Higher expression of miR-133b is associated with better efficacy of erlotinib as the second or third line in non-small cell lung cancer patients

Abstract

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (gefitinib, erlotinib and afatinib) are indicated as first-line therapy in patients with non-small cell lung cancer (NSCLC) whose tumors harbor activating mutations in the EGFR gene. Erlotinib is also used in second and third-line therapy for patients whose tumors have wild type EGFR but to date there are no validated biomarkers useful to identify which patients may benefit from this treatment. The expression level of four miRNAs: miR-133b, -146a, -7 and -21 which target EGFR was investigated by real-time PCR in tumor specimens from NSCLC patients treated with erlotinib administered as the second or third line. We found that miR-133b expression level better discriminated responder from non-responder patients to erlotinib. Higher levels of miR-133b in NSCLCs were associated with longer progression-free survival time of patients. Functional analyses on miR-133b through transfection of a miR-133b mimic in A549 and H1299 NSCLC cell lines indicated that increasing miR-133b expression level led to a decreased cell growth and altered morphology but did not affect sensitivity to erlotinib. The detection of miR-133b expression levels in tumors help in the identification of NSCLC patients with a better prognosis and who are likely to benefit from second and third-line therapy with erlotinib.

Fig 1. MiRNAs expression in relation to response to erlotinib.

A Analysis of miRNA expression was investigated using TaqMan real-time quantitative PCR in NSCLC specimen from "responders" (n = 8) and "non-responders" patients (n = 24). Results are shown as normalized expression: 2^−ΔCt compared with Mann-Whitney test. B ROC curve analysis of miRNA levels to predict response to erlotinib. AUC = Area Under the Curve; CI = Confidence Interval; Thr = Threshold; Se = Sensitivity; Sp = Specificity.

Fig 2. Effects of miR-133b in combination to erlotinib on NSCLC cell lines.

Cells were seeded at 1.5 x 10⁵ cells/well and cultured up to 72 hours. Data are presented as mean ± SEM of three independent experiments. A Cell growth relative to lipofectamine-treated cells. B Photographs of A549 and H1299 after 72 h treatment. Magnification 400X. C Cell shape measured as forward scatter (FSC) shown on the top and side scatter (SSC) shown on the bottom. Note that the axes in each diagram are displayed in relative percent scale. *p<0.05 by the one sample t test with hypothetical value = 100.

Fig 3. Effects of mir-133b and erlotinib on EGFR and EGFR pathway.

A Flow cytometry to evaluate EGFR surface expression. Expression relative to lipofectamine treated cells is shown (mean ± SEM of three independent experiments is shown). *p<0.05 by the one sample t test with hypothetical value = 100. B Western blot assay to detect total EGFR, total ERK, GAPDH and phosphorylated ERK. Cropped blots are displayed. Full length blots are showed in S1 Fig. Exposure time: EGFR 200 sec, GAPDH 90 sec, pERK 420 sec, total EGFR 90 sec.