Frédéric van den Brûle

Distinct epigenetic changes in the stromal cells of breast cancers

Increasing evidence suggests that changes in the cellular microenvironment contribute to tumorigenesis, but the molecular basis of these alterations is not well understood. Although epigenetic modifications of the neoplastic cells in tumors have been firmly implicated in tumorigenesis, it is not known whether epigenetic modifications occur in the non-neoplastic stromal cells. To address this question in an unbiased and genome-wide manner, we developed a new method, methylation-specific digital karyotyping, and applied it to epithelial and myoepithelial cells, stromal fibroblasts from normal breast tissue, and in situ and invasive breast carcinomas. Our analyses showed that distinct epigenetic alterations occur in all three cell types during breast tumorigenesis in a tumor stage– and cell type–specific manner, suggesting that epigenetic changes have a role in the maintenance of the abnormal cellular microenvironment in breast cancer.

DECREASED EXPRESSION OF GALECTIN-3 IS ASSOCIATED WITH PROGRESSION OF HUMAN BREAST CANCER

Galectin‐3, a member of the β‐galactoside‐binding lectin family, is involved in several biological events including binding to the basement membrane glycoprotein laminin. Although the exact role of galectin‐3 during the interactions between cells and laminin is not yet known, it has recently been observed that its expression is down‐regulated at both the protein and the mRNA level in colon cancer tissues in correlation with progression of the disease. This study investigated the possibility that breast cancer cells might also exhibit decreased galectin‐3 expression in association with their aggressiveness. The expression of galectin‐3 was examined by immunoperoxidase staining, using a polyclonal antibody raised against recombinant galectin‐3, in a collection of 98 human breast lesions including 12 fibroadenomas, 15 fibrocystic disease lesions, 22 in situ carcinomas, and 49 infiltrating ductal carcinomas, 19 of which had positive axillary lymph nodes. Normal breast tissue adjacent to the lesions was present in 59 biopsies. Normal breast tissue expressed high levels (3+) of galectin‐3. High expression (2+ to 3+) was also found in most benign lesions examined. The expression of galectin‐3 was significantly decreased in in situ carcinoma and this down‐regulation was more pronounced in invasive ductal carcinoma, particularly when associated with infiltration of axillary lymph nodes. These data constitute the first observation that galectin‐3 is down‐regulated in breast cancer and suggest the decreased expression of this galactoside‐binding lectin is associated with the acquisition of the invasive and metastatic phenotype.

Expression of Galectins in Cancer: A Critical Review

A large body of literature has examined and described galectin expression in cancer. Discrepancies have been observed in the reported data, which hampered clear understanding of the expression profiles. This relates to the use of different types of methods that evaluate either global or specific gene expression in heterogeneous cancer tissue samples, type of antibodies used in immunohistochemistry and procedures of comparison of gene expression. In this manuscript, we review the main data concerning expression of galectins in human cancer. Only galectin-1 and galectin-3, the most abundant and examined galectins, will be examined here. Published in 2004.

Galectin-1 accumulation in the ovary carcinoma peritumoral stroma is induced by ovary carcinoma cells and affects both cancer cell proliferation and adhesion to laminin-1 and fibronectin

Galectin-1 (gal-1) is a 14-kDa laminin-binding galectin involved in several biologic events including regulation of cancer cell proliferation and adhesion to the matrix. In this study, we examined gal-1 expression in 30 human epithelial ovary carcinoma samples by Western and Northern blotting and by immunohistochemistry. Gal-1 mRNA levels were increased in more than 95% of the examined ovary carcinoma samples, compared with a wedge resection of a normal ovary. Immunohistochemical analysis of the samples demonstrated gal-1 expression in cancer epithelial cells from 17 of 30 samples, with a cytoplasmic pattern. Gal-1 immunostaining was significantly increased in the stroma associated with carcinoma cells compared with the normal, noninvaded stroma (p = 0.003). This pattern of expression was confirmed by examination of 12 other frozen epithelial ovary carcinomas, using in situ hybridization. Immunohistochemical staining of the specimens demonstrated colocalization of gal-1, laminin-1, and fibronectin. In vitro experiments were conducted to elucidate the potential biologic role of gal-1 in ovarian cancer progression. Gal-1 protein expression and release was detected in AZ364, SK-OV-3, and AZ224, but not in OVCAR-3, AZ419, and AZ382, human ovary carcinoma cell lines. Incubation of 84BR fibroblasts with conditioned media harvested from the ovary carcinoma cell lines induced an increased expression of gal-1 in the cultured fibroblasts in all cases except AZ419 and SK-OV-3. High concentrations of gal-1 (100 μg/ml) induced significantly decreased cell proliferation in all cell lines, as defined by bromodeoxyuridine incorporation. Additionally, recombinant gal-1 induced a dose-dependent increase in in vitro adhesion of AZ224, SK-OV-3, and AZ382 cells to laminin-1; adhesion to fibronectin was increased by gal-1 in OVCAR-3, AZ224, and SK-OV-3. No effect was observed in the other cases. Our data contribute to define a role for gal-1 during the interactions between human ovary carcinoma cells and host fibroblasts.

Dual activities of galectin-3 in human prostate cancer: tumor suppression of nuclear galectin-3 vs tumor promotion of cytoplasmic galectin-3.

Galectin-3, a multifunctional lectin, is involved during cancer progression. Previous observations showed that both cytosolic expression and nuclear exclusion of galectin-3 in human prostate cancer cells were associated to progression of the disease. In this study, we examined the biological roles of galectin-3 when expressed either in the nucleus or in the cytosol. LNCaP, a galectin-3-negative human prostate cancer cell line, was used to generate transfectants expressing galectin-3 either in the nucleus or in the cytosol. No changes in cell morphology, proliferation, attachment to laminin-1 or androgen dependency were observed. Cytoplasmic galectin-3 induced significantly increased Matrigel invasion, anchorage-independent growth and in vivo tumor growth and angiogenesis, and decreased inducible apoptosis. Surprisingly, nuclear galectin-3 affected these parameters in an opposite fashion with an overall antitumoral activity. Thus, our study demonstrates that galectin-3 exerts opposite biological activities according to its cellular localization: nuclear galectin-3 plays antitumor functions and cytoplasmic galectin-3 promotes tumor progression.

Expression of the 67-kD laminin receptor, galectin-1, and galectin-3 in advanced human uterine adenocarcinoma

Alterations of tumor cell interactions with laminin, a basement membrane glycoprotein, are consistent features of the invasive and metastatic phenotype. Qualitative and quantitative changes in the expression of cell surface laminin-binding proteins have been correlated with the ability of cancer cells to cross basement membranes during the metastatic cascade. Such phenotypic modifications are usually associated with poor prognosis. In this study, the authors examined the possibility that expression of three laminin-binding proteins, the 67-kD laminin receptor (67LR), galectin-1, and galectin-3, is altered in human endometrial cancer in a fashion similar to that reported in other carcinomas, such as breast, colon, and ovarian cancer. Twenty advanced uterine adenocarcinomas were analyzed for expression of these three molecules using immunoperoxidase staining and specific antibodies. The authors found a significant increase in the expression of the 67LR and galectin-1 in cancer cells compared with normal adjacent endometrium (P = .0004 and .0022, respectively). As observed in other carcinomas, a significant down-regulation of galectin-3 expression was found in endometrial cancer cells compared with normal mucosa (P = .02). In the galectin-3 positive tumors, galectin-3 was detected in the cytoplasm and/or nucleus of cancer cells. Interestingly, tumors in which galectin-3 was detected only in the cytoplasm were characterized by deeper invasion of the myometrium than lesions where galectin-3 was found both in nucleus and cytoplasm (P = .02). This study shows an alteration of nonintegrin laminin-binding protein expression in advanced human endometrial cancer. Further studies on larger populations should determine the prognostic value of the detection of these laminin-binding proteins in endometrial carcinoma. Inverse modulation of the 67LR and galectin-3 appears to be a phenotypical feature of invasive carcinoma.

Formation of the 67-kDa laminin receptor by acylation of the precursor

Even though the involvement of the 67‐kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full‐length gene encoding a 37‐kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67‐kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120‐kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP‐40‐lysis buffer whereas the 37LRP is NP‐40‐insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP‐40‐soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67‐ and the 120‐kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross‐reacting molecule. J. Cell. Biochem. 69:244–251, 1998.

Increased Expression of Galectin-1 in Carcinoma-Associated Stroma Predicts Poor Outcome in Prostate Carcinoma Patients

Galectin‐1, a member of the β‐galactoside‐binding galectin family, is a pleiotropic dimeric protein participating in a variety of normal and pathological processes, including cancer progression. Modulation of the interactions with the basement membrane glycoprotein laminin and induction of apoptosis in activated T lymphocytes are well‐known functions of this galectin. In this study, the expression of galectin‐1 was examined in 148 human primary prostate carcinoma samples. Immunohistochemical staining of paraffin sections of prostate tissues revealed that galectin‐1 was not detected in normal, PIN (prostatic intraepithelial neoplasia) or carcinoma cells, but accumulated in the stroma and associated fibroblasts. Galectin‐1 expression was significantly increased in the tumour‐associated stroma compared with the non‐neoplastic gland‐associated stroma in 21.3% of the cases (Mantel–Haenszel test, p=0.001; Wilcoxon signed rank test, p<0.0001). Increased galectin‐1 expression in the cancer‐associated stroma compared to the normal gland‐associated stroma (p=0.03) was identified by multivariate analysis as a strong independent predictor of prostate‐specific antigen (PSA) recurrence, just after the pathological stage (p<0.0001). The association between accumulation of galectin‐1 in the stroma of the malignant tissue and aggressiveness of the tumour adds weight to the body of evidence that identifies a role for galectin‐1 in the acquisition of the invasive phenotype. In addition to modulating cancer cell interactions with laminin, galectin‐1 accumulated around the cancer cells may act as an immunological shield by inducing activated T‐cell apoptosis. This exciting hypothesis warrants further investigation. Copyright

Expression of Lamp‐1 and Lamp‐2 and their interactions with galectin‐3 in human tumor cells

Lysosomal‐membrane‐associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell‐surface expression in tumor cells, with some data indicating that the adhesion of some cancer cells to the extracellular matrix is partly mediated by interactions between Lamps and E‐selectin and between Lamps and galectins (endogenous‐galactoside‐binding lectins). The present study examined the expression of Lamp‐1 and Lamp‐2 and their interactions with galectin‐3 in different human tumor cell lines. Indirect immunofluorescence staining revealed accumulation of Lamp molecules at the edges of A2058 human metastasizing melanoma cells suggesting that these glycoproteins could participate in cell adhesion. Flow cytometry showed the presence of cell‐surface Lamps in A2058, HT1080 (human fibrosarcoma) and CaCo‐2 (human colon‐adenocarcinoma) cells. Treatment with 2 mM sodium butyrate for 24 and 48 hr resulted in a significant increase in Lamps surface expression. A strong binding of A2058 to recombinant galectin‐3 was detected by FACS. The application of 2 and 5 mM butyrate for the same incubation period enhanced galectin‐3 binding to Lamps‐expressing cells. Our results support the idea that Lamps may be considered a new family of adhesive glycoproteins participating in the complex process of tumor invasion and metastasis. Int. J. Cancer 75:105–111, 1998.© 1998 Wiley‐Liss, Inc.

Galectin-1 expression in prostate tumor-associated capillary endothelial cells is increased by prostate carcinoma cells and modulates heterotypic cell–cell adhesion

Besides providing tumors with nutrients, newly formed capillaries constitute a potential escape route for tumor cells favoring metastatic dissemination, and constitute an access for the anti-tumoral host immune cells. Galectin-1, a soluble human lectin, is involved in numerous biological functions including cell–cell and cell–substrate interactions. In addition, galectin-1 is able to induce apoptosis of activated T-lymphocytes. In this study, we have examined galectin-1 expression in capillaries associated to the carcinoma cells or present in the remote non-tumoral stroma of 100 human prostate carcinoma samples by immunoperoxidase staining. Galectin-1 was expressed by endothelial cells from capillaries infiltrating the tumor tissue in 64% (64/100) of the cases. On the contrary, endothelial cells in the adjacent non-tumoral stroma expressed galectin-1 in very few cases (7/100). Increased frequency of galectin-1-positive capillaries in the tumor-associated compared to the tumor-free areas was observed in 63% of the cases. This striking contrast led us to set up an in vitro model to test whether tumor cells could induce galectin-1 expression by endothelial cells. Incubation of human umbilical vein endothelial cells with conditioned media from PC-3 or DU 145 prostate carcinoma cells led to a significant increase of galectin-1 protein expression (+32.97% and 37.91% P < 0.01 and P < 0.05, respectively). PC-3 conditioned medium also induced increased adhesion values of PC-3 cells to the endothelial cells (53.4 ± 4.7 vs. 38.5 ± 3.5 after 30 min; 66.6 ± 7.8 vs. 46.2 ± 6.4 after 60 min). An anti-galectin-1 antiserum abolished this modulation, and recombinant galectin-1 also induced increased adhesion values in a dose-dependent fashion. This effect was specific as no such modulations were observed using normal lymphocytes instead of PC-3 cells. Preferential galectin-1 expression in the endothelial cells close to the cancer cells could provide these latter with increased abilities to interact with the endothelial cells as well as a defense against the host immune system.