Yves St-Pierre

Protein Kinase C-ζ Regulates Transcription of the Matrix Metalloproteinase-9 Gene Induced by IL-1 and TNF-α in Glioma Cells via NF-κB

The regulation of matrix metalloproteinase-9 (MMP-9) expression in glioma cells is one of the key processes in tumor invasion through the brain extracellular matrix. Although some studies have demonstrated the implication of classic protein kinase C (PKC) isoforms in the regulation of MMP-9 production by phorbol esters or lipopolysaccharide, the involvement of specific PKC isoforms in the signaling pathways leading to MMP-9 expression by inflammatory cytokines remains unclear. Here we report that the atypical PKC-ζ isoform participates in the induction of MMP-9 expression by interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) in rat C6 glioma cells. Indeed, zymography and semi-quantitative reverse transcriptase-PCR analysis showed that pretreatment of C6 cells with PKC-ζ pseudosubstrate abolished MMP-9 activity and gene expression induced by IL-1 or TNF-α. Accordingly, IL-1 and TNF-α were able to induce PKC-ζ activity, as demonstrated by in vitro kinase assay using immunoprecipitated PKC-ζ. Furthermore, stable C6 clones overexpressing PKC-ζ, but not PKC-ε, displayed an up-regulation of MMP-9 constitutive expression as well as an increase ofmmp-9 promoter activity. These processes were inhibited by an NF-κB-blocking peptide and completely prevented by NF-κB-binding site mutation in the mmp-9 promoter. Taken together, these results indicate that PKC-ζ plays a key role in the regulation of MMP-9 expression in C6 glioma cells through NF-κB.

Glycoprotein Nonmetastatic B Is an Independent Prognostic Indicator of Recurrence and a Novel Therapeutic Target in Breast Cancer

Purpose: Although the murine orthologue of glycoprotein nonmetastatic B (GPNMB), Osteoactivin, promotes breast cancer metastasis in an in vivo mouse model, its importance in human breast cancer is unknown. We have examined the significance of GPNMB expression as a prognostic indicator of recurrence and assessed its potential as a novel therapeutic target in breast cancer. Experimental Design: The clinical significance of GPNMB expression in breast cancer was addressed by analyzing GPNMB levels in several published gene expression data sets and two independent tissue microarrays derived from human breast tumors. GPNMB-expressing human breast cancer cell lines were further used to validate a toxin-conjugated anti-GPNMB antibody as a novel therapeutic agent. Results: GPNMB expression correlates with shorter recurrence times and reduced overall survival of breast cancer patients. Epithelial-specific GPNMB staining is an independent prognostic indicator for breast cancer recurrence. GPNMB is highly expressed in basal and triple-negative breast cancers and is associated with increased risk of recurrence within this subtype. GPNMB expression confers a more migratory and invasive phenotype on breast cancer cells and sensitizes them to killing by CDX-011 (glembatumumab vedotin), a GPNMB-targeted antibody-drug conjugate. Conclusions: GPNMB expression is associated with the basal/triple-negative subtype and is a prognostic marker of poor outcome in patients with breast cancer. CDX-011 (glembatumumab vedotin) is a promising new targeted therapy for patients with metastatic triple-negative breast cancers, a patient population that currently lacks targeted-therapy options. Clin Cancer Res; 16(7); 2147–56. ©2010 AACR.

Block and Graft Copolymers and Nanogel™ Copolymer Networks for DNA Delivery into Cell

Abstract Self-assembling complexes from nucleic acids and synthetic polymers are evaluated for plasmid and oligonucleotide (oligo) delivery. Polycations having linear, branched, dendritic, block- or graft copolymer architectures are used in these studies. All these molecules bind to nucleic acids due to formation of cooperative systems of salt bonds between the cationic groups of the polycation and phosphate groups of the DNA. To improve solubility of the DNA/polycation complexes, cationic block and graft copolymers containing segments from polycations and non-ionic soluble polymers, for example, poly(ethylene oxide) (PEO) were developed. Binding of these copolymers with short DNA chains, such as oligos, results in formation of species containing hydrophobic sites from neutralized DNA-polycation complex and hydrophilic sites from PEO. These species spontaneously associate into polyion complex micelles with a hydrophobic core from neutralized polyions and a hydrophilic shell from PEO. Such complexes are very small (10-40 nm) and stable in solution despite complete neutralization of charge. They reveal significant activity with oligos in vitro and in vivo. Binding of cationic copolymers to plasmid DNA forms larger (70-200 nm) complexes, which are practically inactive in cell transfection studies. It is likely that PEO prevents binding of these complexes with the cell membranes (“stealth effect”). However attaching specific ligands to the PEO-corona can produce complexes, which are both stable in solution and bind to target cells. The most efficient complexes were obtained when PEO in the cationic copolymer was replaced with membrane-active PEO-b-poly(propylene oxide)-b-PEO molecules (Pluronic 123). Such complexes exhibited elevated levels of transgene expression in liver following systemic administration in mice. To increase stability of the complexes, NanoGel™ carriers were developed that represent small hydrogel particles synthesized by cross-linking of PEI with double end activated PEO using an emulsification/solvent evaporation technique. Oligos are immobilized by mixing with NanoGel™ suspension, which results in the formation of small particles (80 nm). Oligos incorporated in NanoGel are able to reach targets within the cell and suppress gene expression in a sequence-specific fashion. Further, loaded NanoGel particles cross-polarized monolayers of intestinal cells (Caco-2) suggesting potential usefulness of these systems for oral administration of oligos. In conclusion the approaches using polycations for gene delivery for the design of gene transfer complexes that exhibit a very broad range of physicochemical and biological properties, which is essential for design of a new generation of more effective non-viral gene delivery systems.

Evidence for the role of promoter methylation in the regulation of MMP-9 gene expression.

Several studies have reported that elevated MMP-9 expression in lymphoma tissues correlated with tumor stage, grade, or prognosis. Because the DNA methylation pattern is critical for gene expression, detailed methylation analysis using genomic bisulfite sequencing was performed on a series of lymphoma cell lines. We found an inverse correlation between level of methylation of the MMP-9 promoter and the level of MMP-9 expression. Treating lymphoma cells with a DNA methylation inhibitor decreased MMP-9 promoter methylation and increased MMP-9 messenger RNA and protein secretion. This increased expression was potentiated by PMA, a known stimulus of MMP-9 in lymphoma cells. Finally, experiments using in vitro methylated MMP-9 promoter constructs confirmed the fact that DNA methylation exerts suppression on transcriptional activity. The results thus indicate that methylation may contribute to the transcriptional activity of the MMP-9 promoter.

A Novel Function for Galectin-7: Promoting Tumorigenesis by Up-regulating MMP-9 Gene Expression

Metastasis is a multistep process by which cancer cells, after acquiring several capabilities, spread to distinct sites in the body. It is the major cause of death in individuals suffering from cancer. We have recently identified galectin-7 as a new gene associated with the progression of T cell lymphoma toward a metastatic phenotype, suggesting a possible causal relationship. The present study was designed to investigate the role of galectin-7 in lymphoma. We found that the development of thymic lymphoma was accelerated when induced by lymphoma cells overexpressing galectin-7. Moreover, transfection of an expression vector containing the galectin-7 gene in low metastatic lymphoma cells increased their metastatic behavior and confers these cells with the new ability to overcome the resistance of intercellular adhesion molecule-1-deficient mice to lymphoma dissemination. Finally, we provide data suggesting that galectin-7 modulates the aggressive behavior of lymphoma cells by controlling the expression of metastatic genes, such as MMP-9. This hypothesis is based on the following evidence: (a) galectin-7 transfectants have higher levels of MMP-9 expression, (b) addition of beta-lactose completely inhibits expression of MMP-9 by galectin-7 transfectants, and (c) recombinant forms of galectin-7 induces the expression of MMP-9 in both mouse and human lymphoma cells. Our results have uncovered the existence of a previously undescribed activity, the promotion of cancer cell malignancy, to galectin-7.

Emerging features in the regulation of MMP-9 gene expression for the development of novel molecular targets and therapeutic strategies.

Matrix metalloproteinase 9 (MMP-9; gelatinase B) belongs to the subfamily of MMPs that play an important role in tissue remodelling in normal and pathological inflammatory processes. MMP-9 is a major secretion product of macrophages and a component of cytoplasmic granules of neutrophils. The enzyme is also secreted by lymphocytes and stromal cells upon stimulation by inflammatory cytokines, or upon delivery of bi-directional activation signals following integrin-mediated cell-cell or cell-extracellular matrix (ECM) contacts. Since the integrity of the tissue architecture is closely dependent of the delicate balance between MMPs and their inhibitors, excessive production of MMP-9 is linked to tissue damage and degenerative inflammatory disorders. As a consequence, regulation of gene transcription and tissue-specific expression of MMP-9 in normal and diseased states are being actively investigated to pave the way for new therapeutic targets. The objective of this article is to provide an overview of recent developments in the field of mmp-9 gene expression in different cell types, from the triggering of cell-surface receptors, to the activation of cytoplasmic mediators and transcription factors responsible for the activation of MMP-9 promoter. We will then focus on emerging evidence showing that transcription of mmp-9 gene can be controlled by epigenetic mechanisms. The usefulness of targeting the signalling pathways regulating MMP-9 expression for the treatment of inflammatory disorders and other indications will be discussed in light of these findings.

In vitro expression of MMP-2 and MMP-9 in glioma cells following exposure to inflammatory mediators

Progression of glioma is associated with local degenerative processes which are attributed to the activity of gelatinases. As glioma cells are candidate for secretion of these enzymes, we have studied in vitro the potential of cytokines (interleukin-1alpha (IL-1), tumor necrosis factor-alpha (TNFalpha) and transforming growth factor-beta (TGFbeta2)) to regulate the expression of gelatinase A and B (Gels A and B, respectively) in two glioma cells of human (A172) and rat origin (C6). We showed that IL-1 and TNFalpha both induced gene expression and protein secretion of Gel B in both cell lines, as revealed by RT-PCR and gelatin zymography, respectively. In C6 cells, TNFalpha had no effect on Gel A constitutive expression while IL-1 increased its production, but only at high doses. We have also demonstrated that TGFbeta2 inhibited both IL-1- or TNFalpha-induced gene expression and Gel B production in a dose-dependent manner but had no effect on Gel A secretion. The effect of TGFbeta2 on Gel B secretion was reversed by phorbol myristate acetate (PMA). Taken together, these data suggest that IL-1, TNFalpha and TGFbeta2 tightly regulate Gel B secretion in glioma cells, an enzyme which is believed to play an important role in the local invasion of brain tissue by tumor cells.

Acidic extracellular pH increases calcium influx‐triggered phospholipase D activity along with acidic sphingomyelinase activation to induce matrix metalloproteinase‐9 expression in mouse metastatic melanoma

Acidic extracellular pH is a common feature of tumor tissues. We have reported that culturing cells at acidic pH (5.4–6.5) induced matrix metalloproteinase‐9 expression through phospholipase D, extracellular signal regulated kinase 1/2 and p38 mitogen‐activated protein kinases and nuclear factor‐κB. Here, we show that acidic extracellular pH signaling involves both pathways of phospholipase D triggered by Ca2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA‐AM [1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid tetrakis (acetoxymethyl) ester], a chelator of intracellular free calcium, and the voltage dependent Ca2+ channel blockers, mibefradil (for T‐type) and nimodipine (for L‐type), dose‐dependently inhibited acidic extracellular pH‐induced matrix metalloproteinase‐9 expression. Intracellular free calcium concentration ([Ca2+]i) was transiently elevated by acidic extracellular pH, and this [Ca2+]i elevation was repressed by EGTA and the voltage dependent Ca2+ channel blockers but not by phospholipase C inhibitor, suggesting that acidic extracellular pH increased [Ca2+]i through voltage dependent Ca2+ channel. In contrast, SR33557, an L‐type voltage dependent Ca2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated matrix metalloproteinase‐9 induction but did not affect calcium influx. We found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors (perhexiline and desipramine) and siRNA targeting aSMase/smpd1 could inhibit acidic extracellular pH‐induced matrix metalloproteinase‐9 expression. BAPTA‐AM reduced acidic extracellular pH‐induced phospholipase D but not acidic sphingomyelinase acitivity. The acidic sphingomyelinase inhibitors did not affect the phosphorylation of extracellular signal regulated kinase 1/2 and p38, but they suppressed nuclear factor‐κB activity. These data suggest that the calcium influx‐triggered phospholipase D and acidic sphingomyelinase pathways of acidic extracellular pH induced matrix metalloproteinase‐9 expression, at least in part, through nuclear factor‐κB activation.

ICAM-1 isoforms: specific activity and sensitivity to cleavage by leukocyte elastase and cathepsin G

The extracellular moiety of ICAM‐1 consists of five Ig‐like domains, the first and third domains mediating adhesion to integrin ligands. The ICAM‐1 gene, however, gives rise to the expression of five alternative splice variants containing two, three, or four Ig‐like domains. In this work, we have investigated whether the rearrangement of the architecture of ICAM‐1 affects its structural properties and function. We showed that, in contrast to the common form, all alternative isoforms of ICAM‐1 were susceptible to cleavage by leukocyte elastase and cathepsin G. We found that the length of an isoform did not influence the susceptibility to proteolysis. The molecular diversity provided by the skipping of entire Ig domains and the level of expression on the APC, however, significantly influenced their ability to potentiate the proliferation of T cells. Finally, we found that the expression of minor ICAM‐1 isoforms encoding the third Ig‐like domains was sufficient to sustain neutrophil infiltration in the liver and confer exon‐5‐targeted ICAM‐1‐deficient mice susceptibility to LPS‐induced septic shock. These findings not only demonstrate that ICAM‐1 isoforms are fully functional, but support the concept that alternative RNA splicing in the Ig superfamily may fulfill distinct roles during the development of the immune response.

GELATINASE B (MMP-9), BUT NOT ITS INHIBITOR (TIMP-1), DICTATES THE GROWTH RATE OF EXPERIMENTAL THYMIC LYMPHOMA

Dysregulation of metalloproteinase production at tumor sites contributes to the modification of local stromal tissue necessary for tumor development. Gelatinase B (matrix metalloproteinase‐9, MMP‐9) is one of the key enzymes that have been associated with the progression of several tumors. Paradoxically, MMP‐9 expression by tumor cells, most notably by lymphoma cells, is concomitant with the expression of its physiological inhibitor, TIMP‐1. Not only are both genes often co‐expressed in the most aggressive forms of lymphomas but also both are up‐regulated upon contact with stromal cells. Since TIMP‐1 is known to regulate growth in several cell types and some aggressive lymphoma cells express TIMP‐1 constitutively without MMP‐9, it is unclear whether the over‐expression of MMP‐9 is counterbalanced by TIMP‐1 and whether TIMP‐1 expression alone could favor the development of lymphoma. To gain further insight into the respective roles of MMP‐9 and TIMP‐1 in lymphoma, we generated lymphoma cell lines expressing constitutively high levels of MMP‐9 or TIMP‐1 and compared these cells for the ability to form thymic lymphoma in vivo. Moreover, we generated lymphoma cell lines expressing constitutively high levels of both MMP‐9 and TIMP‐1 to reproduce the net physiological balance resulting from the expression of both genes simultaneously and to determine which gene overrides the other. Our results show that mice injected with lymphoma cells expressing MMP‐9 constitutively developed thymic lymphoma more rapidly than those injected with control lymphoma cells. Over‐expression of TIMP‐1 alone did not significantly influence tumor progression of lymphoma nor did it delay the capacity of MMP‐9 to accelerate the development of thymic lymphoma. Int. J. Cancer 82:743–747, 1999.