Elsevier

Experimental Cell Research

Volume 314, Issue 13, 1 August 2008, Pages 2448-2453
Experimental Cell Research

Research Article
Vascular endothelial growth factor-A stimulates Snail expression in breast tumor cells: Implications for tumor progression

https://doi.org/10.1016/j.yexcr.2008.05.004Get rights and content

Abstract

The E-cadherin transcriptional repressor Snail is a prognostic marker for metastatic breast carcinoma, as well as a critical determinant of tumor growth and recurrence. We define a non-angiogenic, autocrine function for the vascular endothelial growth factor-A (VEGF-A) in regulating Snail expression in breast tumor cells. The transfection of well-differentiated breast tumor cells with VEGF-A increases Snail mRNA and protein levels, resulting in reduced E-cadherin expression. Conversely, reducing endogenous VEGF-A expression in poorly differentiated breast tumor cells by siRNA transfection decreases Snail levels. Our studies demonstrate that VEGF and the VEGF receptor Neuropilin-1 increase Snail expression by suppressing the Glycogen Synthase Kinase-3 (GSK-3), an established inhibitor of Snail transcription and protein stability. The VEGF-A neutralizing antibody Avastin® was recently approved by the FDA for the treatment of metastatic breast cancer. We present the provocative finding that beyond its anti-angiogenic activity, Avastin® can reduce Snail expression in breast tumor cells. Collectively, this work describes a novel autocrine function for VEGF in breast tumor cells in driving the expression of Snail, a breast tumor progression factor. Based on our demonstration that Avastin® reduces Snail expression in breast tumor cells, we propose that the treatment of early stage breast cancer patients with Avastin® may impede tumor progression.

Introduction

The transition of a “normal” epithelial cell to a metastatic tumor cell requires that this cell evolve the ability to survive in foreign environments, to migrate and to invade tissue. The E-cadherin transcriptional repressor Snail is unique in its ability to impart on a cell a number of these activities associated with metastatic tumor progression. Strikingly, Snail is a prognostic marker for metastatic breast carcinoma, being expressed in node-positive, but not in low grade, node-negative specimens [1]. Early studies of Snail focused on its ability to induce an epithelial–mesenchymal transition (EMT) by acting as an E-cadherin transcriptional repressor [2], [3]. However, recent studies indicate that Snail also maintains cell survival and promotes cell migration [4]. Furthermore, in mouse models of cancer, Snail is critical for tumor growth and recurrence [5], [6]. Based on its established importance for breast tumor progression, studies identifying the factors that regulate Snail expression in metastatic breast carcinoma are of utmost importance.

We and others established previously that breast tumor cells express the VEGF receptor Neuropilin-1 (NP-1) [7], [8], [9], and that an autocrine VEGF/NP-1 pathway stimulates constitutive PI3-kinase activity in breast tumor cells [7]. Similar to the expression pattern of Snail in breast tumors [1], VEGF-A expression is significantly increased in metastatic compared to non-metastatic breast tumors [10]. Furthermore, recombinant VEGF can stimulate the expression of Snail family members in pancreatic tumor cells [11]. Based on these findings, in the current work, we address the hypothesis that autocrine VEGF-A signaling in breast tumor cells stimulates Snail expression and activity. We also investigate a novel activity for a VEGF-A-directed therapeutic in suppressing tumor cell expression of Snail.

Section snippets

Cell lines

T47D and MDA-MB-435 cells were obtained from Duke University Comprehensive Cancer Center's cell culture facility, and were cultured in RPMI/5% FBS and DMEM/5% FBS, respectively. SUM159 cells were kindly provided by Dr. Stephen Ethier, and cultured in DMEM/F12(1:1)/5% FBS. MCF-7 cells were obtained from Dr. Joseph Geradts (Duke University Medical Center), and were cultured in DMEM/5% FBS.

Antibodies

The following antibodies were used in these studies: Neuropilin-1-neutralizing antibody (R&D Systems-MAB566),

Results/discussion

We investigated the impact of introducing VEGF-A to two well-differentiated breast tumor cell lines in vitro. MCF-7 and T47D cells were transfected transiently with a VEGF-A-expressing or control vector. After 24h, a 5-fold increase in VEGF-A expression levels was observed in VEGF-A transfectants compared to control transfectants (Figs. 1A and B). Control transfectants grew as islands of clustered cells maintaining strong cell–cell adhesion, reflecting the fact that these cells express

Acknowledgments

We thank Dr. Valentina Folgiero for her assistance with this study. In addition, we thank Dr. Arthur Mercurio for his valuable advice on this work. This work was supported by NIH grant CA093855 (R.E.B.) and a pilot award (R.E.B.) from the Duke Comprehensive Cancer Center's Breast and Ovarian Oncology Research Program.

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