[CANCER RESEARCH 61, 1727–1732, February 15, 2001] Up-Regulation of Vascular Endothelial Growth Factor in Breast Cancer Cells by the Heregulin-1-activated p38 Signaling Pathway Enhances Endothelial Cell Migration 1 Shunbin Xiong, Rebecca Grijalva, Lianglin Zhang, Nina T. Nguyen, Peter W. Pisters, Raphael E. Pollock, and Dihua Yu 2 Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 ABSTRACT Heregulin (HRG) belongs to a family of polypeptide growth factors that bind to receptor tyrosine kinases ErbB3 and ErbB4. HRG binding induces ErbB3 and ErbB4 heterodimerization with ErbB2, activating downstream signal transduction. Vascular endothelial growth factor (VEGF) is a pri- mary regulator of physiological angiogenesis and is a major mediator of pathological angiogenesis, such as tumor-associated neovascularization. In this study, we demonstrate that HRG-1 increased secretion of VEGF from breast cancer cells in a time- and dosage-dependent manner and that this increase resulted from up-regulation of VEGF mRNA expression via transcriptional activation of the VEGF promoter. Deletion and mutational analysis revealed that a CA-rich upstream HRG response element located between nucleotide-2249 and -2242 in the VEGF promoter mediated HRG-induced transcriptional up-regulation of VEGF. While investigating the downstream signaling pathways involved in HRG-mediated up-regu- lation of VEGF, we found that HRG activated extracellular signal-regu- lated protein kinases, Akt kinase, and p38 mitogen-activated protein kinase (MAPK). However, only the specific inhibitor of p38 MAPK (SB203580), not extracellular signal-regulated kinase inhibitor PD98059 nor the inhibitor of phosphatidylinositol 3-kinase-Akt pathway (Wort- mannin), blocked the up-regulation of VEGF by HRG. The HRG-stimu- lated secretion of VEGF from breast cancer cells resulted in increased migration of murine lung endothelial cells, an activity that was inhibited by either VEGF-neutralizing antibody or SB203580. These results show that HRG can activate p38 MAPK to enhance VEGF transcription via an upstream HRG response element, leading to increased VEGF secretion and angiogenic response in breast cancer cells. INTRODUCTION VEGF 3 , also known as vascular permeability factor, is an important stimulator of angiogenesis. Normal levels and appropriately timed expression of VEGF are essential for normal development of the vascular system (1–3). VEGF-induced angiogenesis is also essential for the growth of solid tumors (4 – 6). VEGF is highly expressed in solid tumors and is required for the maintenance of tumor blood vessels. Withdrawal of VEGF causes tumor regression (7–9). Re- cently, VEGF was recognized as a survival factor for endothelial cells (10). VEGF expression is regulated by many growth factors and cytokines, such as insulin-like growth factor (11), interleukin-6 (12), transforming growth factor- (13), basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor (14). VEGF expression is increased by hypoxia (4, 15) and inhibited by p53 (16). It is conceivable that the expression and function of VEGF are regulated by many cellular factors during tumor progression. Reveal- ing these factors and their mechanisms of action will enable us to better alter the detrimental consequence of VEGF. HRG, also named neu differentiation factor (17), neuregulin (18), AchR-inducing activity (19), and glial growth factor (20), is a member of the epidermal growth factor-like growth factor family. HRGs are ligands of ErbB3 and/or ErbB4, which belong to the ErbB family of receptor tyrosine kinases. The binding of HRG to its receptors induces either ErbB3 or ErbB4 to form homodimers or to form heterodimers with ErbB2, thus triggering diverse signaling cascades (21). HRGs can induce a variety of cellular responses in different cell types, including proliferation, differentiation, survival, apoptosis, migration, and aggregation (22–26). Data from HRG gene knockout mice dem- onstrated that HRG is essential for the early development of the heart and central nervous system (27). HRG is also known to be involved in breast cancer metastasis and in ErbB2-related and hormone-inde- pendent breast cancer progression (28, 29). HRG has been shown to regulate invasive and metastasis-related properties in breast cancer cell lines (30). However, the role of HRG in breast cancer metastasis remains elusive. HRG has also been implicated in the regulation of gene expression. HRG was reported to stimulate AchR and utrophin gene expression in muscle cells via GA-binding proteins by 2–3 fold compared with that in the absence of HRG (31–35). HRG also up-regulated AchR gene expression about 3-fold in p-19 teratocarcinoma cells and breast cancer cells through Sp1-containing complex (36). The mechanisms of HRG-induced transcriptional regulation in these studies remain unclear. After studying the role of HRG and ErbB2 in breast cancer progression and metastasis, we found that HRG up-regulated VEGF secretion from breast cancer cell lines through transcriptional up- regulation and that it required activation of the p38 MAPK signaling pathway. Furthermore, HRG-1-induced VEGF secretion enhanced mouse endothelial cell migration. Thus, transcriptional up-regulation of VEGF via activation of the p38 MAPK pathway may be one of the mechanisms that contribute to HRG-induced breast cancer metastasis. MATERIALS AND METHODS Cell Culture and Transfection. Breast cancer cell lines were cultured in DMEM/F12 containing 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY). Transient transfection was performed following the man- ufacturer’s instruction for LipofectAmine (Life Technologies, Inc.). Briefly, cells were seeded in 6-well plates overnight. Luciferase reporter gene (3 g/well) and pCMV--gal plasmid (0.2 g/well), used as an internal control, were mixed in 100 l of OPTI-MEM medium, to which 10 l of diluted LipofectAmine was added. The mixture was incubated for 30 min at room temperature. Then, 800 l of OPTI-MEM was added to the mixture, which was transferred onto the cells for 3– 4 h. Cells were incubated in serum-free Received 7/28/00; accepted 12/13/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grants P30-CA16672 (Cancer Center Core Grant) and 2RO1-CA60448 (to D. Y.) from the NIH; DAMD17-98-2-8338 and DAMD17-99-1-9271 (both to D. Y.) from the United States Army Research and Material Command; and The University of Texas M. D. Anderson Breast Cancer Basic Research Program Fund (to D. Y.). 2 To whom requests for reprints should be addressed, at Department of Surgical Oncology, Box 107, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 792-3636; Fax: (713) 794-4830. 3 The abbreviations used are: VEGF, vascular endothelial growth factor; HRG, heregu- lin; nt, nucleotide; AchR, acetylcholine receptor; MAPK, mitogen-activated protein ki- nase; DMEM/F12, 1:1 mixture of Dulbecco’s modified essential medium and Ham’s F12 nutrient mixture; CMV, cytomegalovirus; HRE, heregulin response element; uHRE, upstream heregulin response element; WT, wild-type; MUT, mutant; EGFld, epidermal growth factor-like domain; CM, conditioned media; ERK, extracellular signal-regulated kinase; ActD, actinomycin D; CMX, cycloheximide; MluE, murine lung endothelial; PI-3K, phosphatidylinositol 3-kinase. 1727