Matrix Metalloproteinase-activated Anthrax Lethal Toxin Demonstrates High Potency in Targeting Tumor Vasculature

Abstract

Anthrax lethal toxin (LT), a virulence factor secreted by Bacillus anthracis, is selectively toxic to human melanomas with the BRAF V600E activating mutation because of its proteolytic activities toward the mitogen-activated protein kinase kinases (MEKs). To develop LT variants with lower in vivo toxicity and high tumor specificity, and therefore greater potential for clinical use, we generated a mutated LT that requires activation by matrix metalloproteinases (MMPs). This engineered toxin was less toxic than wild-type LT to mice because of the limited expression of MMPs by normal cells. Moreover, the systemically administered toxin produced greater anti-tumor effects than wild-type LT toward human xenografted tumors. This was shown to result from its greater bioavailability, a consequence of the limited uptake and clearance of the modified toxin by normal cells. Furthermore, the MMP-activated LT had very potent anti-tumor activity not only to human melanomas containing the BRAF mutation but also to other tumor types, including lung and colon carcinomas regardless of their BRAF status. Tumor histology and in vivo angiogenesis assays showed that this antitumor activity is due largely to the indirect targeting of tumor vasculature and angiogenic processes. Thus, even tumors genetically deficient in anthrax toxin receptors were still susceptible to the toxin therapy in vivo. Moreover, the modified toxin also displayed lower immunogenicity compared with the wild-type toxin. All these properties suggest that this MMP-activated antitumor toxin has potential for use in cancer therapy.

Analysis of differential gene expression in colorectal cancer and stroma using fluorescence-activated cell sorting purification

“…Looking at individual genes, upregulation of connective tissue genes was prevalent (e.g., COL6A3, COL1A2, COL12A1, COL5A2, COL3A1, CTHRC1, SULF1), as were genes involved in extracellular matrix function (e.g., LAMA4, PI3, POSTN, TIMP2) and cell adhesion (e.g., TNS1). Genes involved in endothelial function (e.g., TIMP2) and specifically, colon cancer tumour endothelium, such as the anthrax toxin receptor (ANTXR1), were also upregulated (Liu et al, 2008). In contrast, the 31 highly significantly expressed genes in the FACS-purified cells do not display characteristics of stromal gene expression, and may be representative of tumour parenchyma gene expression.…”

Heterodimeric integrin complexes containing β1-integrin promote internalization and lethality of anthrax toxin

“…Diverse cell lines treated with LF-PA show a wide range of sensitivities to the toxin (9,(20)(21)(22)(23)(24)(25), and cell death occurs only in cells that have impaired MAPK kinase function (21). We hypothesized that such phenotypic differences might enable microarray-based identification of other genes whose differentially elevated or reduced expression correlates with differential toxin lethality.…”

“…GABRIEL software (26) separated cancer cell lines (SK-MEL-5, SK-MEL28, M-14, MALME-3M, MDA-MB-435, MDA-N, SK-MEL-2, HL-60, MCF7, SW-620, EKVX, and A549) into two groups, LF-PA-sensitive and LF-PA-resistant, based on previous reports (9,(20)(21)(22)(23)(24)(25). GABRIEL (27) was used for correlation of steady-state levels of expression with toxin lethality.…”

Prodrug Applications for Targeted Cancer Therapy

“…The other is by replacing the native furin cleavage site with recognition sequences for other proteases. Such substitutions have been made with cleavage sites for the ECM proteases uPA and MMP (78)(79)(80)(81)(82). Thus, engineered ATx with transductionally or proteolytically redirected PA acts functionally as a targeted cancer prodrug.…”