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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.


Intracellular GSH levels modulate response to erlotinib. (a) Schematics of the GSH metabolic pathway. White boxes, synthesising enzymes; and grey boxes, catabolic enzymes. (b) Quantitative reverse transcription PCR for GSH pathway enzymes in PC9, PC9ER, H3255 and H1975 cells. Data are relative mRNAs levels in PC9ER (upper panel) and H1975 (lower panel) normalised to those in PC9 and H3255 cells, respectively. (c) PC9 and PC9ER cells were transfected with siRNA targeting GSH-catabolic (grey bars) and synthesising (white bars) enzymes or a non-targeting control (NT) and cell survival to erlotinib (50 nm) monitored by crystal violet staining. Data for the relative survival to erlotinib are normalised to non-targeting control. Survival to erlotinib of PC9ER (e) and H1975 (f) cells treated with ethacrynic acid (EA) or PC9 (g) and H3255 (h) cells treated with buthionine sulphoximine (BSO) was monitored by crystal violet staining. Accompanying changes in GSH levels in PC9ER (d) and PC9 (i) cells were assessed by colorimetric assay. (e–h) Data are the relative responsiveness to erlotinib normalised to vehicle (−; DMSO). (b–i) Data representative of ⩾3 experiments and are average of n=3±s.e.m. Statistics: (e–h) analysis of variance, (b–d, i) Student’s t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S3 and S4.
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fig2: Intracellular GSH levels modulate response to erlotinib. (a) Schematics of the GSH metabolic pathway. White boxes, synthesising enzymes; and grey boxes, catabolic enzymes. (b) Quantitative reverse transcription PCR for GSH pathway enzymes in PC9, PC9ER, H3255 and H1975 cells. Data are relative mRNAs levels in PC9ER (upper panel) and H1975 (lower panel) normalised to those in PC9 and H3255 cells, respectively. (c) PC9 and PC9ER cells were transfected with siRNA targeting GSH-catabolic (grey bars) and synthesising (white bars) enzymes or a non-targeting control (NT) and cell survival to erlotinib (50 nm) monitored by crystal violet staining. Data for the relative survival to erlotinib are normalised to non-targeting control. Survival to erlotinib of PC9ER (e) and H1975 (f) cells treated with ethacrynic acid (EA) or PC9 (g) and H3255 (h) cells treated with buthionine sulphoximine (BSO) was monitored by crystal violet staining. Accompanying changes in GSH levels in PC9ER (d) and PC9 (i) cells were assessed by colorimetric assay. (e–h) Data are the relative responsiveness to erlotinib normalised to vehicle (−; DMSO). (b–i) Data representative of ⩾3 experiments and are average of n=3±s.e.m. Statistics: (e–h) analysis of variance, (b–d, i) Student’s t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S3 and S4.

Mentions: We investigated whether erlotinib-resistant cells differed from their sensitive counterparts in their GSH-metabolic enzymes expression pattern. Quantitative PCR analysis revealed lower messenger RNA (mRNA) levels for GSH-synthesising enzymes (GCLC, GSS and GSR) in erlotinib-resistant cells compared with sensitive ones (Figure 2a and b). In addition, mRNA levels for GCLM, the modulatory subunit of GCLC, were significantly lower in H1975 than in H3255 cells. In contrast, changes in the levels for GSH-catabolic enzymes (GPX1/2/3, GGT and GSTpi/m1/zi) varied greatly between cell line pairs and enzyme subtypes indicating no clear pattern (Figure 2b). Therefore, a reduction in GSH biosynthesis becomes a sound explanation for the decreased GSH levels in EGFRm/T790M erlotinib-resistant cells.


Decreased glutathione biosynthesis contributes to EGFR T790M-driven erlotinib resistance in non-small cell lung cancer
Intracellular GSH levels modulate response to erlotinib. (a) Schematics of the GSH metabolic pathway. White boxes, synthesising enzymes; and grey boxes, catabolic enzymes. (b) Quantitative reverse transcription PCR for GSH pathway enzymes in PC9, PC9ER, H3255 and H1975 cells. Data are relative mRNAs levels in PC9ER (upper panel) and H1975 (lower panel) normalised to those in PC9 and H3255 cells, respectively. (c) PC9 and PC9ER cells were transfected with siRNA targeting GSH-catabolic (grey bars) and synthesising (white bars) enzymes or a non-targeting control (NT) and cell survival to erlotinib (50 nm) monitored by crystal violet staining. Data for the relative survival to erlotinib are normalised to non-targeting control. Survival to erlotinib of PC9ER (e) and H1975 (f) cells treated with ethacrynic acid (EA) or PC9 (g) and H3255 (h) cells treated with buthionine sulphoximine (BSO) was monitored by crystal violet staining. Accompanying changes in GSH levels in PC9ER (d) and PC9 (i) cells were assessed by colorimetric assay. (e–h) Data are the relative responsiveness to erlotinib normalised to vehicle (−; DMSO). (b–i) Data representative of ⩾3 experiments and are average of n=3±s.e.m. Statistics: (e–h) analysis of variance, (b–d, i) Student’s t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S3 and S4.
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fig2: Intracellular GSH levels modulate response to erlotinib. (a) Schematics of the GSH metabolic pathway. White boxes, synthesising enzymes; and grey boxes, catabolic enzymes. (b) Quantitative reverse transcription PCR for GSH pathway enzymes in PC9, PC9ER, H3255 and H1975 cells. Data are relative mRNAs levels in PC9ER (upper panel) and H1975 (lower panel) normalised to those in PC9 and H3255 cells, respectively. (c) PC9 and PC9ER cells were transfected with siRNA targeting GSH-catabolic (grey bars) and synthesising (white bars) enzymes or a non-targeting control (NT) and cell survival to erlotinib (50 nm) monitored by crystal violet staining. Data for the relative survival to erlotinib are normalised to non-targeting control. Survival to erlotinib of PC9ER (e) and H1975 (f) cells treated with ethacrynic acid (EA) or PC9 (g) and H3255 (h) cells treated with buthionine sulphoximine (BSO) was monitored by crystal violet staining. Accompanying changes in GSH levels in PC9ER (d) and PC9 (i) cells were assessed by colorimetric assay. (e–h) Data are the relative responsiveness to erlotinib normalised to vehicle (−; DMSO). (b–i) Data representative of ⩾3 experiments and are average of n=3±s.e.m. Statistics: (e–h) analysis of variance, (b–d, i) Student’s t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S3 and S4.
Mentions: We investigated whether erlotinib-resistant cells differed from their sensitive counterparts in their GSH-metabolic enzymes expression pattern. Quantitative PCR analysis revealed lower messenger RNA (mRNA) levels for GSH-synthesising enzymes (GCLC, GSS and GSR) in erlotinib-resistant cells compared with sensitive ones (Figure 2a and b). In addition, mRNA levels for GCLM, the modulatory subunit of GCLC, were significantly lower in H1975 than in H3255 cells. In contrast, changes in the levels for GSH-catabolic enzymes (GPX1/2/3, GGT and GSTpi/m1/zi) varied greatly between cell line pairs and enzyme subtypes indicating no clear pattern (Figure 2b). Therefore, a reduction in GSH biosynthesis becomes a sound explanation for the decreased GSH levels in EGFRm/T790M erlotinib-resistant cells.

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.