Wnt/β-catenin pathway regulates ABCB1 transcription in chronic myeloid leukemia

Abstract

Background: The advanced phases of chronic myeloid leukemia (CML) are known to be more resistant to therapy. This resistance has been associated with the overexpression of ABCB1, which gives rise to the multidrug resistance (MDR) phenomenon. MDR is characterized by resistance to nonrelated drugs, and P-glycoprotein (encoded by ABCB1) has been implicated as the major cause of its emergence. Wnt signaling has been demonstrated to be important in several aspects of CML. Recently, Wnt signaling was linked to ABCB1 regulation through its canonical pathway, which is mediated by β-catenin, in other types of cancer. In this study, we investigated the involvement of the Wnt/β-catenin pathway in the regulation of ABCB1 transcription in CML, as the basal promoter of ABCB1 has several β-catenin binding sites. β-catenin is the mediator of canonical Wnt signaling, which is important for CML progression.

Methods: In this work we used the K562 cell line and its derived MDR-resistant cell line Lucena (K562/VCR) as CML study models. Real time PCR (RT-qPCR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), flow cytometry (FACS), western blot, immunofluorescence, RNA knockdown (siRNA) and Luciferase reporter approaches were used.

Results: β-catenin was present in the protein complex on the basal promoter of ABCB1 in both cell lines in vitro, but its binding was more pronounced in the resistant cell line in vivo. Lucena cells also exhibited higher β-catenin levels compared to its parental cell line. Wnt1 and β-catenin depletion and overexpression of nuclear β-catenin, together with TCF binding sites activation demonstrated that ABCB1 is positively regulated by the canonical pathway of Wnt signaling.

Conclusions: These results suggest, for the first time, that the Wnt/β-catenin pathway regulates ABCB1 in CML.

Figure 1

EMSA using oligonucleotides of different TCF consensus binding sites in theABCB1promoter and protein extracts from K562 and Lucena cells. EMSAs to verify specific binding using the S4 and S5 oligonucleotides and protein extracts from K562 and Lucena cells. The specificity of the DNA-protein complex is demonstrated by competition reactions with 200-fold excess unlabeled oligonucleotide and by supershift assays. SL – Migration of the probe alone. “+C”- Competition reactions with 200-fold excess unlabeled probe. “+Opt” – Competition reactions with 200-fold excess unlabeled wild-type oligonucleotide for the TCF consensus binding site. EXT K – protein extract from K562 cells. EXT L – protein extract from Lucena cells. “+Smad 8”– Supershift reactions using Smad8 antibody. . “+ βcat” – Supershift reactions using β-catenin antibody. The arrows show specific binding.

Figure 2

ChIP assay forin vivoquantification of β-catenin binding to theABCB1promoter. (A) RT-qPCR quantification of β-catenin binding in K562 and Lucena cells. DNA amplification was quantified in bound and unbound fractions after normalization with protein A unspecific amplification. Normalized fractions were used to calculate the bound/input ratio. (B) Representative agarose gel - qualitative analysis – of ABCB1 promoter amplification for β-catenin ChIP assay. Input: bound and unbound fractions; B: bound; UB: unbound.

Figure 3

β-catenin expression levels in K562 and Lucena cells. (A) RT-qPCR analysis of β-catenin mRNA levels. Raw expression values were normalized to β-actin expression. (B) β-catenin expression by FACS, represented as MRFI. Secondary antibody was used as isotype antibody control. (C) Representative western blot analysis of β-catenin expression. 50 μg of protein extracts from both cell lines were separated SDS-PAGE and probed with anti- β-catenin antibody. α-tubulin was used for constitutive expression. Values represent the means of three independent determinations ± s.d.

Figure 4

Distribution of β-catenin in K562 and Lucena cells. Confocal microscopy of cytospin preparations showing the relative nuclear, membrane and cytoplasmic distribution of β-catenin in K562 and Lucena cells exposed to LiCl 10 mM treatment for 24 h. Nuclei β-catenin density significantly increases upon treatment, compared to untreated cells. Nuclei are stained with DAPI (blue). Micrograph panel is representative of three experiments for each condition.

Figure 5

Western Blot analysis of GSK3 activation. Representative western blot analysis of P-GSK3 from both cell lines protein extracts, with or without LiCl 10 mM treatment. 50 μg of protein extracts were separated SDS-PAGE and probed with anti-P-GSK3 antibody. α-tubulin was used for constitutive expression. For each treatment results, representative of three independent experiments are shown.

Figure 6

Wnt/β-catenin pathway activation increasesABCB1expression. Increases in ABCB1 mRNA levels were evaluated after LiCl 10 mM treatment for 24 h. Total RNA was isolated and used in RT-qPCR to determine changes in ABCB1 mRNA levels after normalization to β-actin expression. Values represent the means of three independent determinations ± s.d.

Figure 7

Expression ofABCB1mRNA levels afterWnt1depletion in CML cell lines. (A) WNT1 siRNA in K562 and Lucena cells. (B) Analysis of ABCB1 mRNA levels after Wnt1 depletion. Total RNA was isolated and used in RT-qPCR analysis to determine changes in ABCB1 mRNA levels after normalization to β-actin expression. All data were presented as fold inductions relative to control group expression (scrambled). Values represent the means of three independent determinations ± s.d.

Figure 8

Expression ofABCB1mRNA levels afterβ-catenindepletion in CML cell lines. (A) β-catenin siRNA in K562 and Lucena cells. (B) Analysis of ABCB1 mRNA levels after β-catenin depletion. Total RNA was isolated and used in RT-qPCR analysis to determine changes in ABCB1 mRNA levels after normalization to β-actin expression. All data were presented as fold inductions relative to control group expression (scrambled). Values represent the means of three independent determinations ± s.d.

Figure 9

ABCB1promoter TCF binding sites transcription activity using reporter plasmid pGL3 basic. (A) Scheme of constructs with TCF binding sites. (B) Luciferase activity reporter assay in K562 cells with or without LiCl 10 mM for 24 h. (C) Luciferase activity reporter assay in Lucena cells with or without LiCl 10 mM for 24 h. All luciferase assay results expressed as relative light units (RLU).