PIAS1 Regulates Breast Tumorigenesis through Selective Epigenetic Gene Silencing

<div><p>Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth <i>in vivo</i>. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing.</p></div

PIAS1 regulates the expression of a panel of tumor suppressor genes.

<p>(<b>a</b>) Real-time quantitative PCR (Q-PCR) assay. MDA-MB231 cells containing control shRNA, PIAS1 shRNA1 or shRNA2 were cultured in DMEM plus 10% FBS (DMEM) or Stem Cell Media (SCM) for 30 h, and total RNA was used for Q-PCR assays with gene-specific primers. The gene names are labeled at the top left of each panel. The data were normalized by beta-Actin (<i>ACTB</i>) and presented as “Relative Expression” as compared to that in control shRNA cells under DMEM condition, which was set as “1” except for the <i>ESR1</i> gene (the expression was not detectable in control shRNA cells). Shown is a representative of 3 independent experiments. Error bars represent SD. ND, not detected. See also Table S1 and Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089464#pone.0089464.s001" target="_blank">File S1</a>. (<b>b</b>) Same as in <b>a</b> except that total RNA from fat pad tumor xenograft samples were used (n = 5). Error bars represent SEM. <i>P</i> values were determined by non-paired <i>t</i>-test.</p

PIAS1 is important for the maintenance of the Tumor Initiating Cells (TICs) in MDA-MB231 cells.

<p>(<b>a</b>) Reduced ALDH⁺ population in PIAS1 knockdown cells using the ALDEFLUOR assay. Cells cultured in Stem Cell Media (SCM) for 25 days were incubated with ALDEFLUOR substrate (BAAA) with or without the specific inhibitor of ALDH, DEAB, to define the ALDH⁺ population (R2). The number indicates the percentage of the ALDH⁺ population. (<b>b</b>) Mammosphere assays. MDA-MB231 control shRNA and PIAS1 shRNA1 and shRNA2 cells were seeded in SCM on 35 mm petri dishes (5,000 cells/dish) and spheres were counted 7 days later. (<b>c</b>) PIAS1 is phosphorylated on Ser90 in response to EGF and Heregulin in breast cancer cells. Various breast cancer cells were starved for 16 h, then either untreated or treated with EGF (100 ng/ml) or Heregulin (15 ng/ml) for indicated time points. Western blot analyses were performed with whole cell extracts, using an antibody specific for Ser90-phosphorylated PIAS1 (anti-pPIAS1) or total PIAS1 (anti-PIAS1). (<b>d</b>) Reconstitution of MDA-MB231 PIAS1 shRNA1 cells with the lenti-viruses encoding the empty vector (Vec), wild type PIAS1 (WT), PIAS1 S90A mutant (S90A), or PIAS1 W372A mutant (W372A). Western blot was performed with whole cell extracts from these cells using anti-PIAS1 or anti-Tubulin. (<b>e</b>) The effect of WT or S90A and W372A PIAS1 mutants on cell proliferation and survival. MDA-MB231 cells as in <b>d</b> were seeded in DMEM supplemented with 10% FBS (left), or Stem Cell Media (SCM) (right) (mean ± SEM). Shown is a representative of 3 independent experiments. <i>P</i> values were determined by paired <i>t</i>-test. (<b>f</b>) Mammosphere assay. MDA-MB231 cells as in <b>d</b> were seeded in SCM at 5,000 cells/dish. Spheres were counted 7 days after plating. (<b>g</b>) ALDEFLUOR assay. MDA-MB231 cells as in <b>d</b> were cultured in SCM for 5 days, and the ALDH⁺ population was determined by the ALDEFLUOR assays. Shown in each panel is a representative of 3 independent experiments. Error bars represent SEM. <i>P</i> values were determined by paired <i>t</i>-test.</p

PIAS regulates the histone marks of the target genes.

<p>(<b>a</b>) Chromatin immunoprecipitation (ChIP) assay. Extracts from MDA-MB231 cells containing control shRNA, or PIAS1 shRNA2 were immunoprecipitated with anti-PIAS1 or IgG. The bound DNA was quantified by Q-PCR with gene-specific primers and presented as “percent of input” (% input). (<b>b</b>) Same as in <b>a</b> except that antibodies specific for acetylated histone H3 (AcH3), histone H3 trimethylated at Lys9 (H3K9me3), or histone H3 trimethylated at Lys27 (H3K27me3) were used, and the levels of these histone marks at the <i>WNT5A</i> loci were quantified. (<b>c</b>) Same as in <b>b</b> except that histone marks at the <i>CCND2</i> promoter were quantified. (<b>d</b>) Same as in <b>b</b> except that the level of H3K9me3 at the heterochromatin region <i>Satellite 2</i> was quantified. (<b>e</b>) Same as in <b>b</b> except that histone marks at the <i>CCND1</i> promoter were quantified. Shown in each panel is a representative of 3 independent experiments. Error bars represent SD.</p

A proposed model of the function of PIAS1 in breast cancer.

<p>In response to growth factor and inflammatory signals, PIAS1 is activated via Ser90 phosphorylation (S90p), and recruited to the target gene promoters. PIAS1 represses the expression of epigenetic gatekeeper genes, such as <i>ESR1</i>, <i>WNT5A</i> and <i>CCND2</i>, by promoting inhibitory histone H3 lysine 9 and lysine 27 trimethylation (H3K9/K27) and DNA methylation (Met), while inhibiting acetylated histone H3 (AcH3). Therefore, PIAS1 promotes tumorigenesis by selective epigenetic gene silencing.</p

PIAS1 is important for tumorigenesis of breast cancer.

<p>(<b>a</b>) PIAS1 protein levels are increased in breast tumor samples as revealed by tissue microarray analysis. Left panel: Representative tissue microarray spot from morphologically normal duct, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Right panel: a box and whisker plot of PIAS1 levels in various tissue samples. Total sample numbers (n) were indicated. <i>P</i> values are determined by non-parametric two-tailed Kruskal Wallis test with alpha level equals 0.05. “+”, the mean of each population; “Δ”, outliers. (<b>b</b>) Western blot analyses were performed with whole cell extracts from MDA-MB231 cells containing a control shRNA or two independent PIAS1 shRNAs (PIAS1 shRNA1 and 2). (<b>c</b>) The growth of MDA-MB231 control shRNA, PIAS1 shRNA1 and shRNA2 cells in DMEM supplemented with 10% FBS (left), or Stem Cell Media (SCM) (right) (mean ± SEM). Shown is a representative of 3 independent experiments. <i>P</i> values were determined by paired <i>t</i>-test. (<b>d</b>) <i>In vivo</i> tumorigenesis studies. MDA-MB231 cells containing a control shRNA or PIAS1 shRNA2 were injected into the female SCID-beige mice subcutaneously (left: 1×10⁶ cells/mouse; n = 4), or in fat pad (right: 2×10⁵ cells/mouse; n = 5). Shown is a representative of 3 independent experiments. Each data point represents mean ± SEM. <i>P</i> values were determined by non-paired <i>t</i>-test.</p

PIAS1-mediated <i>WNT5A</i> suppression is important for the maintenance of breast Tumor Initiating Cells (TICs).

<p>(<b>a</b>) Western blot analyses with whole cell extracts from MDA-MB231 cells containing control shRNA, PIAS1 shRNA1 or shRNA2 cultured in DMEM plus 10% FBS. (<b>b</b>) Western blot analyses with whole cell extracts from MDA-MB231 PIAS1 shRNA2 cells containing either a control shRNA, a WNT5A-specific shRNA (WNT5A shRNA1), or a non-working WNT5A shRNA (WNT5A shRNA2). (<b>c</b>) Mammosphere assay. Cells were seeded in Stem Cell Media (SCM) at 5,000 cells/dish, and spheres were counted 7 days later. Shown is a representative of 3 independent experiments. Error bars represent SEM. <i>P</i> values were determined by paired <i>t</i>-test. (<b>d</b>) Same as in <b>c</b> except that the parental MDA-MB231 cells were used with or without recombinant WNT5A treatment as indicated. (<b>e</b>) Tumorigenesis <i>in vivo</i>. Cells as in <b>b</b> were injected subcutaneously into SCID-beige mice (5×10⁶ cells/mice; n = 6). Shown is a representative of 3 independent experiments. Each data point represents mean ± SEM. <i>P</i> values were determined by non-paired <i>t</i>-test.</p

PIAS1 regulates DNA methylation status of the target genes.

<p>(<b>a</b>) DNA Methylation analyses of the indicated loci were performed by bisulfite conversion of genomic DNA from MDA-MB231 cells containing control shRNA or PIAS1 shRNA2. The <b><i>x</i></b> axis represents the positions of the CpG sites relative to the transcription start site (+1); the <b><i>y</i></b> axis represents the percentage. (<b>b</b>) Chromatin immunoprecipitation (ChIP) assay. Extracts from MDA-MB231 cells containing control shRNA, or PIAS1 shRNA2 were immunoprecipitated with anti-DNMT1, anti-DNMT3A or IgG. The bound DNA was quantified by Q-PCR with <i>CCND2</i> promoter-specific primers and presented as “percent of input” (% input). Shown in each panel is a representative of 3 independent experiments. Error bars represent SD.</p