Analysis of Alternative mRNA Splicing in Vemurafenib-Resistant Melanoma Cells

Alternative splicing (AS) is one of the hallmarks of human cancer. One of the most common mechanisms of vemurafenib resistance in malignant melanoma is AS of BRAF, occurring in 15-30% of patients. The aim of our study was to investigate the transcriptome and AS D04landscape in the isogenic BRAF V600E cell line pair SK-MEL-239, where the vemurafenib-resistant derivative expresses a truncated BRAF transcript that lacks the RAS-binding domain. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. AS analysis, by four different tools, DEXSeq, rMATS, ASpli, and LeafCutter, has identified genes enriched for cell motility and melanin synthesis in vemurafenib-resistant cells. Overlapping predictions for all four tools have been experimentally validated. Our study expands the understanding of melanoma drug resistance from a new perspective and supports the need to investigate in detail the aberrant AS landscape in patients with malignant melanoma. Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.Peer reviewe

Analysis of Alternative mRNA Splicing in Vemurafenib-Resistant Melanoma Cells

Alternative splicing (AS) is one of the hallmarks of human cancer. One of the most common mechanisms of vemurafenib resistance in malignant melanoma is AS of BRAF, occurring in 15-30% of patients. The aim of our study was to investigate the transcriptome and AS D04landscape in the isogenic BRAF V600E cell line pair SK-MEL-239, where the vemurafenib-resistant derivative expresses a truncated BRAF transcript that lacks the RAS-binding domain. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. AS analysis, by four different tools, DEXSeq, rMATS, ASpli, and LeafCutter, has identified genes enriched for cell motility and melanin synthesis in vemurafenib-resistant cells. Overlapping predictions for all four tools have been experimentally validated. Our study expands the understanding of melanoma drug resistance from a new perspective and supports the need to investigate in detail the aberrant AS landscape in patients with malignant melanoma. Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.Peer reviewe

Dynamic regulation of RAS and RAS signaling

RAS proteins regulate most aspects of cellular physiology. They are mutated in 30% of human cancers and 4% of developmental disorders termed Rasopathies. They cycle between active GTP-bound and inactive GDP-bound states. When active, they can interact with a wide range of effectors that control fundamental biochemical and biological processes. Emerging evidence suggests that RAS proteins are not simple on/off switches but sophisticated information processing devices that compute cell fate decisions by integrating external and internal cues. A critical component of this compute function is the dynamic regulation of RAS activation and downstream signaling that allows RAS to produce a rich and nuanced spectrum of biological outputs. We discuss recent findings how the dynamics of RAS and its downstream signaling is regulated. Starting from the structural and biochemical properties of wild-type and mutant RAS proteins and their activation cycle, we examine higher molecular assemblies, effector interactions and downstream signaling outputs, all under the aspect of dynamic regulation. We also consider how computational and mathematical modeling approaches contribute to analyze and understand the pleiotropic functions of RAS in health and disease

Signalling ballet in space and time.

Although we have amassed extensive catalogues of signalling network components, our understanding of the spatiotemporal control of emergent network structures has lagged behind. Dynamic behaviour is starting to be explored throughout the genome, but analysis of spatial behaviours is still confined to individual proteins. The challenge is to reveal how cells integrate temporal and spatial information to determine specific biological functions. Key findings are the discovery of molecular signalling machines such as Ras nanoclusters, spatial activity gradients and flexible network circuitries that involve transcriptional feedback. They reveal design principles of spatiotemporal organization that are crucial for network function and cell fate decisions

Dynamics of receptor and protein transducer homodimerisation

<p>Abstract</p> <p>Background</p> <p>Signalling pathways are complex systems in which not only simple monomeric molecules interact, but also more complex structures that include constitutive or induced protein assemblies. In particular, the hetero-and homo-dimerisation of proteins is a commonly encountered motif in signalling pathways. Several authors have suggested in recent times that dimerisation relates to a series of physical and biological outcomes used by the cell in the regulation of signal transduction.</p> <p>Results</p> <p>In this paper we investigate the role of homodimerisation in receptor-protein transducer interactions. Towards this end, mathematical modelling is used to analyse the features of such kind of interactions and to predict the behaviour of the system under different experimental conditions. A kinetic model in which the interaction between homodimers provokes a dual mechanism of activation (single and double protein transducer activation at the same time) is proposed. In addition, we analyse under which conditions the use of a power-law representation for the system is useful. Furthermore, we investigate the dynamical consequences of this dual mechanism and compare the performance of the system in different simulated experimental conditions.</p> <p>Conclusion</p> <p>The analysis of our mathematical model suggests that in receptor-protein interacting systems with dual mechanism there may be a shift between double and single activation in a way that intense double protein transducer activation could initiate and dominate the signal in the short term (getting a fast intense signal), while single protein activation could control the system in the medium and long term (when input signal is weaker and decreases slowly). Our investigation suggests that homodimerisation and oligomerisation are mechanisms used to enhance and regulate the dynamic properties of the initial steps in signalling pathways.</p

The secret life of kinases: functions beyond catalysis

Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility

When kinases meet mathematics: the systems biology of MAPK signalling

The mitogen activated protein kinase/extracellular signal regulated kinase pathway regulates fundamental cellular function such as cell proliferation, survival, differentiation and motility, raising the question how these diverse functions are specified and coordinated. They are encoded through the activation kinetics of the pathway, a multitude of feedback loops, scaffold proteins, subcellular compartmentalisation, and crosstalk with other pathways. These regulatory motifs alone or in combination can generate a multitude of complex behaviour. Systems biology tries to decode this complexity through mathematical modelling and prediction in order to gain a deeper insight into the inner works of signalling networks

A structured approach for the engineering of biochemical network models, illustrated for signalling pathways

http://dx.doi.org/10.1093/bib/bbn026Quantitative models of biochemical networks (signal transduction cascades, metabolic pathways, gene regulatory circuits) are a central component of modern systems biology. Building and managing these complex models is a major challenge that can benefit from the application of formal methods adopted from theoretical computing science. Here we provide a general introduction to the field of formal modelling, which emphasizes the intuitive biochemical basis of the modelling process, but is also accessible for an audience with a background in computing science and/or model engineering. We show how signal transduction cascades can be modelled in a modular fashion, using both a qualitative approach { Qualitative Petri nets, and quantitative approaches { Continuous Petri Nets and Ordinary Differential Equations. We review the major elementary building blocks of a cellular signalling model, discuss which critical design decisions have to be made during model building, and present ..

Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP

Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK modul

Integration of a Phosphatase Cascade with the MAP Kinase Pathway provides for a Novel Signal Processing Function

We mathematically modeled the receptor-activated MAP kinase signaling by incorporating the regulation through cellular phosphatases. Activation induced the alignment of a phosphatase cascade in parallel with the MAP kinase pathway. A novel regulatory motif was thus generated, providing for the combinatorial control of each MAPK intermediate. This ensured a non-linear mode of signal transmission with the output being shaped by the balance between the strength of input signal, and the activity gradient along the phosphatase axis. Shifts in this balance yielded modulations in topology of the motif, thereby expanding the repertoire of output responses. Thus we identify an added dimension to signal processing, wherein the output response to an external stimulus is additionally filtered through indicators that define the phenotypic status of the cell.Comment: Whole Manuscript 33 pages inclduing Main text, 7 Figures and Supporting Informatio