Fernando Donate

A non–RGD-based integrin binding peptide (ATN-161) blocks breast cancer growth and metastasis in vivo

Purpose: Integrins are expressed by numerous tumor types including breast cancer, in which they play a crucial role in tumor growth and metastasis. In this study, we evaluated the ability of ATN-161 (Ac-PHSCN-NH2), a 5-mer capped peptide derived from the synergy region of fibronectin that binds to α5β1 and αvβ3 in vitro, to block breast cancer growth and metastasis. Experimental design: MDA-MB-231 human breast cancer cells were inoculated s.c. in the right flank, or cells transfected with green fluorescent protein (MDA-MB-231-GFP) were inoculated into the left ventricle of female BALB/c nu/nu mice, resulting in the development of skeletal metastasis. Animals were treated with vehicle alone or by i.v. infusion with ATN-161 (0.05–1 mg/kg thrice a week) for 10 weeks. Tumor volume was determined at weekly intervals and tumor metastasis was evaluated by X-ray, microcomputed tomography, and histology. Tumors were harvested for histologic evaluation. Result: Treatment with ATN-161 caused a significant dose-dependent decrease in tumor volume and either completely blocked or caused a marked decrease in the incidence and number of skeletal as well as soft tissue metastases. This was confirmed histologically as well as radiographically using X-ray and microcomputed tomography. Treatment with ATN-161 resulted in a significant decrease in the expression of phosphorylated mitogen-activated protein kinase, microvessel density, and cell proliferation in tumors grown in vivo. Conclusion: These studies show that ATN-161 can block breast cancer growth and metastasis, and provides a rationale for the clinical development of ATN-161 for the treatment of breast cancer. [Mol Cancer Ther 2006;5(9):2271–80]

Copper Binding by Tetrathiomolybdate Attenuates Angiogenesis and Tumor Cell Proliferation through the Inhibition of Superoxide Dismutase 1

Purpose: A second-generation tetrathiomolybdate analogue (ATN-224; choline tetrathiomolybdate), which selectively binds copper with high affinity, is currently completing two phase I clinical trials in patients with advanced solid and advanced hematologic malignancies. However, there is very little information about the mechanism of action of ATN-224 at the molecular level. Experimental Design: The effects of ATN-224 on endothelial and tumor cell growth were evaluated in cell culture experiments in vitro. The antiangiogenic activity of ATN-224 was investigated using the Matrigel plug model of angiogenesis. Results: ATN-224 inhibits superoxide dismutase 1 (SOD1) in tumor and endothelial cells. The inhibition of SOD1 leads to inhibition of endothelial cell proliferation in vitro and attenuation of angiogenesis in vivo. The inhibition of SOD1 activity in endothelial cells is dose and time dependent and leads to an increase in the steady-state levels of superoxide anions, resulting in the inhibition of extracellular signal-regulated kinase phosphorylation without apparent induction of apoptosis. In contrast, the inhibition of SOD1 in tumor cells leads to the induction of apoptosis. The effects of ATN-224 on endothelial and tumor cells could be substantially reversed using Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, a catalytic small-molecule SOD mimetic. Conclusions: These data provide a distinct molecular target for the activity of ATN-224 and provide validation for SOD1 as a target for the inhibition of angiogenesis and tumor growth.

The Bioreductive Prodrug PR-104A Is Activated under Aerobic Conditions by Human Aldo-Keto Reductase 1C3

PR-104, currently in phase II clinical trials, is a phosphate ester pre-prodrug which is converted in vivo to its cognate alcohol, PR-104A, a prodrug designed to exploit tumor hypoxia. Bioactivation occurs via one-electron reduction to DNA crosslinking metabolites in the absence of oxygen. However, certain tumor cell lines activate PR-104A in the presence of oxygen, suggesting the existence of an aerobic nitroreductase. Microarray analysis identified a cluster of five aldo-keto reductase (AKR) family members whose expressions correlated with aerobic metabolism of PR-104A. Plasmid-based expression of candidate genes identified aldo-keto reductase 1C3 as a novel nitroreductase. AKR1C3 protein was detected by Western blot in 7 of 23 cell lines and correlated with oxic PR-104A metabolism, an activity which could be partially suppressed by Nrf2 RNAi knockdown (or induced by Keap1 RNAi), indicating regulation by the ARE pathway. AKR1C3 was unable to sensitize cells to 10 other bioreductive prodrugs and was associated with single-agent PR-104 activity across a panel of 9 human tumor xenograft models. Overexpression in two AKR1C3-negative tumor xenograft models strongly enhanced PR-104 antitumor activity. A population level survey of AKR1C3 expression in 2,490 individual cases across 19 cancer types using tissue microarrays revealed marked upregulation of AKR1C3 in a subset including hepatocellular, bladder, renal, gastric, and non-small cell lung carcinoma. A survey of normal tissue AKR1C3 expression suggests the potential for tumor-selective PR-104A activation by this mechanism. These findings have significant implications for the clinical development of PR-104.

Pharmacology of the Novel Antiangiogenic Peptide ATN-161 (Ac-PHSCN-NH2): Observation of a U-Shaped Dose-Response Curve in Several Preclinical Models of Angiogenesis and Tumor Growth

Purpose: ATN-161 (Ac-PHSCN-NH2) is an integrin-binding peptide that is currently in phase II trials in cancer patients. This peptide has been shown to have antitumor activity in a number of different preclinical models. Experimental Design: In this study, we examined the binding, biodistribution, and dose and biomarker response of ATN-161 in several animal models. Results: ATN-161 bound to the β subunit of a number of different integrins implicated in tumor growth and progression, which depended on its cysteine thiol. The peptide had antiangiogenic activity in the Matrigel plug model, and this activity could be reversed by inhibitors of protein kinase A, an effector of α5β1-dependent angiogenesis. A labeled analogue of ATN-161, ATN-453, localized to neovessels but not to preexisting vasculature in vivo. The half-life of the peptide when localized to a tumor was much longer than in plasma. Dose-response studies in the Matrigel plug model of angiogenesis or a Lewis lung carcinoma model of tumor growth showed a U-shaped dose-response curve with 1 to 10 mg/kg given thrice a week, being the optimal dose range of ATN-161. Two additional pharmacodynamic models of angiogenesis (dynamic contrast-enhanced magnetic resonance imaging and measurement of endothelial cell progenitors) also revealed U-shaped dose-response curves. Conclusions: The presence of a U-shaped dose-response curve presents a significant challenge to identifying a biologically active dose of ATN-161. However, the identification of biomarkers of angiogenesis that also exhibit this same U-shaped response should allow the translation of those biomarkers to the clinic, allowing them to be used to identify the active dose of ATN-161 in phase II studies.

The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.

Phase I Study of Copper-Binding Agent ATN-224 in Patients with Advanced Solid Tumors

Purpose: Copper chelation reduces the secretion of many angiogenic factors and reduces tumor growth and microvascular density in animal models. ATN-224 is a second-generation analogue of ammonium tetrathiomolybdate. The aim of our phase I study was to reduce serum copper levels, as measured by ceruloplasmin, to 5 to 15 mg/dL (normal 16-60) in 14 to 21 days, to determine the pharmacokinetic profile of ATN-224 and to evaluate dose-limiting toxicities. Patients and Methods: Cohorts of patients were treated with escalating oral doses of ATN-224 until copper depletion followed by a titrated maintenance dose. Results: Eighteen patients received 78 cycles of ATN-224. Mean baseline ceruloplasmin was 39.6 mg/dL. The maximum administered dose was 330 mg/d where grade 3 fatigue was dose-limiting. At the maximum tolerated dose of 300 mg/d, the median time to achieve target ceruloplasmin was 21 days, and toxicities included grade 3 anemia, grade 3 neutropenia, fatigue, and sulfur eructation. ATN-224 treatment caused a significant reduction (>90%) in RBC superoxide dismutase 1 activity and circulating endothelial cells. Pharmacokinetic data indicate greater absorption of ATN-224 and more rapid ceruloplasmin reduction when administered with a proton pump inhibitor. Stable disease of >6 months was observed in 2 patients. Conclusions: Oral ATN-224 is a well-tolerated therapy and at a loading dose of 300 mg/d leads to a reduction of serum ceruloplasmin levels in 80% patients within 21 days. A loading dose of 300 mg/d for 2 weeks followed by a titrated maintenance dose will be the recommended starting dose for phase II study.

Peptides Derived from the Histidine-Proline Domain of the Histidine-Proline-Rich Glycoprotein Bind to Tropomyosin and Have Antiangiogenic and Antitumor Activities

The antiangiogenic activity of the multidomain plasma protein histidine-proline-rich glycoprotein (HPRG) is localized to its histidine-proline–rich (H/P) domain and has recently been shown to be mediated, at least partially, through binding to cell-surface tropomyosin in fibroblast growth factor-2-activated endothelial cells (X. Guan et al., Thromb Haemost, in press). HPRG and its H/P domain, but not the other domains of HPRG, bind specifically and with high affinity to tropomyosin. In this study, we characterize the interaction of the H/P domain with tropomyosin and delineate the region within the H/P domain responsible for that interaction. The H/P domain of HPRG consists mostly of repetitions of the consensus sequence [H/P][H/P]PHG. Applying an in vitro tropomyosin binding assay, we demonstrate that the synthetic peptide HHPHG binds to tropomyosin in vitro and inhibits angiogenesis and tumor growth in vivo. The affinity for tropomyosin increases exponentially upon multimerization of the HHPHG sequence, with a concurrent increase in antiangiogenic activity. Specifically, the tetramer (HHPHG)4 has significant antiangiogenic activity in the Matrigel plug model (IC50 ∼600 nm) and antitumor effects in two syngeneic mouse tumor models. Thus, we show that a 16-mer peptide analogue mimics the antiangiogenic activity of intact HPRG and is also able to inhibit tumor growth, suggesting that cell surface tropomyosin may represent a novel antiangiogenic target for the treatment of cancer.

Histidine-Proline Rich Glycoprotein (HPRG) binds and transduces anti-angiogenic signals through cell surface tropomyosin on endothelial cells

The anti-angiogenic properties of the histidine-proline-rich (H/P) domain of HPRG have recently been described (Juarez JC, et al. Cancer Research 2002; 62: 5344-50). However, the binding site that mediates these properties is unknown. HPRG is evolutionarily, functionally and structurally related to cleaved high molecular weight kininogen (HKa), an anti-angiogenic polypeptide that stimulates apoptosis of proliferating endothelial cells through binding to cell-surface tropomyosin (Zhang J-C, et al. Proc Natl Acad Sci USA 2002; 99: 12224-9). In this study, we demonstrate that HPRG binds with high affinity to FGF-2-stimulated human umbilical vein endothelial cells (HUVEC) and immobilized tropomyosin in a Zn2+ or pH-dependent manner, and that this interaction is mediated by the H/P domain of HPRG. At least two binding sites for HPRG, tropomyosin and heparan sulfate proteoglycans (HSPs), were identified on the surface of FGF-2-activated endothelial cells. Translocation of tropomyosin to the surface of HUVEC occurred in response to FGF-2, and the anti-angiogenic activity of HPRG in a Matrigel plug model was partially inhibited by soluble tropomyosin. These results suggest that HPRG binds to endothelial cell surface tropomyosin which at least partially mediates the antiangiogenic effects of HPRG.

Design of novel N-(2,4-dioxo-1,2,3,4-tetrahydro-thieno[3,2-d]pyrimidin-7-yl)-guanidines as thymidine phosphorylase inhibitors, and flexible docking to a homology model

A novel class of thymidine phosphorylase (TP) inhibitors has been designed based on analogy to the enzyme substrate as well as known inhibitors. Flexible docking studies, using a homology model of human TP, of the designed N-(2,4-dioxo-1,2,3,4-tetrahydro-thieno[3,2-d]pyrimidin-7-yl)-guanidines as well as their synthetic precursors provide insight into the observed experimental trends in binding affinity.

Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268–275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.