Down-Regulation of Yes Associated Protein 1 Expression Reduces Cell Proliferation and Clonogenicity of Pancreatic Cancer Cells

BACKGROUND: The Hippo pathway regulates organ size by inhibiting cell proliferation and promoting cell apoptosis upon its activation. The Yes Associated Protein 1 (YAP1) is a nuclear effector of the Hippo pathway that promotes cell growth as a transcription co-activator. In human cancer, the YAP1 gene was reported as amplified and over-expressed in several tumor types. METHODS: Immunohistochemical staining of YAP1 protein was used to assess the expression of YAP1 in pancreatic tumor tissues. siRNA oligonucleotides were used to knockdown the expression of YAP1 and their effects on pancreatic cancer cells were investigated using cell proliferation, apoptosis, and anchorage-independent growth assays. The Wilcoxon signed-rank, Pearson correlation coefficient, Kendall's Tau, Spearman's Rho, and an independent two-sample t (two-tailed) test were used to determine the statistical significance of the data. RESULTS: Immunohistochemistry studies in pancreatic tumor tissues revealed YAP1 staining intensities were moderate to strong in the nucleus and cytoplasm of the tumor cells, whereas the adjacent normal epithelial showed negative to weak staining. In cultured cells, YAP1 expression and localization was modulated by cell density. YAP1 total protein expression increased in the nuclear fractions in BxPC-3 and PANC-1, while it declined in HPDE6 as cell density increased. Additionally, treatment of pancreatic cancer cell lines, BxPC-3 and PANC-1, with YAP1-targeting siRNA oligonucleotides significantly reduced their proliferation in vitro. Furthermore, treatment with YAP1 siRNA oligonucleotides diminished the anchorage-independent growth on soft agar of pancreatic cancer cells, suggesting a role of YAP1 in pancreatic cancer tumorigenesis. CONCLUSIONS: YAP1 is overexpressed in pancreatic cancer tissues and potentially plays an important role in the clonogenicity and growth of pancreatic cancer cells

Negative regulation of NF-κB by the ING4 tumor suppressor in breast cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer

Generation and characterization of a monoclonal anti-ING4 antibody.

<p>(<b>A</b>) Schematic diagram of ING4 protein containing a nuclear localization signal (NLS) and a plant homeodomain (PHD). Stick figures represent the ING4 fragments that were used to immunize mice. (<b>B</b>) N-terminal (AA 5–147) and C-terminal (AA 173–249) recombinant fragments of ING4 purified from bacteria visualized by SDS-PAGE gel stained with Coomassie Blue. (<b>C</b>) Detection of ING4 and HA-epitope tagged ING4 overexpressed in 293T cells by Western blot using BTIM-4 and anti-HA monoclonal antibody. * denotes smaller ING4-derived protein species recognized by the antibody. (<b>D</b>) Western blot analysis of ING4 protein expression using BTIM-4 antibody in MCF10A breast epithelial cells and T47D breast cancer cells containing pMIG (the vector control), ING4 (ING4 overexpression), shNT (non-targeting shRNA control), or shING4 (shRNA targeting ING4). Tubulin antibody was used as a loading control. (<b>E</b>) MCF10A cells transduced with pMIG (the vector control), ING4 (ING4 overexpression), shNT (non-targeting shRNA control), or shING4 (shRNA targeting ING4), and T47D cells transduced with pMIG (the vector control) or ING4 (ING4 overexpression) were immunostained with BTIM-4 anti-ING4 antibody (red) and visualized using fluorescent microscopy. 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain individual cell nuclei (blue). White scale bars represent 100 µm.</p

Low <i>ING4</i> mRNA expression and ING4-regulated NF-κB-target gene signature are associated with reduced disease-free survival in the GDS806 dataset.

<p>(<b>A</b>) Dot plot analysis of ING4 transcript levels in primary breast tumors from patients who remained disease-free (DF, n = 32) and from those who recurred (REC, n = 28) from the GDS806 dataset (n = 60). (<b>B</b>) Kaplan-Meier analysis of disease-free survival based on ING4 transcript level from the GDS806 dataset. CI, confidence interval. Kaplan-Meier analysis of disease-free survival based on NF-κB-target gene signatures (<b>C</b>) 25-gene or (<b>D</b>) 14-gene signature.</p

Inverse correlation between ING4 and p-p65/RelA (Ser276) antibody staining in breast tumor samples.

<p>(<b>A,B</b>) Hyperplastic breast tissue with moderate atypia from a ductal carcinoma in situ case, showing ING4 score +3 and p-p65 score 0. Inset is a higher magnification image of the region showing ING4 staining in luminal and basal epithelial cells and in stroma cells. (<b>C,D</b>) Invasive ductal carcinoma with ING4 score 0 and p-p65 score +2. (<b>E,F</b>) Grade 3 invasive ductal carcinoma with ING4 score +1 and p-p65 score +3. (bars, 100 µm). (<b>G</b>) Low ING4 expression is more prevalent in the tumors that express high levels of p-p65. Number of ING4-low (solid bars, IHC score <1.5) and ING4-high (slanted-line bars, IHC score ≥1.5) tumors, presented as a percentage of total number of tumors in each subgroup. Total (n = 227, 77 ING4-low, 150 ING4-high), ER+ (n = 156, 55 ING4-low, 101 ING4-high), ER− (n = 63, 18 ING4-low, 45 ING4-high), HER2- (n = 197, 67 ING4-low, 130 ING4-high), HER2+ (n = 27, 9 ING4-low, 18 ING4-high), p-p65-low (n = 166, 48 ING4-low, 118 ING4-high), and p-p65-high (n = 62, 29 ING4-low, 33 ING4-high). <i>P</i> values were determined by Fisher’s Exact test.</p

Tumors with low ING4 and high p-p65 levels are frequently lymph node-positive.

<p>Tumors with low ING4 and high p-p65 levels are frequently lymph node-positive.</p

ING4 represses NF-κB-target gene expression.

<p>(<b>A</b>) ING4 represses expression of a luciferase reporter construct containing an NRE (NF-κB response element) promoter sequence in T47D cells. (<b>B</b>) ING4 represses expression of endogenous NF-κB-target genes, <i>IL6, IL8,</i> and <i>PTGS2</i>, evaluated by RT-qPCR. (<b>C</b>) Heat map of 35 NF-κB-target genes induced by PMA in T47D pMIG (vector) cells (red, 2^nd row). Twenty-seven of 35 genes were repressed by ING4 (green, 4^th row). Starred genes indicate those previously measured in single RT-qPCR assays. (<b>D</b>) ING4 transcript level inversely correlates with NF-κB-target gene expression in breast tumors in two independent gene expression profile data sets, GDS806 (n = 60) and GSE3521 (n = 45). GDS806 contained gene expression data for 25 of the 27 NF-κB-target genes and GSE3521 contained gene expression data for all 27 NF-κB-target genes.</p

Reduced p65/RelA phosphorylation in breast cancer cells overexpressing ING4.

<p>(<b>A</b>) Western blot analysis of T47D cells expressing pMIG (the vector control) or ING4 (ING4 overexpression) for ING4, p65, p-p65 (Ser536), and IκBα. Cells were treated with PMA for 0, 15, 30 or 60 minutes prior to cell fractionation. (<b>B</b>) Western blot analysis of MCF7 cells expressing pMIG (the vector control) or ING4 (ING4 overexpression) for ING4, p65, and p-p65 (Ser536). Cells were treated with PMA for 60 minutes prior to cell fractionation. Histone H3 and Tubulin antibodies were used as loading controls for the nuclear and cytosolic fractions, respectively. (<b>C</b>) ING4 inhibits PMA-induced T47D cell invasion. Cells that invaded through a basement membrane matrix were stained with Hoechst dye and visualized under a fluorescent microscope (picture). Cell numbers were determined by averaging a minimum of 4 images per experiment from at least 6 independent experiments. <i>P</i> value was determined by <i>t</i>-test (p = 0.0011).</p