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. 2019 Sep 24;10(54):5645-5659.
doi: 10.18632/oncotarget.27210.

Dual-Targeting AKT2 and ERK in Cancer Stem-Like Cells in Neuroblastoma

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Free PMC article

Dual-Targeting AKT2 and ERK in Cancer Stem-Like Cells in Neuroblastoma

Kwang Woon Kim et al. Oncotarget. .
Free PMC article

Abstract

Neuroblastoma remains one of the most difficult pediatric solid tumors to treat. In particular, the refractory and relapsing neuroblastomas are highly heterogeneous with diverse molecular profiles. We previously demonstrated that AKT2 plays critical roles in the regulation of neuroblastoma tumorigenesis. Here we hypothesize that targeting AKT2 could block the signal transduction pathways enhanced in chemo- and/or radiation-resistant neuroblastoma cancer stem-like cells. We found cell proliferation and survival signaling pathways AKT2/mTOR and MAPK were enhanced in cisplatin (CDDP)- and radiation-resistant neuroblastoma cells. Blocking these two pathways with specific inhibitors, CCT128930 (AKT2 inhibitor) and PD98059 (MEK inhibitor) decreased cell proliferation, angiogenesis, and cell migration in these resistant cells. We further demonstrated that the resistant cells had a higher sphere-forming capacity with increased expression of stem cell markers CD133, SOX2, ALDH, Nestin, Oct4, and Nanog. Importantly, the tumorsphere formation, which is a surrogate assay for self-renewal, was sensitive to the inhibitors of AKT2 and MAPK. Taken together, our findings suggest that CDDP- and radiation-resistant cancer stem-like neuroblastoma cells might serve as a useful tool to improve the understanding of molecular mechanisms of therapeutic resistance. This may aid in the development of more effective novel treatment strategies and better clinical outcomes in patients with neuroblastoma.

Keywords: AKT2; MAPK; chemotherapy; radiotherapy; resistance.

Conflict of interest statement

CONFLICTS OF INTEREST The authors have no financial disclosures.

Figures

Figure 1
Figure 1. Drug/radiation selected human neuroblastoma cells in a dose-dependent manner.
(A) BE(2)-C and SK-N-AS cells were treated with increasing concentrations of cisplatin (CDDP) for 96 h. Cell survival was measured using Cell Counting Kit-8 (CCK-8). Data are the mean ± SEM (* = p < 0.005). (B) BE(2)-C and SK-N-AS cells were irritated with increasing dose of 137Cs (5 Gy-30 Gy) for 96 h. Cell survival was determined using CCK-8. Data are the mean ± SEM (* = p < 0.005). (C) Drug/radiation surviving cells were generated after 7 days in the BE(2)-C and SK-N-AS cells treated with 5 μM of CDDP and irradiated with 10 Gy of 137Cs respectively. Representative photographs are shown for surviving cells after 7 days treatment of CDDP and 137Cs.
Figure 2
Figure 2. CDDP-R/Rad-R human neuroblastoma cells increase proliferation and inhibit programmed cell death.
(A) CDDP-R/Rad-R BE(2)-C cells and CDDP-R/Rad-R SK-N-AS cells demonstrated increased proliferation when compared with parental BE(2)-C cells and SK-N-AS cells. Data are the mean ± SEM. * Significantly different at p < 0.005 vs. Parental. (B) Cleaved caspased-3 was inhibited in the CDDP-R BE(2)-C cells and CDDP-R SK-N-AS cells when compared with parental BE(2)-C cells and SK-N-AS cells after treatment with CDDP (5 μM) for 48 h. (C) Cleaved caspased-3 was inhibited in the Rad-R BE(2)-C cells and Rad-R SK-N-AS cells when compared with parental BE(2)-C cells and SK-N-AS cells after irritation with 137Cs (10 Gy) for 48 h. β-actin served as a protein loading control.
Figure 3
Figure 3. Activation of GRP-R/AKT2 and MAPK signaling pathways in CDDP-R/Rad-R BE(2)-C cells and CDDP-R/Rad-R SK-N-AS cells.
(A) Levels of GRP-R/p-AK2 and p-MAPK were spontaneously increased in CDDP-R/Rad-R BE(2)-C cells and CDDP-R/Rad-R SK-N-AS cells. β-actin protein was used as a loading control. (B) Parental BE(2)-C cells, CDDP-R/Rad-R BE(2)-C cells, parental SK-N-AS cells, and CDDP-R/Rad-R SK-N-AS cells were treated with DMSO, CCT128930 (AKT2 selective inhibitor, 10 μM), PD98059 (MAPK inhibitor, 100 μM), and combined CCT128930/PD98059 over a time course, and cell proliferation was measured using Cell Counting Kit-8. Data are the mean ± SEM. * Significantly different at p < 0.005 vs. parental. (C) Parental BE(2)-C cells, CDDP-R/Rad-R BE(2)-C cells, parental SK-N-AS cells and CDDP-R/Rad-R SK-N-AS cells were treated with DMSO, CCT128930 (AKT2 selective inhibitor, 10 μM), PD98059 (MAPK inhibitor, 100 μM), and combined CCT128930/PD98059. After 24 h, scratches were made using 200 μl tips. Wound closure was measured from microscopic images at 24 h and 72 h after wounding (100× magnification). (D) Data are representative of the mean distance of unclosure from three independent experiments (* = p < 0.05 vs. DMSO). (E) HUVECs were plated on 24-well plates coated with Matrigel and incubated with cell culture media from parental BE(2)-C cells, CDDP-R/Rad-R BE(2)-C cells, parental SK-N-AS cells and CDDP-R/Rad-R SK-N-AS cells treated with DMSO, CCT128930 (AKT2 selective inhibitor, 10 μM), PD98059 (MAPK inhibitor, 100 μM), and combined CCT128930/PD98059 for 48 h. Tubule staining was performed in triplicate and representative images shown (×200). (F) The numbers of tubule were counted. Values shown are mean ± SEM of three separate experiments (* = p < 0.05 vs. DMSO).
Figure 4
Figure 4. Markers of cancer stem cells in drug/radiation selected cells.
(A) Parental BE(2)-C cells, CDDP-R/Rad-R BE(2)-C cells, parental SK-N-AS cells, and CDDP-R/Rad-R SK-N-AS cells demonstrated increased expression of several stem cell markers, CD133, SOX2, ALDH, Nestin, Oct4, and Nanog, by Western blotting. β-actin served as a protein loading control. (B) Parental BE(2)-C cells, CDDP-R/Rad-R BE(2)-C cells, parental SK-N-AS cells, and CDDP-R/Rad-R SK-N-AS cells demonstrated neurosphere formation at 1 day and 5 days. Representative photographs are shown for floating spheres (scale bar = 100 μm). (C) CDDP-R/Rad-R BE(2)-C cells and CDDP-R/Rad-R SK-N-AS cells were altered to neurosphere formation. Spheres (a diameter >100 μm) were counted using inverted microscopy and quantified. Values shown are mean ± SEM of three separate experiments (* = p < 0.05 vs. Parental).
Figure 5
Figure 5. Signaling pathways in cancer stem-like cells with activated AKT2/mTOR and MAPK in Drug/Radiation selected cells.
(A) Sphere formation selected for subpopulations of CDDP-R/Rad-R BE(2)-C cells and CDDP-R/Rad-R SK-N-AS cells. The neurospheres demonstrated activation of p-AKT2, p-mTOR, p-MAPK, not p-p38 MAPK by Western blotting. β-actin served as a protein loading control. (B) Parental BE(2)-C cells, CDDP-R/Rad-R BE(2)-C cells, parental SK-N-AS cells and CDDP-R/Rad-R SK-N-AS cells were cultured in sphere formation media with DMSO, CCT128930 (AKT2 selective inhibitor, 10 μM), PD98059 (MAPK inhibitor, 100 μM), and combined CCT128930/PD98059 and a limiting dilution analysis was performed. Floating spheres were observed using inverted microscopy. Spheres with a diameter > 100 um were counted and quantified. Data represent frequency of sphere-formation (%) determined by 8-dose dilution and 6 wells/group in DMSO and treatment groups (mean ± SEM; * = p < 0.05 vs. DMSO). (C) Sphere formed from subpopulations of parental BE(2)-C cells, CDDP-R/Rad-R BE(2)-C cells, parental SK-N-AS cells, and CDDP-R/Rad-R SK-N-AS cells demonstrated deactivated levels of p-AKT2/p-mTOR/P-S6R protein and p-MAPK with DMSO, CCT128930 (AKT-2 selective inhibitor, 10 μM), PD98059 (MAPK inhibitor, 100 μM), and combined CCT128930/PD98059 treatments by Western blotting for 48 h. β-actin served as a protein loading control.
Figure 6
Figure 6. A schematic diagram demonstrating dual targeting of AKT2 and MAPK pathways in neuroblastoma cancer stem cells.
AKT2 and MAPK pathways were spontaneously overactivated in CDDP-R/Rad-R BE(2)-C cells and CDDP-R/Rad-R SK-N-AS cells growing up in neurosphere media. Despite targeting AKT2 by CCT128930, the MAPK pathway is still overactivated in neurosphere formation due to the effect of AKT2 inhibition. Therefore, dual-targeting of AKT2 and MAPK is far more effective in neuroblastoma cancer stem-like cells. Our data demonstrated that dual targeting of AKT2 and MAPK, by CCT128930 and PD98059 respectively, provides more effective therapeutic strategies for neuroblastoma cancer stem cells which can initiate a risk of relapse.

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