(239g) Phospho-Proteomics Reveals a Metabolic and Signaling Amplification Loop Leading to Cell Death Following Glucose Deprivation

Introduction: Tumor cells exhibit an altered metabolism compared to non-transformed cells, consuming glucose and glutamine and producing lactate at prodigious rates. One consequence of deregulated tumor metabolism is that cells can become dependent on the supply of metabolic substrates such as glucose or glutamine for viability. Based on evidence that oncogenic signaling can impose this metabolic inflexibility (Choo et al, 2010), as well as the concept that complex networks link metabolism and signal transduction (Wellen et al, 2010), we conducted a systems level investigation into the role of signal transduction underlying cell death following metabolic perturbation in cell systems dependent on glucose for survival.

Materials and Methods: Phospho-tyrosine signaling and viability of U87 glioblastoma cells were assessed by Western blotting and trypan blue exclusion, respectively. Quantitative, label-free mass spectrometry was performed as previously described (Rubbi et al, 2011) on peptides immunoprecipitated using a pan-specific anti-phosphotyrosine antibody (clone 4G10, Millipore). Reactive oxygen species were measured by flow cytometry using DCF-DA. Protein tyrosine phosphatase activity was measured using a substrate dephosphorylation assay.

Results and Discussion: In cells dependent upon glucose for survival, glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling. Increased phospho-tyrosine signaling was not observed in glucose withdrawal-resistant cells. Using unbiased mass spectrometry-based phospho-proteomics, we found that glucose withdrawal initiates a unique signature of phospho-tyrosine activation compared to other stimuli (eg, growth factor stimulation, the phosphatase inhibitor vanadate). Building upon the observation, we demonstrate that glucose withdrawal activates a positive feedback loop whereby reactive oxygen species (ROS) oxidize and inhibit protein tyrosine phosphatases (PTPs), leading to increased tyrosine kinase signaling, which induces further ROS generation. Following glucose deprivation, ROS generation and tyrosine kinase signaling synergize to amplify ROS levels until cells undergo ROS-mediated cell death.

Conclusions: We demonstrate that a metabolic perturbation (ie, glucose starvation) provokes a signaling-based positive feedback loop that amplifies ROS levels until cells surpass a toxicity threshold. This positive feedback loop demonstrates the complex, systems level integration of homeostatic control mechanisms for metabolism (eg, redox balance) and tyrosine kinase signaling (eg, PTPs). Furthermore, this systems integration offers a scaffold for synergistic combinations of therapeutics targeting signaling and metabolism.

References: Choo et al, Mol Cell (2010); 38: 487-499.

Rubbi et al, Sci Signal (2011); 4: ra18.

Wellen et al, Genes Dev (2010); 24: 2784-2799.