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The Histone Methyltransferase WHSC1 Is Regulated by EZH2 and Is Important for Ovarian Clear Cell Carcinoma Cell Proliferation

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The Histone Methyltransferase WHSC1 Is Regulated by EZH2 and Is Important for Ovarian Clear Cell Carcinoma Cell Proliferation Kojima et al. BMC Cancer (2019) 19:455 https://doi.org/10.1186/s12885-019-5638-9 RESEARCHARTICLE Open Access The histone methyltransferase WHSC1 is regulated by EZH2 and is important for ovarian clear cell carcinoma cell proliferation Machiko Kojima1, Kenbun Sone1*, Katsutoshi Oda1, Ryuji Hamamoto2, Syuzo Kaneko2, Shinya Oki1, Asako Kukita1, Hidenori Machino1, Harunori Honjoh1, Yoshiko Kawata1, Tomoko Kashiyama1, Kayo Asada1, Michihiro Tanikawa1, Mayuyo Mori-Uchino1, Tetsushi Tsuruga1, Kazunori Nagasaka3, Yoko Matsumoto1, Osamu Wada-Hiraike1, Yutaka Osuga1 and Tomoyuki Fujii1 Abstract Background: Wolf-Hirschhorn syndrome candidate gene-1 (WHSC1), a histone methyltransferase, has been found to be upregulated and its expression to be correlated with expression of enhancer of zeste homolog 2 (EZH2) in several cancers. In this study, we evaluated the role of WHSC1 and its therapeutic significance in ovarian clear cell carcinoma (OCCC). Methods: First, we analyzed WHSC1 expression by quantitative PCR and immunohistochemistry using 23 clinical OCCC specimens. Second, the involvement of WHSC1 in OCCC cell proliferation was evaluated by MTT assays after siRNA-mediated WHSC1 knockdown. We also performed flow cytometry (FACS) to address the effect of WHSC1 on cell cycle. To examine the functional relationship between EZH2 and WHSC1, we knocked down EZH2 using siRNAs and checked the expression levels of WHSC1 and its histone mark H3K36m2 in OCCC cell lines. Finally, we checked WHSC1 expression after treatment with the selective inhibitor, GSK126. Results: Both quantitative PCR and immunohistochemical analysis revealed that WHSC1 was significantly overexpressed in OCCC tissues compared with that in normal ovarian tissues. MTT assay revealed that knockdown of WHSC1 suppressed cell proliferation, and H3K36me2 levels were found to be decreased in immunoblotting. FACS revealed that WHSC1 knockdown affected the cell cycle. We also confirmed that WHSC1 expression was suppressed by EZH2 knockdown or inhibition, indicating that EZH2 is upstream of WHSC1 in OCCC cells. Conclusions: WHSC1 overexpression induced cell growth and its expression is, at least in part, regulated by EZH2. Further functional analysis will reveal whether WHSC1 is a promising therapeutic target for OCCC. Keywords: Histone methyltransferase, Wolf-Hirschhorn syndrome candidate gene-1, Enhancer of zeste homolog 2, Ovarian clear cell carcinoma, Epigenetic modifier, EZH2 selective inhibitor, H3K36 dimethylation, cell proliferation * Correspondence: [email protected] 1Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kojima et al. BMC Cancer (2019) 19:455 Page 2 of 9 Background compared the expression of WHSC1 in clinical OCCC Ovarian clear cell carcinoma (OCCC) was defined by the samples and normal ovarian tissues. We further blocked World Health Organization as one of the histologic WHSC1 functions in OCCC cells to determine the subtypes of ovarian cancer in 1973. The recent surveil- specific effects on cellular behaviors related to cancer lance and epidemiology showed that the incidence of development and progression. In addition, we analyzed OCCC in the United States is 4.8% in Caucasians, 3.1% the relationship between EZH2 and WHSC1 in OCCC in African American, and 11.1% in Asians. In Japan, the cells. These findings will provide a foundation for further number of cases of OCCC is about 25% that of epithelial investigation of the roles of histone methylation in ovarian cancers, which is higher than that in western carcinogenesis and new effective therapeutic strategies. countries [1]. OCCC is known to be resistant to platinum-based, front-line chemotherapy and shows a Methods worse prognosis compared with serous carcinoma or Tumor samples – endometrioid carcinoma [1 3]. The most frequent and Tumor specimens were acquired from 23 OCCC important genetic alterations observed in OCCC are patients and 3 patients with normal ovaries who under- those that occur in the chromatin remodeling factor went surgery at the University of Tokyo Hospital ARID1A gene, AT rich interactive domain 1A ( ). It has (Additional file 1: Table S1). The specimens were frozen been reported that over 50% of OCCC patients have in liquid nitrogen immediately after collection and then ARID1A mutations in [4]. stored at − 80 °C until RNA extraction. For the use of Histone methylation is one of the important epigenetic specimens in this research, informed consent was modifications, along with histone acetylation, phosphor- obtained from all patients, and the study was approved ylation, ubiquitination, poly ADP-ribosylation, and by the University of Tokyo Genetic Analysis Research sumoylation, and is generally associated with gene Ethics Committee. expression [5]. Many reports have suggested a role of histone methylation dysregulation in carcinogenesis and cancer progression [6]. In addition, several types of Cell lines and EZH2 inhibitor histone methyltransferases have been reported to play OVISE (JCRB1043),OVTOKO (JCRB1048) and RMG-I important roles in tumor progression in many types of (JCRB0172) OCCC cell lines, were obtained from the cancers [7]. For instance, our previous study showed that Japanese Collection of Research Bioresources Cell Bank SUV39H2, a the histone methyltransferase caused thera- (Ibaraki, Osaka, Japan). OVISE and OVTOKO are peutic resistance in cancer cells [8]. ARID1A-mutated cell lines, RMG-Iis ARID1A-wildtype. Wolf-Hirschhorn syndrome candidate 1 (WHSC1) is a Both OVISE and OVTOKO were maintained in SET-domain containing histone methyltransferase [9]. To RPMI1640 medium with 10% fetal bovine serum, ’ activate transcription in various regions of the genome, RMG-Iwas maintained in Ham s F12 with 20% FBS. We WHSC1 specifically catalyzes the dimethylation of lysine used the International Cell Line Authentication Com- 36 of histone H3 (H3K36me2), a histone mark associated mittee (ICLAC) database to confirm that these cell lines with the open chromatin region [10]. Although recent re- were not cross-contaminated or misidentified. In ™ ports suggest that WHSC1 is overexpressed in multiple addition, we used the MycoAlert Mycoplasma Detec- – solid cancers [11, 12], there are no reports regarding its tion Kit (LT07 218, Lonza, Tokyo, Japan) to ensure that expression profile and function in OCCC. there was no mycoplasma contamination before and Enhancer of zeste homolog 2 (EZH2) is one of the most after the study. EZH2 inhibitor GSK126 was purchased widely studied histone methyltransferases in cancer re- from Active Biochemicals (Maplewood, NJ, USA). search. EZH2 tri-methylates H3K27 to silence target gene expression. Increased EZH2 activity is known to have an Quantitative PCR oncogenic effect by repressing tumor suppressor gene ex- RNeasy Mini Kit (Qiagen, Valencia, CA, USA) was used pression [13]. Previously, we reported EZH2 overexpres- for total mRNA extraction according to the manufac- sion in endometrial cancer cell lines and clinical samples. turer’s protocol. We used ReverTra Ace (Toyobo, Osaka, We also found that knockdown of its expression or the Japan) for reverse transcription. WHSC1 and EZH2 use of an EZH2-selective inhibitor could suppress cell mRNA levels were measured by quantitative real-time growth and induce apoptosis [14]. In OCCC, it has been PCR. We designed specific primers for WHSC1, EZH2, reported that EZH2 inhibition has a synthetic lethal effect and GAPDH (Additional file 1: Table S2). Real-time PCR in ARID1A-mutated ovarian cancer cells [15]. was performed using the One-Step SYBR PrimeScript The present study was undertaken to elucidate the in- RT-PCR Kit (TaKaRa Bio, Tokyo, Japan) in a Light Cycler volvement of WHSC1 in OCCC and to evaluate its po- instrument (Roche, Basel, Switzerland). GAPDH (house- tential for therapeutic targeting. To this end, we keeping gene) mRNA levels were used for normalization. Kojima et al. BMC Cancer (2019) 19:455 Page 3 of 9 Western blot analysis Epoch™ Microplate Spectrophotometer (BioTek, Winoo- After treating the OCCC cells with WHSC1-specific siR- ski, VT, USA) [20]. NAs or GSK126 for the indicated times at the indicated concentrations, total protein was extracted and trans- Flow cytometry ferred to a nitrocellulose membrane as previously Cells were fixed in 70% ethanol, and cell cycle analysis described [16, 17]. Primary antibody diluted with block- was performed by staining with propidium iodide ac- ing buffer was added to the membrane and reacted cording to standard protocols. DNA content was mea- overnight at 4 °C. The primary antibodies used in this sured by a. study were anti-WHSC1 (75,359, Abcam, Cambridge, FACS Calibur HG (Becton Dickinson, Franklin Lakes, UK), anti-EZH2 (PA0575, Leica Biosystems, Wetzlar, NJ, USA) and examined using CellQuest Pro ver. 3.1.
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