Céline Keime

mTOR inhibition reverses acquired endocrine therapy resistance of breast cancer cells at the cell proliferation and gene-expression levels.

Activation of the Akt/mammalian target of rapamycin (mTOR) pathway has been shown to be associated with resistance to endocrine therapy in estrogen receptor alpha (ERα)‐positive breast cancer patients. Utmost importance is attached to strategies aimed at overcoming treatment resistance. In this context, this work aimed to investigate whether, in breast cancer cells, the use of an mTOR inhibitor would be sufficient to reverse the resistance acquired after exposure to endocrine therapy. The ERα‐positive human breast adenocarcinoma derived‐MCF‐7 cells used in this study have acquired both cross‐resistance to hydroxy‐tamoxifen (OH‐Tam) and to fulvestrant and strong activation of the Akt/mTOR pathway. Cell proliferation tests in control cells demonstrated that the mTOR inhibitor rapamycin enhanced cell sensitivity to endocrine therapy when combined to OH‐Tam or to fulvestrant. In resistant cells, rapamycin used alone greatly inhibited cell proliferation and reversed resistance to endocrine therapy by blocking the agonist‐like activity of OH‐Tam on cell proliferation and bypassing fulvestrant resistance. Reversion of resistance by rapamycin was associated with increased ERα protein expression levels and modification of the balance of phospho‐ser167 ERα/total ERα ratio. Pangenomic DNA array experiments demonstrated that the cotreatment of resistant cells with fulvestrant and rapamycin allowed the restoration of 40% of the fulvestrant gene‐expression signature. Taken together, data presented herein strongly support the idea that mTOR inhibitor might be one of the promising therapeutic approaches for patients with ERα‐positive endocrine therapy‐resistant breast cancers. (Cancer Sci 2008; 99: 1992–2003)

SAGE analysis of mosquito salivary gland transcriptomes during Plasmodium invasion.

Invasion of the vector salivary glands by Plasmodium is a critical step for malaria transmission. To describe salivary gland cellular responses to sporozoite invasion, we have undertaken the analysis of Anopheles gambiae salivary gland transcriptome using Serial Analysis of Gene Expression (SAGE). Statistical analysis of the more than 160u2003000 sequenced tags generated from four libraries, two from glands infected by Plasmodium berghei, two from glands of controls, revealed that at least 57 Anopheles genes are differentially expressed in infected salivary glands. Among the 37 immune‐related genes identified by SAGE tags, four (Defensin1, GNBP, Serpin6 and Cecropin2) were found to be upregulated during salivary gland invasion, while five genes encoding small secreted proteins display induction patterns strongly reminiscent of that of Cecropin2. Invasion by Plasmodium has also an impact on the expression of genes involved in transport, lipid and energy metabolism, suggesting that the sporozoite may exploit the metabolism of its host. In contrast, protein composition of saliva is predicted to be only slightly modified after infection. This study, which is the first transcriptome analysis of the salivary gland response to Plasmodium infection, provides a basis for a better understanding of Plasmodium/Anopheles salivary gland interactions.

A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome

BackgroundOpen transcriptome analysis methods allow to study gene expression without a priori knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for open transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered.ResultsIn order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method.ConclusionWe found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method.

Identitag, a relational database for SAGE tag identification and interspecies comparison of SAGE libraries

BackgroundSerial Analysis of Gene Expression (SAGE) is a method of large-scale gene expression analysis that has the potential to generate the full list of mRNAs present within a cell population at a given time and their frequency. An essential step in SAGE library analysis is the unambiguous assignment of each 14 bp tag to the transcript from which it was derived. This process, called tag-to-gene mapping, represents a step that has to be improved in the analysis of SAGE libraries. Indeed, the existing web sites providing correspondence between tags and transcripts do not concern all species for which numerous EST and cDNA have already been sequenced.ResultsThis is the reason why we designed and implemented a freely available tool called Identitag for tag identification that can be used in any species for which transcript sequences are available. Identitag is based on a relational database structure in order to allow rapid and easy storage and updating of data and, most importantly, in order to be able to precisely define identification parameters. This structure can be seen like three interconnected modules : the first one stores virtual tags extracted from a given list of transcript sequences, the second stores experimental tags observed in SAGE experiments, and the third allows the annotation of the transcript sequences used for virtual tag extraction. It therefore connects an observed tag to a virtual tag and to the sequence it comes from, and then to its functional annotation when available. Databases made from different species can be connected according to orthology relationship thus allowing the comparison of SAGE libraries between species. We successfully used Identitag to identify tags from our chicken SAGE libraries and for chicken to human SAGE tags interspecies comparison. Identitag sources are freely available on http://pbil.univ-lyon1.fr/software/identitag/ web site.ConclusionsIdentitag is a flexible and powerful tool for tag identification in any single species and for interspecies comparison of SAGE libraries. It opens the way to comparative transcriptomic analysis, an emerging branch of biology.

Unexpected observations after mapping LongSAGE tags to the human genome

BackgroundSAGE has been used widely to study the expression of known transcripts, but much less to annotate new transcribed regions. LongSAGE produces tags that are sufficiently long to be reliably mapped to a whole-genome sequence. Here we used this property to study the position of human LongSAGE tags obtained from all public libraries. We focused mainly on tags that do not map to known transcripts.ResultsUsing a published error rate in SAGE libraries, we first removed the tags likely to result from sequencing errors. We then observed that an unexpectedly large number of the remaining tags still did not match the genome sequence. Some of these correspond to parts of human mRNAs, such as polyA tails, junctions between two exons and polymorphic regions of transcripts. Another non-negligible proportion can be attributed to contamination by murine transcripts and to residual sequencing errors. After filtering out our data with these screens to ensure that our dataset is highly reliable, we studied the tags that map once to the genome. 31% of these tags correspond to unannotated transcripts. The others map to known transcribed regions, but many of them (nearly half) are located either in antisense or in new variants of these known transcripts.ConclusionWe performed a comprehensive study of all publicly available human LongSAGE tags, and carefully verified the reliability of these data. We found the potential origin of many tags that did not match the human genome sequence. The properties of the remaining tags imply that the level of sequencing error may have been under-estimated. The frequency of tags matching once the genome sequence but not in an annotated exon suggests that the human transcriptome is much more complex than shown by the current human genome annotations, with many new splicing variants and antisense transcripts. SAGE data is appropriate to map new transcripts to the genome, as demonstrated by the high rate of cross-validation of the corresponding tags using other methods.

Global transcription analysis of immature avian erythrocytic progenitors: from self-renewal to differentiation.

The molecular mechanisms regulating the cell fate decision between self-renewal and differentiation/apoptosis in stem and progenitor cells are poorly understood. Here, we report the first comprehensive identification of genes potentially involved in the switch from self-renewal toward differentiation of primary, non-immortalized erythroid avian progenitor cells (T2EC cells). We used the Serial Analysis of Gene Expression (SAGE) technique in order to identify and quantify the genome fraction functionally active in a self-renewing versus a differentiating cell population. We generated two SAGE libraries and sequenced a total of 37u2009589 tags, thereby obtaining the first transcriptional profile characterization of a chicken cell. Tag identification was performed using a new relational database (Identitag) developed in the laboratory, which allowed a highly satisfactory level of identification. Among 123 differentially expressed genes, 11 were investigated further and for nine of them the differential expression was subsequently confirmed by real-time PCR. The comparison of tag abundance between the two libraries revealed that only a small fraction of transcripts was differentially expressed. The analysis of their functions argue against a prominent role for a master switch in T2EC cells decision-making, but are in favor of a critical role for coordinated small variations in a relatively small number of genes that can lead to essential cellular identity changes.

SQUAT: A web tool to mine human, murine and avian SAGE data

BackgroundThere is an increasing need in transcriptome research for gene expression data and pattern warehouses. It is of importance to integrate in these warehouses both raw transcriptomic data, as well as some properties encoded in these data, like local patterns.DescriptionWe have developed an application called SQUAT (SAGE Querying and Analysis Tools) which is available at: http://bsmc.insa-lyon.fr/squat/. This database gives access to both raw SAGE data and patterns mined from these data, for three species (human, mouse and chicken). This database allows to make simple queries like In which biological situations is my favorite gene expressed? as well as much more complex queries like: ≪what are the genes that are frequently co-over-expressed with my gene of interest in given biological situations?≫. Connections with external web databases enrich biological interpretations, and enable sophisticated queries. To illustrate the power of SQUAT, we show and analyze the results of three different queries, one of which led to a biological hypothesis that was experimentally validated.ConclusionSQUAT is a user-friendly information retrieval platform, which aims at bringing some of the state-of-the-art mining tools to biologists.

Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays

BackgroundRecent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available.ResultsWe show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested.ConclusionsMAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions.

Selective upregulation of lipid metabolism in skeletal muscle of foraging juvenile king penguins: an integrative study

The passage from shore to marine life of juvenile penguins represents a major energetic challenge to fuel intense and prolonged demands for thermoregulation and locomotion. Some functional changes developed at this crucial step were investigated by comparing pre-fledging king penguins with sea-acclimatized (SA) juveniles (Aptenodytes patagonicus). Transcriptomic analysis of pectoralis muscle biopsies revealed that most genes encoding proteins involved in lipid transport or catabolism were upregulated, while genes involved in carbohydrate metabolism were mostly downregulated in SA birds. Determination of muscle enzymatic activities showed no changes in enzymes involved in the glycolytic pathway, but increased 3-hydroxyacyl-CoA dehydrogenase, an enzyme of the β-oxidation pathway. The respiratory rates of isolated muscle mitochondria were much higher with a substrate arising from lipid metabolism (palmitoyl-l-carnitine) in SA juveniles than in terrestrial controls, while no difference emerged with a substrate arising from carbohydrate metabolism (pyruvate). In vivo, perfusion of a lipid emulsion induced a fourfold larger thermogenic effect in SA than in control juveniles. The present integrative study shows that fuel selection towards lipid oxidation characterizes penguin acclimatization to marine life. Such acclimatization may involve thyroid hormones through their nuclear beta receptor and nuclear coactivators.

Sensitivity and gene expression profile of fresh human acute myeloid leukemia cells exposed ex vivo to AS602868

PurposeThe need for new treatment options for acute myeloid leukemia (AML) is increasing. AS602868 is a novel investigational drug with reported activity on AML cells.MethodsWe studied gene expression profiles in AML blasts exposed to AS602868 in order to better understand its mechanism of action. We analyzed the in vitro cytotoxicity of AS602868 alone or in combination with daunorubicin, etoposide or cytarabine. To document AS602868-induced IKK2 inhibition in fresh AML cells, a flow cytometry analysis of IκB was performed. Finally, the effect of AS602868 on gene expression in fresh AML cells was analyzed.ResultsThe results show that AML cells are globally as sensitive to AS602868 as they are to cytarabine, with large interindividual variations. Combinations with conventional antileukemic agents showed enhanced cytotoxic activity in subsets of patients. IKK2 appeared to be effectively inhibited by 100xa0μM AS602868 in fresh leukemic cells. Gene expression profiling and gene ontology analyses identified several groups of genes induced/inhibited by exposure to AS602868 and/or exhibiting a correlation with sensitivity to this agent in vitro. Of note, the expression of several genes related to immune function was found to be significantly altered after exposure to AS602868.ConclusionThese data suggest that AS602868 is cytotoxic against fresh human AML blasts and provide insights regarding the mechanisms of cytotoxicity.