Role of EZH2 in oral squamous cell carcinoma carcinogenesis

Gene 537 (2014) 197–202

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Role of EZH2 in oral squamous cell carcinoma carcinogenesis Lingbo Zhao a, Yang Yu b, Jie Wu b, Jing Bai b, Yuzhen Zhao b, Chunming Li a, Wenjing Sun b,⁎, Xiumei Wang a,⁎⁎ a b

Department of Dentistry, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China Laboratory of Medical Genetics, Harbin Medical University, Harbin 150081, China

a r t i c l e

i n f o

Article history: Accepted 4 January 2014 Available online 11 January 2014 Keywords: EZH2 Oral squamous cell carcinoma Oncogene

a b s t r a c t Oral squamous cell carcinoma (OSCC) is a common human malignancy with high incidence rate and poor prognosis. Although the polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a crucial role in cell proliferation and differentiation during the occurrence and development progress of several kinds of malignant tumors, the impact of EZH2 on the development and progression of OSCC is unclear. In this study, we demonstrate that EZH2 is overexpressed in OSCC cells and clinical tissue. With in vitro RNAi analysis, we generated stable EZH2 knocking down cell lines from two OSCC cell lines, with two sh-RNAs targeting to EZH2, respectively. We found that knocking down of EZH2 could decrease the proliferation ability and induce apoptosis of OSCC cells. Moreover, we demonstrated that of EZH2 inhibition decreased the migration and metastasis of OSCC cells. In conclusion, the results of the current study demonstrated an association between EZH2 expression and OSCC cell development. We recommend that EZH2 acts as an oncogene and plays an important role in OSCC carcinogenesis. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally. There are 1.6 million new cases diagnoses and 333 000 deaths caused by HNSCC annually, with half are localized in the oral cavity (oral squamous cell carcinoma, OSCC) (Jemal et al., 2009). Oral squamous cell carcinoma has been an important component of the worldwide burden of cancer, with the 5-year survival rate approximately 50%, which is poorer than breast cancer or melanoma (Jemal et al., 2011). The occurrence and development of OSCC are complex involving many genes and pathways. The mechanism of OSCC development remains unclear. Several proto-oncogenes have been reported to be involved in OSCC, including RAS, epithelial growth factor receptor (EGFR), MYC, survivin, and Cyclin D1 (Mishra and Das, 2009; Murugan et al., 2012; Pai, 2009; Abbreviations: OSCC, oral squamous cell carcinoma; HNSCC, head and neck squamous cell carcinoma; EZH2, enhancer of zeste homolog 2; EGFR, epithelial growth factor receptor; H3K27, a specific histone 3 lysine 27; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PVDF, polyvinylidene difluoride; qRT-PCR, quantitative reverse transcription polymerase chain reaction. ⁎ Correspondence to: W. Sun, Laboratory of Medical Genetics, Harbin Medical University, #157 Baojian Road, Nangang District, Harbin 150081, China. Tel./fax: + 86 451 86674798. ⁎⁎ Correspondence to: X. Wang, Department of Dentistry, The Second Affiliated Hospital of Harbin Medical University, #246 Xuefu Road, Nangang District, Harbin 150086, China. Tel.: +86 451 86605544; fax: +86 451 86674798. E-mail addresses: [email protected] (W. Sun), [email protected] (X. Wang). 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2014.01.006

Preuss et al., 2008; Sarkis et al., 2010). Overexpression or mutation of these genes is associated with abnormal cell proliferation and tumor aggressiveness (Choi and Myers, 2008; Scully, 2011). Revealing the molecular mechanisms underlying the pathogenesis and progression of oral squamous cell carcinoma may lead to the development of new and effective strategies for diagnosis, prognostication, early detection, and targeted therapy. The polycomb group protein enhancer of zeste homolog 2 (EZH2), a specific histone 3 lysine 27 (H3K27) methyltransferase, plays a critical role in epigenetic gene silencing and chromatin remodeling. It has a master regulatory function in cell proliferation and differentiation (Hock, 2012; Wu et al., 2011). Overexpression of EZH2 has been related to repression of tumor suppressor genes and derepression of genes involved in metastasis, and has been shown to exert oncogenic effects on various types of tumors, including human breast cancer, prostate cancer, gastric cancer, hepatic carcinoma, bladder cancer, kidney cancer, and ovarian cancer (Alford et al., 2011; Chang et al., 2011; He et al., 2012, 2010; Karanikolas et al., 2009; Kim et al., 2013; Raman, 2005; Sasaki et al., 2008; Wagener et al., 2010). EZH2 promotes the progression of regulating cell cycle and apoptosis in cholangiocarcinoma cells and down-regulation in breast cancer reduces in vivo tumor growth (Gonzalez et al., 2009; Nakagawa et al., 2013). Previous research has shown that overexpression of EZH2 was related to malignant potential and adverse outcomes in OSCCs, while a functional study of the role of EZH2 in the development and progression of OSCC has not yet been investigated (Kidani et al., 2009). The purpose of this study was to investigate the biological function of EZH2 in OSCC. We detected EZH2 in OSCC cells to elucidate the

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function and the influence of EZH2 on cell proliferation, apoptosis, metastasis and invasion of OSCC cells. 2. Materials and methods 2.1. Cell culture and tissue specimens The human OSCC cell lines Tca8113, Tb, Ts, and the human adenoid cystic carcinoma cell lines ACC-M and ACC-2 were cultured in RPMI1640 medium. The human OSCC cell lines CAL27 and SCC-4, and human skin keratinocyte cell line HaCaT were grown in Dulbecco's modified Eagle's medium (DMEM). These cells are all supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories GmbH, Pasching, Australia), at 37 °C in a humidified 5% CO2 atmosphere. Tissues of OSCC and the normal specimens were obtained from surgical specimens immediately after resection from patients. The samples were flash frozen in liquid nitrogen and stored at − 80 °C until use. All specimens were randomly selected at the Second Affiliated Hospital of Harbin Medical University, China. 2.2. RNA isolation and quantitative reverse transcription PCR (qRT-PCR) For EZH2 gene expression analysis, total RNA was isolated using TRIzol reagent (Invitrogen Inc., Carlsbad, USA). The cDNA synthesis was generated from a First-Strand cDNA Synthesis Kit (Promega, Madison, WI, USA), according to the instructions of the supplier. qRT-PCR was performed on LightCycler 480 (Roche Diagnostics Ltd, Rotkreuz, Switzerland). Assays were performed in 20 μl reaction mixtures, using a LightCycler 480 SYBR Green I Master Kit, following the manufacturer's protocol. The primers used to amplify EZH2 were: (F) 5′-CAT GTG CAG CTT TCT GTT CAA-3′ and (R) 5′-AGT CTG GAT GGC TCT CTT GG-3′. The primers used to amplify the β-actin control gene were: (F) 5′-AAA TCT GGC ACC ACA CCT TC-3′ and (R) 5′-GGG GTG TTG AAG GTC TCA AA-3′. All measurements were done in triplicate. The threshold cycle value for each product was determined and normalized to that of the internal control, β-actin. qRT-PCR results were analyzed using LightCycler 480 version 1.5.0 software. 2.3. Immunoblotting analysis Cells were harvested in logarithmic phase and were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA, 50 mM Tris–HCl, pH 7.4). Proteins were separated by 10% SDS-PAGE

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and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were incubated with the anti-EZH2 antibody (Cell Signaling Technology Inc., Danvers, MA, USA) overnight at 4 °C, and then incubated with fluorescent-labeled secondary antibody (Zhongshan Bio-Tech Co., Beijing, China) for 1 h at room temperature. GAPDH was detected as control with the antiGAPDH antibody (KangChen Biotech., Shanghai, China). The membranes were then scanned using the Odyssey infrared imaging system (LICOR, Lincoln, NE, USA). 2.4. Generation of stable oral cancer cell lines knocking down EZH2 Oligonucleotides encoding a siRNA specific for EZH2 were subcloned into pLKO.1-TRC vector (Addgene, Cambridge, MA, USA). The following target sequences of EZH2 have been selected: 5′-AAACAGCTGCCTTA GCTTCA-3′ (sh-EZH2-1), and 5′-GCTAGGTTAATTGGGACCAAA-3′ (sh-EZH2-2). sh-Control is: 5′-CTGGCATCGGTGTGGATGA-3′. The authenticity of these plasmids was confirmed by sequencing. Tca8113 cells and CAL27 cells were transfected with sh-EZH2 or sh-control vector by Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer's instructions. Stable cell lines were established after 2 weeks of G418 (200 μg/ml to Tca8113 cells, and 300 μg/ml to CAL27 cells) selection, and the expression of EZH2 was confirmed by immunoblotting analysis. 2.5. Cell proliferation assay and colony formation assay Two thousand Tca8113 cells and CAL27 cells were seeded in 96-well plates. Then cells were performed with MTS using CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega Corporation, Madison, WI, USA). The OD value of each well was read for every 24 h for continuously 5 days, and each experiment was performed in triplicate. The statistical significance was analyzed using ANOVA for each day, * indicates P b 0.05, ** indicates P b 0.01, and *** indicates P b 0.001. For colony formation assay, six hundred Tca8113 cells and CAL27 cells were plated in 6-well plates. After a 14-day period, cells were washed with PBS, and fixed with 10% methanol for 15 min, and stained with Giemsa staining. Colony formation images were pictured for each well and the numbers of colony were counted with ImageJ software. All experiments were performed in triplicate. The statistical significance was analyzed using ANOVA, * indicates P b 0.05, ** indicates P b 0.01, and *** indicates P b 0.001.

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GAPDH Fig. 1. EZH2 protein and messenger RNA (mRNA) levels are illustrated in OSCC cells and tissue specimens. (A) Cell extracts were prepared from HaCat, CAL27, Tca8113, Ts, Tb, SCC-4, ACCM and ACC-2 cells, and analyzed by immunoblotting analyses with the anti-EZH2 antibody. GAPDH was detected as control. (B, C) EZH2 protein and mRNA levels were determined in 14 tissue specimens from patients with OSCC and in 4 paired OSCC tissue specimens using immunoblotting and qRT-PCR analyses. mRNA level was calculated by using the 2−ΔΔCt method. ** indicates P b 0.01. GAPDH was detected as control.

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2.6. Flow cytometry (FCM) analysis for apoptosis detection For apoptosis detection, OSCC cells were stained with PI and FITC labeled Annexin-V, and analyzed by Flow cytometry (BD Biosciences).

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Statistical difference of the percentage of early apoptotic cells (PI −/ FITC +) in each group was analyzed with Chi-square test, * indicates P b 0.05, ** indicates P b 0.01, and *** indicates P b 0.001. The FCM assays were performed in triplicate.

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Fig. 2. Knocking down of EZH2 decreases cell growth rate in OSCC cells. (A, B) Expression levels of EZH2 were decreased in EZH2 stable knocking down OSCC cell lines. Protein and mRNA levels of EZH2 were detected in stable knocking down Tca8113 and CAL27 cells by qRT-PCR analyses and immunoblotting analyses. mRNA level was calculated by using the 2−ΔΔCt method. GAPDH was detected as control. (C, D) The growth rates of sh-EZH2 Tca8113 and CAL27 cells were decreased compared to control cells with MTS assay. The OD value of the cells was measured every day for 5 days and plotted with mean ± SD. ** indicates P b 0.01, *** indicates P b 0.001 by t-test. (SD, standard deviation). (E, F) Knocking down of EZH2 decreased cell proliferation in stable sh-EZH2 Tca8113 and CAL27 cells with colony formation assay. (E, F) The quantification analyses for E and F; the data are the mean ± SD of colony numbers. ** indicates P b 0.01, and *** indicates P b 0.001 by ANOVA (Dunnett's multiple comparison test).

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2.7. Wound healing assay Cells were seeded in 6-well plates to confluence and the cell monolayer was scraped in three straight lines with a 200 μl pipette tip to create ‘scratches’. Cell debris was removed by PBS and then the culture was re-fed with fresh medium. Photographs of the Tca8113 cell wound area were taken at 0 and 48 h after the scratches, and the CAL27 cell wound area were taken at 0 and 24 h after the scratches. The experiments were performed in triplicate. Statistical significance was determined by ANOVA, * indicates P b 0.05, ** indicates P b 0.01, and *** indicates P b 0.001. 2.8. Cell invasion assay For cell invasion assay, BD Biocoat growth factor-reduced Matrigel Invasion Chambers were rehydrated by adding 0.5 ml medium to the upper chambers for 2 h at 37 °C. After rehydration, the medium was carefully removed, and 5 × 104 cells were added to the upper chambers in triplicate. The lower compartments of invasion chambers were filled with medium alone. After 48 h incubation at 37 °C, cells that had invaded through the filter were fixed with absolute methanol, stained with hematoxylin, counterstained with eosin, and then counted under light microscope. Statistical significance was determined by ANOVA, * indicates P b 0.05, ** indicates P b 0.01, and *** indicates P b 0.001. 3. Results 3.1. EZH2 is overexpressed in oral cancer cells We investigated the expression of EZH2 in oral cancer cells in vitro in seven oral cancer cell lines (CAL27, Tca8113, Ts, Tb, SCC-4, ACC-M, ACC-2) using immunoblotting analysis and human skin keratinocyte cell line

HaCaT was as control (Fig. 1A). EZH2 was highly expressed in CAL27, Tca8113, Ts, Tb, ACC-M and ACC-2 cells. We used CAL27 and Tca8113 cell lines in subsequent studies. In addition, we detected EZH2 expression in OSCC tissue specimens by immunoblotting analysis and qRT-PCR. Compared to controls, EZH2 protein and RNA expression levels in OSCC tissue were all increased (Figs. 1B and C).

3.2. Suppression of EZH2 reduces cell proliferation ability in OSCC cells To further investigate the role of EZH2 in oral squamous carcinoma, we performed the following functional study of EZH2 in Tca8113 and CAL27 cells. We generated stable oral cancer Tca8113 and CAL27 cell lines knocking down EZH2 with RNAi system for elaborating the function of EZH2 in OSCC. EZH2 had lower expression levels in sh-EZH2-1 and sh-EZH2-2 Tca8113 and CAL27 cells compared to the respective sh-control cells by immunoblotting analyses and qRT-PCR (Figs. 2A and B). Thus we used these stable cell lines for further study. We first examined the cell proliferation ability of Tca8113 and CAL27 cells with EZH2 stable knocking down. In the cell proliferation assay, we observed that the growth rate of EZH2 knocking down cells were obviously slower compared to the sh-control cells, with a statistically significant difference from the third day on forward of Tca8113 cells and the second day in CAL27 cells (Figs. 2C and D). In addition, we performed a colony formation assay to further illustrate the cell proliferation ability. Two thousand cells of sh-EZH2 and sh-control Tca8113 and CAL27 cells were seeded in 6-well plates for growing 14 days and photographed (Figs. 2E and F). With the number of colony scored for each group, we found the colony numbers of EZH2 knocking down cells to be significantly less than sh-control cells in both cell lines (Figs. 2E and F). Those results indicate that suppression of EZH2 in OSCC cells may reduce their proliferation ability.

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Fig. 3. Knocking down of EZH2 promotes OSCC cell apoptosis. (A, B) Knocking down of EZH2 induced much percentage of early apoptotic cell in sh-EZH2 Tca8113 and CAL27 groups in FACS assay. * indicates P b 0.05, and *** indicates P b 0.001 by Chi-square test when compared to the control group.

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3.3. Depression of EZH2 promotes cell apoptosis in OSCC cells

Together, EZH2 plays important roles in cell proliferation and inhibition of cell apoptosis in OSCC cells.

Based on the above result, we further assessed the apoptotic profile of the sh-EZH2 cells. With the FACS assay, we found that the percentage of cells in early apoptosis was increased higher in sh-EZH2 cells compared to controls, increasing from 2.91% to 7.65% and 4.05% in Tca8113 cells, and from 8.25% to 11.56% and 21.61% in CAL27 cells, with the statistically significant difference (Figs. 3A and B). The result suggests that knocking down of EZH2 in OSCC cells inhibited cell growth inhibition and activated cell apoptosis.

3.4. Lower expression of EZH2 decreases cell migration and metastasis of OSCC cells Overexpression of EZH2 acts as an indicator of metastasis and migration (Alford et al., 2011; Tang et al., 2012). We examined whether knocking down of EZH2 could decreased these malignant behaviors in OSCC cells. We performed wound healing assay to investigate the effects

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Fig. 4. Knocking down of EZH2 decreases cell migration and metastasis. (A, B) Knocking down of EZH2 decreased Tca8113 and CAL27 cell migration in wound healing assay. The quantification analyses for A and B; the data are the mean ± SD percentage of the remaining area determined by normalizing the area of wound after 24 h or 48 h to the initial wound area at 0 h (set to 100%). *** indicates P b 0.001 by ANOVA. (C, D) Knocking down of EZH2 decreased metastasis in sh-EZH2 Tca8113 and CAL27 groups in cell invasion assay compared to the shcontrol cells, 100× magnifications. Statistical analyses for C and D; the data are the mean ± SD of invaded cells. *** indicates P b 0.001 by ANOVA (Dunnett's multiple comparison test). (SD, standard deviation).

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of lower expression of EZH2 on cell migration in OSCC cells (Figs. 4A and B). Forty eight hours and twenty four hours later after the scratches of Tca8113 and of CAL27 cells respectively, the migration rate of sh-EZH2 cells was lower than that of the sh-control cells, both in Tca8113 and in CAL27 cell lines (Figs. 4A and B). The quantification of wound healing is shown in Figs. 4A and B, implying that lower expression of EZH2 may decrease cell migratory ability. Moreover, metastatic ability is the most aggressive function of cancer cells. Thus, in the cell invasion assay, we examined the metastasis of OSCC cells after EZH2 knocking down. Forty eight hours later after the incubation of cells in invasion chambers, we found that sh-EZH2 cells invaded through the basement membrane were much less than the sh-control cells (Figs. 4C and D). By numerical scoring, the histograms showed that knocking down of EZH2 led to a significantly lower invasion rate of OSCC cells in both cell lines, with the statistically significant difference (Figs. 4C and D), suggesting that suppression of EZH2 decreases metastasis in OSCC cells. Taken together, EZH2 acts as an oncogene in OSCC. It may play an important role in cell proliferation, cell metastasis and inhibition of apoptosis. 4. Discussion Although some clinical studies have investigated EZH2 expression in OSCC (Chen et al., 2013; Kidani et al., 2009), the EZH2 functional research is missing in OSCC. The current study provides the first evidence of the importance of EZH2 expression in OSCC development by in vitro experiment. With the functional study, our results indicated that knocking down of EZH2 inhibited OSCC cell proliferation and activated cell apoptosis in vitro. These results supported previous studies (Borbone et al., 2011; Chang et al., 2011; Li and Zhang, 2013). Moreover, Cao et al. results proved the biological link between EZH2 overexpression and cell proliferation in oral leukoplakia, and that cell-cycle progression had been promoted by EZH2 in early oral tumorigenesis (Cao et al., 2011). These findings are consistent with our findings. As migration and invasion are the developing processes during the cancer progression, we examined the role of EZH2 in migration and metastasis of OSCC cells. With the expression of EZH2 reduced, the migration and invasion rate of knocking down OSCC cells are obviously decreased. Recent results on cell migration and invasion showed that high levels of EZH2 could cause a rise in cell migration and invasion in prostate cancer cells, human epithelial ovarian cancer cells and oral leukoplakia (Tang et al., 2012). Taken together, EZH2 is involved in the migration and invasion behavior of tumor cells. Both our data and that of Kidani et al.'s clarified that EZH2 expression was high in OSCC cell lines and tissue specimens. Kidani et al. identified that EZH2 expression had an association with histological differentiation, clinical stage, tumor size, lymph node metastasis and poor prognosis, suggesting that EZH2 might serve as a prognostic predictive marker in OSCC. Our current studies confirmed that inhibition of EZH2 expression could reduce cell proliferation and invasion in OSCC. Taken together, EZH2 plays an important role in OSCC cell carcinogenesis and may represent a potential therapeutic target. Further in vivo investigations are needed to elucidate these issues. In conclusion, our data provide evidence that EZH2 is associated with cell proliferation, apoptosis, metastasis and invasion in OSCC development. In view of the data presented here and previously reported, we recommend that EZH2 could represent a potential therapeutic target, playing an important role in OSCC cell carcinogenesis. Conflict of interest The authors declare no conflict of interest.

Acknowledgments This work was supported by the Natural Science Foundation of Heilongjiang Province of China (D201175), Leading Talent Echelon Fund for Reserve Leaders of Heilongjiang Province and Postdoctoral Science Foundation of Heilongjiang Province (LRB05-279) (to X.W.). References Alford, S.H., Toy, K., Merajver, S.D., Kleer, C.G., 2011. Increased risk for distant metastasis in patients with familial early-stage breast cancer and high EZH2 expression. Breast Cancer Res. Treat. 132, 429–437. Borbone, E., Troncone, G., Ferraro, A., Jasencakova, Z., Stojic, L., Esposito, F., Hornig, N., Fusco, A., Orlando, V., 2011. Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas. J. Clin. Endocrinol. Metab. 96, 1029–1038. Cao, W., Younis, R.H., Li, J., Chen, H., Xia, R., Mao, L., Chen, W., Ren, H., 2011. EZH2 promotes malignant phenotypes and is a predictor of oral cancer development in patients with oral leukoplakia. Cancer Prev. 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