Inhibition of p70 S6 kinase (S6K1) activity by A77 1726 and its effect on cell proliferation and cell cycle progress

Abstract

Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor-mediated signaling pathway. In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726-induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726-induced cell cycle arrest in the S phase.

Figure 1

Effect of A77 1726 on the PI3K and MAP kinase pathways. A375 cells seeded in a six-well plate were starved in DMEM containing 0.5% FBS for 2 hours and treated with the indicated concentration of A77 1726 for another 2 hours (A) or treated with A77 1726 (100 μM) for the indicated time (B). Cells were harvested and analyzed for protein phosphorylation by specific antibodies as indicated. Protein loading was monitored by stripping membrane and reprobing with antibodies against non-phosphorylated proteins.

Figure 2

Mechanisms of A77 1726–induced feedback activation of the PI3K and MAP kinase pathways. (A) Inability of uridine to reverse the effect of A77 1726 on the PI3K and MAP kinase pathways. A375 cells seeded in a six-well plate were starved in DMEM containing 05% FBS for 2 hours and treated with the indicated concentration of A77 1726 in the absence or presence of uridine (200 μM) for 2 hours. Phosphorylated and total proteins were analyzed by Western blot as described in Figure 1. (B) In vitro S6K1 kinase assay. Leflunomide and A77 1726 diluted at the final concentrations as indicated were premixed with S6K1 for 30 minutes, followed by the addition of peptide substrate of S6K1 and incubation for 1 hour. S6K1 activity was measured by using an ADP-Glo system. The experiment was repeated with similar results. The data represent the mean ± SD from one experiment in triplicate. (C) The effect of the MAP kinase pathway inhibitors on A77 1726–induced feedback activation of the PI3K and MAP kinase pathways. A375 cells seeded in six-well plates were starved in DMEM containing 0.5% FBS for 2 hours and then pretreated with vehicle (0.1% DMSO) or the inhibitors of the MAP kinase pathway (PLX4720, 1 μM; U0126, 10 μM) for 1 hour. Cells were then treated with A77 1726 (100 μM) or rapamycin (20 nM) as indicated for 2 hours. Cells were harvested and analyzed for protein phosphorylation by specific antibodies as indicated. (D–F) The effect of PI3K, mTOR, and IGF-1 receptor inhibitors on A77 1726–induced feedback activation of the PI3K and MAP kinase pathways. A375 cells seeded in six-well plates were starved in DMEM containing 0.5% FBS for 2 hours and then pretreated with vehicle (0.1% DMSO), LY294002 (10 μM) or rapamycin (20 nM) (D), PPP (1 μM) (E), or OSI-906 (0.2 μM) (F) for 1 hour. Cells were then treated with A77 1726 (100 μM) as indicated for 2 hours. Cells were harvested and analyzed for protein phosphorylation by specific antibodies as indicated. Protein loading was monitored by stripping membrane and reprobing with antibodies against non-phosphorylated proteins.

Figure 3

Antiproliferative effect of A77 1726. A375 cells were seeded in 96-well plates (2000 cells per well) and incubated at the indicated concentrations of A77 1726 for 72 hours in the absence or presence of various concentrations of PLX4720 with (A) or without uridine (200 μM) (B). Cell proliferation was analyzed by an ATP-based Cell-Glo assay. Data from one representative of three experiments with similar results were shown.

Figure 4

Effect of S6K1 knockdown on feedback PI3K pathway activation and cell proliferation. (A) S6K1 knockdown. A375 cells seeded in a six-well plate were transfected with scrambled or S6K1 siRNA (2.5 nmol each). After incubation for 48 hours, the cells were harvested and analyzed for S6K1 expression and phosphorylation of the indicated proteins by Western blot. (B) The effect of S6K1 knockdown on cell proliferation. A375 cells seeded in a 96-well plate were transfected with a scrambled control siRNA or S6K1 siRNA. After incubation overnight, the cells were incubated in the absence or presence of A77 1726 (100 or 200 μM) for 72 hours and analyzed for cell proliferation by an ATP-based Cell-Glo assay. Data represent the mean ± SD from one of two experiments in triplicate with similar results. *P < .05, compared to control siRNA-transfected A375 cells.

Figure 5

Effect of exogenous uridine and MAP kinase pathway inhibitors on A77 1726–stimulated S phase entry and cell cycle arrest. (A) Ability of uridine to relieve the cell cycle arrest in the S phase. A375 cells grown in six-well plates were treated with the indicated concentration of A771726 with or without uridine (200 μM) for 24 hours. Cell cycle was analyzed in a flow cytometer as described in the Materials and Methods section. (B and C) Effect of the MAP kinase pathway inhibitors (B) and the PI3K pathway inhibitors (C) on A77 1726–mediated cell cycle arrest in the S phase. A375 cells were treated with A771726 (100 μM) in the absence or presence of 0.1% DMSO, PLX 4720 (1 μM), U0126 (10 μM) (B), or rapamycin (20 nM) and LY294002 (10 μM) (C) for 24 hours. Single-cell suspensions were prepared and analyzed for cell cycle in a flow cytometer.

Figure 6

Effect of exogenous uridine and MAP kinase pathway inhibitors on A77 1726–stimulated BrdU incorporation. (A) Effect of uridine to A77 1726–induced DNA synthesis. A375 cells were treated with the indicated concentration of A771726 in the absence or presence of uridine (200 μM) for 22 hours. After pulsing with BrdU (10 μM) for 2 hours, cells were harvested and analyzed for BrdU incorporation by staining with an Alexa Fluor 488–conjugated anti-BrdU monoclonal antibody followed by flow cytometry. (B and C) Effect of the MAP kinase pathway inhibitors (B) and the PI3K pathway inhibitors (C) on A77 1726–stimulated BrdU incorporation. A375 cells were treated with A77 1726 (100 μM) in the presence of 0.1% DMSO, PLX4720 (1 μM), U0126 (10 μM) (B) or rapamycin (20 nM), and LY294002 (10 μM) (C). After incubation for 22 hours, the cells were pulsed with BrdU for 2 hours. Single-cell suspensions were stained for BrdU incorporation and analyzed for cell cycle in a flow cytometer as described in A.

Figure 7

Mechanisms of action of A77 1726 on cell cycle progress and proliferation. A77 1726 inhibits the activity of S6K1, leading to the feedback activation of the PI3K and MAP kinase pathways through IGF-1 receptor. MAP kinase activation accelerates the entry of cell cycle into the S phase. However, due to the depletion of pyrimidine nucleotide pools through the inhibition of DHO-DHase and CAD activity, DNA replication and chromosome duplication cannot be completed, leading to the stall of cell cycle in the S phase. A77 1726 suppresses cell proliferation by inhibiting DNA and protein synthesis. In some types of cancer, A77 1726 may also inhibit cell proliferation by inhibiting the activity of protein tyrosine kinases.