Molecular map for ERK regulation and sub-cellular localisation.

<p>After RAS activation, ERK cascade can be recruited and activated on plasma membrane with the help of KRS1 scaffold protein (upper part of the figure); activated ERK is then released (in complex with MEK and KSR1) into the cytoplasm, where it can activate some of its cytoplasmic targets (e.g. PLA2G4A protein). Alternatively, activated receptor complex can translocate to late endosomes (left part of the figure), where ERK cascade can be triggered with the help of MP1 scaffold protein; in this case, activated ERK monomers are released into the cytoplasm, from where they can translocate into the nucleus and exert other effects (e.g. induction of DUSP1 phosphatase). This map is a small fraction of the detailed MAPK network built with the software CellDesigner (<a href="http://www.celldesigner.org" target="_blank">www.celldesigner.org</a>) and provided in <i>png</i> and <i>cell</i> formats (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi.1003286.s001" target="_blank">supplementary Dataset S1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi.1003286.s002" target="_blank">S2</a>).</p

FGFR3 activating mutation and role of SPRY.

<p>Simulations were performed under FGFR3 gain-of-function (FGFR3 = 1 and all inputs set to 0, throughout the simulations). Simplified model dynamics are shown as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi-1003286-g004" target="_blank">Figure 4a–b</a>. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#s3" target="_blank">Results</a> are shown for the wild type model (red1 model reduction), as well as for perturbed model versions obtained by disrupting the inhibition of interest.</p

Coherence of model simulations with published experimental evidence.

<p>Asterisks denote facts explicitly related to bladder cancer, whereas unmarked entries correspond to generic or loosely specified mechanisms. Full simulation results can be found in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi.1003286.s003" target="_blank">Dataset S3</a>, with the help of the identifiers provided in the first two columns.</p

Regulatory graph of the MAPK logical model.

<p>Each node denotes a model component. Model inputs, phenotypes and MAPK proteins (ERK, p38, JNK) are denoted in pink, blue and orange, respectively. Green arrows and red T-arrows denote positive and negative regulations, respectively. A comprehensive documentation is provided in the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi.1003286.s008" target="_blank">Table S4</a>, which includes a summary of all modelling assumptions, references (PubMed links) and the specification of the logical rule associated with each component. The source file is further provided as <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi.1003286.s004" target="_blank">supplementary Dataset S4</a>, which can be opened, edited, analysed and simulated with the softare GINsim (www://<a href="http://www.ginsim.org/beta" target="_blank">www.ginsim.org/beta</a>).</p

Coherence of the logical model with well established bladder cancer deregulations.

<p><b>a</b>) Simplified representation of the model dynamics following EGFR over-expression (EGFR = 1 throughout the simulation and all inputs set to 0 throughout the simulation). If p53 is activated first (right branch), an apoptotic attractor is reached, characterised by inactivation of ERK and AKT. If ERK and PI3K are activated first (left branch), then p53 is inactivated and AKT is activated, leading to a proliferative attractor. <b>b</b>) Simplified representation of the model dynamics following FGFR3 activating mutation (FGFR3 = 1 and all inputs set to 0 throughout the simulation). When p53 is activated first (right branch), an apoptotic attractor is reached, characterised by inactivation of ERK and AKT. If ERK is activated first (left and central branches), then p53 is inactivated. When PI3K is also activated (central), a proliferative attractor is reached, characterised by activated AKT. In contrast, when PI3K is not activated (left), the cell fails to make a clear decision at the level of the MAPK network. <b>c</b>) Attractors reached by the model in presence of receptor alterations, coupled with additional common deregulations observed in bladder cancer. Coloured circles denote the phenotypes characterising the attractors reached in each situation (we used the same colour code as in panels a and b, while empty spaces denote the loss of the corresponding branch in the state transition graph). Identifiers in rectangles (e.g.. r3, r4, etc.) point to simulation results reported in more details in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi.1003286.s003" target="_blank">Dataset S3</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi.1003286.s010" target="_blank">Text S2</a>.</p

Regulatory graph of a reduced version of the MAPK model.

<p>The regulatory graph corresponds to the “red1” reduced model version (cf. supplementary <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003286#pcbi.1003286.s007" target="_blank">Table S3</a>, column 1). To obtain this version, the preservation of pink and blue nodes was imposed, along with that of {EGFR, FGFR3, p53, p14, PI3K, AKT, PTEN, ERK}, in order to investigate the effects of perturbations affecting these components. The remaining nodes {FRS2, MSK} were maintained by the reduction algorithm because of the occurrence of auto-regulatory loops during the reduction process. Green arrows and red T-arrows denote positive and negative regulations, respectively, whereas blue arrows denote dual interactions.</p

Analysis of MAPK cross-talks by disruptions of specific interactions.

<p><b>a</b>) Effects of the disruptions of the inhibitions of MEK by AP1 and the phosphatase PPP2CA. <b>b</b>) Effects of the disruption of the inhibition of p38 or of JNK by the phosphatase DUSP1. These simulations were performed after removing the corresponding interactions and blocking the level of the perturbed receptor to level 1 (with all inputs set to 0, throughout the simulation).</p

Additional file 4: Figure S3. of IGF1R activation and the in vitro antiproliferative efficacy of IGF1R inhibitor are inversely correlated with IGFBP5 expression in bladder cancer

IGF1R expression by epithelial cells in normal urothelium and bladder tumors. (a)Anti-IGF1R immunohistochemistry from human protein atlas project ( http://www.proteinatlas.org/ ). 3 examples of representative staining in tumors are presented in the right panel, staining of the two normal samples are presented in the left panel. Scale bar represents 100 μm (b) Haematoxylin-eosin staining of our CIT-series of tumors. Examples of tumors with high and low IGF1R expression assessed by RPPA. (TIFF 7100 kb

Rab and Rab-interacting proteins.

<p>Example of the Rab27 cluster. The Rab27 cluster is comprised of the two <i>RAB27</i> isoforms (<i>RAB27A</i> and <i>RAB27B</i>), the GEF <i>MADD</i>, the GAP <i>TBC1D10A</i> and 12 effector proteins. The other Rab and Rab-interacting proteins are shown in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039469#pone.0039469.s001" target="_blank">Figure S1</a>.</p

Deregulated genes correlated with differentiation markers.

<p>The Pearson correlation coefficient (r) of the expression of the deregulated genes with the expression of urothelial differentiation markers in <i>FGFR3</i>-mutated superficial tumors (TaG1G2) (n = 28 samples) and <i>FGFR3</i>-non-mutated muscle-invasive tumors (T2–4) (n = 63) is presented. Correlation with p<1% (|r| above 0.479 for Ta-T1 tumors, and |r| above 0.323 for <i>FGFR3</i>-non-mutated T2–4 tumors) are written in bold.</p