Identification of <i>cis</i>-elements of the <i>UBC</i> promoter.

<p>(<b>A</b>) Site-directed mutagenesis was carried out with the UBC9 pGL-137/+124 construct. The mutation of a putative transcription factor binding site is indicated by a solid cross. MCF-7 cells cultured in phenol red-free medium in the absence (white bars) or the presence (black bars) of E₂ and and MDA-MB-231 cells (dotted bars) were transfected with the indicated constructs and assayed for luciferase activity after 48 hours. Luciferase activity was expressed as fold change relative to that obtained from promoterless vector <i>pGL</i>-basic, which was arbitrarily set to 1. Values were normalized for transfection efficiency by co-transfection with the <i>Renilla</i> expression plasmid and were expressed as mean ±SD obtained in four separate experiments. *P<0.05, **P<0.01 (Student’s <i>t</i>-test). (<b>B</b>) Enhancement of the <i>UBC9</i> promoter activity by ER-α or NF-YA overexpression. Untreated and E₂-treated MCF-7 cells were transfected with WT, Mut-1, Mut-4 and Mut-5<i>pGL</i>-137/+124 constructs, NF-YA (dotted bars) or ER-α (black bars) expression plasmids. Mock transfected cells were used as a control (white bars). Relative luciferase activity (RLA) was expressed as fold change relative to that obtained from pGL-137/+124 (E₂-), which was arbitrarily set to 1. Values were normalized for transfection efficiency by co-transfection with the <i>Renilla</i> expression plasmid and were expressed as mean ±SD obtained in four separate experiments. **P<0.01 (Student’s <i>t</i>-test).</p

Estrogen Receptor Alpha and Nuclear Factor Y Coordinately Regulate the Transcription of the SUMO-Conjugating <i>UBC9</i> Gene in MCF-7 Breast Cancer Cells

<div><p><i>UBC9</i> encodes a protein that conjugates small ubiquitin-related modifier (SUMO) to target proteins thereby changing their functions. Recently, it was noted that UBC9 expression and activity play a role in breast tumorigenesis and response to anticancer drugs. However, the underlying mechanism is poorly understood. To investigate the transcriptional regulation of the <i>UBC9</i> gene, we identified and characterized its promoter and <i>cis</i>-elements. Promoter activity was tested using luciferase reporter assays. The binding of transcription factors to the promoter was detected by chromatin immunoprecipitation (ChIP), and their functional role was confirmed by siRNA knockdown. <i>UBC9</i> mRNA and protein levels were measured by quantitative reverse transcription PCR and Western blot analysis, respectively. An increased expression of <i>UBC9</i> mRNA and protein was found in MCF-7 breast cancer cells treated with 17β-estradiol (E₂). Analysis of various deletion mutants revealed a 137 bp fragment upstream of the transcription initiation site to be sufficient for reporter gene transcription. Mutations of putative estrogen receptor α (ER-α) (one imperfect estrogen response element, ERE) and/or nuclear factor Y (NF-Y) binding sites (two CCAAT boxes) markedly reduced promoter activity. Similar results were obtained in ER-negative MDA-MB-231 cells except that the ERE mutation did not affect promoter activity. Additionally, promoter activity was stimulated upon E₂ treatment and overexpression of ER-α or NF-YA in MCF-7 cells. ChIP confirmed direct binding of both transcription factors to the <i>UBC9</i> promoter <i>in vivo</i>. Furthermore, <i>UBC9</i> expression was diminished by ER-α and NF-Y siRNAs on the mRNA and protein levels. In conclusion, we identified the proximal <i>UBC9</i> promoter and provided evidence that ER-α and NF-Y regulate <i>UBC9</i> expression on the transcriptional level in response to E₂ in MCF-7 cells. These findings may contribute to a better understanding of the regulation of UBC9 in ER-positive breast cancer and be useful for the development of cancer therapies targeting UBC9.</p> </div

Recruitment of ER-α and NF-Y to the <i>UBC9</i> promoter <i>in vivo</i>.

<p>(<b>A</b>) Schematic representation of the <i>UBC9</i> gene including the proximal promoter with the putative transcription factor binding sites and the negative control region (<i>UBC9</i> exon 7). Primer pairs are indicated by arrows. (<b>B</b>) ChIP assays using anti-ER-α, NF-YA or IgG control antibodies were performed on chromatin isolated from cells cultured in phenol red-free medium in the absence (white bars) or the presence (black bars) of E₂ for 48 hours. The equivalent fraction of the sonicated chromatin was set aside as 'input' DNA (non-immunoprecipitated) before the antibody affinity manipulations. Data were presented as relative amount of immunoprecipitated DNA normalized to input as measured by quantitative PCR assay, and were given as mean ±SD obtained in four separate experiments. **P<0.01 (Student’s <i>t</i>-test). (<b>C</b>) Ethidium bromide staining of the PCR products of the <i>UBC9</i> promoter region (upper panel) and <i>UBC9</i> exon 7 control region (lower panel).</p

UBC9 expression is up-regulated by E₂ in MCF-7 cells.

<p>(<b>A</b>) <i>UBC9</i> and (<b>B</b>) ER-α mRNA expression after treatment of MCF-7 cells with 10 nM E₂ for 12, 24, 48 and 72 hours. Where indicated, 100 nM ICI was also added to the culture medium. Total RNA was isolated and analyzed by real-time RT-PCR. Expression levels were normalized to GAPDH expression and relative to expression in untreated cells, which was arbitrarily set to 1. The data refer to results obtained in four separate experiments performed in triplicate. Bars represent the standard deviation (SD). (<b>C</b>) Up-regulation of UBC9 protein levels after induction with E₂. Total protein was extracted and analyzed by Western blotting. Actin was used as an internal protein loading control.</p

Identification and characterization of the minimal <i>UBC9</i> promoter.

<p>(<b>A</b>) MCF-7 cells cultured in phenol red-free medium in the presence (black bars) or absence (white bars) of E₂ were transfected with the indicated constructs and assayed for luciferase activity after 48 hours. The numbers given for each construct indicate the 5’ and 3’ ends of the <i>UBC9</i> 5’-flanking region; the position numbered +1 corresponds to the transcription initiation site. Luciferase activity was expressed as fold change relative to that obtained from promoter-less vector <i>pGL</i>-basic, which was arbitrarily set to 1. Values were normalized for transfection efficiency by co-transfection with the <i>Renilla</i> expression plasmid and were given as mean ±SD obtained in four separate experiments. **P<0.01 (Student’s <i>t</i>-test). (<b>B</b>) <i>UBC9</i> sequence and putative transcription factor binding sites of the minimal <i>UBC9</i> promoter. Position +1 refers to the transcription initiation site. Putative transcription factor-binding sites predicted by the webtool PromoterSweep [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075695#B30" target="_blank">30</a>], including an imperfect ERE, a CCAAT box and an inverted CCAAT box (iCCAAT) are overlined.</p

Inherited variants in the inner centromere protein (incenp) gene of the chromosomal passenger complex contribute to the susceptibility of er-negative breast cancer

The chromosomal passenger complex (CPC) plays a pivotal role in the regulation of cell division. Therefore, inherited CPC variability could influence tumor development. The present candidate gene approach investigates the relationship between single nucleotide polymorphisms (SNPs) in genes encoding key CPC components and breast cancer risk. Fifteen SNPs in four CPC genes (INCENP, AURKB, BIRC5 and CDCA8) were genotyped in 88 911 European women from 39 case-control studies of the Breast Cancer Association Consortium. Possible associations were investigated in fixed-effects meta-analyses. The synonymous SNP rs1675126 in exon 7 of INCENP was associated with overall breast cancer risk [per A allele odds ratio (OR) 0.95, 95% confidence interval (CI) 0.92-0.98, P = 0.007] and particularly with estrogen receptor (ER)-negative breast tumors (per A allele OR 0.89, 95% CI 0.83-0.95, P = 0.0005). SNPs not directly genotyped were imputed based on 1000 Genomes. The SNPs rs1047739 in the 3E(1) untranslated region and rs144045115 downstream of INCENP showed the strongest association signals for overall (per T allele OR 1.03, 95% CI 1.00-1.06, P = 0.0009) and ER-negative breast cancer risk (per A allele OR 1.06, 95% CI 1.02-1.10, P = 0.0002). Two genotyped SNPs in BIRC5 were associated with familial breast cancer risk (top SNP rs2071214: per G allele OR 1.12, 95% CI 1.04-1.21, P = 0.002). The data suggest that INCENP in the CPC pathway contributes to ER-negative breast cancer susceptibility in the European population. In spite of a modest contribution of CPC-inherited variants to the total burden of sporadic and familial breast cancer, their potential as novel targets for breast cancer treatment should be further investigated

Common breast cancer susceptibility loci are associated with triple-negative breast cancer.

Triple-negative breast cancers are an aggressive subtype of breast cancer with poor survival, but there remains little known about the etiologic factors that promote its initiation and development. Commonly inherited breast cancer risk factors identified through genome-wide association studies display heterogeneity of effect among breast cancer subtypes as defined by the status of estrogen and progesterone receptors. In the Triple Negative Breast Cancer Consortium (TNBCC), 22 common breast cancer susceptibility variants were investigated in 2,980 Caucasian women with triple-negative breast cancer and 4,978 healthy controls. We identified six single-nucleotide polymorphisms, including rs2046210 (ESR1), rs12662670 (ESR1), rs3803662 (TOX3), rs999737 (RAD51L1), rs8170 (19p13.1), and rs8100241 (19p13.1), significantly associated with the risk of triple-negative breast cancer. Together, our results provide convincing evidence of genetic susceptibility for triple-negative breast cancer