Induction of the transcriptional repressor ZBTB4 in prostate cancer cells by drug-induced targeting of microRNA-17-92/106b-25 clusters

Abstract

Androgen-insensitive DU145 and PC3 human prostate cancer cells express high levels of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and treatment of cells with methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (CDODA-Me) inhibited cell growth and downregulated Sp1, Sp3, and Sp4 expression. CDODA-Me (15 mg/kg/d) was a potent inhibitor of tumor growth in a mouse xenograft model (PC3 cells) and also decreased expression of Sp transcription factors in tumors. CDODA-Me-mediated downregulation of Sp1, Sp3, and Sp4 was due to induction of the transcriptional repressor ZBTB4, which competitively binds and displaces Sp transcription factors from GC-rich sites in Sp1-, Sp3-, Sp4-, and Sp-regulated gene promoters. ZBTB4 levels are relatively low in DU145 and PC3 cells due to suppression by miR paralogs that are members of the miR-17-92 (miR-20a/17-5p) and miR-106b-25 (miR-106b/93) clusters. Examination of publically available prostate cancer patient array data showed an inverse relationship between ZBTB4 and miRs-20a/17-5p/106b/93 expression, and increased ZBTB4 in patients with prostate cancer was a prognostic factor for increased survival. CDODA-Me induces ZBTB4 in prostate cancer cells through disruption of miR-ZBTB4 interactions, and this results in downregulation of pro-oncogenic Sp transcription factors and Sp-regulated genes.

Figure 1

Effects of CDODA-Me on prostate cancer cell proliferation, invasion and migration. Cell proliferation (A) and cell cycle progression (B) DU145 or PC3 cells were treated with DMSO or CDODA-Me (1 – 5 μM) for up to 72 hr (A) or 24 hr (B), and the number of cells or their distribution in different phases of the cell cycle were determined as outlined in the Materials and Methods. Apoptosis (C) and migration/invasion (D). Cells were treated with CDODA-Me for 32 hr (apoptosis) or 18 hr (migration/invasion), and the effects on apoptosis, migration and invasion were determined as outlined in the Materials and Methods. Results are expressed as means ± SE for at least three replication determinations, and significantly (p < 0.05) increased (*) or decreased (**) responses are indicated.

Figure 2

CDODA-Me-dependent regulation of Sp transcription factors. Downregulation of Sp proteins by western blot (A) or immunostaining (B). Cells were treated with CDODA-Me for the indicated times and Sp1, Sp3 and Sp4 were analyzed by western blots or Immunostaining (Sp1 only) as described in the Materials and Methods. (C) Decreased Sp-regulated genes. Cells were treated as described in (A) and Sp-regulated genes were analyzed by western blots. (D) Sp1 knockdown alters cell cycle progression. Cells were transfected with siSp1 oligonucleotide and after 48 hr, cells were treated with 2.5 μM CDODA-Me for 24 hr and analyzed by FACS as described in the Materials and Methods. Results in (D) are expressed as means ± SE for three replicate determinations and significant (p < 0.05) increases (*) or decreases (**) compared to control non-specific oligonucleotide (CT) are indicated.

Figure 3

ZBTB4 induction and function. (A) Induction of ZBTB4 mRNA and protein. Cells were treated with CDODA-Me for the indicated times, and mRNA and protein levels were determined by real time PCR and western blots, respectively, as outlined in the Materials and Methods. (B) Effects of ZBTB4 overexpression on Sp-regulated proteins and constructs. ZBTB4 (1 μg/well) was overexpressed alone in PC3 and DU145 cells or in combination with several GC-rich Sp-regulated constructs and proteins, and luciferase activity was determined as described in the Materials and Methods. ZBTB4 overexpression modulates the cell cycle (C) and Sp1 binding to the GC-rich Sp1 promoter in a ChIP assay (D). Cells were transfected with ZBTB4 and after 24 hr (C) or 18 hr (D), cells were analyzed by FACS or in a ChIP assay as described in the Materials and Methods. Results are expressed as means ± SE for at least three replication determinations, and significantly (p < 0.05) increased (*) or decreased (**) responses are indicated.

Figure 4

MiR paralog expression and regulation in prostate cancer cells. (A) Expression in cells and patients. MiR levels in PC3 and DU145 cells were determined by real time PCR as described in the Materials and Methods, and expression in tumors was obtained from publically available results (GSE23022). Effects of CDODA-Me on miR expression (B) and luciferase activity (C). Cells were treated with 2.5 μM CDODA-Me for 24 hr (B) or transfected with promoter construct from MCM7 gene and miR-17-92 cluster (C) promoters. MiR levels and luciferase activities were determined as outlined in the Materials and Methods. (D) MCM7 expression. Cells were treated with 2.5 μM CDODA-Me for 24 hr and MCM7 mRNA levels were determined by real time PCR as outlined in the Materials and Methods. The significance (p < 0.05) of treatment-related decreases are indicated (**), and the level of significance for the patient data (A) is indicated.

Figure 5

MiR mimics and antagomirs and their effects on ZBTB4 and Sp-regulated genes/reporter genes. Interactions with the 3′-UTR of ZBTB4 (A) and induction of ZBTB4 mRNA (B). PC3 cells were transfected with the ZBTB4-UTR-luciferase construct containing the CACUUUA miR binding site (A) and cotransfected with indicated miR mimics (A) or antagomirs (A and B). Luciferase activity or ZBTB4 mRNA expression were determined as outlined in the Materials and Methods. (C) MiRantagomirs decrease luciferase activity (C) and downregulate Sp proteins and Sp-regulated genes (D). Cells were transfected with the antagomirs and, in (C), were cotransfected with GC-rich constructs. Luciferase activity or protein expression was determined as outlined in the Materials and Methods. Results (A – C) are expressed as means ± SE for at least three replicate determinations, and significantly (p < 0.05) induced (*) or decreased (**) responses are indicated.

Figure 6

In vivo studies. Inhibition of tumor size and weights (A), H&E and Sp1, Sp3 and Sp4 staining (B), and miR-106b/miR-92 levels (C) in mice treated with CDODA-Me. Athymic nude mice bearing PC3 cells as xenografts were treated with CDODA-Me (15 mg/kg/d) over a period of 21 days, and tumor volumes were estimated. Tumor weights, H&E and Sp1 staining, and miR mRNA levels were determined as outlined in the Materials and Methods. (D) Mode of action for CDODA-Me. Proposed pathways triggered by CDODA-Me in prostate cancer cells/tumors that lead to downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes/responses. Results are expressed as means ± SE for at least three replication determinations, and significantly (p < 0.05) decreased (*) responses are indicated.