Elsevier

Human Pathology

Volume 43, Issue 9, September 2012, Pages 1425-1435
Human Pathology

Original contribution
High expression of trimethylated histone H3 lysine 4 is associated with poor prognosis in hepatocellular carcinoma

https://doi.org/10.1016/j.humpath.2011.11.003Get rights and content

Summary

Tumor-associated epigenetic alterations including DNA methylation and histone modifications are important determinants in the initiation and progression of hepatocellular cancer (HCC) and represent promising biomarkers and therapeutic targets. Locus-specific trimethylation of histone H3 lysine 4 (H3K4me) is a well-known modification linked to the enhanced transcriptional expression of many genes activated in HCC. Our aim was to assess the cellular expression pattern of H3K4me3 in HCC and its association with clinicopathologic variables and outcome. Expression of H3K4me3 and the histone methyltransferase (HMT) SET and MYND domain-containing protein 3 (SMYD3) was studied by Western blotting and immunohistochemistry in cell lines and tumor tissue microarray from a well-characterized series of HCC patients (n = 168). The optimal cut-point of H3K4me3 expression for prognosis was determined by the X-tile program. The prognostic significance was evaluated using Kaplan-Meier survival estimates and log-rank tests. Tumor tissue microarray from another independent HCC patients cohort (n = 147) was used for validation studies. Expression of H3K4me3 and SMYD3 were enhanced in HCC cell lines. In tumor specimens, enhanced expression of H3K4me3 was correlated with reduced overall survival (P < .0001), especially in early-stage HCC patients (TNM I/II). Furthermore, both univariate and multivariate analyses revealed that H3K4me3 level was a significant and independent predictor of poor survival (hazard ratio, 3.592; 95% confidence interval, 2.302-5.605). In addition, H3K4m3 expression was positively correlated with SMYD3 expression in both testing and validation cohorts (P < .0001). In conclusion, H3K4me3 level defines unrecognized subsets of HCC patients with distinct epigenetic phenotype and clinical outcome and can thus be a novel predictor for poor prognosis of HCC patients, especially at TNM I/II stage.

Introduction

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide, and the long-term prognosis of patients with HCC remains poor despite improvements in surgical techniques and chemotherapies [1]. The clinical course of HCC can vary significantly in tumors with similar characteristics. Accordingly, it is reasonable to subclassify patients with HCC to more accurately predict clinical course and treatment outcome. Biomarkers that can predict clinical outcome of specific cancers are helpful for the development of therapeutic strategies in the earlier stages of the disease, and many biomarkers for prediction and intervention of HCC have been investigated [2]. Although several prognostic biomarkers for HCC have been reported [3], [4], there is still no ideal biomarker for clinical use [5].

In recent years, substantial progress has been made in understanding the molecular pathogenesis of HCC, including the role of histone modifications [6], [7]. Methylation of lysine 4 in histone H3 (H3K4me3) is particularly important for transcriptional activation [8], [9]. Histone 3 methylation was first discovered in the trout testes [10], and modified histone residues were found in 3 states: monomethylated, dimethylated, and trimethylated. The prognostic utility of histone H3K4 modifications has been demonstrated independently for multiple cancers [11], [12], [13], [14]. Trimethylation of lysine 4 in histone 3, a marker associated with active genes, was found to contribute to the alteration of gene expression and tumor progression. More recent studies have shown that H3K4me3 plays an active role in determining the course of various types of human cancers, including multiple myeloma, lung cancer, and prostate cancer [15], [16], [17]. In addition, analysis of c-Myc target gene promoters indicated a strong dependence on H3K4 trimethylation for E-box–dependent c-Myc binding [18]. However, to our knowledge, the cellular expression pattern of H3K4me3 in HCC tumor tissues and its association with clinical outcomes have not been reported.

The SET and MYND domain-containing protein 3 (SMYD3) was found to be overexpressed in the majority of HCC, colon, and breast cancers [19] and promoted dimethylation and trimethylation of histone H3K4, which initiated oncogenesis by activating the transcription of multiple downstream target genes [20], [21]. However, the carcinogenic role of SMYD3 in HCC remains unknown. In the present study, we investigated the predictive value of H3K4me3 and its association with SMYD3 in HCC.

Section snippets

Cell lines

The human hepatic cell line HL-7702 and the HCC cell lines PLC/PRF/5, HepG2, and Huh7 were purchased from the American Type Culture Collection. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) at 37°C, 5% CO2.

Patients cohorts and TMAs

Tissues were obtained from 2 independent HCC patient cohorts. The inclusion criteria of the patient cohorts include (a) having a distinctive pathologic

Cellular H3K4me3 levels in HCC TMAs

Immunostaining for H3K4me3 was observed predominantly in the cell nuclei (representative staining was shown in Fig. 1) and the frequency of immunopositive cells ranged from 0% to 100%. For the 23 uninformative TMA specimens, which included samples with less than 300 cells in the field and samples lost during immunohistochemical analysis, IHC analyses were performed on whole tissue slides. According to the X-tile plots, we dichotomized the testing cohort into low (IHC score ≤67.5%) and high (IHC

Discussion

Here we showed that cellular level of H3K4me3 provides significant and independent prognostic information in 2 large cohorts of patients: higher level of H3K4me3 is correlated with worse prognosis in HCC patients. This result is inconsistent with a previous observation of H3K4me prognostic significance in renal carcinoma [27]. They reported that low levels of H3K4me1-3 were detected in patients with worse prognosis in the univariate analysis, but the significant correlation vanished in a

Acknowledgments

We thank Dr. Sheng-Xue Wei and Dr. Dong-Ping Rao for the assistance in statistical analysis and Zi Yin, Ying-Fei Zhang, and Shao-Wen Jin for the clinicopathologic data review.

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    Grant support: National Natural Science Foundation of China (grant 30700803), Natural Science Foundation of Guangdong Province (grant 10151008901000138).

    1

    These authors contributed equally to this work.

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