Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

Abstract

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS.

Fig. 1.

A large-scale, high-content imaging screen identifies compounds that induce differentiation of human ERMS cells. (A) Schematic of the experimental design. (B) Results for the differentiation screen. Lead compounds that increase the frequency of MF20-expressing RD cells are shown in the magnified box (>3δ). (C–J) Representative images of the MF20 (green) and DAPI (blue) staining in drug-treated cells. Control (DMSO, C) and a negative control compound (sunitinib, G) are shown. Blue, DAPI staining. (K) Quantification of MF20 induction in treated RD cells. Error bars denote ±1 SD. *P < 0.005; **P < 0.001; ***P < 0.0005.

Fig. 2.

A secondary screen identifies lead compounds that suppress ERMS tumor growth in live zebrafish. (A) Schematic of the secondary screen completed in zebrafish transplanted with fluorescent-labeled ERMS. (B–G) Pretreatment images for DMSO (B), sunitinib (C), and BIO (D), with corresponding posttreatment images (E, F, and G, respectively). Tumor volume is indicated by the heat map (Right). (Scale bar, 2 mm.) (H) Summary of tumor volume changes in animals treated with compounds that inhibit RAS-signaling in embryonic zebrafish (blue), representative compounds of major classes of hits identified in the human differentiation screen (yellow), or common hits from both screens (green). Error bars equal SD. *Statistical significance by Student t test, with P < 0.05.

Fig. 3.

Activation of canonical WNT/β-catenin pathway induces differentiation of human ERMS cells. (A) Graphic summary of MF20-positive cells in human ERMS treated with DMSO or BIO. *Statistically significant differences between experimental and control treated cells (P < 0.05). (B) AXIN2 expression assessed by real-time RT-PCR. (C and D) β-Catenin staining (green) in the presence of DMSO (C) or BIO (D). Blue, DAPI. (Scale bars, 50 μM.) (E) Quantification of MF20-positive cells in human ERMS treated with recombinant WNT3a, CTNNB1 siRNA, and doxycycline-inducible CTNNB1S33Y. *Significant difference for bracketed comparisons (P < 0.05, t test). (F) Western blot analysis.

Fig. 4.

BIO activates canonical WNT/β-catenin signaling and induces differentiation of ERMS cells in vivo. (A–D) H&E-stained sections (A and C) and immunohistochemistry for β-catenin (B and D) of ERMS-bearing fish after 7 d of drug treatment with BIO (300 nM) or DMSO (vehicle control). (Scale bar, 25 μM.) (E) Quantitative RT-PCR analysis. (F) Pie charts showing the relative percentage of myf5:GFP⁺/mylz2:mCherry⁻ TPCs (green), myf5:GFP⁺/mylz2:mCherry⁺ (yellow), and late-differentiating myf5:GFP⁻/mylz2:mCherry⁺ cells (red) for three independent ERMS as assessed by FACS (n > 3 animals per experimental arm and ±SD). *Statistically significant differences between DMSO and BIO treatments (Student t test, P < 0.05). (G) Summary of EDU staining of DMSO- or BIO-treated tumors. Each error bar indicates SEM of three tumors from each treatment group. *P < 0.005, Student t test.

Fig. 5.

Activation of canonical WNT/β-catenin pathway reduces self-renewal in human ERMS cells. (A–D) Quantitation of spheres in RD cells plated at limiting dilution in the presence of DMSO and BIO (A), BSA, and WNT3A (B). RD cells were also engineered to stably express control shRNA and CTNNB1-specific shRNA (C) and inducible CTNN1S33Y in the absence or presence of 10 μg/mL doxycycline treatment (D). *P < 0.05, Student t test. Each error bars denote ±1 SD. (E) Summary of serial replating self-renewal assays in the presence of BIO and WNT3A (Left) and of spheres expressing CTNNB1S33Y (Right).