Reactive oxygen generated by Nox1 triggers the angiogenic switch

Jack Arbiser

John Petros

Robert Klafter

Baskaran Govindajaran

Susan Kilroy+2 authors

Departments of ^Dermatology,^Urology,^{Hematology/Oncology, and}^{Pathology Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322;}^{Department of Pathology, Beth Israel Deaconess Hospital, Boston, MA 02215; and}^{Department of Surgical Research, Children's Hospital and Harvard Medical School, Boston, MA 02115}

^{To whom reprint requests should be addressed at: Department of Dermatology, Emory University School of Medicine, 1639 Pierce Drive, 5309 Woodruff Memorial Building, Atlanta, GA 30322. E-mail:}ude.yrome@esibraj or ude.yrome@ebmald.

Communicated by Douglas C. Wallace, Emory University School of Medicine, Atlanta, GA

Received 2001 May 7; Accepted 2001 Nov 27.

Abstract

The reactive oxygen-generating enzyme Nox1 transforms NIH 3T3 cells, rendering them highly tumorigenic and, as shown herein, also increases tumorigenicity of DU-145 prostate epithelial cells. Although Nox1 modestly stimulates cell division in both fibroblasts and epithelial cells, an increased mitogenic rate alone did not account fully for the marked tumorigenicity. Herein, we show that Nox1 is a potent trigger of the angiogenic switch, increasing the vascularity of tumors and inducing molecular markers of angiogenesis. Vascular endothelial growth factor (VEGF) mRNA becomes markedly up-regulated by Nox1 both in cultured cells and in tumors, and VEGF receptors (VEGFR1 and VEGFR2) are highly induced in vascular cells in Nox1-expressing tumors. Matrix metalloproteinase activity, another marker of the angiogenic switch, also is induced by Nox1. Nox1 induction of VEGF is eliminated by coexpression of catalase, indicating that hydrogen peroxide signals part of the switch to the angiogenic phenotype.

Abstract

Reactive oxygen species (ROS; superoxide, hydrogen peroxide, and their metabolites) are conventionally thought of as cytotoxic and mutagenic, and in high levels they induce an oxidative stress response (1, 2). However, recent evidence implicates lower levels of ROS as an intracellular mediator of growth, apoptosis, and senescence (2–6). For example, growth factors including platelet-derived growth factor and epidermal growth factor stimulate H₂O₂ generation through a pathway involving PI 3-kinase and Rac, and elimination of H₂O₂ with antioxidants prevents growth stimulation by these growth factors (4, 7–10).

Reactive oxygen may play a role in neoplastic growth, because a variety of cell lines derived from human cancers demonstrate significantly elevated H₂O₂ (6). NIH 3T3 cells transformed with constitutively active Ras show elevated ROS, and antioxidants such as N-acetyl cysteine reduce the abnormally rapid DNA synthesis in these cells (11, 12). Antioxidants enhance antitumor activity of conventional chemotherapeutic agents in rodents through unknown mechanisms (11). Tumor cells may be inherently more resistant to oxidative stress than normal cells, or oxidative stress may provide a selective advantage in tumor growth.

Nox1 (Mox1 in an earlier terminology), a recently identified (1) homolog of gp91phox, the catalytic subunit of the phagocyte superoxide-generating NADPH-oxidase, constitutively produces both superoxide and H₂O₂ when overexpressed in fibroblasts. Moreover, expression of Nox1 in these cells induces malignant transformation, rendering them highly tumorigenic in athymic mice (1). Decreased expression of endogenous Nox1 decreases proliferation of vascular smooth muscle, implicating Nox1 in normal cell growth. NIH 3T3 cells that stably express Nox1 exhibit modestly increased growth rates, but increased growth alone may be insufficient to account for the marked tumorigenicity of these cells. Coexpression of catalase along with Nox1 reverses the growth phenotype, rendering these cells poorly tumorigenic and indicating that one of the signaling species generated by Nox1 is H₂O₂ (13).

Microscopic dormant tumors are thought to occur relatively frequently, but few progress to form active tumors. Angiogenesis, the process by which tissues recruit and develop new blood vessels, is needed for tumors to grow beyond 1–2 mm in diameter (14). Progression to a growing tumor is characterized by induction in the tumor tissue of angiogenic factors, particularly vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs), and VEGF receptors (VEGFR) in the growing endothelial cells. The conversion to the angiogenic phenotype in previously dormant tumors is known as the “angiogenic switch”. Because Nox1 causes aggressive growth of tumors in vivo that cannot be readily explained based on mitogenic rates alone, we tested the hypothesis that ROS produced by Nox1 triggers the angiogenic switch, permitting vascularization and rapid expansion of the tumor.

Acknowledgments

This work was supported by the American Skin Association and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR47901 (to J.L.A.), Emory Skin Disease Research Core Center Grant P30, National Institutes of Health Grants AR42687 and AR02030 (to J.L.A.), National Institutes of Health Grant CA84138 (to J.D.L.), Department of Defense Grant BC995541 (to J.P.), and Research Scientist Development Award (to J.P.) and Department of Defense Grant DAMD 17-00-1-0080 (to J.P., R.S.A., and J.D.L.). M.M. is supported by Grant 83821 from the American Cancer Society.

Acknowledgments

Abbreviations

ROS	reactive oxygen species
VEGF	vascular endothelial growth factor
VEGFR	VEGF receptor
MMP	matrix metalloproteinase
ISH	in situ hybridization

Abbreviations

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