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

Li Z, Wang Y, Qiu J, Li Q, Yuan C, Zhang W, Wang D, Ye J, Jiang H, Yang J, Cheng J - Oncotarget (2013)

Bottom Line: We found that aberrantly overexpressed EZH2 was associated with pathological grade, cervical nodes metastasis and Ki-67 expression in tongue cancers.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.

View Article: PubMed Central - PubMed

Affiliation: Head Neck Cancer Center, Institute of Stomatology, Affiliated Stomatological Hospital, Nanjing Medical University, Jiangsu, China PRC.

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.

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EZH2 knockdown by siRNAs phenocopys the effects of DZNep exposure in tongue cancer cellsA: 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.
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Figure 6: EZH2 knockdown by siRNAs phenocopys the effects of DZNep exposure in tongue cancer cellsA: 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.

Mentions: Since DZNep functions as a global potent inhibitors of histone methylation and doesn't act as EZH2 antagonist with high specificity, we further examine that whether these anti-cancer properties exerted by DZNep were attributed to its depletion of EZH2. The siRNA-mediated knockdown strategy was exploited to delineate the phenotype changes after endogenous EZH2 was specifically inhibited in tongue cancer cells. As shown in Fig.6A, B, both EZH2 and H3k27me3 were significantly reduced following the EZH2 siRNAs transfection as compared with non-specific control, thus validating the knockdown efficiency of EZH2 siRNA. Subsequently, the relevant phenotype changes of Cal27 and Tca8113 treated with EZH2 siRNAs were further examined. As displayed in Fig.6C-K, our results indicated that siRNA-mediated EZH2 depletion resulted in impaired cell proliferation, reduced migration and invasion, triggered more apoptotic and senescence cells, less CD44-positive cells and enhanced 5-FU chemosensitivity, largely resembling the results derived from DZNep treatment in vitro. Taken together, these findings strongly suggest that the EZH2 serves as a potent oncogenic regulator involving multiple properties of tongue cancer cells and DZNep executes its anticancer functions, at least in part by its pharmacological inhibition of EZH2 in tongue cancer cells.


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

Li Z, Wang Y, Qiu J, Li Q, Yuan C, Zhang W, Wang D, Ye J, Jiang H, Yang J, Cheng J - Oncotarget (2013)

EZH2 knockdown by siRNAs phenocopys the effects of DZNep exposure in tongue cancer cellsA: 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: EZH2 knockdown by siRNAs phenocopys the effects of DZNep exposure in tongue cancer cellsA: 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.
Mentions: Since DZNep functions as a global potent inhibitors of histone methylation and doesn't act as EZH2 antagonist with high specificity, we further examine that whether these anti-cancer properties exerted by DZNep were attributed to its depletion of EZH2. The siRNA-mediated knockdown strategy was exploited to delineate the phenotype changes after endogenous EZH2 was specifically inhibited in tongue cancer cells. As shown in Fig.6A, B, both EZH2 and H3k27me3 were significantly reduced following the EZH2 siRNAs transfection as compared with non-specific control, thus validating the knockdown efficiency of EZH2 siRNA. Subsequently, the relevant phenotype changes of Cal27 and Tca8113 treated with EZH2 siRNAs were further examined. As displayed in Fig.6C-K, our results indicated that siRNA-mediated EZH2 depletion resulted in impaired cell proliferation, reduced migration and invasion, triggered more apoptotic and senescence cells, less CD44-positive cells and enhanced 5-FU chemosensitivity, largely resembling the results derived from DZNep treatment in vitro. Taken together, these findings strongly suggest that the EZH2 serves as a potent oncogenic regulator involving multiple properties of tongue cancer cells and DZNep executes its anticancer functions, at least in part by its pharmacological inhibition of EZH2 in tongue cancer cells.

Bottom Line: We found that aberrantly overexpressed EZH2 was associated with pathological grade, cervical nodes metastasis and Ki-67 expression in tongue cancers.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.

View Article: PubMed Central - PubMed

Affiliation: Head Neck Cancer Center, Institute of Stomatology, Affiliated Stomatological Hospital, Nanjing Medical University, Jiangsu, China PRC.

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.

Show MeSH
Related in: MedlinePlus