Tigatuzumab

TRAIL-activating therapy is promising in treating various cancers, including pancreatic cancer, a highly malignant neoplasm with poor prognosis. However, many pancreatic cancer cells are resistant to TRAIL-induced apoptosis despite their expression of intact death receptors (DRs). Protein O-GlcNAcylation is a versatile posttranslational modification that regulates various biological processes. Elevated protein O-GlcNAcylation has been recently linked to cancer cell growth and survival. In this study, we evaluated the role of protein O-GlcNAcylation in pancreatic cancer TRAIL resistance, and identified higher levels of O-GlcNAcylation in TRAIL-resistant pancreatic cancer cells. With gain- and loss-of-function of the O-GlcNAc-adding enzyme, O-GlcNActransferase (OGT), we determined that increasing O-GlcNAcylation rendered TRAIL-sensitive cells more resistant to TRA-8-induced apoptosis, while inhibiting O-GlcNAcylation promoted TRA-8-induced apoptosis in TRAIL-resistance cells. Furthermore, we demonstrated that OGT knockdown sensitized TRAIL-resistant cells to TRA-8 therapy in a mouse model in vivo. Mechanistic studies revealed direct O-GlcNAc modifications of DR5, which regulated TRA-8-induced DR5 oligomerization. We further defined that DR5 O-GlcNAcylation was independent of FADD, the adapter protein for the downstream death-inducing signaling. These studies have demonstrated an important role of protein O-GlcNAcylation in regulating TRAIL resistance of pancreatic cancer cells; and uncovered the contribution of O-GlcNAcylation to DR5 oligomerization and thus mediating DR-inducing signaling.

The poly(ADP‐ribose) polymerases (PARP) play important roles in repairing damaged DNA during intrinsic cell death. We recently linked PARP‐1 to death receptor (DR)‐activated extrinsic apoptosis, the present studies sought to elucidate the function of cytoplasmic PARP‐1 in pancreatic cancer tumorigenesis and therapy. Using human normal and pancreatic cancer tissues, we analyzed the prevalence of cytoplasmic PARP‐1 expression. In normal human pancreatic tissues, PARP‐1 expression was present in the nucleus; however, cytoplasmic PARP‐1 expression was identified in pancreatic cancers. Therefore, cytoplasmic PARP‐1 mutants were generated by site‐direct mutagenesis, to determine a causative effect of cytoplasmic PARP‐1 on pancreatic cancer tumorigenesis and sensitivity to therapy with TRA‐8, a humanized DR5 antibody. PARP‐1 cytoplasmic mutants rendered TRA‐8 sensitive pancreatic cancer cells, BxPc‐3 and MiaPaCa‐2, more resistant to TRA‐8‐induced apoptosis; whereas wild‐type PARP‐1, localizing mainly in the nucleus, had no effects. Additionally, cytoplasmic PARP‐1, but not wild‐type PARP‐1, increased resistance of BxPc‐3 cells to TRA‐8 therapy in a mouse xenograft model in vivo. Inhibition of PARP enzymatic activity attenuated cytoplasmic PARP‐1‐mediated TRA‐8 resistance. Furthermore, increased cytoplasmic PARP‐1, but not wild‐type PARP‐1, was recruited into the TRA‐8‐activated death‐inducing signaling complex and associated with increased and sustained activation of Src‐mediated survival signals. In contrast, PARP‐1 knockdown inhibited Src activation. Taken together, we have identified a novel function and mechanism underlying cytoplasmic PARP‐1, distinct from nuclear PARP‐1, in regulating DR5‐activated apoptosis. Our studies support an innovative application of available PARP inhibitors or new cytoplasmic PARP‐1 antagonists to enhance TRAIL therapy for TRAIL‐resistant pancreatic cancers.