Daniel Mazal

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)

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.

Immunohistochemical Analysis of MUC5B Apomucin Expression in Breast Cancer and Non-malignant Breast Tissues

A deregulation of several MUC genes (MUC1, MUC2, MUC3, MUC5AC, and MUC6) was previously demonstrated in breast carcinomas. Considering that recently we found the “non-mammary” MUC5B mRNA in primary breast tumors (Berois et al. 2003), we undertook the present study to evaluate the expression profile of MUC5B protein product in breast tissues, using LUM5B-2 antisera raised against sequences within the non-glycosylated regions of this apomucin. Expression of MUC5B by breast cancer cells was confirmed by immunocytochemistry, in situ hybridization, and Western blot on MCF-7 cancer cells. Using an immunohistochemical procedure, MUC5B apomucin was detected in 34/42 (81%) primary breast tumors, in 13/14 (92.8%) samples of non-malignant breast diseases, in 8/19 (42.1%) samples of normal-appearing breast epithelia adjacent to cancer, and in 0/5 normal control breast samples. The staining pattern of MUC5B was very different when comparing breast cancer cells (cytoplasmic) and non-malignant breast cells (predominantly apical and in the secretory material). We analyzed MUC5B mRNA expression using RT-PCR in bone marrow aspirates from 22/42 patients with breast cancer to compare with MUC5B protein expression in the primary tumors. Good correlation was observed because the six MUC5B-positive bone marrow samples also displayed MUC5B expression in the tumor. Our results show, for the first time at the protein level, that MUC5B apomucin is upregulated in breast cancer. Its characterization could provide new insights about the glycobiology of breast cancer cells.

Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.

A novel clinically relevant animal model for studying galectin-3 and its ligands during colon carcinogenesis.

Galectin-3 (Gal-3) is a multifunctional protein that plays different roles in cancer biology. To better understand the role of Gal-3 and its ligands during colon carcino-genesis, we studied its expression in tumors induced in rats treated with 1,2-dimethylhydrazine (DMH) and in human tissues. Normal colon from untreated rats showed no staining using two specific monoclonal antibodies. In contrast, morphologically normal colon from DMH-treated rats and dysplastic aberrant crypt foci were strongly stained, indicating that increased Gal-3 expression is an early event during the neoplastic transformation in colon cells. Gal-3 was weakly expressed in adenocarcinomas. Overall, the Gal-3 expression pattern observed in the DMH rat model closely resembles that displayed by human colon stained with the same antibodies. We also found that Gal-3 phosphorylation diminishes in serines while increasing in tyrosines during rat colon carcinogenesis. Finally, we showed that Gal-3–ligands expression is strikingly similar in rat and human malignant colon and in non-malignant tissues. In conclusion, the DMH-induced rat colon cancer model displays expression patterns of Gal-3 and its ligands very similar to those observed in human samples. This animal model should contribute to clarifying the role of Gal-3 in colon carcinogenesis and also to finding effective preventive cancer agents based on Gal-3 targeting.

Immunobiological characterization of N-nitrosomethylurea-induced rat breast carcinomas: tumoral IL-10 expression as a possible immune escape mechanism

Improvement of immunotherapy-based protocols in cancer requires a better understanding of tumor microenvironment and tumor–host interaction. Stromal and immune cells and molecules such as cytokines, chemokines, growth factors and metalloproteases mediate tumor–host interaction determining, at least in part, tumor development. In the present study, we used an immunohistochemical approach to explore leukocyte sub-populations, cytokine profiles and costimulatory molecule expression in rat N-Nitrosomethylurea (NMU)-induced breast tumors. Our results show a strong leukocyte infiltration mainly composed of macrophages and TCR αβ positive T cells. We observed a weak expression of costimulatory molecules (CD80, CD86) and an absence of inflammatory cytokines (IFNγ, TNFα, IP-10) and lymphocyte activation markers (CD25). Interestingly, this immunosuppressed status could be a consequence of IL-10 expression by malignant cells, as demonstrated by immunohistology and western blot analysis, which seems to be an early event during mammary carcinogenesis. Analysis of a cell line derived from an NMU-induced rat breast tumor showed that this cell line also expresses IL-10. This study shows that the NMU model of rat breast cancer could be used to evaluate different immune based therapies as well as to study the role of IL-10 in breast cancer. Furthermore, this rat breast cancer model shows an immunohistological profile similar to that found in human cancer and the fact that it develops like spontaneously arising malignancies make it interesting as a cancer model in immunobiology.

Polypeptide‑GalNAc‑T6 expression predicts better overall survival in patients with colon cancer

Colorectal carcinoma (CRC) is the second leading cause of cancer mortality worldwide. O-glycosylated mucins at the cell surface of colonic mucosa exhibit alterations in cancer and are involved in fundamental biological processes, including invasion and metastasis. Certain members of the GalNAc-transferase family may be responsible for these changes and are being investigated as novel biomarkers of cancer. In the present study the prognostic significance of GalNAc-T6 was investigated in patients with CRC patients. GalNAc-T6 expression was observed in all three colon cancer cell lines analyzed by reverse transcription-polymerase chain reaction, immunofluorescence and flow cytometry. A cohort of 81 colon cancer specimens was analyzed by immunohistochemical staining using MAb T6.3. It was demonstrated that GalNAc-T6 was expressed in 35/81 (43%) cases of colon cancer but not in the normal colonic mucosa. No association was observed with the clinical-pathologic parameters. However, patients expressing GalNAc-T6 had a significantly increased overall survival (median, 58 months; P<0.001) compared with GalNAc-T6 negative patients, especially those with advanced disease. These results suggest that GalNAc-T6 expression predicts an improved outcome in patients with CRC. The molecular mechanism underlying the less aggressive behavior of colon cancer cells expressing GalNAc-T6 remains to be elucidated.