Marsha A. Moses

Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma

The phenomenon of inhibition of tumor growth by tumor mass has been repeatedly studied, but without elucidation of a satisfactory mechanism. In our animal model, a primary tumor inhibits its remote metastases. After tumor removal, metastases neovascularize and grow. When the primary tumor is present, metastatic growth is suppressed by a circulating angiogenesis inhibitor. Serum and urine from tumor-bearing mice, but not from controls, specifically inhibit endothelial cell proliferation. The activity copurifies with a 38 kDa plasminogen fragment that we have sequenced and named angiostatin. A corresponding fragment of human plasminogen has similar activity. Systemic administration of angiostatin, but not intact plasminogen, potently blocks neovascularization and growth of metastases. We here show that the inhibition of metastases by a primary mouse tumor is mediated, at least in part, by angiostatin.

Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer.

The matrix metalloproteinase (MMP) family of enzymes is comprised of critically important extracellular matrix remodeling proteases whose activity has been implicated in a number of key normal and pathologic processes. The latter include tumor growth, progression, and metastasis as well as the dysregulated angiogenesis that is associated with these events. As a result, these proteases have come to represent important therapeutic and diagnostic targets for the treatment and detection of human cancers. In this review, we summarize the literature that establishes these enzymes as important clinical targets, discuss the complexity surrounding their choice as such, and chronicle the development strategies and outcomes of their clinical testing to date. The status of the MMP inhibitors currently in US Food and Drug Administration approved clinical trials is presented and reviewed. We also discuss the more recent and successful targeting of this enzyme family as diagnostic and prognostic predictors of human cancer, its status, and its stage. This analysis includes a wide variety of human cancers and a number of human sample types including tissue, plasma, serum, and urine.

Reactive oxygen generated by Nox1 triggers the angiogenic switch

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.

Phase II Study of Cediranib, an Oral Pan–Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Patients With Recurrent Glioblastoma

PURPOSE Glioblastoma is an incurable solid tumor characterized by increased expression of vascular endothelial growth factor (VEGF). We performed a phase II study of cediranib in patients with recurrent glioblastoma. METHODS Cediranib, an oral pan-VEGF receptor tyrosine kinase inhibitor, was administered (45 mg/d) until progression or unacceptable toxicity to patients with recurrent glioblastoma. The primary end point was the proportion of patients alive and progression free at 6 months (APF6). We performed magnetic resonance imaging (MRI) and plasma and urinary biomarker evaluations at multiple time points. RESULTS Thirty-one patients with recurrent glioblastoma were accrued. APF6 after cediranib was 25.8%. Radiographic partial responses were observed by MRI in 17 (56.7%) of 30 evaluable patients using three-dimensional measurements and in eight (27%) of 30 evaluable patients using two-dimensional measurements. For the 15 patients who entered the study taking corticosteroids, the dose was reduced (n = 10) or discontinued (n = 5). Toxicities were manageable. Grade 3/4 toxicities included hypertension (four of 31; 12.9%); diarrhea (two of 31; 6.4%); and fatigue (six of 31; 19.4%). Fifteen (48.4%) of 31 patients required at least one dose reduction and 15 patients required temporary drug interruptions due to toxicity. Drug interruptions were not associated with outcome. Changes in plasma placental growth factor, basic fibroblast growth factor, matrix metalloproteinase (MMP) -2, soluble VEGF receptor 1, stromal cell-derived factor-1alpha, and soluble Tek/Tie2 receptor and in urinary MMP-9/neutrophil gelatinase-associated lipocalin activity after cediranib were associated with radiographic response or survival. CONCLUSION Cediranib monotherapy for recurrent glioblastoma is associated with encouraging proportions of radiographic response, 6-month progression-free survival, and a steroid-sparing effect with manageable toxicity. We identified early changes in circulating molecules as potential biomarkers of response to cediranib. The efficacy of cediranib and the predictive value of these candidate biomarkers will be explored in prospective trials.

PPARγ ligands inhibit primary tumor growth and metastasis by inhibiting angiogenesis

Several drugs approved for a variety of indications have been shown to exhibit antiangiogenic effects. Our study focuses on the PPARgamma ligand rosiglitazone, a compound widely used in the treatment of type 2 diabetes. We demonstrate, for the first time to our knowledge, that PPARgamma is highly expressed in tumor endothelium and is activated by rosiglitazone in cultured endothelial cells. Furthermore, we show that rosiglitazone suppresses primary tumor growth and metastasis by both direct and indirect antiangiogenic effects. Rosiglitazone inhibits bovine capillary endothelial cell but not tumor cell proliferation at low doses in vitro and decreases VEGF production by tumor cells. In our in vivo studies, rosiglitazone suppresses angiogenesis in the chick chorioallantoic membrane, in the avascular cornea, and in a variety of primary tumors. These results suggest that PPARgamma ligands may be useful in treating angiogenic diseases such as cancer by inhibiting angiogenesis.

Matrix Metalloproteinase-3 Releases Active Heparin-binding EGF-like Growth Factor by Cleavage at a Specific Juxtamembrane Site

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored precursor that is cleaved to release the soluble mature growth factor. The two forms are active as juxtacrine and paracrine/autocrine growth factors, respectively. The enzymes that process the HB-EGF transmembrane form are unknown. Accordingly, an in vitro assay was established using a fusion protein in which alkaline phosphatase (AP) replaced the transmembrane and cytoplasmic domains of HB-EGF (HB-EGF JM-AP). The fusion protein was anchored to agarose beads coated with anti-AP antibodies. Several matrix metalloproteinases (MMPs) were tested for the ability to release soluble HB-EGF in the in vitrosystem. MMP-3 released soluble 12-kDa immunoreactive and mitogenic HB-EGF within 30 min. On the other hand neither MMP-2 nor MMP-9 had any cleavage activities. A non-cleavable mutant was prepared by replacing the juxtamembrane (JM) region of HB-EGF with the JM region of CD4. The mutant HB-EGF, which in its full-length form was as active a juxtacrine growth factor as was the wild type HB-EGF in vivo, was not cleaved by MMP-3 in the in vitro assay. The C-terminal portion of the cleaved HB-EGF JM-AP that remained attached to the anti-AP beads was N-terminally sequenced and the MMP-3 cleavage site was determined to be Glu151-Asn152, a site within the JM domain. MMP-3 treatment also released soluble HB-EGFin vivo from MC2 cells expressing transmembrane HB-EGF precursor, at a level of about 2-fold above control. It was concluded that MMP-3 cleaves HB-EGF at a specific site in the JM domain and that this enzyme might regulate the conversion of HB-EGF from being a juxtacrine to a paracrine/autocrine growth factor.

The Regulation of Neovascularization by Matrix Metalloproteinases and Their Inhibitors

The process of new capillary formation from pre‐existing vessels, angiogenesis, is a complex physiological event which is strictly controlled, occurring only very rarely under normal conditions. In contrast, there are a number of serious diseases, among them solid tumor growth, rheumatoid arthritis and several eye diseases, which are characterized by unrestricted new capillary growth and which are described as “angiogenic diseases.” One of the key events required for successful angiogenesis is extracellular proteolysis. Increased attention has been focused on the matrix metalloproteinase (MMP) family of enzymes whose activity is a rate‐limiting step in extracellular matrix remodeling. This review will present the accumulating body of evidence, from a number of laboratories, which documents the important role of MMP activity in the regulation of angiogenesis. Taken together, these data suggest that one strategy for controlling the deregulated angiogenesis characteristic of these serious angiogenic diseases may be one which is operative at the level of the control of MMP activity.

Adult mouse epicardium modulates myocardial injury by secreting paracrine factors

The epicardium makes essential cellular and paracrine contributions to the growth of the fetal myocardium and the formation of the coronary vasculature. However, whether the epicardium has similar roles postnatally in the normal and injured heart remains enigmatic. Here, we have investigated this question using genetic fate-mapping approaches in mice. In uninjured postnatal heart, epicardial cells were quiescent. Myocardial infarction increased epicardial cell proliferation and stimulated formation of epicardium-derived cells (EPDCs), which remained in a thickened layer on the surface of the heart. EPDCs did not adopt cardiomyocyte or coronary EC fates, but rather differentiated into mesenchymal cells expressing fibroblast and smooth muscle cell markers. In vitro and in vivo assays demonstrated that EPDCs secreted paracrine factors that strongly promoted angiogenesis. In a myocardial infarction model, EPDC-conditioned medium reduced infarct size and improved heart function. Our findings indicate that epicardium modulates the cardiac injury response by conditioning the subepicardial environment, potentially offering a new therapeutic strategy for cardiac protection.

Regulation of Angiostatin Production by Matrix Metalloproteinase-2 in a Model of Concomitant Resistance

We have previously reported the identification of the endogenous angiogenesis inhibitor angiostatin, a specific inhibitor of endothelial cell proliferation in vitro and angiogenesisin vivo. In our original studies, we demonstrated that a Lewis lung carcinoma (LLC-LM) primary tumor could suppress the growth of its metastases by generating angiostatin. Angiostatin, a 38-kDa internal fragment of plasminogen, was purified from the serum and urine of mice bearing LLC-LM, and its discovery provides the first proven mechanism for concomitant resistance (O’Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M. A., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994)Cell 79, 315–328). Subsequently, we have shown that systemic administration of angiostatin can regress a wide variety of malignant tumors in vivo. However, at the time of our initial discovery of angiostatin, the source of the protein was unclear. We hypothesized that the tumor or stromal cells might produce an enzyme that could cleave plasminogen sequestered by the primary tumor into angiostatin. Alternatively, we speculated that the tumor cells might express angiostatin. By Northern analysis, however, we have found no evidence that the tumor cells express angiostatin or other fragments of plasminogen (data not shown). We now report that gelatinase A (matrix metalloproteinase-2), produced directly by the LLC-LM cells, is responsible for the production of angiostatin, which suppresses the growth of metastases in our original model.

The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients

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.