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 tumorinitiating 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

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.

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 2/2 Deltalike protein (Dlk) 1 hepatic stem/progenitor cells showed de-repression of the Ink4a/Arf locus and displayed impaired growth activity. In contrast, Ink4a/Arf 2/2 Dlk 1 cells gave rise to considerably larger colonies containing a greater number of bipotent cells than wild-type Dlk 1 cells. Although Ink4a/Arf 2/2 Dlk 1 cells did not initiate tumors in recipient nonobese diabetic/severe combined immunodeficiency mice, enforced expression of Bmi1 in Ink4a/Arf 2/2 Dlk 1 cells further augmented their self-renewal capacity and resulted in tumor formation in vivo. Microarray analyses successfully identified five downregulated 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 1 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.

No abstract

Supplementary Figure 2 from The Polycomb Gene Product BMI1 Contributes to the Maintenance of Tumor-Initiating Side Population Cells in Hepatocellular Carcinoma

Supplementary Figure Legends 1-3 from The Polycomb Gene Product BMI1 Contributes to the Maintenance of Tumor-Initiating Side Population Cells in Hepatocellular Carcinoma

Supplementary Figure 1 from The Polycomb Gene Product BMI1 Contributes to the Maintenance of Tumor-Initiating Side Population Cells in Hepatocellular Carcinoma

<div>Abstract<p>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 <i>BMI1</i> 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 <i>BMI1</i> knockdown, indicating that BMI1 is required for the self-renewal of SP cells in culture. More importantly, the <i>BMI1</i> knockdown abolished the tumor-initiating ability of SP cells in nonobese diabetic/severe combined immunodeficiency mice. Derepression of the <i>INK4A</i> and <i>ARF</i> genes that are major targets for BMI1 was not necessarily associated with impaired self-renewal of SP cells caused by <i>BMI1</i> 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. [Cancer Res 2008;68(19):7742–9]</p></div>