Vascular Endothelial Growth Factor (VEGF) Promotes Assembly of the p130Cas Interactome to Drive Endothelial Chemotactic Signaling and Angiogenesis.

p130Cas is a polyvalent adapter protein essential for cardiovascular development, and with a key role in cell movement. In order to identify the pathways by which p130Cas exerts its biological functions in endothelial cells we mapped the p130Cas interactome and its dynamic changes in response to VEGF using high-resolution mass spectrometry and reconstruction of protein interaction (PPI) networks with the aid of multiple PPI databases. VEGF enriched the p130Cas interactome in proteins involved in actin cytoskeletal dynamics and cell movement, including actin-binding proteins, small GTPases and regulators or binders of GTPases. Detailed studies showed that p130Cas association of the GTPase-binding scaffold protein, IQGAP1, plays a key role in VEGF chemotactic signaling, endothelial polarization, VEGF-induced cell migration, and endothelial tube formation. These findings indicate a cardinal role for assembly of the p130Cas interactome in mediating the cell migratory response to VEGF in angiogenesis, and provide a basis for further studies of p130Cas in cell movement.

Navigating the multilayered organization of eukaryotic signaling: a new trend in data integration.

The ever-increasing capacity of biological molecular data acquisition outpaces our ability to understand the meaningful relationships between molecules in a cell. Multiple databases were developed to store and organize these molecular data. However, emerging fundamental questions about concerted functions of these molecules in hierarchical cellular networks are poorly addressed. Here we review recent advances in the development of publically available databases that help us analyze the signal integration and processing by multilayered networks that specify biological responses in model organisms and human cells.

An integrated Bayesian framework for identifying phosphorylation networks in stimulated cells.

One of the primary mechanisms of signal transduction in cells is protein phosphorylation. Upon ligand stimulation a series of phosphorylation events take place which eventually lead to transcription. Different sets of phosphorylation events take place due to different stimulating ligands in different types of cells. Knowledge of these phosphorylation events is essential to understand the underlying signaling mechanisms. We have developed a Bayesian framework to infer phosphorylation networks from time series measurements of phosphosite concentrations upon ligand stimulation. To increase the prediction accuracy we integrated different types of data, e.g., amino acid sequence data, genomic context data (gene fusion, gene neighborhood, and phylogentic profiles), primary experimental evidence (physical protein interactions and gene coexpression), manually curated pathway databases, and automatic literature mining with time series data in our inference framework. We compared our results with data available from public databases and report a high level of prediction accuracy.

A Systems Biology Approach to Investigate Kinase Signal Transduction Networks That Are Involved in Triple Negative Breast Cancer Resistance to Cisplatin.

Triple negative breast cancer (TNBC) remains a therapeutic challenge due to the lack of targetable genetic alterations and the frequent development of resistance to the standard cisplatin-based chemotherapies. Here, we have taken a systems biology approach to investigate kinase signal transduction networks that are involved in TNBC resistance to cisplatin. Treating a panel of cisplatin-sensitive and cisplatin-resistant TNBC cell lines with a panel of kinase inhibitors allowed us to reconstruct two kinase signalling networks that characterise sensitive and resistant cells. The analysis of these networks suggested that the activation of the PI3K/AKT signalling pathway is critical for cisplatin resistance. Experimental validation of the computational model predictions confirmed that TNBC cell lines with activated PI3K/AKT signalling are sensitive to combinations of cisplatin and PI3K/AKT pathway inhibitors. Thus, our results reveal a new therapeutic approach that is based on identifying targeted therapies that synergise with conventional chemotherapies.

An Integrative Computational Approach for a Prioritization of Key Transcription Regulators Associated With Nanomaterial-Induced Toxicity.

A rapid increase of new nanomaterial (NM) products poses new challenges for their risk assessment. Current traditional methods for estimating potential adverse health effect of NMs are complex, time consuming, and expensive. In order to develop new prediction tests for nanotoxicity evaluation, a systems biology approach, and data from high-throughput omics experiments can be used. We present a computational approach that combines reverse engineering techniques, network analysis and pathway enrichment analysis for inferring the transcriptional regulation landscape and its functional interpretation. To illustrate this approach, we used published transcriptomic data derived from mice lung tissue exposed to carbon nanotubes (NM-401 and NRCWE-26). Because fibrosis is the most common adverse effect of these NMs, we included in our analysis the data for bleomycin (BLM) treatment, which is a well-known fibrosis inducer. We inferred gene regulatory networks for each NM and BLM to capture functional hierarchical regulatory structures between genes and their regulators. Despite the different nature of the lung injury caused by nanoparticles and BLM, we identified several conserved core regulators for all agents. We reason that these regulators can be considered as early predictors of toxic responses after NMs exposure. This integrative approach, which refines traditional methods of transcriptomic analysis, can be useful for prioritization of potential core regulators and generation of new hypothesis about mechanisms of nanoparticles toxicity.

An Integrated Global Analysis of Compartmentalized HRAS Signaling.

Modern omics technologies allow us to obtain global information on different types of biological networks. However, integrating these different types of analyses into a coherent framework for a comprehensive biological interpretation remains challenging. Here, we present a conceptual framework that integrates protein interaction, phosphoproteomics, and transcriptomics data. Applying this method to analyze HRAS signaling from different subcellular compartments shows that spatially defined networks contribute specific functions to HRAS signaling. Changes in HRAS protein interactions at different sites lead to different kinase activation patterns that differentially regulate gene transcription. HRAS-mediated signaling is the strongest from the cell membrane, but it regulates the largest number of genes from the endoplasmic reticulum. The integrated networks provide a topologically and functionally resolved view of HRAS signaling. They reveal distinct HRAS functions including the control of cell migration from the endoplasmic reticulum and TP53-dependent cell survival when signaling from the Golgi apparatus.

Fitting mathematical models of biochemical pathways to steady state perturbation response data without simulating perturbation experiments.

Fitting Ordinary Differential Equation (ODE) models of signal transduction networks (STNs) to experimental data is a challenging problem. Computational parameter fitting algorithms simulate a model many times with different sets of parameter values until the simulated STN behaviour match closely with experimental data. This process can be slow when the model is fitted to measurements of STN responses to numerous perturbations, since this requires simulating the model as many times as the number of perturbations for each set of parameter values. Here, I propose an approach that avoids simulating perturbation experiments when fitting ODE models to steady state perturbation response (SSPR) data. Instead of fitting the model directly to SSPR data, it finds model parameters which provides a close match between the scaled Jacobian matrices (SJM) of the model, which are numerically calculated using the model's rate equations and estimated from SSPR data using modular response analysis (MRA). The numerical estimation of SJM of an ODE model does not require simulating perturbation experiments, saving significant computation time. The effectiveness of this approach is demonstrated by fitting ODE models of the Mitogen Activated Protein Kinase (MAPK) pathway using simulated and real SSPR data.

Impact of measurement noise, experimental design, and estimation methods on Modular Response Analysis based network reconstruction.

Modular Response Analysis (MRA) is a method to reconstruct signalling networks from steady-state perturbation data which has frequently been used in different settings. Since these data are usually noisy due to multi-step measurement procedures and biological variability, it is important to investigate the effect of this noise onto network reconstruction. Here we present a systematic study to investigate propagation of noise from concentration measurements to network structures. Therefore, we design an in silico study of the MAPK and the p53 signalling pathways with realistic noise settings. We make use of statistical concepts and measures to evaluate accuracy and precision of individual inferred interactions and resulting network structures. Our results allow to derive clear recommendations to optimize the performance of MRA based network reconstruction: First, large perturbations are favorable in terms of accuracy even for models with non-linear steady-state response curves. Second, a single control measurement for different perturbation experiments seems to be sufficient for network reconstruction, and third, we recommend to execute the MRA workflow with the mean of different replicates for concentration measurements rather than using computationally more involved regression strategies.

Identification of potential new treatment response markers and therapeutic targets using a Gaussian process-based method in lapatinib insensitive breast cancer models.

Molecularly targeted therapeutics hold promise of revolutionizing treatments of advanced malignancies. However, a large number of patients do not respond to these treatments. Here, we take a systems biology approach to understand the molecular mechanisms that prevent breast cancer (BC) cells from responding to lapatinib, a dual kinase inhibitor that targets human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR). To this end, we analysed temporal gene expression profiles of four BC cell lines, two of which respond and the remaining two do not respond to lapatinib. For this analysis, we developed a Gaussian process based algorithm which can accurately find differentially expressed genes by analysing time course gene expression profiles at a fraction of the computational cost of other state-of-the-art algorithms. Our analysis identified 519 potential genes which are characteristic of lapatinib non-responsiveness in the tested cell lines. Data from the Genomics of Drug Sensitivity in Cancer (GDSC) database suggested that the basal expressions 120 of the above genes correlate with the response of BC cells to HER2 and/or EGFR targeted therapies. We selected 27 genes from the larger panel of 519 genes for experimental verification and 16 of these were successfully validated. Further bioinformatics analysis identified vitamin D receptor (VDR) as a potential target of interest for lapatinib non-responsive BC cells. Experimentally, calcitriol, a commonly used reagent for VDR targeted therapy, in combination with lapatinib additively inhibited proliferation in two HER2 positive cell lines, lapatinib insensitive MDA-MB-453 and lapatinib resistant HCC 1954-L cells.

A Bayesian algorithm for detecting differentially expressed proteins and its application in breast cancer research.

Presence of considerable noise and missing data points make analysis of mass-spectrometry (MS) based proteomic data a challenging task. The missing values in MS data are caused by the inability of MS machines to reliably detect proteins whose abundances fall below the detection limit. We developed a Bayesian algorithm that exploits this knowledge and uses missing data points as a complementary source of information to the observed protein intensities in order to find differentially expressed proteins by analysing MS based proteomic data. We compared its accuracy with many other methods using several simulated datasets. It consistently outperformed other methods. We then used it to analyse proteomic screens of a breast cancer (BC) patient cohort. It revealed large differences between the proteomic landscapes of triple negative and Luminal A, which are the most and least aggressive types of BC. Unexpectedly, majority of these differences could be attributed to the direct transcriptional activity of only seven transcription factors some of which are known to be inactive in triple negative BC. We also identified two new proteins which significantly correlated with the survival of BC patients, and therefore may have potential diagnostic/prognostic values.

BGRMI: A method for inferring gene regulatory networks from time-course gene expression data and its application in breast cancer research.

Reconstructing gene regulatory networks (GRNs) from gene expression data is a challenging problem. Existing GRN reconstruction algorithms can be broadly divided into model-free and model-based methods. Typically, model-free methods have high accuracy but are computation intensive whereas model-based methods are fast but less accurate. We propose Bayesian Gene Regulation Model Inference (BGRMI), a model-based method for inferring GRNs from time-course gene expression data. BGRMI uses a Bayesian framework to calculate the probability of different models of GRNs and a heuristic search strategy to scan the model space efficiently. Using benchmark datasets, we show that BGRMI has higher/comparable accuracy at a fraction of the computational cost of competing algorithms. Additionally, it can incorporate prior knowledge of potential gene regulation mechanisms and TF hetero-dimerization processes in the GRN reconstruction process. We incorporated existing ChIP-seq data and known protein interactions between TFs in BGRMI as sources of prior knowledge to reconstruct transcription regulatory networks of proliferating and differentiating breast cancer (BC) cells from time-course gene expression data. The reconstructed networks revealed key driver genes of proliferation and differentiation in BC cells. Some of these genes were not previously studied in the context of BC, but may have clinical relevance in BC treatment.

Integrating network reconstruction with mechanistic modeling to predict cancer therapies.

Signal transduction networks are often rewired in cancer cells. Identifying these alterations will enable more effective cancer treatment. We developed a computational framework that can identify, reconstruct, and mechanistically model these rewired networks from noisy and incomplete perturbation response data and then predict potential targets for intervention. As a proof of principle, we analyzed a perturbation data set targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) pathways in a panel of colorectal cancer cells. Our computational approach predicted cell line-specific network rewiring. In particular, feedback inhibition of insulin receptor substrate 1 (IRS1) by the kinase p70S6K was predicted to confer resistance to EGFR inhibition, suggesting that disrupting this feedback may restore sensitivity to EGFR inhibitors in colorectal cancer cells. We experimentally validated this prediction with colorectal cancer cell lines in culture and in a zebrafish (Danio rerio) xenograft model.

A bayesian framework that integrates heterogeneous data for inferring gene regulatory networks.

Reconstruction of gene regulatory networks (GRNs) from experimental data is a fundamental challenge in systems biology. A number of computational approaches have been developed to infer GRNs from mRNA expression profiles. However, expression profiles alone are proving to be insufficient for inferring GRN topologies with reasonable accuracy. Recently, it has been shown that integration of external data sources (such as gene and protein sequence information, gene ontology data, protein-protein interactions) with mRNA expression profiles may increase the reliability of the inference process. Here, I propose a new approach that incorporates transcription factor binding sites (TFBS) and physical protein interactions (PPI) among transcription factors (TFs) in a Bayesian variable selection (BVS) algorithm which can infer GRNs from mRNA expression profiles subjected to genetic perturbations. Using real experimental data, I show that the integration of TFBS and PPI data with mRNA expression profiles leads to significantly more accurate networks than those inferred from expression profiles alone. Additionally, the performance of the proposed algorithm is compared with a series of least absolute shrinkage and selection operator (LASSO) regression-based network inference methods that can also incorporate prior knowledge in the inference framework. The results of this comparison suggest that BVS can outperform LASSO regression-based method in some circumstances.

Primary cilium-associated genes mediate bone marrow stromal cell response to hypoxia.

Currently there is intense interest in using mesenchymal stem cells (MSC) for therapeutic interventions in many diseases and conditions. To accelerate the therapeutic use of stem cells we must understand how they sense their environment. Primary cilia are an extracellular sensory organelle present on most growth arrested cells that transduce information about the cellular environment into cells, triggering signaling cascades that have profound effects on development, cell cycle, proliferation, differentiation and migration. Migrating cells likely encounter differing oxygen tensions, therefore we investigated the effect of oxygen tension on cilia. Using bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) we found that oxygen tension significantly affected the length of cilia in primary BMSCs. Chronic exposure to hypoxia specifically down-regulated genes involved in hedgehog signaling and re-localized the Smo and Gli2 proteins to cilia. Investigating the effects of chemotactic migration on cilia, we observed significantly longer cilia in migrating cells which was again, strongly influenced by oxygen tension. Finally, using computational modeling we identified links between migration and ciliation signaling pathways, characterizing the novel role of HSP90 and PI3K signaling in regulating BMSC cilia length. These findings enhance our current understanding of BMSC adaptions to hypoxia and advance our knowledge of BMSC biology and cilia regulation.

Integrating Bayesian variable selection with Modular Response Analysis to infer biochemical network topology.

BACKGROUND: Recent advancements in genetics and proteomics have led to the acquisition of large quantitative data sets. However, the use of these data to reverse engineer biochemical networks has remained a challenging problem. Many methods have been proposed to infer biochemical network topologies from different types of biological data. Here, we focus on unraveling network topologies from steady state responses of biochemical networks to successive experimental perturbations. RESULTS: We propose a computational algorithm which combines a deterministic network inference method termed Modular Response Analysis (MRA) and a statistical model selection algorithm called Bayesian Variable Selection, to infer functional interactions in cellular signaling pathways and gene regulatory networks. It can be used to identify interactions among individual molecules involved in a biochemical pathway or reveal how different functional modules of a biological network interact with each other to exchange information. In cases where not all network components are known, our method reveals functional interactions which are not direct but correspond to the interaction routes through unknown elements. Using computer simulated perturbation responses of signaling pathways and gene regulatory networks from the DREAM challenge, we demonstrate that the proposed method is robust against noise and scalable to large networks. We also show that our method can infer network topologies using incomplete perturbation datasets. Consequently, we have used this algorithm to explore the ERBB regulated G1/S transition pathway in certain breast cancer cells to understand the molecular mechanisms which cause these cells to become drug resistant. The algorithm successfully inferred many well characterized interactions of this pathway by analyzing experimentally obtained perturbation data. Additionally, it identified some molecular interactions which promote drug resistance in breast cancer cells. CONCLUSIONS: The proposed algorithm provides a robust, scalable and cost effective solution for inferring network topologies from biological data. It can potentially be applied to explore novel pathways which play important roles in life threatening disease like cancer.