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Decreased glutathione biosynthesis contributes to EGFR T790M-driven erlotinib resistance in non-small cell lung cancer

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ABSTRACT

Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib are novel effective agents in the treatment of EGFR-driven lung cancer, but their clinical impact is often impaired by acquired drug resistance through the secondary T790M EGFR mutation. To overcome this problem, we analysed the metabonomic differences between two independent pairs of erlotinib-sensitive/resistant cells and discovered that glutathione (GSH) levels were significantly reduced in T790M EGFR cells. We also found that increasing GSH levels in erlotinib-resistant cells re-sensitised them, whereas reducing GSH levels in erlotinib-sensitive cells made them resistant. Decreased transcription of the GSH-synthesising enzymes (GCLC and GSS) due to the inhibition of NRF2 was responsible for low GSH levels in resistant cells that was directly linked to the T790M mutation. T790M EGFR clinical samples also showed decreased expression of these key enzymes; increasing intra-tumoural GSH levels with a small-molecule GST inhibitor re-sensitised resistant tumours to erlotinib in mice. Thus, we identified a new resistance pathway controlled by EGFR T790M and a therapeutic strategy to tackle this problem in the clinic.

No MeSH data available.


Expression of EGFRm/T790M decreases intracellular GSH levels and NRF2 activity. (a–d) HEK293 cells were transfected with empty vector control (EV), activated L858R-EGFR or activated/resistant L858R/T790M EGFR-mutant constructs. (a) Quantitative reverse transcription PCR for EGFR, (b) colorimetric assay for GSH levels and (c) cell fractionation followed by SDS-PAGE/western blotting for the indicated proteins were done on stable cell lines. Detection of lamin and tubulin was used as loading controls for nuclear and cytoplasmic fractions, respectively. (d–g) PC9ER cells transfected with an EGFR T790M-specific or NT siRNAs were subjected to (d) treatment with erlotinib before crystal violet staining, (e) colorimetric assay for intracellular GSH levels, (f) quantitative PCR for GSH metabolic enzymes or (g) SDS-PAGE/western blotting. All data representative of ≥3 experiments. (a,b,d–f) Values are average of n=4±s.e.m. Statistics: (a,b) analysis of variance, (d,e,f) Student’s t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S6 and S7.
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fig4: Expression of EGFRm/T790M decreases intracellular GSH levels and NRF2 activity. (a–d) HEK293 cells were transfected with empty vector control (EV), activated L858R-EGFR or activated/resistant L858R/T790M EGFR-mutant constructs. (a) Quantitative reverse transcription PCR for EGFR, (b) colorimetric assay for GSH levels and (c) cell fractionation followed by SDS-PAGE/western blotting for the indicated proteins were done on stable cell lines. Detection of lamin and tubulin was used as loading controls for nuclear and cytoplasmic fractions, respectively. (d–g) PC9ER cells transfected with an EGFR T790M-specific or NT siRNAs were subjected to (d) treatment with erlotinib before crystal violet staining, (e) colorimetric assay for intracellular GSH levels, (f) quantitative PCR for GSH metabolic enzymes or (g) SDS-PAGE/western blotting. All data representative of ≥3 experiments. (a,b,d–f) Values are average of n=4±s.e.m. Statistics: (a,b) analysis of variance, (d,e,f) Student’s t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S6 and S7.

Mentions: Although lower GSH levels and NRF2 activity were associated with T790M-driven erlotinib resistance in our cell lines, this may still be incidental unless the T790M mutation directly induces these changes. We further expressed the active (L858R) or active/resistant (L858R/T790M) EGFR mutants in HEK293 cells that contain low endogenous EGFR levels (Figure 4a). Unlike expression of the L858R-EGFR, expression of the L858R/T790M double-mutant receptor reduced intracellular GSH levels (Figure 4b). This was associated with reduced PALB2 and SQSTM1 expression (Figure 4c). Conversely, transfection with two independent siRNA sequences previously shown to selectively target T790M-mutant EGFR [33]-sensitised PC9ER cells to erlotinib (Figure 4d; Supplementary Figure S7C and D) and increased GSH levels (Figure 4e). The latter correlated with a reversal of changes in the expression pattern of GSH metabolic enzymes observed between PC9 and PC9ER cells (Figure 4f vs Figure 2b) and with increased PALB2, SQSTM1 and NRF2 levels in T790M-silenced cells (Figure 4g). Therefore, lower GSH levels in T790M NSCLC cells are a direct consequence of acquiring this mutation and the accompanying impairment of NRF2 activity.


Decreased glutathione biosynthesis contributes to EGFR T790M-driven erlotinib resistance in non-small cell lung cancer
Expression of EGFRm/T790M decreases intracellular GSH levels and NRF2 activity. (a–d) HEK293 cells were transfected with empty vector control (EV), activated L858R-EGFR or activated/resistant L858R/T790M EGFR-mutant constructs. (a) Quantitative reverse transcription PCR for EGFR, (b) colorimetric assay for GSH levels and (c) cell fractionation followed by SDS-PAGE/western blotting for the indicated proteins were done on stable cell lines. Detection of lamin and tubulin was used as loading controls for nuclear and cytoplasmic fractions, respectively. (d–g) PC9ER cells transfected with an EGFR T790M-specific or NT siRNAs were subjected to (d) treatment with erlotinib before crystal violet staining, (e) colorimetric assay for intracellular GSH levels, (f) quantitative PCR for GSH metabolic enzymes or (g) SDS-PAGE/western blotting. All data representative of ≥3 experiments. (a,b,d–f) Values are average of n=4±s.e.m. Statistics: (a,b) analysis of variance, (d,e,f) Student’s t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S6 and S7.
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fig4: Expression of EGFRm/T790M decreases intracellular GSH levels and NRF2 activity. (a–d) HEK293 cells were transfected with empty vector control (EV), activated L858R-EGFR or activated/resistant L858R/T790M EGFR-mutant constructs. (a) Quantitative reverse transcription PCR for EGFR, (b) colorimetric assay for GSH levels and (c) cell fractionation followed by SDS-PAGE/western blotting for the indicated proteins were done on stable cell lines. Detection of lamin and tubulin was used as loading controls for nuclear and cytoplasmic fractions, respectively. (d–g) PC9ER cells transfected with an EGFR T790M-specific or NT siRNAs were subjected to (d) treatment with erlotinib before crystal violet staining, (e) colorimetric assay for intracellular GSH levels, (f) quantitative PCR for GSH metabolic enzymes or (g) SDS-PAGE/western blotting. All data representative of ≥3 experiments. (a,b,d–f) Values are average of n=4±s.e.m. Statistics: (a,b) analysis of variance, (d,e,f) Student’s t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S6 and S7.
Mentions: Although lower GSH levels and NRF2 activity were associated with T790M-driven erlotinib resistance in our cell lines, this may still be incidental unless the T790M mutation directly induces these changes. We further expressed the active (L858R) or active/resistant (L858R/T790M) EGFR mutants in HEK293 cells that contain low endogenous EGFR levels (Figure 4a). Unlike expression of the L858R-EGFR, expression of the L858R/T790M double-mutant receptor reduced intracellular GSH levels (Figure 4b). This was associated with reduced PALB2 and SQSTM1 expression (Figure 4c). Conversely, transfection with two independent siRNA sequences previously shown to selectively target T790M-mutant EGFR [33]-sensitised PC9ER cells to erlotinib (Figure 4d; Supplementary Figure S7C and D) and increased GSH levels (Figure 4e). The latter correlated with a reversal of changes in the expression pattern of GSH metabolic enzymes observed between PC9 and PC9ER cells (Figure 4f vs Figure 2b) and with increased PALB2, SQSTM1 and NRF2 levels in T790M-silenced cells (Figure 4g). Therefore, lower GSH levels in T790M NSCLC cells are a direct consequence of acquiring this mutation and the accompanying impairment of NRF2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib are novel effective agents in the treatment of EGFR-driven lung cancer, but their clinical impact is often impaired by acquired drug resistance through the secondary T790M EGFR mutation. To overcome this problem, we analysed the metabonomic differences between two independent pairs of erlotinib-sensitive/resistant cells and discovered that glutathione (GSH) levels were significantly reduced in T790M EGFR cells. We also found that increasing GSH levels in erlotinib-resistant cells re-sensitised them, whereas reducing GSH levels in erlotinib-sensitive cells made them resistant. Decreased transcription of the GSH-synthesising enzymes (GCLC and GSS) due to the inhibition of NRF2 was responsible for low GSH levels in resistant cells that was directly linked to the T790M mutation. T790M EGFR clinical samples also showed decreased expression of these key enzymes; increasing intra-tumoural GSH levels with a small-molecule GST inhibitor re-sensitised resistant tumours to erlotinib in mice. Thus, we identified a new resistance pathway controlled by EGFR T790M and a therapeutic strategy to tackle this problem in the clinic.

No MeSH data available.