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

doi:10.1016/j.humpath.2009.01.017

Human Pathology

Volume 40, Issue 9, September 2009, Pages 1304-1311

https://doi.org/10.1016/j.humpath.2009.01.017 Get rights and content

Summary

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 immnuohistochemical 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.

Introduction

Cell-type–specific gene expression profiles are stabilized by changes in chromatin structure and DNA methylation patterns. Polycomb group proteins form multiprotein complexes and serve as a cellular memory system through epigenetic chromatin modifications [1], [2]. So far, 2 major polycomb group complexes have been well characterized. Polycomb repressive complex 1 includes BMI1, RNF110/MEL18, HPH, CBX2/HPC1, Ring1A, and Rind1B, and polycomb repressive complex 2 includes EED, EZH2, and SUZ12. The polycomb repressive complex 1 and polycomb repressive complex 2 possess H2A-K119 ubiquitin E3 ligase activity and H3-K27 methyltransferase activity, respectively. BMI1, which is part of the polycomb repressive complex 1, contributes to the enhancement of RING1-mediated H2A-K119 ubiquitin E3 ligase activity. EZH2, a key molecule of polycomb repressive complex 2, possesses histone methyltransferase activity and causes methylation at lysine residues of histone H3 (H3-K27). Both histone modifications contribute to polycomb group gene silencing. Although no physical associations have been demonstrated between the 2 complexes, H3-K27 methylation serves as a binding site for the recruitment of the polycomb repressive complex 1. Thus, the 2 polycomb group complexes could function in a cooperative manner to maintain gene silencing [1], [2].

Polycomb repressive complex 1 has been implicated in stem cell self-renewal [1], [2], a process by which epigenetic cellular memory is precisely inherited by daughter cells through cell division. Among polycomb repressive complex 1 components, we have demonstrated that BMI1 plays a central role in the inheritance of self-renewal of hematopoietic stem cells in both loss-of-function and gain-of-function analyses [3]. It is well recognized that polycomb repressive complex 1 transcriptionally represses tumor suppressor genes such as the Ink4b-Arf-Ink4a locus, which functions as a barrier to eliminate oncogenic cells by triggering apoptosis or cellular senescence [1], [4]. We have demonstrated that tight repression of Ink4a-Arf in hematopoietic stem cells is essential to maintain the self-renewing capacity of hematopoietic stem cells [5]. On the other hand, there is increasing evidence that up-regulation of polycomb group proteins is deeply involved in tumor development. EZH2 is reportedly involved in the pathogenesis of malignant lymphoma and multiple myeloma [6], [7]. BMI1 was initially identified as a c-myc–cooperating protooncogene in murine lymphomas [8], and increased levels of BMI1 expression has been implicated not only in human lymphoma but also in several types of leukemia [9], [10], [11]. Moreover, coexpression of EZH2 and BMI1 appears to be associated with the degree of malignancy in B-cell non–Hodgkin lymphoma [12]. Of importance, recent studies demonstrated that the increased expression of EZH2 proteins correlates with progression and poor prognosis of solid tumors such as prostate cancer and breast cancer [13], [14], [15].

In the present study, we first examined the cell growth activity of hepatocellular carcinoma cells stably expressing short hairpin RNAs against EZH2 and BMI1 in culture. Next, we conducted immunohistochemical analyses to estimate the expression levels of polycomb group proteins EZH2 and BMI1 in hepatocellular carcinoma. The cumulative survival rates and recurrent rates were analyzed using the Kaplan-Meier method to ask whether these molecules could be novel biologic markers.

Section snippets

Cell culture

The hepatocellular carcinoma cell line HepG2 was cultured in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen).

Lentiviral production and transduction

Lentiviral vectors (CS-H1-short hairpin RNA EF-1α-EGFP and CS-CDF-RfA-ERP) expressing short hairpin RNA that targets human EZH2 (target sequence: 5′-GGAAAGAACGGAAATCTTA-3′), human BMI1 (target sequence: 5′-GAGAAGGAATGGTCCACTT-3′), and luciferase were constructed.

Stable short hairpin RNA-mediated knockdown of EZH2 and BMI1 mediated growth inhibition in HepG2 cells

To examine the basal expression of EZH2 and BMI1 in hepatocellular carcinoma cells, we conducted dual immunocytochemical analyses in HepG2 cells. EZH2 and BMI1 were widely detected in the nucleus and were coexpressed in more than 70% of HepG2 cells (Fig. 1A). To gain insight into the role of the polycomb gene products in HepG2 cells, we performed loss-of-function assays using lentivirus-mediated knockdown techniques. MTS assays showed that cell growth activity was consistently repressed, after

Discussion

Hepatocellular carcinoma is one of the most common malignancies, and chronic viral infection by hepatitis B or C virus is well recognized as a major risk factor [17]. Therapeutic advancements such as nucleotide analogues and interferon has successfully improved hepatitis viremia and reduced the incidence of hepatocellular carcinoma, but the mortality rate of hepatocellular carcinoma remains high in spite of recent advances in cancer therapy [18]. In hepatocarcinogenesis, it appears that

Acknowledgments

The authors thank Dr Huroyuki Miyoshi (RIKEN, Tsukuba, Japan) for providing lentiviral vectors and Dr Yoh Zen (Kanazawa University, Kanazawa, Japan) for technical assistance in immunohistochemical analyses.

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Enhancer of zeste homolog 2 (EZH2), a core component of polycomb repressive component 2 is overexpressed in a variety of cancers and recognized as a therapeutic target molecule. However, EZH2 possesses immunomodulatory functions in the tumor microenvironment (TME). The impact of EZH2 on TME of hepatocellular carcinoma (HCC) using immunocompetent mouse model was evaluated in the present study. UNC1999, an EZH2 inhibitor, impaired growth of the murine HCC cells (H22 cells) and induced apoptosis in a dose-dependent manner. Although UNC1999 significantly inhibited the growth of H22 cells-derived and Hepa1-6 cells-derived tumors in nonobese diabetic/severe combined immunodeficiency mice, its antitumor effect was diminished in allogenic BALB/c and C57BL/6 mice. Flow cytometric analyses of TME cells in BALB/c mice demonstrated a significant decrease in the number of interferon‑γ⁺ CD8⁺ T cells and regulatory T cells and a significant increase in the number of myeloid-derived suppressor cells (MDSCs). Administration of Gr-1 neutralizing antibody concomitant with UNC1999 restored antitumor effect accompanied by an increase in the number of CD8⁺ T cells followed by a decrease in the number of MDSCs. Chemokine antibody array demonstrated an enhanced expression of chemokines responsible for MDSCs recruitment such as C5a, CCL8, and CCL9. In conclusion, the study results demonstrated that EZH2 inhibitor contributed to attenuation of tumor immunity caused by TME arrangement. Combination therapy with EZH2 inhibitors and agents that reduce MDSCs might represent a novel therapeutic strategy for HCC.
MiR-203 Interplays with Polycomb Repressive Complexes to Regulate the Proliferation of Neural Stem/Progenitor Cells
2017, Stem Cell Reports
The polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are two distinct polycomb group (PcG) proteins that maintain the stable silencing of specific sets of genes through chromatin modifications. Although the PRC2 component EZH2 has been known as an epigenetic regulator in promoting the proliferation of neural stem/progenitor cells (NSPCs), the regulatory network that controls this process remains largely unknown. Here we show that miR-203 is repressed by EZH2 in both embryonic and adult NSPCs. MiR-203 negatively regulates the proliferation of NSPCs. One of PRC1 components, Bmi1, is a downstream target of miR-203 in NSPCs. Conditional knockout of Ezh2 results in decreased proliferation ability of both embryonic and adult NSPCs. Meanwhile, ectopic overexpression of BMI1 rescues the proliferation defects exhibited by miR-203 overexpression or EZH2 deficiency in NSPCs. Therefore, this study provides evidence for coordinated function of the EZH2-miR-203-BMI1 regulatory axis that regulates the proliferation of NSPCs.
Prognostic role of high Bmi-1 expression in Asian and Caucasian patients with solid tumors: A meta-analysis
2014, Biomedicine and Pharmacotherapy
Recently, many studies have shown that the B-cell-specific moloney leukemia virus insertion site 1 (Bmi-1) exhibits altered expression in various cancers and may serve as prognostic biomarkers. We performed a meta-analysis to evaluate the prognostic role of Bmi-1 expression in solid cancers. Studies were recruited by searching PubMed, Embase and the Cochrane Library. Thirty-nine articles including 40 studies were involved in this meta-analysis. Our results indicated that the Bmi-1 showed the opposite prognostic effect in Asian and Caucasian populations. High Bmi-1 expression as a negative predictor for overall survival (OS) in Asian patients (HR = 1.96, 95% CI 1.62–2.36), but a positive predictor in Caucasian populations (HR = 0.77, 95% CI 0.63–0.93). Furthermore, we took a further subgroup analysis based on tumor type in these two populations, respectively. In Asian cases, high expression of Bmi-1 was associated with poor OS in oesophageal carcinoma (HR = 1.93, 95% CI 1.52–2.46), gastric cancer (HR = 1.50, 95% CI 1.22–1.85), lung cancer (HR = 1.73, 95% CI 1.05–2.85), cervical cancer (HR = 2.80, 95% CI 2.26–3.47) and colorectal cancer (HR = 3.36, 95% CI 2.19–5.15), rather than in breast cancer and HCC. In Caucasian populations, high expression of Bmi-1 was associated with better OS in breast cancer (HR = 0.70, 95% CI 0.51–0.97), but it showed no significance in oesophageal carcinoma. In conclusion, high Bmi-1 expression was significantly associated with poor survival in Asian patients with oesophageal carcinoma, gastric cancer, lung cancer, colorectal cancer and cervical carcinoma, whereas high level of Bmi-1 can predict better prognosis in Caucasian patients with breast cancer.
Combined aberrant expression of Bmi1 and EZH2 is predictive of poor prognosis in glioma patients
2013, Journal of the Neurological Sciences
Citation Excerpt :
Interestingly, recent studies have demonstrated that EZH2 may be one of the PcG proteins essential for Bmi1 recruitment to the PcG bodies [33], suggesting the closed relationship between Bmi1 and EZH2. In addition, accumulated reports indicated that various tumors, such as esophageal squamous cell carcinoma, prostate cancer, hepatocellular carcinoma, lung cancer, with dual-positive, Bmi1/EZH2 high-expressing tumor cells manifest clinically aggressive disease phenotypes and are significantly more likely to recur after surgery [34–37]. On the basis of above evidence, we here hypothesized that the combined overexpression of Bmi1 and EZH2 may be related to the malignant transformation of gliomas and that it may also reflect the biological aggressiveness of this disease.
Bmi1 and EZH2 are involved in tumorigenesis of gliomas. However, clinicopathologic significance of their expression in gliomas is unknown; especially, the prognostic value of combined expression of Bmi1 and EZH2 has not been explored.
Bmi1 and EZH2 expression in human gliomas and nonneoplastic brain tissues was measured by immunohistochemistry.
Both Bmi1 and EZH2 expressions in glioma tissues were significantly higher than those in corresponding nonneoplastic brain tissues (both P < 0.001). Additionally, the upregulations of Bmi1 and EZH2 proteins were both significantly associated with advanced WHO grades (both P < 0.001) and low KPS (P = 0.008 and 0.01, respectively). Moreover, the overall survival of patients with high Bmi1 protein expression (P = 0.006) or high EZH2 protein expression (P = 0.01) was obviously lower than those with low expressions. More interestingly, glioma patients with combined overexpression of Bmi1 and EZH2 proteins had the shortest overall survival (P < 0.001). Furthermore, multivariate analysis showed that Bmi1n expression (P = 0.02), EZH2 expression (P = 0.03), and combined expression of Bmi1 and EZH2 (P = 0.008), were all independent prognostic factors for overall survival in glioma patients.
Our data suggest for the first time that the combination of Bmi1 and EZH2 overexpression may be a highly sensitive marker for the prognosis in glioma patients.
Genome-wide discovery and validation of diagnostic DNA methylation-based biomarkers for hepatocellular cancer detection in circulating cell free DNA
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Hepatocellular carcinoma (HCC), the most prevalent form of liver cancer, is growing in incidence but treatment options remain limited, particularly for late stage disease. As liver cirrhosis is the principal risk state for HCC development, markers to detect early HCC within this patient population are urgently needed. Perturbation of epigenetic marks, such as DNA methylation (5mC), is a hallmark of human cancers, including HCC. Identification of regions with consistently altered 5mC levels in circulating cell free DNA (cfDNA) during progression from cirrhosis to HCC could therefore serve as markers for development of minimally-invasive screens of early HCC diagnosis and surveillance.
Methods: To discover DNA methylation derived biomarkers of HCC in the background of liver cirrhosis, we profiled genome-wide 5mC landscapes in patient cfDNA using the Infinium HumanMethylation450k BeadChip Array. We further linked these findings to primary tissue data available from TCGA and other public sources. Using biological and statistical frameworks, we selected CpGs that robustly differentiated cirrhosis from HCC in primary tissue and cfDNA followed by validation in an additional independent cohort.
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Conclusion: Our finding that 5mC markers derived from primary tissue did not perform well in cfDNA, compared to those identified directly from cfDNA, reveals potential advantages of starting with cfDNA to discover high performing markers for liquid biopsy development.
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Original contributionDistinct expression of polycomb group proteins EZH2 and BMI1 in hepatocellular carcinoma

Summary

Introduction

Section snippets

Cell culture

Lentiviral production and transduction

Stable short hairpin RNA-mediated knockdown of EZH2 and BMI1 mediated growth inhibition in HepG2 cells

Discussion

Acknowledgments

Cell

Immunity

Cell

Mol Cell

Blood

Blood

Breast

Cancer Cell

Polycomb silencers control cell fate, development and cancer

Nat Rev Cancer

Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all

Nat Rev Mol Cell Biol

Differential impact of Ink4a and Arf on hematopoietic stem cells and their bone marrow microenvironment in Bmi1-deficient mice

J Exp Med

The Polycomb group protein EZH2 is upregulated in proliferating, cultured human mantle cell lymphoma

Br J Haematol

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