Modeling of TGFb pathway dynamics in lung cancer cells

Rationale: Transforming growth factor-β (TGFβ)-induced effects are different, even opposite, depending on the cellular type and conditions. TGFβ signaling has been widely acknowledged to contribute to cancerogenesis via enhancing tumor cell metastasis. Numerous attempts to target TGFβ pathway to reduce cancer spread proved to be ineffective.

Aim: In the current work we aim to use systems biology approach in order to obtain better understanding of information processing in TGFβ/Smad signaling pathway. Identifying emerging systems-level properties that arise from network components would help discovering novel therapeutic strategies in targeting TGFβ pathway.

Methods: Quantitative immunoblot and mass-spectrometry were used to acquire time- and dose-resolved measurements of TGFβ/Smad signaling dynamics in H1975, H838 and H1650 non-small-cell lung adenocarcinoma (NSCLC) cell lines. We also examined expression of multiple downstream EMT-related genes by qRT-PCR. Ordinary differential equation (ODE)-based mathematical model was used to combine Smad signaling and downstream gene expression data.

Results: We show that in all three tested cell lines Smad2/3 phosphorylation peaks at about 1 hour after stimulation, then gradually declines and remains steadily elevated until all ligand is consumed. The dynamics of Snail1, a direct TGFβ-inducible gene, directly mirrors the kinetics of activated Smad2/3, while other EMT-related genes demonstrate delayed activation and more complex dynamics, which cannot be linearly explained by phospho-Smad2/3 dynamics. On the basis of dynamic measurements of the pathway we established mathematical model, which is able to integrate both protein and gene expression data. Additionally, we demonstrate that TGFβ treatment of lung cancer cells greatly enhance their migratory and invasive potential as well as results in pronounced phenotypic changes.

Conclusion: Dynamics of TGFb downstream target genes cannot be linearly predicted from the Smad2/3 activation profile. Presence of multiple regulatory nodes in the core TGFβ/Smad pathway structure might be potentially used for designing novel intervention strategies.

*Presenting author