Fritz Costa

Vascular Targeting and Antiangiogenesis Agents Induce Drug Resistance Effector GRP78 within the Tumor Microenvironment

Therapeutic targeting of the tumor vasculature that destroys preexisting blood vessels of the tumor and antiangiogenesis therapy capitalize on the requirement of tumor cells on an intact vascular supply for oxygen and nutrients for growth, expansion and metastasis to the distal organs. Whereas these classes of agents show promise in delaying tumor progression, they also create glucose and oxygen deprivation conditions within the tumor that could trigger unintended prosurvival responses. The glucose-regulated protein GRP78, a major endoplasmic reticulum chaperone, is inducible by severe glucose depletion, anoxia, and acidosis. Here we report that in a xenograft model of human breast cancer, treatment with the vascular targeting agent, combretastatin A4P, or the antiangiogenic agent, contortrostatin, promotes transcriptional activation of the Grp78 promoter and elevation of GRP78 protein in surviving tumor cells. We further show that GRP78 is overexpressed in a panel of human breast cancer cells that has developed resistance to a variety of drug treatment regimens. Suppression of GRP78 through the use of lentiviral vector expressing small interfering RNA sensitizes human breast cancer cells to etoposide-mediated cell death. Our studies imply that antivascular and antiangiogenesis therapy that results in severe glucose and oxygen deprivation will induce GRP78 expression that could lead to drug resistance.

Development of a Novel Recombinant Disintegrin, Contortrostatin, as an Effective Anti-Tumor and Anti-Angiogenic Agent

Contortrostatin (CN) (Mr 13,500 Da) is a novel homodimeric disintegrin isolated from the venom of Agkistrodon contortrix contortrix (Southern Copperhead) snake and displays two RGD motifs (one on each chain), which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. In previous studies, we have shown that native CN administered in a liposomal formulation exhibits potent anti-angiogenic and tumor growth inhibitory activities. Current isolation of the protein from crude venom is difficult and prohibitively expensive for translation into the clinic. In this report, we describe a method amenable to large-scale production of a soluble monomeric form of recombinant CN with biologic activity; the protein is expressed directly in the cytoplasm of an engineered bacterial system with an expression yield of approximately 20 mg/l of culture. We present here the in vitro assays as well as the anti-tumor and anti-angiogenic evaluation of liposomal recombinant CN in an orthotopic, xenograft model of human breast cancer.

Contortrostatin, a Snake Venom Disintegrin with Anti-Angiogenic and Anti-Tumor Activity

Disintegrins are soluble peptides found in snake venom. They bind to Arg-Gly-Asp (RGD)-responsive integrins with high affinity (nM range) and block integrin function. Contortrostatin (CN), the disintegrin from southern copperhead venom, is a homodimer with a molecular weight of 13,500. Each chain contains 65 amino acids with an Arg-Gly-Asp motif. CN has anti-invasive and anti-adhesive activity on tumor cells and endothelial cells in vitro, and binds to integrins αvβ3, αvβ5, and/or α5β1. In vivo studies using the human metastatic breast cancer cell line MDA-MB-435, in an orthotopic xenograft model in nude mice, revealed that CN has potent anti-tumor and anti-angiogenic activity. Recent studies have employed an intravenous liposomal delivery procedure. Liposomal delivery of CN has also been shown to provide effective in vivo anti-tumor and anti-angiogenic activity in a human ovarian cancer animal model.

Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with ECs ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.

A SNAKE VENOM DISINTEGRIN WITH POTENT ANTITUMOR AND ANTIANGIOGENIC ACTIVITY

In this report we describe an effective method for delivery of a novel snake venom disintegrin, contortrostatin (CN), in an orthotopic, xenograft model of human mammary cancer in immunodeficient mice. Contortrostatin (Mr 13,500) is a homodimeric disintegrin isolated from venom of the southern copperhead snake. Contortrostatin possesses two RGD sites, one in each chain, which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. Although our laboratory has previously described the antitumor activity of CN in a mouse model of human mammary cancer, the method of delivery, daily intratumor injection, was not translatable to clinical application. We now describe a clinically relevant method of administering CN, liposomal delivery (LCN). A unique liposomal system has been designed for intravenous (IV) administration of a biologically active protein with full retention of biological activity. Pharmacokinetics, biodistribution, platelet reactivity, and immunogenicity of LCN were examined and compared with similar characteristics of native, unencapsulated CN. There are several advantages to liposomal delivery of CN: (i) LCN has a significantly prolonged circulatory half-life compared to native CN; (ii) LCN is passively accumulated in the tumor; (iii) LCN has no platelet reactivity; and (iv) LCN is not recognized by the immune system. We have previously demonstrated that antiangiogenic activity is an important component of CNs mechanism of antitumor action. In the present communication we demonstrate that IV delivery of LCN leads to potent antitumor and antiangiogenic activity in the orthotopic, xenograft human mammary cancer model.

Anti-Angiogenesis and Disintegrins

Angiogenesis is a critical process in tumor and disease progression. A number of features are central to both tumor growth and development, and the recruitment and invasion of a growing vascular network supplying the tumor with nutrients and a mechanism of escape to allow meastatic growth. One class of molecules important to both tumor growth and blood vessel recruitment are a family of cell surface receptors identified as integrins. Integrins are α/β heterodimeric glycoproteins in which the different α subunits combine with distinct β subunits resulting in a range of specificities toward various extracellular matrix (ECM) proteins. The RGD sequence found in a number of ECM proteins is recognized by several classes of integrins, allowing for linkage of cytoskeletal proteins associated with the integrins to the ECM which leads to involvement in bi-directional signaling that displays profound effects on cellular functions. Among these integrin mediated interactions are the adhesion of both endothelial cells and cancer cells to ECM proteins, an interaction that is integral to metastasis, tumor growth and angiogensis. Antibodies targeted to integrins have been shown to retard tumor growth and subsequent tumor induced angiogenesis. One concern with this approach is that the antibody targets a single integrin, which may allow the tumor to utilize other non-targeted integrins to circumvent this type of blockage. A more broad spectrum agent is available that binds to and blocks the function of several different integrins at a time, this agents is identified as a disintegrin. Originally purified from the venom of Viperidae family of snakes, a disintegrins role in nature is presumably to block platelet aggregation following envenomation based on interaction of an integrin on the activated platelet surface with an RGD sequence in the disintegrin. It has been observed that integrins overexpressed on some tumor types and angiogenic vasculature have similar affinity for RGD motifs found in ECM proteins. Based on disintegrin structure we have developed a recombinant form of a snake venom disintegrin, which we call vicrostatin (VCN). VCN is a potent anti-angiogenic/anti-tumor agent in in vitro and in vivo laboratory studies. Further development of the recombinant venom derived disintegrin along with new technology looking at additional disintegrin-like proteins may offer a novel therapeutic approach in targeting tumor induced angiogenesis.