The Ras-Raf-MEK-ERK pathway (or ERK pathway) is an important signal transduction system involved in the control of cell proliferation, survival and differentiation. However, the dynamic regulation of the pathway by positive- and negative-feedback mechanisms, in particular the functional role of Raf kinase inhibitor protein (RKIP) are still incompletely understood. RKIP is a physiological endogenous inhibitor of MEK phosphorylation by Raf kinases, but also participates in a positive-feedback loop in which ERK can inactivate RKIP. The aim of this study was to elucidate the hidden dynamics of these feedback mechanisms and to identify the functional role of RKIP through combined efforts of biochemical experiments and in silico simulations based on an experimentally validated mathematical model. We show that the negative-feedback loop from ERK to SOS plays a crucial role in generating an oscillatory behavior of ERK activity. The positive-feedback loop in which ERK functionally inactivates RKIP also enhances the oscillatory activation pattern of ERK. However, RKIP itself has an important role in inducing a switch-like behavior of MEK activity. When overexpressed, RKIP also causes delayed and reduced responses of ERK. Thus, positive- and negative-feedback loops and RKIP work together to shape the response pattern and dynamical characteristics of the ERK pathway.

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP₃binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.

Mathematical modeling and dynamic simulation of signal transduction pathways is a central theme in systems biology and is increasingly attracting attention in the postgenomic era. The estimation of model parameters from experimental data remains a bottleneck for a major breakthrough in this area. This study’s aim is to introduce a new strategy for experimental design based on parameter sensitivity analysis. The approach identifies key parameters/variables in a signal transduction pathway model and can thereby provide experimental biologists with guidance on which proteins to consider for measurement. The article focuses on applying this approach to the TNFα-mediated NF-κB pathway, which plays an important role in immunity and inflammation and in the control of cell proliferation, differentiation, and apoptosis. A mathematical model of this pathway is proposed, and the sensitivity analysis of model parameters is illustrated for this model by employing the Monte Carlo method over a broad range of parameter values.

Epithelial-mesenchymal transition (EMT) is a key event in the generation of invasive tumor cells. A hallmark of EMT is the repression of E-cadherin expression, which is regulated by various signal transduction pathways including extracellular signal-regulated kinase (ERK) and Wnt. These pathways are highly interconnected via multiple coupled feedback loops (CFL). As the function of such coupled feedback regulations is difficult to analyze experimentally, we used a systems biology approach where computational models were designed to predict biological effects that result from the complex interplay of CFLs. Using epidermal growth factor (EGF) and Wnt as input and E-cadherin transcriptional regulation as output, we established an ordinary differential equation model of the ERK and Wnt signaling network containing six feedback links and used extensive computer simulations to analyze the effects of these feedback links in isolation and different combinations. The results show that the feedbacks can generate a rich dynamic behavior leading to various dose-response patterns and have a decisive role in determining network responses to EGF and Wnt. In particular, we made two important findings: first, that coupled positive feedback loops composed of phosphorylation of Raf kinase inhibitor RKIP by ERK and transcriptional repression of RKIP by Snail have an essential role in causing a switch-like behavior of E-cadherin expression; and second, that RKIP expression inhibits EMT progression by preventing E-cadherin suppression. Taken together, our findings provide us with a system-level understanding of how RKIP can regulate EMT progression and may explain why RKIP is downregulated in so many metastatic cancer cells. Cancer Res; 70(17); 6715–24. ©2010 AACR.

In this study, we propose a system-theoretic approach to the analysis and quantitative modeling of the TNFα-mediated NF-κB-signaling pathway. Tumor necrosis factor α (TNFα) is a potent proinflammatory cytokine that plays an important role in immunity and inflammation, in the control of cell proliferation, differentiation, and apoptosis. To date, there have been numerous approaches to model cellular dynamics. The most prominent uses ordinary differential equations (ODEs) to describe biochemical reactions. This approach can provide us with mathematically well-founded and tractable interpretations regarding pathways, especially those best described by enzyme reactions. This work first introduces a graphical method to intuitively represent the TNFα-mediated NF-κB-signaling pathway and then utilizes ODEs to quantitatively model the pathway. The simulation study shows qualitative validation of the proposed model compared with experimental results for this pathway. The proposed system-theoretic approach is expected to be further applicable to predict the signaling behavior of NF-κB in a quantitative manner for any variation of the ligand, TNFα.

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous disease with poor prognosis and no effective targeted therapies. EGFR is highly expressed in basal-like TNBC and is considered as a potential therapeutic target. However, EGFR targeting exerts only marginal clinical benefits, possibly due to activation of compensatory signaling pathways, which are frequently associated with HER3 upregulation. Here we show that concomitant targeting of EGFR and the nonreceptor tyrosine kinases PYK2/FAK synergistically inhibits the proliferation of basal-like TNBC cells in vitro and attenuates tumor growth in a mouse xenograft model. Dual targeting of EGFR and PYK2/FAK inhibited complementary key growth and survival pathways mediated by AKT, S6K, STAT3, and ERK1/2 activation. PYK2 inhibition also abrogated HER3 upregulation in response to EGFR antagonists, thereby circumventing HER3-associated drug resistance. Mechanistically, PYK2 inhibition facilitated the proteasomal degradation of HER3 while inducing upregulation of NDRG1 (N-myc downstream regulated 1 gene). NDRG1 enhanced the interaction of HER3 with the ubiquitin ligase NEDD4, while PYK2, which interacts with NEDD4 and HER3, interfered with NEDD4–HER3 binding, suggesting that the PYK2–NDRG1–NEDD4 circuit has a critical role in receptor degradation, drug response, and resistance mechanism. Our studies offer a preclinical proof of concept for a strategy of cotargeting the EGFR and PYK2/FAK kinases to improve TNBC therapy. Cancer Res; 77(1); 86–99. ©2016 AACR.