Marilyne Labrie

Epigenetic regulation of mmp-9 gene expression.

Matrix metalloproteinase 9 (MMP-9) is one of the most studied enzymes in cancer. MMP-9 can cleave proteins of the extracellular matrix and a large number of receptors and growth factors. Accordingly, its expression must be tightly regulated to avoid excessive enzymatic activity, which is associated with disease progression. Although we know that epigenetic mechanisms play a central role in controlling mmp-9 gene expression, predicting how epigenetic drugs could be used to suppress mmp-9 gene expression is not trivial because epigenetic drugs also regulate the expression of key proteins that can tip the balance towards activation or suppression of MMP-9. Here, we review how our understanding of the biology and expression of MMP-9 could be exploited to augment clinical benefits, most notably in terms of the prevention and management of degenerative diseases and cancer.

Intracellular galectins in cancer cells: Potential new targets for therapy (Review)

Dysregulation of galectin expression is frequently observed in cancer tissues. Such an abnormal expression pattern often correlates with aggressiveness and relapse in many types of cancer. Because galectins have the ability to modulate functions that are important for cell survival, migration and metastasis, they also represent attractive targets for cancer therapy. This has been well-exploited for extracellular galectins, which bind glycoconjugates expressed on the surface of cancer cells. Although the existence of intracellular functions of galectins has been known for many years, an increasing number of studies indicate that these proteins can also alter tumor progression through their interaction with intracellular ligands. In fact, in some instances, the interactions of galectins with their intracellular ligands seem to occur independently of their carbohydrate recognition domain. Such findings call for a change in the basic assumptions, or paradigms, concerning the activity of galectins in cancer and may force us to revisit our strategies to develop galectin antagonists for the treatment of cancer.

Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53

Galectin-7 was initially described as a marker of epithelial differentiation expressed in the stratified epithelium of various tissues. Like other members of the galectin family, its expression level is often significantly altered in cancer cells. In breast cancer, its expression is significantly augmented in aggressive molecular subtypes, most notably in estrogen receptor-negative tumors and in cell lines with a basal-like phenotype. Studies using experimental mouse models have further shown high expression of galectin-7 was sufficient to increase the metastatic behavior of poorly metastatic breast cancer cells, rendering them more resistant to apoptosis. This expression pattern in breast cancer cells is unexpected because galectin-7 was originally identified as a p53-induced gene. To address this paradox, we have examined the molecular mechanisms regulating galectin-7 in breast cancer cells. Our results showed that transfection of breast cancer cells with expression vectors encoding mutant p53 was sufficient to induce galectin-7 at both mRNA and protein levels. Doxorubicin treatment of breast cancer cells harboring a mutant p53 also induced galectin-7. This induction was specific since knockdown of endogenous mutant p53 inhibited doxorubicin-induced galectin-7 expression. The p53-induced galectin-7 expression in breast cancer cells correlated with increased NF-κB activity and was inhibited by NF-κB inhibitors, indicating that the ability of mutant p53 to induce galectin-7 was dependent on NF-κB activity. The implication of NF-κB was further supported by data showing that NF-κB bound to the endogenous galectin-7 promoter and that TNFα-induced galectin-7 expression was abolished by NF-κB inhibitors. Taken together, our data provide an explanation to the observed high galectin-7 expression levels in cancer cells and suggest that galectin-7 could be part of a common pathway used by mutant p53 to promote cancer progression.

Galectin signatures contribute to the heterogeneity of breast cancer and provide new prognostic information and therapeutic targets

Because of their ability to induce local immunosuppression and to confer cancer cells with resistance to apoptosis, members of the galectin family are emerging as a new class of actionable targets in cancer. Unfortunately, we have yet to obtain a clear picture of the galectin signatures in cancer cells and the surrounding tumor microenvironment. The aim of this study was to provide the first detailed analysis of the galectin signature in molecular subtypes of breast cancer. Expression signatures of galectins were obtained at the mRNA and protein levels. A particular attention was paid to stromal versus epithelial staining and to subcellular compartmentalization. Analysis of the stromal signature showed that gal-1, -3, -9-positive stroma were preferentially found in triple-negative (TN) and HER2 subtypes. In cancer cells, gal-1, −3, -8, and -9 showed a dual expression pattern, being found either in the cytosol or in the cytosol and the nucleus. TN patients with gal-8-positive nuclei had significantly better disease-free survival (DFS), distant-disease-free survival (DDFS), and overall survival (OS). In contrast, high expression of nuclear gal-1 correlated with poor DDFS and OS. TNBC patients who were positive for both nuclear gal-1 and gal-8 had 5-year DFS and DDFS of 100%, suggesting a dominance of the gal-8 phenotype. Overall, the results indicate that specific galectin expression signatures contribute to the phenotypic heterogeneity of aggressive subtypes of breast cancer. Our data also suggest that galectins have clinical utility as indicators of disease progression and therapeutic targets in aggressive molecular subtypes of breast cancer.

The CCAAT/enhancer-binding protein beta-2 isoform (CEBPβ-2) upregulates galectin-7 expression in human breast cancer cells

Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ) in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2), the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.

Cytosolic galectin-7 impairs p53 functions and induces chemoresistance in breast cancer cells

BackgroundResistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 (gal-7) was recently shown to be specifically expressed in basal-like but not in luminal subtypes of human breast cancer.MethodsWe generated a mutant form of gal-7 (R74S). Arginine 74 is the structural equivalent of arginine 186 found in human galectin-3. Mutation R186S was previously shown to abolish the biological function of galectin-3.ResultsMutation of arginine 74 induced only limited and local changes to the gal-7 fold. Recombinant forms of R74S and wtgal-7 were also equally effective at forming dimers in solution. Analysis of the thermodynamic parameters by isothermal titration calorimetry (ITC) indicated, however, that binding of lactose to gal-7 was inhibited by the R74S mutation. Using confocal microscopy and electron microscopy, we confirmed the expression of gal-7 in the cytosolic and nuclear compartments of breast cancer cells and the ability of gal-7 to translocate to mitochondria. The mutation at position 74, however, greatly reduced the expression of gal-7 in the nuclear and mitochondrial compartments. Interestingly, cells expressing mutated gal-7 were equally if not even more resistant to drug-induced apoptosis when compared to cells expressing wtgal-7. We also found that both wtgal-7 and R74S inhibited dox-induced PARP-1 cleavage and p53 protein expression. The inhibition of p53 correlated with a decrease in p21 protein expression and CDKN1A mRNA. Furthermore, analysis of nuclear and cytoplasmic fractions showed that both wild type and R74S mutant gal-7 inhibited p53 nuclear translocation, possibly by increasing degradation of cytosolic p53.ConclusionsThese findings pose a challenge to the paradigm that has guided the design of galectin-specific inhibitors for the treatment of cancer. This study suggests that targeting CRD-independent cytosolic gal-7 in breast cancer cells may be a valuable strategy for the treatment of this disease. Our study will thus complement efforts towards improving selectivity of targeted anticancer agents.

A Mutation in the Carbohydrate Recognition Domain Drives a Phenotypic Switch in the Role of Galectin-7 in Prostate Cancer

The observation that galectin-7 (gal-7) is specifically expressed in mammary myoepithelial (basal) cells prompted us to investigate whether this protein is expressed in the basal cells of other tissues. Given that breast and prostate cancer have remarkable underlying biological similarities and given the important roles of basal cells in prostate cancer, we examined the expression patterns and role of gal-7 in human prostate cancer. Using tissue microarray, we found that although gal-7 is readily expressed in basal cells in normal prostate tissue, it is downregulated in prostate cancer (PCa) cells. De novo expression of gal-7 in prostate cancer cells increases their sensitivity to apoptosis in response to etoposide and cisplatin. The assessment of a carbohydrate-recognition domain (CRD)-defective mutant form of gal-7 (R7S) showed that the ability of this protein to modulate apoptosis was independent of its CRD activity. This activity was also independent of its ability to translocate to the mitochondrial and nuclear compartments. However, CRD activity was necessary to inhibit the invasive behaviors of prostate cancer cells. In vivo, gal-7 overexpression in PCa cells led to a modest yet significant reduction in tumor size, while its CRD-defective mutant form significantly increased tumor growth compared to controls. Taken together, these results suggest that although de novo expression of gal-7 may be an interesting means of increasing the tumorigenic phenotypes of PCa cells, alterations in the CRD activity of this protein drive a phenotypic switch in its role in PCa cells. This CRD-independent activity represents a paradigm shift in our understanding of the functions of galectin. The R74S model will be useful to distinguish CRD-dependent and CRD-independent functions of gal-7 in cancer progression.

Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake

Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver.

Design of a peptidic inhibitor that targets the dimer interface of a prototypic galectin

Galectins are small soluble lectins that bind β-galactosides via their carbohydrate recognition domain (CRD). Their ability to dimerize is critical for the crosslinking of glycoprotein receptors and subsequent cellular signaling. This is particularly important in their immunomodulatory role via the induction of T-cell apoptosis. Because galectins play a central role in many pathologies, including cancer, they represent valuable therapeutic targets. At present, most inhibitors have been directed towards the CRD, a challenging task in terms of specificity given the high structural homology of the CRD among galectins. Such inhibitors are not effective at targeting CRD-independent functions of galectins. Here, we report a new class of galectin inhibitors that specifically binds human galectin-7 (hGal-7), disrupts the formation of homodimers, and inhibits the pro-apoptotic activity of hGal-7 on Jurkat T cells. In addition to representing a new means to achieve specificity when targeting galectins, such inhibitors provide a promising alternative to more conventional galectin inhibitors that target the CRD with soluble glycans or other small molecular weight allosteric inhibitors.

Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

Galectins are moving closer to center stage in detecting glycosylation aberration in cancer cells. Here, we have investigated the expression of galectins in ovarian cancer (OC) and examined their potential as biomarkers in tissues and blood plasma samples of high grade serous ovarian carcinoma (HGSC) patients. In tissues, we found that increased protein expression of stromal gal-1 and epithelial gal-8/9 was associated with a poor response to treatment of HGSC patients. Gal-8/9 were both independent predictors of chemoresistance and overall survival (OS), respectively. This galectin signature increased the predictive value of the cancer antigen 125 (CA125) on 5-year disease-free survival (DFS), post-chemotherapy treatment and 5-year OS. In CA125LOW patients, epithelial gal-9 was associated with a lower 5-year OS while stromal gal-1 and epithelial gal-8 were both associated with a lower 5-year DFS. Such negative predictive value of gal-8 and gal-9 was also found using plasma samples. In both cases, high plasma levels of gal-8 and gal-9 was associated with a lower OS and DFS. Overall, these data suggest that galectins may be promising biomarkers to identify subgroups of HGSC patients with poorer prognosis. Our study also contributes to better define the heterogeneity of the disease.