3-Deazaneplanocin A (DZNep), an Inhibitor of the Histone Methyltransferase EZH2, Induces Apoptosis and Reduces Cell Migration in Chondrosarcoma Cells

Objective Growing evidences indicate that the histone methyltransferase EZH2 (enhancer of zeste homolog 2) may be an appropriate therapeutic target in some tumors. Indeed, a high expression of EZH2 is correlated with poor prognosis and metastasis in many cancers. In addition, 3-Deazaneplanocin A (DZNep), an S-adenosyl-L homocysteine hydrolase inhibitor which induces EZH2 protein depletion, leads to cell death in several cancers and tumors. The aim of this study was to determine whether an epigenetic therapy targeting EZH2 with DZNep may be also efficient to treat chondrosarcomas. Methods EZH2 expression was determined by immunohistochemistry and western-blot. Chondrosarcoma cell line CH2879 was cultured in the presence of DZNep, and its growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7 expression using flow cytometry, and by PARP cleavage using western-blot. Cell migration was assessed by wound healing assay. Results Chondrosarcomas (at least with high grade) highly express EZH2, at contrary to enchondromas or chondrocytes. In vitro, DZNep inhibits EZH2 protein expression, and subsequently reduces the trimethylation of lysine 27 on histone H3 (H3K27me3). Interestingly, DZNep induces cell death of chondrosarcoma cell lines by apoptosis, while it slightly reduces growth of normal chondrocytes. In addition, DZNep reduces cell migration. Conclusion These results indicate that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treat chondrosarcomas.

Here, we show that high grade chondrosarcomas express EZH2 protein, and that DZNep reduces its expression and subsequently H3K27me3. Interestingly, DZNep treatment induces apoptosis of chondrosarcoma cell lines whereas it has a weak effect on normal chondrocyte, and reduces cell migration, suggesting that targeting EZH2, for instance using DZNep, may be an innovative therapeutic strategy to treat chondrosarcomas.

Reagents
DZNep was provided by R&D Biosystems (Lille, France) and resuspended in phosphate buffered saline (PBS). Inhibitors and propidium iodide were purchased from Sigma and dissolved in PBS. Oligonucleotides were supplied by Eurogentec (Angers, France).

Human material
This study was approved by the local ethic committee (Comité de protection des personnes Nord Ouest III). Tumoral and normal cartilage was collected from surgical departments of Caen University hospital. All donors signed agreement forms before the surgery, according to local legislations.

Immunohistochemistry
Multiple specimens of chondrosarcomas (n = 7) or enchondromas (n = 8) were fixed, routinely processed and embedded in paraffin. H&E-stained sections from original block were used to select a representative tumor area. 4-mm sections of nondecalcified chondrosarcomas were prepared from paraffin-embedded tumor blocks and placed on superfrost plus slides. After antigen retrieval with pH 6.0 citrate buffer, immunohistochemistry was performed using an automated immunohistochemical staining processor (Autostainer plus, Dako, Glostrup, Denmark). After incubation with primary antibody EZH2 (Cell signaling, 1:100), detection was performed using an indirect biotin avidin system, LSABTM2 detection kit (Dako) according to the manufacturer's instructions.
The normal cartilage was obtained from biopsy of nasal cartilage. Chondrocytes were released by digestion with XIV Pronase (2 mg/ml for 30 minutes, Sigma-Aldrich, St Quentin Fallavier, France) and type I collagenase (2 mg/ml for 15 hours, Invitrogen, Cergy-Pontoise, France). The cells were incubated in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FBS, 0.25 mg/ml fungizone and 10 mM of ciprofloxacin, and then incubated at 37uC in a humidified atmosphere containing 5% CO 2 .

RNA isolation and real-time reverse transcriptionpolymerase chain reaction (RT-PCR)
Total RNA was extracted with Trizol reagent according to the manufacturer's condition (Invitrogen). Samples (2 mg) were treated with DNAse I (Invitrogen, Cergy-Pontoise, France) and the reverse transcriptase was effected with oligo dT and Moloney murine leukemia virus reverse transcriptase. Complementary DNA was diluted (1:100) and stocked at 220uCpending for PCR. This product (5 mL) was mixed with appropriated reverse and forward primers and SYBR Green PCR Master Mix (Applied Biosystems, Villebon sur Yvette, France) in 15 mL volume final. RT-PCR was run in an ABI Prism 7000 sequence detection system apparatus.
Relative expression was calculated according to the 2 2DDCt method [35].

Cell growth experiment
Cells were seeded at 750 cells/cm 2 and treated with DZNep (1 mM) for 14 days. The medium was changed twice during the treatment. Adherent cells were counted each indicated time (4, 7, 9, 14 days

EZH2 is expressed in chondrosarcomas
First, we investigated whether EZH2 is expressed in chondrosarcomas. Immunohistochemistry analysis from patient biopsies showed that EZH2 is expressed in nucleus of high grade chondrosarcomas (for 6/7 samples). Interestingly, we could not detect EZH2 in all enchondromas tested (n = 8) ( figure 1A). Furthermore, by Western-Blot, we found that EZH2 expression was higher in chondrosarcoma cells than normal chondrocytes (figure 1B). Since EZH2 is highly expressed in chondrosarcomas, we hypothesized that these tumors may be a sensitive to EZH2 inhibitors, such as DZNep.

DZNep inhibits EZH2 and reduces H3K27me3
As expected, DZNep treatment reduced EZH2 protein level, and subsequently H3K27me3 level in two chondrosarcoma cell lines, CH2879 and SW1353 ( figure 2A and B). To investigate whether decreased levels of EZH2 protein resulted from transcriptional regulation, we performed quantitative real time-PCR analysis. DZNep treatment had no effect on EZH2 expression at mRNA level (figure 2C).
DZNep selectively induces cytotoxicity in cancerous but not normal cartilage cells

DZNep reduces cell migration
Finally, the effect of DZNep on migration was also examined by wound healing assay (figure 5). We found that compared to control, DZNep reduced the migration of chondrosarcomas.

Discussion
With the advent of systemic chemotherapy in the management of mesenchymal malignancies such as osteosarcoma and Ewing's sarcoma, there has been an increase in the long-term survival of patients. In contrast, chondrosarcomas continue to have a poor prognosis owing to the absence of an effective therapy [36][37][38]. Identifying new drugs that enables to reduce chondrosarcoma growth may improve survival of patients. Here, we identified, 3-Deazaneplanocin A (DZNep), a small molecule EZH2 inhibitor [13,39], as a putative treatment of chondrosarcomas. Indeed, we show that DZNep treatment significantly reduces the EZH2 protein and H3K27 trimethylation level, and induces chondrosarcoma death by apoptosis while it decreases their migration ability.
First, we showed for the first time, that human chondrosarcomas, but not enchondromas, express EZH2 protein. In addition, EZH2 level was more elevated in a grade II and III chondrosarcoma cell lines, SW1353 and CH2879, than in chondrocytes. This agrees with observations in other tumors showing that EZH2 is overexpressed in cancers, including melanoma, lymphoma, and breast and prostate cancers [13,27,33,40]. This high expression of EZH2 is related to poor prognostic in these cancers and tumors [8,41,42]. The potential use of EZH2 expression for improving diagnostic of chondrosarcomas and the correlation between its expression and tumor grade or prognostic for patients is still in process.
Furthermore, we show for the first time that DZNep is efficient to reduce chondrosarcoma growth, survival and migration, in vitro. Numerous studies suggest that DZNep induces death in tumoral cells though EZH2 downregulation and H3K27me3 reduction [13,30,31,43]. In our chondrosarcoma model, we also found that DZNep reduces EZH2 at protein level (but not at mRNA level) and subsequently decreases H3K27me3. This discrepancy between mRNA and protein levels has ever been observed with other tumoral cells [13,44], and can be explained by the mechanism by which DZNep acts on EZH2. Indeed, DZNep acts indirectly by inhibiting an S-adenosylhomocysteine (SAH) hydrolase, which induce an accumulation of SAH, leading to the degradation of EZH2 protein [39].
However, at this point, we cannot ascertain that chondrosarcoma death induced by DZNep is directly due to EZH2 inhibition. Indeed, DZNep is an AdoHcy hydrolase inhibitor and is able to inhibit methylation of another repressive histone marks, such as H4-K20 methylation [13]. More recently, it has been reported, in MCF7 cells, that DZNep also causes a global decrease in most histone modifications, except for H3K9me3 and H3K37me3, implicating that DZNep is effective in decreasing histone modifications with both repressive and active chromatin markers, in a non-selective manner [39,45]. Therefore, the molecular mechanisms of DZNep might be more complex than our current knowledge base, which associates the downregulation of EZH2 with the ability of DZNep to induce tumor cell death.
Contrary to the majority of tumoral cells, we found that DZNep exerts cytotoxicity in chondrosarcomas with a delay. This was also observed in pancreatic tumors [46]. However, similarly to a number of tumor cells, we found that DZNep induced apoptosis in chondrosarcomas, suggesting that the mechanism of death is shared with all tumors cells, whereas here the effect was delayed. We hypothesize that this delay may be due to a slower proliferation kinetic of chondrosarcomas compared to other tumoral cells. Interestingly, normal chondrocytes only show a slight decrease of their growth upon DZNep treatment. Similarly, other report also show that DZNep does not induce apoptosis in normal cells, making it a promising drug candidate for anti-cancer treatment, in particular to treat radioresistant and chemoresistant tumors such as chondrosarcomas.
In conclusion, in this study, we describe the effect of an AdoHcy hydrolase inhibitor, DZNep, on EZH2 expression and subsequent H3K27me3 in chondrosarcomas, as well as its ability to preferentially induce death by apoptosis in tumoral cartilage cells than normal chondrocytes.