Function Of Bmi1 Research Articles

Dysregulation of Bmi1 promotes malignant transformation of hepatic progenitor cells.

Adult hepatic progenitor cells (HPCs) are involved in a wide range of human liver diseases, including hepatocellular carcinoma (HCC). Bmi1 has been reported to have vital roles in stem cell self-renewal and carcinogenesis. We have previously demonstrated that Bmi1 is upregulated in HCC with bile duct tumor thrombi, a subtype of HCC characterized by profuse expression of hepatic stem cell markers. However, the function of Bmi1 in HPCs has not yet been well elucidated. The current study was designed to investigate the effects of Bmi1 on the biological properties of rat HPCs. To accomplish this, Bmi1 was silenced or enhanced in two HPC cell lines (WB-F344 and OC3) by, respectively, using either small interfering RNA against Bmi1 or a forced Bmi1 expression retroviral vector. The biological functions of Bmi1 in HPCs were investigated through cell proliferation assays, colony-formation assays, cell cycle analysis and invasion assays, as well as through xenograft-formation assays. In this study, genetic depletion of Bmi1 repressed cell proliferation, colony formation and invasion in both assessed HPC cell lines relative to controls. Conversely, forced expression of Bmi1 in two HPCs cell lines promoted cell proliferation, colony formation and invasion in vitro. Aldehyde dehydrogenase (ALDH) assay revealed a significant increase in the number of ALDH-positive cells following the forced expression of Bmi1 in HPCs. Most importantly, transplantation of forced Bmi1 expression HPCs into nude mice resulted in the formation of tumors with histological features of poorly differentiated HCC. Taken together, our findings indicate that forced expression of Bmi1 promotes the malignant transformation of HPCs, suggesting Bmi1 might be a potential molecular target for the treatment of HCC.

The Polycomb Repressive Complex 1 Protein BMI1 Is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells

The polycomb repressive complex 1 (PRC1), containing the core BMI1 and RING1A/B proteins, mono-ubiquitinylates histone H2A (H2A(ub)) and is associated with silenced developmental genes at facultative heterochromatin. It is, however, assumed that the PRC1 is excluded from constitutive heterochromatin in somatic cells based on work performed on mouse embryonic stem cells and oocytes. We show here that BMI1 is required for constitutive heterochromatin formation and silencing in human and mouse somatic cells. BMI1 was highly enriched at intergenic and pericentric heterochromatin, co-immunoprecipitated with the architectural heterochromatin proteins HP1, DEK1, and ATRx, and was required for their localization. In contrast, BRCA1 localization was BMI1-independent and partially redundant with that of BMI1 for H2A(ub) deposition, constitutive heterochromatin formation, and silencing. These observations suggest a dynamic and developmentally regulated model of PRC1 occupancy at constitutive heterochromatin, and where BMI1 function in somatic cells is to stabilize the repetitive genome.

A Novel Aspect of Tumorigenesis-BMI1 Functions in Regulating DNA Damage Response.

BMI1 plays critical roles in maintaining the self-renewal of hematopoietic, neural, intestinal stem cells, and cancer stem cells (CSCs) for a variety of cancer types. BMI1 promotes cell proliferative life span and epithelial to mesenchymal transition (EMT). Upregulation of BMI1 occurs in multiple cancer types and is associated with poor prognosis. Mechanistically, BMI1 is a subunit of the Polycomb repressive complex 1 (PRC1), and binds the catalytic RING2/RING1b subunit to form a functional E3 ubiquitin ligase. Through mono-ubiquitination of histone H2A at lysine 119 (H2A-K119Ub), BMI1 represses multiple gene loci; among these, the INK4A/ARF locus has been most thoroughly investigated. The locus encodes the p16INK4A and p14/p19ARF tumor suppressors that function in the pRb and p53 pathways, respectively. Its repression contributes to BMI1-derived tumorigenesis. BMI1 also possesses other oncogenic functions, specifically its regulative role in DNA damage response (DDR). In this process, BMI1 ubiquitinates histone H2A and γH2AX, thereby facilitating the repair of double-stranded DNA breaks (DSBs) through stimulating homologous recombination and non-homologous end joining. Additionally, BMI1 compromises DSB-induced checkpoint activation independent of its-associated E3 ubiquitin ligase activity. We review the emerging role of BMI1 in DDR regulation and discuss its impact on BMI1-derived tumorigenesis.

Knockdown of Bmi1 inhibits bladder cancer cell growth both in vitro and in vivo by blocking cell cycle at G1 phase and inducing apoptosis.

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. However, its role in the initiation and progression of bladder cancer is not clearly known. The present study aimed to investigate the function of Bmi1 in the development of bladder cancer. Bmi1 expression was detected in human bladder cancer tissues and their adjacent normal tissues (n=10) by immunohistochemistry, qRT-PCR and Western blotting, respectively. Bmi1 small interference RNA (siRNA) was synthesized and transfected into human bladder carcinoma cells (EJ) by lipofectamine 2000. The Bmil expression at mRNA and protein levels was measured in EJ cells transfected with Bmil siRNA (0, 80, 160 nmol/L) by qRT-PCR and Western blotting, respectively. Cell viability and Ki67 expression (a marker of cell proliferation) were determined in Bmi1 siRNA-transfected cells by CCK-8 assay and qRT-PCR, respectively. Cell cycle of transfected cells was flow-cytometrically determined. Immunofluorescence and Western blotting were used to detect the expression levels of cell cycle-associated proteins cyclin D1 and cyclin E in the cells. Pro-apoptotic proteins Bax and caspase 3 and anti-apoptotic protein Bcl-2 were detected by Western blotting as well. Additionally, xenograft tumor models were established by inoculation of EJ cells (infected with Bmil shRNA/pLKO.1 lentivirus or not) into nude mice. The tumor volumes were measured every other day for 14 days. The results showed that the Bmil expression was significantly increased in bladder tumor tissues when compared with that in normal tissues (P<0.05). Perturbation of Bmi1 expression by using siRNA could significantly inhibit the proliferation of EJ cells (P<0.05). Bmi1 siRNA-transfected EJ cells were accumulated in G1 phase and the expression levels of cyclin D1 and cyclin E were down-regulated. Bax and caspase-3 expression levels were significantly increased and Bcl-2 levels decreased after Bmi1 knockdown. Tumor volume was conspicuously reduced in mice injected with EJ cells with Bmi1 knockdown. Our findings indicate that Bmi1 is a potential driver oncogene of bladder cancer and it may become a potential treatment target for human bladder cancer.

MiR-300 mediates Bmi1 function and regulates differentiation in primitive cardiac progenitors.

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a polycomb-family transcriptional factor critical for self-renewal in many adult stem cells and human neoplasia. We sought to identify microRNAs regulated by Bmi1 that could play a role in multipotent cardiac progenitor cell (CPC) decisions. We found that miR-300, a poorly characterized microRNA mapping in the Dlk1-Dio3 microRNA cluster, was positively regulated by Bmi1 in CPCs. Forced expression of miR-300 in CPCs promoted an improved stemness signature with a significant increase in Oct4 levels, a reduction in senescence progression and an enhanced proliferative status via p19 activation and inhibition of p16 accumulation. Endothelial and cardiogenic differentiation were clearly compromised by sustained miR-300 expression. Additionally, RNA and protein analysis revealed a significant reduction in key cardiac transcription factors, including Nkx2.5 and Tbx5. Collectively, these results suggest that some functions attributed to Bmi1 are due to induction of miR-300, which decreases the cardiogenic differentiation potential of multipotent CPCs in vitro and promotes self-renewal.

BMI1, ATM and DDR.

BMI1, ATM and DDR.

Abstract 4647: Bioactive tanshinones and tea polyphenols inhibit prostate cancer stem cells by targeting bmi1

Abstract Prostate cancer (CaP) is the most common invasive malignancy and the second leading cause of cancer-related death in U.S. men. Androgen-independent/hormone-refractory CaP remains the primary cause of CaP death. The search for effective therapeutic and preventive strategies, particularly the ways to intervene in the progression of androgen-independent tumors remains the top priority of CaP research. Prostate cancer stem cells (CSCs) are suggested to be responsible for initiation, progression, drug resistance, recurrence and metastasis of CaP. Strategies that target prostate CSCs may significantly prevent and/or delay cancer progression and metastasis. The objective of this study was to identify the potent natural bioactive compounds for targeting prostate CSCs. The sphere-forming efficiencies in LNCaP, DUCaP, PC3 and DU145 prostate cancer cell lines were 2.8%, 15.7%, 1%, and 5.6%, respectively. The expression of several CSC related biomarkers, such as c-met, c-kit, bmi1 and CD44 were increased, whereas the expression of androgen receptor was decreased in prostate spheres. Natural compounds, especially tanshinones (tanshinone I (T1), tanshinone IIA (T2A), cryptotanshinone) and tea compounds (epigallocatechin gallate (EGCG) and theaflavines), inhibited the sphere-forming efficiency of CaP cells with T1 and T2A the most potent. Furthermore, T1 and EGCG significantly downregulated Bmi1 gene expression and protein level. Since Bmi1 has been reported to be a crucial regulator of stem cell self-renewal and malignant transformation, we further studied the functional role of bmi1 in self-renewal of prostate CSCs. Silencing of bmi1 dramatically inhibited the growth and decreased the sphere-forming ability of CaP cells. Animal studies showed that T1 and EGCG inhibited the growth and metastasis of prostate tumors associated with downregulation of bmi1 expression. The results derived from our preliminary studies suggest that bmi1 may play an important role in self-renewal ability of prostate CSCs, and that some natural bioactive agents, such as T1 and EGCG, may inhibit self-renewal of prostate CSCs in part via downregulation of bmi1 gene expression and function. Supported in part by NCI/NIH R21 CA153355. Note: This abstract was not presented at the meeting. Citation Format: Jin-Rong Zhou, Yi Gong, Hamid Abdolmaleky. Bioactive tanshinones and tea polyphenols inhibit prostate cancer stem cells by targeting bmi1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2015-4647

High peritumoral Bmi-1 expression is an independent prognosticator of poor prognosis in renal cell carcinoma.

B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is a transcriptional repressor, which plays important roles in the development of cancers, but the function of Bmi-1 in kidney tumorigenesis and its prognostic values remain unclear. This study aims to investigate prognostic values of the intratumoral and peritumoral expression of Bmi-1 in patients with renal cell carcinoma. Expression of Bmi-1 was assessed by immunohistochemistry in specimens containing paired tumor and peritumoral renal tissue from 241 patients who had undergone curative nephrectomy at Zhongshan Hospital from 2005 to 2007. The association of Bmi-1 expression with clinical and pathological parameters and outcomes was investigated. Specific expression of Bmi-1 was found both in peritumoral and intratumoral tissues. High expression of Bmi-1 in peritumoral but not intratumoral is significantly associated with poor overall survival (OS) (P < 0.001) and relapse-free survival (RFS) (P = 0.003). Furthermore, Bmi-1 expression was identified as an independent prognostic factor for OS, and combination of peritumoral Bmi-1 and tumor node metastasis (TNM) stage had a better power to predict the patients' death and disease recurrence. High peritumoral Bmi-1 expression can serve as an independent prognostic biomarker and could be a novel therapeutic target for renal cell carcinoma (RCC).

Bmi1 enhances skeletal muscle regeneration through MT1-mediated oxidative stress protection in a mouse model of dystrophinopathy.

The Polycomb group (PcG) protein Bmi1 is an essential epigenetic regulator of stem cell function during normal development and in adult organ systems. We show that mild up-regulation of Bmi1 expression in the adult stem cells of the skeletal muscle leads to a remarkable improvement of muscle function in a mouse model of Duchenne muscular dystrophy. The molecular mechanism underlying enhanced physiological function of Bmi1 depends on the injury context and it is mediated by metallothionein 1 (MT1)-driven modulation of resistance to oxidative stress in the satellite cell population. These results lay the basis for developing Bmi1 pharmacological activators, which either alone or in combination with MT1 agonists could be a powerful novel therapeutic approach to improve regeneration in muscle wasting conditions.

P21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells.

Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21.

Reprogramming of mouse somatic cells into pluripotent stem-like cells using a combination of small molecules

Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) by overexpression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc. However, exogenous expression of pluripotency factors raised concerns for clinical applications. Here, we show that iPS-like cells (iPSLCs) were generated from mouse somatic cells in two steps with small molecule compounds. In the first step, stable intermediate cells were generated from mouse astrocytes by Bmi1. These cells called induced epiblast stem cell (EpiSC)-like cells (iEpiSCLCs) are similar to EpiSCs in terms of expression of specific markers, epigenetic state, and ability to differentiate into three germ layers. In the second step, treatment with MEK/ERK and GSK3 pathway inhibitors in the presence of leukemia inhibitory factor resulted in conversion of iEpiSCLCs into iPSLCs that were similar to mESCs, suggesting that Bmi1 is sufficient to reprogram astrocytes to partially reprogrammed pluripotency. Next, Bmi1 function was replaced with Shh activators (oxysterol and purmorphamine), which demonstrating that combinations of small molecules can compensate for reprogramming factors and are sufficient to directly reprogram mouse somatic cells into iPSLCs. The chemically induced pluripotent stem cell-like cells (ciPSLCs) showed similar gene expression profiles, epigenetic status, and differentiation potentials to mESCs.

Molecular cloning, sequence analysis, and function of the intestinal epithelial stem cell marker Bmi1 in pig intestinal epithelial cells1

In the present work, we cloned the full-length cDNA of the pig Bmi1 gene (BMI1 polycomb ring finger oncogene), which has been indicated as an intestinal epithelial stem cell (IESC) marker in other mammals. This paper provides the first report of the function of Bmi1 in pig intestinal epithelial cells and a brief description of its underlying mechanism. Rapid amplification of cDNA ends technology was used to clone the complete pig Bmi1 sequence, and a Bmi1-pcDNA3.1 vector was constructed for transfection into an intestinal porcine epithelial cell line (IPEC-1). The proliferation ability of the cells was estimated using the MTT assay and the EdU incorporation method at different time points after seeding. Cell cycle information was detected by flow cytometry. The mRNA abundances of cell cycle-related genes were also measured. The results indicated that the pig Bmi1 cDNA is 3,193 bp in length and consists of a 981 bp open reading frame, a 256 bp 5´ untranslated region (UTR), and a 1,956 bp 3' UTR. The transcript contains no signal peptides, and there are no transmembrane regions in the pig Bmi1 coded protein, which has a total of 326 AA. The overexpression of the pig Bmi1 in the IPEC-1 cells led to increased cell proliferation and a lower percentage of cells in the G1 and S phases (P < 0.05), along with a higher percentage of cells in the G2 phase (P < 0.05). Furthermore, the gene expression levels of PCNA, Cyclin D1, Cyclin D2, Cyclin B, CDK1, and CDK2 were all elevated (P < 0.05) by Bmi1 overexpression, while the gene expression levels of Cyclin A2 and p21 showed little difference (P > 0.05). Our data suggested that pig Bmi1 can increase the proliferation of IPEC-1 cells by promoting the G1/S transition and the overall cell cycle process.

Ubap2l-Bmi-1-Rnf2 Define a Novel Polycomb Complex Essential For Self-Renewal Of Hematopoietic Stem Cells

The polycomb group protein Bmi-1 is a well known determinant of hematopoietic stem cell function. Bmi-1-/- mice display severe hematopoietic defects, including progressive loss of hematopoietic cells from the bone marrow. Bmi-1 is dispensable for hematopoietic stem cell specification, but essential for their maintenance, an effect attributable to its ability to promote HSC self-renewal. The mechanism by which Bmi-1 regulates this process is not completely understood. Bmi-1 has been shown to repress the INK4A/ARF locus encoding the cell cycle inhibitors p16ink4a and p19arf , to interact with the E4F1 protein and to regulate the DNA damage response pathway, however experimental manipulation of these proteins/pathways only partially rescues the hematopoietic defects of the Bmi-1-/-mice. It thus appears that the mechanism by which Bmi-1 regulates HSC self-renewal remains to be determined.Towards this goal, we purified Bmi-1 containing protein complexes from cellular extracts and identified Bmi-1 interaction partners by mass spectrometry. We observed that the protein Ubap2l, which has never been shown to associate with Bmi-1 and for which no link with polycomb group protein function has been described, was consistently found in Bmi-1-containing protein complexes. Immunoprecipitation experiments revealed that Ubap2l indirectly associates with Bmi-1 via an interaction with the polycomb group protein Rnf2. We then evaluated the possibility that Ubap2l might be involved in the regulation of HSC activity. We observed that Ubap2l transcripts are more abundant in primitive HSC populations compared to total BM. CFC assays performed with BM cells infected with Ubap2l shRNAs revealed that Ubap2l knockdown causes a modest and progressive loss of progenitor activity when cells are kept in culture, with multipotent and bipotent progenitors being substantially more affected than unipotent progenitors. We transplanted these cells in mice and observed a gradual decrease in the percentage of donor derived cells expressing Ubap2l shRNAs in the peripheral blood of the recipient mice, with the most striking effect observed 16 weeks post-transplantation in the BM. Bmi-1 has been shown to regulate the proliferative capacity of both progenitor and stem cells, and its deletion in BM cells is known to dramatically reduce the reconstitution activity of these cells at early time points following transplantation. In contrast, Ubap2l appears to preferentially regulate LTR-HSC activity. We tested the effects of Ubap2l silencing on leukemic cells in vivo and observed that a reduction of Ubap2l levels in these cells had an important impact on their ability to reconstitute recipient mice, suggesting that Ubap2l also plays a role in leukemic stem cell activity. We determined if the mechanism by which Ubap2l regulates HSC activity is related to Bmi-1 function by simultaneously introducing Bmi-1 cDNA and Ubap2l shRNAs in BM cells and found that Bmi-1 is able to rescue the long-term reconstitution defect caused by Ubap2l downregulation in these cells. We observed that Ubap2l silencing does not significantly affect the expression of the known Bmi-1 targets p16ink4a and p19arf, implying that Ubap2l regulates HSC activity via a Bmi-1-dependent mechanism that does not involve repression of the INK4A/ARF locus. One explanation for the two Bmi-1 dependent mechanisms at play in the regulation of HSC activity could be that Bmi-1 is part of two separate protein complexes, each regulating different aspects of hematopoietic cell function. To test this hypothesis, we fractionated cellular extracts and were indeed able to resolve two distinct Bmi-1 containing protein complexes, distinguishable by the presence of Ubap2l.Based on the results we obtained, we propose a model in which two different Bmi-1 containing protein complexes regulate hematopoietic stem cell function. An Ubap2l-independent complex, which is most likely involved in the repression of the INK4A/ARF locus, and could be responsible for the effects of Bmi-1 on multipotent progenitors and STR-HSCs, and an Ubap2l-dependent complex, which operates via a yet to be defined mechanism unrelated to p16Ink4a and p19Arf, and would account for the effects of Bmi-1 on LTR-HSC activity. These results position Ubap2l as a key regulator of LTR-HSC activity and unveil a novel protein complex mediating the effects of Bmi-1 on LTR-HSCs. Disclosures:No relevant conflicts of interest to declare.

Tea bioactive compounds inhibit prostate cancer stem cells via downregulation of Bmi1

Cancer stem cells (CSCs) are suggested to be responsible for tumor initiation, progression, drug resistance, recurrence and metastasis. Strategies that target CSCs may significantly prevent and/or delay cancer progression. The objective of this study was to evaluate the effect of tea bioactive components on targeting prostate CSCs. The effect of tea components on the self‐renewal ability of CSCs was evaluated by sphere‐forming efficiency (SFE) assay, gene expression was quantified by real time PCR, and Bmi1 gene silencing was manipulated by siRNA. The SFEs of LNCaP, DUCaP, PC3 and DU145 human prostate cancer cell lines were 2.8%, 15.7%, 1%, and 5.6%, respectively. The expression of several CSC related biomarkers, such as c‐met, Bmi1 and CD44 were increased, whereas the expression of androgen receptor (AR) was decreased in prostate spheres. Tea compounds, especially epigallocatechin gallate and theaflavines inhibited dose‐dependently the SFEs of prostate cancer cells and downregulated the expression of Bmi1 gene. Gene function assay showed that silencing of Bmi1 dramatically inhibited the growth and decreased the sphere‐forming ability of prostate cancer cells. The results derived from our preliminary studies suggest that Bmi1 may play an important role in self‐renewal ability of prostate CSCs, and that tea bioactive components may have inhibitory effect on prostate CSC self‐renewal in part via downregulation of Bmi1 gene expression and function. Supported in part by R21 CA153355 from NCI/NIH

Bmi1 Enhances Tumorigenicity and Cancer Stem Cell Function in Pancreatic Adenocarcinoma

BackgroundBmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma.MethodsWe measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing.ResultsBmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue.ConclusionsOur results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.

Retinal degeneration depends on Bmi1 function and reactivation of cell cycle proteins

The epigenetic regulator Bmi1 controls proliferation in many organs. Reexpression of cell cycle proteins such as cyclin-dependent kinases (CDKs) is a hallmark of neuronal apoptosis in neurodegenerative diseases. Here we address the potential role of Bmi1 as a key regulator of cell cycle proteins during neuronal apoptosis. We show that several cell cycle proteins are expressed in different models of retinal degeneration and required in the Rd1 photoreceptor death process. Deleting E2f1, a downstream target of CDKs, provided temporary protection in Rd1 mice. Most importantly, genetic ablation of Bmi1 provided extensive photoreceptor survival and improvement of retinal function in Rd1 mice, mediated by a decrease in cell cycle markers and regulators independent of p16(Ink4a) and p19(Arf). These data reveal that Bmi1 controls the cell cycle-related death process, highlighting this pathway as a promising therapeutic target for neuroprotection in retinal dystrophies.

Reprogramming of mouse fibroblasts into induced pluripotent stem cells with Nanog

Oct4–Sox2–Nanog transcriptional networks are critical for the maintenance of embryonic stem (ES) cell self-renewal and induction of pluripotency. However, in transcription factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), Nanog is initially dispensable and Oct4 remains the sole factor that could not be substituted/omitted. Here, we show that mouse fibroblasts could be reprogrammed into iPSCs by Nanog and Bmi1, which replaces Sox2, Klf4, and c-Myc, in the absence of Oct4. Furthermore, we show that in the presence of shh agonists (oxysterol and purmophamine), which replaces the function of Bmi1, a single transcription factor, Nanog is sufficient to reprogram mouse fibroblasts into iPSCs. Nanog-induced iPSCs resemble mESCs in terms of morphology, global gene expression profiles, epigenetic status and pluripotency both in vitro and in vivo. These findings support that Nanog can replace the Oct4 for the somatic cell reprogramming and underlie the mechanisms of Nanog in reprogramming process.

Regulation of hematopoietic stem cells using protein transduction domain–fused Polycomb

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

GFAP-Cre-Mediated Transgenic Activation of Bmi1 Results in Pituitary Tumors

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16INK4A/Rb rather than on regulation of p19ARF/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.

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

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.