Characterization of the Potency and Mechanism of Action of a Novel Dual-Selective Intermolecularly Complemented Version of Anthrax Lethal Toxin (PAU2R200A- PAL1I207R/FP59) in Breast Cancer

Abstract

Wild type Anthrax Lethal toxin is a binary toxin comprised of a protective antigen (PrAg), the cell binding moiety and a lethal factor (LF), the catalytic moiety. Following cell entry, LF cleaves all mitogen- activated protein kinase kinase (MEKs) leading to the inactivation of the MAPK pathway. PrAg/LF causes cell toxicity, and this motivated the development of a new generation of PrAg variants with increased selectivity. The new version of anthrax, PrAgL1 and PrAgU2 were re-engineered where their activation requires the cleavage by specific tumor associated proteases such as MMPs and uPA/uPAR. PrAgL1 and PrAgU2 demonstrated high potency against a wide range of tumors with diminished cell toxicity. To add another layer of selectivity, the existing PrAg variants were combined, and the intermolecularly complementing (IMC) version of anthrax toxin emerged. IMC requires the cleavage with both MMPs and uPA/uPAR system simultaneously for it to be activated and to translocate the catalytic moiety into the cell. In this study, we evaluated the cytotoxicity of the wild type version of anthrax toxin on a normal model of breast cancer MCF-10A and three different breast cancer cell lines MDA-MB-231, MCF-7, and UACC-2087. PrAg/LF was cytotoxic against MDA-MB-231 cells only. For this we chose FP59 as a more potent catalytic moiety to be conjugated with IMC in our further studies. Second, we tested the IMC/FP59 on the panel of breast cancer cell lines. IMC/FP59 was cytotoxic against MDA-MB-231 cells only where PrAgU2R200A/FP59 showed similar cytotoxic effect as IMC/FP59. This indicated that uPA/uPAR is the rate limiting factor for the activation of IMC/FP59. Since IMC/FP59 had cytotoxic effect on MDA-MB-231 cells, the type of cell death was investigated by staining for Annexin V/PI, and there was no evidence of apoptosis. Later we detected uPAR levels in breast cancer cell lines. MDA-MB-231 had the most significant uPAR expression, MCF-7 had a moderate level of expression, and UACC-2087 had negligible expression. Finally, we investigated the levels of MMP-2, MMP-9, and uPAR in UACC-2087 cells after treatment with IMC/LF and IMC/FP59. Results show that UACC-2087 cell line expresses the three proteases and upon treatment, the levels of expression were not consistent. In order to understand the effect of both IMC/LF and IMC/FP59 on the levels of expression of the studied proteases, further experiments must be done. In this study, we demonstrated for first time the potency of the novel, intermolecularly complemented version of anthrax toxin, IMC/FP59 on breast cancer cell lines, its mode of action and mechanism of cell death.