Breast cancer invasion is mediated by beta-N-acetylglucosaminidase (beta-NAG) and associated with a dysregulation in the secretory pathway of cancer cells.

The extracellular matrix is enriched with carbohydrate polymers that mask the protein backbone. This study aims to test the hypothesis that for successful cancer cell invasion the cells must secrete glycosidases to reveal the protein backbone, and then the action of proteases provides the physical space needed for cancer cell movement. Thus, the activity of intracellular and secreted beta-N-acetylglucosaminidase (beta-NAG) was assayed in luminal breast epithelial cells (HB4a) and breast cancer cells (BT474, ZR75-1, MDA-MB-435, MCF7). An increase in the V(max) of beta-NAG was observed in MDA-MB-435 and MCF7 cells. Exoglycosidases are normally located in the lysosomes and function at an acidic pH, but in the cancer cells there was significant enzyme activity at neutral pH. A change in lysosome location and number was observed in the cancer cells, consistent with alterations in the secretory pathway. Finally, applying a cocktail of protease inhibitors resulted in a 20% reduction in invasion of MDA-MB-435 cells through Matrigel after 24 h, and when the cells were treated with protease and beta-NAG inhibitors then cellular invasion was reduced by > 60%. The results suggest combination therapies that inhibit proteases and glycosidases might be a rational way forward for the design of drugs aimed at arresting cellular invasion.

Predictors of axillary lymph node metastasis in breast cancer: a systematic review.

AIMS: To review the established and emerging techniques in axillary lymph node prediction and explore their potential impact on clinical practice. To reliably identify patients in whom axillary lymph node surgery, including SLNB, can be safely omitted. METHODS: Searches of PubMed were made using the search terms "axilla" (or "axillary"), "lymph", "node" and "predictor" (or "prediction"). Articles from abstracts and reports from meetings were included only when they related directly to previously published work. FINDINGS: There are numerous studies in which the predictive utility of biomarkers as determinants of axillary lymph node status have been investigated. Few of these have specifically addressed the attributes of the primary tumour which could offer much potential for the prediction of tumour metastasis to the axillary lymph nodes. CONCLUSIONS: Currently, no single marker is sufficiently accurate to obviate the need for formal axillary staging using SLNB or axillary clearance.

Harnessing changes in cellular glycosylation in new cancer treatment strategies.

The majority of proteins are modified in post-translational events and one of the most common of these is glycosylation. Many reports describe alterations to the normal cellular glycosylation in cancer but detailed knowledge of the underlying structures and mechanisms that result in the altered glycosylation of cancer glycoproteins have been hindered by the inherent complexity of glycans themselves. Improved analytical tools for the study of glycosylation and application of molecular techniques for the characterisation of the genes encoding glycosyltransferases have, however, enabled the structural identification of some of the cancer-associated changes in glycosylation. The observed alterations in protein glycosylation in cancer have led to clinical trials in which glycans on cancer cell-surface proteins are targeted. These new approaches to cancer treatment include immunotherapy and carbohydrate-processing inhibitor-based strategies. Compounds that mimic glycans involved in the metastatic dissemination of cancer are also actively sought. The results that have been obtained and the long-term potential of these new approaches are discussed in this review article.

Proteome analysis enables separate clustering of normal breast, benign breast and breast cancer tissues.

We have used proteomics with cluster analysis for the classification of breast tumour tissues. In our approach, we can distinguish between normal breast, benign breast and breast cancer tissues on the basis of the protein expression profiles. We propose an objective method for the classification of breast tumour specimens.

Proteome and glycosylation mapping identifies post-translational modifications associated with aggressive breast cancer.

Changes in glycosylation of glycoproteins and glycolipids is a common feature of cancer and may influence cancer cell behaviour, perhaps by enabling cell-cell interactions which favour metastasis or by allowing cancer cells to evade immuno-surveillance. Studies to identify glycosylation changes in human cancer have often used immunohistochemical techniques with lectins or antibodies and human tissue sections. Whilst some detailed biochemical studies have been performed there are few clinically relevant studies since the numbers of specimens evaluated are often very small. Using an immuno-histochemical approach, we have found that the lectin HPA from the albumen gland of Helix pomatia detects aggressive metastatic breast cancers. We have sought to identify the breast cancer associated oligosaccharides and the proteins to which they are attached via 2-DE for proteome analysis and HPLC separations for glycosylation mapping. Sixty-nine breast cancer specimens were studied. The HPA staining pattern, clinical treatment and follow-up data were known. Oligosaccharides that related to HPA staining were identified and found in elevated levels in metastatic breast cancer specimens. Human breast and colorectal cancer cells grown in vitro and in a clinically relevant animal model of metastasis also show increased levels of the oligosaccharides. We are now structurally characterising the oligosaccharides and aim to use them as diagnostic tools and targets for immuno-therapy of breast and other solid tumours.

Helix pomatia agglutinin lectin-binding oligosaccharides of aggressive breast cancer.

Predicting long-term outcome after breast-cancer diagnosis remains problematic, particularly for patients with clinically small, axillary lymph node- negative tumours. Evidence suggests that the lectin Helix pomatia agglutinin (HPA) identifies oligosaccharides associated with poor-prognosis cancer. Our aim was to identify oligosaccharides that bind HPA in aggressive breast cancers. Breast-cancer cell lines (MCF-7, BT-549 and BT-20) and a cell line from human milk (HBL-100), which showed a range of HPA-binding intensities, were used to extract HPA-binding glycoproteins. Oligosaccharides were released using anhydrous hydrazine and separated on a range of HPLC matrices. We investigated whether HPA-binding oligosaccharides from cell lines were present in human breast-cancer tissues, using 69 breast-cancer specimens from patients with between 5 and 10 years' follow-up. A monosialylated oligosaccharide was over-expressed in the cell line that bound HPA strongly. Further analysis by normal-phase HPLC showed that the 2-aminobenzamide-conjugated oligosaccharide had a hydrodynamic volume of 4.58 glucose units (HPAgly1). Increased expression of HPAgly1 was associated with HPA staining of breast-cancer specimens (Student's t-test p = 0.025). Analysis of oligosaccharide levels and disease-free survival after treatment for breast cancer indicated a shorter disease-free interval for patients with elevated levels of HPAgly1. This is the first time that histochemical lectin staining has been correlated with biochemical mapping of oligosaccharides. Using this approach, we have identified a monosialylated HPA lectin-binding oligosaccharide present in breast-cancer cells grown in vitro which is elevated in breast-cancer specimens that bind the lectin.

Release and analysis of polypeptides and glycopolypeptides from formalin-fixed, paraffin wax-embedded tissue.

Archival tissue specimens are commonly stored as formalin-fixed, paraffin wax-embedded blocks. Formalin fixation facilitates excellent morphological preservation, and the immunoreactivity of many antigens is preserved, but formalin-induced chemical cross-linking of proteins renders them insoluble and inaccessible to standard biochemical extraction and analytical methods. Thus, biochemical analysis of tissue components identified by histochemistry, with the advantage of long-term clinical follow-up, is precluded. We have applied cyanogen bromide cleavage, a technique used routinely for fragmenting proteins for sequencing experiments, to solubilize transferrin polypeptides and glycopolypeptides from formalin-fixed, paraffin wax-embedded rat liver. Cyanogen bromide cleaves protein specifically at methionine residues, yielding a predictable array of polypeptide fragments. Subsequent oligosaccharide analysis of the transferrin glycopolypeptides by anion exchange chromatography confirmed that, in addition to successful release of polypeptide chains, sialylated oligosaccharide structures remained intact after cyanogen bromide cleavage. This approach may have wide applicability to a range of research interests in which correlation of tissue biochemistry with long-term follow-up is advantageous.

Breast cancer progression is associated with a reduction in the diversity of sialylated and neutral oligosaccharides.

Changes in the oligosaccharides attached to glycoproteins and glycolipids have been observed in a variety of malignancies. To understand the relationship between oligosaccharide expression and breast cancer progression we extracted and mapped the sialylated and neutral oligosaccharides from primary breast tumours of patients treated between 1979 and 1981 at Middlesex and University College Hospitals, London. Tumours from two patient groups were evaluated as short-term and long-term survivors. Short-term survivors developed widespread disease within five years (n = 10) whereas long-term survivors had no sign of cancer after fifteen years (n = 9). Paraffin-wax embedded breast cancer specimens were microdissected, the oligosaccharides were released and mapped by separation on anion-exchange and gel permeation chromatography columns. A decrease in the diversity of sialylated and neutral oligosaccharides and the number of sialylated structures was observed in aggressive breast cancers. Aggressive cancers had elevated levels of a mono- and tri-sialylated oligosaccharide only found in trace levels in non-aggressive cancers.

Oligosaccharide release from frozen and paraffin-wax-embedded archival tissues.

Altered glycosylation is a feature of many solid tissue diseases such as ulcerative colitis, Crohn's disease, cancer, and connective tissue disorders. Conventionally, oligosaccharide changes have been studied by immunohistochemical techniques. We have adapted existing techniques, developed for purified protein preparations, to allow the release of intact oligosaccharides from archival tissues, so that the oligosaccharides may be structurally characterized. In our study, sections cut from paraffin-wax blocks were dewaxed and oligosaccharides were released using hydrazine and labeled with 2-aminobenzamide. Sialylated oligosaccharides were compared by passing through a GlycoSep C divinylbenzene anion exchange resin column and neutral oligosaccharides were compared by passing through a BioGel P4 column. The oligosaccharide profiles obtained from the same fresh frozen versus archival paraffin-wax-embedded, normal liver, and tumor tissues showed remarkable similarity in terms of their sialylated and neutral structures. Matrix-assisted laser desorption ionization mass spectrometry has shown that the oligosaccharides are not affected by fixation in formalin and storage in paraffin wax. The results indicate that while the proteins themselves may be denatured, oligosaccharides are not adversely affected by fixation in formalin and storage in paraffin wax. By applying these methods, oligosaccharides from archival tissues, where the natural history of the disease has been followed, may now be liberated and structurally characterized.

Identification, purification and analysis of a 55 kDa lectin binding glycoprotein present in breast cancer tissue.

The Helix pomatia agglutinin (HPA)-binding glycoproteins from primary breast cancers and their metastases were compared with appropriate normal control tissues on Western blots. From these studies a single glycoprotein of 55 kDa was found to bind HPA in tumours but not in normal control tissues. The glycoprotein was identified by protein sequencing as being homologous to human immunoglobulin heavy chain variable region. Subsequent immunostaining showed it to be immunoglobulin subclass A. IgA1 was purified from both tumour and normal tissue by affinity chromatography. It was demonstrated that IgA1 from tumour tissue bound HPA whereas IgA1 from normal tissue did not. The oligosaccharides were cleaved from the protein backbone and the glycans from the HPA-binding glycoform of IgA1 were compared with those from normal human IgA1. IgA1 from tumour tissue appears to be associated with an HPA-binding glycan which is not present on the normal tissue-derived IgA1.