Mechanism of Regulating Fas Signaling by Promyelocytic Leukemia Protein (PML) and Its Dominant-Negative Mutant PMLRARα

Chemotherapies and irradiation depend on an intact Fas signaling system to eradicate cancer cells. Defective Fas signaling is an important cause of acquired resistance to cancer therapy. If we were able to restore Fas apoptosis or sensitize cancer cells to Fas-mediated apoptosis, we could improve the efficacy of many current cancer therapies. To elucidate defects of Fas signaling in cancer cells, we sought to identify potential modulators of Fas selectively expressed in cancers cells and target them to sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy.

Liquid chromatography tandem mass spectroscopy was used to identify Fas-associated proteins. Co-immunoprecipitation (co-IP) and Western Blot (WB) were used to detect/confirm interactions of PML and PMLRARα with Fas, and components of death-inducing signaling complex (DISC) FADD, c-FLIP, and caspase-8 cleavage in tissues from PML wild-type (WT) and knock-out (KO) mice and acute promyelocytic leukemia (APL) cells. Deletional mutagenesis was used to map protein interacting domains. PML shRNA lentivirus and As2O3 were used to downregulate PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis in response to Fas stimulation. Mice transfected with PMLRARα were monitored for survival after a lethal challenge with agonistic Fas antibody Jo2 and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL.

The promyelocytic leukemia protein (PML) was identified as a Fas-binding protein by mass spectroscopy analysis. Using co-IP/WB analysis of tissues from PML WT and KO mice, we found PML interaction with Fas and FADD in PML WT MEF cells and liver cells but absent in KO MEF and liver cells; PML-Fas complexes were exclusively present in the membrane/cytoplasmic extracts but not in the nuclear extracts. The B-box domain of PML was found to be required for Fas binding. Knockdown of PML was associated with suppressed rates of Fas-mediated apoptosis compared to non-targeted knockdown cells; PML KO cells reconstituted with cytoplasmic PML were sensitized to Fas apoptosis. Furthermore, we found that liver cells from PML KO mouse showed impaired assembly of the Fas death-inducing signaling complex (DISC) in response to Fas activation when compared to PML WT. PML functions are known to be blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in primary human and transgenic mouse APL cells blocked Fas-mediated apoptosis. Blockage of apoptosis was mediated through PMLRARα -dependent recruitment of c-FLIP_L/Sto and exclusion of procaspase-8 from the DISC. PMLRARα effects were also observed in vivo, as expression of PMLRARα protected mice against a lethal effect of agonistic anti-Fas antibody (P<.001). Livers from PMLRARα -transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice.

PML binds to Fas and promotes Fas-mediated apoptosis through enhancing Fas DISC formation while binding of PMLRARα to Fas blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex enriched in c-FLIP. Our data suggest that PML plays a critical role in initiation of Fas signaling; in contrast, the dominant-negative mutant PMLRARα is a confirmed cancer specific inhibitor of Fas-mediated apoptosis. Thus, deficiency of PML or expression of PMLRARα can contribute to cancer development and resistance to chemotherapy. The newly discovered PML-Fas and PMLRARα -Fas complexes can be sites for modulation of apoptosis. Thus, by neutralizing the inhibitory effect of Fas-binding proteins such as PMLRARα and/or promoting positive Fas modulators such as PML, we can improve responses to chemotherapy that depend on activation of death receptors for effective elimination of cancer cells.

No relevant conflicts of interest to declare.