CREB O-GlcNAcylation in TL-om1 T cells. TL-om1 T cells were transfected with either the control or Tax plasmid. Left panel: Total proteins (lysates) or WGA-bound proteins (WGA) were separated by SDS-PAGE and blotted with either an anti-O-GlcNAc or anti-CREB antibody. Tax expression in cell lysates is also shown. Middle and right panels: Total O-GlcNAc signal (middle panel) or WGA/lysate ratio for CREB signal (right panel) in two independents experiments.</p

Effect of Tax on OGA activity and O-GlcNAcylation.

<p>(<b>A</b>) Total OGA activity was measured at two different time-points in extracts from TL-om1 T cells transfected with either the control or Tax plasmid. Results are means ± SEM of 4 independent experiments. The insert shows the expression of OGA, OGT and Tax in the cell extracts. (<b>B</b>) The BRET O-GlcNAc-biosensor is composed of Rluc8 fused to the fimbrial adhesin lectin domain GafD, a known OGT substrate peptide derived from casein kinase II placed between two flexible linkers (GGSGG), followed by the Venus variant of the yellow fluorescent protein (Adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006518#ppat.1006518.ref027" target="_blank">27</a>]). (<b>C</b>) TL-om1 cells were transfected with the O-GlcNAc BRET biosensor and either the control or Tax plasmid. BRET experiments were performed 24h after transfection. BRET measurements were started 5 min after the addition of coelenterazine. Left panel: typical BRET experiment. Middle panel: mean delta BRET (increased BRET signal induced by Tax expression). Results are means ± SEM of 3 independent experiments. Right panel: level of O-GlcNAc proteins evaluated by western-blotting with an anti-O-GlcNAc antibody. (<b>D</b>) Total OGA activity was measured in extracts from HEK-293T cells transfected with either the control or Tax plasmid. Cells were extracted and OGA activity was measured at 30 min. Results are means ± SEM of 3 independent experiments. The insert shows the expression of OGA, OGT and Tax in the cell extracts. (<b>E</b>) HEK-293T cells were transfected with the O-GlcNAc BRET biosensor and either the control or Tax plasmid. BRET experiments were performed 48h after transfection. BRET measurements were started 5 min after the addition of coelenterazine. Left panel: typical BRET experiment. Middle panel: mean delta BRET (increased BRET signal induced by Tax expression). Results are means ± SEM of 3 independent experiments. Right panel: level of O-GlcNAc proteins evaluated by western-blotting with an anti-O-GlcNAc antibody. Statistical significance was calculated using the student’s t test (**: p<0.01; ***: p<0.001; ****: p<0.0001).</p

Mutation of CREB Serine 40 inhibits both Tax-mediated increase in CREB O-GlcNAcylation and LTR activation.

<p>(<b>A</b>) HEK-293T cells were transfected with the control (- Tax) or Tax plasmid (+ Tax) and either the wild-type (wt) or S40A mutant YFP-CREB construct and lysed 48h post-transfection. The amounts of wt and S40A YFP-CREB in the lysate were evaluated by measuring fluorescence emission at 530 nm after excitation at 480 nm. After normalization for fluorescence, O-GlcNAcylated proteins were captured on wheat germ lectin agarose (WGA) beads. Total proteins (Lysates) or WGA-bound proteins (WGA) were separated by SDS-PAGE and blotted with an anti-CREB antibody. Tax expression in cell lysates is also shown. <b>(B)</b> HEK-293T cells were transfected and cell extracts were normalized as in (A), but wt and mutant YFP-CREB proteins were immunoprecipitated using an anti-GFP antibody. The level of O-GlcNAcylation of wt and S40A YFP-CREB was evaluated using an anti-O-GlcNAc antibody. The membranes were then striped and reprobed with an anti-CREB antibody. (<b>C</b>) Effect of wt or S40A mutant YFP-CREB on Tax-mediated LTR activation. Left panel: HEK-293T cells were transfected with U3R-LTR-luciferase and pRL-TK plasmids along with Tax and either a control plasmid or the plasmid coding for wt or S40A YFP-CREB. Luciferase production was then measured two days post-transfection using the dual luciferase assay. Results are from 4 independent experiments performed in duplicates. Statistical significance was analyzed using the Tukey’s multiple comparison test (*: p<0.05; ***: p<0.001; ns: not significant). Right panel: levels of Tax and CREB expression in the transactivation experiments.</p