Dp44mT targets the AKT, TGF-β and ERK pathways via the metastasis suppressor NDRG1 in normal prostate epithelial cells and prostate cancer cells

Abstract

Background: Effective treatment of prostate cancer should be based on targeting interactions between tumour cell signalling pathways and key converging downstream effectors. Here, we determined how the tumourigenic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), tumour-suppressive phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and transforming growth factor-β (TGF-β) pathways are integrated via the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1). Moreover, we assessed how the novel anti-tumour agent, Dp44mT, may target these integrated pathways by increasing NDRG1 expression.

Methods: Protein expression in Dp44mT-treated normal human prostate epithelial cells and prostate cancer cells (PC-3, DU145) was assessed by western blotting. The role of NDRG1 was examined by transfection using an NDRG1 overexpression vector or shRNA.

Results: Dp44mT increased levels of tumour-suppressive PTEN, and decreased phosphorylation of ERK1/2 and SMAD2L, which are regulated by oncogenic Ras/MAPK signalling. Importantly, the effects of Dp44mT on NDRG1 and p-SMAD2L expression were more marked in prostate cancer cells than normal prostate epithelial cells. This may partly explain the anti-tumour selectivity of these agents. Silencing NDRG1 expression increased phosphorylation of tumourigenic AKT, ERK1/2 and SMAD2L and decreased PTEN levels, whereas NDRG1 overexpression induced the opposite effect. Furthermore, NDRG1 silencing significantly reduced the ability of Dp44mT to suppress p-SMAD2L and p-ERK1/2 levels.

Conclusion: NDRG1 has an important role in mediating the tumour-suppressive effects of Dp44mT in prostate cancer via selective targeting of the PI3K/AKT, TGF-β and ERK pathways.

Figure 1

Increase of NDRG1, p-NDRG1 (Ser³³⁰), p-NDRG1 (Thr³⁴⁶), PTEN, p-AKT (Ser⁴⁷³) and AKT protein levels in (A) PrEC, (B) PC-3 and (C) DU145 cells after a 24 h incubation at 37 °C with control medium, DFO (250 μℳ) or Dp44mT (2.5 μℳ). Western blots are typical of three independent experiments, with densitometric analysis representing mean±s.d. Relative to control: *P<0.05, **P<0.01, ***P<0.001.

Figure 2

The effect of iron supplementation on the DFO- or Dp44mT-induced increase in expression of NDRG1, PTEN and p-AKT (Ser⁴⁷³) in (A) PrEC, (B) PC-3 and (C) DU145 cells. Cells were incubated first with either control medium, DFO (250 μℳ) or Dp44mT (2.5 μℳ) for 14 h at 37 °C and then secondly re-incubated with either media alone, DFO (250 μℳ), Dp44mT (2.5 μℳ) or the iron donor, FAC (100 μg ml⁻¹), for a further 14 h at 37 °C. Western blots are typical of three independent experiments, with densitometric analysis representing mean±s.d. Relative to control: *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Incubation of cells with DFO (50, 100 and 250 μℳ) increases p-AKT (Ser⁴⁷³) levels, but in terms of its effect on downstream effectors, has no effect on p-mTOR (Ser²⁴⁴⁸), while decreasing cyclin D1 expression in (A) PrEC, (B) PC-3 and (C) DU145 cells after 24 h at 37 °C. Western blots are typical of three independent experiments, with densitometric analysis representing mean±s.d. Relative to control: *P<0.05, **P<0.01.

Figure 4

Incubation of cells with DFO (250 μℳ) or Dp44mT (2.5 μℳ) for 24 h at 37 °C decreases p-SMAD2L and p-ERK1/2 levels, but has no effect on total ERK1/2, or p-SMAD2C levels in (A) PrEC, (B) PC-3 and (C) DU145 cells. After incubation of DU145 cells with control medium or chelators, p-SMAD2C was barely detectable, but its levels were detected after (D) a 24-h incubation at 37 °C with TGF-β (10 ng ml⁻¹), indicating that the protein is inducible in DU145 cells. Western blots are typical of three independent experiments, with densitometric analysis representing mean±s.d. Relative to control: *P<0.05, **P<0.01, ***P<0.001.

Figure 5

Modulation of NDRG1 expression using (A) two shRNA constructs (sh 1 or sh 2) and the scrambled control, or (B) an overexpression construct (two separate clones: +1 and +2) and the vector control leads to alterations in the levels of PTEN, p-AKT (Ser⁴⁷³), p-SMAD2L, SMAD2 and p-ERK1/2 in DU145 cells compared with the respective controls. Western blots are typical of three independent experiments, with densitometric analysis representing mean±s.d. Relative to control: *P<0.05, **P<0.01, ***P<0.001.

Figure 6

(A) Decreasing NDRG1 expression via shRNA reduces the DFO- or Dp44mT-induced decrease in p-SMAD2L, SMAD2 and p-ERK1/2 levels in DU145 cells. Cells with and without silenced NDRG1 (shNDRG1) were incubated for 24 h at 37 °C with DFO (250 μℳ) or Dp44mT (2.5 μℳ). Western blots are typical of three independent experiments, with densitometric analysis representing mean±s.d. *P<0.05, **P<0.01 significantly different to non-silenced control; ^^P<0.01 significantly different to silenced control; ^#P<0.05, ^##P<0.01 significantly different to respective chelator (DFO or Dp44mT)-treated non-silenced control. (B) Schematic illustrating the effect of the chelators DFO and Dp44mT on key cell signalling pathways in prostate cancer. The studies herein demonstrate that the chelators upregulate NDRG1, which is required for the increase in the expression of the tumour suppressor, PTEN, leading to decreased cell proliferation. While the chelators also initially increase p-AKT, the NDRG1-mediated increase in PTEN may subsequently decrease p-AKT levels. The chelator-mediated increase in NDRG1 expression also reduces levels of oncogenic p-ERK and its downstream target, p-SMAD2L, preventing proliferation and accounting, in part, for the anti-tumour activity of these agents.