KRAS and EGFR Mutations Differentially Alter ABC Drug Transporter Expression in Cisplatin-Resistant Non-Small Cell Lung Cancer

Abstract

Lung carcinoma is still the most common malignancy worldwide. One of the major subtypes of non-small cell lung cancer (NSCLC) is adenocarcinoma (AC). As driver mutations and hence therapies differ in AC subtypes, we theorized that the expression and function of ABC drug transporters important in multidrug resistance (MDR) would correlate with characteristic driver mutations KRAS or EGFR. Cisplatin resistance (CR) was generated in A549 (KRAS) and PC9 (EGFR) cell lines and gene expression was tested. In three-dimensional (3D) multicellular aggregate cultures, both ABCB1 and ABCG2 transporters, as well as the WNT microenvironment, were investigated. ABCB1 and ABCG2 gene expression levels were different in primary AC samples and correlated with specific driver mutations. The drug transporter expression pattern of parental A549 and PC9, as well as A549-CR and PC9-CR, cell lines differed. Increased mRNA levels of ABCB1 and ABCG2 were detected in A549-CR cells, compared to parental A549, while the trend observed in the case of PC9 cells was different. Dominant alterations were observed in LEF1, RHOU and DACT1 genes of the WNT signalling pathway in a mutation-dependent manner. The study confirmed that, in lung AC-s, KRAS and EGFR driver mutations differentially affect both drug transporter expression and the cisplatin-induced WNT signalling microenvironment.

Keywords: ABC drug transporters; AC; EGFR; KRAS; NSCLC; WNT signalling.

Figure 1

Drug transporter analysis of primary human adenocarcinoma (AC) samples and cell lines. (A) Relative mRNA expression (2^−ddCt) levels of ABCB1 and ABCG2 drug transporters in adenocarcinoma patients (n = 12) with different mutational background (EGFR wild type/KRAS wild type; EGFR mutant/KRAS wild type, EGFR wild type/KRAS mutant). mRNA expression is relative to normal, healthy lung tissue. Data are presented as mean ± SD. (B) Relative mRNA expression (2^−ddCt) levels of ABCB1 and ABCG2 in adenocarcinoma cell lines (A549, PC9). mRNA from primary human small airway epithelial cells (SAEC) was the control. The graph shows the mean and SD of biological repeats (n = 3), “*” p < 0.05 and “**” p < 0.002.

Figure 2

Adenocarcinoma morphology, drug transporter gene expression pattern and functional analysis of ABCB1, ABCG2 in 2D cultures. (A) Phase-contrast microscopic images (10×) from solvent control parental A549, cisplatin resistant A549 (A549-CR), solvent control parental PC9 and cisplatin resistant PC9 (PC9-CR). Scale bar is 50 µm. (B) Relative mRNA expression (2-ddCt) of ABCB1 and ABCG2 drug transporters in solvent control parental (A549, PC9) and cisplatin-resistant adenocarcinoma cell lines (A549-CR, PC9-CR) compared to normal, primary human SAEC. The graph shows the mean and SD of biological repeats (n = 3); “*” p < 0.05 and “**” p < 0.002. (C) Functional analysis of ABCB1 and ABCG2 drug transporters in control and cisplatin-treated (CR) A549 and PC9 cell lines, expressed as MAF values. Data presented as representatives of 4 independent measurements.

Figure 3

Formation of the 3D tissue aggregates. (A) Schematic representation of the preparation steps of 3D tissue aggregates using the Greiner Bio-One magnetization core technology with a NanoShuttle™-PL.-kit. (B) Light microscopic picture of a 3D PC9 tissue aggregate (20× magnification).

Figure 4

Mimicking cell composition of the human lung tissue. (A) Relative mRNA expression (2^−ddCt) of ABCB1 and ABCG2 drug transporters in parental (A549, PC9) and cisplatin-resistant AC cell line (A549-CR, PC9-CR) containing 3D aggregate co-cultures. The inner control was CK7. Biological repeats n = 3. (B) 3D aggregate tissue sections were stained with anti-ABCB1 or anti-ABCG2 specific primary antibodies, respectively (green), while EpCAM and CK5 were stained with specific antibodies (red) and nuclei were stained with DAPI (blue). The staining shows a representative of 3 independent experiments. (C) Densitometry of ABCB1 and ABCG2 proteins in A549, A549-CR and PC9, PC9-CR containing aggregate co-cultures. Drug transporter protein intensity quantification was normalized to the respective epithelial marker intensity. (“*” p < 0.05 and “**” p < 0.002).

Figure 5

Effect of clinically relevant chemotherapeutic drugs on CR cell line containing 3D aggregates. (A) Relative mRNA expression (2^−ddCt) of ABCB1 drug transporters in cisplatin-resistant AC cell line (A549-CR, PC9-CR) containing 3D aggregate co-cultures. The inner control was beta actin. (B) Relative mRNA expression of ABCG2 drug transporters in cisplatin-resistant AC cell line (A549-CR, PC9-CR) containing 3D aggregate co-cultures. The inner control was beta actin. (C) Relative mRNA expression (2^−ddCt) of ABCB1 drug transporters in cisplatin-resistant AC cell line (A549-CR, PC9-CR) containing 3D aggregate co-cultures. The inner control was CK7. (D) Relative mRNA expression (2^−ddCt) of ABCG2 drug transporters in cisplatin-resistant AC cell line (A549-CR, PC9-CR) containing 3D aggregate co-cultures. The inner control was CK7. (“*” p < 0.05 and “**” p < 0.002).

Figure 6

Changes in the WNT microenvironment. (A) mRNA expression of WNT signalling pathway genes in A549 3D aggregates relative to PC9 3D aggregates. (B) mRNA expression of WNT signalling pathway genes in A549-CR 3D aggregates relative to A549 3D aggregates. (C) mRNA expression of WNT signalling pathway genes in PC9-CR 3D aggregates relative to PC9 3D aggregates. (D) mRNA expression of WNT signalling pathway genes in A549-CR 3D aggregates relative to PC9-CR 3D aggregates. (The cut-off was at and above a 2-fold increase or decrease in the values detected by the arrays. The full analysis data can be viewed in Figure S3).