Schematic representation of our findings.

<p>A) Inhibition of NF-κB/p65 translocation reduces the expression of EMT-transcription factors that have been shown to regulate target genes of NF-κB. This inhibition increased E-cadherin expression, decreased N-cadherin and MMP11 expression and reduced cell motility and invasiveness potential. B) Then, NF-κB/p65 transcriptionally regulates the promoter regions of TWIST1, SLUG and SIP1, which in turn represses the epithelial marker E-cadherin and activates the mesenchymal markers N-cadherin and MMP11, resulting in induction of the EMT process.</p

NF-kappaB Is Involved in the Regulation of EMT Genes in Breast Cancer Cells - Fig 6

<p><b>Representative scheme of putative NF-κB binding sites located in the SNAIL1 (A), SLUG (B), TWIST1 (C) and SIP1 (D) promoter regions predicted by Tfsitescan, TESS, TFBind, TFSearch and Transfac bioinformatics tools.</b> An alignment of the DNA region showed evolutionarily conservation among metazoan species. Identical nucleotides are in bold. Gray lines indicate regions investigated by chromatin immunoprecipitation. +1: transcription start site.</p

Relative expression of EMT-phenotype markers after NF-κB/p65 signaling inhibition.

<p>The mRNA levels of <i>E-CADHERIN</i>, <i>N-CADHERIN</i> and <i>MMP11</i> were assessed in MDA-MB-231 (A) and HCC-1954 (B) cells at 8, 16 and 24 h of DHMEQ treatment. The data were expressed as the mean ± SD. * = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001.</p

Migration assay.

<p>A representative wound healing assay evaluating cell migration at 24 h after DHMEQ treatment of MDA-MB-231 (A) HCC-1954 (B) and MCF-7 (C) cells is shown. The box plots represent migratory ability as indicated by the percent of wound closure. Magnification x100. The data were expressed as the mean ± SD. * = <i>p</i><0.05, ns = not statistically significant.</p

NF-kappaB Is Involved in the Regulation of EMT Genes in Breast Cancer Cells

<div><p>The metastatic process in breast cancer is related to the expression of the epithelial-to-mesenchymal transition transcription factors (EMT-TFs) SNAIL, SLUG, SIP1 and TWIST1. EMT-TFs and nuclear factor-κB (NF-κB) activation have been associated with aggressiveness and metastatic potential in carcinomas. Here, we sought to examine the role of NF-κB in the aggressive properties and regulation of EMT-TFs in human breast cancer cells. Blocking NF-κB/p65 activity by reducing its transcript and protein levels (through siRNA-strategy and dehydroxymethylepoxyquinomicin [DHMEQ] treatment) in the aggressive MDA-MB-231 and HCC-1954 cell lines resulted in decreased invasiveness and migration, a downregulation of SLUG, SIP1, TWIST1, MMP11 and N-cadherin transcripts and an upregulation of E-cadherin transcripts. No significant changes were observed in the less aggressive cell line MCF-7. Bioinformatics tools identified several NF-κB binding sites along the promoters of SNAIL, SLUG, SIP1 and TWIST1 genes. Through chromatin immunoprecipitation and luciferase reporter assays, the NF-κB/p65 binding on TWIST1, SLUG and SIP1 promoter regions was confirmed. Thus, we suggest that NF-κB directly regulates the transcription of EMT-TF genes in breast cancer. Our findings may contribute to a greater understanding of the metastatic process of this neoplasia and highlight NF-κB as a potential target for breast cancer treatment.</p></div

Relative expression of EMT-related genes after NF-κB/p65 genetic silencing using a siRNA approach.

<p>The mRNA levels of the EMT-inducing factors <i>SNAIL1</i>, <i>SLUG</i>, <i>TWIST1</i>, and <i>SIP1</i> and EMT-phenotype markers <i>E-CADHERIN</i>, <i>N-CADHERIN</i> and <i>MMP11</i> together with NF-κB/p65 inhibition at the protein level were assessed in MDA-MB-231 (A) and HCC-1954 (B) cells (scramble and siNF-κB/p65). Moreover, Slug expression was evaluated at the protein level for MDA-MB-231 by western blot assay in scramble and siNF-κB cells. Ponceau staining was used as a loading control. The data were expressed as the mean ± SD. * = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001.</p

Relative expression of the EMT-inducing factors after NF-κB/p65 signaling inhibition.

<p>The mRNA levels of <i>SNAIL1</i>, <i>SLUG</i>, <i>TWIST1</i>, and <i>SIP1</i> were assessed in MDA-MB-231 (A) and HCC-1954 (B) cells at 8, 16 and 24 h of DHMEQ treatment. NF-κB/p65 inhibition was evaluated at protein levels by western blot assay at 16 and 24 h of DHMEQ treatment. Ponceau staining was used as a loading control. Ctrl: control. The data were expressed as the mean ± SD. * = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001.</p

Increased expression of protease-activated receptor 1 (PAR-1) in human leukemias

Protease-activated receptor 1 (PAR-1) is a G-protein-coupled receptor that is overexpressed in solid tumors, being associated with several pro-tumoral responses including primary growth, invasion, metastasis and angiogenesis. Expression of PAR-1 in human leukemic cell lines is reported but the status of its expression in human leukemic patients is currently unknown. In this study we evaluated the expression pattern of PAR-1 in patients with the four main types of leukemia - chronic lymphocytic leukemia subtype B (B-CLL), acute lymphoblastic leukemia subtype B (B-ALL), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Flow cytometry analyses show that lymphocytes from B-CLL patients express this receptor at similar levels to healthy individuals. On the other hand, it was observed a significant increase in PAR-1 expression in B-ALL lymphocytes as compared to B-CLL and healthy donors. Flow cytometric and real-time PCR demonstrated a significant increase in PAR-1 expression in granulocytes from CML patients in blast phase (CML-BP) but not in chronic phase (CML-CP) as compared to healthy donors. Finally, a significant increase in PAR-1 expression has been also observed in blasts from AML (subtypes M4 and M5) patients, as compared to monocytes or granulocytes from healthy donors. We conclude that PAR-1 might play an important biological role in aggressive leukemias and might offer additional strategies for the development of new therapies. (C) 2010 Elsevier Inc. All rights reserved.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Fundacao de Amparo Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho (FAPERJ)Fundacao do Cance