The pipeline employed to generate context specific signaling networks EGFR_E and EGFR_M of an immortalized breast epithelial cell line (D492) and mesenchymal cell line (D492M).

<p>SBML pathway map from Reactome was converted to mathematical format using the COBRA toolbox. This network was further modified to remove dead ends and include GPRs for modifiers, activators and inhibitors, resulting in the formation of the EGFR_SN network. The EGFR_SN network was constrained with the transcriptomic data of D492 and D492M to form EGFR_E and EGFR_M networks. Flux differences between EGFR_E and EGFR_M were used to further predict metabolic phenotype and target reactions critical for the reversal of mesenchymal to epithelial phenotype.</p

The rules for adding GPRs.

<p>Reaction1 is catalyzed by a modifier which is a multiprotein complex of gene1 and gene2. The combination of both the genes are required for the reaction to take place. Reaction2 is inhibited in presence of inhibitor which is an isoform of gene3 and gene4. Presence of either gene3 or gene4 will inhibit Reaction2. A ‘NOT’ operator is assigned to the GPR of Reaction2 to indicate inhibition. A GPR is not assigned to Reaction 3 in this example.</p

Predicted expression of metabolic genes regulated by AKT in D492 and D492M cells.

<p>Predicted expression of metabolic genes regulated by AKT in D492 and D492M cells.</p

The pipeline used to determine the effects of active AKT signaling on the EMT metabolic network.

<p>The metabolic genes predicted to be altered in D492 and D492M dependent on the AKT signaling network, were used to constrain the EMT metabolic network generated from Recon2, in order to identify downstream metabolic pathways that are affected by AKT activation.</p

Flux differences between epithelial network (EGFR_E) and mesenchymal network (EGFR_M).

<p>A) Probability density estimates for the flux values in selected reactions as obtained by random sampling. The blue curve represents the flux distribution of EGFR_E and the red curve that of EGFR_M. Vertical axis denote probability and flux values are represented on the horizontal axis. AU: arbitrary units B) Relative mean flux for each reaction in EGFR_E and EGFR_M through the AKT, RAS and DAG/IP3 and CaM pathways. Higher flux within reactions in AKT and RAS/MAPK pathways are observed in the EGFR_E network compared to EGFR_M while CaM and DAG/IP3 have higher flux in EGFR_M. Negative values denote higher flux in EGFR_E and positive values denotes higher flux in EGFR_M. Numerical values of these fluxes are given in supplementary file (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004924#pcbi.1004924.s002" target="_blank">S1 File</a>).</p

Sub-network of EGFR_SN network.

<p>A part of the EGFR signaling network representing AKT signaling. Nodes indicate the reacting component and edges denote the reactions. Red nodes indicate inhibitors which were manually added to the model in the form of GPRs (information obtained from the Reactome database), white nodes indicate signaling components present in the original SBML file downloaded from the Reactome database and green nodes indicate exchange reactions which were added to the model to remove dead ends. Node ‘B’ and ‘P’ indicates binding, and phosphorylation, respectively. Edges: → transition, ⟞ inhibition, ⫯ catalysis.</p

Proliferation rate and glycolytic activity are higher in D492 than in D492M cells.

<p>A) Cell proliferation assay demonstrated a higher growth rate of D492 cells compared to D492M cells. B) Spent medium analysis of glucose (dashed lines) and lactate (solid lines) shows higher glucose uptake and lactate secretion in D492 cells (blue) than in D492M cells (red). C) Calculated glucose uptake and lactate secretion rates indicate higher glycolytic flux rates per cell per hour in D492 cells than D492M cells. Data represents results from 3 independent experiments. Error bars represent standard deviation in a single experiment done in triplicate. mM: milli molar, fmol: femto molar.</p

An overview of downstream signaling pathways induced by EGFR signaling.

<p>Three main pathways: AKT, RAS/MAPK and CaM and DAG/IP3 are induced downstream of active EGFR signaling.</p

Comparison of flux distribution in the reactions within AKT, RAS and CaM pathway in different breast epithelial cell lines.

<p>The heat map has been generated using log2 relative mean flux of the reactions within the AKT, RAS and DAG/IP3 and CaM pathways in epithelial and mesenchymal cells of D492, MCF, MCF10A and HMLE cells. Exchange reactions are not included in the heat map. The negative and positive values as depicted on the color scale denote higher flux in reactions of epithelial and mesenchymal cells, respectively. D492M, MCF7_mirna, MCF7_snail, MCF10_snail, MCF10A_tgfb, HMLE_slug, HMLE_twist and HMLE_snail are EMT derived mesenchymal cells.</p

EGFR overexpression in D492M.

<p><b>(A)</b> Real-Time Quantitative Reverse Transcription PCR of EGFR in D492, D492M^EGFR and D492M^Empty, normalized to GAPDH. EGFR transcription level is significantly higher in D492M^EGFR compared to D492M^Empty but does not reach the D492 EGFR transcription level. <b>(B)</b> Protein expression of EGFR, Phospho-p44/42 MAPK (ERK1/2) and Phospho-AKT determined by Western blotting. Overexpression of EGFR in D492M leads to increased protein expression of EGFR in D492M^EGFR compared to D492M^Empty, but EGFR protein expression does not reach the D492 level. Overexpression of EGFR in D492M leads to increased MAPK (Erk1/2) and Akt phosphorylation which is higher than in both D492M^Empty and D492.</p