A positive feedback loop regulates the expression of polycomb group protein BMI1 via WNT signaling pathway

Abstract

Polycomb group protein BMI1 plays an important role in cellular homeostasis by maintaining a balance between proliferation and senescence. It is often overexpressed in cancer cells and is required for self-renewal of stem cells. At present, very little is known about the signaling pathways that regulate the expression of BMI1. Here, we report that BMI1 autoactivates its own promoter via an E-box present in its promoter. We show that BMI1 acts as an activator of the WNT pathway by repressing Dickkopf (DKK) family of WNT inhibitors. BMI1 mediated repression of DKK proteins; in particular, DKK1 led to up-regulation of WNT target c-Myc, which in turn further led to transcriptional autoactivation of BMI1. Thus, a positive feedback loop connected by the WNT signaling pathway regulates BMI1 expression. This positive feedback loop regulating BMI1 expression may be relevant to the role of BMI1 in promoting cancer and maintaining stem cell phenotype.

Figures

FIGURE 1.

BMI1 autoactivates its promoter.A and B, wild type and mutant BMI1 promoter-reporters constructs (as indicated) with pRL-TK plasmid were transiently transfected into MCF7 (A) or MCF10A (B) cells, and luciferase (luc) assays were performed. F and R represent Firefly and Renilla respectively. The error bars represent the means ± S.D. of three independent experiments. C, the qRT-PCR analysis of endogenous BMI1 was performed using primers specific for endogenous BMI1 that amplify the 3′ non-coding region of BMI1 and RNA isolated from an MCF10A series of cells (B0 and Ctrl-i, controls; BMI1, BMI1-overexpressing; BMI-i, BMI1 knockdown cells). Error bars represent ±S.D. *, p < 0.05 (Student's t test).

FIGURE 2.

BMI1 autoactivation involves WNT pathway.A, the heat map of the gene expression profile of the human WNT pathway genes in control and BMI1 shRNA-expressing MCF10A cells. The color scale depicts the relative gene expression between the two cell lines: red indicates an increase with a ΔCT value below the neutral level, and green indicates a decrease with a ΔCT value above the neutral level. The broken arrow indicates c-Myc, whereas the solid arrows represent DKK1, DKK2, and DKK3. B, the LEF/TCF reporter assay was performed to confirm the regulation of the WNT pathway by BMI1. The TOPflash reporter contains wild type LEF/TCF binding sites, whereas the FOPflash reporter contains mutant LEF/TCF binding sites. These constructs were transiently transfected into an MCF10A series, MCF7 control, and MCF7 BMI1 knockdown cells, and the reporter activity was measured as described under “Experimental Procedures.” C, the qRT-PCR analysis of Cyclin D1 and c-Myc was performed using RNA isolated from control, BMI1-overexpressing, and BMI1 knockdown cells as indicated. D, Western blot analysis of WNT pathway-related proteins c-Myc, Cyclin D1, β-catenin, and phospho (p)-β-catenin in the indicated cell types derived from MCF10A and MCF7 was performed using specific antibodies as described under “Experimental Procedures.” E, the ΔPS mutant of BMI1 is more active than wild type BMI1 in activating the WNT pathway and up-regulating its target genes, c-Myc and Cyclin D1. The Western blot analysis of WNT pathway genes (as indicated) was performed using total cell extracts of MCF10A-B0, MCF10A-BMI1WT, and MCF10A-BMI1ΔPS cells (E, top panel). The qRT-PCR analysis of endogenous BMI1 was performed using MCF10A-derived cells (as indicated) and primers specific for the BMI1 coding region and 3′-UTR (designed for detection of endogenous BMI) (E, bottom panel). A representative Western blot and protein quantification data from three different experiments are shown in D and E. Error bars represent ±S.D. *, p < 0.05 (Student's t test). Ctrl, control.

FIGURE 3.

The expression of BMI1 and DKK1 inversely correlates.A, Western blot analysis of BMI1 and qRT-PCR analysis of DKK1 were performed using total cell extract or RNA isolated from MCF10A and various breast cancer cell lines as indicated. The error bars represent the means ±S.D. of three independent assays. The Pearson's correlation coefficient (r) was calculated using Microsoft Excel data analysis tools. B, knockdown of BMI1 up-regulates DKK1, DKK2, and DKK3. qRT-PCR analysis of BMI1 and DKK family members was carried out using specific primers and RNA isolated from an MCF10A series of cells (described in Fig. 1). C, BMI1 regulation of DKK1 was studied in MCF10A control and BMI1-overexpressing MCF10A cells using qRT-PCR and Western blot analysis as described under “Experimental Procedures.” Each experiment was done in triplicates. Error bars represent ±S.D. *, p < 0.05 (Student's t test). D, the expression of BMI1, DKK1, Wnt1, and c-Myc was studied in an MCF10A tumor progression series using a Western blot analysis. A representative Western blot and protein quantification data are shown in C and D. Ctrl, control.

FIGURE 4.

BMI1 regulates DKK1 promoter and binds to it.A, the DKK1 promoter (1 kb upstream) was cloned in pGL4.18 luciferase reporter vector, and its activity was determined as described under “Experimental Procedures.” F and R represent Firefly and Renilla, respectively. Error bars represent ±S.D. *, p < 0.05 (Student's t test). B and C, binding of BMI1 and H3K27me3 to DKK1 promoter was determined using a ChIP assay in the indicated set of cells and four different sets of primers covering 1 kb of promoter region as described under “Experimental Procedures.” Ctrl, control.

FIGURE 5.

DKK1 regulates BMI1 expression.A, DKK1-expressing plasmid (0–5 μg) was transiently transfected into 293T cells, and the expression of DKK1, BMI1, and c-Myc was determined by qRT-PCR analysis (left panel) and Western blot analysis (right panel). GFP-tubulin plasmid was transfected as a loading control with DKK1 plasmid. Ponceau staining of the blot was also done to ascertain equal loading of supernatant extract used for DKK1 probing. B, MCF10A cells with DKK1 knockdown were generated and analyzed for the expression of DKK1, BMI1, and c-Myc using qRT-PCR and Western blot analysis. In each case, a representative Western blot and protein quantification data are shown. Error bars represent ±S.D. *, p < 0.05 (Student's t test). Ctrl, control.

FIGURE 6.

WNT pathway regulates BMI1 expression.A, 293T cells were transiently transfected with a Wnt1-expressing plasmid (0–5 μg), and the expression of Wnt1, BMI1, and c-Myc was determined by qRT-PCR (left panel) and Western blot analysis (right panel). Controls are described in Fig. 5A. A representative Western blot and protein quantification data are shown (right panel). B, promoter-reporter assays using pGL3-Bmi1PrWT and pGL3-Bmi1PrMut vectors were performed in 293T cells transiently transfected to overexpress DKK1 or Wnt1. F and R represent Firefly and Renilla respectively. C, the reporter activity of wild type and mutant BMI1 promoters was determined in MCF7 cells stably expressing DKK1 or Wnt1 (as indicated). Error bars represent ±S.D. *, p < 0.05 (Student's t test). Ctrl, control.

FIGURE 7.

BMI1 and WNT regulators modulate oncogenic activity of each other.A, MCF7 cells expressing a control shRNA (Ctrl-i) or BMI1 shRNA (BMI1-i) were infected with an empty vector (vector) or a Wnt-expressing retroviral vector (Wnt1), and resulting cells were studied for cell proliferation (left panel) and colony formation in soft agar (right panel). B, the ability of BMI1 to overcome DKK1 tumor suppressor activity in MCF7 cells was studied using cell proliferation and colony formation assays in soft agar (as indicated). C, knockdown of DKK1 restores proliferation and colony formation in soft agar in MCF7 cells that express BMI1 shRNA. The combinations of cells expressing a single shRNA (BMI1 or DKK1) or both shRNAs (BMI1 and DKK1) were generated using respective retroviral vectors. Error bars represent ±S.D. *, p < 0.05 (significant); **, p = 0.08 (not significant) (Student's t test).

FIGURE 8.

BMI1 and DKK1 regulate breast cancer stem cell phenotype.A, knockdown of BMI1 results in a decrease in the number of CSCs, which can be restored by Wnt1 overexpression. B, DKK1 decreases the number of CSCs, which are restored by BMI1 overexpression. C, decrease in the number of mammospheres and ALDH-positive fraction of cells in BMI1 knockdown cells is overcome by knocking down the expression of DKK1. Mammosphere formation (upper panel) and ALDEFLUOR (lower panel) assays were carried out using MCF7-derived cells as indicated (upper panel). Assays were performed as described under “Experimental Procedures.” In the case of ALDEFLUOR assays, diethylaminobenzaldehyde (DEAB; ALDH inhibitor)-treated cells were used to set the background. For the mammosphere formation assay, the average diameter of spheres was >50 μm. Error bars represent ±S.D. *, p < 0.05 (significant); **, p > 0.05 (not significant) (Student's t test). Ctrl, control.

FIGURE 9.

Schematic model of autoregulation of BMI1. BMI1 activates the WNT pathway by repressing the expression of DKK family members (DKKs), which leads to up-regulation of c-Myc. c-Myc in turn up-regulates BMI1 via a c-Myc binding site (E-box) that is present in its promoter. BMI1 can also directly activate expression of certain WNT factors (broken line), resulting in up-regulation of c-Myc and ultimately transcriptional up-regulation of BMI1. FZD, Frizzled; LRP, LDL receptor-related protein; DVL, Dishevelled; β-TRCP, β-transducin repeat-containing protein; APC, anaphase-promoting complex.