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. 2013 Dec 4:13:570.
doi: 10.1186/1471-2407-13-570.

NCOA3 is a selective co-activator of estrogen receptor α-mediated transactivation of PLAC1 in MCF-7 breast cancer cells

Meike Wagner, Michael Koslowski, Claudia Paret, Marcus Schmidt, Ozlem Türeci, Ugur Sahin  1
Affiliations

NCOA3 is a selective co-activator of estrogen receptor α-mediated transactivation of PLAC1 in MCF-7 breast cancer cells

Meike Wagner et al. BMC Cancer. .

Abstract

Background: The placenta-specific 1 (PLAC1) gene encodes a membrane-associated protein which is selectively expressed in the placental syncytiotrophoblast and in murine fetal tissues during embryonic development. In contrast to its transcriptional repression in all other adult normal tissues, PLAC1 is frequently activated and highly expressed in a variety of human cancers, in particular breast cancer, where it associates with estrogen receptor α (ERα) positivity. In a previous study, we showed that ERα-signaling in breast cancer cells transactivates PLAC1 expression in a non-classical pathway. As the members of the p160/nuclear receptor co-activator (NCOA) family, NCOA1, NCOA2 and NCOA3 are known to be overexpressed in breast cancer and essentially involved in estrogen-mediated cancer cell proliferation we asked if these proteins are involved in the ERα-mediated transactivation of PLAC1 in breast cancer cells.

Methods: Applying quantitative real-time RT-PCR (qRT-PCR), Western Blot analysis and chromatin immunoprecipitation, we analyzed the involvement of NCOA1, NCOA2, NCOA3 in the ERα-mediated transactivation of PLAC1 in the breast cancer cell lines MCF-7 and SK-BR-3. RNAi-mediated silencing of NCOA3, qRT-PCR, Western blot analysis and ERα activation assays were used to examine the role of NCOA3 in the ERα-mediated regulation of PLAC1 in further detail. Transcript expression of NCOA3 and PLAC1 in 48 human breast cancer samples was examined by qRT-PCR and statistical analysis was performed using Student's t-test.

Results: We detected selective recruitment of NCOA3 but not NCOA1 or NCOA2 to the PLAC1 promoter only in ERα-positive MCF-7 cells but not in ERα-negative SK-BR-3 breast cancer cells. In addition, we demonstrate that silencing of NCOA3 results in a remarkable decrease of PLAC1 expression levels in MCF-7 cells which cannot be restored by treatment with estradiol (E₂). Moreover, significant higher transcript levels of PLAC1 were found only in ERα-positive human breast cancer samples which also show a NCOA3 overexpression.

Conclusions: In this study, we identified NCOA3 as a selective co-activator of ERα-mediated transactivation of PLAC1 in MCF-7 breast cancer cells. Our data introduce PLAC1 as novel target gene of NCOA3 in breast cancer, supporting the important role of both factors in breast cancer biology.

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Figures

Figure 1
Figure 1
Expression of NCOA1, 2 and 3 in ERα-positive MCF-7 and ERα-negative SK-BR-3 breast cancer cells. (A) Real-time RT-PCR quantification of NCOA1, 2 and 3 mRNA expression in MCF-7 and SK-BR-3 breast cancer cells. Shown are mean and standard deviation of three individual experiments. Statistical analysis of NCOA3 mRNA expression compared to NCOA1 and 2 were performed using two-tailed Student’s t-test. P-values ≤ 0.05 (*) were considered statistically significant. (B) Protein expression of NCOA1, 2 and 3 in MCF-7 and SK-BR-3 breast cancer cells was analyzed by Western blot. β-actin was used as a loading control.
Figure 2
Figure 2
Selective E2-dependent recruitment of NCOA3 to the endogenous PLAC1 promoter in ERα positive MCF-7 cells. (A) Chromatin immunoprecipitation (ChIP) was performed with chromatin prepared from MCF-7 and SK-BR-3 cells which were either untreated (C), treated with 17-β-estradiol (E2) alone, with ICI alone or with both compounds (E2/ICI). The promoter region of PLAC1 containing the C/EBPβ and SP1 elements (−348/-198) and a region upstream of the promoter (−1219/-1064) as negative control were analyzed by PCR following immunoprecipitation with the indicated antibodies. Amplification products from soluble chromatin prior to precipitation are shown as control (Input). (B) Quantitative analysis of the recruitment and occupancy shown in (A) determined by real-time RT-PCR. The results corrected by input are shown as fold increase compared to unstimulated cells used as a reference.
Figure 3
Figure 3
ERα-mediated transactivation of PLAC1 is dependent on NCOA3. (A) Quantitative real-time RT-PCR expression analysis of NCOA3 and PLAC1 in MCF-7 and SK-BR-3 cells 48 h after transfection with NCOA3- or PLAC1-specific siRNA duplexes. ns-siRNA, non-silencing siRNA. The results are shown as fold increase compared to untransfected cells used as a reference. (B) Protein expression of NCOA3 in MCF-7 and SK-BR3 cells 48 h after transfection with siRNA duplexes targeting NCOA3 or PLAC1 mRNA. Transfection of cells with PLAC1-specific siRNA was conducted to verify that knockdown of NCOA3 specifically affects PLAC1 mRNA and protein level. (C) Quantitative real-time RT-PCR analysis of PLAC1 expression in E2-depleted (72 h) MCF-7 cells in response to treatment with 100 nM E2, no E2 (C), or E2 and 5 μM ICI (E2/ICI), or ICI alone for 12 h. 48 h prior to treatment, cells were transfected with siRNA duplexes targeting NCOA3. Shown are mean and standard deviation of two independent experiments.
Figure 4
Figure 4
mRNA expression of PLAC1 is elevated in NCOA3 overexpressing and ERα-positive human breast cancer samples. (A)NCOA3 and PLAC1 mRNA expression was examined by quantitative real-time RT-PCR in 48 human breast cancer samples. Relative expression of NCOA3 below 100 000 were considered to be ‘NCOA3 low’ (N = 25), whereas relative expression of NCOA3 over 100 000 were considered to be ‘NCOA3 high’ (N = 23). (B) Evaluation of PLAC1 expression in ERα-positive breast cancer samples with ‘NCOA3 low’ (N = 20) or ‘NCOA3 high’ (N = 13) status. (C) Evaluation of PLAC1 expression in ERα-negative breast cancer samples with ‘NCOA3 low’ (N = 5) or ‘NCOA3 high’ (N = 10) status. Statistical analysis was performed using two-tailed Student’s t-test. P ≤ 0.05 (*) was considered statistically significant.
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