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Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

Sayar N, Karahan G, Konu O, Bozkurt B, Bozdogan O, Yulug IG - Clin Epigenetics (2015)

Bottom Line: TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues.Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells.TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Bilkent University, Faculty of Science, TR-06800 Ankara, Turkey.

ABSTRACT

Background: CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation.

Results: Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells.

Conclusions: TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

No MeSH data available.


Related in: MedlinePlus

TAGLN expression in independent sets of tumor and normal breast tissues. a Examples of IHC staining of TAGLN in normal and tumor tissues. T (red), tumor tissue; S (yellow), stroma, ×100 magnification. b–e Box-plot analyses showing significant downregulation of TAGLN mRNA (left) and protein (right) expression levels in tumors compared to normal breast tissues (b); in grade 3 compared to grade 1 and 2 tumors (c); progesterone receptor (PR) negative compared to positive subtypes (d); and in triple negative compared to any hormone receptor positive (ER and/or PR and/or Her2 positive, Any positive) tumors (e). Horizontal lines: maximum, median, and minimum values. Left panels: qRT-PCR analysis of TAGLN mRNA expression levels (log2 transformed) in a cDNA array (n = 7 normal, n = 41 tumor tissues) relative to geometric means of ACTB and SDHA mRNA reference genes. Right panels: IHC staining of TAGLN protein in a tissue array (n = 67 tumor, n = 7 normal tissues). H-score method was used for staining intensity calculation. *P < 0.05, **P < 0.01, Mann–Whitney Test
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Fig5: TAGLN expression in independent sets of tumor and normal breast tissues. a Examples of IHC staining of TAGLN in normal and tumor tissues. T (red), tumor tissue; S (yellow), stroma, ×100 magnification. b–e Box-plot analyses showing significant downregulation of TAGLN mRNA (left) and protein (right) expression levels in tumors compared to normal breast tissues (b); in grade 3 compared to grade 1 and 2 tumors (c); progesterone receptor (PR) negative compared to positive subtypes (d); and in triple negative compared to any hormone receptor positive (ER and/or PR and/or Her2 positive, Any positive) tumors (e). Horizontal lines: maximum, median, and minimum values. Left panels: qRT-PCR analysis of TAGLN mRNA expression levels (log2 transformed) in a cDNA array (n = 7 normal, n = 41 tumor tissues) relative to geometric means of ACTB and SDHA mRNA reference genes. Right panels: IHC staining of TAGLN protein in a tissue array (n = 67 tumor, n = 7 normal tissues). H-score method was used for staining intensity calculation. *P < 0.05, **P < 0.01, Mann–Whitney Test

Mentions: To confirm downregulation of TAGLN in both protein and mRNA levels in other independent breast tumor tissue panels of larger sample sizes with pathological information, expression of TAGLN was examined in a breast cancer tissue array by IHC staining (BioChain, Cat.No:Z7020005), and in an independent cDNA panel by qRT-PCR (OriGene, Cat.No:BCRT101). Both protein (P = 0.0116) and mRNA expression levels (P = 0.0072) of TAGLN were significantly downregulated in breast tumors in comparison to non-tumor tissues (Fig. 5a, b). Average of relative mRNA expression, ARE(mRNA) ± SEM was 0.4248 ± 0.0792 in normal tissues (n = 7) and 0.1858 ± 0.0387 in tumor tissues (n = 41). Average histochemical score H-score ± SEM was 243.6 ± 25.35 in normal tissues (n = 7), with extensive staining in myoepithelial cells (Fig. 5a), and was 169.8 ± 9.773 in tumor tissues (n = 67). In both the tissue and cDNA arrays, TAGLN expression was downregulated in grade 3 tumors when compared to grade 1 and 2 tumors (P = 0.049 and P = 0.0329, respectively, Fig. 5c), upregulated in progesterone receptor PR (+) tumors (P = 0.0031 and P = 0.0043, respectively, Fig. 5d) and in any hormone receptor positive tumors with respect to triple negative tumor types (P = 0.0063 and P = 0.0489, respectively, Fig. 5e). TAGLN expression levels were not significantly different in estrogen receptor (ER) or human epidermal growth factor receptor 2 (Her2) positive or negative tumors or in different stage groups.Fig. 5


Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

Sayar N, Karahan G, Konu O, Bozkurt B, Bozdogan O, Yulug IG - Clin Epigenetics (2015)

TAGLN expression in independent sets of tumor and normal breast tissues. a Examples of IHC staining of TAGLN in normal and tumor tissues. T (red), tumor tissue; S (yellow), stroma, ×100 magnification. b–e Box-plot analyses showing significant downregulation of TAGLN mRNA (left) and protein (right) expression levels in tumors compared to normal breast tissues (b); in grade 3 compared to grade 1 and 2 tumors (c); progesterone receptor (PR) negative compared to positive subtypes (d); and in triple negative compared to any hormone receptor positive (ER and/or PR and/or Her2 positive, Any positive) tumors (e). Horizontal lines: maximum, median, and minimum values. Left panels: qRT-PCR analysis of TAGLN mRNA expression levels (log2 transformed) in a cDNA array (n = 7 normal, n = 41 tumor tissues) relative to geometric means of ACTB and SDHA mRNA reference genes. Right panels: IHC staining of TAGLN protein in a tissue array (n = 67 tumor, n = 7 normal tissues). H-score method was used for staining intensity calculation. *P < 0.05, **P < 0.01, Mann–Whitney Test
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Fig5: TAGLN expression in independent sets of tumor and normal breast tissues. a Examples of IHC staining of TAGLN in normal and tumor tissues. T (red), tumor tissue; S (yellow), stroma, ×100 magnification. b–e Box-plot analyses showing significant downregulation of TAGLN mRNA (left) and protein (right) expression levels in tumors compared to normal breast tissues (b); in grade 3 compared to grade 1 and 2 tumors (c); progesterone receptor (PR) negative compared to positive subtypes (d); and in triple negative compared to any hormone receptor positive (ER and/or PR and/or Her2 positive, Any positive) tumors (e). Horizontal lines: maximum, median, and minimum values. Left panels: qRT-PCR analysis of TAGLN mRNA expression levels (log2 transformed) in a cDNA array (n = 7 normal, n = 41 tumor tissues) relative to geometric means of ACTB and SDHA mRNA reference genes. Right panels: IHC staining of TAGLN protein in a tissue array (n = 67 tumor, n = 7 normal tissues). H-score method was used for staining intensity calculation. *P < 0.05, **P < 0.01, Mann–Whitney Test
Mentions: To confirm downregulation of TAGLN in both protein and mRNA levels in other independent breast tumor tissue panels of larger sample sizes with pathological information, expression of TAGLN was examined in a breast cancer tissue array by IHC staining (BioChain, Cat.No:Z7020005), and in an independent cDNA panel by qRT-PCR (OriGene, Cat.No:BCRT101). Both protein (P = 0.0116) and mRNA expression levels (P = 0.0072) of TAGLN were significantly downregulated in breast tumors in comparison to non-tumor tissues (Fig. 5a, b). Average of relative mRNA expression, ARE(mRNA) ± SEM was 0.4248 ± 0.0792 in normal tissues (n = 7) and 0.1858 ± 0.0387 in tumor tissues (n = 41). Average histochemical score H-score ± SEM was 243.6 ± 25.35 in normal tissues (n = 7), with extensive staining in myoepithelial cells (Fig. 5a), and was 169.8 ± 9.773 in tumor tissues (n = 67). In both the tissue and cDNA arrays, TAGLN expression was downregulated in grade 3 tumors when compared to grade 1 and 2 tumors (P = 0.049 and P = 0.0329, respectively, Fig. 5c), upregulated in progesterone receptor PR (+) tumors (P = 0.0031 and P = 0.0043, respectively, Fig. 5d) and in any hormone receptor positive tumors with respect to triple negative tumor types (P = 0.0063 and P = 0.0489, respectively, Fig. 5e). TAGLN expression levels were not significantly different in estrogen receptor (ER) or human epidermal growth factor receptor 2 (Her2) positive or negative tumors or in different stage groups.Fig. 5

Bottom Line: TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues.Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells.TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Bilkent University, Faculty of Science, TR-06800 Ankara, Turkey.

ABSTRACT

Background: CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation.

Results: Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells.

Conclusions: TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

No MeSH data available.


Related in: MedlinePlus