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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.


Erlotinib resistance correlates with decreased NRF2 activity. (a, b) Subcellular fractions (a) and total lysates (b) from PC9, PC9ER, H3255 and H1975 cells were analysed by SDS-PAGE/western blotting for the indicated proteins. Detection of lamin and tubulin was used as loading controls for nuclear fractions and total lysates or cytoplasmic fractions, respectively. (c–h) PC9 cells transfected with non-targeting (NT), NRF2 or SQSTM1 siRNAs (c–f) or PC9ER cells transfected with KEAP1 or NT siRNAs (g, h) were treated with erlotinib and survival assessed by crystal violet staining (c, e, g). GSH levels were measured by colorimetric assay (d, f, h). (c–h) Data are average of n=4±s.e.m. Statistics: Student’s t-test,*P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S5 and S6.
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fig3: Erlotinib resistance correlates with decreased NRF2 activity. (a, b) Subcellular fractions (a) and total lysates (b) from PC9, PC9ER, H3255 and H1975 cells were analysed by SDS-PAGE/western blotting for the indicated proteins. Detection of lamin and tubulin was used as loading controls for nuclear fractions and total lysates or cytoplasmic fractions, respectively. (c–h) PC9 cells transfected with non-targeting (NT), NRF2 or SQSTM1 siRNAs (c–f) or PC9ER cells transfected with KEAP1 or NT siRNAs (g, h) were treated with erlotinib and survival assessed by crystal violet staining (c, e, g). GSH levels were measured by colorimetric assay (d, f, h). (c–h) Data are average of n=4±s.e.m. Statistics: Student’s t-test,*P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S5 and S6.

Mentions: GCLC, GSS and GSR are transcriptional targets of NFE2-related factor 2 (NRF2) [29–31], a downstream target of EGFR [32]. We therefore hypothesised that NRF2 activity might be impaired in EGFRm/T790M cells. NRF2’s transcriptional activity requires its nuclear localisation and NRF2 is also degraded through binding to KEAP1, a process counteracted by competitive interaction of the latter protein with PALB2 and/or SQSTM1. Analysis of nucleocytoplasmic fractions and total lysates from our four cell lines revealed that NRF2 or KEAP1 localisation/expression had no differences between PC9 and PC9ER cells, whereas H3255 cells showed higher level of nuclear NRF2 than H1975 cells (Figure 3a; Supplementary Figure S5A). This correlated with increased KEAP1 expression in H1975 as compared with H3255 cells (Figure 3a; Supplementary Figure S5B). Although these results alone may explain the difference in GSH pathway enzymes expression between the latter two cell lines, they cannot account for that seen between PC9 and PC9ER cells. However, SQSTM1 was downregulated in both PC9ER and H1975 cells as compared with their erlotinib-sensitive counterparts (Figure 3b; Supplementary Figure S5C), whereas PALB2 levels were lower in PC9ER as compared with PC9 cells (Figure 3b; Supplementary Figure S5D). Furthermore, NRF2 has been shown to be a transcriptional regulator of SQSTM1 and, indeed, mRNA levels of SQSTM1 were found to be significantly lower in both the resistant cell line pair (Supplementary Figure S5E and F). Hence, inhibition of NRF2 activity through various mechanisms may be linked to erlotinib resistance in NSCLC cells.


Decreased glutathione biosynthesis contributes to EGFR T790M-driven erlotinib resistance in non-small cell lung cancer
Erlotinib resistance correlates with decreased NRF2 activity. (a, b) Subcellular fractions (a) and total lysates (b) from PC9, PC9ER, H3255 and H1975 cells were analysed by SDS-PAGE/western blotting for the indicated proteins. Detection of lamin and tubulin was used as loading controls for nuclear fractions and total lysates or cytoplasmic fractions, respectively. (c–h) PC9 cells transfected with non-targeting (NT), NRF2 or SQSTM1 siRNAs (c–f) or PC9ER cells transfected with KEAP1 or NT siRNAs (g, h) were treated with erlotinib and survival assessed by crystal violet staining (c, e, g). GSH levels were measured by colorimetric assay (d, f, h). (c–h) Data are average of n=4±s.e.m. Statistics: Student’s t-test,*P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S5 and S6.
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Related In: Results  -  Collection

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fig3: Erlotinib resistance correlates with decreased NRF2 activity. (a, b) Subcellular fractions (a) and total lysates (b) from PC9, PC9ER, H3255 and H1975 cells were analysed by SDS-PAGE/western blotting for the indicated proteins. Detection of lamin and tubulin was used as loading controls for nuclear fractions and total lysates or cytoplasmic fractions, respectively. (c–h) PC9 cells transfected with non-targeting (NT), NRF2 or SQSTM1 siRNAs (c–f) or PC9ER cells transfected with KEAP1 or NT siRNAs (g, h) were treated with erlotinib and survival assessed by crystal violet staining (c, e, g). GSH levels were measured by colorimetric assay (d, f, h). (c–h) Data are average of n=4±s.e.m. Statistics: Student’s t-test,*P⩽0.05, **P⩽0.01, ***P⩽0.001. See also Supplementary Figures S5 and S6.
Mentions: GCLC, GSS and GSR are transcriptional targets of NFE2-related factor 2 (NRF2) [29–31], a downstream target of EGFR [32]. We therefore hypothesised that NRF2 activity might be impaired in EGFRm/T790M cells. NRF2’s transcriptional activity requires its nuclear localisation and NRF2 is also degraded through binding to KEAP1, a process counteracted by competitive interaction of the latter protein with PALB2 and/or SQSTM1. Analysis of nucleocytoplasmic fractions and total lysates from our four cell lines revealed that NRF2 or KEAP1 localisation/expression had no differences between PC9 and PC9ER cells, whereas H3255 cells showed higher level of nuclear NRF2 than H1975 cells (Figure 3a; Supplementary Figure S5A). This correlated with increased KEAP1 expression in H1975 as compared with H3255 cells (Figure 3a; Supplementary Figure S5B). Although these results alone may explain the difference in GSH pathway enzymes expression between the latter two cell lines, they cannot account for that seen between PC9 and PC9ER cells. However, SQSTM1 was downregulated in both PC9ER and H1975 cells as compared with their erlotinib-sensitive counterparts (Figure 3b; Supplementary Figure S5C), whereas PALB2 levels were lower in PC9ER as compared with PC9 cells (Figure 3b; Supplementary Figure S5D). Furthermore, NRF2 has been shown to be a transcriptional regulator of SQSTM1 and, indeed, mRNA levels of SQSTM1 were found to be significantly lower in both the resistant cell line pair (Supplementary Figure S5E and F). Hence, inhibition of NRF2 activity through various mechanisms may be linked to erlotinib resistance in NSCLC 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.