Hypoxia Induces VEGF-C Expression in Metastatic Tumor Cells via a HIF-1α-Independent Translation-Mediated Mechanism

SummaryVarious tumors metastasize via lymph vessels and lymph nodes to distant organs. Even though tumors are hypoxic, the mechanisms of how hypoxia regulates lymphangiogenesis remain poorly characterized. Here, we show that hypoxia reduced vascular endothelial growth factor C (VEGF-C) transcription and cap-dependent translation via the upregulation of hypophosphorylated 4E-binding protein 1 (4E-BP1). However, initiation of VEGF-C translation was induced by hypoxia through an internal ribosome entry site (IRES)-dependent mechanism. IRES-dependent VEGF-C translation was independent of hypoxia-inducible factor 1α (HIF-1α) signaling. Notably, the VEGF-C IRES activity was higher in metastasizing tumor cells in lymph nodes than in primary tumors, most likely because lymph vessels in these lymph nodes were severely hypoxic. Overall, this transcription-independent but translation-dependent upregulation of VEGF-C in hypoxia stimulates lymphangiogenesis in tumors and lymph nodes and may contribute to lymphatic metastasis

Assessment of alternatives to environmental toxic formalin for DNA conservation in biological specimens

International audienceOne essential step of museum and clinical specimen preservation is immersion in a fixative fluid to prevent degradation. Formalin is the most largely used fixative, but its benefit is balanced with its toxic and carcinogenic status. Moreover, because formalin-fixation impairs nucleic acids recovery and quality, current museum wet collections and formalin- fixed, paraffin-embedded clinical samples do not represent optimal tanks of molecular information. Our study has been developed to compare formalin to two alternative fixatives (RCL2® and ethanol) in a context of molecular exploitation. Based on a unique protocol, we created mammalian fixed collections, simulated the impact of time on preservationusing an artificial ageing treatment and followed the evolution of specimens’ DNA quality. DNA extraction yield, purity, visual integrity and qualitative and quantitative ability to amplify the Cox1 gene were assessed. Our results show that both RCL2 and ethanol exhibit better performances than formalin. They do not impair DNA extraction yield, and more importantly, DNA alteration is delayed over the preservation step. The use of RCL2 or ethanol as fixative in biological collections may insure a better exploitation of the genetic resources they propose

Rotavirus NSP3 Is a Translational Surrogate of the Poly(A) Binding Protein-Poly(A) Complex

Through its interaction with the 5' translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5'-capped and 3'-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3' GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3' end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCE: To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery

Reversing pancreatic cancer metastasis through pharmacological targeting of pancreatic stellate cell's dialog with M2 macrophages

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Reversing pancreatic cancer immunosuppression through pharmacological targeting of activated pancreatic stellate cells

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Gene Expression Signature Associated with Clinical Outcome in ALK-Positive Anaplastic Large Cell Lymphoma

International audienceAnaplastic large cell lymphomas associated with ALK translocation have a good outcome after CHOP treatment; however, the 2-year relapse rate remains at 30%. Microarray gene-expression profiling of 48 samples obtained at diagnosis was used to identify 47 genes that were differentially expressed between patients with early relapse/progression and no relapse. In the relapsing group, the most significant overrepresented genes were related to the regulation of the immune response and T-cell activation while those in the non-relapsing group were involved in the extracellular matrix. Fluidigm technology gave concordant results for 29 genes, of which FN1, FAM179A, and SLC40A1 had the strongest predictive power after logistic regression and two classification algorithms. In parallel with 39 samples, we used a Kallisto/Sleuth pipeline to analyze RNA sequencing data and identified 20 genes common to the 28 genes validated by Fluidigm technology—notably, the FAM179A and FN1 genes. Interestingly, FN1 also belongs to the gene signature predicting longer survival in diffuse large B-cell lymphomas treated with CHOP. Thus, our molecular signatures indicate that the FN1 gene, a matrix key regulator, might also be involved in the prognosis and the therapeutic response in anaplastic lymphomas

Tracking a refined eIF4E-binding motif reveals Angel1 as a new partner of eIF4E.

International audienceThe initiation factor 4E (eIF4E) is implicated in most of the crucial steps of the mRNA life cycle and is recognized as a pivotal protein in gene regulation. Many of these roles are mediated by its interaction with specific proteins generally known as eIF4E-interacting partners (4E-IPs), such as eIF4G and 4E-BP. To screen for new 4E-IPs, we developed a novel approach based on structural, in silico and biochemical analyses. We identified the protein Angel1, a member of the CCR4 deadenylase family. Immunoprecipitation experiments provided evidence that Angel1 is able to interact in vitro and in vivo with eIF4E. Point mutation variants of Angel1 demonstrated that the interaction of Angel1 with eIF4E is mediated through a consensus eIF4E-binding motif. Immunofluorescence and cell fractionation experiments showed that Angel1 is confined to the endoplasmic reticulum and Golgi apparatus, where it partially co-localizes with eIF4E and eIF4G, but not with 4E-BP. Furthermore, manipulating Angel1 levels in living cells had no effect on global translation rates, suggesting that the protein has a more specific function. Taken together, our results illustrate that we developed a powerful method for identifying new eIF4E partners and open new perspectives for understanding eIF4E-specific regulation

Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome

The 4G family of eukaryotic mRNA translation initiation factors is composed of three members (eIF4GI, eIF4GII, and DAP5). Their specific roles in translation initiation are under intense investigations, but how their respective intracellular amounts are controlled remains poorly understood. Here we show that eIF4GI and eIF4GII exhibit much shorter half-lives than that of DAP5. Both eIF4GI and eIF4GII proteins, but not DAP5, contain computer-predicted PEST motifs in their N-termini conserved across the animal kingdom. They are both sensitive to degradation by the proteasome. Under normal conditions, eIF4GI and eIF4GII are protected from proteasomal destruction through binding to the detoxifying enzyme NQO1 [NAD(P)H:quinone oxidoreductase]. However, when cells are exposed to oxidative stress both eIF4GI and eIF4GII, but not DAP5, are degraded by the proteasome in an N-terminal-dependent manner, and cell viability is more compromised upon silencing of DAP5. These findings indicate that the three eIF4G proteins are differentially regulated by the proteasome and that persistent DAP5 plays a role in cell survival upon oxidative stress