Caspase-8-mediated PAR-4 cleavage is required for TNFα-induced apoptosis

Abstract

The tumor suppressor protein prostate apoptosis response-4 (PAR-4) is silenced in a subset of human cancers and its down-regulation serves as a mechanism for cancer cell survival following chemotherapy. PAR-4 re-expression selectively causes apoptosis in cancer cells but how its pro-apoptotic functions are controlled and executed precisely is currently unknown. We demonstrate here that UV-induced apoptosis results in a rapid caspase-dependent PAR-4 cleavage at EEPD131G, a sequence that was preferentially recognized by caspase-8. To investigate the effect on cell growth for this cleavage event we established stable cell lines that express wild-type-PAR-4 or the caspase cleavage resistant mutant PAR-4 D131G under the control of a doxycycline-inducible promoter. Induction of the wild-type protein but not the mutant interfered with cell proliferation, predominantly through induction of apoptosis. We further demonstrate that TNFα-induced apoptosis leads to caspase-8-dependent PAR-4-cleavage followed by nuclear accumulation of the C-terminal PAR-4 (132-340) fragment, which then induces apoptosis. Taken together, our results indicate that the mechanism by which PAR-4 orchestrates the apoptotic process requires cleavage by caspase-8.

Post-translational regulation of the cleaved fragment of Par-4 in ovarian and endometrial cancer cells

“…We and others have shown that Par-4 is cleaved by caspases 3-8 under apoptotic circumstances [14, 37]. As observed in the current findings, the cleavage did not occur in chemoresistant cancer cells probably because pro-caspases, which did not undergo cleavage, are required to produce the cl-Par-4 fragment.…”

“…In the present study, we showed that cl-Par-4 was localized in both compartments with slight differences between the different cell lines used. The observed localization from our experiments could be related to tissue specificity (endometrial and ovarian), whereas the other manuscripts investigated the commonly used HeLa cervical cancer cells or HEK293 human embryonic kidney cells [14, 37]. It is also interesting to note that in estrogen-dependent cancers (ovarian and endometrium), other studies have shown Par-4 being mainly localized in the cytoplasm [7].…”

“…In the present manuscript, we focused on the ~25kDa cleaved form of Par-4 which has been previously reported as a product of Par-4 following its cleavage by caspase-8 and caspase-3 under apoptotic stimuli [14, 37]. We found that in ovarian and endometrial cancer cells, cl-Par-4 undergoes rapid degradation by the proteasome in baseline conditions but is stabilized upon cisplatin treatment.…”

“…Actually, various papers reported that the translocation of Par-4 to the nucleus was needed to induce apoptosis and stimulate the transcription of diverse genes [9, 57, 58]. Concerning cl-Par-4, it was previously demonstrated that this product was localized in both cytosol/nucleus with a modest trend for nuclear enrichment [14, 37]. In the present study, we showed that cl-Par-4 was localized in both compartments with slight differences between the different cell lines used.…”

A Naturally Generated Decoy of the Prostate Apoptosis Response-4 Protein Overcomes Therapy Resistance in Tumors

“…An in silico search of caspase cleavage sites within Par-4 identified a potential cleavage site at the D131 position (D 131 EEEPD). During the course of these studies, several groups identified D131 as a target for caspase-3 and focused on the role of the 25 kDa fragment, which contains the SAC effector domain of Par-4, but did not specifically identify the 15 kDa fragment (26–28). Pretreatment of H460 cells with TRAIL in the presence of cycloheximide, which is known to inhibit de novo protein biosynthesis, did not inhibit production of the 25 kDa and 15 kDa bands (Figure S3A), indicating that production of these fragments was not dependent on new gene expression events.…”