Inhibition of neuraminidase-1 sialidase activity by interfering peptides impairs insulin receptor activity in vitro and glucose homeostasis in vivo.

Neuraminidases (NEUs) also called sialidases are glycosidases which catalyze the removal of terminal sialic acid residues from glycoproteins, glycolipids, and oligosaccharides. Mammalian NEU-1 participates in regulation of cell surface receptors such as insulin receptor (IR), epithelial growth factor receptor, low-density lipoprotein receptor, and toll-like receptor 4. At the plasma membrane, NEU-1 can be associated with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to constitute the elastin receptor complex. In this complex, NEU-1 is essential for elastogenesis, signal transduction through this receptor and for biological effects of the elastin-derived peptides on atherosclerosis, thrombosis, insulin resistance, nonalcoholic steatohepatitis, and cancers. This is why research teams are developing inhibitors targeting this sialidase. Previously, we developed interfering peptides to inhibit the dimerization and the activation of NEU-1. In this study, we investigated the effects of these peptides on IR activation in vitro and in vivo. Using cellular overexpression and endogenous expression models of NEU-1 and IR (COS-7 and HepG2 cells, respectively), we have shown that interfering peptides inhibit NEU-1 dimerization and sialidase activity which results in a reduction of IR phosphorylation. These results demonstrated that NEU-1 positively regulates IR phosphorylation and activation in our conditions. In vivo, biodistribution study showed that interfering peptides are well distributed in mice. Treatment of C57Bl/6 mice during 8 weeks with interfering peptides induces a hyperglycemic effect in our experimental conditions. Altogether, we report here that inhibition of NEU-1 sialidase activity by interfering peptides decreases IR activity in vitro and glucose homeostasis in vivo.

Pro-differentiating effects of a synthetic flavagline on human teratocarcinomal cancer stem-like cells.

As initiators of the carcinogenic process, cancer stem cells (CSCs) are considered as new targets for anti-cancer therapies. However, these cells are hidden in the cancer bulk and remain relatively insensitive to chemotherapy, which targets their proliferative capacities. Alternatively, growing evidences have pointed out that a differentiation therapy could adversely affect these cells, which consequently should lose their self-renewal properties and become less aggressive. In order to evaluate the differentiation potential of an emerging class of anti-cancer drugs, we used the poorly differentiated teratocarcinomal cell as a model of Oct4-expressing CSC and determined the molecular mechanisms induced by the highly active flavagline FL3. The drug, administrated at sublethal concentration and for long period, was able to downregulate the expression levels of the stemness factors Oct4 and Nanog at both transcriptional and translational levels, concomitantly with a decrease of clonogenicity. The appearance of specific neural markers further demonstrated the differentiation properties of FL3. Interestingly, an expression of active caspase-3 and an upregulation of the expression of the germ cell nuclear factor were observed in treated cells; this suggests that the suppression of Oct4 expression required for the induction of differentiation involves overlapping mechanisms of protein degradation and gene repression. Finally, this study shows that FL3, like all-trans retinoic acid (ATRA), acts as a differentiation inducer of teratocarcinomal cells. Thus, FL3 offers an alternative possibility for cancer treatment since it could target the carcinogenic process by inducing the differentiation of ATRA-resistant and Oct4-expressing CSCs, without toxic side effects on normal cells.

Neuraminidase-1: A Sialidase Involved in the Development of Cancers and Metabolic Diseases.

Sialidases or neuraminidases (NEU) are glycosidases which cleave terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Four types of mammalian sialidases, which are encoded by different genes, have been described with distinct substrate specificity and subcellular localization: NEU-1, NEU-2, NEU-3 and NEU-4. Among them, NEU-1 regulates many membrane receptors through desialylation which results in either the activation or inhibition of these receptors. At the plasma membrane, NEU-1 also associates with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to form the elastin receptor complex. The activation of NEU-1 is required for elastogenesis and signal transduction through this receptor, and this is responsible for the biological effects that are mediated by the elastin-derived peptides (EDP) on obesity, insulin resistance and non-alcoholic fatty liver diseases. Furthermore, NEU-1 expression is upregulated in hepatocellular cancer at the mRNA and protein levels in patients, and this sialidase regulates the hepatocellular cancer cells' proliferation and migration. The implication of NEU-1 in other cancer types has also been shown notably in the development of pancreatic carcinoma and breast cancer. Altogether, these data indicate that NEU-1 plays a key role not only in metabolic disorders, but also in the development of several cancers which make NEU-1 a pharmacological target of high potential in these physiopathological contexts.

Discoidin Domain Receptor 1 Expression in Colon Cancer: Roles and Prognosis Impact.

Extracellular matrix components such as collagens are deposited within the tumor microenvironment at primary and metastatic sites and are recognized to be critical during tumor progression and metastasis development. This study aimed to evaluate the clinical and prognostic impact of Discoidin Domain Receptor 1 (DDR1) expression in colon cancers and its association with a particular molecular and/or morphological profile and to evaluate its potential role as a prognosis biomarker. Immunohistochemical expression of DDR1 was evaluated on 292 colonic adenocarcinomas. DDR1 was highly expressed in 240 (82.2%) adenocarcinomas. High DDR1 immunostaining score was significantly associated, on univariate analysis, with male sex, left tumor location, BRAF wild type status, KRAS mutated status, and Annexin A10 negativity. High DDR1 immunohistochemical expression was associated with shorter event free survival only. Laser capture microdissection analyses revealed that DDR1 mRNA expression was mainly attributable to adenocarcinoma compared to stromal cells. The impact of DDR1 expression on cell invasion was then evaluated by modified Boyden chamber assay using cell types with distinct mutational profiles. The invasion capacity of colon adenocarcinoma is supported by DDR1 expression. Thus, our results showed that DDR1 was highly expressed in most colon adenocarcinomas and appears as an indicator of worse event free survival.

Functional Interplay Between Collagen Network and Cell Behavior Within Tumor Microenvironment in Colorectal Cancer.

Colorectal cancer is the second most common cancer diagnosed in men and the third most commonly occurring in women worldwide. Interactions between cells and the surrounding extracellular matrix (ECM) are involved in tumor development and progression of many types of cancer. The organization of the ECM molecules provides not only physical scaffoldings and dynamic network into which cells are embedded but also allows the control of many cellular behaviors including proliferation, migration, differentiation, and survival leading to homeostasis and morphogenesis regulation. Modifications of ECM composition and mechanical properties during carcinogenesis are critical for tumor initiation and progression. The core matrisome consists of five classes of macromolecules, which are collagens, laminins, fibronectin, proteoglycans, and hyaluronans. In most tissues, fibrillar collagen is the major component of ECM. Cells embedded into fibrillar collagen interact with it through their surface receptors, such as integrins and discoidin domain receptors (DDRs). On the one hand, cells incorporate signals from ECM that modify their functionalities and behaviors. On the other hand, all cells within tumor environment (cancer cells, cancer-associated fibroblasts, endothelial cells, and immune cells) synthesize and secrete matrix macromolecules under the control of multiple extracellular signals. This cell-ECM dialog participates in a dynamic way in ECM formation and its biophysical and biochemical properties. Here, we will review the functional interplay between cells and collagen network within the tumor microenvironment during colorectal cancer progression.

LRP-1 Promotes Colon Cancer Cell Proliferation in 3D Collagen Matrices by Mediating DDR1 Endocytosis.

Low density lipoprotein receptor related protein-1 (LRP-1) is a large ubiquitous endocytic receptor mediating the clearance of various molecules from the extracellular matrix. Several studies have shown that LRP-1 plays crucial roles during tumorigenesis functioning as a main signal pathway regulator, especially by interacting with other cell-surface receptors. Discoïdin Domain Receptors (DDRs), type I collagen receptors with tyrosine kinase activity, have previously been associated with tumor invasion and aggressiveness in diverse tumor environments. Here, we addressed whether it could exist functional interplays between LRP-1 and DDR1 to control colon carcinoma cell behavior in three-dimensional (3D) collagen matrices. We found that LRP-1 established tight molecular connections with DDR1 at the plasma membrane in colon cancer cells. In this tumor context, we provide evidence that LRP-1 regulates by endocytosis the cell surface levels of DDR1 expression. The LRP-1 mediated endocytosis of DDR1 increased cell proliferation by promoting cell cycle progression into S phase and decreasing apoptosis. In this study, we identified a new molecular way that controls the cell-surface expression of DDR1 and consequently the colon carcinoma cell proliferation and apoptosis and highlighted an additional mechanism by which LRP-1 carries out its sensor activity of the tumor microenvironment.

Transmembrane Peptides as Inhibitors of Protein-Protein Interactions: An Efficient Strategy to Target Cancer Cells?

Cellular functions are regulated by extracellular signals such as hormones, neurotransmitters, matrix ligands, and other chemical or physical stimuli. Ligand binding on its transmembrane receptor induced cell signaling and the recruitment of several interacting partners to the plasma membrane. Nowadays, it is well-established that the transmembrane domain is not only an anchor of these receptors to the membrane, but it also plays a key role in receptor dimerization and activation. Indeed, interactions between transmembrane helices are associated with specific biological activity of the proteins as cell migration, proliferation, or differentiation. Overexpression or constitutive dimerization (due notably to mutations) of these transmembrane receptors are involved in several physiopathological contexts as cancers. The transmembrane domain of tyrosine kinase receptors as ErbB family proteins (implicated in several cancers as HER2 in breast cancer) or other receptors as Neuropilins has been described these last years as a target to inhibit their dimerization/activation using several strategies. In this review, we will focus on the strategy which consists in using peptides to disturb in a specific manner the interactions between transmembrane domains and the signaling pathways (induced by ligand binding) of these receptors involved in cancer. This approach can be extended to inhibit other transmembrane protein dimerization as neuraminidase-1 (the catalytic subunit of elastin receptor complex), Discoidin Domain Receptor 1 (a tyrosine kinase receptor activated by type I collagen) or G-protein coupled receptors (GPCRs) which are involved in cancer processes.

Transmembrane Peptides as a New Strategy to Inhibit Neuraminidase-1 Activation.

Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.

Role of LRP-1 in cancer cell migration in 3-dimensional collagen matrix.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a member of Low Density Lipoprotein Receptor (LDLR) family, which is ubiquitously expressed and which is described as a multifunctional endocytic receptor which mediates the clearance of various extracellular matrix molecules including serine proteinases, proteinase-inhibitor complexes, and matricellular proteins. Several studies showed that high LRP-1 expression promotes breast cancer cell invasiveness, and LRP-1 invalidation leads to cell motility abrogation in both tumor and non-tumor cells. Furthermore, our group has reported that LRP-1 silencing prevents the invasion of a follicular thyroid carcinoma despite increased pericellular proteolytic activities from MMP2 and uPA using a 2D-cell culture model. As the use of 3D culture systems is becoming more and more popular due to their promise as enhanced models of tissue physiology, the aim of the present work is to characterize for the first time how the 3D collagen type I matrix may impact the ability of LRP-1 to regulate the migratory properties of thyroid carcinoma using as a model FTC-133 cells. Our results show that inhibition of LRP-1 activity or expression leads to morphological changes affecting cell-matrix interactions, reorganizations of the actin-cytoskeleton especially by inhibiting FAK activation and increasing RhoA activity and MLC-2 phosphorylation, thus preventing cell migration. Taken together, our results suggest that LRP-1 silencing leads to a decrease of cell migratory capacity in a 3D configuration.

AKAP-Lbc: a molecular scaffold for the integration of cyclic AMP and Rho transduction pathways.

A Kinase-anchoring proteins (AKAPs) are a family of functionally related proteins involved in the targeting of the PKA holoenzyme towards specific physiological substrates. We have recently identified a novel anchoring protein expressed in cardiomyocytes, called AKAP-Lbc, that functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) that activates the GTPase RhoA. Here, we discuss the most recent findings elucidating the molecular mechanisms and the transduction pathways involved in the regulation of the AKAP-Lbc signaling complex inside cells. We could show that AKAP-Lbc is regulated in a bi-directional manner by signals that activate or deactivate its Rho-GEF activity. Activation of AKAP-Lbc occurs in response to agonists that stimulate G proteins coupled receptors linked to the heterotrimeric G protein G12, whereas inactivation occurs through mechanisms that require phosphorylation of AKAP-Lbc by anchored PKA and subsequent recruitment of the regulatory protein 14-3-3. Interestingly, we could demonstrate that AKAP-Lbc can form homo-oligomers inside cells and that 14-3-3 can inhibit the Rho-GEF activity of AKAP-Lbc only when the anchoring protein adopts an oligomeric conformation. These findings reveal the molecular architecture of the AKAP-Lbc transduction complex and provide a mechanistic explanation of how upstream signaling pathways can be integrated within the AKAP-Lbc transduction complex to precisely modulate the activation of Rho.

Comparing reagents for efficient transfection of human primary myoblasts: FuGENE 6, Effectene and ExGen 500.

This study compared three different synthetic reagents (FuGENE 6, Effectene and ExGen 500) for the transfection of human primary myoblasts. We examined the efficiency, cytotoxicity and size of the complexes formed in the presence of different amounts of vector and DNA and with variable amounts of serum. Transfection rates were relatively high for primary cells, especially with FuGENE 6 (20%), which appeared to be the best transfection reagent for these cells, even in the presence of 10% serum. Cultured human myoblasts are an interesting tool for studying neuromuscular diseases and are potentially useful for myoblast transfer therapy studies. Moreover, the efficiency of these transfection reagents in a medium containing 10% serum is promising for possible gene therapy protocols for muscle diseases.

Role of ErbB Receptors in Cancer Cell Migration and Invasion.

Growth factors mediate their diverse biologic responses (regulation of cellular proliferation, differentiation, migration and survival) by binding to and activating cell-surface receptors with intrinsic protein kinase activity named receptor tyrosine kinases (RTKs). About 60 RTKs have been identified and can be classified into more than 16 different receptor families. Their activity is normally tightly controlled and regulated. Overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal stimulation by autocrine growth factor loops contribute to constitutive RTK signaling, resulting in alterations in the physiological activities of cells. The ErbB receptor family of RTKs comprises four distinct receptors: the EGFR (also known as ErbB1/HER1), ErbB2 (neu, HER2), ErbB3 (HER3) and ErbB4 (HER4). ErbB family members are often overexpressed, amplified, or mutated in many forms of cancer, making them important therapeutic targets. EGFR has been found to be amplified in gliomas and non-small-cell lung carcinoma while ErbB2 amplifications are seen in breast, ovarian, bladder, non-small-cell lung carcinoma, as well as several other tumor types. Several data have shown that ErbB receptor family and its downstream pathway regulate epithelial-mesenchymal transition, migration, and tumor invasion by modulating extracellular matrix (ECM) components. Recent findings indicate that ECM components such as matrikines bind specifically to EGF receptor and promote cell invasion. In this review, we will present an in-depth overview of the structure, mechanisms, cell signaling, and functions of ErbB family receptors in cell adhesion and migration. Furthermore, we will describe in a last part the new strategies developed in anti-cancer therapy to inhibit ErbB family receptor activation.

The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy.

In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth have been the subject of intensive investigation. It has been known for more than a decade that the small molecular weight GTPase RhoA is involved in the signaling pathways leading to cardiomyocyte hypertrophy. Although some of the hypertrophic pathways activated by RhoA have now been identified, the identity of the exchange factors that modulate its activity in cardiomyocytes is currently unknown. In this study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critical for activating RhoA and transducing hypertrophic signals downstream of alpha1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbc-specific short hairpin RNAs strongly reduces both alpha1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, alpha1-ARs promote AKAP-Lbc activation via a pathway that requires the alpha subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor (GEF) involved in the signaling pathways leading to cardiomyocytes hypertrophy.

Regulation of g protein-coupled receptor signaling by a-kinase anchoring proteins.

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.