Susan A. Brooks

Molecular interactions in cancer cell metastasis.

Metastasis, the process by which cancer cells leave the primary tumour, disseminate and form secondary tumours at anatomically distant sites, is a serious clinical problem as it is disseminated disease, which is often impossible to eradicate successfully, that causes the death of most cancer patients. Metastasis results from a complex molecular cascade comprising many steps, all of which are interconnected through a series of adhesive interactions and invasive processes as well as responses to chemotactic stimuli. In spite of its clinical significance, it remains incompletely understood. This review provides an overview of some of the molecular interactions that are critical to metastasis. It summarises the principle molecular players in the major steps of the metastatic cascade. These are: (1) tumour angiogenesis, (2) disaggregation of tumour cells from the primary tumour mass, mediated by cadherins and catenins, (3) invasion of, and migration through, the basement membrane (BM) and extracellular matrix (ECM) surrounding the tumour epithelium, and subsequent invasion of the BM of the endothelium of local blood vessels. This is mediated through integrins and proteases, including urokinase form of plasminogen activator (uPA), matrix metalloproteinases (MMPs) and cathepsins, (4) intravasation of the tumour cells into the blood vessels prior to hematogeneous dissemination to distant sites, (5) adhesion of the circulating tumour cells to the endothelial cell lining at the capillary bed of the target organ site. This occurs through adhesive interactions between cancer cells and endothelial cells involving selectins, integrins and members of the immunoglobulin superfamily (IgSF), (6) invasion of the tumour cells through the endothelial cell layer and surrounding BM (extravasation) and target organ tissue and (7) the development of secondary tumour foci at the target organ site.

Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

INTRODUCTION Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. MATERIALS AND METHODS Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. RESULTS Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. CONCLUSIONS This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.

Appropriate glycosylation of recombinant proteins for human use: implications of choice of expression system.

One of the commonest and least well understood posttranslational modifications of proteins is their glycosylation. Human glycoproteins are glycosylated with a bewilderingly heterogeneous array of complex N- and O-linked glycans, which are the product of the coordinated activity of enzymes resident in the endoplasmic reticulum and Golgi apparatus of the cell. Glycosylation of proteins is highly regulated and changes during differentiation, development, under different physiological—and cell culture—conditions and in disease. The glycosylation of recombinant proteins, especially those destined for potential administration to human subjects, is of critical importance. Glycosylation profoundly affects biological activity, function, clearance from circulation, and crucially, antigenicity. The cells of nonhuman species do not glycosylate their proteins in the same way as human cells do. In many cases, the differences are profound. Overall, the species most distant to humans in evolutionary terms, such as bacteria, yeasts, fungi, insects and plants—the species used most commonly in expression systems—have glycosylation repertoires least like our own. This review gives a brief overview of human N- and O-linked protein glycosylation, summarizes what is known of the glycosylation potential of the cells of nonhuman species, and presents the implications for the biotechnology industry.

Altered Glycosylation of Proteins in Cancer: What Is the Potential for New Anti-Tumour Strategies

It is becoming increasingly apparent that cell surface oligosaccharides play pivotal roles as recognition molecules in a range of cell communication and adhesion processes. Alterations in cellular glycosylation are also associated with diseases, including cancer, and may have functional significance. This paper gives an overview of the complex topic of cellular glycosylation mechanisms and reviews the well-documented alterations in cellular glycosylation of proteins in malignancy. One particular type of cancer-associated glycosylation change, the incomplete synthesis of O-linked glycans, is highlighted, and its possible functional significance in cancer cell metastatic mechanisms is discussed. The significance that cancer-associated changes in glycoprotein glycosylation may have in new approaches to anti-tumour therapies is explored.

Strategies for Analysis of the Glycosylation of Proteins: Current Status and Future Perspectives

More than half of human proteins are glycosylated by a bewildering array of complex and heterogeneous N- and O-linked glycans. They function in myriad biological processes, including cell adhesion and signalling and influence the physical characteristics, stability, function, activity and immunogenicity of soluble glycoproteins. A single protein may be glycosylated differently to yield heterogenous glycoforms. Glycosylation analysis is of increasing interest in biomedical and biological research, the pharmaceutical and healthcare industry and biotechnology. This is because it is increasingly apparent that glycosylation changes in diseases, such as cancer, making it a promising target for development of clinically useful biomarkers and therapeutics. Furthermore, as the non-human cells employed in expression systems glycosylate their proteins very differently to human cells, and as glycosylation changes unpredictably under changing environmental conditions, glycans analysis for quality control, optimum efficacy and safety of recombinant glycoproteins destined for human therapeutic use is paramount. The complexities of carbohydrate chemistry make analysis challenging and while there are a variety of robust methodologies available for glycan analysis, there is currently a pressing need for the development of new, streamlined, high throughput approaches accessible to non-specialist laboratories.

The passenger strand, miR-21-3p, plays a role in mediating cisplatin resistance in ovarian cancer cells

OBJECTIVE Ovarian cancer is the deadliest gynaecological cancer. A major contributor to the poor survival rate is the development of chemoresistance to platinum-based therapies such as cisplatin and carboplatin. Here we aimed to test the role of miRNAs in the acquisition of drug resistance in ovarian cancer. METHODS We used microarrays to measure miRNA levels in the ovarian cancer cell line A2780 and its cisplatin-resistant derivative CP70. The role of miRNAs and the mRNA targets were tested using transfected miRNA mimics and siRNAs, respectively. Potential in vivo significance was investigated by analysing RNA levels in cohorts of ovarian cancer patients. RESULTS We identified several miRNAs that are increased in cisplatin-resistant cells. We show that most of these do not directly contribute to cisplatin resistance. Interestingly, miR-21-3p, the passenger strand of the known oncomiR, directed increased resistance to cisplatin in a range of ovarian cell lines. This effect was specific to the star strand, as miR-21-5p had the opposite effect and actually increased sensitivity of A2780 cells to cisplatin. We identify NAV3 as a potential target of miR-21-3p and show that knockdown of NAV3 increases resistance. Exosomes released by CP70 cells were also capable of increasing resistance in A2780 cells, although this was independent of miR-21-3p. Finally, we use publically available transcriptomic data to demonstrate that miR-21-3p is raised, while NAV3 is reduced, in ovarian tumours that are resistant to platinum treatment. CONCLUSION Our data suggest that miR-21-3p can induce cisplatin resistance in ovarian tumours, potentially by targeting the NAV3 gene.

Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins.

A major challenge for the biotechnology industry is to engineer the glycosylation pathways of expression systems to synthesize recombinant proteins with human glycosylation. Inappropriate glycosylation can result in reduced activity, limited half-life in circulation and unwanted immunogenicity. In this review, the complexities of glycosylation in human cells are explained and compared with glycosylation in bacteria, yeasts, fungi, insects, plants and nonhuman mammalian species. Key advances in the engineering of the glycosylation of expression systems are highlighted. Advances in the challenging and technically complex field of glycan analysis are also described. The emergence of a new generation of expression systems with sophisticated engineering for humanized glycosylation of glycoproteins appears to be on the horizon.

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.

Molecular pathogenesis of follicular cell derived thyroid cancers

Thyroid cancers are the most common endocrine malignancy. Radiation exposure, family history of thyroid cancer and some inherited conditions are the most important predisposing factors for the development of thyroid cancer. Three mitogenic signalling pathways have been described in the thyroid cell, which are influenced by various stimulatory and inhibitory hormones, growth factors and neurotransmitters. Various proto-oncogenes and oncogenes like ras, braf, trk, met and RET also play a role in the signal transduction systems. Two theories have been described in thyroid cancer pathogenesis, the foetal cell carcinogenesis theory and the more common, multistep carcinogenesis theory. The multistep carcinogenesis theory is now the accepted model in many human cancers, including thyroid cancer. The early events of tumour formation are the consequence of activation of either various growth factors or the proto-oncogenes like ras, met or ret. This results in the formation of differentiated thyroid cancers like the papillary, follicular or Hurthle cell cancers. The later stages of tumour formation involve further activation of proto-oncogenes and loss or inactivation of tumour suppressor genes like p53. Based on this theory, follicular carcinomas are generated from follicular adenomas and papillary carcinomas from precursor cells generated from thyrocytes. Anaplastic carcinoma may develop from papillary or follicular carcinoma by dedifferentiation. In this review article, we highlight the molecular pathogenesis of thyroid tumours.