S-nitrosylation homeostasis in plants: key enzymes and regulatory pathways

The nitrosylated form of glutathione (GSNO) has been acknowledged to be the most important nitrosylating agent of the plant cell, and the tuning of its intracellular concentration is of pivotal importance for photosynthetic life. During my time as a PhD student, I focused my attention on the enzymatic systems involved in the degradation of GSNO. Hence, we decided to study the structural and catalytic features of alcohol dehydrogenases (GSNOR and ADH1) from the model land plant Arabidopsis thaliana (At). These enzymes displayed a very similar 3D structure except for their active site which might explain the extreme catalytic specialization of the two enzymes. They share NAD(H) as a cofactor, but only AtGSNOR was able to catalyze the reduction of GSNO whilst being ineffective in oxidizing ethanol. Moreover, our study on the enzyme from the unicellular green alga Chlamydomonas reinhardtii (Cr) revealed how this S-nitrosoglutathione reductase (GSNOR) specifically use NADH to catalyze GSNO reduction and how its activity responds to thiol-based post-translational modifications. Contextually, the presence of NADPH-dependent GSNO-degrading systems in algal protein extract was highlighted and resulted to be relatively efficient in this model organism. This activity could be ascribed to several proteins whose contribution has not been defined yet. Intriguingly, protein extract from GSNOR null mutants of Arabidopsis displayed an increased NADPH-dependent ability to degrade GSNO and our quantitative proteome profiling on the gsnor mutant revealed the overexpression of two class 4 aldo-keto reductases (AKR), specifically AtAKR4C8 and AtAKR4C9. Later, all four class 4 AKRs showed to possess a NADPH-dependent GSNO-degrading activity. Finally, we initiated a preliminary analysis to determine the kinetic parameters of several plant proteins, including GSNOR, AKR4Cs, and thioredoxins. These data suggested GSNOR to be the most effective enzyme in catalyzing GSNO reduction because of its extremely high catalytic proficiency compared to NADPH-dependent systems.

Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations.

The present study investigated promoter hypermethylation of TP53 regulatory pathways providing a potential link between epigenetic changes and mitochondrial DNA (mtDNA) alterations in breast cancer patients lacking a TP53 mutation. The possibility of using the cancer-specific alterations in serum samples as a blood-based test was also explored. Triple-matched samples (cancerous tissues, matched adjacent normal tissues and serum samples) from breast cancer patients were screened for TP53 mutations, and the promoter methylation profile of P14(ARF), MDM2, TP53 and PTEN genes was analyzed as well as mtDNA alterations, including D-loop mutations and mtDNA content. In the studied cohort, no mutation was found in TP53 (DNA-binding domain). Comparison of P14(ARF) and PTEN methylation patterns showed significant hypermethylation levels in tumor tissues (P < 0.05 and <0.01, respectively) whereas the TP53 tumor suppressor gene was not hypermethylated (P < 0.511). The proportion of PTEN methylation was significantly higher in serum than in the normal tissues and it has a significant correlation to tumor tissues (P < 0.05). mtDNA analysis revealed 36.36% somatic and 90.91% germline mutations in the D-loop region and also significant mtDNA depletion in tumor tissues (P < 0.01). In addition, the mtDNA content in matched serum was significantly lower than in the normal tissues (P < 0.05). These data can provide an insight into the management of a therapeutic approach based on the reversal of epigenetic silencing of the crucial genes involved in regulatory pathways of the tumor suppressor TP53. Additionally, release of significant aberrant methylated PTEN in matched serum samples might represent a promising biomarker for breast cancer.

“Reconstruction of Gene Regulatory Network from Expression Profile of Plasma RNA Data of Colorectal Cancer Patients using Soft Computing Techniques”

Microarray data analysis is one of data mining tool which is used to extract meaningful information hidden in biological data. One of the major focuses on microarray data analysis is the reconstruction of gene regulatory network that may be used to provide a broader understanding on the functioning of complex cellular systems. Since cancer is a genetic disease arising from the abnormal gene function, the identification of cancerous genes and the regulatory pathways they control will provide a better platform for understanding the tumor formation and development. The major focus of this thesis is to understand the regulation of genes responsible for the development of cancer, particularly colorectal cancer by analyzing the microarray expression data. In this thesis, four computational algorithms namely fuzzy logic algorithm, modified genetic algorithm, dynamic neural fuzzy network and Takagi Sugeno Kang-type recurrent neural fuzzy network are used to extract cancer specific gene regulatory network from plasma RNA dataset of colorectal cancer patients. Plasma RNA is highly attractive for cancer analysis since it requires a collection of small amount of blood and it can be obtained at any time in repetitive fashion allowing the analysis of disease progression and treatment response.

Preimplantation development regulatory pathway construction through a text-mining approach

Background: The integration of sequencing and gene interaction data and subsequent generation of pathways and networks contained in databases such as KEGG Pathway is essential for the comprehension of complex biological processes. We noticed the absence of a chart or pathway describing the well-studied preimplantation development stages; furthermore, not all genes involved in the process have entries in KEGG Orthology, important information for knowledge application with relation to other organisms. Results: In this work we sought to develop the regulatory pathway for the preimplantation development stage using text-mining tools such as Medline Ranker and PESCADOR to reveal biointeractions among the genes involved in this process. The genes present in the resulting pathway were also used as seeds for software developed by our group called SeedServer to create clusters of homologous genes. These homologues allowed the determination of the last common ancestor for each gene and revealed that the preimplantation development pathway consists of a conserved ancient core of genes with the addition of modern elements. Conclusions: The generation of regulatory pathways through text-mining tools allows the integration of data generated by several studies for a more complete visualization of complex biological processes. Using the genes in this pathway as “seeds” for the generation of clusters of homologues, the pathway can be visualized for other organisms. The clustering of homologous genes together with determination of the ancestry leads to a better understanding of the evolution of such process.

An Update On The Pharmacogenetics Of Treating Hypertension.

Hypertension is a leading cause of cardiovascular mortality, but only one third of patients achieve blood pressure goals despite antihypertensive therapy. Genetic polymorphisms may partially account for the interindividual variability and abnormal response to antihypertensive drugs. Candidate gene and genome-wide approaches have identified common genetic variants associated with response to antihypertensive drugs. However, there is no currently available pharmacogenetic test to guide hypertension treatment in clinical practice. In this review, we aimed to summarize the recent findings on pharmacogenetics of the most commonly used antihypertensive drugs in clinical practice, including diuretics, angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers, beta-blockers and calcium channel blockers. Notably, only a small percentage of the genetic variability on response to antihypertensive drugs has been explained, and the vast majority of the genetic variants associated with antihypertensives efficacy and toxicity remains to be identified. Despite some genetic variants with evidence of association with the variable response related to these most commonly used antihypertensive drug classes, further replication is needed to confirm these associations in different populations. Further studies on epigenetics and regulatory pathways involved in the responsiveness to antihypertensive drugs might provide a deeper understanding of the physiology of hypertension, which may favor the identification of new targets for hypertension treatment and genetic predictors of antihypertensive response.Journal of Human Hypertension advance online publication, 28 August 2014; doi:10.1038/jhh.2014.76.

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts, This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones, This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types, This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

The cellular basis of a congenital heart defect Drosophila

RESUMO: Mutações em genes envolvidos na formação do coração e anomalias em qualquer etapa deste processo causam frequentemente malformações cardíacas, que representam o tipo mais comum de defeitos em neonatais, afetando cerca de 1% dos nascimentos por ano. Assim, estima-se que 20 milhões de pessoas sejam portadoras de um defeito cardíaco congénito. O coração da Drosophila melanogaster (mosca-da-fruta), denominado vaso dorsal, é um órgão relativamente simples que actua como uma bomba muscular, contraindo automaticamente para permitir a circulação da hemolinfa através do corpo. A formação do vaso dorsal na mosca é muito semelhante ao desenvolvimento do coração em vertebrados, representando por isso, um poderoso modelo para estudar a rede de genes e os padrões regulatórios relacionados com o desenvolvimento deste órgão. Anteriormente, nós identificámos um gene em Drosophila, darhgef10, fortemente expresso no coração em desenvolvimento e cuja deleção induz anormalidades cardíacas subtis mas prevalentes. Os mutantes para darhgef10 são viáveis e férteis no ambiente controlado de laboratório. Este trabalho teve como objectivos caracterizar fenotipicamente os mutantes nulos para darhgef10, determinar a localização subcelular da proteína dArhgef10 e investigar a base celular subjacente ao defeito no alinhamento dos cardioblastos observado nos mutantes. Os nossos resultados revelaram que a deleção de darhgef10 provoca uma severa redução da viabilidade, sem no entanto comprometer o tempo de desenvolvimento e a longevidade. Por outro lado, o aumento da expressão de darhgef10 em músculos, glândulas salivares e no disco imaginal do olho afeta drasticamente a integridade destes tecidos. A expressão ectópica de darhgef10 in vitro e in vivo revelou que a proteína está localiza no citoplasma com enriquecimento junto à membrana celular, com associação à actina F. Live imaging de embriões mutantes para darhgef10 revelou que os defeitos observados no coração podem estar associados a um defeito na adesão dos músculos alary e/ou das células pericardiais ao vaso dorsal. O homólogo humano de darhgef10, ARHGEF10, também é expresso no coração e está associação a uma maior susceptibilidade para a ocorrência de acidentes vasculares cerebrais aterotrombóticos, sugerindo que o que aprendemos sobre darhgef10 em Drosophila pode ter implicações do ponto de vista clínico para a saúde humana. ----------------------------- ABSTRACT: Mutations in genes controlling heart development and abnormalities in any of its steps frequently cause cardiac malformations, the most common type of birth defects in humans, affecting nearly 1% of births per year. Hence around 20 million adults are expected to live with a congenital heart defect. The Drosophila melanogaster heart, called dorsal vessel, is a relatively simple organ that acts as a muscular pump contracting automatically to allow the circulation of hemolymph. Drosophila heart formation shares many similarities with heart development in vertebrates providing a powerful system to study gene networks and regulatory pathways involved in heart development. We have previously identified a Drosophila gene, darhgef10, which is strongly expressed in the developing heart and when deleted, leads to flies with highly prevalent yet subtle heart abnormalities, compatible with unchallenged life in the laboratory. Our aims were to phenotypically characterize homozygous null darhgef10 mutants, characterize the subcellular localization of dArhgef10 and to study the cellular basis of the misaligned cardioblasts defect. We found that about half of darhgef10 mutants die during development. However, the survivors surprisingly have a nearly normal developmental time, adult locomotor behavior and total lifespan. Detection of transgene-derived dArhgef10 protein in vitro and in vivo using custom antibodies revealed a cytosolic protein slightly enriched in the cellular membranes and associated with F-actin. Tissue-specific darhgef10 expression disrupts the normal morphology of developing muscles, salivary glands and the eye. Live imaging of darhgef10 mutant embryos revealed that heart defect could be caused by a reduced capacity of attachment of pericardial cells and/or alary muscle to dorsal vessel. The human homolog of darhgef10 is also expressed in the heart and is a susceptibility gene for atherothrombotic stroke, suggesting that what we learn about the function of this gene and its phenotypes in Drosophila could have implications to human health.

Giving SBML support to ByoDyn

Report for the scientific stay at the California Institute of Technology during the summer of 2005. ByoDyn is a tool for simulating the dynamical expression of gene regulatory networks (GRNs) and for parameter estimation in uni- and multicellular models. A software support was carried out describing GRNs in the Systems Biology Markup Language (SBML). This one is a computer format for representing and storing computational models of biochemical pathways in software tools and databases. Supporting this format gives ByoDyn a wide range of possibilities to study the dynamical properties of multiple regulatory pathways.

Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling.

Continuous turnover of epithelia is ensured by the extensive self-renewal capacity of tissue-specific stem cells. Similarly, epithelial tumour maintenance relies on cancer stem cells (CSCs), which co-opt stem cell properties. For most tumours, the cellular origin of these CSCs and regulatory pathways essential for sustaining stemness have not been identified. In murine skin, follicular morphogenesis is driven by bulge stem cells that specifically express CD34. Here we identify a population of cells in early epidermal tumours characterized by phenotypic and functional similarities to normal bulge skin stem cells. This population contains CSCs, which are the only cells with tumour initiation properties. Transplants derived from these CSCs preserve the hierarchical organization of the primary tumour. We describe beta-catenin signalling as being essential in sustaining the CSC phenotype. Ablation of the beta-catenin gene results in the loss of CSCs and complete tumour regression. In addition, we provide evidence for the involvement of increased beta-catenin signalling in malignant human squamous cell carcinomas. Because Wnt/beta-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas.

Role of ENaC in skin wound healing and phenotypic analysis of GILZ-deficient mice

In my first project, I analyzed the role of the amiloride-sensitive epithelial sodium channel ENaC) in the skin during wound healing. ENaC is present in the skin and a function in keratinocyte differentiation and barrier formation has been demonstrated. Previous findings suggested, that ENaC might be implicated in keratinocyte migration, although its role in wound healing was not analyzed yet. Using skin-specific (K14-Cre) conditional ENaC knockout and overexpressing mice, I determined the wound closure kinetic and performed morphometric measurements. The time course of wound repair was not significantly different in knockouts or transgenics when compared to control mice and the morphology of the closing wound was not altered. In my second project, I studied the glucocorticoid-induced leucine zipper (GILZ, Tsc22d3). GILZ is widely expressed and an important role has been predicted in immunity, adipogenesis and renal sodium handling. Mice were generated that constitutively lack all the functional domains of the Gilz gene. In these mice, the expression of GILZ mRNA transcripts and protein were completely abolished in all tissues tested. Surprisingly, knockout mice survived. To test whether GILZ mimicks glucocorticoid action, we studied its implication in T- and B- cell development and in a model of sepsis. We measured cytokine secretion in different inflammatory models, like in peritoneal and bone marrow-derived macrophages, in splenocytes and a model of sepsis. In all our experiments, cytokine secretion from GILZ- deficient cells was not different from controls. From 6 months onwards, knockout mice contained significantly less body fat and were lighter. Following sodium and water deprivation experiments, water and salt homeostasis was preserved. Sterility of knockout males was associated with a severe testis dysplasia, smaller seminiferous tubules, the number of Sertoli and germ cell was reduced while increased apoptosis, but not cell proliferation, was evidenced. The interstitial Leydig cell population was augmented, and higher plasma FSH and testosterone levels were found. Interestingly, the expression of the target gene Ppar2 was diminished in the testis and in the liver, but not in the skin, kidney or fat. Tsc22d1 mRNA transcript level was found to be upregulated in testis, but not in the kidney or fat tissue. In most tissue, excepted the testis, GILZ-deficient mice reveal functional redundancy amongst members of the Tsc22d family or genes involved in the same regulatory pathways. In summary, contrarily to the published in vitro data, GILZ does not play a crucial role attributed in immunology or inflammation, but we identified a novel function in spermatogenesis. -- Dans mon premier projet, j'ai analysé le rôle du canal épithélial sodique sensible à l'amiloride (ENaC) dans la cicatrisation de la peau. ENaC est présent dans la peau et il a une fonction dans la différenciation des kératinocytes et dans la formation de la barrière. Des études suggèrent qu'ENaC pourrait être impliqué dans la migration des kératinocytes, cependant, son rôle dans la cicatrisation n'a pas encore été étudié. A l'aide de souris qui surexpriment ou qui sont knockout pour ENaC, spécifiquement dans la peau (K14-Cre), j'ai analysé le temps de clôture de la cicatrice et j'ai aussi étudié la morphologie de la plaie guérissant. Chez les souris qui surexpriment ou chez les knockouts, la vitesse de fermeture et la morphologie de la cicatrice étaient identiques aux souris contrôles. Dans mon second projet, j'ai étudié le glucocorticoid-induced leucine zipper (GILZ, Tsc22d3). GILZ est largement exprimé et un rôle important a été prédit dans l'immunité, l'adipogénèse et le transport sodique rénal. Des souris ont été générées dont les domaines fonctionnels du gène Gilz sont éliminés. L'expression de GILZ en ARNm et protéine a été complètement abolie dans tous les tissus testés. Étonnamment, ces souris knockout survivent. Afin de tester si GILZ imite les effets des glucocorticoïdes, nous avons étudié son implication dans le développement des cellules T et B ainsi qu'un modèle de septicémie. Nous avons mesuré la sécrétion de cytokines à partir de différents modèles d'inflammation tels que des macrophages péritonéaux ou de moelle, de splénocytes ou encore d'un modèle de septicémie. Dans toutes nos expériences, la sécrétion de cytokines de cellules GILZ-déficientes était semblable. Dès 6 mois, les knockouts contenaient significativement moins de graisses et étaient plus légères. Suite à une privation sodique et aqueuse, l'homéostasie du sel et de l'eau était préservée. Les mâles knockouts présentaient une stérilité accompagnée d'une dysplasie testiculaire sévère, de tubules séminifères étaient plus petits et contenaient un nombre réduit de cellules de Sertoli et de cellules germinales. L'apoptose était augmentée dans ces cellules mais pas la prolifération cellulaire. Le nombre de cellules de Leydig était aussi plus élevé, ainsi que la FSH et la testostérone. L'expression du gène cible Pparγ2 était diminuée dans le testicule et le foie, mais pas dans la peau, le rein ou le tissu adipeux. L'ARNm de Tsc22d1 était plus exprimé dans le testicule, mais pas dans le rein ou le tissu adipeux. Dans la plupart des tissus, sauf le testicule, les souris knockouts révélaient une redondance fonctionnelle des autres membres de la famille Tsc22d ou de gènes impliqués dans les mêmes voies de régulation. En résumé, contrairement aux données in vitro, GILZ ne joue pas un rôle essentiel en immunologie, mais nous avons identifié une nouvelle fonction dans la spermatogénèse.

Concerted action of ENaC, Nedd4-2, and Sgk1 in transepithelial Na(+) transport.

The epithelial Na(+) channel (ENaC), located in the apical membrane of renal aldosterone-responsive epithelia, plays an essential role in controlling the Na(+) balance of extracellular fluids and hence blood pressure. As of now, ENaC is the only Na(+) transport protein for which genetic evidence exists for its involvement in the genesis of both hypertension (Liddle's syndrome) and hypotension (pseudohypoaldosteronism type 1). The regulation of ENaC involves a variety of hormonal signals (aldosterone, vasopressin, insulin), but the molecular mechanisms behind this regulation are mostly unknown. Two regulatory proteins have gained interest in recent years: the ubiquitin-protein ligase neural precursor cell-expressed, developmentally downregulated gene 4 isoform Nedd4-2, which negatively controls ENaC cell surface expression, and serum glucocorticoid-inducible kinase 1 (Sgk1), which is an aldosterone- and insulin-dependent, positive regulator of ENaC density at the plasma membrane. Here, we summarize present ideas about Sgk1 and Nedd4-2 and the lines of experimental evidence, suggesting that they act sequentially in the regulatory pathways governed by aldosterone and insulin and regulate ENaC number at the plasma membrane.

Pregnancy-induced chromatin remodeling in the breast of postmenopausal women.

Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation-associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full-term pregnancy.

Transcriptional regulation of metabolism.

Our understanding of metabolism is undergoing a dramatic shift. Indeed, the efforts made towards elucidating the mechanisms controlling the major regulatory pathways are now being rewarded. At the molecular level, the crucial role of transcription factors is particularly well-illustrated by the link between alterations of their functions and the occurrence of major metabolic diseases. In addition, the possibility of manipulating the ligand-dependent activity of some of these transcription factors makes them attractive as therapeutic targets. The aim of this review is to summarize recent knowledge on the transcriptional control of metabolic homeostasis. We first review data on the transcriptional regulation of the intermediary metabolism, i.e., glucose, amino acid, lipid, and cholesterol metabolism. Then, we analyze how transcription factors integrate signals from various pathways to ensure homeostasis. One example of this coordination is the daily adaptation to the circadian fasting and feeding rhythm. This section also discusses the dysregulations causing the metabolic syndrome, which reveals the intricate nature of glucose and lipid metabolism and the role of the transcription factor PPARgamma in orchestrating this association. Finally, we discuss the molecular mechanisms underlying metabolic regulations, which provide new opportunities for treating complex metabolic disorders.

Pharmacogenetics of CYP1A2 activity and inducibility in smokers and exsmokers.

BACKGROUND: There is a high interindividual variability in cytochrome P4501A2 (CYP1A2) activity and in its inducibility by smoking, only poorly explained by known CYP1A2 polymorphisms. We aimed to study the contribution of other regulatory pathways, including transcription factors and nuclear receptors, toward this variability. METHODS: CYP1A2 activity was determined by the paraxanthine/caffeine ratio in 184 smokers and in 113 of them who were abstinent for 4 weeks. Participants were genotyped for 22 polymorphisms in 12 genes. RESULTS: A significant influence on CYP1A2 inducibility was observed for the NR1I3 rs2502815 (P=0.0026), rs4073054 (P=0.029), NR2B1 rs3818740 (P=0.0045), rs3132297 (P=0.036), AhR rs2282885 (P=0.040), rs2066853 (P=0.019), NR1I1 rs2228570 (P=0.037), and NR1I2 rs1523130 (P=0.044) polymorphisms. Among these, the NR1I3 rs2502815 (P=0.0045), rs4073054 (P=0.048), and NR2B1 rs3818740 (P=0.031) also influenced CYP1A2 basal activity. CONCLUSION: This is the first in-vivo demonstration of the influence of genes involved in CYP1A2 regulatory pathways on its basal activity and inducibility by smoking. These results need to be confirmed by other studies.

The CD4-like molecule LAG-3, biology and therapeutic applications.

IMPORTANCE OF THE FIELD: Promising immunotherapeutic agents targeting co-stimulatory pathways are currently being tested in clinical trials. One player in this array of regulatory pathways is the LAG-3/MHC class II axis. The lymphocyte activation gene-3 (LAG-3) is a negative co-stimulatory receptor that modulates T cell homeostasis, proliferation and activation. A recombinant soluble dimeric form of LAG-3 (sLAG-3-Ig, IMP321) shows adjuvant properties and enhances immunogenicity of tumor vaccines. Recent clinical trials produced encouraging results, especially when the human dimeric soluble form of LAG-3 (hLAG-3-Ig) was used in combination with chemotherapy. AREAS COVERED IN THIS REVIEW: The biological relevance of LAG-3 in vivo. Pre-clinical data demonstrating adjuvant properties, as well as the improvement of tumor immunity by sLAG-3-Ig. Recent advances in the clinical development of the therapeutic reagent IMP321, hLAG-3-Ig, for cancer treatment. WHAT THE READER WILL GAIN: This review summarizes preclinical and clinical data on the biological functions of LAG-3. TAKE HOME MESSAGE: The LAG-3 inhibitory pathway is attracting attention, in the light of recent studies demonstrating its role in T cell unresponsiveness, and Treg function after chronic antigen stimulation. As a soluble recombinant dimer, the sLAG-3-Ig protein acts as an adjuvant for therapeutic induction of T cell responses, and may be beneficial to cancer patients when used in combination therapies.