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TGF-β1 induces epigenetic silence of TIP30 to promote tumor metastasis in esophageal carcinoma.

Bu F, Liu X, Li J, Chen S, Tong X, Ma C, Mao H, Pan F, Li X, Chen B, Xu L, Li E, Kou G, Han J, Guo S, Zhao J, Guo Y - Oncotarget (2015)

Bottom Line: Here, we found TIP30 was decreased in cells undergoing EMT induced by TGF-β1, an occurrence that was related to promoter hypermethylation.Mechanically, TIP30 silencing induced the nuclear translocation and transcriptional activation of β-catenin in an AKT-dependent manner, which further resulted in the initiation of EMT.Loss of TIP30 correlated with nuclear β-catenin and aberrant E-cadherin expression.

View Article: PubMed Central - PubMed

Affiliation: PLA General Hospital Cancer Center Key Lab, Medical School of Chinese PLA, Beijing, P.R. China.

ABSTRACT
TGF-β1, a potent EMT (epithelial-mesenchymal transition) inducer present in the tumor microenvironment, is involved in the metastasis and progression of various carcinomas, including esophageal squamous cell carcinoma (ESCC). TIP30 (30kDa HIV-1 Tat interacting protein) is a putative tumor metastasis suppressor. Here, we found TIP30 was decreased in cells undergoing EMT induced by TGF-β1, an occurrence that was related to promoter hypermethylation. TGF-β1 induced TIP30 hypermethylation via increasing DNMT1 and DNMT3A expression, which could be restored by TGF-β antibodies. In our in vitro and in vivo studies, we showed that silence of TIP30 led to EMT, enhanced migrative and invasive abilities of ESCC cells, promoted tumor metastasis in xenografted mice; alternatively, overexpression of TIP30 inhibited TGF-β1-induced EMT, and metastatic abilities of ESCC cells. Mechanically, TIP30 silencing induced the nuclear translocation and transcriptional activation of β-catenin in an AKT-dependent manner, which further resulted in the initiation of EMT. Consistently, TIP30 was frequently methylated and downregulated in ESCC patients. Loss of TIP30 correlated with nuclear β-catenin and aberrant E-cadherin expression. TIP30 was a powerful marker in predicting the prognosis of ESCC. Taken together, our results suggest a novel and critical role of TIP30 involved in TGF-β1-induced activation of AKT/β-catenin signaling and ESCC metastasis.

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The reverse correlation of TIP30 and TGF-β1 levels in ESCC cell linesKYSE30 and KYSE450 cells were treated with 5ng/ml TGF-β1 or BSA for 48 hours, (A) morphologies of KYSE30 and KYSE450 were shown by phase-contrast microscopy (magnification, × 200); (B) invasion and migration assay were performed, total number of invaded and migrated cells were quantified and compared to the control samples; *P < 0.05; (C) the mRNA expression levels of EMT-related genes as well as TIP30 were determined by QRT-PCR (E-cad, E-cadherin; N-cad, N-cadherin; Fn1, Fibronectin 1; Vim, Vimentin; *P < 0.05). (D) The expressions of TIP30 mRNA were examined in 6 ESCC cell lines and a normal esophageal mucosa cell line Het-1A by QRT-PCR (upper); TGF-β1 concentrations in the cell culture supernatant were measured by specific enzyme-linked immunosorbent assay (ELISA) and normalized to the total number of cells (lower). Data are expressed as pg/ml of TGF-β1 per 105 cells. (E) KYSE30 cells were stimulated with TGF-β1 at indicated concentrations or for defined intervals, and then QRT-PCR and Western blots were performed to determine the expression level of TIP30. (F) ESCC cells were treated with anti-TGF-β antibody (5ng/ml) for 3 days, and then the expression of TIP30 mRNA was determined by QRT-PCR. Each bar represented the mean ±sd. of samples measured in triplicate, and each experiment was repeated at least three times.
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Figure 1: The reverse correlation of TIP30 and TGF-β1 levels in ESCC cell linesKYSE30 and KYSE450 cells were treated with 5ng/ml TGF-β1 or BSA for 48 hours, (A) morphologies of KYSE30 and KYSE450 were shown by phase-contrast microscopy (magnification, × 200); (B) invasion and migration assay were performed, total number of invaded and migrated cells were quantified and compared to the control samples; *P < 0.05; (C) the mRNA expression levels of EMT-related genes as well as TIP30 were determined by QRT-PCR (E-cad, E-cadherin; N-cad, N-cadherin; Fn1, Fibronectin 1; Vim, Vimentin; *P < 0.05). (D) The expressions of TIP30 mRNA were examined in 6 ESCC cell lines and a normal esophageal mucosa cell line Het-1A by QRT-PCR (upper); TGF-β1 concentrations in the cell culture supernatant were measured by specific enzyme-linked immunosorbent assay (ELISA) and normalized to the total number of cells (lower). Data are expressed as pg/ml of TGF-β1 per 105 cells. (E) KYSE30 cells were stimulated with TGF-β1 at indicated concentrations or for defined intervals, and then QRT-PCR and Western blots were performed to determine the expression level of TIP30. (F) ESCC cells were treated with anti-TGF-β antibody (5ng/ml) for 3 days, and then the expression of TIP30 mRNA was determined by QRT-PCR. Each bar represented the mean ±sd. of samples measured in triplicate, and each experiment was repeated at least three times.

Mentions: TGF-β1 is a classic EMT inducer in many types of epithelial tumors, including ESCC. As shown in Fig. 1A, KYSE30 and KYSE450 cells had an epithelial-like morphology. After treatment with TGF-β1, cells underwent a morphologic change from a cobblestone-like cell morphology to a spindle-like, fibroblastic morphology, accompanied with increased cell invasion and migration ability (Fig. 1A and 1B). To better characterize TGF-β1-induced EMT, we examined the mRNA expressions of EMT-related genes and TIP30 (Fig. 1C). We found that besides typical molecular changes of EMT, TIP30 expression was significantly decreased upon TGF-β1 treatment in ESCC cells. To correlate the endogenous expression levels of TIP30 with the levels of TGF-β1, we detected the mRNA expressions of TIP30 (Fig. 1D, upper) and the secretion levels of TGF-β1 (Fig. 1D, lower) in 6 ESCC cell lines and normal esophageal mucosa cell line Het-1A. These results reveal a strong inverse correlation between TIP30 expression and TGF-β1 level (Spearman's r=0.93, P=0.03). Further, qRT-PCR and Western blots showed that TGF-β1 decreased overall TIP30 expression levels in both dose-dependent (Fig. 1E, left) and time-dependent (Fig. 1E, right) manners in KYSE30 cells. In contrast, mRNA expressions of TIP30 were restored in all silenced cell line when treated with anti-TGF-β antibody (Fig. 1F). All the above suggested that TIP30 expression was downregulated by TGF-β1 in ESCC cells.


TGF-β1 induces epigenetic silence of TIP30 to promote tumor metastasis in esophageal carcinoma.

Bu F, Liu X, Li J, Chen S, Tong X, Ma C, Mao H, Pan F, Li X, Chen B, Xu L, Li E, Kou G, Han J, Guo S, Zhao J, Guo Y - Oncotarget (2015)

The reverse correlation of TIP30 and TGF-β1 levels in ESCC cell linesKYSE30 and KYSE450 cells were treated with 5ng/ml TGF-β1 or BSA for 48 hours, (A) morphologies of KYSE30 and KYSE450 were shown by phase-contrast microscopy (magnification, × 200); (B) invasion and migration assay were performed, total number of invaded and migrated cells were quantified and compared to the control samples; *P < 0.05; (C) the mRNA expression levels of EMT-related genes as well as TIP30 were determined by QRT-PCR (E-cad, E-cadherin; N-cad, N-cadherin; Fn1, Fibronectin 1; Vim, Vimentin; *P < 0.05). (D) The expressions of TIP30 mRNA were examined in 6 ESCC cell lines and a normal esophageal mucosa cell line Het-1A by QRT-PCR (upper); TGF-β1 concentrations in the cell culture supernatant were measured by specific enzyme-linked immunosorbent assay (ELISA) and normalized to the total number of cells (lower). Data are expressed as pg/ml of TGF-β1 per 105 cells. (E) KYSE30 cells were stimulated with TGF-β1 at indicated concentrations or for defined intervals, and then QRT-PCR and Western blots were performed to determine the expression level of TIP30. (F) ESCC cells were treated with anti-TGF-β antibody (5ng/ml) for 3 days, and then the expression of TIP30 mRNA was determined by QRT-PCR. Each bar represented the mean ±sd. of samples measured in triplicate, and each experiment was repeated at least three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385840&req=5

Figure 1: The reverse correlation of TIP30 and TGF-β1 levels in ESCC cell linesKYSE30 and KYSE450 cells were treated with 5ng/ml TGF-β1 or BSA for 48 hours, (A) morphologies of KYSE30 and KYSE450 were shown by phase-contrast microscopy (magnification, × 200); (B) invasion and migration assay were performed, total number of invaded and migrated cells were quantified and compared to the control samples; *P < 0.05; (C) the mRNA expression levels of EMT-related genes as well as TIP30 were determined by QRT-PCR (E-cad, E-cadherin; N-cad, N-cadherin; Fn1, Fibronectin 1; Vim, Vimentin; *P < 0.05). (D) The expressions of TIP30 mRNA were examined in 6 ESCC cell lines and a normal esophageal mucosa cell line Het-1A by QRT-PCR (upper); TGF-β1 concentrations in the cell culture supernatant were measured by specific enzyme-linked immunosorbent assay (ELISA) and normalized to the total number of cells (lower). Data are expressed as pg/ml of TGF-β1 per 105 cells. (E) KYSE30 cells were stimulated with TGF-β1 at indicated concentrations or for defined intervals, and then QRT-PCR and Western blots were performed to determine the expression level of TIP30. (F) ESCC cells were treated with anti-TGF-β antibody (5ng/ml) for 3 days, and then the expression of TIP30 mRNA was determined by QRT-PCR. Each bar represented the mean ±sd. of samples measured in triplicate, and each experiment was repeated at least three times.
Mentions: TGF-β1 is a classic EMT inducer in many types of epithelial tumors, including ESCC. As shown in Fig. 1A, KYSE30 and KYSE450 cells had an epithelial-like morphology. After treatment with TGF-β1, cells underwent a morphologic change from a cobblestone-like cell morphology to a spindle-like, fibroblastic morphology, accompanied with increased cell invasion and migration ability (Fig. 1A and 1B). To better characterize TGF-β1-induced EMT, we examined the mRNA expressions of EMT-related genes and TIP30 (Fig. 1C). We found that besides typical molecular changes of EMT, TIP30 expression was significantly decreased upon TGF-β1 treatment in ESCC cells. To correlate the endogenous expression levels of TIP30 with the levels of TGF-β1, we detected the mRNA expressions of TIP30 (Fig. 1D, upper) and the secretion levels of TGF-β1 (Fig. 1D, lower) in 6 ESCC cell lines and normal esophageal mucosa cell line Het-1A. These results reveal a strong inverse correlation between TIP30 expression and TGF-β1 level (Spearman's r=0.93, P=0.03). Further, qRT-PCR and Western blots showed that TGF-β1 decreased overall TIP30 expression levels in both dose-dependent (Fig. 1E, left) and time-dependent (Fig. 1E, right) manners in KYSE30 cells. In contrast, mRNA expressions of TIP30 were restored in all silenced cell line when treated with anti-TGF-β antibody (Fig. 1F). All the above suggested that TIP30 expression was downregulated by TGF-β1 in ESCC cells.

Bottom Line: Here, we found TIP30 was decreased in cells undergoing EMT induced by TGF-β1, an occurrence that was related to promoter hypermethylation.Mechanically, TIP30 silencing induced the nuclear translocation and transcriptional activation of β-catenin in an AKT-dependent manner, which further resulted in the initiation of EMT.Loss of TIP30 correlated with nuclear β-catenin and aberrant E-cadherin expression.

View Article: PubMed Central - PubMed

Affiliation: PLA General Hospital Cancer Center Key Lab, Medical School of Chinese PLA, Beijing, P.R. China.

ABSTRACT
TGF-β1, a potent EMT (epithelial-mesenchymal transition) inducer present in the tumor microenvironment, is involved in the metastasis and progression of various carcinomas, including esophageal squamous cell carcinoma (ESCC). TIP30 (30kDa HIV-1 Tat interacting protein) is a putative tumor metastasis suppressor. Here, we found TIP30 was decreased in cells undergoing EMT induced by TGF-β1, an occurrence that was related to promoter hypermethylation. TGF-β1 induced TIP30 hypermethylation via increasing DNMT1 and DNMT3A expression, which could be restored by TGF-β antibodies. In our in vitro and in vivo studies, we showed that silence of TIP30 led to EMT, enhanced migrative and invasive abilities of ESCC cells, promoted tumor metastasis in xenografted mice; alternatively, overexpression of TIP30 inhibited TGF-β1-induced EMT, and metastatic abilities of ESCC cells. Mechanically, TIP30 silencing induced the nuclear translocation and transcriptional activation of β-catenin in an AKT-dependent manner, which further resulted in the initiation of EMT. Consistently, TIP30 was frequently methylated and downregulated in ESCC patients. Loss of TIP30 correlated with nuclear β-catenin and aberrant E-cadherin expression. TIP30 was a powerful marker in predicting the prognosis of ESCC. Taken together, our results suggest a novel and critical role of TIP30 involved in TGF-β1-induced activation of AKT/β-catenin signaling and ESCC metastasis.

Show MeSH
Related in: MedlinePlus