Inferring Intracellular Signal Transduction Circuitry from Molecular Perturbation Experiments.

The development of network inference methodologies that accurately predict connectivity in dysregulated pathways may enable the rational selection of patient therapies. Accurately inferring an intracellular network from data remains a very challenging problem in molecular systems biology. Living cells integrate extremely robust circuits that exhibit significant heterogeneity, but still respond to external stimuli in predictable ways. This phenomenon allows us to introduce a network inference methodology that integrates measurements of protein activation from perturbation experiments. The methodology relies on logic-based networks to provide a predictive approximation of the transfer of signals in a network. The approach presented was validated in silico with a set of test networks and applied to investigate the epidermal growth factor receptor signaling of a breast epithelial cell line, MFC10A. In our analysis, we predict the potential signaling circuitry most likely responsible for the experimental readouts of several proteins in the mitogen-activated protein kinase and phosphatidylinositol-3 kinase pathways. The approach can also be used to identify additional necessary perturbation experiments to distinguish between a set of possible candidate networks.

RhoC GTPase Is a Potent Regulator of Glutamine Metabolism and N-Acetylaspartate Production in Inflammatory Breast Cancer Cells.

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including (13)C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement.

The importance of accurately correcting for the natural abundance of stable isotopes.

The use of isotopically labeled tracer substrates is an experimental approach for measuring in vivo and in vitro intracellular metabolic dynamics. Stable isotopes that alter the mass but not the chemical behavior of a molecule are commonly used in isotope tracer studies. Because stable isotopes of some atoms naturally occur at non-negligible abundances, it is important to account for the natural abundance of these isotopes when analyzing data from isotope labeling experiments. Specifically, a distinction must be made between isotopes introduced experimentally via an isotopically labeled tracer and the isotopes naturally present at the start of an experiment. In this tutorial review, we explain the underlying theory of natural abundance correction of stable isotopes, a concept not always understood by metabolic researchers. We also provide a comparison of distinct methods for performing this correction and discuss natural abundance correction in the context of steady state 13C metabolic flux, a method increasingly used to infer intracellular metabolic flux from isotope experiments.

Follow-the-leader cell migration requires biased cell-cell contact and local microenvironmental signals.

Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.

Unraveling the complex regulatory relationships between metabolism and signal transduction in cancer.

Cancer cells exhibit an altered metabolic phenotype, known as the Warburg effect, which is characterized by high rates of glucose uptake and glycolysis, even under aerobic conditions. The Warburg effect appears to be an intrinsic component of most cancers and there is evidence linking cancer progression to mutations, translocations, and alternative splicing of genes that directly code for or have downstream effects on key metabolic enzymes. Many of the same signaling pathways are routinely dysregulated in cancer and a number of important oncogenic signaling pathways play important regulatory roles in central carbon metabolism. Unraveling the complex regulatory relationship between cancer metabolism and signaling requires the application of systems biology approaches. Here we discuss computational approaches for modeling protein signal transduction and metabolism as well as how the regulatory relationship between these two important cellular processes can be combined into hybrid models.

Temporal patterns of conditions and symptoms potentially associated with ovarian cancer.

BACKGROUND: The late stage at which ovarian cancer is typically diagnosed and its subsequent high mortality have been attributed to a lack of symptoms in its early stages. This study examined the temporal patterns of prediagnostic ovarian cancer symptoms and conditions among women with and without ovarian cancer. METHODS: We identified 920 ovarian cancer cases from 1998-2002 claims and encounters from Thomson Healthcare's Medstat MarketScan Commercial Claims and Encounters and Medicare Supplemental Databases. These were matched with 2760 comparison women based on age, geographic region, Medicare eligibility, and health plan type. The rates of ovarian cancer-related symptoms, conditions, and procedures recorded in the claims data were compared between the two groups using chi-square and Student's t tests. RESULTS: In the 270 to 31 days prior to the case diagnosis dates, cases had nearly five times more recorded abdominal symptoms (36.2% vs. 7.5%), 3.5 times more recorded female genital symptoms (9.8% vs. 2.7%), and 1.5-2 times more recorded gastrointestinal symptoms (7.7% vs. 3.5%), urethra/urinary tract disorders (12.7% vs. 6.4%), and menopausal disorders (12.4% vs. 7.5%) than the comparison women. However, when the data were examined in 30-day increments for these five diagnosed conditions, the rates for cases and comparison women only started to diverge as the cases' diagnosis drew closer-60-90 days prior. CONCLUSIONS: The presence of ovarian cancer-related symptoms and conditions prior to diagnosis among cases was documented in claims data; however, this increase was most pronounced in the 2-3 months prior to diagnosis. It is likely that physicians will see similar symptoms and conditions for women with and without ovarian cancer during most of the 9 months prior to the cases' diagnosis.

Adipocytes promote pancreatic cancer cell proliferation via glutamine transfer.

Adipocytes promote progression of multiple cancers, but their role in pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC) is poorly defined. Nutrient transfer is a mechanism underlying stromal cell-cancer crosstalk. We studied the role of adipocytes in regulating in vitro PanIN and PDAC cell proliferation with a focus on glutamine metabolism. Murine 3T3L1 adipocytes were used to model adipocytes. Cell lines derived from PKCY mice were used to model PanIN and PDAC. Co-culture was used to study the effect of adipocytes on PanIN and PDAC cell proliferation in response to manipulation of glutamine metabolism. Glutamine secretion was measured with a bioanalyzer. Western blotting was used to study the effect of PanIN and PDAC cells on expression of glutamine-related enzymes in adipocytes. Adipocytes promote proliferation of PanIN and PDAC cells, an effect that was amplified in nutrient-poor conditions. Adipocytes secrete glutamine and rescue PanIN and PDAC cell proliferation in the absence of glutamine, an effect that was glutamine synthetase-dependent and involved PDAC cell-induced down-regulation of glutaminase expression in adipocytes. These findings suggest glutamine transfer as a potential mechanism underlying adipocyte-induced PanIN and PDAC cell proliferation.

Inferring the Effects of Honokiol on the Notch Signaling Pathway in SW480 Colon Cancer Cells.

In a tumor cell, the development of acquired therapeutic resistance and the ability to survive in extracellular environments that differ from the primary site are the result of molecular adaptations in potentially highly plastic molecular networks. The accurate prediction of intracellular networks in a tumor remains a difficult problem in cancer informatics. In order to make truly rational patient-driven therapeutic decisions, it will be critical to develop methodologies that can accurately infer the molecular circuitry in the cells of a specific tumor. Despite enormous heterogeneity, cellular networks elicit deterministic digital-like responses. We discuss the use and limitations of methodologies that model molecular networks in cancer cells as a digital circuit. We also develop a network model of Notch signaling in colon cancer using a novel reverse engineering logic-based method and published western blot data to elucidate the interactions likely present in the circuits of the SW480 colon cancer cell line. Within this framework, we make predictions related to the role that honokiol may be playing as an anti-cancer drug.

Computational modelling of cell chain migration reveals mechanisms that sustain follow-the-leader behaviour.

Follow-the-leader chain migration is a striking cell migratory behaviour observed during vertebrate development, adult neurogenesis and cancer metastasis. Although cell-cell contact and extracellular matrix (ECM) cues have been proposed to promote this phenomenon, mechanisms that underlie chain migration persistence remain unclear. Here, we developed a quantitative agent-based modelling framework to test mechanistic hypotheses of chain migration persistence. We defined chain migration and its persistence based on evidence from the highly migratory neural crest model system, where cells within a chain extend and retract filopodia in short-lived cell contacts and move together as a collective. In our agent-based simulations, we began with a set of agents arranged as a chain and systematically probed the influence of model parameters to identify factors critical to the maintenance of the chain migration pattern. We discovered that chain migration persistence requires a high degree of directional bias in both lead and follower cells towards the target. Chain migration persistence was also promoted when lead cells maintained cell contact with followers, but not vice-versa. Finally, providing a path of least resistance in the ECM was not sufficient alone to drive chain persistence. Our results indicate that chain migration persistence depends on the interplay of directional cell movement and biased cell-cell contact.

Logic-based models in systems biology: a predictive and parameter-free network analysis method.

Highly complex molecular networks, which play fundamental roles in almost all cellular processes, are known to be dysregulated in a number of diseases, most notably in cancer. As a consequence, there is a critical need to develop practical methodologies for constructing and analysing molecular networks at a systems level. Mathematical models built with continuous differential equations are an ideal methodology because they can provide a detailed picture of a network's dynamics. To be predictive, however, differential equation models require that numerous parameters be known a priori and this information is almost never available. An alternative dynamical approach is the use of discrete logic-based models that can provide a good approximation of the qualitative behaviour of a biochemical system without the burden of a large parameter space. Despite their advantages, there remains significant resistance to the use of logic-based models in biology. Here, we address some common concerns and provide a brief tutorial on the use of logic-based models, which we motivate with biological examples.

Kinase inhibitors can produce off-target effects and activate linked pathways by retroactivity.

BACKGROUND: It has been shown in experimental and theoretical work that covalently modified signaling cascades naturally exhibit bidirectional signal propagation via a phenomenon known as retroactivity. An important consequence of retroactivity, which arises due to enzyme sequestration in covalently modified signaling cascades, is that a downstream perturbation can produce a response in a component upstream of the perturbation without the need for explicit feedback connections. Retroactivity may, therefore, play an important role in the cellular response to a targeted therapy. Kinase inhibitors are a class of targeted therapies designed to interfere with a specific kinase molecule in a dysregulated signaling pathway. While extremely promising as anti-cancer agents, kinase inhibitors may produce undesirable off-target effects by non-specific interactions or pathway cross-talk. We hypothesize that targeted therapies such as kinase inhibitors can produce off-target effects as a consequence of retroactivity alone. RESULTS: We used a computational model and a series of simple signaling motifs to test the hypothesis. Our results indicate that within physiologically and therapeutically relevant ranges for all parameters, a targeted inhibitor can naturally induce an off-target effect via retroactivity. The kinetics governing covalent modification cycles in a signaling network were more important for propagating an upstream off-target effect in our models than the kinetics governing the targeted therapy itself. Our results also reveal the surprising and crucial result that kinase inhibitors have the capacity to turn "on" an otherwise "off" parallel cascade when two cascades share an upstream activator. CONCLUSIONS: A proper and detailed characterization of a pathway's structure is important for identifying the optimal protein to target as well as what concentration of the targeted therapy is required to modulate the pathway in a safe and effective manner. We believe our results support the position that such characterizations should consider retroactivity as a robust potential source of off-target effects induced by kinase inhibitors and other targeted therapies.