Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer
- Xiaoxue Zhang†1,
- Dan Liu†1,
- Mengchen Li1,
- Canhui Cao1,
- Dongyi Wan1,
- Bixin Xi1,
- Wenqian Li1,
- Jiahong Tan1,
- Ji Wang1,
- Zhongcai Wu1,
- Ding Ma1 and
- Qinglei Gao1Email author
© The Author(s). 2017
Received: 30 September 2016
Accepted: 13 January 2017
Published: 23 January 2017
Epigenetics has been known to play a critical role in regulating the malignant phenotype. This study was designed to examine the expression of DOT1L (histone 3 lysine 79 methyltransferase) and H3K79 methylation in normal ovarian tissues and ovarian tumors and to explore the function of DOT1L and its underline mechanisms in ovarian cancer.
The expression of DOT1L and H3K79 methylation in 250 ovarian tumor samples and 24 normal ovarian samples was assessed by immunohistochemistry. The effects of DOT1L on cell proliferation in vitro were evaluated using CCK8, colony formation and flow cytometry. The DOT1L-targeted genes were determined using chromatin immune-precipitation coupled with high-throughput sequencing (ChIP-seq) and ChIP-PCR. Gene expression levels were measured by real-time PCR and immunoblotting. The effects of DOT1L on tumor growth in vivo were evaluated using an orthotopic ovarian tumor model.
DOT1L expression and H3K79 methylation was significantly increased in malignant ovarian tumors. High DOT1L expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage, histologic grade, and lymphatic metastasis. DOT1L was an independent prognostic factor for the overall survival (OS) and progression-free survival (PFS) of ovarian cancer, and higher DOT1L expression was associated with poorer OS and PFS. Furthermore, DOT1L regulates the transcription of G1 phase genes CDK6 and CCND3 through H3K79 dimethylation; therefore, blocking DOT1L could result in G1 arrest and thereby impede the cell proliferation in vitro and tumor growth in vivo.
Our findings first demonstrate that DOT1L over-expression has important clinical significance in ovarian cancer and also clarify that it drives cell cycle progression through transcriptional regulation of CDK6 and CCND3 through H3K79 methylation, suggesting that DOT1L might be potential target for prognostic assessment and therapeutic intervention in ovarian cancer.
Ovarian cancer (OC) is the fourth most common cause of cancer-related deaths in women, with an estimated 200,000 cases and 125,000 deaths occurring annually worldwide [1, 2]. Despite current advances in surgery and adjuvant chemotherapy, the clinical outcome of patients with OC remains uncertain, with the 5-year survival rate being ~30% for advanced-stage disease. The poor survival rate is largely attributed to the poor understanding of the OC initiation and progression . The discovery of novel biomarkers with high sensitivity and specificity for the early diagnosis and target therapies of OC will help to improve the prognosis of this disease.
Epigenetic changes, such as DNA methylation and histone post-translational modifications, are major contributing factors for carcinogenesis . Given that epigenetic changes are reversible, there is a hope that reversing epigenetic malfunction in cancer cells could render them normal, launching a new and rapidly expanding field of cancer epigenetic therapeutics . Disruptor of telomeric silencing-1-like (DOT1L), a human homologue of DOT1 in yeast, is the only histone methyltransferase that produces methylated H3K79 (H3K79me x , x = 1, 2, or 3). Globally, H3K79 methylation is enriched in actively transcribing genes . In addition to transcription, Dot1 plays an important role in cell cycle regulation and the DNA damage response . Recent studies have revealed that DOT1L is involved in the initiation and maintenance of mixed lineage leukemia (MLL)-rearranged leukemia, by associating with MLL fusion partner proteins, e.g., AF4, AF9, AF10, or ENL, resulting in abnormal H3K79 methylation and overexpression of a number of MLL target genes (e.g., HoxA9 and Meis1) [8–10]. Consequently, DOT1L is a drug target for MLL-rearranged leukemia [11, 12]. Furthermore, a few recent publications have reported that DOT1L plays crucial roles not only in leukemia, but also in solid tumors, such as breast cancer, esophageal squamous cell carcinoma, colorectal cancer, prostate cancer, and gastric cancer [13–19]. Moreover, DOT1L inhibitors have shown effective suppression of proliferation, migration, and invasion of breast cancer .
To date, the potential role of DOT1L and its malignancies in OC have not been assessed. In this study, the prognostic significance of DOT1L expression in OC for clinicopathological parameters and patient survival was evaluated. Moreover, it is very significant that we identified DOT1L as a promising therapeutic target for OC both in vitro and in vivo experiments.
Patients and samples
A total of 274 formalin-fixed paraffin-embedded tissue samples were included in this study, comprising 24 normal ovarian tissues, 15 benign ovarian tumor, 7 borderline ovarian tumor, and 228 malignant ovarian tumor samples. These samples were obtained from patients diagnosed with ovarian carcinoma who underwent intitial surgery at the Gynecology Department of Huazhong University of Science and Technology Affiliated Tongji Hospital from 2003 to 2013. The tissue samples were obtained with informed consent from patients. None of the patients had any prior history of chemoradiation, radiation, or hormonal therapy before the surgery. Clinical information of the patients’ was recorder in detail, and the diagnoses were confirmed by at least two pathologists. The histopathologic subtypes of the 7 borderline ovarian tumor and 228 malignant ovarian tumor samples included 211 serous cystadenocarcinomas, 5 mucinous cystadenocarcinomas, 12 endometrioid adenocarcinomas, 4 ovarian germ-cell tumors, 2 ovarian clear-cell carcinomas, and 1 ovarian transitional cell carcinoma. There were 20 cases in stage I, 17 cases in stage II, 159 cases in stage III, 29 cases in stage IV, and 10 cases remained unknown, according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. With regard to histological grading, 40 cases had grade 1, 58 cases had grade 2, 120 cases had grade 3, and 17 cases remained unknown. The median age of patients (16–80 years old) was 54 years old. Among the 235 OC tissues, the total of 206 cases has been followed up. The present study was with the approval and support of the Ethics or Institutional Review Board of Tongji Hospital.
Formalin-fixed, paraffin-embedded tissue blocks were retrieved from the archive and were analyzed by IHC described previously . In short, The Avidin-Biotin Complex (ABC) Vectastain Kit (Zsgb-Bio, Beijing, China) was used and the DOT1L polyclonal antibody (1:250; ab64077; Abcam), H3K79me2 polyclonal antibody (1:200; ab3594; Abcam), cyclin D3 antibody (1:100; ab63535; Abcam), and the CDK6 polyclonal antibody (1:500; GTX103992; GeneTex) were used as primary antibodies incubated with tissue sections (2 mm) after heat-induced epitope retrieval (10 mM sodium citrate buffer of pH 6.0), followed by incubation with a secondary antibody conjugated to peroxidase (1:100; Dako). Detection was performed using diaminobenzidine for 8 min, and the slides were counterstained with hematoxylin.
The scoring system that was used incorporated both the intensity of the scoring (0 = absent, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive tumor cells (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, 4 = 76–100%). Points for the intensity and the percentage of staining were added and assigned as overall score according to Kamat and M. Caroline [22, 23]. Two investigators scored all slides for DOT1L and H3K79 methylation staining and followed the criterions of double-blind trials. Based on the overall score, the DOT1L and H3K79 methylation staining results were classified into low expression (≤4) and high expression (>4).
Cells and transfection
Human epithelial ovarian cell lines SK-OV-3, TOV21G, OV90, and CaOV3 were purchased from the American Type Culture Collection (Manassas, VA, USA). SK-OV-3 and CaOV3 were cultured in McCoy’s 5A Medium (Boster, Wuhan, China), while TOV21G and OV90 in mixed MCDB105 Medium (Sigma-Aldrich Co., St Louis, MO, USA) and M199 Medium (Gibco, Invitrogen, Carlsbad, CA, USA) (1:1) supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cells were transduced with CMV-Fluc-IRES-RFP lentiviral particles (GeneChem, Shanghai, China). RFP/luciferase-expressing cells were isolated by FACS and used in living imaging. ShDOT1L-1 and shDOT1L-2 shRNA lentiviral particles (GeneChem) were used to knock-down DOT1L expression, targeting 5′-CGGATCTCAAGCTCGCTAT-3′ and 5′-AGTGCTCGAATTGAGAGAA-3′, respectively. ShCON, the short hairpin RNA (shRNA) not targeting any known gene, was used as control. SK-OV-3 and TOV21G cells were transduced with DOT1L shRNA lentiviral particles, or control shRNA lentiviral particles. The cells with stable transfection were selected with puromycin.
Assay of gene expression by real-time RT-PCR
Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA). RT-PCR analysis was carried out in a CFX96 Touch™ Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and iQ™ SYBR®Green Supermix (Bio‑Rad Laboratories, Inc., Hercules, CA, USA), which contained 5 ng complementary DNA (cDNA) and 10 pM of each primer. The primer sequences used for PCR are shown in Additional file 1: Table S1. The relative messenger RNA (mRNA expression) levels were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR protocol is as follows: denaturation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 10 sec; annealing at 60 °C for 30 sec; and extension at 72 °C for 30 sec. Each sample was assayed in triplicate, and relative mRNA expression levels were calculated using the △△Ct method.
Western blot analysis
Western blot assay was performed following the protocol as previously described [24, 25]. Briefly, the cells were harvested and lysed using ice-cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, and 0.5% sodium deoxycholate). Following centrifugation at 10,000×g for 15 min at 4 °C, the proteins in the supernatants were quantified by Bradford method and separated using 10% SDS-PAGE gel and electrotransferred from the gel to a nitrocellulose membrane (Merck & Co., Inc., Whitehouse Station, NJ, USA). Following blocking with 5% skimmed milk in phosphate-buffered saline, the membranes were immunoblotted with the primary antibodies, namely, CDK6 (GeneTex, San Antonio, Texas, USA), DOT1L, H3, H3K79me2, cyclin D3, and GAPDH (Abcam, Cambridge, UK) at 4 °C overnight. Specifically bound HRP-conjugated secondary antibodies were detected using an ECL detection system (ChemiDocTM XRS+ machine, Bio-Rad Laboratories). Protein levels of GAPDH and H3 were employed as loading controls. Densitometric analyses were performed using ImageJ software. Relative quantification was carried out after normalization to the band intensities of GAPDH or H3. A Mann-Whitney test was performed to assess the difference of protein expression between groups. Each experiment was performed in triplicate and repeated at least 3 times.
In vitro cell viability
Cell proliferation was evaluated using a colorimetric assay as described previously . Cell viability was measured by a commercially Cell Counting Kit (CCK-8, Beyotime Institute of Biotechnology, China). The indicated cells were seeded into 96-well plates at a density of 104 cells per well. The drugs were added when the plates were seeded. After incubation for 3, 6, 9, and 12 days, cell proliferation in 96-well plates was measured and 10 μL CCK-8 reagent was added to each well and incubated at 37 °C for 4 h. Absorbance was measured at 450 nm by using a spectrophotometer. Each experiment was performed in triplicate and repeated at least 3 times.
Colony formation assay
The colony formation assay was performed on the indicated cells. For each group, 800 survived cells/well were incubated in 6-well plates, containing 2.5 ml complete medium per well, followed by incubation for 10 days. At the indicated time point, cells were washed twice with PBS, treated with crystal violet for 10 min, washed, then counted, and measured. All the experiments were performed at least 3 times. The colonies with >40 cells were subsequently counted under a phase contrast microscope at ×40 magnification.
Cell cycle and cell apoptosis analyses
Cell cycle and cell apoptosis analyses were determined by flow cytometry (BD, UA) [27, 28]. In this experiment, the SK-OV-3 and TOV21G cells were treated with 10 μM SGC0946 and EPZ004777 for 12 days, respectively. Then, the cells treated above were harvested and washed with phosphate-buffered saline (PBS). For cell cycle analysis, the pellets were fixed with ice-cold 70% ethanol overnight at 4 °C. Cell pellets were then suspended in 300 μl PBS containing 100 μg/ml RNase for 30 min at room temperature, and then 10 μg/ml propidium iodide (Sigma-Aldrich Co., St Louis, MO, USA). The distribution of cells in the different phases of the cell cycle was analyzed from DNA histograms using BD Cell Quest software (Becton Dickinson Biosciences, San Diego, CA, USA). For cell apoptosis analysis, cells were resuspended in 500 μl binding buffer (Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences)), containing 5 μl of annexin V-fluorescein isothiocyanate (FITC) stock and 5 μl of 20 μg/ml PI for determination of phosphatidylserine (PS) exposure on the outer plasma membrane. After incubation for 10 min at room temperature in a light-protected area, the cells were quantified by flow cytometry. Viable cells were annexin V- and PI-negative.
ChIP-qPCR and ChIP-Seq
ChIP assays were performed following the protocols as previously described [13, 29, 30]. In brief, crosslinking was performed with 1% formaldehyde at room temperature for 10 min and the reaction was stopped by treatment with 0.125 M glycine. The cell pellets were resuspended in 200 ml of SDS lysis buffer and sonicated using a Bioruptor (Cosmo Bio Co. Ltd, Tokyo, Japan). The cell lysates were then immunoprecipitated with specific antibodies overnight at 4 °C and further incubated with salmon sperm DNA coupled to protein A-agarose (Millipore) for 2 h at 4 °C. The precipitates were washed, eluted, and then reverse-crosslinked with 20 ml 5 M NaCl by incubating at 65 °C overnight. DNA fragments were precipitated from the eluate and dissolved in ddH2O. Eluted DNA fragments were analyzed by qPCR, or subjected to sequencing using next-generation Solexa sequencing.
Female NOD-SCID mice (4 weeks old) were purchased from Beijing HFK Bio-Technology Co. Ltd. (Beijing, China). The studies were approved by the Committee on Ethics of Animal Experiments of Tongji Medical College. The mice were maintained in the accredited animal facility of Tongji Medical College. Upon injection of SK-OV-3 cells transduced with CMV-Fluc-IRES-RFP lentiviral particles into the ovarian bursa bilaterally of female mice, we have developed an orthotopic ovarian tumor model. SGC0946 (10 mg/kg per mouse) was administered intraperitoneal twice a week for 6 weeks. The size of tumor was monitored in living mice by optical imaging of luciferase activity using the IVIS Spectrum system (Caliper, Xenogen, USA). After anaesthetization with 3% pentobarbital sodium, mice were imaged 10 min after intraperitoneal injection of 100 mg/kg D-luciferin. In the experimental metastasis model [31, 32], tumor cells were injected into mice via the tail vein (2 × 106 cells/mouse). At each time option, mice were monitored using the IVIS SPECTRUM system according to the method above. Total flux (photons/s) was quantified using Living Image version 4.3.1 software.
Chi-squared tests were used to evaluate whether DOT1L expression was correlated with clinicopathological factors of OC. Overall survival (OS) and progression-free survival (PFS) curves were plotted using the Kaplan-Meier analysis followed by two-sided log-rank tests. The univariate and multivariate Cox proportional hazards modeling was used to evaluate prognostic significance. For cell-based assays, differences between groups were assessed by two sided Student’s t tests, unless indicated otherwise. Data were analyzed using SPSS 18.0 statistic software (SPSS Inc., Chicago, IL). Each experiment was repeated at least 3 times with comparable results, unless indicated otherwise. Significance was assessed at the level of P < 0.05. (*P < 0.05, **P < 0.01, and ***P < 0.001, ****P < 0.0001).
DOT1L and H3K79 methylation expression in normal ovarian tissues and benign, borderline, and malignant ovarian tumors
Association between DOT1L expression and clinicopathological parameters of OC
Association of DOT1L expression with the clinicopathological features of patients with borderline and malignant ovarian tumors
Age at diagnosis
Preoperative CA125 (U/ml)
High expression of DOT1L as an independent prognostic marker of OC
Multivariate Cox regression analysis of the clinicopathogical parameters for overall survival and progression free survival
Progression free survival
FIGO stage (early stage vs. advanced stage)
Histologic grade (well vs. poor and moderate)
Ascites (no vs. yes)
LN metastasis (no vs. yes)
Age (≤50 vs. >50 years)
Preoperative CA125 (U/ml) (≤600 vs. >600)
DOT1L expression (low vs. high)
Knocking down of DOT1L inhibits the proliferation of ovarian cancer cells and induces G1 phase arrest
DOT1L regulates the transcription of G1 arrest genes CDK6 and CCND3 through H3K79 dimethylation
Effect of inhibition of DOT1L pharmacologically on tumor progression in vivo
This is the first study to examine DOT1L expression and H3K79 methylation in ovarian cancer. In human, DOT1L is involved in the oncogenesis of several leukemia subtypes, mostly characterized by chromosomal translocations involving the mixed lineage leukemia (MLL) gene . To identify the role of DOT1L in ovarian cancer, we performed immunohistochemistry and found that DOT1L expression was significantly increased in malignant ovarian cancer. In addition, H3K79 methylation was also significantly increased in malignant ovarian cancer, indicating the correlation of DOT1L and H3K79 methylation in ovarian cancer. Higher DOT1L expression was associated with advanced FIGO stage, poor histopathological grade, and lymph node metastasis of OC patients. Importantly, DOT1L expression was identified as an independent prognostic factor for OC, which raised the possibility that DOT1L might be correlated with malignant behavior of ovarian cancer. As expected, our in vitro experiment demonstrated that blocking DOT1L in ovarian cancer could lead to the decrease of ovarian cancer cell viability and colony-forming ability.
DOT1L has a critical role in regulating gene transcription, development, cell cycle progression, somatic reprogramming, and DNA damage repair [37–39]. Consistent with previous finding that DOT1L siRNA-transfected human lung cancer cells undergo G1 cell cycle arrest , we showed here that knocking down of DOT1L in ovarian cancer cells could result in G1 arrest and therefore impede the cell proliferation. It is worth mentioning that asynchronized cells were used for cell cycle analysis in this paper, due to the reason that DOT1L might be related with DNA replication [38, 39]. Most importantly, the present study provides mechanistic insight into the inhibition of ovarian cancer cell proliferation by blocking DOT1L via downregulation of CDK6 and CCND3 through H3K79 dimethylation, providing a novel mechanism of epigenetic regulation of DOT1L-mediated transcription of cell cycle genes. The D-type cyclins (cyclins D1, D2, and D3) are the first cyclins to be induced as cells enter the G1 phase of the cell cycle [34, 41, 42]. D-type cyclins associate with and activate cyclin-dependent kinases (CDK), such as CDK4 and CDK6 . Specially, cyclin D3 was identified as a direct target of Notch1 that promotes cell-cycle progression and proliferation both in peripheral and leukemic T cells . The data presented in this report demonstrated that DOT1L regulation of cell cycle was through CDK6 and CCND3. Although several studies reported that some histone modifications transcriptionally regulated the expression of cell cycle regulators such as CDK inhibitors to govern cell cycle progression [40, 44, 45], in fact, our data together with the described molecular functions identified CDK6 and cyclin D3 as transcriptionally regulated genes by DOT1L. Given the fact that the cell-cycle arrest network is complex, because the regulators involved act redundantly, have multiple substrates, and provide feedback control , the phenomenon was explainable.
The dynamic interplays among different chromatin post-transcription modifications control gene expression and provide novel opportunities for targeted combination therapies in multiple cancer types. Molecular targeting of specific histone methyltransferase is emerging as a new direction for cancer therapy . Of the known histone methyltransferase enzymes, DOT1L is a promising target due to it being the only known H3K79 histone methyltransferase, its unique non-SET catalytic domain, and its role in promoting and maintaining MLL leukemogenesis [48, 49]. It is interesting to note that small molecule DOT1L inhibitors have been recently developed, and one of the DOT1L inhibitors is already under investigation in a phase I clinical trial in patients with MLL fusion gene-driven leukemia . Fortunately, our data showed that inhibition of DOT1L enzymatic activity with EPZ004777 and SGC0946 treatment could suppress ovarian cancer cell proliferation in vitro, and furthermore, administration of SGC0946 in vivo resulted in significant inhibition of tumor progression in the ovarian orthotopic xenograft mice model. Consequently, DOT1L could have strong prospects for therapeutic target in ovarian cancer.
Our findings first demonstrated that OC patients expressing high level of DOT1L have poorer OS and PFS compared with those with low DOT1L expression, and DOT1L expression can be regarded as an independent prognostic factor. Furthermore, DOT1L regulates the transcription of G1 phase genes CDK6 and CCND3 through H3K79 dimethylation, thereby knocking down of DOT1L could result in G1 arrest and therefore impede the cell proliferation and tumor progression both in vitro and in vivo. Our results suggest that DOT1L might be potential target for prognostic assessment and therapeutic intervention in ovarian cancer.
We thank Dr. Liangle Yang (Department of Occupational and Environmental Health and Ministry of Education Key Lab for Environment and Health, Tongji Medical College, Huazhong University of Sicence and Technology, Wuhan, China) for his kind help in the statistical analysis.
This work was supported by the National Natural Science Foundation of China (81572570, 81502250, 81372801, 81472783, 81230038).
Availability of data and materials
QG and DL designed the research. XZ analyzed the data and prepared the typescript. The other authors provided the subject data. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study was approved by the Ethics or Institutional Review Board of Tongji Hospital, and written informed consent was obtained from all subjects in accordance with the Declaration of Helsinki.
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