Modeling and Analysis of the Eukaryotic Hypoxic Response and Angiogenesis

Eukaryotes initiate a complex program in response to low levels of extracellular oxygen. The hypoxic response, which is mediated in part by the action of the HIF1α transcription factor and reactive oxygen species (ROS), involves the integration of several intracellular signaling axes. While the hypoxic response is vital to normal physiology and development, it is also a prevalent feature of angiogenesis. In this study, we developed a family of mechanistic mathematical models of the signaling pathways involved in the eukaryotic hypoxic response. We cataloged the molecular modules mediating different aspects and branches of the hypoxic response and integrated these modules into a single comprehensive model. The integrated hypoxia model (548 molecular species interconnected by 920 interactions), was analyzed to determine critical points of network failure and possible sources of crosstalk and redundancy. Model parameters were estimated by comparing simulations with experimental data. We identified a population of models, consistent with data, using a novel multi-objective optimization framework combined with cross-validation. EHR model analysis revealed a two-phased response within EHR. Initial activity of NF-κβ and AP-1 mediated by VEGF and IL-8 signaling cascades lead to autocrine mediated signal amplification (∼24-48 hrs) in both VEGF (less significant) and IL-8 (more significant) signals. This phased behavior was validated with experimental studies on MDA-MB231 cells. Structural analysis using extreme pathways identified modes of crosstalk within VEGF and IL-8 signaling e.g., via p38MAPK, MAPK and PKC, and modes of redundancy e.g., divergent sources of transcription factor (NF-κβ, AP-1) activation. On the other hand, sensitivity analysis suggested that regulators of HIF-1α stability e.g., PHD and FIH along with NF-κβ and AP-1 activation were critical components of the hypoxia program. Computational gene knockdown(overexpression) studies identified network configurations that either amplified or destroyed the hypoxic response and modulated the levels of phenotypic markers. Ultimately, analysis of the model population suggested that interdiction of both NF-κβ and AP-1 regulatory blocks within EHR was required to disrupt the hypoxic program. These configurations represent experimentally testable hypothesis and potentially new strategies to manipulate the hypoxia program.

Figure: The Eukaryotic Hypoxic Response: Low cellular oxygen marks the increased stability of HIF1α and ROS production. Via the role of transcription factors like AP1, NF-κβ and HIF1, VEGF and IL8 sig- naling cascades lead to the critical balance mediating tumor (regular cell) growth and angiogenic signaling. Nomenclature: HIF1 - Hypoxia Inducible Factor 1, VEGFa - Vascular Endothelial Growth Factor A, VEGFR2 - Vascular Endothelial Growth Factor Receptor 2, IL8 - Interleukin -8, CXCR1 - IL-8 (or CXCL8) chemokine receptors, PKC - Protein Kinase C, PI3K - Phosphoinositide 3-kinase, p38MAPK - p38 Mitogen- Activated Protein Kinases, p50 p65 - Nuclear Factor κ -light-chain-enhancer of activated B cells. Species (proteins and protein-protein complexes) and corresponding interactions making up the Eukaryotic Hypoxic Response (EHR) model (Top Left).