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. Invitro 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.

Inhibition of p70 S6 kinase (S6K1) activity by A77 1726, the active metabolite of leflunomide, induces autophagy through TAK1-mediated AMPK and JNK activation

“…Our recent study showed that A77 1726 induces the feedback activation of the PI-3 and MAP kinase pathways; and that PLX4720, an inhibitor of Raf kinase, and U0126, a MEK inhibitor, block A77 1726-induced phosphorylation of ERK1/2 T202/Y204 and MEK1/2 S217/S221 [32]. Here we found that these inhibitors did not change the levels of LC3-II lipidation in the presence of A77 1726 (Figure 3E).…”

Leflunomide/teriflunomide inhibit Epstein-Barr virus (EBV)-induced lymphoproliferative disease and lytic viral replication

“…Thus these drugs also inhibit the proliferation of EBV-transformed B cells through “off-target” mechanisms. Although the precise mechanism(s) for these off-target effects on EBV-transformed B cells remain to be determined, they likely reflect the previously described ability of higher dose leflunomide/teriflunomide treatment to inhibit multiple different cellular tyrosine kinases, as well as AKT, S6 Kinase, NF-KB and STAT3 signaling [ 16 , 23 , 52 ]. Interestingly, however, we found increased, rather than decreased, NF-kappa B signaling in teriflunomide treated LCLs, presumably due to the increased expression of LMP1.…”

Inactivation/deficiency of DHODH induces cell cycle arrest and programed cell death in melanoma

“…Since cancerous cells need more nutrient substance to produce new cells, metabolic signaling pathways are suggested to be essential during carcinogenesis [ 33 ]. As a rate-limiting enzyme of nucleotide metabolism pathway, DHODH seemed to contribute to malignant characteristics of various tumors, including multiple myeloma [ 16 ], chronic lymphocytic leukemia [ 34 – 37 ], neuroblastoma [ 38 ], colon cancer [ 9 ], prostate cancer [ 39 ], and so on.…”

“…Importantly, DHODH inhibition by leflunomide induced a significant decrease in melanoma growth both in vitro and in vivo studies [ 18 ]. Several other studies also showed that teriflunomide could suppress growth of melanoma cells [ 16 , 19 ]. However, the mechanisms underlying remained to be further explored.…”