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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.


Related in: MedlinePlus

Changes in NO levels modulate erlotinib response. (a) NO levels in PC9 and PC9ER cells were compared by fluorescence-activated cell sorting (FACS) using DAF-FM. Left: FACS profile; right: fold changes in geometric mean. (b) PC9ER cells transfected with non-targeting (NT) or NOS1 siRNAs or (c) PC9 and PC9ER cells treated±an NO-trap were exposed to a dose range of erlotinib. Cell survival was determined by crystal violet staining. Statistics: Student’s t-test, *P⩽0.05.
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fig5: Changes in NO levels modulate erlotinib response. (a) NO levels in PC9 and PC9ER cells were compared by fluorescence-activated cell sorting (FACS) using DAF-FM. Left: FACS profile; right: fold changes in geometric mean. (b) PC9ER cells transfected with non-targeting (NT) or NOS1 siRNAs or (c) PC9 and PC9ER cells treated±an NO-trap were exposed to a dose range of erlotinib. Cell survival was determined by crystal violet staining. Statistics: Student’s t-test, *P⩽0.05.

Mentions: As GSH buffers reactive oxidative species, we investigated whether lower GSH levels in erlotinib-resistant cells associated with elevated reactive oxidative species. We performed flow cytometry analysis in the presence of dihydroethidine and DAF-FM (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) to detect superoxide and nitric oxide (NO) species, respectively. Erlotinib-resistant cells showed an increase in NO species (Figure 5a), although they did not show increased superoxide levels. To assess whether this could influence erlotinib resistance, we first silenced the expression of the three NO synthases, NOS1–3. Although siRNA-mediated downregulation of NOS2 and 3 did not impact on erlotinib resistance (not shown), NOS1 silencing sensitised PC9ER cells to erlotinib (Figure 5b). Next, we quenched cellular NO in erlotinib-resistant cells with the NO-trap carboxy-PTIO and revealed that this partially re-sensitised PC9ER cells to erlotinib (Figure 5c). Although these data suggest a role for NO in erlotinib resistance, the levels of changes observed as compared with those seen earlier (Figures 2 and 3) suggest that changes in NO are not solely responsible for resistance downstream of decreased GSH levels.


Decreased glutathione biosynthesis contributes to EGFR T790M-driven erlotinib resistance in non-small cell lung cancer
Changes in NO levels modulate erlotinib response. (a) NO levels in PC9 and PC9ER cells were compared by fluorescence-activated cell sorting (FACS) using DAF-FM. Left: FACS profile; right: fold changes in geometric mean. (b) PC9ER cells transfected with non-targeting (NT) or NOS1 siRNAs or (c) PC9 and PC9ER cells treated±an NO-trap were exposed to a dose range of erlotinib. Cell survival was determined by crystal violet staining. Statistics: Student’s t-test, *P⩽0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037574&req=5

fig5: Changes in NO levels modulate erlotinib response. (a) NO levels in PC9 and PC9ER cells were compared by fluorescence-activated cell sorting (FACS) using DAF-FM. Left: FACS profile; right: fold changes in geometric mean. (b) PC9ER cells transfected with non-targeting (NT) or NOS1 siRNAs or (c) PC9 and PC9ER cells treated±an NO-trap were exposed to a dose range of erlotinib. Cell survival was determined by crystal violet staining. Statistics: Student’s t-test, *P⩽0.05.
Mentions: As GSH buffers reactive oxidative species, we investigated whether lower GSH levels in erlotinib-resistant cells associated with elevated reactive oxidative species. We performed flow cytometry analysis in the presence of dihydroethidine and DAF-FM (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) to detect superoxide and nitric oxide (NO) species, respectively. Erlotinib-resistant cells showed an increase in NO species (Figure 5a), although they did not show increased superoxide levels. To assess whether this could influence erlotinib resistance, we first silenced the expression of the three NO synthases, NOS1–3. Although siRNA-mediated downregulation of NOS2 and 3 did not impact on erlotinib resistance (not shown), NOS1 silencing sensitised PC9ER cells to erlotinib (Figure 5b). Next, we quenched cellular NO in erlotinib-resistant cells with the NO-trap carboxy-PTIO and revealed that this partially re-sensitised PC9ER cells to erlotinib (Figure 5c). Although these data suggest a role for NO in erlotinib resistance, the levels of changes observed as compared with those seen earlier (Figures 2 and 3) suggest that changes in NO are not solely responsible for resistance downstream of decreased GSH levels.

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