Eduardo Osinaga

Targeting Galectin-1 Overcomes Breast Cancer-Associated Immunosuppression and Prevents Metastatic Disease

Galectin-1 (Gal1), an evolutionarily conserved glycan-binding protein, contributes to the creation of an immunosuppressed microenvironment at sites of tumor growth. In spite of considerable progress in elucidating its role in tumor-immune escape, the mechanisms underlying the inhibitory functions of Gal1 remain obscure. Here, we investigated the contribution of tumor Gal1 to tumor growth, metastasis, and immunosuppression in breast cancer. We found that the frequency of Gal1(+) cells in human breast cancer biopsies correlated positively with tumor grade, while specimens from patients with benign hyperplasia showed negative or limited Gal1 staining. To examine the pathophysiologic relevance of Gal1 in breast cancer, we used the metastatic mouse mammary tumor 4T1, which expresses and secretes substantial amounts of Gal1. Silencing Gal1 expression in this model induced a marked reduction in both tumor growth and the number of lung metastases. This effect was abrogated when mice were inoculated with wild-type 4T1 tumor cells in their contralateral flank, suggesting involvement of a systemic modulation of the immune response. Gal1 attenuation in 4T1 cells also reduced the frequency of CD4(+)CD25(+) Foxp3(+) regulatory T (T(reg)) cells within the tumor, draining lymph nodes, spleen, and lung metastases. Further, it abrogated the immunosuppressive function of T(reg) cells and selectively lowered the expression of the T-cell regulatory molecule LAT (linker for activation of T cells) on these cells, disarming their suppressive activity. Taken together, our results offer a preclinical proof of concept that therapeutic targeting of Gal1 can overcome breast cancer-associated immunosuppression and can prevent metastatic disease.

Heme oxygenase-1 inhibits rat and human breast cancer cell proliferation: mutual cross inhibition with indoleamine 2,3-dioxygenase

Heme oxygenase‐1 (HO‐1) is the rate limiting enzyme of heme catabolism whereas indoleamine 2,3 dioxygenase (IDO) catabolizes tryptophan through the kynurenine pathway. We analyzed the expression and biological effects of these enzymes in rat and human breast cancer cell lines. We show that rat (NMU and 13762) but not human cells (MCF‐7 and T47D) express HO‐1. When overexpressed, we found this enzyme to have anti‐proliferative and proapoptotic effects by antioxidant mechanisms in these four cell lines. We show that IDO is expressed by rat and human breast cancer cells. IDO inhibition with 1‐MT and siRNA leads to diminished proliferation in rat cells. In contrast, HO‐1 negative human cell lines increase proliferation upon IDO inhibition. Since we also demonstrate that IDO inhibits the anti‐proliferative HO‐1, we propose that IDO has opposite effects on proliferation depending on the coexpression or not of HO‐1. We also describe that HO‐1 inhibits IDO at the post‐translational level through heme starvation. In vivo, we show that rat normal breast expresses HO‐1 and IDO. In contrast, N‐nitrosomethylurea‐induced breast adenocarcinomas only express IDO. In conclusion, we show that HO‐1/IDO cross‐regulation modulates apoptosis and proliferation in rat and human breast cancer cells.—Hill, M., Pereira, V., Chauveau, C., Zagani, R., Remy, S., Tesson, L., Mazal, D., Ubillos, L., Brion, R., Ashgar, K., Mashreghi, M. F., Kotsch, K., Moffett, J., Doebis, C., Seifert, M., Boczkowski, J., Osinaga, E., Anegon, I. Heme oxygenase‐1 inhibits rat and human breast cancer cells proliferation: mutual cross inhibition with indoleamine 2,3‐dioxygenase. FASEB J. 19, 1957–1968 (2005)

Antibody-dependent cell cytotoxicity synapses form in mice during tumor-specific antibody immunotherapy

Antibody-dependent cell cytotoxicity (ADCC) plays a critical role in monoclonal antibody (mAb)-mediated cancer therapy. ADCC, however, has not been directly shown in vivo but inferred from the requirement for IgG Fc receptors (FcγR) in tumor rejection in mice. Here, we investigated the mechanism of action of a Tn antigen-specific chimeric mAb (Chi-Tn), which binds selectively to a wide variety of carcinomas, but not to normal tissues, in both humans and mice. Chi-Tn mAb showed no direct toxicity against carcinomas cell lines in vitro but induced the rejection of a murine breast tumor in 80% to 100% of immunocompetent mice, when associated with cyclophosphamide. Tumor rejection was abolished in Fc receptors-associated γ chain (FcR-γ)-deficient mice, suggesting a role for ADCC. Indeed, tumor cells formed stable conjugates in vivo with FcR-γ chain-expressing macrophages and neutrophils in Chi-Tn mAb-treated but not in control mAb-treated mice. The contact zone between tumor cells and ADCC effectors accumulated actin, FcγR and phospho-tyrosines. The in vivo formed ADCC synapses were organized in multifocal supra-molecular activation clusters. These results show that in vivo ADCC mediated by macrophages and neutrophils during tumor rejection by Chi-Tn mAb involves a novel type of multifocal immune synapse between effectors of innate immunity and tumor cells.

Analysis of the fine specificity of Tn-binding proteins using synthetic glycopeptide epitopes and a biosensor based on surface plasmon resonance spectroscopy

Using synthetic Tn (GalNAc‐O‐Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti‐Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.

UDP-N-Acetyl-d-Galactosamine: Polypeptide N-Acetylgalactosaminyltransferase-6 as a New Immunohistochemical Breast Cancer Marker:

Mucin O-glycosylation is characterized in cancer by aberrant expression of immature carbohydrate structures (Tn, T, and sialyl-Tn antigens). The UDP-N-acetyl-d-galactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T) family enzymes regulate the initial steps of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. Considering that we recently found the ppGalNAc-T6 mRNA expressed in breast carcinomas, we produced a highly specific monoclonal antibody (MAb T6.3) to assess the expression profile of ppGalNAc-T6 protein product in breast tissues. The expression of ppGalNAc-T6 by breast carcinoma cells was confirmed on MCF-7 and T47D cell lines. In formalin-fixed tissues, ppGalNAc-T6 expression was observed in 60/74 (81%) breast cancers, 21/23 (91.3%) adjacent ductal carcinoma in situ (DCIS), 4/20 benign breast lesions (2/2 sclerosing adenosis and 2/13 fibroadenoma), and in 0/5 normal breast samples. We observed a statistically significant association of ppGalNAc-T6 expression with T1 tumor stage. This fact, as well as the observation that ppGalNAc-T6 was strongly expressed in sclerosing adenosis and in most DCIS, suggests that ppGalNAc-T6 expression could be an early event during human breast carcinogenesis. Considering that an abnormal O-glycosylation greatly contributes to the phenotype and biology of breast cancer cells, ppGalNAc-T6 expression could provide new insights about breast cancer glycobiology.

The crystal structure of a plant lectin in complex with the Tn antigen

The structure of the tetrameric Vicia villosa isolectin B4 (VVLB4) in complex with a cancer antigen, the Tn glycopeptide (GalNAc‐O‐Ser), was determined at 2.7 Å resolution. The N‐acetylgalactoside moiety of the ligand binds to the primary combining site of VVLB4 in a similar way as observed for other Gal/GalNAc‐specific plant lectins. The amino acid moiety of the Tn antigen is largely exposed to the solvent and makes few contacts with the protein. The structure of the complex provides a framework to understand the differences in the strength of VVLB4 binding to different sugars and emphasizes the role of a single protein residue, Tyr127, as a structural determinant of Tn‐binding specificity.

Silencing survivin gene expression promotes apoptosis of human breast cancer cells through a caspase‐independent pathway

Survivin is recognized as an attractive target in cancer therapy because of its selective overexpression in the majority of tumors. Upregulated expression of this protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival. In this study, we assessed the efficacy of two survivin‐specific small interfering RNA (siRNA) constructs to inhibit T47D human breast cancer cell growth. After siRNA transfection, T47D cells showed a significant reduction in proliferation and survival exhibiting clear signs of apoptosis. pSil_1 that targeted exon 1 exhibited a stronger inhibitory effect on cell growth, and increased cell apoptosis compared to pSil_30 that targeted exon 4. Cell apoptosis was found to be mediated by translocation of the mitochondrial apoptosis inducing factor (AIF), while no changes were observed in caspase‐3 activation and Bid cleavage. Thus, silencing survivin expression using siRNA strategies represents a suitable therapeutic approach to selectively modulate the survival and growth of human breast cancer cells. J. Cell. Biochem. 105: 381–390, 2008.

Mucin-type O-glycosylation in helminth parasites from major taxonomic groups: Evidence for widespread distribution of the TN antigen (Galnac-SER/THR) and identification of UDP-Galnac: Polypeptide N-acetylgalactosaminyltransferase activity

This article focuses on the initiation pathway of mucin-type O-glycosylation in helminth parasites. The presence of the GalNAc-O-Ser/Thr structure, also known as Tn antigen, a truncated determinant related to aberrant glycosylation in mammal cells, and the activity of the UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferase (ppGaNTase), the enzyme responsible for its synthesis, were studied in species from major taxonomic groups. Tn reactivity was determined in extracts from Taenia hydatigena, Mesocestoides corti, Fasciola hepatica, Nippostrongylus brasiliensis, and Toxocara canis using the monoclonal antibody 83D4. The Tn determinant was revealed in all preparations, and multiple patterns of Tn-bearing glycoproteins were observed by immunoblotting. Additionally, the first evidence that helminth parasites express ppGaNTase activity was obtained. This enzyme was studied in extracts from Echinococcus granulosus, F. hepatica, and T. canis by measuring the incorporation of UDP-(3H)GalNAc to both deglycosylated ovine syalomucin (dOSM) and synthetic peptide sequences derived from tandem repeats of human mucins. Whereas significant levels of ppGaNTase activity were detected in all the extracts when dOSM was used as a multisite acceptor, it was only observed in F. hepatica and E. granulosus extracts when mucin-derived peptides were used, suggesting that T. canis ppGaNTase enzyme(s) may represent a member of the gene family with a more restricted specificity for worm O-glycosylation motifs. The widespread expression of Tn antigen, capable of evoking both humoral and cellular immunity, strongly suggests that simple mucin-type O-glycosylation does not constitute an aberrant phenomenon in helminth parasites.

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 6 (ppGalNAc-T6) mRNA as a potential new marker for detection of bone marrow-disseminated breast cancer cells.

The evaluation of disseminated epithelial tumor cells in patients with early stages of breast cancer has generated considerable interest because of its potential association with poor clinical outcome. Considering that O‐glycosylation pathways are frequently altered in breast cancer, we performed this work to evaluate the potential usefulness of UDP‐N‐acetyl‐D‐galactosamine:polypeptide N‐acetylgalactosaminyltransferases (ppGalNAc‐Ts) (a family of glycosyltransferases which catalyze the first key step of mucin‐type O‐glycosylation) to detect disseminated cells in bone marrow samples from patients with operable breast cancer. Using RT‐PCR assays, we studied the gene expression of 9 enzymes (ppGalNAc‐T1–T9). Among the ppGalNAc‐Ts expressed by breast tumors (‐T1, ‐T2, ‐T3, ‐T6 and ‐T7), the best specificity (negative results on all PBMN cell samples from healthy donors) was shown for ppGalNAc‐T6. Thus, we selected this enzyme as a target gene for further evaluation. ppGalNAc‐T6 mRNA was found in 22/25 (88%) breast cancer samples, in all 3 human breast cancer cell lines evaluated (MCF‐7, ZR75‐1 and T47D), in 1/30 (3%) PBMN cells and 0/19 bone marrow samples obtained from patients without cancer. Using this method, 22/61 (36%) patients with breast cancer, who underwent curative surgery, showed positive ppGalNAc‐T6 mRNA in bone marrow aspirates obtained prior to surgery, including 11/34 patients with stage‐I or ‐II, without histopathological lymph node involvement. In a preliminary follow‐up evaluation, 19/61 patients experienced recurrence of the disease. ppGalNAc‐T6 was positive in 11/19 (57.9%) of these patients. Interestingly, in the group of patients without lymph node involvement, disease recurrence was observed in 54.5% of patients who showed ppGalNAc‐T6 mRNA‐positive bone marrow aspirates and only in 4.3% of patients when ppGalNAc‐T6 was negative (p = 0.014). These results indicate that ppGalNAc‐T6 mRNA could be a specific marker applicable to the molecular diagnosis of breast cancer cells dissemination.

Detection of bone marrow-disseminated breast cancer cells using an RT-PCR assay of MUC5B mRNA.

The evaluation of disseminated epithelial tumor cells in breast cancer patients has generated considerable interest due to its potential association with disease recurrence. Our work was performed to analyze the usefulness of 5 mucin genes expression (MUC2, MUC3, MUC5B, MUC6 and MUC7), using RT‐PCR assays, to detect disseminated cancer cells in patients with operable breast cancer. The highest frequencies of positive RT‐PCR tests in breast tumor extracts were observed for MUC5B (7/15) and MUC7 (5/12). The best specificity, negative results on all peripheral blood mononuclear (PBMN) cell samples from healthy donors, were shown for MUC2, MUC5B and MUC6 RT‐PCR assays. Thus, we selected MUC5B as a target gene for further evaluation. Using a nested RT‐PCR, MUC5B mRNA transcripts were detected in 16/31 primary breast tumors (but not in 36 samples of normal PBMN cells) and in the human MCF‐7 breast cancer cell line but not in BT20, MDA, T47D and ZR‐75 breast cancer cell lines, indicating that MUC5B mRNA is expressed in a population of breast cancer cells. Using this method, 9/46 patients (19.5%) who underwent curative surgery showed positive MUC5B mRNA in bone marrow aspirates obtained prior to surgery, including 5/24 patients (20.8%) with stage I or II breast cancer, without histopathologic lymph node involvement. These results indicate that MUC5B mRNA could be a specific marker applicable to the molecular diagnosis of breast cancer cell dissemination. A comparative evaluation between MUC5B mRNA, cytokeratin 19 (CK19) mRNA and carcinoembryonic antigen (CEA) mRNA in all bone marrow aspirates suggests a putative complementation for molecular detection of disseminated carcinoma cells. Considering that breast cancer is characterized by a great phenotypic heterogeneity, the use of multimarker approach could contribute to tumor cell detection in bone marrow and blood.