Bmi1 promotes hepatic stem cell expansion and tumorigenicity in both Ink4a/Arf-dependent and -independent manners in mice.

UNLABELLED: We previously reported that forced expression of Bmi1 (B lymphoma Moloney murine leukemia virus insertion region 1 homolog) in murine hepatic stem/progenitor cells purified from fetal liver enhances their self-renewal and drives cancer initiation. In the present study, we examined the contribution of the Ink4a/Arf tumor suppressor gene locus, one of the major targets of Bmi1, to stem cell expansion and cancer initiation. Bmi1(-/-) Delta-like protein (Dlk)(+) hepatic stem/progenitor cells showed de-repression of the Ink4a/Arf locus and displayed impaired growth activity. In contrast, Ink4a/Arf(-/-) Dlk(+) cells gave rise to considerably larger colonies containing a greater number of bipotent cells than wild-type Dlk(+) cells. Although Ink4a/Arf(-/-) Dlk(+) cells did not initiate tumors in recipient nonobese diabetic/severe combined immunodeficiency mice, enforced expression of Bmi1 in Ink4a/Arf(-/-) Dlk(+) cells further augmented their self-renewal capacity and resulted in tumor formation in vivo. Microarray analyses successfully identified five down-regulated genes as candidate downstream targets for Bmi1 in hepatic stem/progenitor cells. Of these genes, enforced expression of sex determining region Y-box 17 (Sox17) in Dlk(+) cells strongly suppressed colony propagation and tumor growth. CONCLUSION: These results indicate that repression of targets of Bmi1 other than the Ink4a/Arf locus plays a crucial role in the oncogenic transformation of hepatic stem/progenitor cells. Functional analyses of Bmi1 target genes would be of importance to elucidate the molecular machinery underlying hepatic stem cell system and explore therapeutic approaches for the eradication of liver cancer stem cells.

The polycomb group gene product Ezh2 regulates proliferation and differentiation of murine hepatic stem/progenitor cells.

BACKGROUND & AIMS: Polycomb group proteins initiate and maintain gene silencing through chromatin modifications and contribute to the maintenance of self-renewal in a variety of stem cells. Among polycomb repressive complexes (PRCs), PRC2 initiates gene silencing by methylating histone H3 lysine 27, and PRC1 maintains gene silencing through mono-ubiquitination of histone H2A lysine 119. We have previously shown that Bmi1, a core component of PRC1, tightly regulates the self-renewal of hepatic stem/progenitor cells. METHODS: In this study, we conducted lentivirus-mediated knockdown of Ezh2 to characterise the function of Ezh2, a major component of PRC2, in hepatic stem/progenitor cells. RESULTS: Loss of Ezh2 function in embryonic murine hepatic stem/progenitor cells severely impaired proliferation and self-renewal capability. This effect was more prominent than that of Bmi1-knockdown and was partially abrogated by the deletion of both Ink4a and Arf, major targets of PRC1 and PRC2. Importantly, Ezh2-knockdown but not Bmi1-knockdown promoted the differentiation and terminal maturation of hepatocytes, followed by the up-regulation of several transcriptional regulators of hepatocyte differentiation. CONCLUSIONS: Our findings indicate that Ezh2 plays an essential role in the maintenance of both the proliferative and self-renewal capacity of hepatic stem/progenitor cells and the full execution of their differentiation.

Distinct expression of polycomb group proteins EZH2 and BMI1 in hepatocellular carcinoma.

Polycomb gene products play a crucial role in the development of highly malignant phenotypes and aggressive cancer progression in a variety of cancers; however, their role in hepatocellular carcinoma remains unclear. First, we analyzed the impact of EZH2 and BMI1 modulation on cell growth of HepG2 cells. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays revealed marked growth inhibition after EZH2 or BMI1 knockdown. In addition, simultaneous knockdown of these 2 genes further augmented cell growth inhibitory effects. Next, we conducted immunohistochemical assessment of 86 hepatocellular carcinoma surgical specimens, evaluating the correlation between EZH2 and BMI1 protein expression and clinicopathologic features. High-level EZH2 and BMI1 expression was detected in 57 (66.3%) and 52 tumor tissues (60.5%), respectively. Among these, 48 tumor tissues (55.8%) showed colocalization of EZH2 and BMI1 in almost all tumor cells. The cumulative recurrence rate, but not survival rate, was significantly higher in patients positive for EZH2 (P = .029) and BMI1 (P = .039) than in their negative counterparts, as determined by Kaplan-Meier analysis. These data indicate that EZH2 and BMI1 may cooperate in initiation and progression of hepatocellular carcinoma.

The polycomb gene product BMI1 contributes to the maintenance of tumor-initiating side population cells in hepatocellular carcinoma.

Side population (SP) cell analysis and sorting have been successfully applied to hepatocellular carcinoma (HCC) cell lines to identify a minor cell population with cancer stem cell properties. However, the molecular mechanisms operating in SP cells remain unclear. The polycomb gene product BMI1 plays a central role in the self-renewal of somatic stem cells in a variety of tissues and organs and seems to be implicated in tumor development. In this study, we determined the critical role of BMI1 in the maintenance of cancer stem cells with the SP phenotype in HCC cell lines. BMI1 was preferentially expressed in SP cells in Huh7 and PLC/PRF/5 HCC cells compared with the corresponding non-SP cells. Lentiviral knockdown of BMI1 considerably decreased the number of SP cells in both Huh7 and PLC/PRF/5 cells. Long-term culture of purified SP cells resulted in a drastic reduction in the SP subpopulation upon the BMI1 knockdown, indicating that BMI1 is required for the self-renewal of SP cells in culture. More importantly, the BMI1 knockdown abolished the tumor-initiating ability of SP cells in nonobese diabetic/severe combined immunodeficiency mice. Derepression of the INK4A and ARF genes that are major targets for BMI1 was not necessarily associated with impaired self-renewal of SP cells caused by BMI1 knockdown. In conclusion, our findings define an important role for BMI1 in the maintenance of tumor-initiating SP cells in HCC. BMI1 might be a novel therapeutic target for the eradication of cancer stem cells in HCC.