heparitin-sulfate and Breast-Neoplasms

Glycosaminoglycans are integral part of the dynamic extracellular matrix (ECM) network that control crucial biochemical and biomechanical signals required for tissue morphogenesis, differentiation, homeostasis and cancer development. Breast cancer cells communicate with stromal ones to modulate ECM mainly through release of soluble effectors during cancer progression. The intracellular cross-talk between cell surface receptors and estrogen receptors is important for the regulation of breast cancer cell properties and production of ECM molecules. In turn, reorganized ECM-cell surface interface modulates signaling cascades, which regulate almost all aspects of breast cell behavior. Heparan sulfate chains present on cell surface and matrix proteoglycans are involved in regulation of breast cancer functions since they are capable of binding numerous matrix molecules, growth factors and inflammatory mediators thus modulating their signaling. In addition to its anticoagulant activity, there is accumulating evidence highlighting various anticancer activities of heparin and nano-heparin derivatives in numerous types of cancer. Importantly, heparin derivatives significantly reduce breast cancer cell proliferation and metastasis in vitro and in vivo models as well as regulates the expression profile of major ECM macromolecules, providing strong evidence for therapeutic targeting. Nano-formulations of the glycosaminoglycan heparin are possibly novel tools for targeting tumor microenvironment. In this review, the role of heparan sulfate/heparin and its nano-formulations in breast cancer biology are presented and discussed in terms of future pharmacological targeting.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Communication; Cell Line, Tumor; Cell Proliferation; Cytokines; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Heparin; Heparitin Sulfate; Humans; Intercellular Signaling Peptides and Proteins; Protein Binding; Signal Transduction; Stromal Cells

Breast cancer is defined as a cancer originating in tissues of the breast, frequently in ducts and lobules. During the last 30 years, studies to understand the biology and to treat breast tumor improved patients' survival rates. These studies have focused on genetic components involved in tumor progression and on tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are involved in cell signaling, adhesion, extracellular matrix assembly, and growth factors storage. As a central molecule, HSPG regulates cell behavior and tumor progression. HS accompanied by its glycosaminoglycan counterparts regulates tissue homeostasis and cancer development. These molecules present opposite effects according to tumor type or cancer model. Studies in this area may contribute to unveil glycosaminoglycan activities on cell dynamics during breast cancer exploring these polysaccharides as antitumor agents. Heparanase is a potent tumor modulator due to its protumorigenic, proangiogenic, and prometastatic activities. Several lines of evidence indicate that heparanase is upregulated in all human sarcomas and carcinomas. Heparanase seems to be related to several aspects regulating the potential of breast cancer metastasis. Due to its multiple roles, heparanase is seen as a target in cancer treatment. We will describe recent findings on the function of HSPGs and heparanase in breast cancer behavior and progression.

Topics: Breast Neoplasms; Disease Progression; Female; Glucuronidase; Heparitin Sulfate; Humans; Models, Biological; Neoplasm Metastasis

This review summarizes a series of studies demonstrating that heparan sulfate proteoglycans act to promote the growth and metastasis of myeloma and breast tumors, two tumors that home to, and grow within, bone. Much of the growth-promoting effect of proteoglycans in these tumors may reside in the shed form of syndecan-1 that acts to favorably condition the tumor microenvironment. Moreover, the interplay between heparan sulfate and the extracellular enzyme heparanase-1 also has important regulatory implications. Recent studies indicate that the activity of heparanase, which likely releases heparin sulfate-bound growth factors and generates highly active heparan sulfate fragments, also promotes growth and metastasis of myeloma and breast tumors. Understanding the role of heparan sulfate and heparanase in the regulation of tumor behavior may lead to new therapeutic approaches for treating cancer.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Proliferation; Glucuronidase; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Models, Biological; Multiple Myeloma; Neoplasm Metastasis; Osteolysis

The destruction of the epithelial basement membrane is widely regarded as a clear criterion for invasive malignant tumor growth. Since, however, defects in the basement membrane may also occur in non-invasive conditions, such as inflammatory and proliferative lesions, and since it has been shown that particularly in highly differentiated squamous cell carcinomas a continuous basement membrane is mimicked by the presence of isolated components, this criterion seems to be of minor value for the diagnosis of malignancy. Despite these drawbacks, the immunolocalization of basement membrane material may still be of differential diagnostic significance in certain situations. This holds particularly true for invasive (ductal) breast carcinomas, which usually completely lack a basement membrane. Accordingly, sclerosing adenosis can be distinguished from invasive carcinoma, as a distinction can be made between neoplastic (malignant) tubular formations and reactive lesions.

Topics: Basement Membrane; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Collagen; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Laminin; Neoplasm Invasiveness; Prognosis; Proteoglycans

We report the second case of an acquired heparin-like anticoagulant in a patient with disseminated breast carcinoma. All but one of the small numbers of other cases have also been associated with an underlying malignancy. We comment on the distinction between an immunoglobulin and proteoglycan causing the antithrombin effect and suggest points of interest for consideration in any future cases together with a review of treatment options.

Topics: Anticoagulants; Blood Coagulation Disorders; Breast Neoplasms; Female; Gastrointestinal Hemorrhage; Heparin; Heparitin Sulfate; Humans; Middle Aged; Thrombin Time

Leukocyte arylsulphatase A (AS-A) was shown to be significantly high in newly-diagnosed breast cancer patients. Previous reports imply a connection between serum interleukin-6 (IL-6) and breast cancer, possibly through a modulation of enzymes involved in estrogen synthesis. Abnormal distribution of heparan sulphate proteoglycans (HSPGs) in malignant breast epithelial cells suggests that they play a key role in the regulation of cell growth. Estradiol is believed to be effective in modulating glycosaminoglycans (GAGs) and their depolymerizing enzymes. Therefore, in this study, attempts were made to evaluate the activity of leukocyte arylsulphatase A, serum interleukin-6, urinary GAGs and heparan sulphate (HS) in response to tamoxifen (TAM) therapy in mastectomised breast cancer patients. Thirty-four patients (aged 30-82 years) were administered TAM (20 mg twice daily). Blood and urine samples of each patient were collected three times (at the beginning, and in third and sixth month of TAM therapy), and biochemical parameters were measured. There was no difference between baseline leukocyte AS-A activity and that measured after three months. At the end of six months, enzyme activity was significantly higher than the former values (p=0.022), but within the reference intervals reported in the literature. Although this increase might imply a normalization, the duration of TAM therapy is not long enough to make a decision about either regression or aggravation of the disease. TAM did not have any effect on serum IL-6, urinary HS and GAG levels which may be due to insensitivity of these variables to TAM during the short period of therapy. Both urinary GAG and HS levels measured at sixth month exhibited a positive correlation with the baseline level of leukocyte AS-A (p=0.005 and 0.009, respectively), suggesting that positive responses to the drug might be seen in patients with low AS-A activity.

Topics: Adult; Aged; Aged, 80 and over; Anticarcinogenic Agents; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cerebroside-Sulfatase; Chemotherapy, Adjuvant; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Interleukin-6; Leukocytes; Middle Aged; Tamoxifen

Breast cancer continues to be a serious public health problem. The role of the hedgehog pathway in normal development of the mammary gland as well as in carcinogenesis and progression of breast cancer is the subject of intense investigation, revealing functional interactions with cell surface heparan sulfate. Nevertheless, its influence on breast cancer prognosis, and its relation to specific sulfation motifs in heparan sulfate have only been poorly studied in large patient cohorts. Using the public database KMplotter that includes gene expression and survival data of 3951 patients, we found that the higher expression of SHH, HHAT, PTCH1, GLI1, GLI2, and GLI3 positively influences breast cancer prognosis. Stratifying patients according to the expression of hormone receptors, histological grade, lymph node metastasis, and systemic therapy, we observed that GLI1, GLI2, and GLI3 expression, as well as co-expression of SHH and ELP1 were associated with worse relapse-free survival in patients with HER2-positive tumors. Moreover, GLI1 expression in progesterone receptor-negative tumors and GLI3 expression in grade 3 tumors correlated with poor prognosis. SHH, in a panel of cell lines representing different breast cancer subtypes, and HHAT, PTCH1, GLI1, GLI2, and GLI3 were mostly expressed in cell lines classified as HER2-positive and basal-like. Expression of SHH, HHAT, GLI2, and GLI3 was differentially affected by overexpression of the heparan sulfate sulfotransferases HS2ST1 and HS3ST2 in vitro. Although high HS2ST1 expression was associated with poor prognosis in KMplotter analysis, high levels of HS3ST2 were associated with a good prognosis, except for ER-positive breast cancer. We suggest the GLI transcription factors as possible markers for the diagnosis, treatment, and prognosis of breast cancer especially in HER2-positive tumors, but also in progesterone receptor-negative and grade-3 tumors. The pathway interaction and prognostic impact of specific heparan sulfate sulfotransferases provide novel perspectives regarding a therapeutical targeting of the hedgehog pathway in breast cancer.

Topics: Breast Neoplasms; Female; Hedgehog Proteins; Heparitin Sulfate; Humans; Nerve Tissue Proteins; Patched-1 Receptor; Prognosis; Transcriptional Elongation Factors; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2; Zinc Finger Protein Gli3

In tumor development, the degradation of heparan sulfate (HS) by heparanase (HPSE) is associated with cell-surface and extracellular matrix remodeling as well as the release of HS-bound signaling molecules, allowing cancer cell migration, invasion and angiogenesis. Because of their structural similarity with HS, sulfated polysaccharides are considered a promising source of molecules to control these activities. In this study, we used a depolymerisation method for producing λ-carrageenan oligosaccharides (λ-CO), with progressive desulfation over time. These were then used to investigate the influence of polymeric chain length and degree of sulfation (DS) on their anti-HPSE activity. The effects of these two features on λ-CO anticoagulant properties were also investigated to eliminate a potential limitation on the use of a candidate λ-CO as a chemotherapeutic agent. HPSE inhibition was mainly related to the DS of λ-CO, however this correlation was not complete. On the other hand, both chain length and DS modulated λ-CO activity for factor Xa and thrombin IIa inhibition, two enzymes that are involved in the coagulation cascade, and different mechanisms of inhibition were observed. A λ-carrageenan oligosaccharide of 5.9 KDa was identified as a suitable anticancer candidate because it displayed one of the lowest anticoagulant properties among the λ-CO produced, while showing a remarkable inhibitory effect on MDA-MB-231 breast cancer cell migration.

Topics: Anticoagulants; Antineoplastic Agents; Breast Neoplasms; Carrageenan; Cell Line, Tumor; Cell Movement; Drug Screening Assays, Antitumor; Enzyme Assays; Female; Glucuronidase; Heparitin Sulfate; Humans; Oligosaccharides

The members of the slit homolog (SLIT) and roundabout homolog (ROBO) families have emerged as important signaling molecules in tumor metastasis. This study analyzed their role in regulating breast cancer (BC) cell motility and chemotaxis and assessed expression of ROBO1 in brain metastases (BMs) of breast, lung, and colon cancer, and in peritoneal metastases (PMs) of ovarian cancer.. The BC cell line MDA-MB231 was subjected to scratch, motility, and chemotaxis assays using heparin and a purified recombinant N-terminal SLIT2 fragment. Protein expression was assessed in primary tumors and metastases by immunohistochemistry.. Exposure to SLIT2 induced MDA-MB231 cell motility, but no significant chemotaxis without the presence of heparin. ROBO1 was expressed in 4/5 primary BC and in 18/21 BC-derived BM samples; 7/9 BM primary lung cancer samples also stained positive. In contrast, BMs from colorectal cancer were negative for ROBO1. Primary ovarian cancer and ovarian PM showed ROBO1 expression in 0/6 and in only 2/6 samples, respectively, whereas SLIT2 was observed in 1/6 primary cancer and in 6/6 PMs samples.. SLIT2 can induce BC cell motility and chemotaxis, but the latter requires the presence of heparin. BM expression of ROBO1 is a common feature of some, but not all cancer types. SLIT2 expression appears to be a general feature of ovarian cancer-derived PMs.

Topics: Brain Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Female; Heparitin Sulfate; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Nerve Tissue Proteins; Ovarian Neoplasms; Peritoneal Neoplasms; Receptors, Immunologic; Roundabout Proteins

Heparanase (HPSE) is an endo-beta-glucuronidase that degrades heparan sulfate (HS) chains on proteoglycans. The oligosaccharides generated by HPSE promote angiogenesis, tumor growth and metastasis. Heparanase-2 (HPSE2), a close homolog of HPSE, does not exhibit catalytic activity. Previous studies have demonstrated that serum or plasma from breast cancer patients showed increased expression of both heparanases in circulating lymphocytes. The aim of this study was to better understand the mechanisms involved in the upregulation of heparanases in circulating lymphocytes.. Lymphocytes collected from healthy women were incubated in the presence of MCF-7 breast cancer cells (co-culture) to stimulate HPSE and HPSE2 overexpression. The protein level of heparanases was evaluated by immunocytochemistry, while mRNA expression was determined by quantitative RT-PCR.. The medium obtained from co-culture of MCF-7 cells and circulating lymphocytes stimulated the expression of HPSE and HPSE2. Previous treatment of the co-culture medium with an anti-heparan sulfate proteoglycan antibody or heparitinase II inhibited the upregulation of heparanases in circulating lymphocytes. The addition of exogenous heparan sulfate (HS) enhanced the expression of both heparanases. Moreover, the co-cultured cells, as well as MCF-7 cells, secreted a higher number of exosomes expressing an increased level of HS compared to that of the exosomes secreted by circulating lymphocytes from women who were not affected by cancer.. The results revealed that HS is likely responsible for mediating the expression of heparanases in circulating lymphocytes. HS secreted by tumor cells might be carried by exosome particles, confirming the key role of tumor cells, as well as secreted HS, in upregulating the expression of heparanases, suggesting a possible mechanism of crosstalk between tumor cells and circulating lymphocytes.

Topics: Breast Neoplasms; Cell Communication; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glucuronidase; Heparitin Sulfate; Humans; Lymphocyte Activation; Lymphocytes; MCF-7 Cells; Receptor Cross-Talk

Heparan sulfate 3-O-sulfotransferases (HS3STs) catalyze the final maturation step of heparan sulfates. Although seven HS3ST isozymes have been described in human, 3-O-sulfation is a relatively rare modification, and only a few biological processes have been described to be influenced by 3-O-sulfated motifs. A conflicting literature has recently reported that HS3ST2, 3A, 3B and 4 may exhibit either tumor-promoting or anti-oncogenic properties, depending on the model used and cancer cell phenotype. Hence, we decided to compare the consequences of the overexpression of each of these HS3STs in the same cellular model. We demonstrated that, unlike HS3ST3A, the other three isozymes enhanced the proliferation of breast cancer MDA-MB-231 and BT-20 cells. Moreover, the colony forming capacity of MDA-MB-231 cells was markedly increased by the expression of HS3ST2, 3B and 4. No notable difference was observed between the three isozymes, meaning that the modifications catalyzed by each HS3ST had the same functional impact on cell behavior. We then demonstrated that overexpression of HS3ST2, 3B and 4 was accompanied by increased activation of c-Src, Akt and NF-κB and up-regulation of the anti-apoptotic proteins survivin and XIAP. In line with these findings, we showed that HS3ST-transfected cells are more resistant to cell death induction by pro-apoptotic stimuli or NK cells. Altogether, our findings demonstrate that HS3ST2, 3B and 4 share the same pro-tumoral activity and support the idea that these HS3STs could compensate each other for loss of their expression depending on the molecular signature of cancer cells and/or changes in the tumor environment.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; Killer Cells, Natural; Signal Transduction; Sulfotransferases

Breast cancer remains a leading cause of cancer-related mortality in women. In recent years, regulation of genes involved in heparan sulphate (HS) biosynthesis have received increased interest as regulators of breast cancer cell adhesion and invasion. The exostosin (EXT) proteins are glycosyltransferases involved in elongation of HS, a regulator of intracellular signaling, cell-cell interactions, and tissue morphogenesis. The EXT family contains five members: EXT1, EXT2, and three EXT-like (EXTL) members: EXTL1, EXTL2, and EXTL3. While the expression levels of these enzymes change in tumor cells, little is known how this changes the structure and function of HS. In the present study, we investigated gene expression profiles of the EXT family members, their glycosyltransferase activities and HS structure in the estrogen receptor (ER), and progesterone receptor (PR) positive MCF7 cells, and the ER, PR, and human epidermal growth factor receptor-2 (HER2) negative MDA-MB-231 and HCC38 epithelial breast carcinoma cell lines. The gene expression profiles for MDA-MB-231 and HCC38 cells were very similar. In both cell lines

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; N-Acetylglucosaminyltransferases; RNA, Messenger; Transcriptome

Heparan sulfate (HS) proteoglycan chains are key components of the breast tumor microenvironment that critically influence the behavior of cancer cells. It is established that abnormal synthesis and processing of HS play a prominent role in tumorigenesis, albeit mechanisms remain mostly obscure. HS function is mainly controlled by sulfotransferases, and here we report a novel cellular and pathophysiological significance for the 3-O-sulfotransferase 3-OST3A (HS3ST3A), catalyzing the final maturation step of HS, in breast cancer. We show that 3-OST3A is epigenetically repressed in all breast cancer cell lines of a panel representative of distinct molecular subgroups, except in human epidermal growth factor receptor 2-positive (HER2+) sloan-kettering breast cancer (SKBR3) cells. Epigenetic mechanisms involved both DNA methylation and histone modifications, producing different repressive chromatin environments depending on the cell molecular signature. Gain and loss of function experiments by cDNA and siRNA transfection revealed profound effects of 3-OST3A expression on cell behavior including apoptosis, proliferation, response to trastuzumab in vitro and tumor growth in xenografted mice. 3-OST3A exerted dual activities acting as tumor-suppressor in lumA-michigan cancer foundation (MCF)-7 and triple negative-MD Anderson (MDA) metastatic breast (MB)-231 cells, or as an oncogenic factor in HER2+-SKBR3 cells. Mechanistically, fluorescence-resonance energy transfer-fluorescence-lifetime imaging microscopy experiments indicated that the effects of 3-OST3A in MCF-7 cells were mediated by altered interactions between HS and fibroblast growth factor-7 (FGF-7). Further, this interplay between HS and FGF-7 modulated downstream ERK, AKT and p38 cascades, suggesting that altering 3-O-sulfation affects FGFR2IIIb-mediated signaling. Corroborating our cellular data, a clinical study conducted in a cohort of breast cancer patients uncovered that, in HER2+ patients, high level expression of 3-OST3A in tumors was associated with reduced relapse-free survival. Our findings define 3-OST3A as a novel regulator of breast cancer pathogenicity, displaying tumor-suppressive or oncogenic activities in a cell- and tumor-dependent context, and demonstrate the clinical value of the HS-O-sulfotransferase 3-OST3A as a prognostic marker in HER2+ patients.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; DNA Methylation; Female; Heparitin Sulfate; Humans; MCF-7 Cells; Mice; Prognosis; Receptor, ErbB-2; Signal Transduction; Sulfotransferases; Xenograft Model Antitumor Assays

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.Heparan sulfate (HS) of specific length, i.e., hexasaccharide (HS06), but not longer or shorter sequences, selectively inhibit cancer stem cells (CSCs) through isoform specific activation of p38 mitogen-activated protein kinase. These findings will have major implication for developing chemical probes to decipher complex signaling events that govern cancer stem cells. Additionally, there are direct implications for designing glycosaminoglycan based cancer therapies to selectively target CSCs that escape killing by traditional chemotherapy threatening cancer relapse.

Topics: Breast Neoplasms; Cell Self Renewal; Colorectal Neoplasms; Drug Discovery; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; Neoplastic Stem Cells; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Signal Transduction; Transcription Factor 4; Tumor Cells, Cultured

Dysregulation of microRNA is strongly implicated in the chemoresistance of cancer. In this study, we found that miR-149 was downregulated and involved in chemoresistance in adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM). Downregulation of miR-149 was related to hypermethylation of its 5'-UTR; this methylation also affected the expression of the glypican 1 gene, which is both the host and the target gene of miR-149. Furthermore, we found that miR-149 modulated chemoresistance through targeting the expression of GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST1). With downregulated miR-149, NDST1 expression was increased in chemoresistant MCF-7/ADM cells versus control MCF-7 wild-type cells. The increased NDST1 then activated a heparan sulfate-related pathway involving activation of heparanase. Finally, expression of miR-149 and NDST1 was confirmed in clinical chemoresistant samples of breast cancers receiving anthracycline/taxane-based chemotherapies. The high expression of NDST1 was also an unfavorable predictor for distant relapse-free survival in Her2 and basal breast cancers. Taken together, our findings demonstrate that miR-149 is regulated by methylation, and is a modulator of cancer chemoresistance by targeting NDST1.

Topics: 5' Untranslated Regions; Antibiotics, Antineoplastic; Blotting, Western; Breast Neoplasms; DNA Methylation; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; MicroRNAs; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfotransferases; Tumor Cells, Cultured

The present analyses were undertaken to define the mechanisms by which fetuin-A modulates cellular adhesion. FLAG-tagged fetuin-A was expressed in breast carcinoma and HEK-293T cells. We demonstrated by confocal microscopy that fetuin-A co-localizes with histone H2A in the cell nucleus, forms stable complexes with histones such as H2A and H3 in solution, and shuttles histones to exosomes. The rate of cellular adhesion and spreading to either fibronectin or laminin coated wells was accelerated significantly in the presence of either endogenous fetuin-A or serum derived protein. More importantly, the formation of focal adhesion complexes on surfaces coated by laminin or fibronectin was accelerated in the presence of fetuin-A or histone coated exosomes. Cellular adhesion mediated by histone coated exosomes was abrogated by heparin and heparinase III. Heparinase III cleaves heparan sulfate from cell surface heparan sulfate proteoglycans. Lastly, the uptake of histone coated exosomes and subsequent cellular adhesion, was abrogated by heparin. Taken together, the data suggest a mechanism where fetuin-A, either endogenously synthesized or supplied extracellularly can extract histones from the nucleus or elsewhere in the cytosol/membrane and load them on cellular exosomes which then mediate adhesion by interacting with cell surface heparan sulfate proteoglycans via bound histones.

Topics: alpha-2-HS-Glycoprotein; Breast Neoplasms; Cell Adhesion; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cytoplasm; Exosomes; Female; Fibronectins; Focal Adhesions; HEK293 Cells; Heparin; Heparitin Sulfate; Histones; Humans; Laminin; Polysaccharide-Lyases; Proteoglycans

The chemokine CCL2 (MCP-1) has been identified as a prominent tumor-promoting factor in breast cancer. The major source for CCL2 is in the tumor cells; thus, identifying the mechanisms regulating CCL2 release by these cells may enable the future design of modalities inhibiting CCL2 secretion and consequently reduce tumorigenicity. Using cells deficient in expression of glycosaminoglycans (GAGs) and short hairpin RNAs reducing heparan sulfate (HS) and chondroitin sulfate (CS) expression, we found that intracellular HS and CS (=GAGs) partly controlled the trafficking of CCL2 from the Golgi toward secretion. Next, we determined the secretion levels of GFP-CCL2-WT and GFP-CCL2-variants mutated in GAG-binding domains and/or in the 40s loop of CCL2 ((45)TIVA(48)). We have identified partial roles for R18+K19, H66, and the (45)TIVA(48) motif in regulating CCL2 secretion. We have also demonstrated that in the absence of R24 or R18+K19+(45)TIVA(48), the secretion of CCL2 by breast tumor cells was almost abolished. Analyses of the intracellular localization of GFP-CCL2-mutants in the Golgi or the endoplasmic reticulum revealed particular intracellular processes in which these CCL2 sequences controlled its intracellular trafficking and secretion. The R24, (45)TIVA(48) and R18+K19+(45)TIVA(48) domains controlled CCL2 secretion also in other cell types. We propose that targeting these chemokine regions may lead to reduced secretion of CCL2 by breast cancer cells (and potentially also by other malignant cells). Such a modality may limit tumor growth and metastasis, presumably without affecting general immune activities (as discussed below).

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Breast Neoplasms; Cell Line; Cell Line, Tumor; Chemokine CCL2; Chondroitin Sulfates; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Intracellular Space; Mutation; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport; Recombinant Fusion Proteins

Heparansulfate-proteoglycans (HSPGs) interact via their polyanionic heparansulfate (HS) side chains with a variety of proteins on the cell surface or within the extracellular matrix membrane. The large number of heparin/HS binding proteins form a highly interconnected functional network, which has been termed as the heparin/HS interactome and is functionally linked to physiological and pathological processes. The aim of this study was to investigate the global effect of these protein-HSPG interactions on the tumourigenicity of two breast cancer cell lines (MCF-7 and MDA-MB-231). Cancer cells were cultured in serum-free medium and treated with a concentration of heparin which was capable of modulating HS/ligand interaction. Microarray analysis of MCF-7 cells cultured under these conditions showed that expression of 105 of 1,357 genes potentially related to the pathogenesis of breast neoplasm was significantly altered by heparin treatment. The changes in gene expression correlated with a less tumourigenic phenotype, including reduction of cell adhesive, invasive and migratory properties. These effects were associated with an inhibition of the PI3K/Akt and Raf/MEK/ERK signalling pathways. The modulatory effect of heparin on HS-associated activity was confirmed with one example of heparin/HS interactomes, transforming growth factor β (TGFβ). The innate TGFβ activity of MCF-7 cells was reduced by heparin treatment, with specific interruption of the TGFβ-Smad signalling pathway. The pro-tumourigenic contribution of the heparin/HS interactomes was verified in cells in which HSPG synthesis was blocked using β-xyloside. In conclusion, the interaction between cell surface HPSGs and innate heparin/HS interactomes makes a significant contribution to the tumourigenicity.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Movement; Culture Media, Serum-Free; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Heparin; Heparitin Sulfate; Humans; Ligands; MAP Kinase Signaling System; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Protein Binding; Proteoglycans; Proto-Oncogene Proteins c-akt; Signal Transduction; Transforming Growth Factor beta

Trastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components--heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)--in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab.. The cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). The profile of sulfated glycosaminoglycans was also investigated by [35S]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate.. Breast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. The addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. In addition, HS shedding was increased significantly in these resistant cells.. Trastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. In addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Glucuronidase; Glycosaminoglycans; Heparitin Sulfate; Humans; MCF-7 Cells; Protein Binding; Protein Transport; Receptor, ErbB-2; Syndecan-1; Trastuzumab

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

The 67LR (67 kDa laminin receptor) enables cells to interact with components of the extracellular matrix. The molecule is derived from the 37LRP (37 kDa laminin receptor precursor); however, the precise molecular mechanism of this conversion is unknown. Recombinant 37LRP, expressed in and purified from Escherichia coli, bound to human laminin in a SPR (surface plasmon resonance) experiment. 67LR isolated from human breast-cancer-derived cells in culture was also shown to bind to laminin by SPR. However, the kinetics of association are qualitatively different. 37LRP, but not 67LR, binds to heparan sulfate. The binding of 37LRP to heparan sulfate did not affect the interaction of 37LRP with laminin. In contrast, heparan sulfate reduces the extent of binding of laminin to 67LR. Taken together, these results show that 37LRP has some of the biological activities of 67LR, even prior to the conversion event. However, the conversion affects the sites of interaction with both laminin and heparan sulfate.

Topics: Breast Neoplasms; Cell Line, Tumor; Escherichia coli; Extracellular Matrix; Heparitin Sulfate; Humans; Laminin; Protein Precursors; Receptors, Laminin; Recombinant Proteins; Surface Plasmon Resonance

A major limitation in biopharmaceutical development is selectively targeting drugs to diseased tissues. Growth factors and viruses have solved this problem by targeting tissue-specific cell-surface heparan sulfates. Neuregulin (NRG), a growth factor important in both nervous system development and cancer, has a unique heparin-binding domain (HBD) that targets to cell surfaces expressing its HER2/3/4 receptors (Esper, R. M., Pankonin, M. S., and Loeb, J. A. (2006) Brain Res. Rev. 51, 161-175). We have harnessed this natural targeting ability of NRG by fusing the HBD of NRG to soluble HER4. This fusion protein retains high affinity heparin binding to heparin and to cells that express heparan sulfates resulting in a more potent NRG antagonist. In vivo, it is targeted to peripheral nerve segments where it blocks the activity of NRG as a Schwann cell survival factor. The fusion protein also efficiently blocks autocrine and paracrine signaling and reduces the proliferation of MCF10CA1 breast cancer cells. These findings demonstrate the utility of the HBD of NRG in biopharmaceutical targeting and provide a new way to block HER signaling in cancer cells.

Topics: Animals; Apoptosis; Autocrine Communication; Breast Neoplasms; Cell Proliferation; Chick Embryo; CHO Cells; Cricetinae; Cricetulus; ErbB Receptors; Female; Heparin; Heparitin Sulfate; Humans; Molecular Sequence Data; Neuregulin-1; Paracrine Communication; Phosphorylation; Receptor, ErbB-4; Schwann Cells

Conditionally replicating adenoviruses (CRAds) that replicate in tumor but less in normal cells are promising anticancer agents. A major determinant of their potency is their capacity for infecting target cells. The primary receptor for serotype 5 adenovirus (Ad5), the most widely used serotype in gene therapy, is the coxsackie-adenovirus receptor (CAR). CAR is expressed variably and often at low levels in various tumor types including advanced breast cancer. We generated a novel p16/retinoblastoma pathway-dependent CRAd, Ad5.pK7-Delta24, with a polylysine motif in the fiber C-terminus, enabling CAR-independent binding to heparan sulfate proteoglycans (HSPG). Ad5.pK7-Delta24 mediated effective oncolysis of all breast cancer cell lines tested. Further, we utilized noninvasive, fluorescent imaging for analysis of antitumor efficacy in an orthotopic model of advanced hormone refractory breast cancer. A therapeutic benefit was seen following both intratumoral and intravenous delivery. Murine biodistribution similar to Ad5, proven safe in trials, suggests feasibility of clinical safety testing. Interestingly, upregulation of CAR was seen in low-CAR M4A4-LM3 breast cancer cells in vivo, which resulted in better than expected efficacy also with an isogenic CRAd with an unmodified capsid. These results suggest utility of Ad5.pK7-Delta24 and the orthotopic model for further translational studies.

Topics: Adenoviridae; Animals; Breast Neoplasms; Cell Line, Tumor; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Flow Cytometry; Gene Expression; Gene Targeting; Genetic Therapy; Genetic Vectors; Green Fluorescent Proteins; Heparitin Sulfate; Humans; Immunohistochemistry; Male; Mice; Mice, Nude; Models, Animal; Neoplasm Transplantation; Oncolytic Virotherapy; Protein Binding; Receptors, Virus; Transduction, Genetic; Virus Replication

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.

Topics: Acetylation; Animals; Bacterial Capsules; Betacellulin; Blotting, Western; Breast Neoplasms; Cattle; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Escherichia coli; Heparin; Heparitin Sulfate; Humans; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Magnetic Resonance Spectroscopy; Molecular Weight; Polysaccharides, Bacterial; Swine

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I(125) CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Movement; Chemokine CXCL12; Chemokines, CXC; Female; Gene Expression Regulation, Neoplastic; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Humans; Immunohistochemistry; Iodine Radioisotopes; Lung Neoplasms; Mice; Mice, SCID; Oligosaccharides; Polymers; Radioligand Assay; Receptors, CXCR4; Tinzaparin

Heparan sulphate is a sulphated glycosaminoglycan and is able to bind to and regulate the activity of many growth and signalling factors. We have previously shown that its expression is correlated with tumour grade and cell proliferation in breast phyllodes tumours. In this study, we examined the use of heparan sulphate as a biomarker of invasive ductal carcinoma and the effects of differentially sulphated heparan species on breast cancer cell behaviour. Immunohistochemistry using the 10E4 monoclonal antibody was carried out on 32 paraffin-embedded breast cancer specimens and paired non-cancerous breast tissues to compare the expression patterns of heparan sulphate. Upregulated expression of the sulphated 10E4 epitope in heparan sulphate was detected in both epithelial and stromal compartments of breast cancer compared with normal mammary tissues, with a 2.8X increase in immunoreactivity score. To determine the effects of differentially sulphated heparan sulphate molecules on breast cancer behaviour, cultured breast carcinoma cells were treated with chlorate, a competitive inhibitor of glycosaminoglycan sulphation, and two different heparan sulphate species. Inhibition of glycosaminoglycan sulphation resulted in a significant increase in cancer cell adhesion and a reduction in cell migration, together with upregulated expression of focal adhesion kinase and paxillin. Both porcine intestine- and bovine kidney-derived heparan sulphate species could block the change in cell adhesion. However, the former heparan sulphate species completely abolished, while the latter exacerbated, the chlorate-induced decrease in cell migration. The results show that heparan sulphate is a useful biomarker of breast invasive ductal carcinoma. Different sulphation patterns of heparan sulphate residues have differential effects in regulating breast cancer cellular behaviour, and this may be exploited to develop heparan sulphate into a useful target for treatment of breast carcinoma.

Topics: Animals; Breast Neoplasms; Cattle; Cell Adhesion; Cell Line, Tumor; Cell Movement; Female; Heparitin Sulfate; Humans; Swine

Heparin/heparan sulfate (HS) proteoglycans are found in the extracellular matrix (ECM) and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs) interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF), cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin.. Our results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC) to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities.. Our results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Chemokine CCL21; Chemokine CXCL12; Chemokines, CC; Chemokines, CXC; Culture Media, Conditioned; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Heparin; Heparitin Sulfate; Humans; Interleukin-8; Neoplasm Proteins; Protein Binding; Recombinant Fusion Proteins; Serum Albumin, Bovine; Sulfatases; Sulfotransferases; Vascular Endothelial Growth Factor A

We previously identified HSulf-1 as a down-regulated gene in several tumor types including ovarian, breast, and hepatocellular carcinomas. Loss of HSulf-1, which selectively removes 6-O-sulfate from heparan sulfate, up-regulates heparin-binding growth factor signaling and confers resistance to chemotherapy-induced apoptosis. Here we report that HSulf-1 expression in MDA-MB-468 breast carcinoma clonal lines leads to reduced proliferation in vitro and reduced tumor burden in athymic nude mice in vivo. Additionally, xenografts derived from HSulf-1-expressing stable clones of carcinoma cells showed reduced vessel density, marked necrosis, and apoptosis, indicative of inhibition of angiogenesis. Consistent with this observation, HSulf-1-expressing clonal lines showed reduced staining with the endothelial marker CD31 in Matrigel plug assay, indicating that HSulf-1 expression inhibits angiogenesis. More importantly, HSulf-1 expression in the xenografts was associated with a reduced ability of vascular endothelial cell heparan sulfate to participate in a complex with fibroblast growth factor 2 (FGF-2) and its receptor tyrosine kinase FGF receptor 1c. In vitro, short hairpin RNA-mediated down-regulation of HSulf-1 in human umbilical vein endothelial cells (HUVEC) resulted in an increased proliferation mediated by heparan sulfate-dependent FGF-2, hepatocyte growth factor, and vascular endothelial growth factor 165 (VEGF165) but not by heparan sulfate-independent VEGF121. HSulf-1 down-regulation also enhanced downstream signaling through the extracellular signal-regulated kinase pathway compared with untreated cells. Consistent with the role of heparan sulfate glycosaminoglycan sulfation in VEGF-mediated signaling, treatment of HUVEC cells with chlorate, which inhibits heparan sulfate glycosaminoglycan sulfation and therefore mimics HSulf-1 overexpression, led to an attenuated VEGF-mediated signaling. Collectively, these observations provide the first evidence of a novel mechanism by which HSulf-1 modulates the function of heparan sulfate binding VEGF165 in proliferation and angiogenesis.

Topics: Animals; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Endothelial Cells; Female; Fibroblast Growth Factor 2; Heparitin Sulfate; Humans; Mice; Mice, Nude; Neovascularization, Pathologic; Ovarian Neoplasms; Receptor, Fibroblast Growth Factor, Type 1; RNA, Small Interfering; Sulfotransferases; Transfection; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Phyllodes tumors are fibroepithelial neoplasms typified by stromal proliferation. We have previously shown the role of pathologic parameters and the prognostic significance of p53 and CD117 protein expression in these tumors. In this study, we evaluated the expression of heparan sulfate, which has been implicated in many biological processes such as cell adhesion, embryogenesis, and tumorigenesis (including malignant transformation of mammary cells) in 232 breast phyllodes tumors. We used a monoclonal antibody, 10E4, to examine the localization of heparan sulfate in phyllodes tumors by immunohistochemistry. The immunoreactivity of both epithelial and stromal components was examined and analyzed with pathological parameters and other immunohistochemical markers, including p53, MIB1, bcl2, and CD117. Stromal 10E4 expression was significantly associated with tumor grade, stromal p53, and MIB1 expression in proliferating cells, suggesting that heparan sulfate may participate in malignant tumor growth.

Topics: Antibodies, Antinuclear; Antibodies, Monoclonal; Biomarkers, Tumor; Breast Neoplasms; Cell Proliferation; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; Immunohistochemistry; Ki-67 Antigen; Phyllodes Tumor; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-kit; Retrospective Studies; Stromal Cells; Tissue Array Analysis; Tumor Suppressor Protein p53

An important class of carbohydrates studied within the field of glycobiology, heparin and heparan sulfate (HS) have been implicated in a diverse array of biological functions. Changes in their sulfation pattern and domain organization have been associated with different pathological situations such as viral infectivity, tumor growth, and metastasis. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. The method was applied to assay for the heparin substrate specificity of a newly discovered human extracellular endosulfatase, HSulf-2, which has been implicated in tumorigenesis. This new protocol incorporates 12 known heparin disaccharides, including three sets of isomers. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues.

Topics: Animals; Biological Assay; Breast Neoplasms; Cattle; Disaccharides; Heparin; Heparitin Sulfate; Humans; Intestinal Mucosa; Kidney; Lung; Protein Binding; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Fast Atom Bombardment; Sulfatases; Swine

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.

Topics: Animals; Biological Transport; Breast Neoplasms; Cell Line, Tumor; CHO Cells; Cricetinae; Glioma; Glucuronidase; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Humans; Kinetics; Recombinant Proteins; Transfection

Topics: Adenocarcinoma; Angiogenesis Inducing Agents; Basement Membrane; Breast Neoplasms; Disease Progression; Enzyme Inhibitors; Female; Glucuronidase; Growth Substances; Heparitin Sulfate; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Catalytic; Transfection

A tumor-homing peptide, F3, selectively binds to endothelial cells in tumor blood vessels and to tumor cells. Here, we show that the cell surface molecule recognized by F3 is nucleolin. Nucleolin specifically bound to an F3 peptide affinity matrix from extracts of cultured breast carcinoma cells. Antibodies and cell surface biotin labeling revealed nucleolin at the surface of actively growing cells, and these cells bound and internalized fluorescein-conjugated F3 peptide, transporting it into the nucleus. In contrast, nucleolin was exclusively nuclear in serum-starved cells, and F3 did not bind to these cells. The binding and subsequent internalization of F3 were blocked by an antinucleolin antibody. Like the F3 peptide, intravenously injected antinucleolin antibodies selectively accumulated in tumor vessels and in angiogenic vessels of implanted "matrigel" plugs. These results show that cell surface nucleolin is a specific marker of angiogenic endothelial cells within the vasculature. It may be a useful target molecule for diagnostic tests and drug delivery applications.

Topics: Active Transport, Cell Nucleus; Antibodies; Antigens, Surface; Biomarkers, Tumor; Blood Vessels; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Culture Media, Serum-Free; Endocytosis; Endothelium, Vascular; Epitopes; Heparitin Sulfate; Humans; Neovascularization, Pathologic; Nucleolin; Phosphoproteins; RNA-Binding Proteins

Patients with malignancy often present with a variety of coagulation abnormalities which may ultimately lead to recurrent arterial and venous thromboses. Recently the presence of antiphospholipid antibodies in cancer patients has been proposed as one of the potential mechanisms promoting hypercoagulability. Here we report two consecutive patients with localized tumors, one suffering from breast cancer and another presenting with colorectal cancer, who experienced dramatic exacerbation of the antiphospholipid antibody syndrome (APAS) within 4 weeks after surgery. In the first patient who had also received one course of adjuvant chemotherapy, major ischemic stroke and recurrent venous thromboembolism were paralleled by the development of ulcerative livedoid vasculitis and pancytopenia, constituting the diagnosis of systemic lupus erythematosus with secondary APAS. In the second patient, progressive thrombotic occlusion of the superior and inferior vena cava was associated with bilateral pulmonary embolism, acute renal failure, and disabling soft tissue edema. Although not fulfilling the classic criteria of "catastrophic" APAS, the clinical features were life threatening and appeared to be refractory to oral anticoagulation with phenprocoumon. In addition, a diagnosis of Trousseau's syndrome was unlikely due to missing evidence of gross metastatic disease. Besides a suggested treatment strategy comprising high doses of low-molecular-weight heparin, potential pathogenic mechanisms are discussed in consideration of a recently proposed "thrombotic storm," which may cause multiple thromboses after an initial provocation in patients with known hypercoagulability.

Topics: Adult; Antiphospholipid Syndrome; Breast Neoplasms; Chemotherapy, Adjuvant; Chondroitin Sulfates; Colorectal Neoplasms; Dermatan Sulfate; Drug Combinations; Female; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Humans; Lupus Erythematosus, Systemic; Middle Aged; Neoplasms; Stroke; Thromboembolism; Thrombophilia

The growth of the malignant human mammary MDA-MB-231 cells is stimulated by fibroblast growth factor-1 (FGF-1) but not by FGF-2. When these cells are cultured in the presence of chlorate, an inhibitor of heparan sulfate (HS) sulfation, their proliferation is stimulated by both FGF-1 and FGF-2. We analyzed the interactions of FGF-1 and FGF-2 with HS purified from the cell layer and the culture medium of control and chlorate-treated MDA-MB-231 cells. The HS from the cell layer bound FGF-1 with faster association kinetics than the HS from the culture medium, and so had a higher affinity for FGF-1. Chlorate treatment had no significant effect on the FGF-1 binding kinetics of the HS. In contrast to FGF-1, chlorate treatment of the cells significantly altered the FGF-2 binding kinetics. The HS from untreated cells possessed two binding sites for FGF-2, one with fast association kinetics (k(ass) 470,000 to 610,000 M(-1) s(-1)) and a high affinity (K(d) 46 to 70 nM) and one with slower association kinetics (k(ass) 74,000 to 100,000 M(-1) s(-1)) and a lower affinity (K(d) 290 to 400 nM). HS from chlorate-treated cells possessed just a single binding site for FGF-2 with fast association kinetics (k(ass) 270,000 to 290,000 M(-1) s(-1)) and a high affinity (K(d) 41 to 57 nM). These results show that there is a relationship between the binding kinetics of FGFs and their ability to stimulate cell growth.

Topics: Artifacts; Breast Neoplasms; Cell Division; Cell Survival; Chlorates; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Heparitin Sulfate; Humans; Kinetics; Recombinant Proteins; Tumor Cells, Cultured

To explore how heparan sulfate (HS) controls the responsiveness of the breast cancer cell lines MCF-7 and MDA-MB-231 to fibroblast growth factors (FGFs), we have exposed them to HS preparations known to have specificity for FGF-1 (HS glycosaminoglycan (HSGAG A)) or FGF-2 (HSGAGB). Proliferation assays confirmed that MCF-7 cells were highly responsive to FGF-2 complexed with GAGB, whereas migration assays indicated that FGF-1/HSGAGA combinations were stimulatory for the highly invasive MDA-MB-231 cells. Quantitative polymerase chain reaction for the levels of FGF receptor (FGFR) isoforms revealed that MCF-7 cells have greater levels of FGFR1 and that MDA-MB-231 cells have greater relative levels of FGFR2. Cross-linking demonstrated that FGF-2/HSGAGB primarily activated FGFR1, which in turn up-regulated the activity of mitogen-activated protein kinase; in contrast, FGF-1/HSGAGA led to the phosphorylation of equal proportions of both FGFR1 and FGFR2, which in turn led to the up-regulation of Src and p125(FAK). MDA-MB-231 cells were particularly responsive to vitronectin substrates in the presence of FGF-1/HSGAGA, and blocking antibodies established that they used the alpha(v)beta(3) integrin to bind to it. These results suggest that the clustering of particular FGFR configurations on breast cancer cells induced by different HS chains leads to distinct phenotypic behaviors.

Topics: Breast Neoplasms; Cell Division; Cell Movement; Culture Media, Serum-Free; Drug Interactions; Epithelial Cells; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Heparitin Sulfate; Humans; Mitogen-Activated Protein Kinases; Phenotype; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor; Receptors, Vitronectin

The MCF-7 breast cancer cells exhibit remarkable growth enhancement in response to basic fibroblast growth factor (FGF-2) stimulation in a dose dependent manner. To investigate the involvement of proteoglycans on control of FGF-2 induced proliferation, polysaccharide chains were degraded by specific enzymes. Our results showed that MCF-7 cells were unsensitive to FGF-2 after enzymatic degradation of heparin sulfate proteoglycans (HSPG) by heparinase. After metabolic inhibition of sulphation by sodium chloride, radiolabelled proteoglycans were purified and quantified by ion exchange chromatography. Sodium chlorate treatment reduced by 70% sulfation of proteoglycans. This decrease of sulphation totally inhibited FGF-2-mediated proliferation. The sulphated glycosaminoglycans which were critical in FGF-2-induced proliferation were strictly HSPG, as an addition of heparin in cell culture medium can restore FGF-2 mitogenic activity. In contrast, other glycosaminoglycans (chondroitin sulfate/hyaluronic acid) did not show any effect. These results provide clear evidence for the critical role of HSPG in FGF-2-induced proliferation on MCF-7 breast cancer cells.

Topics: Breast Neoplasms; Cell Division; Chlorates; Female; Fibroblast Growth Factor 2; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Proteoglycans; Tumor Cells, Cultured

Topics: Breast Neoplasms; Disaccharides; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Molecular Structure; Receptors, Fibroblast Growth Factor; Tumor Cells, Cultured

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of 125I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10-30 mM; higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.

Topics: Breast Neoplasms; Cell Differentiation; Cell Division; Chlorates; Female; Fibroblast Growth Factor 2; Heparan Sulfate Proteoglycans; Heparin Lyase; Heparitin Sulfate; Humans; Mitogens; Polysaccharide-Lyases; Proteoglycans; Sulfates; Tumor Cells, Cultured

Breast tumor cells have been shown to be responsive to calcium in that external calcium modifies cell calcium, shape and growth. In order to highlight some of the numerous mechanisms by which calcium is operating, we investigated its influence on the cell microenvironment and particularly its effect on membrane-associated heparan sulfate proteoglycans. The breast cancer cells MCF-7 were grown either at low (0.04 mM) or high (2.5 mM) calcium concentration. After 3 days of culture, cells were labeled with Na2(35)SO4 for 24 h and cell-associated proteoglycans extracted and purified. We showed that calcium enhances approximately twofold the synthesis of sulfated proteoglycans and, among these sulfated proteoglycans, chemical treatments indicated a specific two- to threefold increase of heparan sulfate proteoglycans. In view of the increasing implication of heparan sulfate proteoglycans in numerous mechanisms such as cell-cell contact, cell-matrix interactions and cell growth control, it appears that calcium may be a target for modulating metastatic and growth processes in breast tumor cells.

Topics: Breast Neoplasms; Calcium; Cell Membrane; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Proteoglycans; Tumor Cells, Cultured

The histological and histochemical findings and the clinical course of 160 cases of breast cancer were compared with the biochemical values of the content and pattern of glycosaminoglycans (GAG) in the tumour tissue. For this purpose the cases were divided into 4 groups of increasing malignancy of the tumours, based on the morphological data and the clinical course after radical mastectomy (4-9 years follow-up). Samples of tumour tissue were examined biochemically-analytically for their total content of glycosaminoglycans and for their mg/g values of hyaluronic acid (H), chondroitin-4- and chondroitin-6-sulphates (CA + CC), dermatan sulphate (CB), heparan sulphate (HpS) and component (K). The statistical processing by means of the method of "discriminant analysis" of the corresponding GAG values in relation to the degree of malignancy displayed by the tumours yielded the following results: 1) the glycosaminoglycan patterns of the mammary carcinoma have characteristic features in common for the large majority of cases; 2) an exception of about 5% of the cases developed carcinomata with a very high (up to 100%) content of component. These cases generally have a more favourable clinical course (group 1); 3) the higher the ratio of hyaluronic acid to chondroitin sulphates the better the prognosis (groups 1 and 2, respectively). A ratio of hyaluronic acid to chondroitin sulphates under 0.5 indicates a poor prognosis (group 3 and 4, respectively); 4) high values of heparan sulphate and small ratios of hyaluronic acid to heparan sulphate also signal an unfavourable prognosis (groups 3 or 4). We consider that the above results are a means of defining the degree of malignancy of the breast tumours.

Topics: Breast Neoplasms; Chondroitin Sulfates; Discriminant Analysis; Female; Follow-Up Studies; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Mastectomy, Radical; Prognosis; Survival Analysis

The proteoglycans secreted by a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal human breast cell line (HBL-100). The physicochemical characteristics of these proteoglycans were established by hexosamine analysis, chemical and enzymatic degradations, and dissociative cesium chloride density gradient centrifugation, and by gel filtration before and after alkaline beta-elimination. Both cell lines secreted approximately 70% of the synthesized proteoglycans, which were composed of 20% heparan sulfate and 80% chondroitin sulfate proteoglycans. The MDA cell line secreted large hydrodynamic size (major) and small hydrodynamic size heparan sulfate proteoglycan. In contrast HBL cells secreted only one species having a hydrodynamic size intermediate to the above two. The chondroitin sulfate proteoglycans from MDA medium were slightly larger than the corresponding polymers from HBL medium. All proteoglycans except the small hydrodynamic size heparan sulfate proteoglycan from MDA medium were of high buoyant density. The proteoglycans of both cell lines contained significant proportions of disulfide-linked lower molecular weight components which were more pronounced in the proteoheparan sulfate polymers, particularly those from MDA medium, than in chondroitin sulfate proteoglycans. The glycosaminoglycans of heparan sulfate proteoglycans from MDA medium were more heterogeneous than those from HBL medium. The glycosaminoglycan chains of large hydrodynamic size heparan sulfate proteoglycans from MDA medium were larger in size than those from HBL medium while small hydrodynamic size heparan sulfate proteoglycans contained shorter glycosaminoglycan chains. In contrast to the glycosaminoglycans derived from chondroitin sulfate proteoglycans of both MDA and HBL medium were comparable in size. The heparan sulfate as well as chondroitin sulfate proteoglycans of both cell lines contained both neutral (di- and tetrasaccharides) and sialylated (tri- to hexasaccharides) O-linked oligosaccharides.

Topics: Adenocarcinoma; Breast; Breast Neoplasms; Carbohydrate Conformation; Cell Line; Centrifugation, Density Gradient; Chondroitin Sulfates; Chromatography, Gel; Disulfides; Epithelium; Female; Galactosamine; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Humans; Molecular Weight; Oligosaccharides; Proteoglycans

The distribution of basement membrane (BM) markers, type IV collagen, laminin (LM), heparan sulphate proteoglycan (HSP) and fibronectin (FN) has been studied by indirect immunofluorescence using specific antibodies, in tumoural pathology. The disrupted pattern of BM by these markers in severe dysplastic lesions of the breasts, the bronchi and uterine cervix provides evidence for malignancy. In invasive carcinomas, there is generally a loss of these BM components, with FN persisting in the stroma. The loss of these markers in BM is concomitant and superimposable in double staining studies. In embryonic tumours, the presence of BM markers is related to a mesenchymal differentiation of malignant cells with pericellular FN and/or maturation towards organoid structures with BM. In sarcomas, there is a loss of the pericellular BM staining around most transformed muscular and Schwann cells and adipocytes. The persistence of this labelling in some well-differentiated areas can help to diagnose the nature of the sarcoma. The persistence of intercellular filaments of FN corresponds to the mesenchymal and/or sarcomatous nature of undifferentiated anaplastic proliferations.

Topics: Basement Membrane; Breast Neoplasms; Cell Transformation, Neoplastic; Collagen; Female; Fibronectins; Fluorescent Antibody Technique; Heparitin Sulfate; Humans; Laminin; Lung Neoplasms; Male; Neoplasms; Neoplasms, Germ Cell and Embryonal; Sarcoma

The distribution of four basement membrane components, type IV collagen (C IV), laminin (LM), heparan sulfate proteoglycan (HSP) and fibronection (FN) has been studied by indirect immunofluorescence using specific antibodies, in benign and malignant proliferations of the mammary gland and in soft tissue tumors. In breast carcinomas, specially intraductal cancers, there is a progressive and concomitant loss of these macromolecules around tumoral cells, preceding an overt tumoral invasion. In sarcomas, FN is frequently seen between malignant cells but the regular pericellular labeling observed around normal muscular cells, Schwann cells and adipocytes is absent. Nevertheless, the persistance of some pericellular staining with anti-C IV, anti-LM, anti-HSP and anti-FN antisera, in most differentiated territories of liposarcomas, leiomyosarcomas and neurifibrosarcomas can help to the diagnosis of such lesions.

Topics: Basement Membrane; Breast Neoplasms; Collagen; Female; Fibronectins; Fluorescent Antibody Technique; Heparitin Sulfate; Humans; Laminin; Neoplasms; Sarcoma

Glycosaminoglycans have been characterized from a normal human breast cell line (HBL-100) and two different cell lines from human breast carcinoma (MDA-MB-231 and MCF-7). The glycosaminoglycans were labeled by exposure of cell cultures to [3H]glucosamine and [35S]sulfate and then isolated from both spent media and cells by pronase digestion and cetylpyridinium chloride fractionation. They were further characterized by (a) hexosamine composition, (b) controlled-pore glass exclusion chromatography, (c) reactivity with specific enzymes (hyaluronidase chondroitinase, heparitinase, and heparinase), (d) nitrous acid degradation, and (e) DEAD-Sephadex chromatography. The results indicate that the HBL-100 line synthesizes mainly hyaluronic acid, most of which is secreted into the medium. Chondroitin sulfate and heparan sulfate are the predominant glycosaminoglycans synthesized by the cancer lines; both are found mainly in the spent medium, but the hyaluronic acid synthesized by the MDA-MB-231 line remains cell associated. The cell-associated heparan sulfate had a molecular weight in excess of 13,000 and may contain linkages susceptible to testicular hyaluronidase. The MCF-7 cells produce significantly lower amounts of glycosaminoglycans than do the other two lines.

Topics: Breast; Breast Neoplasms; Cell Line; Chondroitin Sulfates; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Neoplasms, Experimental

The correlation between the content of individual glycosaminoglycans and the histological patterns are studied on breast tumor tissues. The myxomatous stroma of intracanalicular fibroadenoma contained a large amount of glycosaminoglycans, which were mainly hyaluronic acid. The chondroitin 4- and 6-sulfate level was also high. As the supporting stroma of this tumor became denser and more fibrous, the level of hyaluronic acid content was reduced. In the case of pericanalicular fibroadenoma, glycosaminoglycans were small in amount and the levels of hyaluronic acid and chondroitin sulfate were low, but the ratio of dermatan sulfate content was higher. In the case of gynecomastia, the conent was almost the same as that of pericanalicular fibroadenoma. Scirrhous carcinoma tissues contained a relatively large amount of hyaluronic acid and chondroitin sulfate. No remarkable differences in heparan sulfate content were observed in any one of the breast tumors tested. Dermatan sulfate-chondroitin sulfate copolymers were detected in all the tumors. The presence of dermatan sulfate seemed to have an intimate relation with the fibrogenesis in the interstitial stromal element of the tumor tissues.

Topics: Adenocarcinoma, Scirrhous; Adenofibroma; Adult; Breast Neoplasms; Chondroitin Sulfates; Dermatan Sulfate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Middle Aged; Phyllodes Tumor