Small molecule inhibitor regorafenib inhibits RET signaling in neuroblastoma cells and effectively suppresses tumor growth in vivo

Abstract

Neuroblastoma (NB), the most common extracranial pediatric solid tumor, continues to cause significant cancer-related morbidity and mortality in children. Dysregulation of oncogenic receptor tyrosine kinases (RTKs) has been shown to contribute to tumorigenesis in various human cancers and targeting these RTKs has had therapeutic benefit. RET is an RTK which is commonly expressed in NB, and high expression of RET correlates with poor outcomes in patients with NB. Herein we report that RET is required for NB cell proliferation and that the small molecule inhibitor regorafenib (BAY 73-4506) blocks glial cell derived neurotrophic factor (GDNF)-induced RET signaling in NB cells and inhibits NB growth both in vitro and in vivo. We found that regorafenib significantly inhibited cell proliferation and colony formation ability of NB cells. Moreover, regorafenib suppressed tumor growth in both an orthotopic xenograft NB mouse model and a TH-MYCN transgenic NB mouse model. Finally, regorafenib markedly improved the overall survival of TH-MYCN transgenic tumor-bearing mice. In summary, our study suggests that RET is a potential therapeutic target in NB, and that using a novel RET inhibitor, like regorafenib, should be investigated as a therapeutic treatment option for children with NB.

Keywords: RET; chemotherapy; neuroblastoma; regorafenib; tyrosine kinase inhibitor.

Figure 1. High expression of RET predicts poor outcome in patients with NB

A large patient cohort (498 patients, SEQC-498-RPM data set) with annotated clinical and long term follow up data was used to analyze the outcomes in patients with NB. (A) Analysis of RET expression in NB patients from stage 1 to metastatic stage 4 demonstrates higher levels of RET in stage 4 metastatic subgroup compared with other groups (P values were calculated by Student T-test). High expression of RET correlates with MYCN amplification status (B), high-risk disease (C), and lower long-term survival probability (D) in patients with NB. This Kaplan–Meier survival curve was generated (P <0.001) comparing tumors with high (n = 174) versus low (n = 324) expression of RET. (E) RET knockdown decreased cell proliferation in a panel of NB cells. Protein immunoblotting analysis demonstrated RET knockdown in IMR-32, NGP, NB-19, SH-SY5Y and SK-N-AS cells by lenti-virus-mediated expression of shRNAs and anchorage-dependent cell proliferation images of those NB cells were shown.

Figure 2. Regorafenib abrogates RET-mediated PI3K/AKT/mTOR signaling in NB cells

(A) NGP, SH-SY5Y, and SK-N-AS cells were treated with 10 μM of regorafenib for 0–8 hrs, and treated cells were harvested at the end of treatment, subjected to SDS-PAGE, and immunoblotted with the indicated antibodies. (B, C) NGP and SH-SY5Y cells (B) or IMR-32, NGP, NB-19, SH-SY5Y and SK-N-AS cells (C) were starved with serum-free medium and then treated with increasing doses of regorafenib for 2 hrs prior to GDNF stimulation. Cell pellets were collected and analyzed by immunoblotting. β-Actin was used as a loading control for whole cell extracts in all samples.

Figure 3. RET inhibitor regorafenib shows cytotoxic effects on NB cell lines

(A) Six NB cell lines (IMR-32, NGP, NB-19, CHLA-255, SH-SY5Y, and SK-N-AS) were treated with the indicated concentrations of regorafenib for 72 hrs. Cell viability was then measured by CCK-8 assays and data were represented as % vehicle ± S.D. P <0.05 (^*), or P <0.001 (^***) (Student’s t-test, two-tailed). (B) IC50s of regorafenib on each NB cell line was calculated by using Prism 5.0, based on the data collected in the cell viability assays. (C) Morphologic changes of the six NB cell lines treated with increasing concentrations of regorafenib for 72 hrs.

Figure 4. Regorafenib suppresses anchorage-independent growth of NB cell lines

(A) A panel of six NB cell lines were seeded in six-well plates with soft agar. The cells were then treated with indicated concentrations of regorafenib and grown for two to three weeks. Cells were stained with crystal violet and photographed. (B) Cell colonies from (A) were counted and colony numbers were represented as % vehicle ± S.D. P <0.01 (^**) or P <0.001 (^***) (Student’s t-test, two-tailed).

Figure 5. Regorafenib inhibits tumor growth in an orthotopic xenograft NB mouse model

(A, B) Photos of NGP (A) and SH-SY5Y (B) xenografted tumors and corresponding kidney controls from the DMSO control group and the regorafenib treated group (30 mg/kg) were taken at the end of treatment (21 days). (C, D) NGP-derived tumor weights (C) from control (N=4) and treatment (N=4) groups and SH-SY5Y-derived tumor weights (D) from control (N=3) and treatment (N=3) groups, were presented. P <0.05 (^*) and P <0.01 (^**) (Student’s t-test, two-tailed). (E, F) Mice bearing NGP and SH-SY5Y xenografted tumors were treated with either regorafenib (30 mg/kg) or an equal volume of DMSO by i.p. injection daily for two days and the mice were sacrificed and the tumors were harvested and lysed for immunoblotting with the indicated antibodies. β-Actin was measured as a loading control.

Figure 6. Regorafenib prevents tumor development and improves overall survival rate in the TH-MYCN transgenic NB mouse model

(A) Treatment strategy schema for this study. (B, C) Pictures of tumors and corresponding kidneys at the end of treatment (B) and tumor weights (C) shown in the control (N=2) and treated (N=2) groups. Experiments were performed with two mice in each group. P <0.05 (^*) (Student’s t-test, two-tailed). (D) Six-week old homozygous TH-MYCN transgenic mice were treated with either 30 mg/kg of regorafenib or an equal volume of DMSO by i.p. injection daily for two days. Then tumors were harvested and lysed for immunoblotting with the indicated antibodies. β-Actin was detected as a loading control. (E) Homozygous TH-MYCN transgenic mice from control (N=4) and treatment (N=4) groups were treated as in (A), and then tumors were removed after four weeks of treatment. Death dates of the tumor bearing mice were collected and the Kaplan-Meier survival curves were generated by using Prism 5.0 software. P <0.01 (^**) (MCLR test).