Downregulation of TRAIL-Receptor 1 Increases TGFβ Type II Receptor Expression and TGFβ Signalling Via MicroRNA-370-3p in Pancreatic Cancer Cells

Abstract

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 β (TGFβ). Transfection of cells with an artificial miR-370-3p decreased the levels of TGFβ-RII. We further show that transient expression of the miR-370 mimic decreased TGFβ1-induced expression of SERPINE1 encoding plasminogen activator-inhibitor 1 and partially relieved TGFβ1-induced growth inhibition. Moreover, stable TRAIL-R1 knockdown in Colo357 cells increased TGFβ1-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 TGFβ1. Taken together, our study reveals that TRAIL-R1 through regulation of miR-370 can decrease the sensitivity of PDAC cells to TGFβ and therefore represents a potential tumour suppressor in late-stage PDAC.

Keywords: TGFβ; TGFβ receptor II; TRAIL; TRAIL-receptor 1; microRNA; pancreatic ductal adenocarcinoma; signalling.

Figure 1

Downregulation of tumour necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIL-R1) decreases levels of mature miR-370-3p but not of pri-miR-370. (A) Quantitative PCR analyses detecting changes in the levels of mature miR-370-3p in Panc1 cells transfected for 40 h with a control-siRNA (ctrl.-si) or siRNA against TRAIL-R1 (TRAIL-R1-si). (B) Panc1 cells transfected with ctrl.-si were stimulated with anti-TRAIL (10 µg/mL), TRAIL (10 ng/mL) or left untreated. Levels of mature miR-370-3p were quantified by qPCR. (C,D) QPCR analyses of pri-miR-370 levels in Panc1 cells transiently transfected with ctrl.-si or TRAIL-R1-si (C) or in ctrl.-si transfected cells with and without treatment with TRAIL (10 ng/mL) or anti-TRAIL (1 µg/mL) (D). Shown are the mean ± SD of five biological replicates (n = 5), with each one analysed in technical duplicates. The asterisks (*) indicate significance (p < 0.05); n.s.: not significant.

Figure 2

Ectopic expression of miRNA-370-3p in PDAC cells decreases the abundance of TGFβ-RII. Panc1 cells were transfected with 50 nM of an artificial miR-370-3p (miRNA-370-3p mimic) for the indicated periods of time. The levels of TGFβ-RII were analysed by Western blotting in whole cell lysates. Detection of β-actin served as a loading control. The graph underneath the blot shows results from densitometric quantification of band intensities from three independent experiments (mean ± SD, n = 3). The asterisks (*) indicate significance (p < 0.05) relative to respective untreated control.

Figure 3

Knockdown of TRAIL-R1 increases the abundance of TGFβ-RII in Panc1 cells. Panc1 cells were transfected with siRNA against TRAIL-R1 or with control siRNA for 72 h without (A) or with (B) exposure to a neutralizing antibody against TRAIL (anti-TRAIL, 10 µg/mL) or (C) recombinant TRAIL (10 ng/mL). The expression of TRAIL-R1 and TGFβ-RII was analysed by Western blotting in whole cell lysates. As control for equal gel loading, levels of β-actin were determined in parallel. The blots shown are representative of three independent experiments yielding very similar results. (D) Densitometry-based quantification of the Western blots shown in (A). Data were compiled from three independent experiments and represent the mean ± SD (n = 3). (E) Densitometry-based quantification of the Western blots shown in (B). (F) Densitometry-based quantification of the Western blots shown in (C). The asterisks (*) in (D–F) indicate significance relative to the ctrl.-siRNA; n.s.: not significant.

Figure 4

Knockdown of TRAIL-R1 might increase the activation of Smad2 and JNK. Panc1 cells have been transfected with control siRNA (ctrl.-siRNA) or siRNA against TRAIL-R1 (TRAIL-R1-siRNA) for 72 h prior to treatment with TGFβ1 (0.2 ng/mL) for 50 min. Western blot analysis of (A) p-Smad2C and (B) p-JNK. Detection of β-actin served as control for equal loading. In (A) in the Smad2/3 panel, the upper band represents Smad2. The blots in (A,B) are representative of three independent experiments all yielding, albeit to a varying extent, an induction of p-Smad2C and p-JNK levels, respectively, in TRAIL-R1-siRNA over ctrl.-siRNA transfected cells. (C) Densitometry-based quantification of the p-Smad2 bands shown in (A). (D) Densitometry-based quantification of the p-JNK bands shown in (B).

Figure 4

Knockdown of TRAIL-R1 might increase the activation of Smad2 and JNK. Panc1 cells have been transfected with control siRNA (ctrl.-siRNA) or siRNA against TRAIL-R1 (TRAIL-R1-siRNA) for 72 h prior to treatment with TGFβ1 (0.2 ng/mL) for 50 min. Western blot analysis of (A) p-Smad2C and (B) p-JNK. Detection of β-actin served as control for equal loading. In (A) in the Smad2/3 panel, the upper band represents Smad2. The blots in (A,B) are representative of three independent experiments all yielding, albeit to a varying extent, an induction of p-Smad2C and p-JNK levels, respectively, in TRAIL-R1-siRNA over ctrl.-siRNA transfected cells. (C) Densitometry-based quantification of the p-Smad2 bands shown in (A). (D) Densitometry-based quantification of the p-JNK bands shown in (B).

Figure 5

Transfection with a miR-370-3p mimic decreases TGFβ1-induced expression of PAI-1 and SLUG. Panc1 cells were transiently transfected with 50 nM of control (ctrl) miR or a miR-370-3p mimic and 48 h later remained unstimulated (0) or were stimulated with TGFβ1 (5 ng/mL) for 24 or 72 h (PAI-1), or 24 h (SLUG). Cells were examined for PAI-1- and SLUG- expression by qPCR. Data represent the mean ± SD of three independent experiments (n = 3). The asterisks (*) indicate significance relative to control (p < 0.05).

Figure 6

Transfection with a miR-370-3p mimic decreases TGFβ1-induced growth inhibition. Panc1 cells were transiently transfected twice on two consecutive days with 50 nM of control (ctrl) miR or a miR-370-3p mimic and 24 h after the second round of transfection remained unstimulated (0) or were stimulated with TGFβ1 (5 ng/mL) for 72 h. Following TGFβ1 stimulation, cells were detached and viable cells counted. Data represent the mean ± SD of three independent experiments (n = 3). The number of non-TGFβ1-treated control cells were set arbitrarily at 100%. The asterisk (*) indicates significance (p < 0.05).

Figure 7

Knockdown of TRAIL-R1 in Colo357 cells enhances TGFβ1-mediated induction of PAI-1 expression. Colo357 cells stably transduced with a TRAIL-R1 shRNA, or control (ctrl.) shRNA remained unstimulated (0) or were stimulated with TGFβ1 (5 ng/mL) for 24 h. Cells were assayed for PAI-1 mRNA expression by qPCR. Data represent the mean ± SD of three independent experiments (n = 3). The asterisk (*) indicates significance relative to control (p < 0.05).

Figure 8

Ectopic expression of a miR-370-3p mimic partially rescues Colo357 cells from the TRAIL-R1 shRNA-induced TGFβ1 hyperstimulation of PAI-1 expression. Colo357 cells with stable expression of a TRAIL-R1 shRNA were transiently transfected with 50 nM of control (ctrl.) miR or a miR-370-3p mimic and 48 h later stimulated with TGFβ1 (5 ng/mL) for 24 h. Data represent the mean±SD of three independent experiments (n = 3). The asterisk (*) indicates significance relative to control (p < 0.05).