Negative control of TRAIL-R1 signaling by transforming growth factor β1 in pancreatic tumor cells involves Smad-dependent down regulation of TRAIL-R1

Pancreatic ductal adenocarcinoma (PDAC) is characterized by both, overexpression of transforming growth factor (TGF)β and resistance of the tumor cells to many apoptosis-inducing stimuli. The latter negatively impacts the outcome of therapeutic efforts and represents one important mechanism which tumor cells utilize to escape the immune surveillance. Since TGFβ acts as a tumor promoter in advanced tumor stages and suppression of apoptosis is a known driver of tumor progression, it is possible that TGFβ functions as a crucial determinant of tumor cell sensitivity to apoptosis in PDAC. Here, we have studied the impact of TGFβ on TNF-related apoptosis inducing ligand (TRAIL)-induced signaling in PDAC cells. In TGFβ-responsive Panc1 and Colo357 cells, TGFβ1 reduced total and plasma membrane-associated levels of TRAIL-R1 but not those of TRAIL-R2. Consistent with the known predominant role of TRAIL-R1 in TRAIL-mediated signaling in PDAC, TGFβ1 inhibited TRAIL-induced DISC formation and apoptosis as well as phosphorylation of MAPKs and IκBα. Similarly, it also reduced signaling of TRAIL-R1 following its specific activation with an agonistic antibody. In contrast, specific TRAIL-R2 signaling remained unchanged. The TGFβ1 effect on TRAIL-R1 expression was mimicked by ectopic expression of a kinase-active version of the TGFβ type I receptor ALK5 (ALK5-T204D) but not by ALK5 double mutant lacking the ability to phosphorylate Smad proteins (RImL45-T204D). Moreover, TGFβ regulation of TRAIL-R1 was absent in two PDAC cell lines lacking the Smad4 gene DPC4 and siRNA-mediated silencing of Smad4 in Smad4-positive Panc1 cells abolished the TGFβ-mediated decrease in TRAIL-R1 expression, together showing that ALK5/Smad4 signaling is crucial for TGFβ regulation of TRAIL-R1 expression. Our results suggest a novel tumor-promoting function of TGFβ1. By downregulating TRAIL-R1, TGFβ1 may not only promote tumor escape from immune surveillance but also negatively impact on TRAIL- or TRAIL-R1-based therapy regimens for treatment of PDAC.