GALNTs: master regulators of metastasis-associated epithelial-mesenchymal transition (EMT)?

In humans, the UDP-N-α-D galactosamine:polypeptide N-acetylgalactosaminyltransferases family (ppGalNAc-Ts, GalNAc-Ts or GALNTs) comprises 20 isoenzymes. They are responsible for the initial synthesis of α-GalNAc1,3-O-Ser/Thr, or Tn antigen, at initiation of mucin type O-linked glycosylation. This structure is normally extended by the further sequential action of glycosytransferases to build more complex linear or branched O-linked structures, but in cancers it is frequently left unelaborated, and its presence is often associated with poor patient prognosis. Altered levels of GALNT expression or distribution have also been extensively reported in a wide range of cancers. These changes would be predicted to result in marked alterations in GalNAc O-linked glycosylation, including altered levels of site specific O-linked glycosylation and changes in the glycan structures formed, including, potentially, exposure of truncated O-glycans such as Tn antigen. Many reports have demonstrated that altered levels of specific GALNTs have prognostic significance in cancers, or shown that they are associated with changes in cell behaviour, including proliferation, migration, invasion or growth and metastasis in animal models. We have previously reviewed how deregulation of GALNTs in several epithelial cancers is a feature of different stages metastasis. Here we consider evidence that changes in GALNT expression, and therefore consequent alterations in GalNAc O-linked glycosylation, may directly influence molecules implicated in aspects of epithelial-mesenchymal transition (EMT), a fundamental aspect of cancer metastasis, during which epithelial cancer cells lose their cell-cell junctions, apical-basal polarity and adhesive interactions with basement membrane and become mesenchymal, with a spindle-shaped morphology and increased migratory capacity.

Utilising extracellular vesicles for early cancer diagnostics: benefits, challenges and recommendations for the future.

To increase cancer patient survival and wellbeing, diagnostic assays need to be able to detect cases earlier, be applied more frequently, and preferably before symptoms develop. The expansion of blood biopsy technologies such as detection of circulating tumour cells and cell-free DNA has shown clinical promise for this. Extracellular vesicles released into the blood from tumour cells may offer a snapshot of the whole of the tumour. They represent a stable and multifaceted complex of a number of different types of molecules including DNA, RNA and protein. These represent biomarker targets that can be collected and analysed from blood samples, offering great potential for early diagnosis. In this review we discuss the benefits and challenges of the use of extracellular vesicles in this context and provide recommendations on where this developing field should focus their efforts to bring future success.

Isolation of Viable Glycosylation-Specific Cell Populations for Further In Vitro or In Vivo Analysis Using Lectin-Coated Magnetic Beads.

The glycans displayed on the cell surface are highly heterogeneous and their function in cell recognition, identity, signaling, adhesion, and behavior is increasingly recognized. Moreover, as it is yet incompletely understood, it is a topic of significant current interest. Lectins (naturally occurring carbohydrate-binding proteins) are very useful tools for exploring cellular glycosylation. Cell populations, within or between different tissues or species, and in development, health and disease, exhibit different glycosylation and thus distinct lectin-binding characteristics. Even monoclonal cell populations of established cell lines feature subpopulations with strikingly different glycosylation characteristics, and these differences may reflect differences in behavior or function. By separating cell populations on the basis of their cell surface glycosylation, the functional significance of glycosylation can be investigated in in vitro or in vivo models. Also, factors affecting glycosylation, which are also incompletely understood, can be explored or manipulated. In the protocol given here, cells can be separated into subpopulations on the basis of their recognition by a specific biotinylated lectin of choice immobilized on avidin-coated magnetic beads. Importantly, the protocol has been optimized such that lectin-binding and non-binding cells remain viable such that they can be further cultured, if necessary, for subsequent investigations.

The extended ppGalNAc-T family and their functional involvement in the metastatic cascade.

O-linked glycosylation of proteins begins with the attachment of a single N-acetylgalactosamine (GalNAc) residue to a serine or threonine residue of the polypeptide and glycosylation of proteins can dramatically change their properties, interactions and activities. This initial attachment is catalysed by members of a family of 20 isoenzymes, the UDP-N-α-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases or ppGalNAc-Ts. Why such a large family of isoenzymes are required to perform, apparently, a single function has been the subject of intense interest. The ppGalNAc-Ts, in fact, have overlapping, but distinct, substrate specificities and are differentially expressed in different cells and tissues and under different conditions of differentiation and development, allowing subtle and complex control of cellular glycosylation. Intriguingly, there is a growing body of evidence showing that altered expression of members of this transferase family are a common feature of many types of cancer and, crucially, that the resulting aberrant glycosylation has functional effects. Here, we review what is known of the expression and distribution of these intriguing transferases in health and in malignancy and, for the first time, bring together what is known of the functional and molecular effects of their disregulation in each step of the complex cascade of cancer metastasis.

Meta-analysis using a novel database, miRStress, reveals miRNAs that are frequently associated with the radiation and hypoxia stress-responses.

Organisms are often exposed to environmental pressures that affect homeostasis, so it is important to understand the biological basis of stress-response. Various biological mechanisms have evolved to help cells cope with potentially cytotoxic changes in their environment. miRNAs are small non-coding RNAs which are able to regulate mRNA stability. It has been suggested that miRNAs may tip the balance between continued cytorepair and induction of apoptosis in response to stress. There is a wealth of data in the literature showing the effect of environmental stress on miRNAs, but it is scattered in a large number of disparate publications. Meta-analyses of this data would produce added insight into the molecular mechanisms of stress-response. To facilitate this we created and manually curated the miRStress database, which describes the changes in miRNA levels following an array of stress types in eukaryotic cells. Here we describe this database and validate the miRStress tool for analysing miRNAs that are regulated by stress. To validate the database we performed a cross-species analysis to identify miRNAs that respond to radiation. The analysis tool confirms miR-21 and miR-34a as frequently deregulated in response to radiation, but also identifies novel candidates as potentially important players in this stress response, including miR-15b, miR-19b, and miR-106a. Similarly, we used the miRStress tool to analyse hypoxia-responsive miRNAs. The most frequently deregulated miRNAs were miR-210 and miR-21, as expected. Several other miRNAs were also found to be associated with hypoxia, including miR-181b, miR-26a/b, miR-106a, miR-213 and miR-192. Therefore the miRStress tool has identified miRNAs with hitherto unknown or under-appreciated roles in the response to specific stress types. The miRStress tool, which can be used to uncover new insight into the biological roles of miRNAs, and also has the potential to unearth potential biomarkers for therapeutic response, is freely available at http://mudshark.brookes.ac.uk/MirStress.