Ornithine Decarboxylase Inhibition by α-Difluoromethylornithine Activates Opposing Signaling Pathways via Phosphorylation of Both Akt/Protein Kinase B and p27Kip1 in Neuroblastoma

Abstract

Ornithine decarboxylase (ODC) is a key enzyme in mammalian polyamine biosynthesis that is up-regulated in various types of cancer. We previously showed that treating human neuroblastoma (NB) cells with the ODC inhibitor A-difluoromethylornithine (DFMO) depleted polyamine pools and induced G 1 cell cycle arrest without causing apoptosis. However, the precise mechanism by which DFMO provokes these changes in NB cells remained unknown. Therefore, we further examined the effects of DFMO, alone and in combination with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or Akt/protein kinase B (PKB) inhibitor IV, on the regulation of cell survival and cell cycle-associated pathways in LAN-1 NB cells. In the present study, we found that the inhibition of ODC by DFMO promotes cell survival by inducing the phosphorylation of Akt/PKB at residue Ser 473 and glycogen synthase kinase-3B at Ser 9 . Intriguingly, DFMO also induced the phosphorylation of p27Kip1 at residues Ser 10 (nuclear export) and Thr 198 (protein stabilization) but not Thr 187 (proteasomal degradation). The combined results from this study provide evidence for a direct cross-talk between ODC-dependent metabolic processes and well-established cell signaling pathways that are activated during NB tumorigenesis. The data suggest that inhibition of ODC by DFMO induces two opposing pathways in NB: one promoting cell survival by activating Akt/PKB via the PI3K/Akt pathway and one inducing p27 Kip1 /retinoblastomacoupled G 1 cell cycle arrest via a mechanism that regulates the phosphorylation and stabilization of p27 Kip1 . This study presents new information that may explain the moderate efficacy of DFMO monotherapy in clinical trials and reveals potential new targets for DFMO-based combination therapies for NB treatment. [Cancer Res 2008;68(23):9825-31]

S-adenosylmethionine limitation induces p38 mitogen activated protein kinase and triggers cell cycle arrest in G1

“…AdoMetDC consumes SAM to produce aminopropyl groups for polyamine synthesis. It was thus possible that SAM depletion decreases polyamine pools and induces polyaminedepletion phenotypes, which are characterized by Akt activation, p27-dependent G1 arrest and p53-dependent apoptosis (Koomoa et al, 2009;Koomoa et al, 2008;Wallick et al, 2005). However, we did not detect any significant upregulation of p21, p27 or p53 levels during SAM checkpoint activation (supplementary material Fig.…”

“…In the case of methionine-free medium, RPMI 1640 medium was replaced with RPMI 1640 medium deficient in methionine, cysteine, and glutamine (R9016, US biological) and was supplemented with L-cysteine and L-glutamine at the same concentration as in standard RPMI 1640 medium. Medium supplemented with spermidine contained 1 mM aminoguanidine to inhibit serum polyamine oxidase (Koomoa et al, 2008). MK2 inhibitor MK2III was from Calbiochem (EMD Millipore, KGaA, Darmstadt, Germany).…”

Trial Watch: Immunogenic cell death inducers for anticancer chemotherapy

“…The efficacy of bortezomib is being assessed in individuals with (1) various hematological malignancies, who often receive bortezomib as part of multimodal chemo-or immunotherapeutic regimens (NCT02037256; NCT02112916; NCT02208037; NCT02312102); (2) neuroblastoma, who are treated with bortezomib plus difluoromethylornithine (a hitherto experimental inhibitor of polyamine biosynthesis), 148,149 (NCT02139397); and (3) various solid tumors, who receive bortezomib as standalone therapeutic agent or combined with standard chemotherapy (NCT02211755; NCT02220049) ( Table 2). …”

Small Molecule Drugs and Targeted Therapies for Neuroblastoma

“…In looking towards future clinical application, targeted therapies may be useful in combination with conventional chemotherapeutic agents in order to achieve synergistic effects, or alternatively, the combination of two targeted therapeutics may also prove efficacious. For example, recent studies have shown that DFMO acts synergistically with PI3 kinase inhibitors to increase apoptosis in neuroblastoma cells (Koomoa, Yco et al, 2008). Overall, despite the existing challenges in the discovery of new drugs, an increased understanding of cancer genetics is allowing the development of powerful drug or drug combinations that may increase the selectivity and safety of chemotherapy by selective killing of cancer cells and protecting normal cells.…”