Characterization of NF-κB as a regulatory element in distal part of promoter.

<p><b>A)</b> Luciferase reporter gene assay for the effect of NF-κB and Twist-1 on the activity of <i>KIAA1199</i> promoter: Lysates of COS-1 cells transfected with cDNAs as indicated were examined by Dual Luciferase activity assay. Wild type and substitute mutation (pro-1.4 kb swap NF-κB^4th) at distal region of NF-κB binding site were used. <b>B)</b> ChIP assay for identification of the interaction between NF-κB (p65) and <i>KIAA1199</i> promoter. Top panel: A schematic diagram of four putative NF-κB binding sites relative to +1 site. Primer sets A, B, and C were designed for NF-κB binding site IV and I + II, and an area of the first <i>KIAA1199</i> intron, respectively. Middle and low panels: ChIP PCR in HeLa cells transfected with GFP control and P65 cDNAs. Anti-p-65 antibody and normal IgG (control) were used for immunoprecipitation. Results were calculated according to the bound/input ratio.</p

Elevated expression of <i>KIAA1199</i> in human cancers.

<p>A) By mining Oncomine and GEO databases, <i>KIAA1199</i> expression pattern in more than 40 microarray data sets shows significant alteration (P<0.01). Representative data are presented. High <i>KIAA1199</i> expression in various human cancers.1-breast cancer n:27, normal n:7, p = 6.97E-4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Radvanyi1" target="_blank">[41]</a>; 2-colon cancer n:36, normal n:24, p = 1.7E-4<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Skrzypczak1" target="_blank">[42]</a>; 3-gastric cancer n:26, normal n:31, p = 3.69E-13<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-DErrico1" target="_blank">[43]</a>; 4-lung cancer n:45, normal n:65, p = 8.59E-9<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Hou1" target="_blank">[44]</a>; 5-head&neck cancer n:41, normal n:13, p = 2.35E-7<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Ginos1" target="_blank">[45]</a>; 6-ovarian cancer n:6, normal n:4, p = 5.92E-4<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Adib1" target="_blank">[46]</a>; 7-pancreatic cancer n:11, normal n:11, p = 0.001 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Grutzmann1" target="_blank">[47]</a>. <b>B)</b> Expression of <i>KIAA1199</i> in human cancer cell lines: Human prostate (LNCaP and Du145), and breast cancer (MCF-7 and MDA-MB-231) cell lines were examined by real time RT-PCR using <i>KIAA1199</i> specific primers. The expression of <i>KIAA1199</i> was normalized by house-keeping genes (HPRT-1 and GAPDH). The relative levels of genes were determined using the ΔΔCt method. Each bar represents the mean ± S.E (*<0.05).</p

Transcriptional and Epigenetic Regulation of <em>KIAA1199</em> Gene Expression in Human Breast Cancer

<div><p>Emerging evidence has demonstrated that upregulated expression of <em>KIAA1199</em> in human cancer bodes for poor survival. The regulatory mechanism controlling <em>KIAA1199</em> expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human <em>KIAA1199</em> promoter in terms of regulation of <em>KIAA1199</em> gene expression. A 3.3 kb fragment of human genomic DNA containing the 5′-flanking sequence of the <em>KIAA1199</em> gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic <em>KIAA1199</em> promoter containing a TATA-box close to the transcription start site. A combination of 5′-primer extension study with 5′RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human <em>KIAA1199</em> gene. Bioinformatics analysis suggested that the 1.4 kb <em>KIAA1199</em> promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for <em>KIAA1199</em> gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these <em>cis</em>- and <em>trans</em>-acting elements in controlling <em>KIAA1199</em> gene expression. Finally, we found that upregulated <em>KIAA1199</em> expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of <em>KIAA1199</em> expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling <em>KIAA1199</em> gene expression in human cancer.</p> </div

Determination of the transcription start site of the human <i>KIAA1199</i>. A)

<p>5′ primer extension analysis: Mapping of the <i>KIAA1199</i> mRNA start site by non-radioactive 5′ primer extension analysis was performed using the reverse primer located between +254 and +234 in the first exon (shown by underlie arrow in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone-0044661-g002" target="_blank">Figure 2D</a>). <b>B)</b> Identification of the transcription start site(s) of the <i>KIAA1199</i> mRNA: 5′ RLM-RACE was performed followed by DNA sequencing analysis. <i>DNA sequencing chromatogram</i> represents a longest transcript and percentages of the alternative transcription start sites are given. C) CAGE data analysis: One major transcription start site was identified in the CAGE analysis viewer (CTSS:Cage Transcription Start Site). <b>D)</b> Nucleotide sequence of the 5′-flanking region of the <i>KIAA1199</i>: +1 was given to the transcription start site of <i>KIAA1199</i>. According to the +1, TATA-box was identified between −31 and −27; GC-box was identified between −248 and −243. Start site of CpG island was shown with gray box in the upstream region of promoter and nucleotides in the CpG island are shown by italics.</p

Sequence alignment of the <i>KIAA1199</i> promoter between human and mouse genomes and putative transcription factor-binding sites.

<p>Putative transcription factor-binding motifs are underlined and TATA/GC boxes and transcription start site are shown in the box. The asterisks mark the fully conserved sequences across the species.</p

Suppression of <i>KIAA1199</i> expression in less invasive cancer cells by DNA methylation.

<p><b>A)</b> Bioinformatics analysis of the CpG island of <i>KIAA1199</i>: Two major subregions within the CpG island were examined by MSP method for MCF-7 and MDA-MB-231 cells. F: forward primers and R: reverse primers. <b>B)</b> Pyrosequencing analysis for quantitation of methylation of cytosine residues in the second subregion of the CpG island between MCF-7 and MDA-MB-231 cells: The percentage of methylation for 36 CG pairs between +525 and +1059 was shown individually (Left panel). Average methylation rate was calculated for MCF-7 and MDA-MB-231 cells (p<0.001) (Right panel). <b>C)</b> Effect of 4 days treatment with 5′-azaon <i>KIAA1199</i> expression: Real time RT PCR was performed in MCF-7 and MDA-MB-231 cells treated with 5′-aza using <i>KIAA1199</i> specific primers. Housekeeping genes were used to normalize the gene expression. <b>D)</b> Methylation status of <i>KIAA1199</i> in human breast cancer specimens: Human invasive breast cancer cells as well as normal breast epithelial cells were harvested by LCM and pyrosequencing was performed using bisulfite-treated DNA. The average methylation level in normal cells was found higher than in cancer cells. A pair of normal breast epithelial cells and breast cancer cells from patients A and B was labeled. n: case number. <b>E)</b><i>KIAA1199</i> expression in human breast cancer specimens: Total RNA from micro-dissected human breast cancer cells as well as normal breast epithelial cells was examined by real time RT-PCR using <i>KIAA1199</i> specific primers. Housekeeping genes were used to normalize the gene expression. A pair of normal breast epithelial cells and breast cancer cells from patients A and B was labeled. n: case number. <b>F)</b> Hypomethylation of <i>KIAA1199</i> in invasive human breast cancer specimens: Bisulfite-treated DNA from paired normal and cancer cells of breast cancer specimens harvested by laser microdissection technique was examined by a pyrosequencing approach. Representative methylation profile between the +932 and +1025 was shown for the benign and invasive cells of single patients.</p

Requirement of AP-1 binding element in the <i>KIAA1199</i> promoter.

<p><b>A)</b> A schematic diagram of mutations at the AP-1 binding site: A site-directed mutagenesis was carried out to generate either a deletion mutant by removing the AP-1 consensus sequence (GAGT) or a substitute mutation within the pro-1.4 promoter construct. The relative promoter activities of the mutations (ratio of firefly luciferase over Renilla luciferase) were then compared to the activity of the wild type pro-1.4 promoter construct (defined as arbitrary value of 100) in COS-1, MDA-MB-231, and MCF-7 cells. <i>Error bars indicate</i> mean +/− S.E. <b>B)</b> Binding of nuclear proteins to the AP-1 site in the <i>KIAA1199</i> promoter: EMSA was carried out using a biotinylated double-stranded oligonucleotide (50 bp) containing the AP-1 binding site and nuclear extracts from MDA-MB-231 cells. Where indicated, binding was competed with 50–200 fold excess amounts of unlabeled probe. DNA-protein complexes formed are indicated as Shift-1 and Shift-2. <b>C)</b> Determination of specific binding between AP1 and the <i>KIAA1199</i> promoter: EMSA was performed using a biotinylated double-stranded oligonucleotide (50 bp) containing the AP-1 binding site and nuclear extracts from MDA-MB-231 cells. Where indicated, biotinylated probe along with anti-C-Jun antibody or biotinylated probe containing mutated site of AP-1 consensus sequence were incubated with nuclear extracts from MDA-MB-231 cells. <b>D)</b> ChIP assay for analysis of association between endogenous AP-1 and the KIAA199 promoter sequence: A strong relation between AP-1 and DNA sequence was shown by 20% bound/input ratio as compared to the unrelated intron region. Normal rabbit IgG was used as a negative control. Results were calculated according to the bound/input ratio.</p

Analysis of the transcriptional activity of the <i>KIAA1199</i> promoter.

<p><b>A</b>) <i>Left panel</i>: A schematic description of the <i>KIAA1199</i> reporter constructs containing fragments of three different lengths cloned into the pGL3-basic vector. The numbers in the names of the constructs indicate their respective lengths in nucleotides relative to the main transcription start site. <i>Right three panels</i>: Normalized firefly luciferase activity from each construct: Lysates from cells transfected with the different reporter gene constructs together with R<i>enilla luciferase reporter were examined for luciferase activities.</i> The relative promoter activities (ratio of firefly luciferase over Renilla luciferase) were compared to the activity of the pro-1.4 promoter construct (defined as arbitrary value of 100). <i>Error bars indicate</i> mean +/− S.E. <b>B</b>) <i>Top panel</i>: A ladder of PCR fragments for generating deletion mutants from the pro-1.4 promoter. <i>Left lower panel</i>: A schematic description of the pro-1.4 kb and truncations of the <i>KIAA1199</i> reporter constructs cloned in pGL3-basic vector. <i>Right lower three panels</i>: Normalized firefly luciferase activity from each construct: Lysates from cells transfected with the different reporter gene constructs together with R<i>enilla luciferase reporter were examined for luciferase activities. The firefly luciferase value of each sample has been normalized to its renilla luciferase value. Error bars indicate</i> mean +/− S.E.</p

Simultaneous determination of invasion and cytotoxicity.

<p>HT1080 cells expressing GFP were assembled in the 3-D invasion assay in the presence of (A) DMSO, (B) anti-β1 integrin antibody (5 μg/ml), (C) staurosporine (STS) (500 nM), or (D) paclitaxel (Taxol, 1 μM) for 18 hours. The cells were stained with Hoechst and propidium iodide (PI) for 30 minutes followed by microscopic examination. Decreased cell invasion is observed upon treatment with anti-β1 integrin antibody, STS, and taxol. However, STS and taxol show higher levels of PI staining, indicative of increased cell death. Numbers in the top right corners represent the total number of invasive cells (Hoechst images) or cells that died after invading (PI images) within the field. Scale bar = 100 μm.</p

Cell scattering assays for monitoring cell invasive ability.

<p>A) a. Single-cell scattering assay: Single, isolated LNCaP cells were mixed with collagen and examined daily. MT1-GFP-expressing cells gradually displayed a scattering growth pattern after 6 days in culture as compared to the aggregated growth of GFP-expressing cells. b. Aggregate scattering assay: SK-3^rd cells were cultured for 12 days to form mammospheres, transferred and embedded in collagen, and then cultured under normoxia or hypoxia. A scattered pattern was seen after 3 days under hypoxia. c. Microcarrier bead scattering assay: MT1-GFP expressing LNCaP cells were cultured with beads for 24 hours, then transferred and embedded into collagen. Invaded cells were seen as a scattered pattern at day 3. Scale bars = 20 μm. B) Assessment of cancer cell invasiveness using novel 3-D invasion assay: LNCaP cells expressing vector control (a) or MT1-MMP (b), human prostate cancer PC3 cells (c), and Du145 cells (d) were suspended in collagen, dotted into the well of a 96-well plate, and covered with collagen followed by media. After 18-h incubation, PC3 and Du145, as well as MT1-MMP expressing LNCaP cells, showed increased numbers of invaded cells as compared to LNCaP vector control cells. Scale bar = 50 μm.</p