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Bioinformatics, Computational and Systems Biology

Bioinformatics, Computational and Systems Biology - Poster Session E

Poster A5 - The Interplay of EGF Stimulation and Metabolism on Vacuolization in EGFR-Overexpressing Glioblastoma Cells

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by high heterogeneity, contributing to poor patient prognosis. While several forms of heterogeneity exist, genomic alterations of receptor tyrosine kinases are considered the most abundant and targetable oncogenic drivers. Mutations of the epidermal growth factor receptor (EGFR) are highly prevalent which limits the efficacy of EGFR-targeted therapies. Beyond point mutations, EGFR amplification and the mutant EGFRvIII which is characterized by the truncation of the extracellular ligand binding domain are among the most common alterations in GBM. Interestingly, EGFRvIII frequently co-occurs with EGFR amplification at the tumor-level, but not necessarily at a cell-level suggesting cooperative mechanisms that promote tumor progression. EGFRvIII exhibits a lower, constitutive kinase activity that decreases extracellular signal-regulated kinase 1 and 2 (Erk1/2) and increases AKT signaling relative to EGFR wildtype cells. While EGFRvIII-mediated phenotypic changes have been well studied, whether these changes can help provide EGFR amplified cells survival advantages remain underexplored.
EGFR amplification is detected in 40-50% of GBM cases, and results in EGFR overexpression. EGFR amplification can promote excessive vacuolization through apparent macropinocytosis in GBM cells, and if remains unresolved progresses to methuosis, a form of non-apoptotic cell death. Macropinocytosis can occur through either nutrient deprivation or receptor tyrosine kinase mediated Ras activation (Figure 1A). Although these two pathways are known to independently contribute to macropinosome formation, the potential synergistic effect has remained understudied. Here we investigated how changes in nutrient availability and EGF signaling interact and contribute to vacuolization.

Materials and

Methods:  To study EGFR associated signaling, human U251 GBM cells were transduced with a doxycycline-inducible EGFR system. High expressing EGFR cells were isolated from the top 15% EGFR expressing cells using fluorescence assisted cell sorting (FACS). Parental U251 cells acted as negative controls as indicated. All cells were maintained in DMEM +10% FBS +1% penicillin/streptomycin with EGFR sorted cells cultured in media containing 200 ng/mL zeomycin.
For vacuole visualization, cells were seeded at a density of 2500 cells/cm2 and allowed to attach overnight. EGFR expression was induced with 500 ng/mL doxycycline after initial seeding. Serum-deprivation was conducted for 24h simultaneously with doxycycline-induction in FBS-absent media. EGF stimulation (100 ng/ml) and 2-DG mediated glycolytic inhibition (25 mM) were induced 24h after serum-starvation as indicated. Further for glucose deprivation studies, cells were cultured in DMEM with no glucose for 24h after a 24h serum deprivation.
Vacuolization was assessed through brightfield microscopy. Images were taken at 4h and 24h timepoints after EGF and 2-DG treatments and compared on a ThermoFisher EVOS M5000 microscope using a 20x objective. Image analysis was conducted through the manual categorization of cells using the Cell Counter plugin within ImageJ. The presence of EGFR and changes in downstream signaling effectors was confirmed via western blot analysis

Results, Conclusions, and Discussions: Prior studies have demonstrated that EGFR stimulation can promote excessive vacuolization in EGFR overexpressing cells. Additionally, glycolysis inhibition with 2-DG treatment saw vacuoles present irrespective of EGF stimulation (Figure 1B). However, metabolic inhibition studies were conducted in serum-containing conditions that can provide additional cytokine-mediated signaling effects.
To isolate the glycolytic and EGF stimulated effects, we cultured parental and unsorted EGFR-overexpressing U251 cells in serum-deprived conditions for 48h. Cells showed no changes in the fraction of vacuolized cells but displayed an elongated morphology after 24h. This suggested that the vacuolization phenotype was not dependent on serum presence.
Repeating the glycolytic inhibition study in serum-free conditions, we did not observe changes in vacuolization contrary to prior experiments with serum. The lack of vacuole formation in serum-free conditions was also observed when cells were cultured in glucose-free media. Together, these results suggest that growth factors found within FBS may impact vacuole formation associated with metabolic stress.
Given these results we next treated serum-deprived parental and unsorted EGFR overexpressing cells with 2-DG and EGF concurrently to determine if EGF found within serum promotes vacuolization. Increased vacuolization was only observed in 2-DG treated cells with serum, while EGF stimulation and glycolysis inhibition in serum-deprived conditions did not induce vacuolization. However, given the variability in EGFR expression in our unsorted EGFR-overexpressing cells, we sorted the top 15% of overexpressing EGFR cells to ensure robust EGF-induced signaling. Parental control cells showed limited vacuolization at both 4h and 24h timepoints during EGF and EGF with 2-DG treatment. For sorted EGFR cells both EGF stimulated cells and EGF and 2-DG treated cells demonstrated similar vacuolization at the 4h timepoint. However, after 24h the EGF stimulated cells had decreased vacuolization, while the combined EGF and 2-DG treated cells demonstrated prolonged vacuolization (Figure 1C). Western Blot analysis verified both EGFR expression and changes in signaling pathways. Together, our results suggest crosstalk between 2-DG induced glycolytic inhibition and EGF stimulated EGFR activation in vacuole formation in highly expressing EGFR cells. Further, our results suggest that serum-associated effects ought to be considered when studying the interplay between metabolism and growth factor associated signaling.

Acknowledgements (Optional):  This work was funded by NCI U54 CA274499.