The polycomb group protein EZH2 is a novel therapeutic target in tongue cancer

Abstract

EZH2, a core member of the Polycomb Repressor Complex 2 (PRC2), mediates transcriptional silencing by catalyzing the trimethylation of histone 3 lysine 27 (H3K27), which plays key roles in cancer initiation and progression. Here, we investigated the expression pattern and biological roles of EZH2 in tongue tumorigenesis by loss-of-function assays using small interference RNA and EZH2 inhibitor DZNep. Also we determined the therapeutic efficiency of DZNep against tongue cancer in vivo. We found that aberrantly overexpressed EZH2 was associated with pathological grade, cervical nodes metastasis and Ki-67 expression in tongue cancers. Elevated EZH2 correlated with shorter overall survival and showed significant and independent prognostic importance in patients with tongue cancer. Both genetic and pharmacological depletion of EZH2 inhibited cell proliferation, migration, invasion and colony formation and decreased CD44+ subpopulation probably in part through modulating p16, p21 and E-caherin. Moreover, DZNep enhanced the anticancer effects of 5-Fluorouracil. Furthermore, intratumoral EZH2 inhibition induced by DZNep intraperitoneal administration significantly attenuated tumor growth in a tongue cancer xenograft model. Taken together, our results indicate that EZH2 serves as a key driver with multiple oncogenic functions during tongue tumorigenesis and a new biomarker for tongue cancer diagnosis and prognostic prediction. These findings open up possibilities for therapeutic intervention against EZH2 in tongue cancer.

Fig 1. Overexpression of EZH2 and its prognostic significance in TSCC

A: EZH2 mRNA levels were determined by real-time RT-PCR in four tongue SCC cell lines and normal tongue mucosa. B: EZH2 and its associated H3K27me protein levels were measured by western blotting in TSCC lines and normal tongue mucosa. Representative images of WB are shown. C: EZH2 protein levels were determined by western blotting in three pairs of tongue SCC and adjacent non-tumor tissue samples. D: Localization of EZH2 protein was identified by immunofluorescence staining (EZH2 was predominantly detected in nucleus). A representative immunofluorescence image is shown. E: EZH2 expression in human tongue SCC specimens was determined by immunohistochemistrical staining. (a: negative expression in normal tongue mucosa(×200); b: low expression in tongue SCC(×200); c: high expression in tongue SCC(×200); d: high magnification of EZH2 high expression(×400)). F: Overall survival analyses of patients with high or low expression of EZH2 were estimated by Kaplan-Meier method and compared with log-rank test. Data showed here are mean ± SD from three independent experiments, *p<0.05,**p<0.01, Student's t-test.

Fig 2. DZNep inhibits endogenous EZH2 by proteosome-mediated protein degradation

A: DZNep inhibits EZH2 in a dose-and time-dependent manner in both Cal27 and Tca8113 cells. Representative images of WB are shown. B: H3k27me3 was simultaneously reduced following DZNep exposure (5μM, 72h) in both Cal27 and Tca8113 cells. Representative images of WB are shown. C: Real-time RT-PCR assay for EZH2 mRNA levels following DZNep exposure (5μM, 24h) in both Cal27 and Tca8113 cells. D: EZH2 protein was determined by Western blotting after Cal27 was exposed to DZNep or/and the proteosome inhibitor MG132 for 24h. Representative image of WB were shown. Data showed here are mean ± SD from three independent experiments, Student's t-test.

Fig 3. DZNep inhibits cell growth and migration and invasion, while induces cell apoptosis in tongue cancer cells

A: Cell proliferation was significantly impaired after DZNep treatment for 48h as measured by MTT assay. B: The percentages of apoptotic cells were remarkably increased after DZNep treatment as determined by flow cytometry. C: Migration potentials of Cal27 treated with DZNep and vehicle were determined by wound healing assay. D: Invasion potentials of Cal27 treated with DZNep and vehicle were determined by modified Boyden chamber assay. E: The protein abundances of several downstream effectors or targets of EZH2 before and after DZNep exposure were measured by Western blotting. Representative images of WB are shown. Data showed here are mean ± SD from three independent experiments, **p<0.01, Student's t-test.

Fig 4. DZNep reduces colony formation and CD44+subpoputation in tongue cancer cells

A: The number and size of colonies formed by Cal27 pretreated with DZNep or vehicle as stained by crystal violet. B: The percentage of CD44 positive subpopulation in DZNep or vehicle treated Cal27 cells was determined by FACS. C, D: The mRNA levels of several common markers for cancer stem cell isolation were quantified using real-time RT-PCR assay. Data showed here are mean ± SD from three independent experiments, *p<0.05, **p<0.01, Student's t-test.

Fig 5. DZNep induces cell senescence and enhanced 5-FU chemosensititvity in tongue cancer cells

A, B: SA-β-gal staining positive cells were determined and compared after Ca27 and Tca8113 were treated with DZNep or vehicle for 72h. C, D: Cell proliferation was significantly inhibited by DZNep and 5-FU alone or in combination as assayed by MTT. E, F: The percentages of cell undergoing apoptosis after DZNep or vehicle treatment were measure by flow cytometry and compared accordingly. (Data are mean ± SD from two independent experiments, *p<0.05, **p<0.01, Student's t-test.)

Fig 6. EZH2 knockdown by siRNAs phenocopys the effects of DZNep exposure in tongue cancer cells

A: EZH2 mRNA levels after siEZH2 or negative control siRNA transfection in both cell lines as quantified by real-time RT-PCR assay. B: The abundance of EZH2, H3K27me3 and other associated downstream effectors were determined by western blotting following siEZH2 transfection. Representative images of WB are shown. C: Cell proliferation was measured after both cells were transiently transfected with siEZH2 or negative control siRNA. D: Cell cycle distribution of Cal27 followed by siEZH2 and negative control siRNA treatment as measured by flow cytometry. E: Migration potential of Cal27 treated with siEZH2 and negative control siRNA was determined by wound healing assay. F: The percentages of apoptotic cells were remarkably increased after siEZH2 treatment as determined by flow cytometry. G: Invasion potentials of Cal27 and Tca8113 treated with siEZH2 and negative control siRNA were determined by modified Boyden chamber assay. H: The number and size of colonies formed by Cal27 pretreated with siEZH2 or negative control siRNA as stained by crystal violet. I: SA-β-gal staining positive cells were determined and compared after Ca27 treated with siEZH2 or negative control siRNA for 72h. J, K,L: The percentages of CD44 positive subpopulation after Cal27 and Tca8113 transfected with siEZH2 or negative control siRNA for 72h were determined by FACS. The transcriptional level of several common markers for cancer stem cell isolation were quantified using real-time RT-PCR assay. Data are mean ± SD from three independent experiments, *p<0.05, **p<0.01, Student's t-test.

Fig 7. DZNep inhibits tumor growth by inducing intratumoral EZH2 reduction in the TSCC xenograft mouse model

A: Time schedule for establishing the TSCC xenograft mouse model and subsequent DZNep administration (2mg/kg body weight, once every 3 days). B: Tumor xenograft samples in mice bearing Cal27 and Tca8113 cells followed by DZNep intraperitoneal injection or vehicle injection. Representative images of mouse in each group are shown (n=6 each group). C: Tumor weights (when sacrificed) and tumor volumes were compared between the mice which received DZNep or vehicle (n=6 each group). D: HE staining, Ki-67 and active caspase-3 immunohistochemical staining in tissue samples derived from mice inoculated with Cal27. E: EZH2 protein and its associated targets were determined by western blotting in samples from mice bearing Cal27 tumors. Data showed here are mean ± SD from three independent experiments, **p<0.01, Student's t-test.