Extracellular matrix gene expression profiling using microfluidics for colorectal carcinoma stratification.

In cancer, biomarkers have many potential applications including generation of a differential diagnosis, prediction of response to treatment, and monitoring disease progression. Many molecular biomarkers have been put forward for different diseases but most of them do not possess the required specificity and sensitivity. A biomarker with a high sensitivity has a low specificity and vice versa. The inaccuracy of the biomarkers currently in use has led to a compelling need to identify more accurate markers with diagnostic and prognostic significance. The aim of the present study was to use a novel, droplet-based, microfluidic platform to evaluate the prognostic value of a panel of thirty-four genes that regulate the composition of extracellular matrices in colorectal carcinoma. Our method is a novel approach as it uses using continuous-flowing Polymerase Chain Reaction for the sensitive detection and accurate quantitation of gene expression. We identified a panel of relevant extracellular matrix genes whose expression levels were measured by real-time quantitative polymerase chain reaction using Taqman® reagents in twenty-four pairs of matched colorectal cancer tumour and associated normal tissue. Differential expression patterns occurred between the normal and malignant tissue and correlated with histopathological parameters and overall surgical staging. The findings demonstrate that a droplet-based microfluidic quantitative PCR system enables biomarker classification. It was further possible to sub-classify colorectal cancer based on extracellular matrix protein expressing groups which in turn correlated with prognosis.

RACK1 promotes neurite outgrowth by scaffolding AGAP2 to FAK.

RACK1 binds proteins in a constitutive or transient manner and supports signal transmission by engaging in diverse and distinct signalling pathways. The emerging theme is that RACK1 functions as a signalling switch, recruiting proteins to form distinct molecular complexes. In focal adhesions, RACK1 is required for the regulation of FAK activity and for integrating a wide array of cellular signalling events including the integration of growth factor and adhesion signalling pathways. FAK is required for cell adhesion and migration and has a well-established role in neurite outgrowth and in the developing nervous system. However, the mechanism by which FAK activity is regulated in neurons remains unknown. Using neuronal cell lines, we determined that differentiation of these cells promotes an interaction between the scaffolding protein RACK1 and FAK. Disruption of the RACK1/FAK interaction leads to decreased neurite outgrowth suggesting a role for the interaction in neurite extension. We hypothesised that RACK1 recruits proteins to FAK, to regulate FAK activity in neuronal cells. To address this, we immunoprecipitated RACK1 from rat hippocampus and searched for interacting proteins by mass spectrometry. We identified AGAP2 as a novel RACK1-interacting protein. Having confirmed the RACK1-AGAP2 interaction biochemically, we show RACK1-AGAP2 to localise together in the growth cone of differentiated cells, and confirm that these proteins are in complex with FAK. This complex is disrupted when RACK1 expression is suppressed using siRNA or when mutants of RACK1 that do not interact with FAK are expressed in cells. Similarly, suppression of AGAP2 using siRNA leads to increased phosphorylation of FAK and increased cell adhesion resulting in decreased neurite outgrowth. Our results suggest that RACK1 scaffolds AGAP2 to FAK to regulate FAK activity and cell adhesion during the differentiation process.

Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration.

BACKGROUND: Cell migration is a fundamental biological process and has an important role in the developing brain by regulating a highly specific pattern of connections between nerve cells. Cell migration is required for axonal guidance and neurite outgrowth and involves a series of highly co-ordinated and overlapping signalling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) has an essential role in development and is the most highly expressed kinase in the developing CNS. FAK activity is essential for neuronal cell adhesion and migration. RESULTS: The objective of this study was to optimise a protocol for the differentiation of the neuroblastoma cell line, SH-SY5Y. We determined the optimal extracellular matrix proteins and growth factor combinations required for the optimal differentiation of SH-SY5Y cells into neuronal-like cells and determined those conditions that induce the expression of FAK. It was confirmed that the cells were morphologically and biochemically differentiated when compared to undifferentiated cells. This is in direct contrast to commonly used differentiation methods that induce morphological differentiation but not biochemical differentiation. CONCLUSIONS: We conclude that we have optimised a protocol for the differentiation of SH-SY5Y cells that results in a cell population that is both morphologically and biochemically distinct from undifferentiated SH-SY5Y cells and has a distinct adhesion and spreading pattern and display extensive neurite outgrowth. This protocol will provide a neuronal model system for studying FAK activity during cell adhesion and migration events.

Tools used to study how protein complexes are assembled in signaling cascades.

Most proteins do not function on their own but as part of large signaling complexes that are arranged in every living cell in response to specific environmental cues. Proteins interact with each other either constitutively or transiently and do so with different affinity. When identifying the role played by a protein inside a cell, it is essential to define its particular cohort of binding partners so that the researcher can predict what signaling pathways the protein is engaged in. Once identified and confirmed, the information might allow the interaction to be manipulated by pharmacological inhibitors to help fight disease. In this review, we discuss protein-protein interactions and how they are essential to propagate signals in signaling pathways. We examine some of the high-throughput screening methods and focus on the methods used to confirm specific protein-protein interactions including; affinity tagging, co-immunoprecipitation, peptide array technology and fluorescence microscopy.