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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

Effect of TAGLN overexpression and silencing on breast cell lines. a Western blot analysis of TAGLN in breast carcinoma and non-tumorigenic breast cell lines. b–c Western blot shows TAGLN silencing in MCF10A (b) and MCF12A (c) cells (upper panels). 2D colony formation assays (middle and lower panels) show increased colony formation in TAGLN silenced (siTAGLN) MCF10A (b) and MCF12A (c) cells compared to controls (siCTRL). d–e Western blot analyses of TAGLN overexpression and subsequent colony formation assays in MDA-MB-361 (d, upper panel), and MDA-MB-157 (e, left panel) cell clones. Overexpression of ectopic TAGLN (TAGLNvec) and levels of endogenous TAGLN (TAGLNint) were determined with TAGLN specific antibody in clones. TAGLN overexpression caused higher cell proliferation in MDA-MB-361 cells (e), while did not affect MDA-MB-157 cells (f). Each assay was conducted at least in quadruplicates. Two sample t test was used for comparisons. Error bars represent standard errors. **P < 0.01
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Fig6: Effect of TAGLN overexpression and silencing on breast cell lines. a Western blot analysis of TAGLN in breast carcinoma and non-tumorigenic breast cell lines. b–c Western blot shows TAGLN silencing in MCF10A (b) and MCF12A (c) cells (upper panels). 2D colony formation assays (middle and lower panels) show increased colony formation in TAGLN silenced (siTAGLN) MCF10A (b) and MCF12A (c) cells compared to controls (siCTRL). d–e Western blot analyses of TAGLN overexpression and subsequent colony formation assays in MDA-MB-361 (d, upper panel), and MDA-MB-157 (e, left panel) cell clones. Overexpression of ectopic TAGLN (TAGLNvec) and levels of endogenous TAGLN (TAGLNint) were determined with TAGLN specific antibody in clones. TAGLN overexpression caused higher cell proliferation in MDA-MB-361 cells (e), while did not affect MDA-MB-157 cells (f). Each assay was conducted at least in quadruplicates. Two sample t test was used for comparisons. Error bars represent standard errors. **P < 0.01

Mentions: Since TAGLN was frequently hypermethylated and consistently downregulated in both the BC cell lines and three independent panels of tumor tissues, we analyzed the functional role of TAGLN in BC and NTB cells. TAGLN gene was silenced in MCF10A and MCF12A cells with abundant expression (Fig. 6a–c), and was overexpressed it in MDA-MB-361 and MDA-MB-157 BC cell lines (Fig. 6d, e) that expressed TAGLN at zero to intermediate levels, respectively (Fig. 6a). 2D colony formation assays were performed to test the effect of TAGLN on proliferation potentials of these cells. Compared to control siRNA (siCTRL), siTAGLN transfected MCF10A and MCF12A cells formed significantly (P = 0.0013 and P = 0.0016, respectively) higher number of colonies (Fig. 6b, c, middle and lower panels). Colony formation capabilities of MDA-MB-361 cells, but not MDA-MB-157 cells, were significantly impaired when they overexpressed TAGLN protein (Fig. 6d, middle and lower panels and Fig. 6e, right panel). To examine possible effects of TAGLN on migration and invasion potentials of BC cells, we performed in vitro wound healing and Matrigel invasion assays in normally motile MDA-MB-157 cells; however, TAGLN overexpression in these cells did not affect either wound closure or Matrigel invasion capacities of these cells (Additional file 5: Figure S3).Fig. 6


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)

Effect of TAGLN overexpression and silencing on breast cell lines. a Western blot analysis of TAGLN in breast carcinoma and non-tumorigenic breast cell lines. b–c Western blot shows TAGLN silencing in MCF10A (b) and MCF12A (c) cells (upper panels). 2D colony formation assays (middle and lower panels) show increased colony formation in TAGLN silenced (siTAGLN) MCF10A (b) and MCF12A (c) cells compared to controls (siCTRL). d–e Western blot analyses of TAGLN overexpression and subsequent colony formation assays in MDA-MB-361 (d, upper panel), and MDA-MB-157 (e, left panel) cell clones. Overexpression of ectopic TAGLN (TAGLNvec) and levels of endogenous TAGLN (TAGLNint) were determined with TAGLN specific antibody in clones. TAGLN overexpression caused higher cell proliferation in MDA-MB-361 cells (e), while did not affect MDA-MB-157 cells (f). Each assay was conducted at least in quadruplicates. Two sample t test was used for comparisons. Error bars represent standard errors. **P < 0.01
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Fig6: Effect of TAGLN overexpression and silencing on breast cell lines. a Western blot analysis of TAGLN in breast carcinoma and non-tumorigenic breast cell lines. b–c Western blot shows TAGLN silencing in MCF10A (b) and MCF12A (c) cells (upper panels). 2D colony formation assays (middle and lower panels) show increased colony formation in TAGLN silenced (siTAGLN) MCF10A (b) and MCF12A (c) cells compared to controls (siCTRL). d–e Western blot analyses of TAGLN overexpression and subsequent colony formation assays in MDA-MB-361 (d, upper panel), and MDA-MB-157 (e, left panel) cell clones. Overexpression of ectopic TAGLN (TAGLNvec) and levels of endogenous TAGLN (TAGLNint) were determined with TAGLN specific antibody in clones. TAGLN overexpression caused higher cell proliferation in MDA-MB-361 cells (e), while did not affect MDA-MB-157 cells (f). Each assay was conducted at least in quadruplicates. Two sample t test was used for comparisons. Error bars represent standard errors. **P < 0.01
Mentions: Since TAGLN was frequently hypermethylated and consistently downregulated in both the BC cell lines and three independent panels of tumor tissues, we analyzed the functional role of TAGLN in BC and NTB cells. TAGLN gene was silenced in MCF10A and MCF12A cells with abundant expression (Fig. 6a–c), and was overexpressed it in MDA-MB-361 and MDA-MB-157 BC cell lines (Fig. 6d, e) that expressed TAGLN at zero to intermediate levels, respectively (Fig. 6a). 2D colony formation assays were performed to test the effect of TAGLN on proliferation potentials of these cells. Compared to control siRNA (siCTRL), siTAGLN transfected MCF10A and MCF12A cells formed significantly (P = 0.0013 and P = 0.0016, respectively) higher number of colonies (Fig. 6b, c, middle and lower panels). Colony formation capabilities of MDA-MB-361 cells, but not MDA-MB-157 cells, were significantly impaired when they overexpressed TAGLN protein (Fig. 6d, middle and lower panels and Fig. 6e, right panel). To examine possible effects of TAGLN on migration and invasion potentials of BC cells, we performed in vitro wound healing and Matrigel invasion assays in normally motile MDA-MB-157 cells; however, TAGLN overexpression in these cells did not affect either wound closure or Matrigel invasion capacities of these cells (Additional file 5: Figure S3).Fig. 6

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