Urokinase-activated Stat1 mediates antiproliferative effect in vascular smooth muscle cells cocultered with monocytes

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Nicotine, IFN-γ and retinoic acid mediated induction of MUC4 in pancreatic cancer requires E2F1 and STAT-1 transcription factors and utilize different signaling cascades

BACKGROUND: The membrane-bound mucins are thought to play an important biological role in cell–cell and cell–matrix interactions, in cell signaling and in modulating biological properties of cancer cell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remaining undetectable in the normal pancreas, thus indicating a potential role in pancreatic cancer pathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are not yet fully understood. Smoking is strongly correlated with pancreatic cancer and in the present study; we elucidate the molecular mechanisms by which nicotine as well as agents like retinoic acid (RA) and interferon-γ (IFN-γ) induce the expression of MUC4 in pancreatic cancer cell lines CD18, CAPAN2, AsPC1 and BxPC3. RESULTS: Chromatin immunoprecipitation assays and real-time PCR showed that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-γ and RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1 and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction of MUC4; nicotine-mediated induction of MUC4 appeared to require α7-nicotinic acetylcholine receptor subunit. Further, Src and ERK family kinases also mediated the induction of MUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4 was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells, suggesting that induction of MUC4 by nicotine and other agents might contribute to the genesis and progression of pancreatic cancer. CONCLUSIONS: Our studies show that agents that can promote the growth and invasion of pancreatic cancer cells induce the MUC4 gene through multiple pathways and this induction requires the transcriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signaling pathways appear to be involved in the induction of this gene. It appears that targeting these signaling pathways might inhibit the expression of MUC4 and prevent the proliferation and invasion of pancreatic cancer cells

Mammalian lysine histone demethylase KDM2A regulates E2F1-mediated gene transcription in breast cancer cells.

It is established that histone modifications like acetylation, methylation, phosphorylation and ubiquitination affect chromatin structure and modulate gene expression. Lysine methylation/demethylation on Histone H3 and H4 is known to affect transcription and is mediated by histone methyl transferases and histone demethylases. KDM2A/JHDM1A/FBXL11 is a JmjC-containing histone demethylase that targets mono- and dimethylated Lys36 residues of Histone H3; its function in breast cancer is not fully understood. Here we show that KDM2A is strongly expressed in myoepithelial cells (MEPC) in breast cancer tissues by immunohistochemistry. Ductal cells from ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) show positive staining for KDM2A, the expression decreases with disease progression to metastasis. Since breast MEPCs have tumor-suppressive and anti-angiogenic properties, we hypothesized that KDM2A could be contributing to some of these functions. Silencing KDM2A with small interfering RNAs demonstrated increased invasion and migration of breast cancer cells by suppressing a subset of matrix metalloproteinases (MMP-2, -9, -14 and -15), as seen by real-time PCR. HUVEC cells showed increased angiogenic tubule formation ability in the absence of KDM2A, with a concomitant increase in the expression of VEGF receptors, FLT-1 and KDR. KDM2A physically bound to both Rb and E2F1 in a cell cycle dependent manner and repressed E2F1 transcriptional activity. Chromatin immunoprecipitation (ChIP) assays revealed that KDM2A associates with E2F1-regulated proliferative promoters CDC25A and TS in early G-phase and dissociates in S-phase. Further, KDM2A could also be detected on MMP9, 14 and 15 promoters, as well as promoters of FLT1 and KDR. KDM2A could suppress E2F1-mediated induction of these promoters in transient transfection experiments. These results suggest a regulatory role for KDM2A in breast cancer cell invasion and migration, through the regulation of E2F1 function

A model depicting KDM2A function in a mammalian cell.

<p>It can be implied that KDM2A regulates Rb-E2F1 function in the progression of the cell cycle. In a quiescent state, KDM2A represses E2F1 functions on various promoters. Addition of VEGF or serum dissociates KDM2A from these promoters facilitating various cellular processes by transcriptional activation leading to enhanced angiogenesis, invasion and migration of cells.</p

Myoepithelial cells of the breast express KDM2A.

<p>(A) Immunohistochemical staining of KDM2A on human breast cancer tissue microarray. Immunostaining was performed using rabbit anti-human KDM2A antibody and representative images of KDM2A expression in normal breast, Ductal Carcinoma in situ, Infiltrating Ductal Carcinoma with and without metastasis, lymph node metastasis are shown. Magnification is 200X, scale bar  = 50 µm. (B) Enlarged images of normal and cancer breast tissue sections. Arrows indicate positively stained myoepithelial cells. Scale bar  = 50 µm. (C) Quantitative analysis of KDM2A in breast tissue microarray. The Immunostaining of KDM2A was quantified by using semi quantitative scoring method based on cellularity and intensity of expression. The means of two independent arrays are shown. All <i>p</i>-values were calculated using a two-sided Student <i>t-</i>test.</p

KDM2A represses transcriptional activity of MMPs and VEGF receptors by inhibiting E2F1-mediated transcription.

<p>(A) ChIP assay demonstrates occupancy of KDM2A on MMP9, -14, -15, KDR and FLT-1 similar to E2F1. (B, C, D, E) Transient transfection experiments in MCF-7 cells showed that E2F1 induces MMP2 (B), MMP-9 (C), MMP-14 (D) and MMP-15 (E) promoters, and this was repressed by co-transfection of KDM2A or Rb large pocket. (F, G) Transient transfection experiments in MDA-MB-231 cells demonstrate that KDM2A represses the transcriptional activity of E2F1 on KDR (F) and FLT-1 (G) promoters.</p

KDM2A interacts with Rb and E2F1 in a cell cycle specific manner.

<p>(A) GST pull down assay showing KDM2A binding to Rb and E2F1 <i>in vitro</i>. ³⁵S-lysate lane has 1/10^th protein loaded. (B) MCF-7 cells were serum starved for 48 hr and serum stimulated for 2 hr, 4 hr, 6 hr, 8 hr and 18 hr. Western blotting showing protein expression of KDM2A, Rb, E2F1 and actin. There is no significant change in the expression of KDM2A and E2F1 at different time points when normalized to actin levels. Rb shows increasing hyperphosphorylation from 6 hr to 18 hr of serum stimulation. (C) KDM2A interacts with Rb and E2F1 <i>in vivo</i> in MCF-7 cells as seen by immunoprecipitation-western blot experiment. Interaction of KDM2A with E2F1 decreases at 8 hr of serum stimulation. (D) Chromatin Immunoprecipitation (ChIP) assays showing KDM2A occupancy on E2F1-regulated proliferative promoters, CDC25A and TS. KDM2A occupies the promoters at all time points from 0 hr to 6 hr, reduction is seen at 8 hr and complete absence of KDM2A from the promoters is observed at 18 hr of serum stimulation. (E) KDM2A significantly represses E2F1-mediated E2.Luc transcription in a dose-dependent manner in Renilla luciferase assay.</p

KDM2A co-localizes with E2F1 at 6 hr of serum stimulation.

<p>Quiescent MCF-7 cells were serum starved for 48 hr and serum stimulated for 6 hr and 18 hr. Cells were fixed, permeabilized for 5 min with 0.2% Triton X-100/PBS and immunostained for E2F1 (anti-mouse IgG, green) and KDM2A (anti-rabbit IgG, red). Cells were visualized by confocal microscopy. E2F1 was predominantly localized in the nucleus (upper panels), while KDM2A was more ubiquitously distributed in the cells (middle panels). Co-localization of E2F1 and KDM2A was observed in the nucleus at 6 hr of serum stimulation and disappeared totally by 18 hr (right panels). Images were captured at 630X oil using DM16000 inverted Leica TCS SP5 tandem scanning confocal microscope. Scale bar  = 200 µm. Pearson's correlation for co-localization at 6 hr was 1.0.</p

Silencing KDM2A enhances angiogenic tubulogenesis.

<p>(A) HUVEC cells were transfected with either control siRNA or KDM2A siRNA (75 pmol). 24 hr later, cells were plated on matrigel in complete media (asynchronous) or with or without 100 ng/ml VEGF. Images were captured 18 hr later using Leica inverted microscope and representative images are shown. (B) Tubule sprouting points were estimated using Image Pro software. Ablation of KDM2A showed significant increase (2-fold, p<0.01) in the number of sprouting points. (C) Real-Time showing increased mRNA expression of FLT-1 (2.2±0.11-fold, p<0.01) and KDR (1.7±0.9-fold, p<0.05) receptors upon silencing KDM2A expression. Simultaneous decrease in KDM2A mRNA levels (p<0.01) is seen with 75 pmol of KDM2A siRNA compared to control siRNA.</p