PML and PMLRARα Interact with Fas to Regulate Fas-Mediated Apoptosis In Vivo

Altered Fas signaling leads to resistance to various anticancer therapies. The presence of at least low level expression of Fas is required for the transformed phenotype of many cells. In order for Fas to offer this complex regulation and to prevent inadvertent Fas activation, we propose that Fas signaling is subjected to tight regulation and this regulation could produce resistance to apoptosis of cancer cells.

Cell lines NB4 (PMLRARα positive), HL60, U937/PR9 (PMLRARα expression driven by metalothionine promoter), or transfected HEK293 cells were collected and cell extracts were prepared for immunoprecipitation and Western blot analysis with the indicated antibodies. Cellular fractionation was performed using the nuclear/cytosol fractionation kit (BioVision, Mountain View, CA), according to the manufacturer's instructions. We expressed PMLRARα in C57B6 female mice by transfection of PMLRARα plasmid or empty-vector plasmid (12 mice per group) by tail vein inoculation using a hydrodynamic transfection method. Twenty-four hours after transfection, mice were challenged intraperitoneally with 2 μg/g mouse weight of agonistic Fas antibody Jo2, and monitored for survival. Liver tissues were stained with TUNEL assay and antibodies targeting apoptosis proteins. Differences between two groups were assessed using the 2-tailed student's t test and a P value < 0.05 was considered to be statistically significant.

In this study, we identified, by mass spectrometry analysis of Fas-associated proteins, the proapoptotic promyelocytic leukemia protein (PML) as a Fas-interacting protein. PML is a ubiquitously expressed tumor suppressor, which is downregulated or mutated in over 50% of all cancers. PML promotes apoptosis especially under conditions of cell stress. The function of PML is blocked by its dominant-negative form promyelocytic leukemia - retinoic acid receptor α (PMLRARα). Our focus on PMLRARα showed that it interacts with Fas in acute promyelocytic leukemia-derived cell lines and APL primary cells. Because PMLRARα is considered to be a nuclear transcription factor, we fractionated cell compartment proteins and identified PMLRAR α-Fas complexes present only in the cytoplasmic fraction. Binding of PML to Fas was mapped to the B-box domain of PML and death domain of Fas. PMLRARα blockage of apoptosis was shown in U937/PR9 and NB4 cells, in which PMLRARα recruited c-FLIP_L/S and excluded procaspase 8 from the Fas death-inducing signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P<.001). The protected mice tissues contained Fas-PMLRARα-cFLIP complexes. Knockdown of PML in 293 cells compared to non-targeted knockdown control cells showed lower rates of CH-11-induced apoptosis, suggesting a facilitating role of PML in Fas mediated apoptosis. To examine the prevalence of PML-Fas complexes in non cancer tissues, we screened over 6 mouse tissues and all contained PML-Fas complexes.

Together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL through the accumulation of c-FLIP- Fas complexes. Furthermore, the common expression of PML-Fas complexes suggests Fas signaling is routinely regulated in the cytoplasm by PML, but susceptible to the blocking effects of PMLRARα in cancer.

Karp:Schering Merck: Research Funding.