Tetsuhiro Chiba

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell–like properties†‡

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti‐apoptotic properties compared with those of non‐SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 × 10 3 SP cells were sufficient for tumor formation, whereas an injection of 1 × 10 6 non‐SP cells did not initiate tumors. Re‐analysis of SP cell–derived tumors showed that SP cells generated both SP and non‐SP cells and tumor‐initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non‐SP cells, and several so‐called “stemness genes” were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy. (HEPATOLOGY 2006;44:240–251.)

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.

Ezh2 augments leukemogenicity by reinforcing differentiation blockage in acute myeloid leukemia

EZH2, a catalytic component of the polycomb repressive complex 2, trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Although EZH2 is overexpressed in various cancers, including some hematologic malignancies, the role of EZH2 in acute myeloid leukemia (AML) has yet to be examined in vivo. In the present study, we transformed granulocyte macrophage progenitors from Cre-ERT;Ezh2(flox/flox) mice with the MLL-AF9 leukemic fusion gene to analyze the function of Ezh2 in AML. Deletion of Ezh2 in transformed granulocyte macrophage progenitors compromised growth severely in vitro and attenuated the progression of AML significantly in vivo. Ezh2-deficient leukemic cells developed into a chronic myelomonocytic leukemia-like disease with a lower frequency of leukemia-initiating cells compared with the control. Chromatin immunoprecipitation followed by sequencing revealed a significant reduction in the levels of trimethylation at H3K27 in Ezh2-deficient leukemic cells, not only at Cdkn2a, a known major target of Ezh2, but also at a cohort of genes relevant to the developmental and differentiation processes. Overexpression of Egr1, one of the derepressed genes in Ezh2-deficient leukemic cells, promoted the differentiation of AML cells profoundly. Our findings suggest that Ezh2 inhibits differentiation programs in leukemic stem cells, thereby augmenting their leukemogenic activity.

Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells.

Objective: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. Methods: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody. Results: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, α2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, α7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes. Conclusions: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.

The Hbo1-Brd1/Brpf2 complex is responsible for global acetylation of H3K14 and required for fetal liver erythropoiesis

The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.

3-Deazaneplanocin A is a promising therapeutic agent for the eradication of tumor-initiating hepatocellular carcinoma cells.

Recent advances in stem cell biology have identified tumor‐initiating cells (TICs) in a variety of cancers including hepatocellular carcinoma (HCC). Polycomb group gene products such as BMI1 and EZH2 have been characterized as general self‐renewal regulators in a wide range of normal stem cells and TICs. We previously reported that Ezh2 tightly regulates the self‐renewal and differentiation of murine hepatic stem/progenitor cells. However, the role of EZH2 in tumor‐initiating HCC cells remains unclear. In this study, we conducted loss‐of‐function assay of EZH2 using short‐hairpin RNA and pharmacological inhibition of EZH2 by an S‐adenosylhomocysteine hydrolase inhibitor, 3‐deazaneplanocin A (DZNep). Both EZH2‐knockdown and DZNep treatment impaired cell growth and anchorage‐independent sphere formation of HCC cells in culture. Flow cytometric analyses revealed that the two approaches decreased the number of epithelial cell adhesion molecule (EpCAM)+ tumor‐initiating cells. Administration of 5‐fluorouracil (5‐FU) or DZNep suppressed the tumors by implanted HCC cells in non‐obese diabetic/severe combined immunodeficient mice. Of note, however, DZNep but not 5‐FU predominantly reduced the number of EpCAM+ cells and diminished the self‐renewal capability of these cells as judged by sphere formation assays. Our findings reveal that tumor‐initiating HCC cells are highly dependent on EZH2 for their tumorigenic activity. Although further analyses of TICs from primary HCC would be necessary, pharmacological interference with EZH2 might be a promising therapeutic approach to targeting tumor‐initiating HCC cells.

Bmi1 promotes hepatic stem cell expansion and tumorigenicity in both Ink4a/Arf‐dependent and ‐independent manners in Mice

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. (Hepatology 2010)

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.

Metformin, a Diabetes Drug, Eliminates Tumor-Initiating Hepatocellular Carcinoma Cells

Metformin has been widely used as an oral drug for diabetes mellitus for approximately 60 years. Interestingly, recent reports showed that metformin exhibited an anti-tumor action in a wide range of malignancies including hepatocellular carcinoma (HCC). In the present study, we investigated its impact on tumor-initiating HCC cells. Metformin suppressed cell growth and induced apoptosis in a dose-dependent manner. Flow cytometric analysis showed that metformin treatment markedly reduced the number of tumor-initiating epithelial cell adhesion molecule (EpCAM)+ HCC cells. Non-adherent sphere formation assays of EpCAM+ cells showed that metformin impaired not only their sphere-forming ability, but also their self-renewal capability. Consistent with this, immunostaining of spheres revealed that metformin significantly decreased the number of component cells positive for hepatic stem cell markers such as EpCAM and α-fetoprotein. In a xenograft transplantation model using non-obese diabetic/severe combined immunodeficient mice, metformin and/or sorafenib treatment suppressed the growth of tumors derived from transplanted HCC cells. Notably, the administration of metformin but not sorafenib decreased the number of EpCAM+ cells and impaired their self-renewal capability. As reported, metformin activated AMP-activated protein kinase (AMPK) through phosphorylation; however its inhibitory effect on the mammalian target of rapamycin (mTOR) pathway did not necessarily correlate with its anti-tumor activity toward EpCAM+ tumor-initiating HCC cells. These results indicate that metformin is a promising therapeutic agent for the elimination of tumor-initiating HCC cells and suggest as-yet-unknown functions other than its inhibitory effect on the AMPK/mTOR pathway.