PTEN inhibits BMI1 function independently of its phosphatase activity

Abstract

Background: PTEN is the second most mutated tumor suppressor gene other than p53. It suppresses tumorigenesis by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate (PIP3) to phosphatidylinositol (4,5)-biphosphate (PIP2), thereby directly inhibiting phosphatidylinositol 3 kinase (PI3K)-mediated tumorigenic activities. Consistent with this model of action, cytosolic PTEN is recruited to the plasma membrane to dephosphorylate PIP3. While nuclear PTEN has been shown to suppress tumorigenesis by governing genome integrity, additional mechanisms may also contribute to nuclear PTEN-mediated tumor suppression. The nuclear protein BMI1 promotes stem cell self-renewal and tumorigenesis and PTEN inhibits these events, suggesting that PTEN may suppress BMI1 function.

Results: We investigated whether PTEN inhibits BMI1 function during prostate tumorigenesis. PTEN binds to BMI1 exclusively in the nucleus. This interaction does not require PTEN's phosphatase activity, as phosphatase-deficient PTEN mutants, PTEN/C124S (CS), PTEN/G129E (GE), and a C-terminal PTEN fragment (C-PTEN) excluding the catalytic domain, all associate with BMI1. Furthermore, the residues 186-286 of C-PTEN are sufficient for binding to BMI1. This interaction reduces BMI1's function. BMI1 enhances hTERT activity and reduces p16(INK4A) and p14(ARF) expression. These effects were attenuated by PTEN, PTEN(CS), PTEN(GE), and C-PTEN. Furthermore, knockdown of PTEN in DU145 cells increased hTERT promoter activity, which was reversed when BMI1 was concomitantly knocked-down, indicating that PTEN reduces hTERT promoter activity via inhibiting BMI1 function. Conversely, BMI1 reduces PTEN's ability to inhibit AKT activation, which can be attributed to its interaction with PTEN in the nucleus, making PTEN unavailable to dephosphorylate membrane-bound PIP3. Furthermore, BMI1 appears to co-localize with PTEN more frequently in clinical prostate tissue samples from patients diagnosed with PIN (prostatic intraepithelial neoplasia) and carcinoma compared to normal prostate epithelium. While PTEN co-localized with BMI1 in 2.4% of normal prostate epithelial cells, co-localization was observed in 37.6% and 18.5% of cells in PIN and carcinoma, respectively. Collectively, we demonstrate that PTEN inhibits BMI1 function via binding to BMI1 in a phosphatase independent manner.

Conclusion: We demonstrate that nuclear PTEN reduces BMI1 function independently of its phosphatase activity. It was recently observed that nuclear PTEN also suppresses tumorigenesis. Our results, therefore, provide a plausible mechanism by which nuclear PTEN prevents tumorigenesis.

Figures

Figure 1

PTEN binds to BMI1. (A) 293T cells were transiently transfected with FLAG-tagged BMI1 and HA-tagged PTEN. Cell lysates were prepared and immunoprecipitated with anti-PTEN (top panel) and anti-FLAG (M2) (middle panel) antibodies. The precipitates and lysates (bottom panel) were analyzed by western blot using the indicated antibodies. The * symbol indicates endogenous PTEN. (Note: the reason why endogenous PTEN was not detected in the lysate panel was attributable to a low level of endogenous PTEN in 293T cells). (B) DU145 cell lysates were cross-linked with DSP, immunoprecipitated with anti-BMI1 antibody or control IgG, and analyzed by western blot for PTEN and BMI1. Twenty percent of cell lysate used for immunoprecipitation was also analyzed by western blot. (C) Co-localization between PTEN and BMI1. Ectopic PTEN and ectopic BMI1 in 293T cells and their respective endogenous proteins in MCF-7 and DU145 cells were examined by double immunofluorescent (IF) staining. Nuclei were counter-stained with DAPI (blue). Scale bar represents 10 μM.

Figure 2

Characterization of the interaction between PTEN and BMI1 proteins. (A) PTEN binds to BMI1 independently of its phosphatase activity. 293T cells were transiently transfected with BMI1, PTEN, PTEN(C124S) (C124S), PTEN(G129E) (G129E), a C-terminal PTEN fragment (residues 186-403) (C-PTEN) for 48 hours. Cell lysates were prepared and immunoprecipitated with anti-PTEN and anti-FLAG (M2) (for ectopic BMI1) antibodies. The precipitates and lysates were analyzed by western blot using the indicated antibodies. The # and * symbols indicate endogenous PTEN and a possible oligomer of C-PTEN, respectively. (B) Mapping the BMI1 binding motif of the PTEN protein. A set of PTEN truncation mutants were constructed. Their interaction with BMI1 was examined. C2: C2 domain. The + and - symbols indicate binding or not-binding of individual PTEN proteins to BMI1. (C) C2 binds to BMI1. FLAG-tagged BMI1 and HA-tagged C2 were transfected into 293T cells as indicated. BMI1 was immunoprecipitated with an anti-FLAG antibody (M2) or a control IgG (IgG), followed by western blot examination for BMI1 and C2. 20% of the cell lysates that were used for immunoprecipitations were also analyzed. The * symbols indicate background bands. (D) C2N binds to BMI1. C2N, C2C, and C-tail (left panel) and their GFP fusion counterparts (right panel) were co-transfected with either an empty vector (-) or FLAG-tagged BMI1 as indicated, followed by immunoprecipitation with M2 or control IgG (IgG) and then western blot (WB) with the indicated antibodies. The respective cell lysates were shown at the bottom panels.

Figure 3

PTEN inhibits BMI1 function. (A) DU145 cells were stably transfected with pBabe or pBabe-BMI1 retrovirus, followed by transiently transfected with pLHCX (empty vector) and pLHCX-PTEN retrovirus for 48 hours. The expression of FLAG-tagged BMI1, HA-tagged PTEN, p16INK4A, p14ARF, and actin was examined by western blot using specific antibodies. The relative p14ARF and p16INK4A expression was normalized against the respective actin and then expressed as fold changes of p14ARF and p16INK4A in DU145 cells co-infected with pBabe and pLHCX. The experiment was repeated at least three times by three individuals with identical results and representatives are shown. This information was presented under the p14 and p16 panels. Symbols * and ** show statistical significance (p < 0.05 and p < 0.01, respectively), in comparison to pBabe/pLHCX infected cells, determined by Student's t-Test (2-tails). (B) DU145 cells were stably transfected with pBabe and pBabe-BMI1 retrovirus, followed by assaying for hTERT activity using TRAP assay following the manufacturer's procedure. (C) 293T cells were transiently transfected with PTEN, PTEN(G129E) (G129E), PTEN(C124S) (C124S), C-terminal PTEN fragment (residues 186-403) (C-term), and BMI1 as indicated together with a hTERT promoter driven luciferase construct plus a β-Gal construct for 48 hours. Luciferase and β-Gal enzymatic activities were determined. Luciferase activities were normalized against β-Gal activities. Each transfection was carried out in triplicate and the experiment was repeated three times. **: p < 0.01 (in comparison to pcDNA); ++: p < 0.01 (in comparison to BMI1).

Figure 4

Nuclear PTEN reduces BMI1 function. (A) Interaction of nuclear PTEN with BMI1. Chimpanzee PTEN and the indicated mutants PTEN/1-375 and PTEN/1-375(K13A) were transfected without and with FLAG-tagged BMI1 in 293T cells, followed by immunoprecipitation of BMI1 using an anti-FLAG (α-FLAG) and then immunobloted (IB) with the indicated antibodies. Control IgG did not precipitate either BMI1 or PTEN (data not shown). (B) Nuclear PTEN inhibits BMI1 function. DU145 cells were transiently expressed with PTEN/1-375 (top panel) or PTEN/1-375(K13A) (bottom panel). Cells were then double IF stained for ectopic PTEN mutants using an anti-HA antibody (red) or endogenous p14ARF (green). Nuclei were counter-stained with DAPI (blue). More than 200 transfected cells were randomly counted. Typical images of 1-375 and 1-375(K13A) were shown and the related quantification was discussed (see Discussion for details).

Figure 5

PTEN reduces hTERT promoter activity via inhibiting BMI1 function. DU145 cells were transfected with 100 nM PTEN siRNA, BMI1 siRNA, PTEN siRNA plus BMI1 siRNA, and the respective control (Ctrl) siRNA (Dharmcon) using LipofectAMINE2000 (Invitrogene) for 48 hours following our published procedure [52]. The expression of individual proteins was examined by western blot using specific antibodies (top panel). These cells were transfected with a pGL3-hTERTmin-Luc reporter, a lacZ vector, and plus the indicated siRNAs for 48 hours. Luciferase activities were determined and normalized against the respective lacZ activity. Experiments were carried out in triplicate and were repeated three times. Average data of these independent experiments is shown. Luciferase activities in PTEN siRNA (p < 0.001) and BMI1 siRNA (p < 0.05) cells are significantly different from that in Ctrl siRNA cells (bottom panel).

Figure 6

BMI1 reduces PTEN's ability to inactivate the PI3K-AKT pathway. DU145 cells were infected by PTEN, BMI1, or the respective empty retrovirus (-) as indicated. Cells were selected in the respective antibiotics for a few days to achieve 100% infection. The expression of AKT phosphorylation (AKT-P), total AKT (AKT), ectopic BMI1, ectopic PTEN, and actin was determined by western blot using the respective antibodies. The relative levels of AKT-P were quantified.

Figure 7

Co-localization between PTEN and BMI1 in primary prostate cancer tissues. Double IF staining for BMI1 (red) and PTEN (green) in normal prostatic gland (normal), PIN, PTEN-negative carcinoma, and PTEN-positive carcinoma. Nuclei were counter-stained with DAPI (blue). Images were captured with a confocal microscope. Scale bar represents 20 μM.