title: PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells.
abstract: OBJECTIVE
  AKT plays a pivotal role in the signal transduction of cancer cells. MK2206, an AKT inhibitor, has been shown to be an effective anti-cancer drug to a variety of cancer cell lines. However, some cancer cells acquire resistance to MK2206 and new strategies to suppress these cell lines remain to be developed.
  
  
  METHODS
  Acquired MK-2206-resistant neuroblastoma (NB) cell sublines were induced by stepwise escalation of MK-2206 exposure (4-12 weeks). MTT assay was used to validate cell proliferation. Flow cytometry was performed for cell cycle analysis. Western blot assay was used for cell signaling study.
  
  
  RESULTS
  MK2206 (5-10 µmol) significantly suppressed cell growth of MK2206 non-resistant NB cells (LAN-1, KP-N-SIFA, NB-19 and SK-N-DZ), but is less efficient in inhibiting that of resistant sublines, even after 2-week MK2206-free incubation. MK2206 acted in mTOR-S6K dependent and independent methods. MK-2206 resistant sublines (LAN-1-MK, KP-N-SIFA-MK, and SK-N-DZ-MK) showed lower IC50 of GSK2334470 (PDK1 inhibitor). The cell growth of all sublines was prohibited by AZD8805 (mTOR inhibitor), with IC50 of AZD8805 3-10 times lower than MK2206 non-resistant cells. The signaling profiles of these resistant sublines were characterized by elevated PDK1-mTOR-S6K activity, accompanying by low phosphorylation of AKT compared with non-resistant counterparts. GSK2334470 and AZD8055 effectively inhibited phosphorylation of PDK1 and mTOR, respectively, and induced higher G0-G1 ratio in LAN-1-MK than that in LAN-1 as well. PDK1 and mTOR inhibitors effected on phosphorylation of GSK3β in some of resistant sublines.
  
  
  CONCLUSIONS
  NB cells can acquire MK2206 resistance after exposure for 4-12 weeks. Resistant cells feature reliance on PDK1-mTOR-S6K pathway and are more sensitive to PDK1 and mTOR inhibitors than the non-resistant counterparts. Thus, suppression of PDK1-mTOR-S6K signaling pathway is an effective way to overcome the MK2206 resistance, and this may be a promising strategy for targeted therapy.
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan ; Department of Pediatrics, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai, 200092 China.
authors.forename: Lei
authors.initials: L
authors.lastname: Qi
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan.
authors.forename: Hidemi
authors.initials: H
authors.lastname: Toyoda
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan ; Department of Pediatrics, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai, 200092 China.
authors.forename: Dong-Qing
authors.initials: DQ
authors.lastname: Xu
authors.affiliation: Department of Child Health Nursing, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Sakurai
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan.
authors.forename: Keishirou
authors.initials: K
authors.lastname: Amano
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kihira
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Hori
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Azuma
authors.affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Komada
keywords: Akt; Cell growth; MK2206; Neuroblastoma; PDK1; mTOR