A comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells

Abstract

The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1) have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins and their post-translational modifications sites, and over 1,000 biochemical interactions. Using computer simulations of the model, we find it is able to reproduce a number of cellular phenomena. Furthermore, the model predicts that overexpression of Src results in increased endocytosis of EGFR in the absence/low amount of the epidermal growth factor (EGF). Our subsequent laboratory experiments also suggest increased internalization of EGFR upon Src overexpression under EGF-deprived conditions, further supporting this model-generated hypothesis.

Figure 1. Graph representation of the model.

The purpose of this graph is to visualize the complexity of the model, rather than to read the individual interactions. This graph representation of the model was generated in Gephi (www.gephi.org).

Figure 2. Validation of the MEC model against known cellular phenomena.

A) EGF-dependent stimulation of survival signals via activation of Akt. B) Activation of Erk by EGF . Although a positive relationship between Erk and the ligand can be seen, the activation Erk by EGF seems to exhibit a more complex dynamics than the ones seen in the other diagrams (i.e., the positive relationship is exhibited when EGF activity levels are 0–40%). This is, however, not surprising given the complex interactions and cross-communication within the ErbB signaling family. C) Activating mutations of known protooncogenes such as Ras result in growth factor-independent activation of Erk . D) Erk dependency on signaling via integrins by extracellular matrix (ECM) . E and F) Activation of Rac and Cdc42 by ECM . G) Positive relationship between Adenylyl Cyclase (AC) and the G-Protein Coupled Receptor ligand alpha-s , . H) Hierarchy of ErbB receptor dimerization. The panel on the left represents a system with EGFR expression alone and, thus, the formation of EGFR homodimers. The expression of ErbB2 (middle panel) results in the shift of the formation (activity) from EGFR homodimers (EGFR-EGFR) to the formation of EGFR-ErbB2 heterodimers. Furthermore, the expression of ErbB 1–3 results in the dominant formation of ErbB2–3 heterodimers , . Note that the references refer to classical, qualitative input-output relationships (not necessarily quantitative dose–response curves), and the dose-response curves presented here are intended to demonstrate how the computational model qualitatively reproduces the referenced input-output relationships over a range of stimulus signals.

Figure 3. EGFR internalization effects by Src overexpression.

Experiments were conducted under conditions for which the expression of ErbB2–4 was turned off and the virtual cell stimulated with EGF. A) Stimulation of the virtual cell with low levels of EGF (0–5%). B) EGF was introduced to the model in randomly selected high activity levels (60–70% ON). * p<0.05 (Student's t-test; n = 300; error bars represent the standard error of the mean). Note: PM corresponds to EGFR homodimers on the plasma membrane, CCP represents the homodimer in clathrin-coated pits, CCV – clathrin-coated vesicles, and MVB – EGFR homodimer in late endosome/multivescicular bodies.

Figure 4. Src overexpression does not affect total EGFR levels.

Parental 76N-TERT cells or its EGFR or EGFR + Src transductants were EGF deprived by culture in the EGF-deficient D3 medium for 48 hrs and cell lysates were prepared. 50 µg aliquots of cell lysate protein were run on an 8% SDS PAGE gel and immunoblotted with anti-EGFR (top panel) or anti-Src (middle panel). Membrane was re-probed with anti-beta actin (bottom panel) to ensure equal loading.

Figure 5. Surface expression of EGFR is reduced in Src-transfected cells.

FACS analysis of the cell surface expression of EGFR in MEC transductants shows (in row 2) that Src overexpression leads to a reduction in the level of EGFR at the cell surface despite higher total EGFR levels detected biochemically (shown in Figure 3). Cells were EGF-deprived by culture in D3 medium for 48 h, after which single cell suspensions were prepared with trypsin/EDTA. Live cells were stained with isotype-matched control monoclonal antibodies or with anti-EGFR monoclonal antibody for 1 hr.., washed and incubated with the secondary antibody (PE-conjugated) for 45 min., and analyzed by FACS to determine cell surface levels of EGFR. Numbers in the right top corner of each FACS panel indicate median fluorescence channel intensity (higher numbers indicating higher levels and vice versa). Initial experiments (not shown) established the specificity of antibodies.