The switch from a quiescent tumor to an invasive tumor is accompanied by the acquisition of angiogenic properties. This phenotypic change likely requires a change in the balance of angiogenic stimulators and angiogenic inhibitors. The nature of the angiogenic switch is not known. Here, we show that introduction of activated H-ras into immortalized endothelial cells is capable of activating the angiogenic switch. Angiogenic switching is accompanied by up-regulation of vascular endothelial growth factor and matrix metalloproteinase (MMP) bioactivity and downregulation of tissue inhibitor of MMP. Furthermore, we show that inhibition of phosphatidylinositol-3-kinase leads to partial inhibition of tumor angiogenesis, thus demonstrating that activated H-ras activates tumor angiogenesis through two distinct pathways. Finally, we show evidence for two forms of tumor dormancy.

Purpose: Having previously shown that the binding of neutrophil gelatinase-associated lipocalin (NGAL) to matrix metalloproteinase-9 (MMP-9) protects this extracellular matrix remodeling enzyme from autodegradation, we hypothesized that the addition of NGAL to breast cancer cells, which do not express this protein but do express MMP-9, might result in a more aggressive phenotype in vivo. Based on our previous reports that MMPs can be detected in the urine of cancer patients, we also asked whether MMP-9/NGAL could be detected in the urine of breast cancer patients and whether it might be predictive of disease status. Experimental Design: Clones of MCF-7 human breast cancer cells differentially expressing NGAL were generated by stable transfection with human NGAL expression constructs.The established clones were then implanted s.c. in immunodeficient mice and tumor growth was monitored. In addition, we analyzed the urine of individuals with breast cancer and age-matched, sex-matched controls using gelatin zymography for the presence of MMP-9/NGAL. Results: Increased NGAL expression resulted in significant stimulation of tumor growth. Immunohistochemical analysis of MCF-7 tumors revealed that the NGAL-overexpressing ones exhibited increased growth rates that were accompanied by increased levels of MMP-9, increased angiogenesis, and an increase in the tumor cell proliferative fraction. In addition, MMP-9/NGAL complex was detected in 86.36% of the urine samples from breast cancer patients but not in those from healthy age and sex-matched controls. Conclusions: These findings suggest, for the first time, that NGAL may play an important role in breast cancer in vivo by protecting MMP-9 from degradation thereby enhancing its enzymatic activity and facilitating angiogenesis and tumor growth. Clinically, these data suggest that the urinary detection of MMP-9/NGAL may be useful in noninvasively predicting disease status of breast cancer patients.Matrix metalloproteinases (MMP) are a family of zincdependent endopeptidases that collectively degrade most of the molecular components of the basement membrane. Disassembly of the basement membrane by MMPs represents one of the most important hallmarks of cancer progression, from angiogenesis to local growth, invasion, and distant metastasis formation (1). Endogenous inhibitors of MMPs, the tissue inhibitor of metalloproteinases, have been shown to suppress tumor growth and angiogenesis in a variety of in vivo systems, although they differ significantly with respect to the specific angiogenic processes that they inhibit (2, 3). The overproduction of MMPs at tumor sites by tumor cells or by the surrounding stromal cells has been associated with the metastatic phenotype and poor prognosis (4 -7).We have previously reported the detection of intact MMPs in the urine of cancer patients with a variety of cancers and have shown that these urinary MMPs serve as independent predictors of disease status (8,9). MMPs detected in these urine samples included MMP-9 (gelatinase B, EC3.4...