Abstract 2083: The stress oncoprotein LEDGF/p75 attenuates oxidative stress-induced necrosis but not apoptosis in prostate cancer cells

The lens epithelium-derived growth factor p75 (LEDGF/p75) is emerging as a stress response oncoprotein in human cancer. Our group and others showed previously that this transcription coactivator is overexpressed in major human cancers, including prostate cancer, and protects against lysosomal cell death and chemotherapeutic drugs. Although LEDGF/p75 is considered a novel anti-apoptotic protein due to its ability to attenuate lysosomal cell death, previous studies from our group demonstrated its inactivation by caspases during apoptosis. We hypothesized that LEDGF/p75 promotes resistance to stress-induced caspase-independent cell death, but not to apoptosis, in cancer cells. To test this hypothesis, we established PC3 and RWPE-2 prostate cancer cell lines stably overexpressing LEDGF/p75, and evaluated its ability to protect these cells against non-apoptotic and apoptotic insults. LEDGF/p75 significantly attenuated cytotoxicity induced by tert-butyl hydroperoxide (TBHP), a strong inducer of oxidative stress. Characterization of TBHP-induced cell death showed that it was caspase-independent since it could not be inhibited with the broad caspase inhibitor z-VAD-fmk and did not involve caspase-3 activation. Furthermore, TBHP-treated cells displayed a necrotic morphology characterized by extensive cytoplasmic fragmentation and shrunken, non-fragmented nuclei. Necrosis was also confirmed by the appearance in immunoblots of a 45 kD cleavage fragment of DNA topoisomerase that was previously established by our group as a marker of necrosis. Overexpression of LEDGF/p75 attenuated the generation of reactive oxygen species (ROS) in TBHP-treated cells. Interestingly, LEDGF/p75 also attenuated ROS in cells treated with the classical apoptosis inducers staurosporine and TRAIL, but failed to protect against these apoptotic insults. These results indicated that the ability of LEDGF/p75 to attenuate ROS is not sufficient for its pro-survival function. Analysis of LEDGF/p75 integrity by immunoblotting analysis showed that the protein remained intact in cells treated with TBHP, but was cleaved into its signature 65 kd apoptotic fragment in cells treated with staurosporine and TRAIL. Our group has shown previously that this caspase-generated fragment lacks pro-survival activity. Taken together, these results strongly suggest that overexpression of LEDGF/p75 can protect tumor cells from caspase-independent cell death processes in which this protein remains structurally intact. T