Biochemical, Enzymatic, and Computational Characterization of Recurrent Somatic Mutations of the Human Protein Tyrosine Phosphatase PTP1B in Primary Mediastinal B Cell Lymphoma.

Human protein tyrosine phosphatase 1B (PTP1B) is a ubiquitous non-receptor tyrosine phosphatase that serves as a major negative regulator of tyrosine phosphorylation cascades of metabolic and oncogenic importance such as the insulin, epidermal growth factor receptor (EGFR), and JAK/STAT pathways. Increasing evidence point to a key role of PTP1B-dependent signaling in cancer. Interestingly, genetic defects in PTP1B have been found in different human malignancies. Notably, recurrent somatic mutations and splice variants of PTP1B were identified in human B cell and Hodgkin lymphomas. In this work, we analyzed the molecular and functional levels of three PTP1B mutations identified in primary mediastinal B cell lymphoma (PMBCL) patients and located in the WPD-loop (V184D), P-loop (R221G), and Q-loop (G259V). Using biochemical, enzymatic, and molecular dynamics approaches, we show that these mutations lead to PTP1B mutants with extremely low intrinsic tyrosine phosphatase activity that display alterations in overall protein stability and in the flexibility of the active site loops of the enzyme. This is in agreement with the key role of the active site loop regions, which are preorganized to interact with the substrate and to enable catalysis. Our study provides molecular and enzymatic evidence for the loss of protein tyrosine phosphatase activity of PTP1B active-site loop mutants identified in human lymphoma.

Lactosamine-Based Derivatives as Tools to Delineate the Biological Functions of Galectins: Application to Skin Tissue Repair.

Galectins have been recognized as potential novel therapeutic targets for the numerous fundamental biological processes in which they are involved. Galectins are key players in homeostasis, and as such their expression and function are finely tuned in vivo. Thus, their modes of action are complex and remain largely unexplored, partly because of the lack of dedicated tools. We thus designed galectin inhibitors from a lactosamine core, functionalized at key C2 and C3' positions by aromatic substituents to ensure both high affinity and selectivity, and equipped with a spacer that can be modified on demand to further modulate their physico-chemical properties. As a proof-of-concept, galectin-3 was selectively targeted. The efficacy of the synthesized di-aromatic lactosamine tools was shown in cellular assays to modulate collective epithelial cell migration and to interfere with actin/cortactin localization.

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin-3 Inhibitor.

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent-field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3'-mono- and 2,3'-diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin-3 for 2'-arylamido derivatives over ureas, thioureas, and amines and that of galectin-7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin-3 versus galectin-1 and galectin-7.

Galectin-7 in Epithelial Homeostasis and Carcinomas.

Galectins are small unglycosylated soluble lectins distributed both inside and outside the cells. They share a conserved domain for the recognition of carbohydrates (CRD). Although galectins have a common affinity for ß-galatosides, they exhibit different binding preferences for complex glycans. First described twenty years ago, galectin-7 is a prototypic galectin, with a single CRD, able to form divalent homodimers. This lectin, which is mainly expressed in stratified epithelia, has been described in epithelial tissues as being involved in apoptotic responses, in proliferation and differentiation but also in cell adhesion and migration. Most members of the galectins family have been associated with cancer biology. One of the main functions of galectins in cancer is their immunomodulating potential and anti-angiogenic activity. Indeed, galectin-1 and -3, are already targeted in clinical trials. Another relevant function of galectins in tumour progression is their ability to regulate cell migration and cell adhesion. Among these galectins, galectin-7 is abnormally expressed in various cancers, most prominently in carcinomas, and is involved in cancer progression and metastasis but its precise functions in tumour biology remain poorly understood. In this issue, we will focus on the physiological functions of galectin-7 in epithelia and present the alterations of galectin-7 expression in carcinomas with the aim to describe its possible functions in tumour progression.

The Parkinsonism-associated protein DJ-1/Park7 prevents glycation damage in human keratinocyte.

Reducing sugars and dicarbonyls form covalent adducts with proteins through a nonenzymatic process known as glycation, which inactivates proteins, is increased in diabetic patients and is associated with diabetic complications, including retinopathy, cataracts, nephropathy, neuropathy, cardiomyopathy and skin defects. We recently characterized DJ-1/Park7 as a protein deglycase that repairs proteins from glycation by glyoxal and methylglyoxal, two major glycating agents which are responsible for up to 65% of glycation events. In this study, we investigated the ability of DJ-1 to prevent protein glycation in keratinocytes. Glycation of collagen and keratinocyte proteins was tested by measuring ultraviolet absorption and fluorescence emission. Protein glycation in HaCaT keratinocytes was investigated by immunodetection with anti-advanced glycation endproduct antibodies, after DJ-1 depletion or overexpression. In vitro, DJ-1 prevented glycation of collagen and keratinocyte protein extracts. In cell culture, DJ-1 depletion by small interfering RNAs resulted in a 3-fold increase in protein glycation levels. Moreover, protein glycation levels were decreased several-fold in cells overexpressing DJ-1 after addition of the Nrf2 inducer sulforaphane or after transfection with a DJ-1 plasmid. Thus, the DJ-1 deglycase plays a major role in preventing protein glycation in eukaryotic cells and might be important for preventing skin glycation.

[Galectins, a class of unconventional lectins]. / Les galectines - Des lectines pas comme les autres.

Galectins constitute a family of soluble animal lectins defined by their evolutionary conserved carbohydrate recognition domain and their affinity for ß-galactosides containing glycoconjugates. Each galectin is characterized by a specific spatio-temporal distribution and a unique set of ligands and molecular partners. Interestingly, galectins are found both extracellularly and intracellularly and modulate various cellular processes. Knock-out mutant mice for galectins-1, 3 or 7 are viable but display a wide range of defects under various stress conditions. Indeed, galectins are multifunctional proteins involved in cell-cell and cell-extracellular matrix interactions, organization of membrane domains, cell signalling and also in intracellular trafficking, apoptosis, regulation of cell cycle. Galectins represent potential therapeutic targets, especially in the context of cancer and inflammatory diseases.

Interplay between EGFR, E-cadherin, and PTP1B in epidermal homeostasis.

Maintaining epithelial homeostasis is crucial to allow embryo development but also the protective barrier which is ensured by the epidermis. This homeostasis is regulated through the expression of several molecules among which EGFR and E-cadherin which are of major importance. Indeed, defects in the regulation of these proteins lead to abnormalities in cell adhesion, proliferation, differentiation, and migration. Hence, regulation of these two proteins is of the utmost importance as they are involved in numerous skin pathologies and cancers. In the last decades it has been described several pathways of regulation of these two proteins and notably several mechanisms of cross-regulation between these partners. In this review, we aimed to describe the current understanding of the regulation of EGFR and interactions between EGFR and E-cadherin and, in particular, the implication of these cross-regulations in epithelium homeostasis. We pay particular attention to PTP1B, a phosphatase involved in the regulation of EGFR.

Link between the EZH2 noncanonical pathway and microtubule organization center polarization during early T lymphopoiesis.

EZH2 plays an essential role at the ß-selection checkpoint of T lymphopoiesis by regulating histone H3 lysine 27 trimethylation (H3K27me3) via its canonical mode of action. Increasing data suggest that EZH2 could also regulate other cellular functions, such as cytoskeletal reorganization, via its noncanonical pathway. Consequently, we investigated whether the EZH2 noncanonical pathway could be involved in early T-cell maturation, which requires cell polarization. We observed that EZH2 localization is tightly regulated during the early stages of T-cell development and that EZH2 relocalizes in the nucleus of double-negative thymocytes enduring TCRß recombination and ß-selection processes. Furthermore, we observed that EZH2 and EED, but not Suz12, colocalize with the microtubule organization center (MTOC), which might prevent its inappropriate polarization in double negative cells. In accordance with these results, we evidenced the existence of direct or indirect interaction between EED and α-tubulin. Taken together, these results suggest that the EZH2 noncanonical pathway, in association with EED, is involved in the early stages of T-cell maturation.

Identification of a new regulation pathway of EGFR and E-cadherin dynamics.

E-cadherin and EGFR are known to be closely associated hence regulating differentiation and proliferation notably in epithelia. We have previously shown that galectin-7 binds to E-cadherin and favors its retention at the plasma membrane. In this study, we shed in light that galectin-7 establishes a physical link between E-cadherin and EGFR. Indeed, our results demonstrate that galectin-7 also binds to EGFR, but unlike the binding to E-cadherin this binding is sugar dependent. The establishment of E-cadherin/EGFR complex and the binding of galectin-7 to EGFR thus lead to a regulation of its signaling and intracellular trafficking allowing cell proliferation and migration control. In vivo observations further support these results since an epidermal thickening is observed in galectin-7 deficient mice. This study therefore reveals that galectin-7 controls epidermal homeostasis through the regulation of E-cadherin/EGFR balance.

Calpain activation is required for homocysteine-mediated hepatic degradation of inhibitor I kappa B alpha.

Hepatic steatosis is a clinical feature observed in severe hyperhomocysteinemic patients. In mice, cystathionine beta synthase (CBS) deficiency, the most common cause of severe hyperhomocysteinemia, is also associated with steatosis, fibrosis and inflammation. Proinflammatory cytokines usually induce apoptosis. However, hyperhomocysteinemia does not increase apoptosis in liver of CBS-deficient mice compared to wild type mice. The aim of the study was to analyze the activation state of the NF-kappaB pathway in liver of CBS-deficient mice and to investigate its possible involvement in anti-apoptotic signals. We analyzed the level of I kappaB alpha in liver of CBS-deficient mice. A co-culture of primary hepatocytes and Kupffer cells was also used in order to investigate how I kappaB alpha degradation occurs in response to homocysteine. We found lower I kappaB alpha level not only in liver of CBS-deficient mice but also in hepatocyte/Kupffer cell co-culture. The homocysteine-mediated I kappaB alpha enhanced proteolysis occurred via calcium-dependent calpains, which was supported by an increased level of calpain activity and a reduced expression of calpastatin in liver of CBS-deficient mice. Intraperitoneal administration of the inhibitor PDTC normalized the expression of two genes induced by NF-kappaB activation, heme oxygenase-1 and cellular inhibitor of apoptosis 2. Moreover, PDTC administration induced an increase of caspase-3 activity in liver of CBS-deficient mice. Our results suggest that hyperhomocysteinemia induces calpain-mediated I kappaB alpha degradation which is responsible for anti-apoptotic signals in liver.

E-cadherin dynamics is regulated by galectin-7 at epithelial cell surface.

Re-epithelialisation of wounded epidermis is ensured by collective cell migration of keratinocytes. Efficient collective migration requires the maintenance of intercellular adhesion, notably through adherens junctions, to favour cell communication, support tension forces and coordinated movement . Galectin-7, a soluble lectin expressed in stratified epithelia, has been previously implicated in cell migration and intercellular adhesion. Here, we revealed a new function of galectin-7 in the control of directionality and collective behaviour in migrating keratinocytes. Consistently, we identified galectin-7 as a direct partner of E-cadherin, a key component of adherens junctions. Unexpectedly, this interaction does not require glycosylation motifs. Focusing on the underlying mechanisms, we showed that galectin-7 stabilizes E-cadherin at the plasma membrane, restraining its endocytosis. Interestingly, galectin-7 silencing decreases E-cadherin-mediated intercellular adhesion. Consequently, this study not only identifies a new stabilizer of adherens junctions but also emphasises the importance of the interplay between E-cadherin turnover and intercellular adhesion strength.

Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair.

BACKGROUND: The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. METHODS: We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. RESULTS: The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. CONCLUSION: These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.

Galectins in epithelial functions.

Galectins are a family of animal lectins comprising 15 members in vertebrates. These proteins are involved in many biological processes including epithelial homeostasis and tumor progression by displaying intracellular and extracellular activities. Hence Galectins can be found either in the cytoplasm or the nucleus, associated with membranes or in the extracellular matrix. Current studies aim at understanding the roles of Galectins in cell-cell and cell-matrix adhesion, cellular polarity and motility. This review discusses recent progress in defining the specificities and mechanisms of action of Galectins as cell regulators in epithelial cells. Physiological, cellular and molecular aspects of Galectin specificities will be treated successively.

Oncogenic ß-catenin triggers an inflammatory response that determines the aggressiveness of hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Its pathogenesis is frequently linked to liver inflammation. Gain-of-function mutations in the gene encoding ß-catenin are frequent genetic modifications found in human HCCs. Thus, we investigated whether inflammation was a component of ß-catenin-induced tumorigenesis using genetically modified mouse models that recapitulated the stages of initiation and progression of this tumoral process. Oncogenic ß-catenin signaling was found to induce an inflammatory program in hepatocytes that involved direct transcriptional control by ß-catenin and activation of the NF-κB pathway. This led to a specific inflammatory response, the intensity of which determined the degree of tumor aggressiveness. The chemokine-like chemotactic factor leukocyte cell-derived chemotaxin 2 (LECT2) and invariant NKT (iNKT) cells were identified as key interconnected effectors of liver ß-catenin-induced inflammation. In genetic deletion models lacking the gene encoding LECT2 or iNKT cells, hepatic ß-catenin signaling triggered the formation of highly malignant HCCs with lung metastasis. Thus, our results identify inflammation as a key player in ß-catenin-induced liver tumorigenesis. We provide strong evidence that, by activating pro- and antiinflammatory mediators, ß-catenin signaling produces an inflammatory microenvironment that has an impact on tumoral development. Our data are consistent with the fact that most ß-catenin-activated HCCs are of better prognosis.

Delivery of mengovirus-derived RNA replicons into tumoural liver enhances the anti-tumour efficacy of a peripheral peptide-based vaccine.

Hepatocellular carcinoma is a deadly cancer with growing incidence for which immunotherapy is one of the most promising therapeutic approach. Peptide-based vaccines designed to induce strong, sustained CD8+ T cell responses are effective in animal models and cancer patients. We demonstrated the efficacy of curative peptide-based immunisation against a unique epitope of SV40 tumour antigen, through the induction of a strong CD8+ T cell-specific response, in our liver tumour model. However, as in human clinical trials, most tumour antigen epitopes did not induce a therapeutic effect, despite inducing strong CD8+ T cell responses. We therefore modified the tumour environment to enhance peptide-based vaccine efficacy by delivering mengovirus (MV)-derived RNA autoreplicating sequences (MV-RNA replicons) into the liver. The injection of replication-competent RNA replicons into the liver converted partial tumour regression into tumour eradication, whereas non-replicating RNA had no such effect. Replicating RNA replicon injection induced local recruitment of innate immunity effectors (NK and NKT) to the tumour and did not affect specific CD8+ T cell populations or other myelolymphoid subsets. The local delivery of such RNA replicons into tumour stroma is therefore a promising strategy complementary to the use of peripheral peptide-based vaccines for treating liver tumours.

CCR5 deficiency decreases susceptibility to experimental cerebral malaria.

Infection of susceptible mouse strains with Plasmodium berghei ANKA (PbA) is a valuable experimental model of cerebral malaria (CM). Two major pathologic features of CM are the intravascular sequestration of infected erythrocytes and leukocytes inside brain microvessels. We have recently shown that only the CD8+ T-cell subset of these brain-sequestered leukocytes is critical for progression to CM. Chemokine receptor-5 (CCR5) is an important regulator of leukocyte trafficking in the brain in response to fungal and viral infection. Therefore, we investigated whether CCR5 plays a role in the pathogenesis of experimental CM. Approximately 70% to 85% of wild-type and CCR5+/- mice infected with PbA developed CM, whereas only about 20% of PbA-infected CCR5-deficient mice exhibited the characteristic neurologic signs of CM. The brains of wild-type mice with CM showed significant increases in CCR5+ leukocytes, particularly CCR5+ CD8+ T cells, as well as increases in T-helper 1 (Th1) cytokine production. The few PbA-infected CCR5-deficient mice that developed CM exhibited a similar increase in CD8+ T cells. Significant leukocyte accumulation in the brain and Th1 cytokine production did not occur in PbA-infected CCR5-deficient mice that did not develop CM. Moreover, experiments using bone marrow (BM)-chimeric mice showed that a reduced but significant proportion of deficient mice grafted with CCR5+ BM develop CM, indicating that CCR5 expression on a radiation-resistant brain cell population is necessary for CM to occur. Taken together, these results suggest that CCR5 is an important factor in the development of experimental CM.

Amino acid modifications in the wild type sequence p53 232-240 overcome the poor immunogenicity of this self tumour epitope.

A major limitation in antigen-specific cancer vaccines is that most of the tumour antigens that are potent candidates for broad applicability originate from self proteins. The peptides presented by tumour cells are derived from tissue-specific differentiation proteins, from proteins altered by genetic mutation or by non mutated proteins that are normally silent in most adult tissues. As a consequence, T-cell responses elicited against those antigens are rather weak. Several data showed that amino acid modifications could enhance the immunogenicity of such antigens by priming T-cells that have escaped central tolerance based on a poor avidity. In this regard, this strategy could be powerful for inducing immunity against tumours. The present report focuses on the murine wild type epitope p53 232-240 that is poorly immunogenic. It shows that substitution of the two cysteine residues by serine or amino butyric acid derivatives and substitution of the two methionine residues by norleucine residues resulted in enhanced stability of the MHC/peptide complex. The MHC binding affinity of analogue peptides was enhanced between 10 and 100 fold. They were also potent immunogens, stronger than was the original wild type epitope; T-cell responses were increased up to 50 times. Moreover, the effector T-cells elicited by three of these peptides cross reacted with the natural epitope. These observations have important implications for strategies that use the modified-peptide epitope.

Inflammation and cancer, the mastocytoma P815 tumor model revisited: triggering of macrophage activation in vivo with pro-tumorigenic consequences.

Subcutaneous in vivo injections of cells of the mastocytoma line P815 in syngenic DBA/2 mice induce locally fast growing solid tumors. These have been used extensively as a cancer model to analyze and manipulate the relationship between tumor cells and host's immune defenses. We report that progression of P815 tumors in vivo was accompanied by a burst (Days 5-7) of local inflammatory cells recruitment and angiogenesis observed histologically, corroborated in vivo by MRI with gadolinium, overtranscription of macrophage activation marker genes, secretion of TNF-alpha by regional lymph node cells and concomitant systemic inflammation. No substantial overtranscriptions of either VEGF or IL-10 or TGF-beta genes were observed. Induction of COX-2 gene was a late event. To establish a possible relationship between the tumor-induced local, regional and systemic increase of pro-inflammatory mediators and progression of tumors in vivo, we carried out experiments deliberately modulating the inflammatory status of the recipient animals. Pretreatment of recipient animals by i.p. injection of thioglycolate accelerated P815 tumor growth. At the opposite, treatment of mice with either a COX-1 + COX-2 inhibitor (aspirin, 1 mg/day/mouse) or a specific COX-2 inhibitor (celecoxib, 0.13 mg/day/mouse) for 2 weeks after injection of tumor cells, significantly reduced the size and growth rate of tumors compared to control mice. Experiments carried out in vitro indicated that peritoneal macrophages from untreated animals were strongly activated by live P815 cells and by P815 membrane preparations. The tumor-induced inflammatory reaction could establish a local micro environment favoring tumor progression. The P815 tumor model might be helpful to recognize important factors controlling host/tumor relationship.