Crizotinib sensitizes the erlotinib resistant HCC827GR5 cell line by influencing lysosomal function

Abstract

In non-small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR-inhibitor erlotinib and the cMET-inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live-cell microscopy. We observed additive effects in EBC-1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR-inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live-cell microscopy with a pH-sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib-erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions.

Keywords: EGFR; cMET; crizotinib; erlotinib; lysosomes; tyrosine kinase inhibitors.

Figure 1

The methodology of FIJI analysis: Fluorescent images were taken of live cells simultaneously treated with Lysotracker Red, pHrodogreen and sunitinib to image pH‐differences and drug uptake. Here we provide guiding images to clarify our analytical method. Images of the Lysotracker Red and pHrodoGreen channel are shown. Yellow markings represent the regions of interest. (a) Lysotracker Red with yellow markings depicting six selected cells for analysis; (b) pHrodoGreen image with yellow markings for six selected cells; (c) pHrodoGreen with markings for six selected cells with markings for lysosomes; (d) pHrodoGreen image with six selected cells with deleted lysosomal intensity; (e) pHrodoGreen image of lysosomes of the six selected cells

Figure 2

Effect of the erlotinib–crizotinib combination. This figure combines the results of different experiments, evaluating the effect of erlotinib, crizotinib or the combination of growth inhibition, protein phosphorylation, cell cycle arrest, and apoptosis. (a) Combination indexes (CIs) for erlotinib plus crizotinib after 72 hr of treatment. The upper line represents an antagonistic CI > 1.2, the lower bar represents a synergistic CI < 0.8. (b) Protein expression as determined by western blot analysis after treatment with erlotinib and crizotinib (pictures result from the same blot). The insert shows an included positive control To exclude that the lack of signal was due to lack of expression or an issue with the western blot analysis itself. (c) Cell cycle distribution was measured by flow cytometry after propidium iodide staining. (d) Apoptosis after 24 hr treatment, as assessed by the analysis of the sub‐G1 fraction. Control: 0.1% dimethyl sulfoxide, E: 10 µM erlotinib, C: 5 µM crizotinib, Combo: 10 µM erlotinib + 5 µM crizotinib. *p < .05 as compared to control, **p < .01, ***p < .001 as compared to control

Figure 3

Effect of the erlotinib‐crizotinib combination on cell migration and spheroids. (a) Representative images of migration assay after 0 and 8 hr for HCC827 and 0 and 16 hr for HCC827GR5. (b) Statistical evaluation of results of the wound‐healing assay on the HCC827 cell line 8 hr after scratch induction and treatment. The percentages of scratch closure for control, erlotinib, crizotinib or erlotinib + crizotinib treated cells were compared with a one‐way analysis of variance (ANOVA) in GraphPad Prism. (c) Statistical evaluation of the results of the wound‐healing assay on the HCC827GR5 cell line 16 hr after scratch induction and treatment. The percentages of scratch closure for control, erlotinib, crizotinib or erlotinib + crizotinib treated cells were compared with a one‐way ANOVA in GraphPad Prism. (d) Effect of treatment on 3D growth. Bars represent mean ± standard error of mean after 7 days of treatment of three separate tests. Day‐3 represents spheroids immediately before the start of treatment, and Day‐7 after 96 hr of treatment. Control: 0.1% dimethyl sulfoxide, E: 10 µM erlotinib, C: 5 µM crizotinib, Combo: 10 µM erlotinib + 5 µM crizotinib. *p < .05 as compared to control, ***p < .001 as compared to control

Figure 4

Effect of treatment on protein phosphorylation or protein cleavage. Cells were treated with 0.1% dimethyl sulfoxide (DMSO) as control, 10 µM erlotinib, 5 µM crizotinib, or a combination of both for 24 hr. Phosphorylation/protein cleavage was assessed by Pathscan assay. Relative fluorescent units were used and quantified with the Odyssey imaging system. (a) Pathscan assay HCC827; (b) Pathscan assay HCC827GR5. Control: 0.1% DMSO, E: 10 µM erlotinib, C: 5 µM crizotinib, Combo: 10 µM erlotinib + 5 µM crizotinib

Figure 5

Effect of erlotinib on the intracellular and lysosomal accumulation of crizotinib and on the role of pH. Cells were treated with 0.1% dimethyl sulfoxide as control, 10 µM erlotinib, 5 µM crizotinib or their combination for 24 hr. Bafilomycin (50 nM) was used to perturb the lysosomal function. (a) Effect of erlotinib on the intracellular crizotinib concentration in pmol/µg protein. (b) Intracellular erlotinib concentration in fmol/µg protein. Bars represent mean ± standard error of mean of three separate tests. (c) Quantification of pHrodogreen intensity in relative fluorescent units with FIJI from d, e. All the p‐values are summarized in Table S1. (d, e) Intracellular effect of drugs on HCC827 and HCC827GR5, respectively, cells were stained for 1 hr with 5 µM sunitinib and 0.5 µM Lysotracker Red, and for 30 min with pHrodoGreen. Each sample was divided in multiple focus planes (z‐stack). Z‐stacks were imaged using a Leica TCS SP8 STED 3× microscope. Image panels of d from left to right: sunitinib staining of HCC827 control cells; sunitinib staining of HCC827 treated with 10 µM erlotinib and 5 µM crizotinib; Phrodogreen and Lysotracker red staining of HCC827 control cells; Phrodogreen and Lysotracker red staining of HCC827 treated with 10 µM erlotinib and 5 µM crizotinib. Image panels of e from left to right: sunitinib staining of HCC827GR5 control cells; sunitinib staining of HCC827GR5 treated with 10 µM erlotinib and 5 µM crizotinib; Phrodogreen and Lysotracker red staining of HCC827GR5 control cells; Phrodogreen and Lysotracker red staining of HCC827GR5 treated with 10 µM erlotinib and 5 µM crizotinib. Control: 0.1% DMSO, E: 10 µM erlotinib, C: 5 µM crizotinib, Combo: 10 µM erlotinib + 5 µM crizotinib, B: 50 nM Bafilomycin A1. *p < .05 as compared to control, **p < .01; ***p < .001 as compared to control