Targeting of AKT/ERK/CTNNB1 by DAW22 as a potential therapeutic compound for malignant peripheral nerve sheath tumor

Abstract

Malignant peripheral nerve sheath tumors (MPNSTs) are an aggressive form of soft tissue neoplasm with extremely poor prognosis and no effective medical options currently available. MPNSTs can occur either sporadically or in association with the neurofibromatosis type 1 (NF1) syndrome. Importantly, activation of RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, and WNT/CTNNB1 signaling pathways has been reported in both NF1-related and late-stage sporadic MPNSTs. In this study, we found that DAW22, a natural sesquiterpene coumarin compound isolated from Ferula ferulaeoides (Steud.) Korov., could inhibit cell proliferation and colony formation in five established human MPNST cancer cell lines. Further molecular mechanism exploration indicated that DAW22 could target the main components in the MPNST tumorigenic pathways: namely suppress phosphorylation of AKT and ERK, and reduce levels of non-phospho (active) CTNNB1. Using the xenograft mouse model transplanted with human MPNST cancer cell line, daily treatment with DAW22 for 25 days was effective in reducing tumor growth. These results support DAW22 as an alternative therapeutic compound for MPNST treatment by affecting multiple signaling transduction pathways in its disease progression.

Keywords: AKT; DAW22; ERK; MPNST; apoptosis.

Figure 1

DAW22 inhibited cell proliferation of sporadic and NF1‐related MPNST cell lines. A, Chemical structure of DAW22. B, MTS cell viability assay was performed on MPNST cell lines after exposure to indicated concentrations of DAW22 for 48 h. C, Concentrations of DAW22 inducing 50% growth inhibition (IC₅₀) in sporadic and NF1‐related MPNST cell lines. ST8814 and T265 cell lines were more resistant to DAW22, compared with S462, S462‐TY, and STS‐26T cell lines. DAW22 IC₅₀ values were calculated using GraphPad Prism (Version 6). D, Colony formation analyses of five MPNST cell lines after DAW22 treatment. These five cell lines were seeded on six‐well plate at a density of 1000 cells per well, treated with 30 and 45 μmol/L DAW22 for 2 wk, and stained with crystal violet. Values were presented as mean ± SEM of three independent experiments. **P < 0.01, compared with vehicle control.

Figure 2

Cell cycle analyses and morphological changes in DAW22‐treated MPNST cell lines. Negative effect of DAW22 on cell cycle of MPNST cancer cell lines STS‐26T (A) and S462 (B). Cells were seeded on six‐well plate and treated with 30 and 60 μmol/L of DAW22 for 24 h. Cell cycle was analyzed by propidium iodide staining and flow cytometry. C, Morphological changes observed in DAW22‐treated MPNST cell lines. Cell shrinkage, rounding, and loss of adhesion in culture medium were observed, indicating cellular damage or cell death. Scale bars, 10 μm.

Figure 3

Induced apoptosis in DAW22‐treated MPNST cells lines. Cells were exposed with different concentrations of DAW22 for 48 h. Western blot analyses were performed to detect levels of both full‐length (FL) and cleaved (CF) versions of CASP3 (A) and PARP (B). Quantitative analyses of CF relative to its FL shown in (A) and (B). Values were expressed as mean ± SEM of three independent blots. *P < 0.05; **P < 0.01, compared with vehicle control. ACTB loading only shown in (B). Western blot images shown in (A) and (B) were representative results showing similar trend from at least three independent experiments.

Figure 4

DAW22 reduced phosphorylation of AKT, ERK, and non‐phospho (active) CTNNB1 in MPNST cell lines. Cells were treated with different concentrations of DAW22 for 48 h. Levels of phosphorylated AKT/ERK, total AKT/ERK, and active CTNNB1 were detected by Western blot analyses, as shown in (A), (B), and (C). Quantitative analyses of phosphorylated protein relative to its total protein shown in (A) and (B), while active CTNNB1 relative to ACTB was shown in (C). Values were expressed as mean ± SEM of three independent blots. *P < 0.05; **P < 0.01, compared with vehicle control. ACTB loading only shown in (C). Western blot images shown in (A), (B), and (C) were representative results showing similar trend from at least three independent experiments.

Figure 5

In vivo anti‐cancer effect of DAW22 on STS‐26T‐transplanted xenograft mouse model. A, Quantitative analyses of tumor volume in mice from vehicle‐treated and DAW22‐treated groups. Six‐week‐old nude mice were engrafted with STS‐26T cells and treated with DAW22 (60 mg/kg/d) 1 wk after transplantation. DAW22 was introduced by intraperitoneal injection once daily for 25 d. B, Body weights from both vehicle‐treated and DAW22‐treated groups showed no significant differences for the entire treatment period of 25 d. C, Representative images of STS‐26T subcutaneous tumor xenografts at experimental end point. Scale bar, 1 cm. D, Significant reduction in tumor weights from DAW22‐treated group compared with vehicle‐treated animals. Values were expressed as mean ± SEM; *P < 0.05. E, Protein was isolated from transplanted xenograft tumors from both vehicle‐treated and DAW22‐treated groups. Expression levels of phosphorylated AKT/ERK, total AKT/ERK, and active CTNNB1 were evaluated by Western blot analyses.

Figure 6

DAW22 targets multiple signaling pathways involved in MPNST disease progression. DAW22 inhibits expression of phosphorylated ERK, AKT, and non‐phosphorylated (active) CTNNB1. This contributes to the induction of apoptosis by DAW22 in MPNST in vitro and in vivo.