Polycomb group protein EZH2, frequently overexpressed in malignant tumors, is the catalytic subunit of polycomb repressive complex 2 (PRC2). PRC2 interacts with HDACs in transcriptional silencing and relates to tumor suppressor loss. We examined the expression of HDAC isoforms (HDAC 1 and 2) and EZH2, and evaluated the possible use of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and EZH2 repressor for gallbladder carcinoma. We used 48 surgically resected gallbladders and cultures of human gallbladder epithelial cells (HGECs), gallbladder carcinoma (TGBC2TKB), and cholangiocarcinoma (HuCCT-1 and TFK-1) cell lines for examination. Immunohistochemically, EZH2 was overexpressed in gallbladder carcinoma, especially poorly differentiated carcinoma, but not in normal epithelium. In contrast, HDAC1/2 were expressed in both carcinoma and normal epithelium in vivo. This pattern was verified in cultured cells; EZH2 was highly expressed only in TGBC2TKB, whereas HDAC1/2 were expressed in HGECs and TGBC2TKB. Interestingly, SAHA treatment caused significant cell number decline in three carcinoma cells, and this effect was synergized with EZH2 siRNA treatment; however, HGECs were resistant to SAHA. In TGBC2TKB cells, the expression of EZH2 and HDAC1/2 were decreased by SAHA treatment, and p16INK4a, E-cadherin, and p21were simultaneously activated; however, no such findings were obtained in HGECs, suggesting that the effect of SAHA depends on the EZH2-mediated tumor suppressor loss. In conclusion, this study suggests a possible mechanism by which carcinoma cells but not normal cells are sensitive to SAHA and indicates the efficacy of this new anticancer agent in combination with EZH2 repression in gallbladder carcinoma. (Cancer Sci 2009)
Gallbladder carcinoma is an aggressive and depressing disease, particularly affecting older people and women.(1–3) It is usually difficult to detect gallbladder carcinoma in the early stage, because characteristic signs or symptoms are lacking and high risk factors have not been established in a majority of patients. Although complete surgical resection is the only curative therapy, invasion to surrounding organs or metastases is usually found in a majority of gallbladder carcinoma patients at the time of diagnosis. The current chemotherapy for patients with advanced gallbladder carcinoma is limited, and the development and exploration of new anticancer agents is necessary.(4,5)
Polycomb group proteins are epigenetic chromatin modifiers involved in cancer development.(6,7) PcG proteins exist in at least two separate protein complexes, polycomb repressive complex 2 (PRC2) and PRC1. The PRC2 complex involves embryonic ectoderm development and EZH2.(8,9) EZH2 is a transcriptional repressor involved in controlling cell growth and proliferation and an abnormal expression of EZH2 may be involved in the tumorigenesis process and provides proliferative advantages to eukaryotic cells through interaction with the pathways of key elements that control cell growth arrest and differentiation.(10,11) In addition, various studies have identified EZH2 as a potential marker to distinguish aggressive from indolent or benign cancers.(12–15)
PRC2 is known to interact with HDAC proteins.(8,9) Recent studies have disclosed that HDACs have many protein substrates involved in the regulation of gene expression, cell proliferation, and cell death. Acetylation and deacetylation of histones play an important role in the transcription regulation of cells,(16,17) and the acetylation status of histones and non-histone proteins is determined by HDACs.(18) Inhibition of HDACs causes the accumulation of acetylated forms of these proteins with alterations of their function, and the CDK inhibitor p21 (WAF/CIP1) is one of the most common genes induced by HDAC inhibitor.(4) In human cells, PRC2 interacts with class I HDAC, which includes HDAC1 and 2,(8,9,18) and recent data suggest that transient interactions likely provide functional synergy between the silencing enzymes for tumor suppressor genes in vivo. The precise mechanisms of this synergy are not yet clear. Functional links between EZH2 and HDACs contribute to an emerging view that all of these types of epigenetic silencing machinery contribute to abnormal control of gene expression in malignant cells.
HDAC inhibitors are a group of recently discovered targeted anticancer agents. HDAC inhibitors induce different phenotypes in various transformed cells, including growth arrest, activation of extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, mitotic cell death, and senescence.(19) In comparison, normal cells are relatively more resistant to HDAC inhibitor-induced cell death;(20,21) however, the mechanism underlying this difference is not well understood.
Among HDAC inhibitors, SAHA is one of the most advanced in clinical fields as an anticancer agent, which interacts directly with the catalytic site of HDAC-like protein and inhibits its enzymatic activity.(18) SAHA inhibits all 11 members of the class I and II HDAC family, and causes specific modifications in the pattern of acetylation and methylation of lysines in histones H3 and H4 associated with the proximal promoter of the p21 gene.(18) Although considerable progress has been made in elucidating the role of HDACs and the effects of HDAC inhibitors, these areas are still in the early stages of discovery.
Several previous studies reported abnormal promoter methylation of important genes including p16INK4a and E-cadherin in gallbladder carcinoma,(22,23) however, there have been few studies addressing alteration of epigenetic chromatin modification by PcG and HDACs in gallbladder carcinoma.(24,25) In this study using human gallbladders and cultures of gallbladder epithelial cells and the gallbladder carcinoma cell line, we assessed the expression pattern of EZH2 and class I HDACs (HDAC 1 and 2). We considered the possible mechanism by which carcinoma cells are sensitive to SAHA and the possible efficacy of this new anticancer agent in therapeutic treatment in gallbladder carcinoma.
Chromatin alterations are causally related to the development and progression of malignant tumors. The most well-characterized alteration is CpG DNA hypermethylation, which contributes to tumor suppressor loss through epigenetic silencing,(29) and epigenetic modification of histones is also implicated in oncogenesis.(30) A common biological function of PRC2 is transcriptional silencing of differentiation genes, and frequent targets of PRC2 are transcription factors and signaling components with key roles in cell fate decisions in a wide variety of organisms. PRC2 contains a conserved catalytic subunit, EZH2, which contains the signature domain providing the methylation active site,(31) and EZH2 levels are abnormally elevated in malignant tissues, with the highest EZH2 levels correlating with advanced stages and poor prognosis.(10,12,15) This study showed that EZH2 was overexpressed in gallbladder carcinoma, particularly poorly differentiated carcinoma, but its expression was faint and infrequent in non-neoplastic epithelium. Culture studies also showed EZH2 mRNA and protein expression in gallbladder carcinoma cells, but not in normal cells. Furthermore, downregulation of EZH2 by siRNA induces significant growth inhibitory effect in gallbladder carcinoma cells and cholangiocarcinoma cells to various degrees. EZH2 siRNA also increased the expression of E-cadherin, which is supposed to control invasiveness of carcinoma, by chromatin modification. Taken together, EZH2 may be related to factors controlling cell proliferation, differentiation, and invasiveness in gallbladder carcinoma as reported in other types of cancers.(10–15,27,28)
PRC2 is known to interact with HDAC proteins.(8,9) Recent studies disclosed that HDACs have many protein substrates involved in the regulation of gene expression, cell proliferation, and cell death. Acetylation and deacetylation of histones play an important role in transcription regulation of eukaryotic cells.(16,17) Recently, several reports showed that HDACs are strongly expressed in cancerous tissue, and the expression of class I HDACs is an independent prognostic marker in various cancers, such as ovarian, colorectal and cervical cancer.(32–34) The present study showed that HDAC1/2 were expressed in the nuclei of non-neoplastic epithelial cells and carcinoma cells in the gallbladder, although nuclear staining was slightly weaker in the former than in the latter. This pattern was verified in cultured normal epithelial and carcinoma cells, indicating that HDAC1/2 expression level is a little higher in cancerous tissue; however, its expression is not specific to carcinoma cells in the gallbladder.
Both epigenetic changes and genetic alterations to DNA sequence in the malignant cell genome might contribute to disease progression. Once the DNA sequence is changed by mutation, it is difficult to restore the gene; however, epigenetic changes can potentially be reserved with inhibitors that block the relevant chromatin-modifying enzymes. Epigenetic silencing of the tumor suppressor gene in carcinoma has inspired potential therapeutic strategies that use inhibitors of epigenetic enzymes. Many inhibitors target either DNA methyltransferases or HDACs.(16) HDAC inhibitors have been found to have profound anticancer effects in clinical trials.(5) Among HDAC inhibitors, SAHA is one of the most advanced in clinical fields as an anticancer agent. SAHA has shown significant anticancer activity in tumor-bearing animals and in phases I and II clinical trials with little evidence of adverse effects on normal cells.(5)
It was found in this study that SAHA treatment reduced the number of gallbladder carcinoma cells and cholangiocarcinoma cells, suggesting that carcinoma cells are sensitive to SAHA treatment. However, this treatment had no effect on the number of normal epithelial cells in spite of their HDAC expression. The reason why carcinoma cells are more sensitive to SAHA compared to normal cells remains speculative. High expression levels of EZH2 might be related to high sensitivity to SAHA in carcinoma cells, as discussed below. SAHA significantly repressed the EZH2 level in carcinoma cells. These differences in sensitivity to SAHA-induced cell reduction of carcinoma cells compared with normal cells appeared not to be caused by a difference in the expression levels of HDACs, because normal cells and the normal epithelium of the gallbladder has relatively weak but significant expression levels of HDAC1/2, and moreover, it was found that the expression level of HDACs was significantly repressed in normal cells and also carcinoma cells by SAHA treatment.
Histone deacetylase inhibitors are known to alter gene expression followed by the expression of the proteins responsible for composing the transcription factor complex to which HDACs are recruited.(8,9) We therefore examined whether tumor suppressor genes are activated by SAHA treatment by evaluating p21 activation in gallbladder carcinoma cells and normal cells by SAHA treatment. It was found that p21 was expressed in carcinoma cells but not in normal cells, indicating that HDACs forcibly repress the p21 gene in carcinoma cells, that inhibition of HDACs by SAHA treatment re-activated the p21 gene, and that HDACs are not responsible for p21 gene repression in normal cells. One hypothesis is that p21 is not forcibly repressed in normal epithelial cells, and HDACs do not act as a p21 gene suppressor; therefore, HDAC inhibition does not result in activation of the p21 gene in normal cells.
Almost all carcinomas have multiple alterations in the expression and/or structure of proteins that regulate cell proliferation and death. The multiple alterations of tumor suppressor genes in cancer cells might explain why transformed cells are more sensitive than normal cells to the HDAC inhibitor. EZH2 are reported to mediate tumor suppressor genes, such as p16INK4A and E-cadherin.(28,35,36) In this context, the effect of HDAC inhibitor might be linked to EZH2 expression. In the link between EZH2 and HDACs it has been reported that PRC2-mediated transcriptional silencing is impeded by the HDAC inhibitor, trichostatin A (TSA).(12,15) EZH2 is reported to have a SET domain, which is responsible for histone methylation, and this activity was abrogated in the presence of TSA.(12,15,16) Interestingly, the present study showed that SAHA decreased EZH2 expression itself in carcinoma cells, in addition to HDAC repression. Similar downregulation of EZH2 by HDAC inhibitor has been reported by Fiskus et al.(37) Whether the mechanism by which HDAC inhibitors such as SAHA deplete EZH2 level is transcriptional, post-transcriptional, or increased protein degradation remains to be addressed in future study. This double-repression effect might be an important mechanism in the anticancer effect of SAHA and the reason why normal cells are resistant to SAHA treatment. Normal cells express HDAC1/2 in gallbladder epithelial cells, but not EZH2. Whereas gallbladder carcinoma cells, which have both HDAC and EZH2 expression, work together as transcriptional repressors of tumor suppressor genes, and SAHA repressed these epigenetic enzymes together and re-activates tumor suppressor genes. From this point of view, the effect of SAHA on gallbladder carcinoma might be associated with EZH2 expression, rather than HDAC expression.
As epigenetic enzymes often synergize in vivo, there is also great interest in testing combined inhibitor treatments that target more than one epigenetic enzyme, suggesting that combination of an HDAC inhibitor and other anticancer agents might be very attractive therapeutic strategies. It was found in this study that the combination of SAHA and EZH2 siRNA decreased cell numbers more than either single treatment. It was disclosed that cell cycle arrest, not apoptosis, might be related to the synergistic effect of the combined treatment. Furthermore, this study showed that SAHA and/or EZH2 siRNA treatment affect trimetylated and acetylated levels at the p16INK4a and E-cadherin promoter and the combined treatment increased the expression levels of p16INK4a and E-cadherin. Although the mechanism how the addition of EZH2 siRNA enhanced antitumor activity of SAHA remains to be clarified, EZH2 downregulation might affect other members of PRC2(37) and HDAC activity, which result in the enhanced antitumor effect of SAHA. Similar to HDACs, EZH2 histone methyltransferase has emerged as a key target in potential epigenetic strategies; however, specific inhibitors of EZH2 histone methyltransferase have not been described. The most encouraging inhibitory agent of PRC2 reported so far is deazaneplanocin A, which can deplete PRC2 subunits in breast cancer cell lines and reactivate PRC2-silenced genes.(38) However, as this type of inhibitor might affect many processes that require methyl transfer, there are concerns about its specificity in potential therapy. The availability of histone methyltransferase inhibitors specific for EZH2, EZH2 inhibitor, and HDAC inhibitor should expand the repertoire of new possibilities in combined epigenetic therapy.
In conclusion, we showed that EZH2 is overexpressed in carcinoma cells, and HDAC1/2 were clearly expressed in both carcinoma cells and normal epithelial cells of the gallbladder. This expression pattern was also followed in cultured cells. SAHA treatment reduced the expression levels of HDAC1/2 and EZH2 in cultured carcinoma cells along with their reduced cell numbers, and this effect was synergized when treated with EZH2 siRNA. Because gallbladder carcinoma shows increased expressions of EZH2 and HDAC1/2, therapeutic SAHA treatment with EZH2 repressors, such as EZH2-specific methyltransferase inhibitor, is a promising therapeutic approach in gallbladder carcinoma.