Multipathway Model Enables Prediction of Kinase Inhibitor Cross-Talk Effects on Migration of Her2-Overexpressing Mammary Epithelial Cells

Abstract

Small-molecule kinase inhibitors often modulate signaling pathways other than the one targeted, whether by direct "off-target" effects or by indirect "pathway cross-talk" effects. The presence of either or both of these classes of complicating factors impedes the predictive understanding of kinase inhibitor consequences for cell phenotypic behaviors involved in drug efficacy responses. To address this problem, we offer an avenue toward comprehending how kinase inhibitor modulations of cell signaling networks lead to altered cell phenotypic responses by applying a quantitative, multipathway computational modeling approach. We show that integrating measurements of signals across three key kinase pathways involved in regulating migration of human mammary epithelial cells, downstream of ErbB system receptor activation by epidermal growth factor (EGF) or heregulin (HRG), significantly improves prediction of cell migration changes resulting from treatment with the small-molecule inhibitors 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) and 2Ј-amino-3Ј-methoxyflavone (PD98059) for both normal and HER2-overexpressing cells. These inhibitors are primarily directed toward inhibition of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) but are known to exhibit off-target effects; moreover, complex crosstalk interactions between the PI3K/Akt and MEK/extracellular signal-regulated kinase (Erk) pathways are also appreciated. We observe here that treatment with LY294002 reduces migration of HRG-stimulated cells but not EGF-stimulated cells, despite comparable levels of reduction of Akt phosphorylation under both conditions, demonstrating that the target inhibition effect is not unilaterally predictive of efficacy against cell phenotypic response. Consequent measurement of levels of Erk and p38 phosphorylation, along with those for EGF receptor phosphorylation, after LY294002 treatment revealed unintended modulation of these nontargeted pathways. However, when these measurements were incorporated into a partial least-squares regression model, the cell migration responses to treatment were successfully predicted. Similar success was found for the same multipathway model in analogously predicting PD98059 treatment effects on cell migration. We conclude that a quantitative, multipathway modeling approach can provide a significant advance toward comprehending kinase inhibitor efficacy in the face of off-target and pathway cross-talk effects.

Epidermal Growth Factor–induced Enhancement of Glioblastoma Cell Migration in 3D Arises from an Intrinsic Increase in Speed But an Extrinsic Matrix- and Proteolysis-dependent Increase in Persistence

“…As noted earlier, there is increasing interest in ErbB inhibitors as cancer therapeutics, with specific focus on reducing motility in order to diminish invasiveness (Willmarth and Ethier, 2006;Rosano et al, 2007;Kumar et al, 2008) in particular for treating glioblastoma (Lal et al, 2002;Lamszus et al, 2005;Huang et al, 2007b). However, the effects of ErbB signaling network activation on cell migration are complex and highly dependent on extracellular matrix conditions (Ware et al, 1998;Maheshwari et al, 1999;Harms et al, 2005;Zaman et al, 2006); even in the absence of chemotactic concentration gradients, EGF stimulation can increase or decrease the speed of migration while simultaneously increasing or decreasing directional persistence of migration, depending on the substratum.…”

Amplified Ras-MAPK signal states correlate with accelerated EGFR internalization, cytostasis and delayed HER2 tumor onset in Fer-deficient model systems

“…EGFR internalization was assessed by tracking surface and internalized levels of biotin-EGF over time using immunocytochemistry. 53 Surface biotin-EGF levels were assessed in a parallel set of experiments by fixing cells in 3% paraformaldehyde, 0.25% glutaraldehyde for 30 min at 37°C. Internalized biotin-EGF was assessed by acid-stripping receptor-bound EGF followed by membrane permeabilization and fixation.…”

Quantitative modeling perspectives on the ErbB system of cell regulatory processes

“…It does not, of course, describe the mechanisms by which the network state is translated into transcriptional, metabolic, and cytoskeletal processes executing the observed proliferation and migration behavior, but instead merely the "information-processing algorithm" characterizing multi-pathway regulatory network operation upstream of those processes. This perspective was tested directly by Kumar et al [42], who used an analogous partial least-squares modeling approach to predict how modulation of key kinase pathways by pharmacological inhibitors alters migration behavior of HER2-overexpressing mammary epithelial cell migration responses to EGF and HRG. The signal measurements were limited to Western blot assays for phosphorylation state of ERK, Akt, and p38, along with that of EGFR (2 phosphosites), to demonstrate utility for commonly-accessible experimental data-sets.…”