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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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TIP30 was frequently methylated and downregulated in ESCC(A) The CpG island region of TIP30 is shown. The transcriptional start site is denoted as +1. The region used in MSP is underlined. (B) Methylation patterns of TIP30 promoter were determined by MSP analysis (upper) and BGS (lower) in 6 ESCC cell lines and a normal esophageal mucosa cell line Het-1A (M, methylated; U, unmethylated; the black circle indicates the methylated cytosine while the white circle indicates the unmethylated cytosine in the dinucleotide CpG). (C) MSP analysis the methylation pattern of TIP30 promoter in KYSE450 and KYSE150 cells after treatment with 5ng/ml of 5-Aza-2_dC for 3 days. (D) ESCC cells were treated with 5ng/ml of 5-Aza-2_dC for 3 days; the expression of TIP30 mRNA was determined by QRT-PCR (error bar indicate SD, standard deviation; *P < 0.05). (E) Representative results of MSP analysis of TIP30 genes in tumor tissues (T) and 8 normal esophageal mucosa tissues (N). (F) Representative results of BGS analysis of TIP30 genes in tumor tissues. Data shown represent three different experiments.
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Figure 2: TIP30 was frequently methylated and downregulated in ESCC(A) The CpG island region of TIP30 is shown. The transcriptional start site is denoted as +1. The region used in MSP is underlined. (B) Methylation patterns of TIP30 promoter were determined by MSP analysis (upper) and BGS (lower) in 6 ESCC cell lines and a normal esophageal mucosa cell line Het-1A (M, methylated; U, unmethylated; the black circle indicates the methylated cytosine while the white circle indicates the unmethylated cytosine in the dinucleotide CpG). (C) MSP analysis the methylation pattern of TIP30 promoter in KYSE450 and KYSE150 cells after treatment with 5ng/ml of 5-Aza-2_dC for 3 days. (D) ESCC cells were treated with 5ng/ml of 5-Aza-2_dC for 3 days; the expression of TIP30 mRNA was determined by QRT-PCR (error bar indicate SD, standard deviation; *P < 0.05). (E) Representative results of MSP analysis of TIP30 genes in tumor tissues (T) and 8 normal esophageal mucosa tissues (N). (F) Representative results of BGS analysis of TIP30 genes in tumor tissues. Data shown represent three different experiments.

Mentions: There is a typical CpG island spanning the transcription start site of TIP30 (Fig. 2A), as we described previously [15]. To explore whether hypermethylation of TIP30 is involved in the decreased expression of TIP30, we examined the methylation status of TIP30 in 6 ESCC cell lines and normal esophageal mucosa cell line Het-1A (Fig. 2B). Methylation-specific PCR (MSP) results showed that the TIP30 promoter was unmethylated in normal esophageal mucosa cell Het-1A and KYSE30 cells which had abundant TIP30 mRNA expression. In contrast, TIP30 was completely methylated in KYSE150 cells, which had undetectable TIP30 expression. Partial methylation of TIP30 was found in the remaining ESCC cells, which had both methylated and unmethylated alleles. To confirm the MSP results, we further examined TIP30 promoter methylation by conducting bisulfite genomic sequencing (BGS) analysis of 18 individual CpG sites within its CpG island (Fig. 2B lower). The result revealed that promoter of TIP30 was frequently methylated in ESCC cells. ESCC cell lines with methylated TIP30 were treated with DNA demethylating agent 5-Aza-2′dC, and then MSP and QRT-PCR were performed. The results showed that treatment with 5-Aza-2′dC decreased the methylated MSP products (Fig. 2C) and increased TIP30 mRNA expression (Fig. 2D). Together, these data demonstrate that hypermethylation of CpG islands results in epigenetic silence of TIP30 in ESCC cell lines.


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)

TIP30 was frequently methylated and downregulated in ESCC(A) The CpG island region of TIP30 is shown. The transcriptional start site is denoted as +1. The region used in MSP is underlined. (B) Methylation patterns of TIP30 promoter were determined by MSP analysis (upper) and BGS (lower) in 6 ESCC cell lines and a normal esophageal mucosa cell line Het-1A (M, methylated; U, unmethylated; the black circle indicates the methylated cytosine while the white circle indicates the unmethylated cytosine in the dinucleotide CpG). (C) MSP analysis the methylation pattern of TIP30 promoter in KYSE450 and KYSE150 cells after treatment with 5ng/ml of 5-Aza-2_dC for 3 days. (D) ESCC cells were treated with 5ng/ml of 5-Aza-2_dC for 3 days; the expression of TIP30 mRNA was determined by QRT-PCR (error bar indicate SD, standard deviation; *P < 0.05). (E) Representative results of MSP analysis of TIP30 genes in tumor tissues (T) and 8 normal esophageal mucosa tissues (N). (F) Representative results of BGS analysis of TIP30 genes in tumor tissues. Data shown represent three different experiments.
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Figure 2: TIP30 was frequently methylated and downregulated in ESCC(A) The CpG island region of TIP30 is shown. The transcriptional start site is denoted as +1. The region used in MSP is underlined. (B) Methylation patterns of TIP30 promoter were determined by MSP analysis (upper) and BGS (lower) in 6 ESCC cell lines and a normal esophageal mucosa cell line Het-1A (M, methylated; U, unmethylated; the black circle indicates the methylated cytosine while the white circle indicates the unmethylated cytosine in the dinucleotide CpG). (C) MSP analysis the methylation pattern of TIP30 promoter in KYSE450 and KYSE150 cells after treatment with 5ng/ml of 5-Aza-2_dC for 3 days. (D) ESCC cells were treated with 5ng/ml of 5-Aza-2_dC for 3 days; the expression of TIP30 mRNA was determined by QRT-PCR (error bar indicate SD, standard deviation; *P < 0.05). (E) Representative results of MSP analysis of TIP30 genes in tumor tissues (T) and 8 normal esophageal mucosa tissues (N). (F) Representative results of BGS analysis of TIP30 genes in tumor tissues. Data shown represent three different experiments.
Mentions: There is a typical CpG island spanning the transcription start site of TIP30 (Fig. 2A), as we described previously [15]. To explore whether hypermethylation of TIP30 is involved in the decreased expression of TIP30, we examined the methylation status of TIP30 in 6 ESCC cell lines and normal esophageal mucosa cell line Het-1A (Fig. 2B). Methylation-specific PCR (MSP) results showed that the TIP30 promoter was unmethylated in normal esophageal mucosa cell Het-1A and KYSE30 cells which had abundant TIP30 mRNA expression. In contrast, TIP30 was completely methylated in KYSE150 cells, which had undetectable TIP30 expression. Partial methylation of TIP30 was found in the remaining ESCC cells, which had both methylated and unmethylated alleles. To confirm the MSP results, we further examined TIP30 promoter methylation by conducting bisulfite genomic sequencing (BGS) analysis of 18 individual CpG sites within its CpG island (Fig. 2B lower). The result revealed that promoter of TIP30 was frequently methylated in ESCC cells. ESCC cell lines with methylated TIP30 were treated with DNA demethylating agent 5-Aza-2′dC, and then MSP and QRT-PCR were performed. The results showed that treatment with 5-Aza-2′dC decreased the methylated MSP products (Fig. 2C) and increased TIP30 mRNA expression (Fig. 2D). Together, these data demonstrate that hypermethylation of CpG islands results in epigenetic silence of TIP30 in ESCC cell lines.

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