Miriam Dwek

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 immuno‐histochemical 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.

Cell surface glycan–lectin interactions in tumor metastasis

The development of secondary cancers, metastases, requires that a multitude of events are completed in an ordered and sequential manner. This review focuses on the role of cell surface glycans and their binding partners in the metastatic process. A common feature of metastasis is that the steps require adhesive interactions; many of these are mediated by cell surface glycans and their interactions with endogenous carbohydrate binding proteins (lectins). Aberrant glycosylation is a key feature of malignant transformation and the glycans involved influence the adhesive interactions of cancer cells often providing favorable conditions for tumor dissemination. This review focuses on glycans on the cancer cell surface and their association with endogenous lectins. In particular, E-cadherin and siglec-mediated disaggregation of tumor cells from the primary tumor mass; integrins, laminin and CD44-mediated invasion and migration of tumor cells through the connective tissue; the involvement of heparan sulphate in tumor angiogenesis and C-/S-type lectin interactions with the vasculature. The potential role of glycans in cancer cell evasion of immune surveillance is considered.

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.

The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer.

There has been considerable interest in understanding the epitopes that bind the lectin Helix pomatia agglutinin (HPA) in breast cancer as the lectin has been shown to identify glycosylation changes associated with the development of metastatic disease. HPA has previously been shown to recognize aberrant O-linked α-N-acetylgalactosamine (GalNAcα)/mucin glycosylation in cancer, including exposed Tn epitopes. However, recent glycan-array analysis reported that diverse epitopes are also recognized by the lectin, e.g. consortium for functional glycomics (CFG) data: GalNAcα1,3Gal; β-GalNAc; GlcNAcβ1,4Gal. The intriguing observations from the CFG array led to this study, in which HPA-binding epitopes were localized and characterized in an in vitro model of breast cancer metastasis. HMT3522 (benign disease), BT474 (primary cancer) and T47D/MCF7 (metastatic cancer) cells were assessed in confocal microscopy-based co-localization studies and a glycoproteomic analysis based on 2-dimensional electrophoresis (2DE), western blotting and mass spectrometry was adopted. HPA binding correlated with levels of integrin α6, transcription factors heterogeneous nuclear ribonuclear protein (HnRNP) H1, HnRNP D-like, HnRNP A2/B1 as well as heat shock protein 27 (Hsp27), glial fibrillary acidic protein and enolase 1 (ENO1). These glycoproteins were non-detectable in the non-metastatic breast cancer cell lines. The recognition of HnRNPs, Hsp27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Integrin α6 was the most abundant HPA glycoprotein in the breast cancer cells with a metastatic phenotype; this concurred with previous findings in colorectal cancer. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represents a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype.

Functionalization of single-walled carbon nanotubes and their binding to cancer cells

Background Single-walled carbon nanotubes (SWCNTs) have novel properties including their nanoscale size and ease of cellular uptake. This makes them useful for drug delivery, and their photo-thermal effects make them potentially useful in a wide range of applications, particularly the treatment of solid tumors. The poor solubility of SWCNTs has, however, been an issue that may potentially limit the utility of SWCNTs for cancer treatment. Functionalization of the surface of the tubes may be an approach to overcome this problem. Methods SWCNTs were refluxed in HNO3/H2SO4 (1:3) at 120°C for 120 minutes. Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), contact angle measurements, and near infrared (NIR) light exposure were used to assess the functionalization process. The attachment of a carbohydrate-binding protein (lectin) labeled with fluorescein isothiocyanate to the functionalized SWCNTs enabled evaluation of the functionalization step via confocal microscopy. The lectin from Helix pomatia, (Helix pomatia agglutinin [HPA]), can detect changes in protein glycosylation associated with aggressive metastatic cancer. The interaction between the lectin HPA alone and HPA conjugated to the functionalized SWCNTs with human breast cancer cells (MCF-7) was measured using a quartz crystal microbalance biosensor. Results Following the functionalization process, TEM images showed a layer had formed on the surface of the SWCNTs. In the FTIR experiment, results illustrated the presence of the −COOH group on the functionalized SWCNTs. Contact angle measurements showed that upon functionalization the hydrophilicity of the SWCNTs increased. The temperature increase in the liquid (supernatant) surrounding the functionalized SWCNTs following exposure to light in the NIR (808 nm) was greater than for non-functionalized SWCNTs. The biosensor work showed that HPA binds with high affinity (nanomolar range) to human breast cancer cells; HPA-binding properties to MCF-7 cells were retained following conjugation to the functionalized SWCNTs. Conclusion Treating pure SWCNTs with HNO3/H2SO4 (1:3) at 120°C for 120 minutes is an effective method for functionalization of SWCNTs. HPA linked to SWCNTs is a suitable candidate for the delivery of the functionalized SWCNTs to cancer cells.

Conjugation of quantum dots on carbon nanotubes for medical diagnosis and treatment

Cancer is one of the leading causes of death worldwide and early detection provides the best possible prognosis for cancer patients. Nanotechnology is the branch of engineering that deals with the manipulation of individual atoms and molecules. This area of science has the potential to help identify cancerous cells and to destroy them by various methods such as drug delivery or thermal treatment of cancer. Carbon nanotubes (CNT) and quantum dots (QDs) are the two nanoparticles, which have received considerable interest in view of their application for diagnosis and treatment of cancer. Fluorescent nanoparticles known as QDs are gaining momentum as imaging molecules with life science and clinical applications. Clinically they can be used for localization of cancer cells due to their nano size and ability to penetrate individual cancer cells and high-resolution imaging derived from their narrow emission bands compared with organic dyes. CNTs are of interest to the medical community due to their unique properties such as the ability to deliver drugs to a site of action or convert optical energy into thermal energy. By attaching antibodies that bind specifically to tumor cells, CNTs can navigate to malignant tumors. Once at the tumor site, the CNTs enter into the cancer cells by penetration or endocytosis, allowing drug release, and resulting in specific cancer cell death. Alternatively, CNTs can be exposed to near-infrared light in order to thermally destroy the cancer cells. The amphiphilic nature of CNTs allows them to penetrate the cell membrane and their large surface area (in the order of 2600 m2/g) allows drugs to be loaded into the tube and released once inside the cancer cell. Many research laboratories, including our own, are investigating the conjugation of QDs to CNTs to allow localization of the cancer cells in the patient, by imaging with QDs, and subsequent cell killing, via drug release or thermal treatment. This is an area of huge interest and future research and therapy will focus on the multimodality of nanoparticles. In this review, we seek to explore the biomedical applications of QDs conjugated to CNTs, with a particular emphasis on their use as therapeutic platforms in oncology.

A sensitive assay to measure biomarker glycosylation demonstrates increased fucosylation of prostate specific antigen (PSA) in patients with prostate cancer compared with benign prostatic hyperplasia.

BACKGROUND Prostate specific antigen (PSA) measurement is used for the diagnosis of prostate cancer (PCa) but the test lacks specificity due to the number of false positive readings. The glycosylation of PSA is altered in PCa but studies in this area have been limited to few clinical samples and/or require advanced laboratory facilities. An assay to assess PSA glycosylation was established using equipment available in most routine biomedical testing laboratories. METHODS Serum samples from patients with PCa or benign prostatic hyperplasia (BPH) were used. PSA (range 4-10 ng/ml) was affinity purified, separated and probed with the lectin Ulex europaeus (UEA-1; specific for α1,2 linked fucose). An enzyme-linked immunosorbent lectin assay (ELLA) with colorimetric detection was devised and PSA fucosylation assessed in a further independent set of 26 samples. RESULTS Free PSA (fPSA) from PCa patients showed a significant increase in fucosylation compared with fPSA from patients with BPH. The ELLA was 92% specific and 69% sensitive for PCa over BPH. In comparison, fPSA measurement was 70% specific and 56% sensitive (threshold set to 25% tPSA) for PCa over BPH. CONCLUSIONS Changes in glycosylation of PSA were identified using 50 μl of serum with PSA in the range of 4-10 ng/ml, this represents a more specific and sensitive test for PCa based on fucosylation changes of fPSA.

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.

Metastasis Research Protocols

Basement membranes, specialized extracellular matrices composed of collagens, laminins, and proteoglycans, form thin, continuous sheetlike structures that separate epithelial tissues from adjacent connective tissues. The crossing of basement membranes by cancer cells is a crucial aspect of metastasis—it must occur in order that cancer cells can invade lymphatic or blood vessels during dissemination and also when they penetrate into the target organ tissue where they will eventually colonize to form secondary tumors. The assay system described in this chapter utilizes the solubilized basement membrane preparation MatrigelTM and measures the ability of cells to attach to the matrix, invade into and through the matrix, and migrate towards a chemoattractant. It is technically straightforward and requires no specialist equipment and provides a useful tool for assessing the invasive ability of cancer cells, exploring the functional role of specific cell surface molecules/receptors in this process and screening for inhibitors of invasive ability, thus contributing to current knowledge of the molecular events occurring during the invasive process.

A novel approach to determining the affinity of protein-carbohydrate interactions employing adherent cancer cells grown on a biosensor surface

The development of biological agents for the treatment of solid tumours is an area of considerable activity. We are pursuing carbohydrate-binding proteins (lectins) in a strategy aimed at targeting cancer-associated changes in glycosylation. To evaluate lectin-cancer cell interactions we developed a novel cell biosensor in which binding events take place at the cell surface, more closely mimicking an in vivo system. Metastatic, SW620, and non-metastatic, SW480, colorectal cancer cells were grown on the surface of a tissue-culture compatible polystyrene coated biosensor chip and housed in a quartz crystal microbalance (QCM) apparatus, the kinetics of binding of a diverse range of lectins was evaluated. The lectin Helix pomatia agglutinin (HPA) has been shown to bind aggressive metastatic cancer and was produced in recombinant form (His- and RFP-tagged). The affinity of HPA was in the nanomolar range to the metastatic SW620 cells but was only in the micromolar range to the non-metastatic SW480. Overall, the dissociation constant (K(D)) of the lectins tested in the new cell biosensor system was an order of magnitude lower (nanomolar range) than has generally been reported with systems such as QCM/SPR. This new cell-biosensor enables molecular interactions to be studied in a more relevant environment. An intrinsic problem with developing new biological therapies is the difficulty in determining the affinity with which proteins will interact with intact cell surfaces. This methodology will be of interest to researchers developing new biological approaches for targeting cell surfaces in a wide range of diseases, including cancer.