PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells
Lei Qi1, Hidemi Toyoda2, Dong-Qing Xu1
1Department 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.
Purpose:
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.
Experimental Design:
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.
Conclusion:
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.
Insights
Neuroblastoma cells can develop resistance to the AKT inhibitor MK2206. Targeting the PDK1-mTOR-S6K pathway offers a promising strategy to overcome this resistance in cancer therapy.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- AKT signaling is crucial for cancer cell transduction.
- MK2206, an AKT inhibitor, shows efficacy against various cancer cell lines.
- Acquired resistance to MK2206 necessitates novel therapeutic strategies.
Purpose of the Study:
- To investigate the mechanisms of MK2206 resistance in neuroblastoma (NB) cells.
- To identify alternative therapeutic targets for MK2206-resistant NB.
- To evaluate the efficacy of PDK1 and mTOR inhibitors in overcoming MK2206 resistance.
Main Methods:
- Induction of MK2206-resistant NB cell sublines through stepwise MK2206 escalation (4-12 weeks).
- Assessment of cell proliferation using MTT assays.
- Cell cycle analysis via flow cytometry.
- Investigation of cell signaling pathways using Western blot assays.
Main Results:
- MK2206 effectively inhibited non-resistant NB cells but showed limited efficacy against resistant sublines.
- Resistant NB sublines exhibited increased sensitivity to PDK1 (GSK2334470) and mTOR (AZD8805) inhibitors.
- Resistant cells displayed elevated PDK1-mTOR-S6K pathway activity with reduced AKT phosphorylation.
- PDK1 and mTOR inhibitors induced cell cycle arrest in resistant cells.
Conclusions:
- Neuroblastoma cells acquire MK2206 resistance within 4-12 weeks of exposure.
- Resistant cells depend on the PDK1-mTOR-S6K pathway and are more vulnerable to PDK1 and mTOR inhibitors.
- Inhibiting the PDK1-mTOR-S6K pathway is a viable strategy to overcome MK2206 resistance and enhance targeted cancer therapy.
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