The pro-apoptotic tumor suppressor protein prostate apoptosis response-4 (PAR-4) is silenced in a well-defined subset of human cancers including lung, prostate and breast cancer 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 poorly understood.In the present study it is demonstrated that UV- or TNFα-induced apoptosis results in a rapid Caspase-8-specific PAR-4 cleavage at EEPD131↓G, which separates the unstructured N-terminal part from the C-terminal region that contains the NLS, SAC and LZ domains. I further demonstrate that TNFα-mediated hydrolysis of PAR-4 requires Caspase-8 and leads to nuclear accumulation of the C-terminal cleavage fragment of the tumor suppressor and thereby induces apoptosis. Taken together, these results indicate that Caspase-8-mediated cleavage induced nuclear translocation of the C-terminal part of PAR-4 is critical in regulating cell death triggered by TNFα.Recent evidence implicates down-regulation of PAR-4 as a critical step for breast cancer recurrence. Down-regulation of PAR-4 allows tumor cells to survive tumor regression following targeted therapy and chemotherapy and is both a necessary and sufficient step for tumor recurrence. Hence, PAR-4 expression was analyzed in a panel of breast cancer cell lines and low PAR-4 expression was observed in the majority of triple negative breast cancer (TNBC) cells. Interestingly, Caspase-8-mediated PAR-4 cleavage was also observed in TNBC cells following DNA-damage-induced apoptosis using genotoxic drugs. I further demonstrate that loss of PAR-4 in TNBC cells mediates resistance to DNA-damage induced apoptosis and surprisingly also interferes with Caspase-8 activation, indicating that PAR-4 is capable to amplify Caspase-8 activation via an unknown feedback mechanism.Low PAR-4 expression promotes tumor cell survival following therapy but the pathways controlling PAR-4 expression are not identified. Using an unbiased mass spectrometry approach I was able to identify nine physical PAR-4 interaction partners out of which two belong to the family of E3 ubiquitin-protein ligases (MYCBP2 and UBR5) and one to the family of deubiquitylases (USP7). These novel PAR-4 binding proteins suggest that down-regulation of PAR-4 might be specifically linked to disordered ubiquitin signaling in cancer.In summary, the results provide evidence that the mechanism by which PAR-4 orchestrates the apoptotic process requires cleavage by Caspase-8 following TNFα- or genotoxic drug-induced apoptosis and functions by cleavage-induced nuclear translocation of the C-terminal part
