The role of Globo H and SSEA-4 in the development and progression of cancer, and their potential as therapeutic targets.

Glycans, chains of sugar molecules found conjugated to cell proteins and lipids, contribute to their growth, movement and differentiation. Aberrant glycosylation is a hallmark of several medical conditions including tumorigenesis. Glycosphingolipids (GSLs), consisting of glycans conjugated to a lipid (ceramide) core, are found in the lipid bilayer of eukaryotic cell membranes. GSLs, play an active role in cell processes. Several GSLs are expressed by human embryonic stem cells and have been found to be overexpressed in several types of cancer. In this review, we discuss the data, hypotheses and perspectives related to the GSLs Globo H and SSEA-4.

Novel monoclonal antibody L2A5 specifically targeting sialyl-Tn and short glycans terminated by alpha-2-6 sialic acids.

Incomplete O-glycosylation is a feature associated with malignancy resulting in the expression of truncated glycans such as the sialyl-Tn (STn) antigen. Despite all the progress in the development of potential anti-cancer antibodies, their application is frequently hindered by low specificities and cross-reactivity. In this study, a novel anti-STn monoclonal antibody named L2A5 was developed by hybridoma technology. Flow cytometry analysis showed that L2A5 specifically binds to sialylated structures on the cell surface of STn-expressing breast and bladder cancer cell lines. Moreover, immunoblotting assays demonstrated reactivity to tumour-associated O-glycosylated proteins, such as MUC1. Tumour recognition was further observed using immunohistochemistry assays, which demonstrated a high sensitivity and specificity of L2A5 mAb towards cancer tissue, using bladder and colorectal cancer tissues. L2A5 staining was exclusively tumoural, with a remarkable reactivity in invasive and metastasis sites, not detectable by other anti-STn mAbs. Additionally, it stained 20% of cases of triple-negative breast cancers, suggesting application in diseases with unmet clinical needs. Finally, the fine specificity was assessed using glycan microarrays, demonstrating a highly specific binding of L2A5 to core STn antigens and additional ability to bind 2-6-linked sialyl core-1 probes. In conclusion, this study describes a novel anti-STn antibody with a unique binding specificity that can be applied for cancer diagnostic and future development of new antibody-based therapeutic applications.

Enhancement of metastatic ability by ectopic expression of ST6GalNAcI on a gastric cancer cell line in a mouse model.

ST6GalNAcI is a sialyltransferase responsible for the synthesis of sialyl Tn (sTn) antigen which is expressed in a variety of adenocarcinomas including gastric cancer especially in advanced cases, but the roles of ST6GalNAcI and sTn in cancer progression are largely unknown. We generated sTn-expressing human gastric cancer cells by ectopic expression of ST6GalNAcI to evaluate metastatic ability of these cells and prognostic effect of ST6GalNAcI and sTn in a mouse model, and identified sTn carrier proteins to gain insight into the function of ST6GalNAcI and sTn in gastric cancer progression. A green fluorescent protein-tagged human gastric cancer cell line was transfected with ST6GalNAcI to produce sTn-expressing cells, which were transplanted into nude mice. STn-positive gastric cancer cells showed higher intraperitoneal metastatic ability in comparison with sTn-negative control, resulting in shortened survival time of the mice, which was mitigated by anti-sTn antibody administration. Then, sTn-carrying proteins were immunoprecipitated from culture supernatants and lysates of these cells, and identified MUC1 and CD44 as major sTn carriers. It was confirmed that MUC1 carries sTn also in human advanced gastric cancer tissues. Identification of sTn carrier proteins will help understand mechanisms of metastatic phenotype acquisition of gastric cancer cells by ST6GalNAcI and sTn.

Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells.

Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.

Elevated Golgi pH in breast and colorectal cancer cells correlates with the expression of oncofetal carbohydrate T-antigen.

Altered glycosylation has turned out to be a universal feature of cancer cells, and in many cases, to correlate with altered expression or localization of relevant glycosyltransferases. However, no such correlation exists between observed enzymatic changes and the expression of the oncofetal Thomsen-Friedenreich (T)-antigen, a core 1 (Gal-beta1 --> 3-GalNAc-ser/thr) carbohydrate structure. Here we report that T-antigen expression, instead, correlates with elevated Golgi pH in cancer cells. Firstly, using a Golgi-targeted green fluorescent protein (GT-EGFP) as a probe, we show that the medial/trans-Golgi pH (pHG) in a high proportion of breast (MCF-7) and colorectal (HT-29, SW-48) cancer cells is significantly more alkaline (pHG > or = 6.75) than that of control cells (pHG 5.9-6.5). The pH gradient between the cytoplasm and the Golgi lumen is also markedly reduced in MCF-7 cells, suggesting a Golgi acidification defect. Secondly, we show that T-antigen expression is highly sensitive to changes in Golgi pH, as only a 0.2 pH unit increase was sufficient to increase T-antigen expression in control cells. Thirdly, we found that T-antigen expressing MCF-7 cells have 0.3 pH units more alkaline Golgi pH than non-expressing MCF-7 cells. Fourthly, in all cell types examined, we observed significant correlation between the number of T-antigen expressing cells and cells with a markedly elevated Golgi pH (pHG > or = 6.75). Consistent with these observations in cultured cells, cells in solid tumors also heterogenously expressed the T-antigen. Thus, elevated Golgi pH appears to be directly linked to T-antigen expression in cancer cells, but it may also act as a more general factor for altered glycosylation in cancer by affecting the distribution of Golgi-localized glycosyltransferases.

Vascular endothelial growth factor secretion by tumor-infiltrating macrophages essentially supports tumor angiogenesis, and IgG immune complexes potentiate the process.

Tumor growth requires neoangiogenesis. Members of the vascular endothelial growth factor (VEGF) family play an important role as angiogenic promoters in malignant tumors. Tumor cells and stromal cells are sources of VEGF in the tumor. We tested the relevance of the tumor-infiltrating macrophage (TIM) contribution as a source of VEGF in the tumor environment and the role of the local immune complexes in inducing the TIM release of VEGF. Colon and breast carcinoma biopsies were studied with immunoperoxidase staining of CD11b, sialyl-Tn (sTn) antigen (Ag), and gamma immunoglobulin (IgG). The presence of TIM containing phagosomes positive for both IgG and sTn Ag was observed in all tumors, showing that TIMs endocytosed local immune complexes. Reverse transcription-PCR analysis of macrophage (MO) mRNA showed VEGF-A and -B, but not VEGF-C or -D. That pattern was not modified by the presence of tumor cells. In vitro, the interaction of tumor cells and MO promoted the secretion of MO VEGF. The MO secretion of VEGF was augmented when tumor cells were added to cocultures containing MOs and polymorphonuclear cells. Immune complexes formed with tumor sTn Ag and IgG induced a 5-fold increase of MO VEGF secretion. In vivo, TIMs and neoangiogenesis were associated. In vivo experiments with severe combined immunodeficient and athymic nude (nu/nu) mice showed increased number of TIMs, increased tumor angiogenesis, and faster tumor growth in mice with significant serum anti-sTn IgG. This study demonstrates that VEGF secreted by TIMs represents an essential support for tumor angiogenesis and growth, certainly influenced by the humoral antitumor immune response.

The roles of terminal sugar residues of surface glycans in the metastatic potential of human hepatocarcinoma.

PURPOSE: The roles of terminal sialyl and fucosyl residues in cell surface glycans in the metastatic potential of H7721 cells, a human hepatocarcinoma cell line, were studied. METHODS: Neuraminidase and alpha-L-fucosidase were used to remove the sialyl and fucosyl residues, respectively. Cell adhesion to fibronectin (Fn), laminin (Ln), and human umbilical vein epithelial cell (HUVEC), as well as chemotactic cell migration and invasion, were selected as the parameters of metastatic potential ex vivo. RESULTS: Sialyl residue is not essential for cell adhesion to Fn, but is important in cell adhesion to Ln and invasion, and is crucial in cell adhesion to HUVEC and migration. In contrast, fucosyl residue contributes more than sialyl residue to cell adhesion to Fn and Ln, but less to adhesion to HUVEC, and is not essential in chemotactic cell migration and invasion. Cell adhesion to HUVEC, migration, and invasion were inhibited by the monoclonal antibody of sialyl Lewis X, but not by the antibody of non-sialyl Lewis X. CONCLUSION: Terminal sialyl residues on cell surface glycans are more important than fucosyl residues in mediating cell adhesion to HUVEC and cell migration/invasion, but the reverse is true in cell adhesion to Fn and Ln.

The role of Thomsen-Friedenreich antigen in adhesion of human breast and prostate cancer cells to the endothelium.

Interactions of metastatic cancer cells with vasculatory endothelium are critical during early stages of cancer metastasis. Understanding the molecular underpinnings of these interactions is essential for the development of new efficacious cancer therapies. Here we demonstrate that cancer-associated carbohydrate T antigen plays a leading role in docking breast and prostate cancer cells onto endothelium by specifically interacting with endothelium-expressed beta-galactoside-binding protein, galectin-3. Importantly, T antigen-bearing glycoproteins are also capable of mobilizing galectin-3 to the surface of endothelial cells, thus priming them for harboring metastatic cancer cells. The T antigen-mediated, tumor-endothelial cell interactions could be efficiently disrupted using synthetic compounds either mimicking or masking this carbohydrate structure. High efficiency of T antigen-mimicking and T antigen-masking inhibitors of tumor cell adhesion warrants their further development into antiadhesive cancer therapeutics.

Role of carcinoembryonic antigen in the progression of colon cancer cells that express carbohydrate antigen.

Carcinoembryonic antigen (CEA) has been reported to promote the metastatic potential in some experimental tumors. Adhesion molecules are known to play an important role in the process of metastasis. Cytokines, including interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), which are produced by Kupffer cells, induce endothelial cells to express adhesion molecules. As a result, the present study was designed to investigate whether the interaction between CEA and Kupffer cells accelerated the metastatic potential of tumors in the liver. Kupffer cells isolated from the liver of male BALB/c mice were cultured with CEA, either with or without the addition of a cytokine inhibitor. The levels of IL-1beta and TNF-alpha were examined in a culture medium. An adhesion assay of colon cancer cell lines to human umbilical vein endothelial cells was also performed. When CEA was added to the Kupffer cell culture medium, cytokines were produced. Elevated levels of cytokines appeared to lead to increased rates of adhesion of cancer cells to endothelial cells. However, these phenomena were blocked by the addition of cytokine inhibitors. CEA stimulated Kupffer cells to produce cytokines. An elevated number of cytokines have been proven to promote the expression of adhesion molecules in endothelial cells. These processes are therefore considered to contribute to the metastasis of malignant cells to the liver. These results suggest that cytokine inhibitors may therefore play an important role in the inhibition of hepatic metastasis.

Opposite effects on human colon cancer cell proliferation of two dietary Thomsen-Friedenreich antigen-binding lectins.

Increased cell surface expression of the Thomsen-Friedenreich antigen (TF antigen, Galbeta1-3GalNAcalpha-) is a common feature in malignant and pre-malignant epithelia. Our previous studies have shown that dietary TF-binding lectins from peanut (Arachis hypogea) and edible mushroom (Agaricus bisporus) produce marked but different effects on human intestinal epithelial cell proliferation. This study investigates the proliferative effects of the other two known dietary TF-binding lectins: jacalin (Artocarpus integrifolia, JAC) and amaranth lectin (Amaranthus caudatus, ACA). JAC produced dose-dependent and non-cytotoxic inhibition of proliferation in HT29 human colon cancer cells with maximal effects of 46 +/- 4% at 20 microg/ml, whereas ACA produced dose-dependent stimulation of proliferation with maximal effects of 22 +/- 3% at 20 microg/ml when assessed both by incorporation of [3H]thymidine into DNA and by cell counting. The lectin-mediated effects were inhibitable by the presence of appropriate Galbeta1-3GalNAc-expressing glycoproteins but differences existed between JAC and ACA in their patterns of inhibition by such substances. Ligand binding equilibrium studies using iodinated lectins revealed different Kd of the two lectins for HT29 cell surface glycoproteins. Lectin blots of cell membrane extracts showed different binding patterns in all the four TF-binding lectins. These results provide further evidence that dietary TF-binding lectins can have marked effects on the proliferation of human malignant gastro-intestinal epithelial cells and hence may play a role in intestinal cancer development, and also show that the biological effects of dietary lectins cannot be predicted solely from their carbohydrate binding properties.

Specific role of T and Tn tumor-associated antigens in adhesion between a human breast carcinoma cell line and a normal human breast epithelial cell line.

The possibility that tumor-associated antigens T and Tn act as adhesion molecules between normal and malignant breast epithelial cells at the early stages of recognition in the metastatic pathway was examined in vitro. The adhesive specificity of the antigens was assessed by means of in vitro adhesion tests between a carcinomatous breast cancer cell line (ZR75-30) and a normal epithelial breast cell line (HLB100) using both monoclonal antibodies and lectins specific as well as nonspecific for each antigen. Adhesion assay was performed using monolayers of the normal cell line prepared on plastic culture plates and the tumor cell line labeled with a fluorescent dye as a probe. The adhesion between the two cell types occurred with significant specificity via T and Tn antigens (P<0.001), and was temperature-dependent. The results suggest that at the early stages of recognition by tumor cells in the metastatic process, T and Tn antigens play a role as adhesion molecules between the tumor cells and adjacent normal cells.

Cancer-associated glycosphingolipid antigens: their structure, organization, and function.

Experimental and human cancers are often characterized by the presence of tumor-associated glycosphingolipid (GSL) antigens defined by monoclonal antibodies. Major progress has been made during the past two decades on structural identification of these antigens. None of these structures are truly 'tumor-specific'. However, many of the antibodies show preferential or 'specific' reactivity with tumors, based on organizational differences of membrane GSLs in tumor cells versus normal cells. Clustered GSL antigens organized with transducer molecules in microdomain have been found recently to comprise a structural and functional unit involved in tumor cell adhesion coupled with signal transduction. Some of the GSL antigens have been identified as adhesion molecules recognized by carbohydrate-binding proteins or by complementary carbohydrates on target cells. Such adhesion, coupled with signaling, may initiate the metastatic process. Elucidating the mechanism of this initial adhesion/signaling step may lead to discovery of therapeutic agents that disrupt adhesion ('antiadhesion therapy') or normalize signaling ('ortho-signaling therapy'). Tumor-associated GSL antigens are also a target in immunotherapy of tumors, including development of antitumor vaccines.

[Carbohydrate chains and hematogenous metastasis: a review].

It is well known that the expression of carbohydrate chains on cell changes during the development and progression of carcinoma. Some carbohydrate chains have been used in clinical practice as tumor markers. Some, such as sialyl Lewis a (CA 19.9), sialyl Lewis X, and Tn, are known to function as adhesion molecules. However, their role in the development of hematogenous metastasis is still controversial. In this paper, many investigations concerning this issue are reviewed. Clinical trials for treatment of patients with advanced cancer using carbohydrate chains are also mentioned.

Role of sialosyl Lewis(a) in adhesion of colon cancer cells--the antisense RNA approach.

To study whether the adhesion of colon cancer cells to E-selectin can be directly affected by changes in the expression level of sialosyl Le(a) antigen we created a specific loss-of-function phenotype. A stable subclone (CX-1.1) with high expression of sialosyl Le(a) structure, obtained from a heterogenous population of colon carcinoma CX-1 cells, was transfected with an expression vector containing a fragment of cDNA for alpha1,3/4-fucosyltransferase in antisense orientation. After transfection, the cell line was isolated which did not express sialosyl Le(a) antigen and lacked the alpha1,3/4-fucosyltransferase activity, despite an unchanged level of mRNA specific for this enzyme. It was found that the specific lack of expression of sialosyl Le(a) carbohydrate structure on the surface of colon cancer cells completely abolished their adhesion to E-selectin. To evaluate which cellular glycoconjugates carry sialosyl Le(a) antigen, glycoproteins as well as glycolipids of CX-1.1 cells were analysed for the expression of this structure. Anti-sialosyl Le(a) antibodies detected multiple glycoprotein bands with apparent molecular masses of 65-280 kDa on western blots, and an intense band representing sialosyl Le(a)-ganglioside on a thin-layer chromatogram. Using O-sialoglycoprotease from Pasteurella haemolytica and an alkaline beta-elimination procedure, it was shown that protein-linked sialosyl Le(a) structures are carried mostly by mucin-type glycoproteins. However, treatment of CX-1.1 cells with O-sialoglycoprotease did not decrease either their binding to E-selectin-expressing Chinese hamster ovary cells, or binding of anti-sialosyl Le(a) antibodies to the cell surface. These results suggested that cleavage of sialomucins uncovered cryptic sialosyl Le(a)-ganglioside, which was inaccessible for the antibody and E-selectin in untreated cells. This hypothesis was confirmed to some extent by the higher accessibility of gangliosides to galactose oxidase on the surface of O-sialoglycoprotease-treated CX-1.1 cells, comparing to untreated cells. We propose that glycoproteins as well as gangliosides carrying sialosyl Le(a) structures, when properly exposed and present in high density on surface of cancer cells, can effectively support the adhesion of cancer cells to E-selectin.

Metastatic potential of human CX-1 colon adenocarcinoma cells is dependent on the expression of sialosyl Le(a) antigen.

Several lines of evidence indicate that sialosyl Le(a), tumor-associated carbohydrate antigen present on human colon carcinoma cells, is involved in formation of metastases. To study the role of this carbohydrate structure in development of metastases, we have used the clone of human colon carcinoma CX-1 cells transfected with antisense expression vector containing fragment of cDNA for alpha1,3/4-fucosyltransferase (FT III), which is involved in synthesis of sialosyl Le(a) tetrasaccharide. It has been reported previously that, in contrast to the parental cells, the antisense-transfected CX-1.1AS5 cells do not express sialosyl Le(a) and do not adhere to E-selectin-expressing CHO cells. In the present work we have studied the formation of liver metastases by CX-1.1AS5 cells after their orthotopic or intrasplenic implantation into athymic nu/nu mice. After orthotopic implantation of sialosyl Le(a)-negative colon carcinoma CX-1.1AS5 cells, the number of mice with liver metastases was markedly lower (21% of mice) in comparison with their number after implantation of the parental CX-1.1 cells (86% of mice). However, no differences in ability to form colonies in liver were observed between parental CX-1.1 cells and antisense-transfected CX-1.1AS5 cells after intrasplenic inoculation. The liver metastases were formed in 89% and 84% of mice, respectively. Our data support the thesis on the importance of sialosyl Le(a) antigen expression in the development of liver metastases by colon cancer cells, and indicate the role of transplantation route and primary tumor localization in formation of metastases.

Histo-blood group A/B versus H status of human carcinoma cells as correlated with haptotactic cell motility: approach with A and B gene transfection.

In a search for the molecular basis of ABH status of tumors as correlated with malignancy, we studied various malignancy-related phenotypes of high H/Le(y)-expressing tumor cell lines in comparison with phenotypes of the same lines transfected with histo-blood group A or B genes. A and B gene transfectants, prepared independently from different H-active parental cells, showed A or B activity and abolition of H activity. All A and B gene transfectants, regardless of source, were characterized by significantly reduced Matrigel-dependent haptotactic motility. The level of haptotaxis of all transfectants was similar to that of parental cells in the presence of antibodies against human integrin subunits alpha3, alpha6, or beta1. These subunits showed high expression of A or B epitope in the A and B gene transfectants. Enhancement versus reduction of malignancy, associated with deletion versus induction of A/B epitopes, may be due in part to enhanced haptotaxis sustained by alpha3, alpha6, and beta1 integrin receptors, the activities of which are regulated by H or A/B glycosylation. These phenotypic changes provide a rationale for the deletion of A and B epitopes as one criterion defining human tumor malignancy.

Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy.

Aberrant glycosylation is a hallmark of cancer cells. The blood group precursors T (Thomsen-Friedenreich) and Tn epitopes are shielded in healthy and benign-diseased tissues but uncovered in approx. 90% of carcinomas. T and Tn glycoproteins are specific, autoimmunogenic pancarcinoma antigens. These antigens may also be found in neoplastic blood cells (and on LTV-II infected T lymphocytes). Fundamental chemical and physical aspects of these glycoproteins of primary carcinomas are discussed first. Tn and T epitopes are cell and tissue adhesion molecules, essential in invasion by and metastasis of carcinoma which includes adherent and proliferative phases. These properties are then delineated next, followed by consideration of pathophysiological and clinical aspects of these antigens. Immunohistochemical studies of the extent of Tn antigen expression in primary breast carcinoma demonstrate it highly significant correlation with clinicopathological tumor stages, and hence its value as a reliable prognosticator. On the other hand, there is no significant, prognostically useful association between T antigen expression and clinical disease course. Everyone has "preexisting" anticarcinoma anti-Tn and anti-T antibodies, induced predominantly by the intestinal flora, while cellular immune responses to T and Tn epitopes are evoked only by carcinomas and some lymphomas. Carcinoma dedifferentiation leading to predominance of Tn over T epitopes is described, as is the role of Tn and T epitopes in very early, including preclinical, carcinoma detection. The highest sensitivities in carcinoma detection are for preclinical and the earliest clinical stages. Obviously, preclinical carcinoma detection is of practical importance. T/anti-T tests detected preclinical carcinoma in 77% of 48 patients long (mean 6 years) before their biopsy/X-ray results became positive. There were no false predictions of carcinoma in 38 control persons with benign diseases (observation average 4.8 years). These findings open a novel window for both curative approaches and pathophysiological studies. The autoimmunogenicity of carcinoma T/Tn antigen led us more than two decades ago to begin intradermal vaccination of patients with advanced breast carcinoma of stages IV-IIb, predominately after modified radical mastectomy and sometimes lumpectomy plus axillary dissection always followed by adjuvant radio/chemotherapy. The vaccine consists of human group O red blood cell membrane derived, HLA-free T/Tn antigen containing as adjuvant Ca3(PO4)2 plus a trace of phosphoglycolipid A hyperantigen, i.e., S typhi vaccine (USP), which itself has T and Tn specificities. Our efforts have now for up to 20 years remained successful in a large majority of the 32 patients. All 32 patients survived at least 5 years; 10-year survival was statistically highly significantly improved (5-year survival: P < 1 x 10(-7); 10-year survival: P < 1 x 10(-5)) compared to statistics of the United States National Cancer Institute. Because these vaccinations are successful, their extension to large populations with major types of carcinomas should be considered, and even immunological carcinoma prevention may be contemplated.