Predicted transcription factors binding sites in −923/−565 core promoter and their relevance in breast cancer.

<p>Predicted transcription factors binding sites in −923/−565 core promoter and their relevance in breast cancer.</p

Estradiol reverses the TNF-mediated increase of <i>ST8SIA1</i> mRNA expression in ER-positive MCF-7 and in ER-negative Hs578T expressing ERα.

<p>ERα-expressing Hs578T and MCF-7 cells were treated with 10⁻¹⁰ M estradiol and/or 40 ng/mL TNF for 12h. <i>ST8SIA1</i> mRNA expression was determined by qPCR. Results were normalized to the expression of <i>RPLP0</i> and reported to the expression of <i>ST8SIA1</i> in cells treated with vehicle (0.1% ethanol). Data are means +/− SD of n ≥3 experiments. *: p<0.05 <i>vs</i>. untreated (vehicle).</p

Primers used for 5′-RACE, qPCR and plasmid constructions.

1<p>Previously used by Kang <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062559#pone.0062559-Kang1" target="_blank">[25]</a>.</p>2<p>Provided by Invitrogen.</p>3<p>Designed using the NCBI primer design software (<a href="http://www.ncbi.nlm.nih.gov/tools/primer-blast/" target="_blank">http://www.ncbi.nlm.nih.gov/tools/primer-blast/</a>).</p>4<p>Designed using the QuickChange primer design software (<a href="http://labtools.stratagene.com/QC" target="_blank">http://labtools.stratagene.com/QC</a>).</p><p>Restriction site sequences inserted for cloning are underlined and the mutated nucleotides in primers used for site directed mutagenesis are in bold.</p

Promoter activity of the 5′ flanking region of GD3S T1 transcript in Hs578T cells.

<p>(A) Location of the restriction sites used to generate the different deletions of the genomic sequence between −2307 and the ATG site (+1) in E1 exon. The positions of EREs and NFκB binding site in the core promoter are indicated. Arrowheads show the position of TSS from −345 to −20 bp upstream the ATG. (B) On the left, schematic representation of the different constructs inserted in pGL3basic upstream the luciferase gene. Luc indicates the Firefly luciferase coding sequence. On the right, luminescence detected in luciferase assays. Transfection efficiencies were normalized with the co-transfected plasmid expressing <i>Renilla</i> luciferase and luciferase activities are expressed compared to empty pGL3basic activity. The data are means +/− S.D. of n ≥3 experiments.</p

Estradiol-mediated repression of <i>ST8SIA1</i> promoter activity do not involve ERE element but NFκB transcription factor.

<p>(A) Effect of mutations of ERE and NFκB putative sites on −923/−565 core promoter activity. After 48h of culture in steroid-free medium, Hs578T were transfected with pGL3(−923/−565), either native or mutated on ERE binding sites, pcDNA-ERα and/or pCMV-p65 and pCMV-p50. Transfection with empty expression vectors was used as negative controls. The following day, cells were treated for 12 h with 10⁻¹⁰ M estradiol and/or 40 ng/mL TNF. On the left: schematic representation of the sequence transfected in Hs578T cells. Black circles indicate the mutated ERE sequences and black triangle indicates the mutated NFκB site. On the right: relative luciferase activity of the core promoter in Hs578T cells treated or not with 10⁻¹⁰ M estradiol. Transfection efficiencies were normalized with the co-transfected plasmid expressing <i>Renilla</i> luciferase and reported to the luciferase activity in cells transfected with native pGL3(−923/−565) treated with vehicle. Vehicle: 0.1% ethanol. Each bar represents the mean +/− S.D. of n ≥3 experiments. *: p<0.05. (B) Effect of estradiol on p50 and p65 NFκB subunits nuclear expression in ERα expressing Hs578T and MCF-7 cells. Nuclear proteins extracted from ERα-transfected Hs578T and MCF-7 cells treated as previously described in A were used for immunoblotting with anti-p50 or anti-p65 mAbs. Histone H2B expression was used as a loading control.</p

Hypothetical mechanism of GD3S repression by estradiol.

<p>GD3S gene (<i>ST8SIA1</i>) is activated by the canonical NFκB pathway after TNF stimulation. In ER-positive breast cancer cells, GD3S expression is repressed by estradiol (E2)-ERα complex. Repression of NFκB transport to the nucleus could be achieved via the activation of PI3K <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062559#pone.0062559-Ghisletti1" target="_blank">[44]</a>.</p

Reporter plasmid constructions.

<p>Reporter plasmid constructions.</p

<i>In vivo</i> binding of p65 subunit to <i>ST8SIA1</i> promoter by Chromatin Immunoprecipitation in breast cancer cells.

<p>ChIP were performed as described in the Material and Methods from ERα-expressing Hs578T cells and MCF-7 cells treated 12 h with 40 ng/mL TNF in presence or in absence of 10⁻¹⁰ nM estradiol. PCRs were carried out with specific pairs of primers covering the NFκB binding site (−773/−769). PCR products were analyzed on 2% Agarose gel (A) or <i>via</i> qPCR (B). Bars indicate percent enrichment compared to the input DNA and were normalized to the goat isotype control. Each bar represents the mean +/− S.D. of n ≥2 experiments. *: p<0.05.</p

Estradiol represses <i>ST8SIA1</i> mRNA expression in ER-positive MCF-7 and in ER-negative Hs578T expressing ERα.

<p>(A) Effect of estradiol and Tamoxifen on GD3S mRNA expression in MCF-7 cells. After 48h of culture in steroid-free medium, MCF-7 cells were treated with 10⁻¹⁰ M estradiol and/or 10⁻⁶ M TAM for 24h. (B) Effect of estradiol on GD3S mRNA expression in Hs578T cells transfected with ERα coding vector. After 48h of culture in steroid-free medium, Hs578T cells were transfected with pcDNA-ERα or pcDNA empty vector (control). 24h after transfection, cells were treated for 24h with 10⁻¹⁰ M estradiol. For (A) and (B), <i>ST8SIA1</i> or <i>PS2</i> (positive control) mRNA expression was determined by qPCR. Results were normalized to the expression of <i>RPLP0</i> and reported to the expression of <i>ST8SIA1</i> or <i>PS2</i> in cells treated with vehicle (0.1% ethanol). Data are means +/− SD of n ≥3 experiments. * p<0.05 <i>vs</i>. untreated (vehicle).</p

Identification of human GD3S transcripts in breast cancer tumors and Hs578T cells.

<p>(A) Schematic representation of the 5′-RACE strategy. First strand cDNA synthesis was performed with a <i>ST8SIA1</i> specific primer. cDNA was dC-tailed at 3′end and amplified by PCR using Anchor Primer and GSP1. Nested PCR was performed using AUAP and GSP2 primers. (B) Schematic representation of the main 5′-ends of GD3S transcripts expressed in Hs578T cells and breast cancer tumor samples (#137, #142 and #148). The size of intronic sequences between E2 and the different first exon are shown. Position of PCR primers used for specific amplification of T1 transcript is indicated by black arrows. (C) qPCR analysis of T1 transcript (grey) and total <i>ST8SIA1</i> (black) expression, related to <i>HPRT</i>, in 20 ER-negative IDC samples and 2 breast cancer cell lines (Hs578T and MCF-7).</p