Coupled Stochastic Spatial and Non-Spatial Simulations of ErbB1 Signaling Pathways Demonstrate the Importance of Spatial Organization in Signal Transduction

Background

The ErbB family of receptors activates intracellular signaling pathways that control cellular proliferation, growth, differentiation and apoptosis. Given these central roles, it is not surprising that overexpression of the ErbB receptors is often associated with carcinogenesis. Therefore, extensive laboratory studies have been devoted to understanding the signaling events associated with ErbB activation.

Methodology/Principal Findings

Systems biology has contributed significantly to our current understanding of ErbB signaling networks. However, although computational models have grown in complexity over the years, little work has been done to consider the spatial-temporal dynamics of receptor interactions and to evaluate how spatial organization of membrane receptors influences signaling transduction. Herein, we explore the impact of spatial organization of the epidermal growth factor receptor (ErbB1/EGFR) on the initiation of downstream signaling. We describe the development of an algorithm that couples a spatial stochastic model of membrane receptors with a nonspatial stochastic model of the reactions and interactions in the cytosol. This novel algorithm provides a computationally efficient method to evaluate the effects of spatial heterogeneity on the coupling of receptors to cytosolic signaling partners.

Conclusions/Significance

Mathematical models of signal transduction rarely consider the contributions of spatial organization due to high computational costs. A hybrid stochastic approach simplifies analyses of the spatio-temporal aspects of cell signaling and, as an example, demonstrates that receptor clustering contributes significantly to the efficiency of signal propagation from ligand-engaged growth factor receptors.     

SRC Homology 2 Domain Binding Sites in Insulin, IGF-1 and FGF receptor mediated signaling networks reveal an extensive potential interactome

ABSTRACT: Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains.     

Single cell profiling of potentiated phospho-protein networks in cancer cells

Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cancer cell survival and pathology. Profiles of cancer cell signaling networks might therefore identify mechanisms by which such cells interpret environmental cues for continued growth. Using multiparameter flow cytometry, we monitored phospho-protein responses to environmental cues in acute myeloid leukemia at the single cell level. By exposing cancer cell signaling networks to potentiating inputs, rather than relying upon the basal levels of protein phosphorylation alone, we could discern unique cancer network profiles that correlated with genetics and disease outcome. Strikingly, individual cancers manifested multiple cell subsets with unique network profiles, reflecting cancer heterogeneity at the level of signaling response. The results revealed a dramatic remodeling of signaling networks in cancer cells. Thus, single cell measurements of phospho-protein responses reveal shifts in signaling potential of a phospho-protein network, allowing for categorizing of cell network phenotypes by multidimensional molecular profiles of signaling.     

Nonparametric simulation of signal transduction networks with semi-synchronized update

Simulating signal transduction in cellular signaling networks provides predictions of network dynamics by quantifying the changes in concentration and activity-level of the individual proteins. Since numerical values of kinetic parameters might be difficult to obtain, it is imperative to develop non-parametric approaches that combine the connectivity of a network with the response of individual proteins to signals which travel through the network. The activity levels of signaling proteins computed through existing non-parametric modeling tools do not show significant correlations with the observed values in experimental results. In this work we developed a non-parametric computational framework to describe the profile of the evolving process and the time course of the proportion of active form of molecules in the signal transduction networks. The model is also capable of incorporating perturbations. The model was validated on four signaling networks showing that it can effectively uncover the activity levels and trends of response during signal transduction process.     

Protein kinase sensors: an overview of new designs for visualizing kinase dynamics in single plant cells

Protein kinase dynamics play key roles in regulation of cell differentiation, growth, development and in diverse cell signaling networks. Protein kinase sensors enable visualization of protein kinase activity in living cells and tissues in time and space. These sensors have therefore become important and powerful molecular tools for investigation of diverse kinase activities and can resolve long-standing and challenging biological questions. In the present Update, we review new advanced approaches for genetically encoded protein kinase biosensor designs developed in animal systems together with the basis of each biosensor’s working principle and components. In addition, we review recent first examples of real time plant protein kinase activity biosensor development and application. We discuss how these sensors have helped to resolve how stomatal signal transduction in response to elevated CO₂ merges with abscisic acid signaling downstream of a resolved basal SnRK2 kinase activity in guard cells. Furthermore, recent advances, combined with the new strategies described in this Update, can help deepen the understanding of how signaling networks regulate unique functions and responses in distinct plant cell types and tissues and how different stimuli and signaling pathways can interact.

New genetically encoded biosensor design approaches and visualization of protein kinase activity in living plant cells and tissues in time and space are reviewed.     

Tumor-induced disruption of proximal TCR-mediated signal transduction in tumor-infiltrating CD8+ lymphocytes inactivates antitumor effector phase

The presence in cancer tissue of Ag-specific, activated tumor infiltrating CD8(+) T cells proves that tumors express Ags capable of eliciting immune response. Therefore, in general, tumor escape from immune-mediated clearance is not attributable to immunological ignorance. However, tumor-infiltrating lymphocytes are defective in effector phase function, demonstrating tumor-induced immune suppression that likely underlies tumor escape. Since exocytosis of lytic granules is dependent upon TCR-mediated signal transduction, it is a reasonable contention that tumors may induce defective signal transduction in tumor infiltrating T cells. In this review, we consider the biochemical basis for antitumor T cell dysfunction, focusing on the role of inhibitory signaling receptors in restricting TCR-mediated signaling in tumor-infiltrating lymphocytes.     

“Topological Significance” Analysis of Gene Expression and Proteomic Profiles from Prostate Cancer Cells Reveals Key Mechanisms of Androgen Response

Background

The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer.

Methodology/Principal Findings

We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of “topological significance”. This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a “second phase” response, not directly dependent on the initial androgen stimulus.

Conclusions/Significance

We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an interconnected network module containing many alternative routes from inputs to outputs. If the whole network is involved, a precisely formulated combination therapy may be required to fight the tumor growth effectively.     

G-protein-coupled enzyme cascades have intrinsic properties that improve signal localization and fidelity

G-protein-coupled enzyme cascades are used by eukaryotic cells to detect external signals and transduce them into intracellular messages that contain biological information relevant to the cell's function. Since G-protein-coupled receptors that are designed to detect different kinds of external signals can generate the same kind of intracellular response, effective signaling requires that there are mechanisms to increase signal specificity and fidelity. Here we examine the kinetic equations for the initial three stages in a generic G-protein-coupled cascade and show that the physical properties of the transduction pathway result in two intrinsic features that benefit signaling. 1), The response to a single activated receptor is naturally confined to a localized spatial domain, which could improve signal specificity by reducing cross talk. 2), The peak of the response generated by such a signaling domain is limited. This saturation effect reduces trial-to-trial variability and increases signaling fidelity by limiting the response to receptors that remain active for longer than average. We suggest that this mechanism for reducing response fluctuations may be a contributing factor in making the single photon responses of vertebrate retinal rods so remarkably reproducible.     

Simulated Evolution of Signal Transduction Networks

Signal transduction is the process of routing information inside cells when receiving stimuli from their environment that modulate the behavior and function. In such biological processes, the receptors, after receiving the corresponding signals, activate a number of biomolecules which eventually transduce the signal to the nucleus. The main objective of our work is to develop a theoretical approach which will help to better understand the behavior of signal transduction networks due to changes in kinetic parameters and network topology. By using an evolutionary algorithm, we designed a mathematical model which performs basic signaling tasks similar to the signaling process of living cells. We use a simple dynamical model of signaling networks of interacting proteins and their complexes. We study the evolution of signaling networks described by mass-action kinetics. The fitness of the networks is determined by the number of signals detected out of a series of signals with varying strength. The mutations include changes in the reaction rate and network topology. We found that stronger interactions and addition of new nodes lead to improved evolved responses. The strength of the signal does not play any role in determining the response type. This model will help to understand the dynamic behavior of the proteins involved in signaling pathways. It will also help to understand the robustness of the kinetics of the output response upon changes in the rate of reactions and the topology of the network.     

Receptor cross-talk in angiogenesis: Mapping environmental cues to cell phenotype using a stochastic, Boolean signaling network model

Cancer invasion and metastasis depend on tumor-induced angiogenesis, the means by which cancer cells attract and maintain a blood supply. During angiogenesis, cellular processes are tightly coordinated by signaling molecules and their receptors. Understanding how endothelial cells synthesize multiple biochemical signals can catalyze the development of novel therapeutic strategies to combat cancer. This study is the first to propose a signal transduction model highlighting the cross-talk between key receptors involved in angiogenesis, namely the VEGF, integrin, and cadherin receptors. From experimental data, we construct a network model of receptor cross-talk and analyze its dynamics. We identify relationships between receptor activation combinations and cellular function, and show that cross-talk is crucial to phenotype determination. The network converges to a unique set of output states that correspond to known cell phenotypes: migratory, proliferating, quiescent, apoptotic, and it predicts one phenotype that challenges the “go or grow” hypothesis. Finally, we use the model to study protein inhibition and to suggest molecular targets for anti-angiogenic therapies.     

Modeling specificity in the yeast MAPK signaling networks

Cells sense several kinds of stimuli and trigger corresponding responses through signaling pathways. As a result, cells must process and integrate multiple signals in parallel to maintain specificity and avoid erroneous cross-talk. In this study, we focus our theoretical effort on understanding specificity of a model network system in yeast, Saccharomyces cerevisiae, which contains three mitogen-activated protein kinase (MAPK) signal transduction cascades that share multiple signaling components. The cellular response to the pheromone, the filamentous growth and osmotic pressure stimuli in yeast is described and an integrative mathematical model for the three MAPK cascades is developed using available literature and experimental data. The theoretical framework for analyzing the specificity of signaling networks [Bardwell, L., Zou, X.F., Nie, Q., Komarova, N.L., 2007. Mathematical models of specificity in cell signaling. Biophys. J. 92, 3425-3441] is extended to include multiple interacting pathways with shared components. Simulations are also performed with any one stimulus, with any two simultaneous stimuli, and with the simultaneous application of the three stimuli. The interactions between the three pathways are systematically investigated. Moreover, the specificity and fidelity of this model system are calculated using our newly developed concept under different stimuli or with specific mutants. Our simulated and calculated results demonstrate that the yeast MAPK signaling network can achieve specificity and fidelity by filtering out spurious cross-talk between the relevant pathways through different mechanisms, such as scaffolding, cross-inhibiting, and feedback control. Proof that Pbs2 and Hog1 are essential for the maintenance of signaling specificity is presented. Our studies provide novel insights into integration of relevant signaling pathways in a biological system and the mechanisms conferring specificity in cellular signaling networks.     

Crosstalk between Phosphoinositide 3-kinase/Akt signaling pathway with DNA damage response and oxidative stress in cancer

The phosphatidylinositol 3-kinases (PI3K)/Akt signaling pathway is one of the well-characterized and most important signaling pathways activated in response to DNA damage. This review discusses the most recent discoveries on the involvement of PI3K/Akt signaling pathway in cancer development, as well as stimulation of some important signaling networks involved in the maintenance of cellular homeostasis upon DNA damage, with an exploration of how PI3K/Akt signaling pathway contributes to the regulation of modulators and effectors underlying DNA damage response, the intricate, protein-based signal transduction network, which decides between cell cycle arrest, DNA repair, and apoptosis, the elimination of irreparably damaged cells to maintain homeostasis. The review continues by looking at the interplay between cell cycle checkpoints, checking the repair of damage inflicted to the DNA before entering DNA replication to facilitate DNA synthesis, and PI3K/Akt signaling pathway. We then investigate the challenges the cells overcome to ameliorate damages induced by oxidative activities, for example, the recruitment of many pathways and factors to maintain integrity and hemostasis. Finally, the review provides a discussion of how cells use the PI3K/Akt signaling pathway to regulate the balance between these networks.     

The Cbl interactome and its functions

Cbl proteins are ubiquitin ligases and multifunctional adaptor proteins that are implicated in the regulation of signal transduction in various cell types and in response to different stimuli. Cbl-associated proteins can assemble together at a given time or space inside the cell, and such an interactome can form signal competent networks that control many physiological processes. Dysregulation of spatial or temporal constraints in the Cbl interactome results in the development of human pathologies such as immune diseases, diabetes and cancer.     

Stimulus‐dependent dynamics of p53 in single cells

Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input–output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to γ‐radiation. We further found that while γ‐triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input‐sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus‐dependent dynamics of p53, including excitability and input‐duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network.     

Mechanobiology of solid tumors

Mechanical features of cancer cells emerge as a distinct trait during development and progression of solid tumors. Herein, we discuss recent key findings regarding the impact of various types of mechanical stresses on cancer cell properties. Data suggest that different mechanical forces, alterations of matrix rigidity and tumor microenvironment facilitate cancer hallmarks, especially invasion and metastasis. Moreover, a subset of mechanosensory proteins are responsible for mediating mechanically induced oncogenic signaling and response to chemotherapy. Delineating cancer dynamics and decoding of respective signal transduction mechanisms will provide new therapeutic strategies against solid tumors in the future.     

The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus

Poxviruses demonstrate strict species specificity in vivo that range from narrow to broad, however the fundamental factors that mediate the basis of poxvirus tropism remain poorly understood. It is generally believed that most, if not all, poxviruses can efficiently bind and enter a wide range of mammalian cells and all of the known host anti-viral pathways that block viral replication in nonpremissive cells operate downstream of virus entry. A productive poxvirus infection is heavily dependent upon the production of a vast array of host modulatory products that specifically target and manipulate both extracellular immune response pathways of the host, as well as intracellular signal transduction pathways of the individually infected cells. The unique pathogenesis and host tropism of specific poxviruses can be attributed to the broad diversity of host modulatory proteins they express. Myxoma virus (MV) is a rabbit-specific poxviruses that encodes multiple host range factors, including an ankyrin-repeat protein M-T5, which functions to regulate tropism of MV for rabbit lymphocytes and some human cancer cells. At the molecular level, M-T5 binds and alters at least two distinct cellular proteins: Akt and cullin-1. The direct interaction between M-T5 and Akt was shown to be a key restriction determinant for MV tropism in a spectrum of human cancer cells making MV an excellent oncolytic candidate. Thus, the intricate relationship between viral encoded proteins and components of the host cell signaling networks can have profound impact on poxvirus tropism. The lessons we continue to learn from poxvirus host range factors like M-T5 will provide further insights into the factors that regulate poxvirus tropism and the mechanisms by which poxviruses micromanipulate the signaling pathways of the infected cell.