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

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.


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Systemic EA administration re-sensitises PC9ER mouse xenografts to erlotinib. Nude mice (n=10/condition) were injected subcutaneously with PC9ER cells and treatment started when tumours reached 100 mm3. (a) Tumour volume and (b) animals survival were monitored for 27 days. (a) Data are average±s.e.m. (b) End-point events occur when tumour volumes ⩾300 mm3. Log-rank test, Pab<0.01, Pbc<0.01. (c) Following the last treatment, intratumoral GSH levels were measured ex vivo by colorimetric assay. Statistics: (a) analysis of variance, (c) Student’s t-test, *P<0.05; **P<0.01. GSH-synthesising enzymes expression is decreased in EGFRm/T790M patient tumours. mRNA levels for the indicated enzymes were compared by quantitative PCR in two patients before (pre-T790M) and after (post-T790M) the onset of T790M-mediated erlotinib resistance (d) or by RNA-Seq in four pairs of unrelated patients with (Pt1-4) or without (Pt5-8) T790M (e). Data in T790M samples are normalised to those in the corresponding non-T790M samples. (f) Model of changes occurring downstream of T790M EGFR.
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fig6: Systemic EA administration re-sensitises PC9ER mouse xenografts to erlotinib. Nude mice (n=10/condition) were injected subcutaneously with PC9ER cells and treatment started when tumours reached 100 mm3. (a) Tumour volume and (b) animals survival were monitored for 27 days. (a) Data are average±s.e.m. (b) End-point events occur when tumour volumes ⩾300 mm3. Log-rank test, Pab<0.01, Pbc<0.01. (c) Following the last treatment, intratumoral GSH levels were measured ex vivo by colorimetric assay. Statistics: (a) analysis of variance, (c) Student’s t-test, *P<0.05; **P<0.01. GSH-synthesising enzymes expression is decreased in EGFRm/T790M patient tumours. mRNA levels for the indicated enzymes were compared by quantitative PCR in two patients before (pre-T790M) and after (post-T790M) the onset of T790M-mediated erlotinib resistance (d) or by RNA-Seq in four pairs of unrelated patients with (Pt1-4) or without (Pt5-8) T790M (e). Data in T790M samples are normalised to those in the corresponding non-T790M samples. (f) Model of changes occurring downstream of T790M EGFR.

Mentions: The GST inhibitor EA restored GSH levels and erlotinib sensitivity in EGFRm/T790M cells in vitro (Figure 2). EA is still used as a diuretic in humans for conditions including high blood pressure and heart failure [34]. Hence, we hypothesised that co-administration of physiologically relevant doses of EA might improve the responsiveness of EGFRm/T790M tumours to erlotinib in vivo. PC9 or PC9ER cells were injected subcutaneously in nude mice and tumours were left to grow to 100 mm3. The animals were then treated daily with erlotinib and EA alone or in combination. Co-administration of the drugs greatly inhibited tumour growth with 60% of the animals showing tumour volumes ⩽300 mm3 at 25 days, whereas those treated with either drug alone showed more extensive disease (Figure 6a). This was associated with increased survival (Figure 6b) and intra-tumoural GSH levels in combination-treated animals (Figure 6c). EA did not have any effect on erlotinib sensitivity of PC9 xenografts in agreement with the lack of further added sensitisation to erlotinib obtained with this inhibitor in vitro (Supplementary Figure S7E). Thus, co-administration of EA is probably a viable strategy for the management of erlotinib-resistant cancers in humans.


Decreased glutathione biosynthesis contributes to EGFR T790M-driven erlotinib resistance in non-small cell lung cancer
Systemic EA administration re-sensitises PC9ER mouse xenografts to erlotinib. Nude mice (n=10/condition) were injected subcutaneously with PC9ER cells and treatment started when tumours reached 100 mm3. (a) Tumour volume and (b) animals survival were monitored for 27 days. (a) Data are average±s.e.m. (b) End-point events occur when tumour volumes ⩾300 mm3. Log-rank test, Pab<0.01, Pbc<0.01. (c) Following the last treatment, intratumoral GSH levels were measured ex vivo by colorimetric assay. Statistics: (a) analysis of variance, (c) Student’s t-test, *P<0.05; **P<0.01. GSH-synthesising enzymes expression is decreased in EGFRm/T790M patient tumours. mRNA levels for the indicated enzymes were compared by quantitative PCR in two patients before (pre-T790M) and after (post-T790M) the onset of T790M-mediated erlotinib resistance (d) or by RNA-Seq in four pairs of unrelated patients with (Pt1-4) or without (Pt5-8) T790M (e). Data in T790M samples are normalised to those in the corresponding non-T790M samples. (f) Model of changes occurring downstream of T790M EGFR.
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fig6: Systemic EA administration re-sensitises PC9ER mouse xenografts to erlotinib. Nude mice (n=10/condition) were injected subcutaneously with PC9ER cells and treatment started when tumours reached 100 mm3. (a) Tumour volume and (b) animals survival were monitored for 27 days. (a) Data are average±s.e.m. (b) End-point events occur when tumour volumes ⩾300 mm3. Log-rank test, Pab<0.01, Pbc<0.01. (c) Following the last treatment, intratumoral GSH levels were measured ex vivo by colorimetric assay. Statistics: (a) analysis of variance, (c) Student’s t-test, *P<0.05; **P<0.01. GSH-synthesising enzymes expression is decreased in EGFRm/T790M patient tumours. mRNA levels for the indicated enzymes were compared by quantitative PCR in two patients before (pre-T790M) and after (post-T790M) the onset of T790M-mediated erlotinib resistance (d) or by RNA-Seq in four pairs of unrelated patients with (Pt1-4) or without (Pt5-8) T790M (e). Data in T790M samples are normalised to those in the corresponding non-T790M samples. (f) Model of changes occurring downstream of T790M EGFR.
Mentions: The GST inhibitor EA restored GSH levels and erlotinib sensitivity in EGFRm/T790M cells in vitro (Figure 2). EA is still used as a diuretic in humans for conditions including high blood pressure and heart failure [34]. Hence, we hypothesised that co-administration of physiologically relevant doses of EA might improve the responsiveness of EGFRm/T790M tumours to erlotinib in vivo. PC9 or PC9ER cells were injected subcutaneously in nude mice and tumours were left to grow to 100 mm3. The animals were then treated daily with erlotinib and EA alone or in combination. Co-administration of the drugs greatly inhibited tumour growth with 60% of the animals showing tumour volumes ⩽300 mm3 at 25 days, whereas those treated with either drug alone showed more extensive disease (Figure 6a). This was associated with increased survival (Figure 6b) and intra-tumoural GSH levels in combination-treated animals (Figure 6c). EA did not have any effect on erlotinib sensitivity of PC9 xenografts in agreement with the lack of further added sensitisation to erlotinib obtained with this inhibitor in vitro (Supplementary Figure S7E). Thus, co-administration of EA is probably a viable strategy for the management of erlotinib-resistant cancers in humans.

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.


Related in: MedlinePlus