Disruption of TGF-β signaling induces demethylation of E-cadherin promoter and reverses mesenchymal phenotype in ovarian cancer

Lin Yu Chen, Rui-Lan Huang, Han Lin Chou, Jian Liang Chou, Wei-Ting Deng, Hui Wen Yang, Ru Inn Lin, Chin Yang Li, Huey-Jen Lin, Tim Hui Ming Huang, Chien Chih Chiu, Hung-Cheng Lai, Michael W Y Chan

研究成果: 雜誌貢獻文章

摘要

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLThe TGF-β signaling pathway plays an important role in controlling cell growth and differentiation. In advanced ovarian cancer, frequent TGF-β-induced metastasis or epithelial-mesenchymal transition (EMT) can be observed. This phenomenon is often associated with epigenetic silencing of epithelial marker, E-cadherin which can also be observed in ovarian cancer cell lines that demethylation treatment restored E-cadherin expression. We recently hypothesized that long term activation of TGF-β signaling may induce EMT phenotype by epigenetic silencing of E-cadherin and that inhibition of the signaling may restore E-cadherin and reverse EMT in ovarian cancer (Chou et al., Expert Opin Ther Targets 2010). In this study, we cloned the cDNA of the inhibitory SMAD, SMAD7 from a human immortalized ovarian surface epithelial cell, IOSE into pcDNA3.1 mammalian expression vector. The inhibitory effect of this SMAD7 expression vector on TGF-β signaling has been confirmed by reporter assay. We then stably transfected the SMAD7 expression vector into a mesenchymal ovarian cancer cell, CP70 in which E-cadherin is silenced by complete promoter methylation. Cells over-expressing SMAD7 showed up-regulation of SMAD7 and a decrease in SMAD2 phosphorylation while the control cells maintained a hyperphosphorylation of SMAD2 thus suggesting that TGF-β signaling is disrupted in SMAD7-overexpressing cells. We further examined the expression of E-cadherin from passage 5 up to 30 of the stable transfectants. Surprisingly, stable restoration of E-cadherin can only be observed from passage 20 and thereafter, of the SMAD7-overexpressing cells, while E-cadherin remained silence in the control cells. To investigate if this restoration is due to promoter demethylation of E-cadherin, we performed bisulphite pyrosequencing on the E-cadherin promoter CpG island spanning -586 to -12 of the region. Compared with control cells, consistent demethylation of E-cadherin promoter can be observed at 2 CpG sites located at -214 and -235 of the promoter such that gradual demethylation occurred from passage 5 to 30 of the SMAD7-overexpressing cells (passage 20, methylation% control vs SMAD7: 91% vs 67% at -235; 80% vs 52% at -214); while the rest of the CpG sites remained heavily methylated. This demethylation may be due to down-regulation of transcriptional repressor, TWIST after SMAD7 transfection. Additionally, one of the SMAD7 stable expression clones with highest restoration of E-cadherin showed decreased migration and invasion ability as determined by wound healing and invasion assay. Taken together, disruption of TGF-β signaling can induce demethylation of E-cadherin promoter and reverse EMT phenotype in ovarian cancer. The therapeutic potential of targeting TGF-β signaling pathway in inhibiting metastasis of ovarian cancer deserves further investigation.Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3433. doi:10.1158/1538-7445.AM2011-3433
原文繁體中文
頁(從 - 到)3433
頁數1
期刊Cancer Research
71
發行號8 Supplement
出版狀態已發佈 - 十一月 13 2014
對外發佈Yes

引用此文

Chen, L. Y., Huang, R-L., Chou, H. L., Chou, J. L., Deng, W-T., Yang, H. W., ... Chan, M. W. Y. (2014). Disruption of TGF-β signaling induces demethylation of E-cadherin promoter and reverses mesenchymal phenotype in ovarian cancer. Cancer Research, 71(8 Supplement), 3433.

Disruption of TGF-β signaling induces demethylation of E-cadherin promoter and reverses mesenchymal phenotype in ovarian cancer. / Chen, Lin Yu; Huang, Rui-Lan; Chou, Han Lin; Chou, Jian Liang; Deng, Wei-Ting; Yang, Hui Wen; Lin, Ru Inn; Li, Chin Yang; Lin, Huey-Jen; Huang, Tim Hui Ming; Chiu, Chien Chih; Lai, Hung-Cheng; Chan, Michael W Y.

於: Cancer Research, 卷 71, 編號 8 Supplement, 13.11.2014, p. 3433.

研究成果: 雜誌貢獻文章

Chen, LY, Huang, R-L, Chou, HL, Chou, JL, Deng, W-T, Yang, HW, Lin, RI, Li, CY, Lin, H-J, Huang, THM, Chiu, CC, Lai, H-C & Chan, MWY 2014, 'Disruption of TGF-β signaling induces demethylation of E-cadherin promoter and reverses mesenchymal phenotype in ovarian cancer', Cancer Research, 卷 71, 編號 8 Supplement, 頁 3433.
Chen, Lin Yu ; Huang, Rui-Lan ; Chou, Han Lin ; Chou, Jian Liang ; Deng, Wei-Ting ; Yang, Hui Wen ; Lin, Ru Inn ; Li, Chin Yang ; Lin, Huey-Jen ; Huang, Tim Hui Ming ; Chiu, Chien Chih ; Lai, Hung-Cheng ; Chan, Michael W Y. / Disruption of TGF-β signaling induces demethylation of E-cadherin promoter and reverses mesenchymal phenotype in ovarian cancer. 於: Cancer Research. 2014 ; 卷 71, 編號 8 Supplement. 頁 3433.
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abstract = "Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLThe TGF-β signaling pathway plays an important role in controlling cell growth and differentiation. In advanced ovarian cancer, frequent TGF-β-induced metastasis or epithelial-mesenchymal transition (EMT) can be observed. This phenomenon is often associated with epigenetic silencing of epithelial marker, E-cadherin which can also be observed in ovarian cancer cell lines that demethylation treatment restored E-cadherin expression. We recently hypothesized that long term activation of TGF-β signaling may induce EMT phenotype by epigenetic silencing of E-cadherin and that inhibition of the signaling may restore E-cadherin and reverse EMT in ovarian cancer (Chou et al., Expert Opin Ther Targets 2010). In this study, we cloned the cDNA of the inhibitory SMAD, SMAD7 from a human immortalized ovarian surface epithelial cell, IOSE into pcDNA3.1 mammalian expression vector. The inhibitory effect of this SMAD7 expression vector on TGF-β signaling has been confirmed by reporter assay. We then stably transfected the SMAD7 expression vector into a mesenchymal ovarian cancer cell, CP70 in which E-cadherin is silenced by complete promoter methylation. Cells over-expressing SMAD7 showed up-regulation of SMAD7 and a decrease in SMAD2 phosphorylation while the control cells maintained a hyperphosphorylation of SMAD2 thus suggesting that TGF-β signaling is disrupted in SMAD7-overexpressing cells. We further examined the expression of E-cadherin from passage 5 up to 30 of the stable transfectants. Surprisingly, stable restoration of E-cadherin can only be observed from passage 20 and thereafter, of the SMAD7-overexpressing cells, while E-cadherin remained silence in the control cells. To investigate if this restoration is due to promoter demethylation of E-cadherin, we performed bisulphite pyrosequencing on the E-cadherin promoter CpG island spanning -586 to -12 of the region. Compared with control cells, consistent demethylation of E-cadherin promoter can be observed at 2 CpG sites located at -214 and -235 of the promoter such that gradual demethylation occurred from passage 5 to 30 of the SMAD7-overexpressing cells (passage 20, methylation{\%} control vs SMAD7: 91{\%} vs 67{\%} at -235; 80{\%} vs 52{\%} at -214); while the rest of the CpG sites remained heavily methylated. This demethylation may be due to down-regulation of transcriptional repressor, TWIST after SMAD7 transfection. Additionally, one of the SMAD7 stable expression clones with highest restoration of E-cadherin showed decreased migration and invasion ability as determined by wound healing and invasion assay. Taken together, disruption of TGF-β signaling can induce demethylation of E-cadherin promoter and reverse EMT phenotype in ovarian cancer. The therapeutic potential of targeting TGF-β signaling pathway in inhibiting metastasis of ovarian cancer deserves further investigation.Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3433. doi:10.1158/1538-7445.AM2011-3433",
author = "Chen, {Lin Yu} and Rui-Lan Huang and Chou, {Han Lin} and Chou, {Jian Liang} and Wei-Ting Deng and Yang, {Hui Wen} and Lin, {Ru Inn} and Li, {Chin Yang} and Huey-Jen Lin and Huang, {Tim Hui Ming} and Chiu, {Chien Chih} and Hung-Cheng Lai and Chan, {Michael W Y}",
year = "2014",
month = "11",
day = "13",
language = "繁體中文",
volume = "71",
pages = "3433",
journal = "Cancer Research",
issn = "0008-5472",
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TY - JOUR

T1 - Disruption of TGF-β signaling induces demethylation of E-cadherin promoter and reverses mesenchymal phenotype in ovarian cancer

AU - Chen, Lin Yu

AU - Huang, Rui-Lan

AU - Chou, Han Lin

AU - Chou, Jian Liang

AU - Deng, Wei-Ting

AU - Yang, Hui Wen

AU - Lin, Ru Inn

AU - Li, Chin Yang

AU - Lin, Huey-Jen

AU - Huang, Tim Hui Ming

AU - Chiu, Chien Chih

AU - Lai, Hung-Cheng

AU - Chan, Michael W Y

PY - 2014/11/13

Y1 - 2014/11/13

N2 - Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLThe TGF-β signaling pathway plays an important role in controlling cell growth and differentiation. In advanced ovarian cancer, frequent TGF-β-induced metastasis or epithelial-mesenchymal transition (EMT) can be observed. This phenomenon is often associated with epigenetic silencing of epithelial marker, E-cadherin which can also be observed in ovarian cancer cell lines that demethylation treatment restored E-cadherin expression. We recently hypothesized that long term activation of TGF-β signaling may induce EMT phenotype by epigenetic silencing of E-cadherin and that inhibition of the signaling may restore E-cadherin and reverse EMT in ovarian cancer (Chou et al., Expert Opin Ther Targets 2010). In this study, we cloned the cDNA of the inhibitory SMAD, SMAD7 from a human immortalized ovarian surface epithelial cell, IOSE into pcDNA3.1 mammalian expression vector. The inhibitory effect of this SMAD7 expression vector on TGF-β signaling has been confirmed by reporter assay. We then stably transfected the SMAD7 expression vector into a mesenchymal ovarian cancer cell, CP70 in which E-cadherin is silenced by complete promoter methylation. Cells over-expressing SMAD7 showed up-regulation of SMAD7 and a decrease in SMAD2 phosphorylation while the control cells maintained a hyperphosphorylation of SMAD2 thus suggesting that TGF-β signaling is disrupted in SMAD7-overexpressing cells. We further examined the expression of E-cadherin from passage 5 up to 30 of the stable transfectants. Surprisingly, stable restoration of E-cadherin can only be observed from passage 20 and thereafter, of the SMAD7-overexpressing cells, while E-cadherin remained silence in the control cells. To investigate if this restoration is due to promoter demethylation of E-cadherin, we performed bisulphite pyrosequencing on the E-cadherin promoter CpG island spanning -586 to -12 of the region. Compared with control cells, consistent demethylation of E-cadherin promoter can be observed at 2 CpG sites located at -214 and -235 of the promoter such that gradual demethylation occurred from passage 5 to 30 of the SMAD7-overexpressing cells (passage 20, methylation% control vs SMAD7: 91% vs 67% at -235; 80% vs 52% at -214); while the rest of the CpG sites remained heavily methylated. This demethylation may be due to down-regulation of transcriptional repressor, TWIST after SMAD7 transfection. Additionally, one of the SMAD7 stable expression clones with highest restoration of E-cadherin showed decreased migration and invasion ability as determined by wound healing and invasion assay. Taken together, disruption of TGF-β signaling can induce demethylation of E-cadherin promoter and reverse EMT phenotype in ovarian cancer. The therapeutic potential of targeting TGF-β signaling pathway in inhibiting metastasis of ovarian cancer deserves further investigation.Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3433. doi:10.1158/1538-7445.AM2011-3433

AB - Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLThe TGF-β signaling pathway plays an important role in controlling cell growth and differentiation. In advanced ovarian cancer, frequent TGF-β-induced metastasis or epithelial-mesenchymal transition (EMT) can be observed. This phenomenon is often associated with epigenetic silencing of epithelial marker, E-cadherin which can also be observed in ovarian cancer cell lines that demethylation treatment restored E-cadherin expression. We recently hypothesized that long term activation of TGF-β signaling may induce EMT phenotype by epigenetic silencing of E-cadherin and that inhibition of the signaling may restore E-cadherin and reverse EMT in ovarian cancer (Chou et al., Expert Opin Ther Targets 2010). In this study, we cloned the cDNA of the inhibitory SMAD, SMAD7 from a human immortalized ovarian surface epithelial cell, IOSE into pcDNA3.1 mammalian expression vector. The inhibitory effect of this SMAD7 expression vector on TGF-β signaling has been confirmed by reporter assay. We then stably transfected the SMAD7 expression vector into a mesenchymal ovarian cancer cell, CP70 in which E-cadherin is silenced by complete promoter methylation. Cells over-expressing SMAD7 showed up-regulation of SMAD7 and a decrease in SMAD2 phosphorylation while the control cells maintained a hyperphosphorylation of SMAD2 thus suggesting that TGF-β signaling is disrupted in SMAD7-overexpressing cells. We further examined the expression of E-cadherin from passage 5 up to 30 of the stable transfectants. Surprisingly, stable restoration of E-cadherin can only be observed from passage 20 and thereafter, of the SMAD7-overexpressing cells, while E-cadherin remained silence in the control cells. To investigate if this restoration is due to promoter demethylation of E-cadherin, we performed bisulphite pyrosequencing on the E-cadherin promoter CpG island spanning -586 to -12 of the region. Compared with control cells, consistent demethylation of E-cadherin promoter can be observed at 2 CpG sites located at -214 and -235 of the promoter such that gradual demethylation occurred from passage 5 to 30 of the SMAD7-overexpressing cells (passage 20, methylation% control vs SMAD7: 91% vs 67% at -235; 80% vs 52% at -214); while the rest of the CpG sites remained heavily methylated. This demethylation may be due to down-regulation of transcriptional repressor, TWIST after SMAD7 transfection. Additionally, one of the SMAD7 stable expression clones with highest restoration of E-cadherin showed decreased migration and invasion ability as determined by wound healing and invasion assay. Taken together, disruption of TGF-β signaling can induce demethylation of E-cadherin promoter and reverse EMT phenotype in ovarian cancer. The therapeutic potential of targeting TGF-β signaling pathway in inhibiting metastasis of ovarian cancer deserves further investigation.Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3433. doi:10.1158/1538-7445.AM2011-3433

M3 - 文章

VL - 71

SP - 3433

JO - Cancer Research

JF - Cancer Research

SN - 0008-5472

IS - 8 Supplement

ER -