Nkx-2.5 Regulates MDR1 Expression via Its Upstream Promoter in Breast Cancer Cells

Abstract

Background: Increased expression of MDR1 gene is one of the major mechanisms responsible for multidrug resistance in cancer cells. Two alternative promoters, upstream and downstream, are responsible for transcription of MDR1 gene in the human. However, the molecular mechanism regarding the transactivation of MDR1 upstream promoter (USP) has not been determined.

Methods: Dual-luciferase reporter gene assays were used to assess the effect of Nkx-2.5 on MDR1 USP activity using reporter plasmids for human MDR1 USP and its mutants. MDR1 mRNA level was examined by quantitative real-time PCR. The direct binding of Nkx-2.5 to the USP of MDR1 was evaluated by promoter enzyme immunoassays and chromatin immunoprecipitation assays.

Results: Nkx-2.5 significantly stimulates the transactivation of MDR1 USP and increases MDR1 mRNA expression in MCF7 breast cancer cells. Reporter gene assays with deleted MDR1 USPs showed that the Nkx-2.5-binding site is located between positions -71 and +12. Mutation of the Nkx-2.5-binding site at nucleotide +4 to +10 markedly reduced the Nkx-2.5-mediated activation of MDR1 USP activity. A promoter binding immunoassay and a chromatin immunoprecipitation assay revealed that Nkx-2.5 binds directly to the region +4/+10 of human MDR1 USP.

Conclusion: The results in the present study show Nkx-2.5 is a positive regulator for the transactivation of MDR1 USP in MCF7 breast cancer cells. Our findings will help elucidate the regulatory mechanism responsible for the multidrug resistant cancer phenotype.

Keywords: Breast Cancer; MDR1; Nkx-2.5; Transcriptional Regulation.

Fig. 1. Human MDR1 upstream promoter activity in MCF7 breast cancer cells. (A) Schematic diagram for the two transcription initiation sites of the human MDR1 gene. Numbers indicate number of exon in the human MDR1 gene. (B) MCF7 cells were transfected with the pMDR1-U (−1391) or plasmids harboring a series of 5′ deletion mutants of the human MDR1 upstream promoter, and their promoter activities were analyzed using reporter gene assays. Relative luciferase activities were normalized as fold value versus pMDR1-U (−1391). Results are expressed as the means ± SDs. of at least three independent experiments.

USP = upstream promoter, DSP = downstream promoter, LUC, luciferase gene. ^*P < 0.05, ^**P < 0.01; as determined by one-way ANOVA with Tukey's post-hoc test.

Fig. 2. Nkx-2.5 increased MDR1 upstream promoter activity in MCF7 cells. (A) Schematic representation of the nt −71 to +13 region of MDR1 upstream promoter. Numbers indicate positions from the transcriptional start site in the human MDR1 upstream promoter. The binding sites of putative transcription factors, which were identified using the TFBIND program (http://tfbind.hgc.jp), are underlined. (B) MCF7 cells were co-transfected with the pMDR1-U construct and the indicated transcription factors or control vector (pcDNA3.1), and reporter gene assays were performed. The relative luciferase activities were expressed as fold value versus the pcDNA3.1 vector. Results are expressed as the means ± SDs of at least three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; as determined using the unpaired Student's t-test. (C) The pMDR1-U (−1391) or pGL3/basic vector was co-transfected with Nkx-2.5 expression vector or pcDNA3.1 vector into MCF7 cells. Relative luciferase activities were normalized as fold values versus pMDR1-U (−1391) in the absence of Nkx-2.5. Results are expressed as the means ± SDs of at least three independent experiments. ^*P < 0.05; as determined by the unpaired Student's t-test. (D) The pMDR1-U (−1391) construct was co-transfected with the indicated amount of Nkx-2.5 expression vector into MCF7 cells. Relative luciferase activities were normalized as fold values versus pMDR1-U (−1391) in the absence of Nkx-2.5. Results are expressed as the means ± SDs of at least three independent experiments. ^***P < 0.001 as determined by one-way ANOVA with Tukey's post-hoc test. (E) MCF7 cells were infected with retrovirus encoding Nkx-2.5-FLAG or retrovirus from pMxs-IRES-puro vector (Control), and Nkx-2.5 expression was examined by immunoblotting using anti-FLAG antibody. (F) Real-time PCR analysis of MDR1 mRNA in MCF7 cells infected with retrovirus encoding Nkx-2.5 or retrovirus from pMXs-IRES-puro vector. Results are expressed as the means ± SDs of at least three independent experiments. ^**P < 0.01 as determined by the unpaired Student's t-test.

Fig. 3. Identification of the Nkx-2.5-binding site in MDR1 upstream promoter. (A) pMDR1-U (−1391) and plasmids harboring a series of 5′ deletion mutants of human MDR1 upstream promoter were co-transfected with Nkx-2.5 expression vector or control vector (pcDNA3.1) into MCF7 cells. Relative luciferase activities were expressed as fold over that of each promoter in the presence of pcDNA3.1 vector. Results are expressed as the means ± SDs of at least three independent experiments. ^*P < 0.05, ^**P < 0.01; as determined by one-way ANOVA with Tukey's post-hoc test. (B) Schematic representation of pMDR1-U (−1391/+136) with mutations in the Nkx-2.5-binding site. (C) pMDR1-U (−1391) or a mutated MDR1 upstream promoter construct were co-transfected with plasmid encoding Nkx-2.5 or control vector (pcDNA3.1) into MCF7 cells. Relative luciferase activities were expressed as fold over that of each promoter in the presence of pcDNA3.1 vector. Results are expressed as the means ± SDs of at least three independent experiments. ^**P < 0.01; as determined by the unpaired Student's t-test.

Fig. 4. Nkx-2.5 directly bound to the Nkx-2.5-binding site at nt +4 to +10 region of human MDR1 upstream promoter. (A) Schematic representation of biotinylated probes used in promoter enzyme immunoassay. (B) Streptavidin-coated microplates were conjugated with biotin-labeled oligonucleotides containing Wt or Mut Nkx-2.5-binding sites and then incubated with nuclear extracts from MCF7 cells transfected with plasmid encoding Nkx-2.5. After incubation for 2 hours, Nkx-2.5-DNA complex formation was analyzed colorimetrically using anti-Nkx-2.5 and HRP-conjugated secondary antibodies. Results are presented as the means ± SDs (n = 3). (C) Soluble chromatin was prepared from MCF7 cells and immunoprecipitated with anti-Nkx-2.5 antibody or an isotype-matched IgG. Immunoprecipitated chromatin was subjected to PCR using primers specific for the Nkx-2.5-responsive element in MDR1 upstream promoter (upper panel). As a negative control, primers for the distal region (nt −1062 to −1221) of MDR1 upstream promoter were used (lower panel).

Wt = wild-type, Mut = mutated. ^**P < 0.01; as determined by one-way ANOVA with Tukey's post-hoc test.

Fig. 5. Nkx-2.5 knockdown leads to reduction in MDR1 expression in breast cancer cells. (A) Nkx-2.5 expression was analyzed in MCF7, MDA-MB-231, and 4T1-luc cells by Western blot. A representative result is shown. (B, C) MDA-MB-231 cells were transfected with Nkx-2.5 siRNAs or control siRNA, and the expressions of Nkx-2.5 (B) and MDR1 mRNA (C) were analyzed by real-time PCR. Results are expressed as the means ± SDs of at least three independent experiments.

^*P < 0.05, ^**P < 0.01, ^***P < 0.001; as determined by one-way ANOVA with Tukey's post-hoc test.