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Updated: May 16, 2026
Live-Cell Förster Resonance Energy Transfer Imaging of Metabolically Regulated Akt Activation Dynamics in HepG2 Cells
Published on: May 23, 2025
Heterogeneous kinetics of AKT signaling in individual cells are accounted for by variable protein concentration
René Meyer1, Lorenza A D'Alessandro, Sandip Kar
1Division of Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance Heidelberg, Germany.
Abstract:
In most solid cancers, cells harboring oncogenic mutations represent only a sub-fraction of the entire population. Within this sub-fraction the expression level of mutated proteins can vary significantly due to cellular variability limiting the efficiency of targeted therapy. To address the causes of the heterogeneity, we performed a systematic analysis of one of the most frequently mutated pathways in cancer cells, the phosphatidylinositol 3 kinase (PI3K) signaling pathway. Among others PI3K signaling is activated by the hepatocyte growth factor (HGF) that regulates proliferation of hepatocytes during liver regeneration but also fosters tumor cell proliferation. HGF-mediated responses of PI3K signaling were monitored both at the single cell and cell population level in primary mouse hepatocytes and in the hepatoma cell line Hepa1_6. Interestingly, we observed that the HGF-mediated AKT responses at the level of individual cells is rather heterogeneous. However, the overall average behavior of the single cells strongly resembled the dynamics of AKT activation determined at the cell population level. To gain insights into the molecular cause for the observed heterogeneous behavior of individual cells, we employed dynamic mathematical modeling in a stochastic framework. Our analysis demonstrated that intrinsic noise was not sufficient to explain the observed kinetic behavior, but rather the importance of extrinsic noise has to be considered. Thus, distinct from gene expression in the examined signaling pathway fluctuations of the reaction rates has only a minor impact whereas variability in the concentration of the various signaling components even in a clonal cell population is a key determinant for the kinetic behavior.
Insights
Targeted cancer therapy is limited by heterogeneous protein expression. Extrinsic noise, not intrinsic noise, drives this variability in the phosphatidylinositol 3-kinase (PI3K) pathway, primarily due to fluctuating signaling component concentrations.
Area of Science:
- Molecular Biology
- Systems Biology
- Cancer Research
Background:
- Oncogenic mutations in solid cancers often affect only a subset of cells, leading to heterogeneous protein expression.
- This cellular variability can significantly limit the efficacy of targeted cancer therapies.
- The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently mutated in cancer and plays a role in cell proliferation.
Purpose of the Study:
- To investigate the causes of heterogeneity in the HGF-mediated PI3K signaling pathway.
- To understand the molecular basis for variable AKT activation at the single-cell level.
- To determine the relative contributions of intrinsic and extrinsic noise to signaling dynamics.
Main Methods:
- Systematic analysis of PI3K signaling in primary mouse hepatocytes and Hepa1_6 hepatoma cells.
- Monitoring HGF-mediated PI3K signaling at both single-cell and population levels.
- Employing dynamic mathematical modeling within a stochastic framework.
Main Results:
- HGF-mediated AKT responses exhibited significant heterogeneity at the single-cell level.
- Average single-cell behavior mirrored population-level AKT activation dynamics.
- Intrinsic noise alone could not explain the observed kinetic behavior; extrinsic noise was crucial.
- Variability in signaling component concentrations, rather than reaction rate fluctuations, was a key determinant of kinetic behavior.
Conclusions:
- Extrinsic noise, stemming from variations in signaling component concentrations, is a major driver of heterogeneity in the PI3K pathway.
- Understanding noise sources is critical for improving targeted therapy efficacy in cancers with heterogeneous signaling.
- This study highlights the importance of considering molecular noise in signaling pathway dynamics for cancer treatment strategies.
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