Targeting the miR-200c/LIN28B axis in acquired EGFR-TKI resistance non-small cell lung cancer cells harboring EMT features

Abstract

MicroRNA (miR)-200 family members (miR-200s) are frequently silenced in advanced cancer and have been implicated in the process of epithelial-to-mesenchymal transition (EMT). We previously reported that miR-200s were silenced through promoter methylation in acquired EGFR-tyrosine kinase inhibitor (TKI) resistant non-small cell lung cancer (NSCLC) cells harboring EMT features. In this study, we examined the functional role of miR-200s in NSCLC cells and investigated a novel approach to overcoming acquired EGFR-TKI resistance. In the analysis of NSCLC cell lines, each of the miR-200s expression-silenced cell lines showed promoter methylation. Significant correlations between miR-200c silencing and several oncogenic pathway alterations, including EMT-changes and LIN28B overexpression, were observed in the database analysis. In addition, EGFR-wild type cell lines had lower miR-200s expression levels than EGFR-mutant cell lines. The introduction of miR-200c using pre-miR-200c caused LIN28B suppression in cells with acquired EGFR-TKI resistance that harbored EMT features. Interestingly, both the introduction of miR-200c and the knockdown of LIN28B produced an antitumor effect in acquired EGFR-TKI resistance cells, whereas these manipulations were not effective in parental cells. The miR-200c/LIN28B axis plays an important role in cells with acquired resistance to EGFR-TKI that harbor EMT features and might be a useful therapeutic target for overcoming resistance.

Figure 1. Expression and methylation statuses of miR-200s in 34 NSCLC cell lines and HBEC-5KT.

The miR-200a, miR-200b, and miR-200c expression statuses as determined using qRT-PCR and the miR-200ba429 and miR-200c141 methylation statuses as determined using methylation-specific PCR (MSP) in 34 NSCLC cell lines and HBEC-5KT are shown. The miR-200s expression levels in HBEC-5KT were set at 1, and the expression levels in the NSCLC cells were shown relative to those in the HBEC-5KT cell line. For the MSP assay, each circle represents the promoter methylation status (white circle, unmethylated; gray circle, partially methylated; black circle, methylated).

Figure 2. Functional analysis of miR-200s in NSCLC cells.

(A) Correlation between the miR-200c expression level and CDH1 expression in 28 NSCLC cell lines. Each circle represents NSCLC cell lines. Closed circle, miR-200c low expression group; Opened circle, miR-200c high expression group. (B) Correlation between the miR-200c expression level and ZEB1 expression in 28 NSCLC cell lines. (C) Correlation between the miR-200c expression level and LIN28B expression in 28 NSCLC cell lines. (D) Correlation between EGFR-mutation statuses and miR-200s expressions in 34 NSCLC cell lines. WT, wild type; mut, mutation.

Figure 3. Antitumor effect of miR-200c introduction in parental and acquired EGFR-TKI resistance cells.

(A) Relative miR-200c expression level using qRT-PCR in parental HCC4006 cells and acquired EGFR-TKI-resistant HCC4006-GR cells. (B) EMT-related protein expression level using western blotting in HCC4006 and HCC4006-GR cells. The blots of whole membrane are presented in Supplementary Fig. S5. (C) Cell viability after miR-200c transfection in HCC4006 and HCC4006-GR cells using MTT assay.

Figure 4. Forced miR-200c expression leads to suppression of LIN28B expression.

(A) LIN28 expression level as determined using qRT-PCR in HCC4006 and HCC4006-GR cells. ND, not-determined. The LIN28B expression level in H1299 was set at 1, and the relative expression levels in HCC4006 parental and resistance cell lines were shown. (B) EMT-related proteins and LIN28B expression level using western blotting after pre-miR-200c or miR-Scramble transfection in HCC4006 and HCC4006-GR cells. The blots of whole membrane are presented in Supplementary Fig. S5.

Figure 5. Antitumor effect of LIN28B knockdown in parental and acquired EGFR-TKI resistance cells.

(A) Cell viability after LIN28B knockdown in HCC4006 and HCC4006-GR cells as determined using an MTT assay. (B) Expression of the apoptosis marker c-PARP and EMT marker after LIN28B knockdown as determined using western blotting. The blots of whole membrane are presented in Supplementary Fig. S5. (C) Diagram of the mechanism of the EGFR-TKI-mediated miR-200s/LIN28B interaction causing an EMT and acquired EGFR-TKI resistance in EGFR-mut NSCLC cells.

Figure 6. MiR-200c downregulation and LIN28B upregulation after EGFR-TKI resistance acquisition on NSCLC tissues.

(A) Mir-200c expression level as determined using qRT-PCR in NSCLC patients. (B) E-cadherin, vimentin and LIN28B expression level as determined by immunohistochemistry. The images of a representative patient are shown.