RESUMO
The accumulation of perturbations in signalling pathways resulting in an apoptosis-insensitive phenotype is largely responsible for the desperate prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). Accumulating evidence suggests that the death receptors TRAIL-R1 and TRAIL-R2 play important roles in PDAC biology by acting as either tumour suppressors through induction of cell death or tumour promoters through induction of pro-inflammatory signalling, invasion and metastasis. TRAIL-R2 can also associate with nuclear proteins and alter the maturation of micro RNAs (miRs). By genome-wide miR profiling and quantitative PCR analyses we now demonstrate that knockdown of TRAIL-R1 in PDAC cells decreased the level of mature miR-370 and led to an increased abundance of the type II receptor for transforming growth factor ß (TGFß). Transfection of cells with an artificial miR-370-3p decreased the levels of TGFß-RII. We further show that transient expression of the miR-370 mimic decreased TGFß1-induced expression of SERPINE1 encoding plasminogen activator-inhibitor 1 and partially relieved TGFß1-induced growth inhibition. Moreover, stable TRAIL-R1 knockdown in Colo357 cells increased TGFß1-induced SERPINE1 expression and this effect was partially reversed by transient expression of the miR-370 mimic. Finally, after transient knockdown of TRAIL-R1 in Panc1 cells there was a tendency towards enhanced activation of Smad2 and JNK1/2 signalling by exogenous TGFß1. Taken together, our study reveals that TRAIL-R1 through regulation of miR-370 can decrease the sensitivity of PDAC cells to TGFß and therefore represents a potential tumour suppressor in late-stage PDAC.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is characterized by both, overexpression of transforming growth factor (TGF)ß 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 TGFß acts as a tumor promoter in advanced tumor stages and suppression of apoptosis is a known driver of tumor progression, it is possible that TGFß functions as a crucial determinant of tumor cell sensitivity to apoptosis in PDAC. Here, we have studied the impact of TGFß on TNF-related apoptosis inducing ligand (TRAIL)-induced signaling in PDAC cells. In TGFß-responsive Panc1 and Colo357 cells, TGFß1 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, TGFß1 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 TGFß1 effect on TRAIL-R1 expression was mimicked by ectopic expression of a kinase-active version of the TGFß type I receptor ALK5 (ALK5-T204D) but not by ALK5 double mutant lacking the ability to phosphorylate Smad proteins (RImL45-T204D). Moreover, TGFß 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 TGFß-mediated decrease in TRAIL-R1 expression, together showing that ALK5/Smad4 signaling is crucial for TGFß regulation of TRAIL-R1 expression. Our results suggest a novel tumor-promoting function of TGFß1. By downregulating TRAIL-R1, TGFß1 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.