Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer

Abstract

Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis.

Figures

Figure 1. Bmi1 cooperates with PTEN haploinsufficiency in prostate cancer progression.

(A) Schematic representation of the ROSA26 Lox-STOP-Lox Bmi1 allele (Bmi1LSL). (B) PIN- and carcinoma-free mice at the indicated ages. n = 29 (PbCre4); 36 (PbCre4;Bmi1LSL); 26 (PbCre4;PtenloxP/+); 24 (PbCre4;Bmi1LSL;PtenloxP/+). (C) Prostate histopathology of 1-year-old animals. PbCre4 controls (n = 21) appeared mostly normal. PbCre4;Bmi1LSL (n = 25) and PbCre4;PtenloxP/+ (n = 19) mice developed atypical hyperplasia (AH) and PIN lesions with incomplete penetrance, whereas only PbCre4;Bmi1LSL;PtenloxP/+ mice (n = 16) developed prostate adenocarcinoma. (D–G) H&E-stained prostate sections. (D) Normal epithelium in PbCre4 controls. (E) PIN lesion (asterisk) in PbCre4;Bmi1LSL mice. (F) PIN lesion (asterisk) in PbCre4;PtenloxP/+ mice. (G) Adenocarcinoma in PbCre4;Bmi1LSL;PtenloxP/+ animals. Black arrowhead, mitotic figure; white arrowheads, blood vessels. Insets are enlarged 5-fold. (H–O) Immunohistochemical expression analysis of Bmi1-phosphorylated Akt (H–K) and pAkt (S473) (L–O). Scale bars: 100 μm.

Figure 2. Secretory luminal epithelial phenotype of PbCre4;Bmi1LSL;PtenloxP/+ prostate cancer cells.

Immuno­histochemical staining of formalin-fixed paraffin-embedded prostate tissue sections. (A–D) Cytokeratin 14 (CK14) and (E–H) p63, marking basal cells. (I–L) Synaptophysin, marking neuroendocrine cells. (M–P) Cytokeratin 8 (CK8), (Q–T) androgen receptor (AR), and (U–X) E-cadherin, marking secretory luminal cells. Insets are enlarged 5-fold; in X, insets show both tumor (T) and normal prostatic duct (N). Scale bar: 100 μm.

Figure 3. Bmi1 expression and activation of PI3K/Akt pathway correlate in high-grade prostate cancer clinical samples.

(A) Representative immunohistochemical staining for Bmi1 and pAkt on adjacent sections of formalin-fixed, paraffin-embedded human prostate cancer specimens. Scale bar: 100 μm. (B) Immunohistochemical assessment of Bmi1 and pAkt expression status in 168 human prostate tumors categorized according to histological or clinical grade (see Table 1). Bmi1+pAkt+ tumors were associated with high Gleason scores (P < 0.001) and with advanced pathological stage (P = 0.002). (C) Immunohistochemical assessment of Bmi1 and pAkt expression status in 168 human prostate tumors categorized according to both histological and clinical grade (see Table 1). Bmi1+pAkt+ tumors were associated with worsening of both Gleason score and pathological stage (i.e., Gleason ≥ 8 and pT3; P = 0.001).

Figure 4. The Pten/PI3K/pAkt axis modulates Bmi1 phosphorylation.

(A) Western blot analysis and relative quantification of pBmi1/Bmi1 ratio in Jurkat, PC3, and LNCaP Pten-deficient pAkt-high cells and in MCF7, DU145, and U2OS Pten-proficient pAkt-low cells. (B) Western blot showed that retrovirus-induced expression of PTEN in LNCaP cells inhibited pAkt and pBmi1. (C) Bmi1 phosphorylation was positively modulated by pAkt level. Bmi1 phosphorylation and pAkt and PTEN status in DU145, PC3, LNCaP, and PNT1a prostate cell lines either left untreated or treated with 5 μM Akt inhibitor VIII (isozyme selective, Akt-1/2; Calbiochem) for 12 hours and refreshed 2 hours prior to lysis. pBmi1/Bmi1 ratios, quantified using ImageJ, are indicated within blots (2 separate experiments shown). The 3 lower panels corresponded to experiment 2. Phosphorylation status of the Akt substrate GSK-3β (pSer9) is shown as a control for Akt activity. Tubulin was used as a loading control.

Figure 5. Bmi1 is phosphorylated by Akt.

(A) In vitro Akt kinase assay with recombinant active Akt and Bmi1-WT/Ring1B or Bmi1-3A/Ring1B complex. Reactions were carried out for 40 minutes at 30°C in the presence of [γ-32P]ATP. GST and GST-GSK3α/β crosstide were used as negative and positive controls, respectively; phosphorylated proteins were visualized by autoradiography. A Coomassie-stained gel demonstrating equal loading is also shown. (B) Time course Akt kinase assay with recombinant active Akt and Bmi1-WT/Ring1B or Bmi1-3A/Ring1B complex. Equal amounts of substrate proteins and Akt were used in each assay. (C) Western blot analysis showing that HA-tagged Bmi1-3A was not phosphorylated in LNCaP cells.

Figure 6. Effect of Bmi1 phosphorylation on H2A ubiquitination and cellular senescence.

(A) Bmi1/Ring1B recombinant complex was first treated with AP or Na3V04-inactivated AP, then assayed for ubiquitin ligase activity on purified nucleosomes. Dephosphorylation of Bmi1 reduced Bmi1/Ring1B E3 ligase activity on H2A (Ub-H2A); Mel18/Ring1B is shown as a positive control. Autopolyubiquitination of Bmi1/Ring1B and Mel18/Ring1B complexes is also shown. (B) Bmi1/Ring1b complex was first incubated with Akt or inactive Akt (inAkt), then assayed for ubiquitin ligase activity as in A. (C) Global Ub-H2A levels were not affected in doxycycline-treated (Dox) LNCaP-tet-shBmi1 cells expressing Bmi1-3A, nor in the presence of Akt inhibitor. (D) Phosphorylation of Bmi1 was not required for repression of cellular senescence. Bmi1–/– MEFs were infected with a control retrovirus (EV; empty vector) or with retroviruses encoding Bmi1-WT or Bmi1-3A. 3T3 assay was performed over a period of 32 days; Western blot against Bmi1, p16INK4A, and tubulin (loading control) is also shown.

Figure 7. Phosphorylation of Bmi1 stimulates H2A ubiquitination at DNA DSBs.

(A) Phosphorylation of Bmi1 was required for H2A ubiquitination at laser scissors–induced DNA breaks. Bmi1–/–;Ink4a/Arf–/– MEFs were reconstituted with either Bmi1-WT or Bmi1-3A, then treated with UV laser scissors in the presence or absence of Akt inhibitor. Cells were then processed for immunofluorescence using antibodies to 53BP1 and either Bmi1 or Ub-H2A. Percent cells with laser scissors–induced 53BP1 localization is also shown, demonstrating colocalization of Bmi1 and Ub-H2A with 53BP1 in each of these conditions. (B) Western blot showed that global UV irradiation (20 J/m2) induced phosphorylation of both Bmi1 and Akt.

Figure 8. Phosphorylation of Bmi1 enhances HR and genomic instability.

(A) Phosphorylation of Bmi1 was required for efficient DNA DSB repair by HR. The HR GFP reporter is stably integrated into the genome of U2OS cell line. SceGFP is a GFP gene that contains an I-SceI endonuclease site within the coding region. Cleavage of the I-SceI site in vivo and repair by HR directed by the downstream iGFP repeat results in GFP+ cells. HR GFP U2OS cells were transfected with scrambled (Sc) siRNA or Bmi1 siRNA reconstituted with constructs expressing mouse empty vector, Bmi1-WT, or Bmi1-3A. Where indicated, cells were also transfected with an I-SceI expression vector. After 24 hours, the percentage of GFP+ cells was measured by flow cytometry. Data are means ± SD of 3 independent experiments. *P = 0.006 versus Bmi1-WT, unpaired t test. (B) Frequency of spontaneous SCEs in doxycycline-treated LNCaP-tet-shBmi1 prostate cancer cells infected with retroviruses expressing Bmi1-WT, Bmi1-3A, or control empty vector. SCEs were increased 2.3-fold with Bmi1-WT, but not Bmi1-3A, overexpression (*P < 0.0001, unpaired t test). Akt inhibition (Akti) suppressed Bmi1-WT–induced SCEs. Data are means ± SEM. Examples of SCE events (arrowheads) are shown in the inset, which depicts subsets of chromosomes in a metaphase spread labeled for SCE analysis (original magnification, ×200). (C) Immunohistochemistry for γH2AX revealed greater DNA damage in tumor areas of PbCre4;Bmi1LSL;PtenloxP/+ mutant prostates (n = 27) and Bmi1+pAkt+ human samples (n = 77). Scale bars: 100 μm.

Figure 9. Phosphorylation of Bmi1 regulates its oncogenic potential.

(A) Ink4a/Arf–/– MEFs were infected with the indicated retroviral constructs and assayed for anchorage-independent growth (soft agar) for 4 weeks. Data (mean ± SD) represent number of colonies from 2 independent experiments performed in triplicate. (B) Bmi1 reconstitution in human prostate cancer cells. LNCaP-tet-shBmi1 cells infected with the indicated retroviral constructs were incubated for 8 days in the presence of doxycycline and then stained with crystal violet; cell growth was estimated by OD quantification of the crystal violet at 590 nm. Data are mean ± SD of 3 independent experiments. (C) LNCaP-tet-shBmi1 cells infected with the indicated retroviral constructs were injected subcutaneously into nude mice. Tumor size was measured after 4 weeks. Data are means ± SE (n ≥ 12). P = 0.008, empty vector versus Bmi1-WT; P = 0.002, Bmi1-3A versus Bmi1-WT. (D) Percent tumor-bearing mice 3 months after orthotopic transplantation with LHMshP hPrECs (n = 11 per group). (E) Kaplan-Meier analysis of overall lymphoma-free survival. Eμ-Myc fetal liver hematopoietic cells were infected with the indicated retroviral constructs and injected intravenously to reconstitute lethally irradiated recipient mice (n = 5 per group). P = 0.062, empty vector versus Bmi1-3A; P = 0.006, empty vector versus Bmi1-WT; P = 0.026, Bmi1-3A versus Bmi1-WT. *P < 0.05.

Figure 10. Model for Bmi1 and PTEN/PI3K/Akt collaboration in prostate cancer.

Schematic model illustrates the synergy of Bmi1 and PTEN/PI3K/Akt pathways in prostate tumorigenesis. PTEN loss activates the PI3K/Akt pathway and positively modulates p53 levels, leading to the activation of PTEN-induced cellular senescence (PICS). Bmi1 counters cellular senescence by repressing Ink4a/Arf. Upon Akt-mediated phosphorylation, Bmi1 acts mainly through an Ink4a/Arf-independent mechanism to promote prostate carcinogenesis in collaboration with the PI3K/Akt pathway.