Inhibition of Tumor Angiogenesis by the Matrix Metalloproteinase–Activated Anthrax Lethal Toxin in an Orthotopic Model of Anaplastic Thyroid Carcinoma

Abstract

Patients with anaplastic thyroid carcinoma (ATC) typically succumb to their disease months after diagnosis despite aggressive therapy. A large percentage of ATCs have been shown to harbor the V600E B-Raf point mutation, leading to the constitutive activation of the mitogen-activated protein kinase pathway. ATC invasion, metastasis, and angiogenesis are in part dependent on the gelatinase class of matrix metalloproteinases (MMP). The explicit targeting of these two tumor markers may provide a novel therapeutic strategy for the treatment of ATC. The MMP-activated anthrax lethal toxin (LeTx), a novel recombinant protein toxin combination, shows potent mitogen-activated protein kinase pathway inhibition in gelatinase-expressing V600E B-Raf tumor cells in vitro. However, preliminary in vivo studies showed that the MMP-activated LeTx also exhibited dramatic antitumor activity against xenografts that did not show significant antiproliferative responses to the LeTx in vitro. Here, we show that the MMP-activated LeTx inhibits orthotopic ATC xenograft progression in both toxin-sensitive and toxin-resistant ATC cells via reduced endothelial cell recruitment and subsequent tumor vascularization. This in turn translates to an improved long-term survival that is comparable with that produced by the multikinase inhibitor sorafenib. Our results also indicate that therapy with the MMP-activated LeTx is extremely effective against advanced tumors with well-established vascular networks. Taken together, these results suggest that the MMP-activated LeTx-mediated endothelial cell targeting is the primary in vivo antitumor mechanism of this novel toxin. Therefore, the MMP-activated LeTx could be used not only in the clinical management of V600E B-Raf ATC but potentially in any solid tumor. Mol Cancer Ther; 9(1); 190-201. ©2010 AACR.

Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities

“…This unique system relies upon proteolytic activation by tumor over-expressed proteases, MMPs and uPA, and we and others have demonstrated that administration of these protease-activated PrAg derivatives, in conjunction with various cytotoxins, can be used to elicit significant in vivo anti-tumor responses in diverse cancer models including melanomas, carcinomas and sarcomas (Alfano et al, 2010; Liu et al, 2003b; Liu et al, 2008; Rono et al, 2006; Schafer et al, 2011; Su et al, 2007). …”

“…Employing this principle, we have previously generated versions of PrAg requiring activation by MMPs (PrAg-L1) (Liu et al, 2000), uPA (PrAg-U2) (Liu et al, 2001), or co-localized MMP/uPA activities (IC-PrAg, consisting of two separate proteins, PrAg-L1-I210A and PrAg-U2-R200A) (Liu et al, 2005). Multiple studies have been performed, as summarized in Table 1, demonstrating that when these engineered PrAgs are co-administered with various cytotoxins, that significant anti-tumor activity towards established tumors can be achieved in a variety of syngraft, xenograft and orthotopic models, highlighting the potential broad-spectrum therapeutic utility of these agents (Alfano et al, 2010; Liu et al, 2003b; Liu et al, 2005; Liu et al, 2008; Rono et al, 2006; Schafer et al, 2011; Su et al, 2007; Wein et al, 2013). These toxin combinations exert there anti-tumor effects through a variety of means including both direct tumor cell killing and killing of cellular components of the tumor stroma, including tumor vasculature (Alfano et al, 2008; Alfano et al, 2010; Liu et al, 2003b; Liu et al, 2008; Schafer et al, 2011).…”

Diving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody-Targeted Anti-Tumor Toxins

“…The efficient endosomal escape of lethal factor and edema factor has been used for the delivery of fusion proteins containing the protective antigen-binding moiety of lethal factor to deliver other enzymes to the cytosol. These fusion proteins have been successfully utilized for various tumor-therapy approaches [30,42,43], the delivery of reporter proteins [44] and the delivery of anti-apoptotic Bcl-x L [45].…”

“…The elevated expression of certain matrix metalloproteases in the tumor environment can be utilized to activate the toxins at the tumor site [30] and their use in combination with other strategies present attractive possibilities. For instance, targeted protein toxins can be administered as an adjunct treatment for surgery or directly when combined with radio-or chemotherapy [2,3].…”

Cytotoxicity of human recombinant arginase I (Co)-PEG5000 in the presence of supplemental L-citrulline is dependent on decreased argininosuccinate synthetase expression in human cells

“…Coadministration of HuArgI (Co)-PEG5000 with L-citrulline may improve the therapeutic index by protecting normal tissues unable to rescue arginine from ornithine. Furthermore, because amino acid depletion can induce cell cycle arrest, protein synthesis inhibition, and apoptosis, we measured the inhibition of both cellular DNA and protein synthesis, as well as overall growth inhibition measured by metabolic conversion of tetrazolium salt to a formazan [19,29].…”