Environmental stress affects the activity of metabolic and growth factor signaling networks and induces autophagy markers in MCF7 breast cancer cells

Abstract

Phosphoproteomic techniques are contributing to our understanding of how signaling pathways interact and regulate biological processes. This technology is also being used to characterize how signaling networks are remodeled during disease progression and to identify biomarkers of signaling pathway activity and of responses to cancer therapy. A potential caveat in these studies is that phosphorylation is a very dynamic modification that can substantially change during the course of an experiment or the retrieval and processing of cellular samples. Here, we investigated how exposure of cells to ambient conditions modulates phosphorylation and signaling pathway activity in the MCF7 breast cancer cell line. About 1.5% of 3,500 sites measured showed a significant change in phosphorylation extent upon exposure of cells to ambient conditions for 15 min. The effects of this perturbation in modifying phosphorylation patterns did not involve random changes due to stochastic activation of kinases and phosphatases. Instead, exposure of cells to ambient conditions elicited an environmental stress reaction that involved a coordinated response to a metabolic stress situation, which included: (1) the activation of AMPK; (2) the inhibition of PI3K, AKT, and ERK; (3) an increase in markers of protein synthesis inhibition at the level of translation elongation; and (4) an increase in autophagy markers. We also observed that maintaining cells in ice modified but did not completely abolish this metabolic stress response. In summary, exposure of cells to ambient conditions affects the activity of signaling networks previously implicated in metabolic and growth factor signaling. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000472.

Fig. 1.

Overview of phosphorylation events modulated by exposure of MCF7 cells to ambient conditions. A, MCF7 cells were seeded in 75 cm² flask at 45–55% confluence and kept overnight in the incubator to reach a 65–75% confluence. Then cells were left in the incubator (Incu) or exposed to ambient conditions at room temperature conditions (21 ± 2 °C) for 15 min (RT 15 min) or 2 h (RT 2h). Thin lines indicate periods when the cells were kept in the incubator and thick lines indicate periods when the cells were kept at room temperature. The experiment was performed twice and 2 independent replicates per condition were analyzed in each experiment for a total of four biological replicates. After exposure to ambient stress, cells were harvested and lysed, protein cell extracts were digested with trypsin and peptides were desalted by reverse phase. Phosphopeptides were enriched using TiO₂ and analyzed in an Orbitrap XL. MS and MS/MS data were used for peptide identification using Mascot and MS data were used for quantification using Pescal. B, Frequency of phosphopeptide fold changes showing that the average of log₂-fold differences centers around zero. C, Volcano plot showing the fold difference between cells left in the incubator or exposed to ambient conditions for 15 or 120 min. Fold difference in peptide abundance is represented as the log₂ (a positive value for the log₂ of the fold difference indicates the increased abundance of a phosphorylated peptide at ambient conditions) and p value as -log₁₀ (a significance log₁₀ p value <1.3 corresponds to a linear p value of < 0.05). A ±0.8-fold change (log₂) threshold was selected. Red data points represent phosphopeptides showing p values <0.01 and orange dots phosphopeptides showing p values <0.05 and >0.01. D, Number of phosphopeptides whose phosphorylation was increased or decreased (Log2 fold change >0.8 or <−0.8, respectively) as a function of p value threshold.

Fig. 2.

Phosphorylation of proteins in pathways significantly enriched after exposure of cells to RT conditions. Phosphopeptides regulated by exposure of cells to ambient conditions in proteins linked to pathways enriched by these conditions.

Fig. 3.

Environmental stress inhibits the PI3K-AKT axis and activates GSK3β. MCF7 cells were exposed to ambient conditions at RT or in ice (0 °C) for 15 min or 2 h. Cells were then harvested and processed for phosphoproteomic analysis by MS or by Western blot. A, Phosphopeptides significantly affected by exposure of cells to RT conditions and linked to AKT decrease their abundance (top panel) whereas those linked GSK3β increase (bottom panel). Replicates per condition (N) = 4, F₁₅ = average fold over control (Incu) in log₂ after 15 min exposition to RT, F_2h = average fold-over control (Incu) in log₂ after 2 h exposition to RT, P₁₅ = p value of a t test comparing control (Incu) and 15 min exposition to RT. P_2h = p value of a t test comparing control (Incu) and 2 h exposition to RT. B, Ambient conditions reduced the phosphorylation of AKT and its substrates.

Fig. 4.

Environmental stress modulates the phosphorylation of regulators of protein synthesis. Samples were treated and processed as in Fig. 2. A, Effect of RT on phosphopeptides linked to mTOR and S6RPK. Replicates per condition (N) = 4, F₁₅ = average fold-over control (Incu) in log₂ after 15 min exposition to RT, F_2h = average fold-over control (Incu) in log₂ after 2 h exposition to RT, P₁₅ = p value of a t test comparing control (Incu) and 15 min exposition to RT. P_2h = p value of a t test comparing control (Incu) and 2 h exposition to RT. B, Ambient conditions differentially regulate the phosphorylation of regulators of protein synthesis.

Fig. 5.

Environmental stress inhibits ERK1/2 phosphorylation and activity. Samples were treated and processed as in Fig. 2. A, Phosphopeptides quantified by MS, significantly affected by exposure of cells to RT conditions and linked to ERK1/2. Replicates per condition (N) = 4, F₁₅ = average fold-over control (Incu) in log₂ after 15 min exposition to RT, F_2h = average fold-over control (Incu) in log₂ after 2 h exposition to RT, P₁₅ = p value of a t test comparing control (Incu) and 15 min exposition to RT. P_2h = p value of a t test comparing control (Incu) and 2 h exposition to RT. B, WB analysis of phosphorylated ERK 1/2as a function of environmental stress.

Fig. 6.

Environmental stress regulates the phosphorylation of AMPK and induces its activity. Samples were treated and processed as in Fig. 2. A, Phosphopeptides significantly affected by exposure of cells to RT conditions and linked to AMPK. Replicates per condition (N) = 4, F₁₅ = average fold-over control (Incu) in log₂ after 15 min exposition to RT, F_2h = average fold-over control (Incu) in log₂ after 2 h exposition to RT, P₁₅ = p value of a t test comparing control (Incu) and 15 min exposition to RT. P_2h = p value of a t test comparing control (Incu) and 2 h exposition to RT. B, RT and ice regulate the phosphorylation of AMPK and induce the phosphorylation of its substrates.

Fig. 7.

Environmental stress triggers an autophagic response. Samples were treated as in Fig. 2 and analyzed for autophagy markers by immunofluorescence. A, Representative images showing that WIPI2 (in red) and LC3 (in green) puncta increase after exposure of cells to room RT conditions whereas exposure to ice only induce LC3 puncta. B, Quantification of WIPI2 and LC3 puncta in images from cells exposed to RT or ice conditions. Charts show puncta number and puncta area measured by Metamorph software (detailed in Materials and Methods). Data represent average ± S.E. of seven different fields per condition in one representative experiment. The experiment was performed twice with similar results.

Fig. 8.

Environmental stress regulates the phosphorylation of proteins with known roles in the process of autophagy. Heatmap showing phosphopeptides significantly affected by exposure of cells to ambient conditions and linked to autophagy. Replicates per condition (N) = 4, F₁₅ = average fold-over control (Incu) in log₂ after 15 min exposition to RT, F_2h = average fold over control (Incu) in log₂ after 2 h exposition to RT, P₁₅ = p value of a t test comparing control (Incu) and 15 min exposition to RT. P_2h = p value of a t test comparing control (Incu) and 2 h exposition to RT.