Mechanistic target of rapamycin (mTOR) regulates self-sustained quiescence, tumor indolence, and late clinical metastasis in a Beclin-1-dependent manner.

Self-sustained quiescence (SSQ) has been characterized as a stable but reversible non-proliferative cellular state that limits the cloning of cultured cancer cells. By developing refined clonogenic assays, we showed here that cancer cells in SSQ can be selected with anticancer agents and that culture at low cell density induced SSQ in pancreas and prostate adenocarcinoma cells. Pre-culture of cells in 3D or their pretreatment with pharmacological inhibitors of mechanistic target of rapamycin (mTOR) synergize with low cell density for induction of SSQ in a Beclin-1-dependent manner. Dissociated pancreatic adenocarcinoma (PAAD) cells rendered defective for SSQ by down-regulating Beclin-1 expression exhibit higher tumor growth rate when injected subcutaneously into mice. Conversely, dissociated PAAD cells in SSQ promote the formation of small indolent tumors that eventually transitioned to a rapid growth phase. Ex vivo clonogenic assays showed that up to 40% of clonogenic cancer cells enzymatically dissociated from resected fast-growing tumors could enter SSQ, suggesting that SSQ could significantly impact the proliferation of cancer cells that are naturally dispersed from tumors. Remarkably, the kinetics of clinical metastatic recurrence in 124 patients with pancreatic adenocarcinoma included in the TGCA-PAAD project could be predicted from Beclin-1 and Cyclin-A2 mRNA levels in their primary tumor, Cyclin A2 mRNA being a marker of both cell proliferation and mTOR complex 1 activity. Overall, our data show that SSQ is likely to promote the late development of clinical metastases and suggest that identifying new agents targeting cancer cells in SSQ could help improve patient survival.

Transient exposure to androgens induces a remarkable self-sustained quiescent state in dispersed prostate cancer cells.

Cellular quiescence is a reversible cell growth arrest that is often assumed to require a persistence of non-permissive external growth conditions for its maintenance. In this work, we showed that androgen could induce a quiescent state that is self-sustained in a cell-autonomous manner through a "hit and run" mechanism in androgen receptor-expressing prostate cancer cells. This phenomenon required the set-up of a sustained redox imbalance and TGFß/BMP signaling that were dependent on culturing cells at low density. At medium cell density, androgens failed to induce such a self-sustained quiescent state, which correlated with a lesser induction of cell redox imbalance and oxidative stress markers like CDKN1A. These effects of androgens could be mimicked by transient overexpression of CDKN1A that triggered its own expression and a sustained SMAD phosphorylation in cells cultured at low cell density. Overall, our data suggest that self-sustained but fully reversible quiescent states might constitute a general response of dispersed cancer cells to stress conditions.

Post-transcriptional gene silencing activity of human GIGYF2.

In mammalian post-transcriptional gene silencing, the Argonaute protein AGO2 indirectly recruits translation inhibitors, deadenylase complexes, and decapping factors to microRNA-targeted mRNAs, thereby repressing mRNA translation and accelerating mRNA decay. However, the exact composition and assembly pathway of the microRNA-induced silencing complex are not completely elucidated. As the GYF domain of human GIGYF2 was shown to bind AGO2 in pulldown experiments, we wondered whether GIGYF2 could be a novel protein component of the microRNA-induced silencing complex. Here we show that full-length GIGYF2 coimmunoprecipitates with AGO2 in human cells, and demonstrate that, upon tethering to a reporter mRNA, GIGYF2 exhibits strong, dose-dependent silencing activity, involving both mRNA destabilization and translational repression.

Oriented Bioconjugation of Unmodified Antibodies to Quantum Dots Capped with Copolymeric Ligands as Versatile Cellular Imaging Tools.

Distinctive optical properties of inorganic quantum dot (QD) nanoparticles promise highly valuable probes for fluorescence-based detection methods, particularly for in vivo diagnostics, cell phenotyping via multiple markers or single molecule tracking. However, despite high hopes, this promise has not been fully realized yet, mainly due to difficulties at producing stable, nontoxic QD bioconjugates of negligible nonspecific binding. Here, a universal platform for antibody binding to QDs is presented that builds upon the controlled functionalization of CdSe/CdS/ZnS nanoparticles capped with a multidentate dithiol/zwitterion copolymer ligand. In a change-of-paradigm approach, thiol groups are concomitantly used as anchoring and bioconjugation units to covalently bind up to 10 protein A molecules per QD while preserving their long-term colloidal stability. Protein A conjugated to QDs then enables the oriented, stoichiometrically controlled immobilization of whole, unmodified antibodies by simple incubation. This QD-protein A immobilization platform displays remarkable antibody functionality retention after binding, usually a compromised property in antibody conjugation to surfaces. Typical QD-protein A-antibody assemblies contain about three fully functional antibodies. Validation experiments show that these nanobioconjugates overcome current limitations since they retain their colloidal stability and antibody functionality over 6 months, exhibit low nonspecific interactions with live cells and have very low toxicity: after 48 h incubation with 1 µM QD bioconjugates, HeLa cells retain more than 80% of their cellular metabolism. Finally, these QD nanobioconjugates possess a high specificity for extra- and intracellular targets in live and fixed cells. The dithiol/zwitterion QD-protein A nanoconjugates have thus a latent potential to become an off-the-shelf tool destined to unresolved biological questions.

SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy.

Metastasis involves the dissemination of single or small clumps of cancer cells through blood or lymphatic vessels and their extravasation into distant organs. Despite the strong regulation of metastases development by a cell dormancy phenomenon, the dormant state of cancer cells remains poorly characterized due to the difficulty of in vivo studies. We have recently shown in vitro that clonogenicity of prostate cancer cells is regulated by a dormancy phenomenon that is strongly induced when cells are cultured both at low cell density and in a slightly hypertonic medium. Here, we characterized by RT-qPCR a genetic expression signature of this dormant state which combines the presence of both stemness and differentiation markers. We showed that both TFGß/BMP signaling and redox imbalance are required for the full induction of this dormancy signature and cell quiescence. Moreover, reconstruction experiments showed that TFGß/BMP signaling and redox imbalance are sufficient to generate a pattern of genetic expression displaying all characteristic features of the dormancy signature. Finally, we observed that low cell density was sufficient to activate TGFß/BMP signaling and to generate a slight redox imbalance thus priming cells for dormancy that can be attained with a co-stimulus like hypertonicity, most likely through an increased redox imbalance. The identification of a dual regulation of dormancy provides a framework for the interpretation of previous reports showing a restricted ability of BMP signaling to regulate cancer cell dormancy in vivo and draws attention on the role of oxidative stress in the metastatic process.

Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes.

Fluorescence imaging of cells and subcellular compartments is an essential tool to investigate biological processes and to evaluate the development and progression of diseases. In particular, protein-protein interactions can be monitored by Förster resonance energy transfer (FRET) between two proximal fluorophores that are attached to specific recognition biomolecules such as antibodies. We investigated the membrane expression of E- and N-cadherins in three different cell lines used as model systems to study epithelial to mesenchymal transition (EMT) and a possible detection of circulating tumour cells (CTCs). EMT is a key process in cancer metastasis, during which epithelial markers (such as E-cadherin) are down-regulated in the primary tumour whereas mesenchymal markers (such as N-cadherin) are up-regulated, leading to enhanced cell motility, intravasation, and appearance of CTCs. Various FRET donor-acceptor pairs and protein recognition strategies were utilized, in which Lumi4-Tb terbium complexes (Tb) and different organic dyes were conjugated to several distinct E- and N-cadherin-specific antibodies. Pulsed excitation of Tb at low repetition rates (100 Hz) and time-gated (TG) imaging of both the Tb-donor and the dye-acceptor photoluminescence (PL) allowed efficient detection of the EMT markers as well as FRET in the case of sufficient donor-acceptor proximity. Efficient FRET was observed only between two E-cadherin-specific antibodies and further experiments indicated that these antibodies recognized the same E-cadherin molecule, suggesting a limited accessibility of cadherins when they are clustered at adherens junctions. The investigated Tb-to-dye FRET systems provided reduced photobleaching compared to the AlexaFluor 488-568 donor-acceptor pair. Our results demonstrate the applicability and advantages of Tb-based TG FRET for efficient and stable imaging of antibody-antibody interactions on different cell lines. They also reveal the limitations of interpreting colocalization on cell membranes in the case of lacking FRET signals.

A dormant state modulated by osmotic pressure controls clonogenicity of prostate cancer cells.

Cell dormancy constitutes a limiting step of the metastatic process by preventing the proliferation of isolated cancer cells disseminated at distant sites from the primary tumor. The study of cancer cell dormancy is severely hampered by the lack of biological samples so that the mechanisms that regulate cell dormancy have not been extensively explored. In this work, we describe the rapid induction in vitro of a dormant state in prostate cancer cells by exposure to a slightly hypertonic growth medium. This quiescence is observed only when cells are seeded at low density and, once established, requires additional stimuli besides osmotic pressure to be reversed. Media conditioned by cells grown at high density can partially prevent or reverse dormancy, a phenomenon which can be reproduced with citric acid. In addition to this role of small metabolites, inactivation of the p53 and smad pathways also counters the entry into dormancy, whereas exposure to activin A induces it to some extent. Thus, this easily inducible dormancy reproduces several features associated with the dormancy of stem cells and cancer cells in vivo.

The RNA-binding protein Unr prevents mouse embryonic stem cells differentiation toward the primitive endoderm lineage.

The maintenance of embryonic stem cells (ESCs) pluripotency depends on key transcription factors, chromatin remodeling proteins, and microRNAs. The roles of RNA-binding proteins are however poorly understood. We report that the cytoplasmic RNA-binding protein Unr prevents the differentiation of ESCs into primitive endoderm (PrE). We show that unr knockout (unr(-/-) ) ESCs spontaneously differentiate into PrE, and that Unr re-expression in unr(-/-) ESCs reverses this phenotype. Nevertheless, unr(-/-) ESCs retain pluripotency, producing differentiated teratomas, and the differentiated unr(-/-) ESCs coexpress the PrE inducer Gata6 and the pluripotency factors Oct4, Nanog, and Sox2. Interestingly, in the differentiated unr(-/-) ESCs, Nanog and Sox2 exhibit a dual nuclear and cytoplasmic localization. This situation, that has never been reported, likely reflects an early differentiation state toward PrE. Finally, we show that Unr destabilizes Gata6 mRNAs and we propose that the post-transcriptional repression of Gata6 expression by Unr contributes to the stabilization of the ESCs pluripotent state.

Mitochondria associate with P-bodies and modulate microRNA-mediated RNA interference.

P-bodies are cytoplasmic granules that are linked to mRNA decay, mRNA storage, and RNA interference (RNAi). They are known to interact with stress granules in stressed cells, and with late endosomes. Here, we report that P-bodies also interact with mitochondria, as previously described for P-body-related granules in germ cells. The interaction is dynamic, as a large majority of P-bodies contacts mitochondria at least once within a 3-min interval, and for about 18 s. This association requires an intact microtubule network. The depletion of P-bodies does not seem to affect mitochondria, nor the mitochondrial activity to be required for their contacts with P-bodies. However, inactivation of mitochondria leads to a strong decrease of miRNA-mediated RNAi efficiency, and to a lesser extent of siRNA-mediated RNAi. The defect occurs during the assembly of active RISC and is associated with a specific delocalization of endogeneous Ago2 from P-bodies. Our study reveals the possible involvement of RNAi defect in pathologies involving mitochondrial deficiencies.

Unravelling the ultrastructure of stress granules and associated P-bodies in human cells.

Stress granules are cytoplasmic ribonucleoprotein granules formed following various stresses that inhibit translation. They are thought to help protecting untranslated mRNAs until stress relief. Stress granules are frequently seen adjacent to P-bodies, which are involved in mRNA degradation and storage. We have previously shown in live cells that stress granule assembly often takes place in the vicinity of pre-existing P-bodies, suggesting that these two compartments are structurally related. Here we provide the first ultrastructural characterization of stress granules in eukaryotic cells by electron microscopy. Stress granules resulting from oxidative stress, heat-shock or protein overexpression are loosely organised fibrillo-granular aggregates of a moderate electron density, whereas P-bodies are denser and fibrillar. By in situ hybridization at the electron microscopic level, we show that stress granules are enriched in poly(A)(+) mRNAs, although these represent a minor fraction of the cellular mRNAs. Finally, we show that, despite close contact with P-bodies, both domains remain structurally distinct and do not interdigitate.

Nucleocytoplasmic traffic of CPEB1 and accumulation in Crm1 nucleolar bodies.

The translational regulator CPEB1 plays a major role in the control of maternal mRNA in oocytes, as well as of subsynaptic mRNAs in neurons. Although mainly cytoplasmic, we found that CPEB1 protein is continuously shuttling between nucleus and cytoplasm. Its export is controlled by two redundant NES motifs dependent on the nuclear export receptor Crm1. In the nucleus, CPEB1 accumulates in a few foci most often associated with nucleoli. These foci are different from previously identified nuclear bodies. They contain Crm1 and were called Crm1 nucleolar bodies (CNoBs). CNoBs depend on RNA polymerase I activity, indicating a role in ribosome biogenesis. However, although they form in the nucleolus, they never migrate to the nuclear envelope, precluding a role as a mediator for ribosome export. They could rather constitute a platform providing factors for ribosome assembly or export. The behavior of CPEB1 in CNoBs raises the possibility that it is involved in ribosome biogenesis.

Translationally repressed mRNA transiently cycles through stress granules during stress.

In mammals, repression of translation during stress is associated with the assembly of stress granules in the cytoplasm, which contain a fraction of arrested mRNA and have been proposed to play a role in their storage. Because physical contacts are seen with GW bodies, which contain the mRNA degradation machinery, stress granules could also target arrested mRNA to degradation. Here we show that contacts between stress granules and GW bodies appear during stress-granule assembly and not after a movement of the two preassembled structures. Despite this close proximity, the GW body proteins, which in some conditions relocalize in stress granules, come from cytosol rather than from adjacent GW bodies. It was previously reported that several proteins actively traffic in and out of stress granules. Here we investigated the behavior of mRNAs. Their residence time in stress granules is brief, on the order of a minute, although stress granules persist over a few hours after stress relief. This short transit reflects rapid return to cytosol, rather than transfer to GW bodies for degradation. Accordingly, most arrested mRNAs are located outside stress granules. Overall, these kinetic data do not support a direct role of stress granules neither as storage site nor as intermediate location before degradation.

[Regulations through small RNAs]. / Les régulations par les petits ARN.

For a long time RNA molecules have been viewed as simple intermediates between DNA and proteins as conveyed by the name "messenger RNA". However, the similarity between RNA and DNA creates multiple opportunities for regulatory interactions between genes and their transcripts. Over the last ten years a large body of studies in different eukaryotes has shown that indeed RNA molecules play major roles in the control of gene expression. The first pathway to be analyzed has been the control of translation and degradation of messenger RNA by RNA interference and the related processes. This has led to the realization that regulatory RNA molecules have two specific features : a size around twenty nucleotides and the fact that at one point they have been double stranded. The field of action of small regulatory RNA is however much broader and also covers several levels of genomic organisation such as chromatin compaction, gene rearrangement and spatial organisation of the nucleus. Thus small regulatory RNA are involved at all levels of gene expression and are central to cellular regulations.

[RNA interference in mammalian cells]. / L'interférence par l'ARN dans les cellules de mammifère.

RNA interference was the first regulation by small RNA to be described in detail. It was initially identified in C. elegans as a sequence-specific post-transcriptional silencing induced by double stranded RNA. There are two main steps in the process, the cleavage of long double stranded RNA molecules into small interfering RNA of about twenty nucleotides and the incorporation of these small molecules into a protein complex to which it confers a sequence specific interaction with RNA substrates. The "classical" RNA interference is associated with the cleavage and the subsequent degradation of the targeted RNA which in its simplest form can be carried out by a single protein (Argonaute 2 in mammals) and a small interfering RNA. The cleavage requires a near perfect complementarity between the substrate and the small guide present in the complex; this sequence specificity and the catalytic nature of the process create an almost ideal tool to silence any gene for which the sequence is known. However several considerations limit the efficacy and the specificity of this process. Foremost is our current inability to restrict the activity of small regulatory RNA to this RNA cleavage pathway which leads to the activation of other cellular regulations some of which have a lower level of sequence specificity than RNA interference. A better understanding of these regulatory pathways will be necessary in order to achieve the specific and efficient silencing that experimentalists dream of.

Cholesterol contributes to the organization of tetraspanin-enriched microdomains and to CD81-dependent infection by malaria sporozoites.

Tetraspanins constitute a family of widely expressed integral membrane proteins that associate extensively with one another and with other membrane proteins to form specific membrane microdomains distinct from conventional lipid rafts. So far, because of the lack of appropriate tools, the functionality of these microdomains has remained largely unknown. Here, using a new monoclonal antibody that only binds to the tetraspanin CD81 associated with other tetraspanins, we show that membrane cholesterol contributes to the organization of tetraspanin microdomains on the surface of live cells. Furthermore, our data demonstrate involvement of host membrane cholesterol during infection by Plasmodium yoelii and Plasmodium falciparum sporozoites, which both depend on host CD81 expression for invasion, but not during CD81-independent infection by Plasmodium berghei sporozoites. Our results unravel a functional link between CD81 and cholesterol during infection by malaria parasites, and illustrate that tetraspanin microdomains constitute a novel type of membrane microdomains that could be used by pathogens for infection.

Heat shock-independent induction of multidrug resistance by heat shock factor 1.

The screening of two different retroviral cDNA expression libraries to select genes that confer constitutive doxorubicin resistance has in both cases resulted in the isolation of the heat shock factor 1 (HSF1) transcription factor. We show that HSF1 induces a multidrug resistance phenotype that occurs in the absence of heat shock or cellular stress and is mediated at least in part through the constitutive activation of the multidrug resistance gene 1 (MDR-1). This drug resistance phenotype does not correlate with an increased expression of heat shock-responsive genes (heat shock protein genes, or HSPs). In addition, HSF1 mutants lacking HSP gene activation are also capable of conferring multidrug resistance, and only hypophosphorylated HSF1 complexes accumulate in transduced cells. Our results indicate that HSF1 can activate MDR-1 expression in a stress-independent manner that differs from the canonical heat shock-activated mechanism involved in HSP induction. We further provide evidence that the induction of MDR-1 expression occurs at a posttranscriptional level, revealing a novel undocumented role for hypophosphorylated HSF1 in posttranscriptional gene regulation.

High frequency trans-splicing in a cell line producing spliced and polyadenylated RNA polymerase I transcripts from an rDNA-myc chimeric gene.

The 2G1MycP2Tu1 cell line was obtained following transfection of human colon carcinoma cells from the SW613-S cell line with a plasmid carrying a genomic copy of the human MYC gene. 2G1MycP2Tu1 cells produce MYC mRNAs and proteins of abnormal size. In order to analyze the structure of these abnormal products, a cDNA library constructed using RNA isolated from these cells was screened with a MYC probe. Fifty clones were studied by DNA sequencing. The results indicated that a truncated copy of the MYC gene had integrated into an rDNA transcription unit in 2G1MycP2Tu1 cells. This was confirmed by northern blot analysis, PCR amplification on genomic DNA and fluorescent in situ hybridization (FISH) experiments on metaphase chromosomes. 2G1MycP2Tu1 cells produce hybrid rRNA-MYC RNA molecules that are polyadenylated and processed by splicing reactions involving natural and cryptic splice sites. These transcripts are synthesized by RNA polymerase I, as confirmed by actinomycin D sensitivity experiments, suggesting that 3' end processing and splicing are uncoupled from transcription in this case. 2G1MycP2Tu1 cells also produce another type of chimeric mRNAs consisting of correctly spliced exons 2 and 3 of the MYC gene fused to one or more extraneous 5' exons by proper splicing to the acceptor sites of MYC exon 2. These foreign exons belong to 33 different genes, which are located on 14 different chromosomes. These observations and the results of FISH and Southern blotting experiments lead us to conclude that trans-splicing events occur at high frequency in 2G1MycP2Tu1 cells.

[RNA interference: towards a functional genomics in mammalian cells?]. / L'interférence par l'ARN: vers une génomique fonctionnelle chez les mammifères ?

The discovery of the induction of RNA degradation by double stranded RNA in C. elegans, "RNA interference", makes it possible to envision systematic studies of gene function in mammalian cells. Indeed, in spite of the existence in mammals of the interferon response to double stranded RNA, the introduction of small interfering RNA can induce a sequence specific inhibition of gene expression either through RNA degradation or by blocking translation. Although the inhibition is transient and usually not complete, strategies have been developed to achieve long term gene silencing. The issue of target specificity is still not completely clear and will probably constitute a limitation of this approach. However, because of the unprecedented ease with which large scale screens can be performed, RNA interference has already established itself as the tool of choice to initiate functional genomics in mammalian cells.

Coupled amplification and degradation of exogenous RNA injected in amphibian oocytes.

The early development of amphibians takes place in the absence of significant transcription and is controlled at the post-transcriptional level. We have reported that in vitro synthesized transcripts injected into axolotl fertilized eggs or oocytes were not continuously degraded as their abundance apparently fluctuated over time, with detected amounts sometimes higher than initial injected amounts. To further characterize this phenomenon, we have co-injected RNA chain terminators to prevent RNA synthesis. This led to the suppression of fluctuations and to a regular decrease in the amount of transcripts that appeared to be more stable in the presence of inhibitors. These observations indicate a coupling between RNA synthesis and an accelerated degradation. Throughout the time course, cRNA molecules could be detected, and their abundance increased in the early phase of the kinetics, supporting the implication of an RNA-dependent RNA polymerase in an asymmetric amplification process. Finally, when the fate of the injected transcripts was investigated in individual oocytes, we observed an absolute increase in abundance in some but not all oocytes, supporting the existence of a limiting step in the initiation of the RNA amplification stochastic process.

Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus.

Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (-/+) or both (-/-) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr(+/+), unr(+/-), and unr(-/-) cell lines. Translation directed by the HRV IRES was severely impaired in unr(-/-) cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr(-/-) cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.