alpha-Neup5Ac-(2--3)-beta-D-Galp-(1--4)-[alpha-L-Fucp-(1--3)]-D-GlcpNAc and Breast-Neoplasms

-Invasive micropapillary carcinoma (IMPC) is a distinct variant of mammary carcinoma in which tumor cells are arranged in morulelike clusters devoid of fibrovascular cores and situated within empty stromal spaces. Identification of IMPC can be achieved by the assessment of morphologic features in conjunction with the characteristic "inside-out" staining pattern of epithelial membrane antigen and sialyl Lewis X highlighted by immunohistochemical analysis. Although recognizing micropapillary architecture is often not challenging, the criteria for distinguishing between mixed and pure IMPC remain imprecise. Some mucin-producing carcinomas can also have micropapillary histology, but there is no consensus on whether these tumors are variants of IMPC or mucinous carcinomas. The molecular genetic studies demonstrate that IMPCs have distinct molecular genetic profiles, supporting the theory that they constitute distinct pathologic entities. However, genomic analyses have not identified any specific genomic aberration that may explain the distinctive morphology and clinical behavior of IMPC.. -To provide an overview on the current concepts in the diagnosis and pathogenesis of IMPC of the breast, incorporating recent molecular genetic advances and prognosis-based reclassification.. -PubMed search and the cited references were reviewed.. -The recent evolution of prognosis-based reclassification and molecular genetic advances has enhanced our knowledge of the pathogenesis of IMPC of the breast. Additional studies might reveal consistent molecular alterations that underlie the formation of the inside-out growth pattern, and they might elucidate the molecular mechanisms responsible for the unfavorable clinical behavior of IMPC.

Topics: Breast; Breast Neoplasms; Carcinoma, Papillary; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; MicroRNAs; Neoplasm Invasiveness; Oligosaccharides; Sialyl Lewis X Antigen

Breast cancer spreading to different organs have been related to different molecules and mechanisms, but cutaneous metastasis remains unexplored. Increasing evidence showed that MUC1 and some of its carbohydrate associated antigens may be implicated in breast cancer metastasis. In this study we analyzed these tumor markers in order to identify breast cancer cutaneous metastatic profiles. A cohort of 26 primary tumors from breast cancer patients with cutaneous metastases were included; also, cutaneous and lymphatic node metastatic samples and primary tumors from breast cancer patients without metastases were analysed. Immunohistochemical (IHC) studies demonstrated that both underglycosylated MUC1 (uMUC1) and sialyl Lewis x (sLex) to be positively associated with cutaneous metastatic primary tumors (p < 0.05). Notably, a high percentage of tumors with cutaneous metastases were characterized as triple negative and Her2+ tumors (37.5 % and 29 %, respectively). Some discordant results were found between primary tumors and their matched cutaneous metastases. To determine if MUC1 variants may be carriers of carbohydrate antigens, subcellular fractions from a cutaneous metastatic lesion were obtained, immunoprecipitated and analyzed by Western blot. We found that the isolated uMUC1 with a molecular weight of>200 kDa was also the site for binding of anti-sLex MAb; in coincidence, a high correlation of positive IHC expression of both markers was observed. Our findings confirm that breast cancer cutaneous metastases were associated to highly malignant primary tumors and sustain the hypothesis that u-MUC1 and sLe x may drive breast cancer cutaneous metastases.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Middle Aged; Mucin-1; Sialyl Lewis X Antigen; Skin Neoplasms

Bone metastases are common and devastating to cancer patients. Existing treatments do not specifically target the disease sites and are therefore ineffective and systemically toxic. Here we present a new strategy to treat bone metastasis by targeting both the cancer cells ("the seed"), and their surrounding niche ("the soil"), using stem cells engineered to home to the bone metastatic niche and to maximise local delivery of multiple therapeutic factors.. We used mesenchymal stem cells engineered using mRNA to simultaneously express P-selectin glycoprotein ligand-1 (PSGL-1)/Sialyl-Lewis X (SLEX) (homing factors), and modified versions of cytosine deaminase (CD) and osteoprotegerin (OPG) (therapeutic factors) to target and treat breast cancer bone metastases in two mouse models, a xenograft intratibial model and a syngeneic model of spontaneous bone metastasis.. We first confirmed that MSC engineered using mRNA produced functional proteins (PSGL-1/SLEX, CD and OPG) using various in vitro assays. We then demonstrated that mRNA-engineered MSC exhibit enhanced homing to the bone metastatic niche likely through interactions between PSGL-1/SLEX and P-selectin expressed on tumour vasculature. In both the xenograft intratibial model and syngeneic model of spontaneous bone metastasis, engineered MSC can effectively kill tumour cells and preserve bone integrity. The engineered MSC also exhibited minimal toxicity in vivo, compared to its non-targeted chemotherapy counterpart (5-fluorouracil).. Our combinatorial targeting of both the cancer cells and the niche represents a simple, safe and effective way to treat metastatic bone diseases, otherwise difficult to manage with existing strategies. It can also be applied to other cell types (e.g., T cells) and cargos (e.g., genome editing components) to treat a broad range of cancer and other complex diseases. FUND: National Institutes of Health, National Cancer Institute of the National Institutes of Health, Department of Defense, California Institute of Regenerative Medicine, National Science Foundation, Baylx Inc., and Fondation ARC pour la recherche sur le cancer.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Engineering; Cell Line, Tumor; Cytosine Deaminase; Female; Genetic Therapy; Humans; Membrane Glycoproteins; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Osteoprotegerin; P-Selectin; RAW 264.7 Cells; RNA, Messenger; Sialyl Lewis X Antigen; Xenograft Model Antitumor Assays

Distant metastases account for the majority of cancer-related deaths in breast cancer. The rate and site of metastasis differ between estrogen receptor (ER)-negative and ER-positive tumours, and metastatic fate can be very diverse even within the ER-negative group. Characterisation of new pro-metastatic markers may help to identify patients with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metastasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist in carbohydrate molecules, such as sialyl-Lewis x antigen (sLex), borne by glycoproteins or glycolipids on the cancer cell surface. We have previously demonstrated that the molecular scaffold presenting sLex to selectins (e.g. glycolipid vs. glycoproteins) was crucial for these interactions to occur. Moreover, we reported that detection of sLex alone in breast carcinomas was only of limited prognostic value. However, since sLex was found to be carried by several glycoproteins in cancer cells, we hypothesized that the combination of the carbohydrate with its carriers could be more relevant than each marker independently. In this study, we addressed this question by analysing sLex expression together with two glycoproteins (BST-2 and LGALS3BP), shown to interact with E-selectin in a carbohydrate-dependent manner, in a cohort of 249 invasive breast cancers. We found both glycoproteins to be associated with distant metastasis risk and poorer survival. Importantly, concomitant high expression of BST-2 with sLex defined a sub-group of patients with ER-negative tumours displaying higher risks of liver and brain metastasis and a 3-fold decreased survival rate.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Carrier Proteins; Disease-Free Survival; E-Selectin; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; GPI-Linked Proteins; Humans; Lewis X Antigen; Ligands; Middle Aged; Neoplasm Metastasis; Prognosis; Sialyl Lewis X Antigen

Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa) patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLe(x) expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of 'triple-negative' breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments.

Topics: Anti-Inflammatory Agents; Breast Neoplasms; Cell Adhesion; Cell Aggregation; Cell Line, Tumor; Cell Proliferation; Cytokines; E-Selectin; Endothelium; Female; Humans; Hyaluronan Receptors; Inflammation Mediators; Interleukin-6; Metformin; Neoplasm Metastasis; Neoplastic Cells, Circulating; Oligosaccharides; Phenotype; Sialyl Lewis X Antigen; Spheroids, Cellular; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

CSLEX is a type II carbohydrate antigen that interacts with the CSLEX-1 monoclonal antibody. CSLEX in combination with carbohydrate antigen 15-3 may be more useful than Carcinoembryonic Antigen with carbohydrate antigen 15-3 as tumor markers for monitoring of breast cancer.. The serum levels of tumor markers, including CSLEX, were measured in 480 consecutive breast cancer patients with or without metastasis who visited the outpatient clinic of the Division of Breast and Endocrine Surgery, Shinshu University Hospital, between April 2007 and September 2007.. Serum levels of each of the tumor markers correlated significantly with the status of metastasis (P < 0.01). Combinations of Carcinoembryonic Antigen and carbohydrate antigen 15-3, Carcinoembryonic Antigen and Nation Cancer Center-Stomach-439, Carcinoembryonic Antigen and CSLEX, carbohydrate antigen 15-3 and Nation Cancer Center-Stomach-439, and carbohydrate antigen 15-3 and CSLEX levels also correlated significantly with the status of metastasis (P < 0.01). The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were almost the same for CSLEX and Nation Cancer Center-Stomach-439, which are both type II carbohydrate antigens. The cutoff indexes of serum CSLEX and Nation Cancer Center-Stomach-439 for detection of breast cancer metastasis were 38.8 ± 52.7-fold and 22.1 ± 27.8-fold, respectively (P = 0.16).. These data suggest that the diagnostic values of CSLEX and Nation Cancer Center-Stomach-439 are similar in single or combined use. However, the cutoff index of serum CSLEX tended to be higher than that of Nation Cancer Center-Stomach-439, which may make CSLEX more useful for detection of breast cancer metastasis.

Topics: Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Middle Aged; Oligosaccharides; Prognosis; ROC Curve; Sensitivity and Specificity; Sialyl Lewis X Antigen

Metastatic breast cancer (MBC) is currently an incurable condition that is primarily treated with palliative measures. Isolation of circulating tumor cells (CTCs) from the peripheral blood of these patients provides a predictive prognostic indicator, independent of the type of therapy, site of occurrence and biological characteristics of the primary disease. It has been well established that glycosylation processing pathways are disturbed in cancer, leading to alterations in the glycan content of glycoproteins.. The bi-, tri- and tetraantennary glycans containing sialyl Lewis x (sLe(x)) epitopes (A2F1G1, A3F1G1, A4F1G1 and A4F2G2) were quantified using normal phase high-performance liquid chromatography in combination with exoglycosidase array digestions in the glycan pools released from sera of 27 patients with advanced breast cancer (16 with CTCs <5/7.5 ml and 11 with CTCs ≥5/7.5 ml) and 13 healthy women.. The levels of all these glycans were significantly higher in patients with CTCs ≥5/7.5 ml compared with patients with CTCs <5/7.5 ml.. As high levels of glycans containing sLe(x) epitopes were associated with CTCs, their measurement may provide a new noninvasive approach for determining prognosis in women with MBC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Case-Control Studies; Cell Count; Chromatography, High Pressure Liquid; Female; Humans; Middle Aged; Neoplasm Metastasis; Neoplastic Cells, Circulating; Oligosaccharides; Prognosis; Sialyl Lewis X Antigen

Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLe(x)) and sialyl Lewis A (sLe(a)) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin-Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin-Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis.

Topics: Breast Neoplasms; CA-19-9 Antigen; Cell Adhesion; Cell Line, Tumor; E-Selectin; Endothelium, Vascular; Female; Gangliosides; Humans; Ligands; Oligosaccharides; Peptide Hydrolases; Sialyl Lewis X Antigen

Glycosylation of the tumour cell surface is of importance in metastasis formation as indicated by lectin-binding studies. In particular, binding of the lectin HPA is associated with metastasis formation, both in clinical studies and in xenograft models of breast and colon cancer. Here we examined if there is an association between the HPA-positive glycotopes of metastasizing cancer cells and selectin-binding properties.. Glycotope expression of human breast and colon cancer cells (MCF7, T47D, HBL100, HT29, SW480) grown in culture and xenografted into SCID mice were investigated by histochemical analysis.. HPA binding was observed in metastasizing breast and colon cancers and not in non-metastasizing ones. In colon cancer, E-selectin binding and expression of the selectin ligands CD15s and CA19-9 was higher in metastatic HT29 than in non-metastatic SW480 cells, especially when cells were grown in vitro. In breast cancer, E-selectin binding, CD15s and CA19-9 expression were independent of the metastatic potential. P-Selectin binding was slightly higher in metastasizing breast cancer cells (MCF7, T47D) than in non-metastasizing HBL100 cells.. Binding to E-selectin and expression of E-selectin ligands of colon cancer cells grown in vitro is associated with metastasis formation in a xenograft model. However, analysis of selectin ligands is of limited predictive value for the metastatic potential of breast cancer cells in our xenograft model.

Topics: Animals; Breast Neoplasms; CA-19-9 Antigen; Colonic Neoplasms; E-Selectin; Female; Glycosylation; Humans; Immunoenzyme Techniques; Lectins; Lewis X Antigen; Mice; Mice, Inbred BALB C; Mice, SCID; P-Selectin; Sialyl Lewis X Antigen

The glycome acts as an essential interface between cells and the surrounding microenvironment. However, changes in glycosylation occur in nearly all breast cancers, which can alter this interaction. Here, we report that profiles of glycosylation vary between ER-positive and ER-negative breast cancers. We found that genes involved in the synthesis of sialyl-Lewis x (sLe(x); FUT3, FUT4, and ST3GAL6) are significantly increased in estrogen receptor alpha-negative (ER-negative) tumors compared with ER-positive ones. SLe(x) expression had no influence on the survival of patients whether they had ER-negative or ER-positive tumors. However, high expression of sLe(x) in ER-positive tumors was correlated with metastasis to the bone where sLe(x) receptor E-selectin is constitutively expressed. The ER-positive ZR-75-1 and the ER-negative BT20 cell lines both express sLe(x) but only ZR-75-1 cells could adhere to activated endothelial cells under dynamic flow conditions in a sLe(x) and E-selectin-dependent manner. Moreover, L/P-selectins bound strongly to ER-negative MDA-MB-231 and BT-20 cell lines in a heparan sulfate (HS)-dependent manner that was independent of sLe(x) expression. Expression of glycosylation genes involved in heparan biosynthesis (EXT1 and HS3ST1) was increased in ER-negative tumors. Taken together, our results suggest that the context of sLe(x) expression is important in determining its functional significance and that selectins may promote metastasis in breast cancer through protein-associated sLe(x) and HS glycosaminoglycans.

Topics: beta-Galactoside alpha-2,3-Sialyltransferase; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Line; Cell Line, Tumor; E-Selectin; Female; Fucosyltransferases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycomics; Heparitin Sulfate; Human Umbilical Vein Endothelial Cells; Humans; Lewis X Antigen; N-Acetylglucosaminyltransferases; Neoplasm Metastasis; Neoplasms, Hormone-Dependent; Oligonucleotide Array Sequence Analysis; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; Sialyl Lewis X Antigen; Sialyltransferases; Sulfotransferases

To investigate the possible roles of E-selectin and its ligand, Sialyl Lewis X, in lymph node metastasis of invasive micropapillary carcinoma of the breast, 100 cases of invasive micropapillary carcinoma and 97 cases of invasive ductal carcinoma were analyzed immunohistochemically for the expression of E-selectin and Sialyl Lewis X, along with CD34, to measure the microvessel density of invasive micropapillary carcinoma. We found that the number of E-selectin-positive vessels was greater in invasive micropapillary carcinoma than in invasive ductal carcinoma, and it was significantly correlated with the histological grade, the number of positive lymph nodes, and the microvessel density of invasive micropapillary carcinoma. The Sialyl Lewis X expression of invasive micropapillary carcinoma was higher than that of invasive ductal carcinoma, which was also associated with lymph node metastasis. In invasive micropapillary carcinoma, the Sialyl Lewis X expression was predominantly in the stroma-facing surface of the cell clusters and the adjacent stroma, while in invasive ductal carcinoma it was largely intracytoplasmic or intercellular. These findings suggested that E-selectin and Sialyl Lewis X might play an important role in lymph node metastasis in invasive micropapillary carcinoma. The expression pattern of Sialyl Lewis X in invasive micropapillary carcinoma suggested that the reversal of cell polarity of invasive micropapillary carcinoma might be as an important factor for the morphogenesis and possibly the pathogenesis, especially their higher rates of lymph node metastasis.

Topics: Adenocarcinoma, Papillary; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Polarity; E-Selectin; Female; Humans; Lymph Nodes; Lymphatic Metastasis; Microvessels; Neoplasm Invasiveness; Oligosaccharides; Sialyl Lewis X Antigen

One of the most urgent requirements in breast cancer is the development of a blood-based test for early detection and prognosis. Previously published results found a significant difference between specific glycan levels in patients with advanced breast cancer and healthy controls. The aim of this investigation was to address a more clinically relevant problem, i.e., whether the measurement of specific glycans could identify women with aggressive disease at an early stage. In order to reduce potential bias in this study, blood samples from patients were collected, stored and analyzed in a similar manner. Agalactosyl biantennary glycans (FA2) and glycans containing the sialyl Lewis x epitope (A3F1G1 and A2F1G1) were measured using high throughput normal-phase high-performance liquid chromatography in combination with exoglycosidase digestions in sera from 52 patients with early breast cancer (21 with lymph node-negative and 20 with lymph node-positive disease) and 134 women with benign breast disease. The combined levels of the glycans were significantly higher in patients with lymph node metastases compared to women without these metastases. Lymph node status is the single most important determinant of survival in early stage breast cancer. As high levels of these glycans were associated with nodal metastases, their measurement may provide a new non-invasive approach to determining prognosis in women with newly diagnosed breast cancer.

Topics: Adenocarcinoma, Mucinous; Axilla; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Chromatography, High Pressure Liquid; Female; Humans; Lewis X Antigen; Lymph Nodes; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Polysaccharides; Prognosis; Sialyl Lewis X Antigen

Sialyl Lewis x (sLex) carbohydrate antigen acts as an adhesion molecule expressed on the surface of cancer cells and is the most important ligand of the selectins present on endothelial cells. sLex expression was correlated to the metastatic potential of breast cancer. The aim of the present work was to evaluate the prognostic value of sLex in a series of breast carcinomas with long-term follow-up. A total of 127 consecutive patients with primary breast cancer were enrolled and followed for a median of 140 months. Tumor grade, mitotic index, histotype, vascular invasion, and tumor extension and sLex were recorded and used in multivariate analysis. sLex antigen was expressed in 37 specimens (21%). Survival was similar for sLex-positive and sLex-negative tumors (62% vs 60%) for overall survival and for disease-free survival (59% vs 56%). Expression of sLex antigen in breast cancer is not associated with breast cancer survival.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Disease-Free Survival; Female; Humans; Immunohistochemistry; Middle Aged; Oligosaccharides; Prognosis; Sialyl Lewis X Antigen

Glycosylation of proteins plays multiple roles in cell-cell and cell-matrix interactions. Fucose is a monosaccharide associated with glycosylation events and is known to be over-expressed in many malignant tumors. By using alpha-L-fucosidase (alpha-L-fase), a glycosidase that specifically removes alpha-L-fucose (alpha-L-f), we have examined the potential effects of defucosylation on tumor functions, focusing on tumor progression in the context of the interaction of tumor cells with the extracellular microenvironment. In this submission, we report that alpha-L-fase treatment decreases, in static assays, tumor cell adhesion to a wide variety of ECM components including fibronectin, laminin, collagen I, hyaluronic acid and the complex human biomatrix, HuBiogel(R). By immunofluorescence, co-localization of beta1 integrin and alpha-L-f was found to decrease accordingly. Sialyl Lewis X, an alpha-L-f-containing tetrasaccharide, which modulates the rolling of leukocytes and tumor cells on endothelium, was found to be diminished on human breast cancer cells after alpha-L-fase treatment. Using a dynamic flow chamber system, we were able to determine that defucosylation impaired the rolling of mammary cancer cells on human umbilical vein endothelial cells while significantly increasing their flow speed. Further, the rolling capability of these defucosylated tumor cells was also impaired on purified E and P-selectin matrices. Based on these data, we hypothesize that decreased fucosylation impairs the interaction between tumor cells and their external milieu, which in turn, affects key cell functions modulating tumor progression. Building on our previous studies which demonstrated alpha-L-fase decreased tumor cell invasion while significantly reducing MMP-9 activity, when added to the fact that decreased adhesion on HUVEC occurs in the presence of alpha-L-fase also leads us to propose that defucosylation may modulate metastasis, and thus provides a promising additional glycobiotic target for novel therapies.

Topics: alpha-L-Fucosidase; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; E-Selectin; Endothelial Cells; Female; Fucose; Glycoproteins; Humans; Intercellular Adhesion Molecule-1; Oligosaccharides; P-Selectin; Sialyl Lewis X Antigen

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLe(x)). SLe(x) oligosaccharide on tumor cells can be recognized by E- and P-selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E-selectin reactivity, which was sLe(x) dependent, P-selectin reactivity with this cell line was sLe(x)-independent. The sLe(x)-Neg variant of the 4T1 cell line with markedly diminished expression of sLe(x) and lack of sLe(a), provided a unique opportunity to characterize P-selectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E-selectin binding. We observed that P-selectin binding was Ca(2+)-independent and sulfation-dependent. We found that P-selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P-selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P-selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P-selectin-reactive ligands on the surface of human MDA-MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease.

Topics: Animals; Breast Neoplasms; Calcium; Cell Line, Tumor; Cell Membrane; Chondroitin Sulfates; Fucosyltransferases; Glycosaminoglycans; Heparin; Humans; Ligands; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligosaccharides; P-Selectin; Proteoglycans; Sialyl Lewis X Antigen; Transfection

Serum CA15-3 has been one of the most reliable tumor markers used in monitoring breast cancer patients; however, its sensitivity in detecting metastases is limited. To increase its sensitivity, the combined measurement of other tumor markers with CA15-3 was investigated.. Serum CA15-3, carcinoembryonic antigen (CEA) and sialyl Lewis X (CSLEX) were simultaneously measured in a prospective series of 455 postoperative breast cancer patients with or without metastasis. The diagnostic parameters sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for detecting metastases were compared. The correlation of values between pairs of tumor markers was analyzed. The efficacy of combined measurement of two different tumor markers was also evaluated.. The sensitivity for detecting metastases was 61.5, 56.9 and 52.3%; specificity was 97.2, 93.6 and 96.2%; PPV was 78.4, 59.7 and 69.4%; NPV was 93.8, 92.9 and 92.4%; and accuracy was 92.1, 88.8 and 89.9% for CA15-3, CEA and CSLEX, respectively. The values for CA15-3 were significantly correlated with those for CEA (P < 0.001) but not those for CSLEX. The combined measurement of CSLEX and CA15-3 increased the sensitivity by 17.0% but that of CEA and CA15-3 increased the sensitivity by only 10.8%. All diagnostic parameters for the combined measurement of CSLEX and CA15-3 were higher than those for the combined measurement of CEA and CA15-3.. These findings suggest that CSLEX may be more useful than CEA in combination with CA15-3 in monitoring breast cancer patients. The results of this study suggest that CSLEX may be more useful than CEA in combination with CA15-3 in monitoring breast cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carcinoembryonic Antigen; Female; Humans; Middle Aged; Mucin-1; Neoplasm Metastasis; Oligosaccharides; Prospective Studies; Reproducibility of Results; Sensitivity and Specificity; Sialyl Lewis X Antigen

To assess the significance of expression of sialylated carbohydrate antigens and nm23-H1 gene in metastasis and prognosis of breast cancer.. Tissue specimens from 102 cases of primary breast cancer were stained with antibodies against sialyl Lewis A (SleA) and salyl Lewis X (SleX), and nm23-H1 proteins by immunohistochemical methods.. Of the 102 cases, the positive cases of SleA and SleX were 24.5% (25/102) and 59.89% (61/102),respectively; the reduced expression of nm23-H1 was showed in 37.3% (38/102) of the cases. The positive expression of SleX and the reduced expression of nm23-H1 gene were significantly associated with lymph node involvement. Among the 100 patients who underwent curative surgery, the disease-free survival rate was significantly correlated with nm23-H1 and SleX expression, respectively,but not with SleA expression. In multivariate analysis using Cox regression model, combination assay of nm23 H1 and SleX expression emerged as independent prognostic factors.. These results suggest that nm23-H1 gene and SleX may be involved in the metastatic process in human breast cancer, and immunohistochemical detection of SleX and nm23-H1 may be used as a biologic marker of prognosis.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; CA-19-9 Antigen; Female; Gangliosides; Humans; Middle Aged; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Oligosaccharides; Prognosis; Sialyl Lewis X Antigen; Survival Rate

Inflammatory breast carcinoma (IBC) is characterized by florid tumor emboli within lymphovascular spaces called lymphovascular invasion. These emboli have a unique microscopic appearance of compact clumps of tumor cells retracted away from the surrounding endothelial cell layer. Using a human SCID model of IBC (MARY-X), we, in previous studies, demonstrated that the tumor cell embolus (IBC spheroid) forms on the basis of an intact and overexpressed E-cadherin/alpha,beta-catenin axis that mediates tumor cell-tumor cell adhesion. In the present study we examine the mechanism behind the apparent lack of binding of the tumor embolus to the surrounding endothelium. We find that this lack of tumor cell binding is because of markedly decreased sialyl-Lewis(x/a) (sLe(x/a)) carbohydrate ligand-binding epitopes on its overexpressed MUC1 and other surface molecules that bind endothelial E-selectin. Decreased sLe(x/a) is because of decreased alpha3/4-fucosyltransferase activity in MARY-X. The decreased sLe(x/a) fail to confer electrostatic repulsions between tumor cells, which further contributes to the compactness of the MARY-X spheroid by allowing the E-cadherin homodimeric interactions to go unopposed. MARY-X spheroids were retrovirally transfected with FucT-III cDNA, significantly raising their levels of fucosyltransferase activity and surface sLe(x/a). In parallel experiments, enzymatic transfers with a milk alpha1,3-fucosyltransferase and an alpha2,3-sialyltransferase (ST3GalIV) were performed on the MARY-X spheroids and increased surface sLe(x/a). The addition of sLe(x/a) by either manipulation caused disadherence of the MARY-X spheroids and the disruption of the E-cadherin homodimers mediating cell adhesion. Our findings support the cooperative relationship of sLe(x/a) underexpression and E-cadherin overexpression in the genesis of the lymphovascular embolus of IBC.

Topics: Animals; Breast Neoplasms; CA-19-9 Antigen; Cadherins; Female; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Neoplastic Cells, Circulating; Oligosaccharides; Sialyl Lewis X Antigen; Transplantation, Heterologous

The prognostic value of altered blood group factor and Lewis-related carbohydrate antigen expression in breast cancers has not been fully determined.. To this end, breast carcinoma samples from 87 radical mastectomy patients with primary cancer were analyzed by immunohistochemistry for the ABH factors, Le(a), sialyl Le(a), Le(x), and sialyl Le(x).. It was found that ABH, Le(a), sialyl Le(a), Le(x), and sialyl Le(x) antigens were expressed in 25 (21.8%), 26 (22.6%), 26 (22.6%), 36 (31.3%), and 37 specimens (32.2%), respectively. Tumors with lymph node metastasis expressed Le(x) or sialyl Le(x) antigens more frequently than those without lymph node metastasis ( P=0.0020 or P=0.039, respectively). The survival time of patient s after surgery was significantly shorter for those whose tumors expressed Le(x) or sialyl Le(x) than for those without Le(x)- or sialyl Le(x)-positive tumors ( P=0.0028 and P=0.0029, respectively). Cox's multiple regression analysis revealed that sialyl Le(x) expression was an independent prognostic factor for patient survival regardless of primary tumor (T factor) and lymph node (N factor) status (hazards ratio, 3.80).. Thus, expression of sialyl Le(x) antigen in tumor cells is associated with poor prognosis in patients with breast cancer and must be considered in the design of future therapeutic trials.

Topics: ABO Blood-Group System; Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Lewis Blood Group Antigens; Middle Aged; Oligosaccharides; Prognosis; Prospective Studies; Sialyl Lewis X Antigen; Survival Analysis

The adhesion of circulating cancer cells to vascular endothelium is an important step in the hematogenous metastasis of cancer. Until recently, it has been believed that carbohydrate antigens are expressed on cancer cells, and E-selectin is expressed on endothelial cells to effect this adhesion. We investigated the gene expression of fucosyl-transferase (Fuc-T) and sialyltransferase (ST), which are involved in the synthesis of sialyl Lewisx (s-Lex) in breast cancer by using Northern blot analysis. The concentration of s-Lex in the cancerous portion was increased, compared to that in the adjacent non-cancerous portion. A correlation was found between the concentration of s-Lex and the amount of Fuc-T VI message in 9 cases of breast cancer tissue. Expression of the Fuc-T III message was found in only one case who expressed s-Lea. No expression of the Fuc-T V or VII message was observed. There was no relationship between the concentration of s-Lex and the amount of ST3N and ST4 transcripts. Similar findings were obtained from an analysis using cell lines derived from human breast cancer. When Fuc-T VI gene was transfected to MCF-7 cells, the expression of s-Lex was markedly induced on MCF-7 cells, and the attachment of cancer cells to endothelial cells was enhanced. These findings suggest that Fuc-T VI is chiefly involved in the synthesis of s-Lex on breast cancer cells.

Topics: Adult; Aged; Blotting, Northern; Breast Neoplasms; Cell Adhesion; Coculture Techniques; E-Selectin; Endothelium, Vascular; Female; Fucosyltransferases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Lewis Blood Group Antigens; Middle Aged; Oligosaccharides; Polymerase Chain Reaction; Sialyl Lewis X Antigen; Sialyltransferases; Umbilical Veins

Sialyl Lewis X serves as a ligand for selectins and is proposed to be implicated in hematogenous metastasis of cancers. When a cultured human breast cancer cell line, MCF-7, which does not express sialyl Lewis X, was transfected with human fucosyltransferase VI cDNA, a strong expression of sialyl Lewis X was induced on transfectant cells. The transfectant cells were found to be also reactive to the antibody NCC-ST-439, which was initially raised against human gastric cancer cells and later was shown to recognize a tumor-associated carbohydrate antigen in breast, gastric, and colon cancers. This suggested that the antigen recognized by NCC-ST-439 is closely related to sialyl Lewis X. Subsequent studies indicated that NCC-ST-439 specifically reacts to NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6GalNAcalpha1 -->R, the sialyl Lewis X on the mucin GlcNAcbeta1-->6 GalNAcalpha structure. The antibody was not reactive to the conventional sialyl Lewis X determinants on straight and/or branched polylactosamine structures including NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->3Galbeta1-->4 Glcbeta1-->R and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6Galbeta1-->4 Glcbeta1-->R. This was in clear contrast to most of the known anti-sialyl Lewis X antibodies, which do not discriminate internal structures carrying the sialyl Lewis X determinant. On the other hand, the newly generated monoclonal antibody GSC154-27 had a specificity completely the reverse of the specificity of NCC-ST-439 in that it was strongly reactive to the conventional sialyl Lewis X determinants in straight and branched polylactosamine structures, while far less reactive to the sialyl Lewis X determinant on the mucin GlcNAcbeta1-->6GalNAcalpha core structure. A set of these two antibodies would be useful in discriminating the molecular species of sialyl Lewis X expressed by malignant cells and in studying their functional significance.

Topics: Antibodies, Monoclonal; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carbohydrate Sequence; Cell Adhesion; Disaccharides; DNA, Complementary; Epitopes; Female; Flow Cytometry; Humans; Molecular Sequence Data; Mucins; Oligosaccharides; Prognosis; Sialyl Lewis X Antigen; Transfection; Tumor Cells, Cultured

The expression of blood group antigens A, B, and H, as well as sialylated and nonsialylated forms of Lewis(a) and Lewis(x), was studied using immunohistochemical methods in normal and tumor tissues in the following cohort of patients: 51 patients with primary breast carcinoma, 13 with metastatic lymph node lesions, and 16 with benign tumors of the breast. As a control, normal tissue was obtained from a similar group of 22 patients with breast cancer. The noncancerous tissues expressed the same A/B/H antigens as the patients' red blood cells and also usually expressed Lewis-related antigens. Seventy-six percent of primary carcinomas failed to express the appropriate A/B/H antigens, and in one blood group A patient the tumor tissue expressed B antigen. In the metastatic lesions, Lewis(a)/sialyl Lewis(a) expression was reduced when compared with the primary tumors, but Lewis(x)/sialyl Lewis(x) antigens were still expressed. These results suggest a possible relationship between the metastatic behavior of the tumor and expression of the blood group antigens.

Topics: Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; CA-19-9 Antigen; Carbohydrate Sequence; Gangliosides; Humans; Immunohistochemistry; Lewis Blood Group Antigens; Lewis X Antigen; Lymph Nodes; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

Tumor cells can invade and generate metastasis via either lymphatics or blood vessels. When tumor cells are circulating in the blood, they must adhere to the vessel wall, which is lined by endothelium, before they can extravasate and form new metastases. Several families of adhesion molecules have been identified to play a role in the extravasation cascade. Selectins and their sialyl Lewis(x) and/or sialyl Lewis(a) (sLe(x) and sLe(a), respectively) containing ligands play an initiating role in this cascade; we have now analyzed their role in the generation of metastatic breast carcinoma lesions. We examined expression of endothelial E- and P-selectin, expression of epithelial and endothelial sLe(x) and sLe(a) normal tissues compared with primary and metastatic breast in carcinoma lesions within individual patients. While normal breast epithelial cells do not express sLe(x) or sLe(a), epithelial expression of these oligosaccharide epitopes was enhanced in primary breast carcinoma lesions. Furthermore, epithelial expression levels of sLe(x) and/or sLe(a) were even higher in most patients (9 of 12) who had metastatic compared with primary lesions. We show that endothelia in primary lesions express more sLe(x) than in normal tissue and that metastatic lesions express even higher amounts of sLe(x) compared with primary lesions. The expression of P- and E-selectin was also greatly enhanced in tumor-bearing tissue compared with normal tissue. Our data support the hypothesis that while they are circulating in the blood, sLe(x)- and/or sLe(a)-expressing carcinoma cells have a higher probability for extravasation at sites where the endothelium expresses E- and P-selectin and for generation of new metastases.

Topics: Adult; Biomarkers, Tumor; Breast Neoplasms; CA-19-9 Antigen; E-Selectin; Endothelium; Epithelium; Female; Gangliosides; Humans; Middle Aged; Oligosaccharides; P-Selectin; Sialyl Lewis X Antigen

The cancer-associated epitope defined by the monoclonal antibody (MAb) 9H8 was shown to be closely related to the T antigen (Thomsen-Friedenreich antigen) by its sensitivity to 0-glycanase treatment of a mucin glycopeptide known to express this epitope. The reactivity with this glycopeptide increased upon neuraminiclase treatment, and among several MAbs tested for ability to block binding of the 9H8 antibody, the one specific for the T antigen was the most efficient. Out of 41 serum samples from breast-cancer patients, 11 showed elevated levels of the 9H8 epitope, and several sera also showed elevated levels of the cancer-associated carbohydrate epitopes sialyl-Lewis a and sialyl-Lewis x. By the use of antibodies specific for the MUC1 apoprotein (Ma552 and HMFG-2) it could be shown that these epitopes were attached to the MUC1 apoprotein in at least 4 of the cases. By combining antibodies specific to 9H8, sialyl-Lewis a and sialyl-Lewis x in catcher and tracer positions in several types of immunofluorometric assays, it was shown that the 9H8 epitope was rarely co-expressed with sialyl-Lewis a or sialyl-Lewis x epitopes an the same molecule, though all were expressed on MUC1 mucins. In fact, they can be considered as mutually exclusive epitopes, suggesting that these sera contained different populations of MUC1 mucins distinguishable by different sets of oligosaccharides. The existence of mutually exclusive carbohydrate epitopes on different MUC1 mucins in one and the same patient should be taken into account when designing immunoassays exploiting MUC1-reactive antibodies.

Topics: Antibodies; Antibodies, Monoclonal; Blotting, Western; Breast Neoplasms; CA-19-9 Antigen; Carbohydrate Sequence; Electrophoresis; Epitopes; Female; Fluoroimmunoassay; Gangliosides; Humans; Molecular Sequence Data; Mucin-1; Sialyl Lewis X Antigen