Elucidation of the natural resistance mechanism of MCF7 cells to anthrax toxin
Anthrax toxin is a set of three proteins secreted by the bacterium Bacillus anthracis, the causative agent of anthrax. Two of these, edema factor (EF) and lethal factor (LF), are the toxic components. The third, protective antigen (PA) is the receptor-binding protein that delivers EF and LF to cytosol. EF, an adenylyl cyclase, exerts its toxicity by making excessive amounts of cAMP inside cells. LF, a metalloprotease, harms cells by cleaving MAP kinase kinases. Two different anthrax toxin receptors are known, tumor endothelial marker 8 (TEM8) and capillary morphogenesis gene 2 protein (CMG2). The two receptors have 5 splice variants each, which include both functional and nonfunctional forms. The receptors are ubiquitous, and therefore cellular resistance to anthrax toxin is an exceedingly rare phenomenon. We recently found that the human breast cancer cell line MCF7 represents this rare phenomenon; the cell line exhibits natural resistance to anthrax toxin. The focus of this work was to study the mechanism that accounts for MCF7 resistance to the toxin.
To be toxic, anthrax toxin requires normal operation of the endocytic pathway. To determine whether MCF7 has any general defect in endocytic pathways, we tested it for sensitivity to diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (ETA). But MCF7 proved fully sensitive to DT and ETA, suggesting the cell line has no general defect in endocytic pathways, and that its resistance is specific for anthrax toxin. We next assessed the expression of anthrax toxin receptors by nested and real-time quantitative PCR. Sequencing of the amplicons revealed that the cell line has normal transcripts for three isoforms known to function as anthrax toxin receptors -TEM8-V1, CMG2-488, and CMG2-489. For comparison we used HEK293, a cell line nearly 1000-fold more sensitive to anthrax toxin than MCF7, and A431, a cell line nearly 100-fold more sensitive than MCF7. While HEK293 had greater levels of the transcripts, CMG2-488 and -489 transcripts appeared at about the same level in A431 and MCF7. Thus, this analysis revealed no remarkable differences that could account for MCF7 resistance. MCF7 failed to bind PA or bound little of it, consistent with its resistance. Thus, the reason MCF7 is resistant to anthrax toxin is its inability to bind the toxin. Overall these results suggest that either the receptor proteins are not expressed in MCF7, or the cell line lacks an as-yet unknown factor required for normal functioning of the receptors. More work is under way to address these questions.