Abstract B091: Disruption of cancer stemness by targeting the JNK-STAT3 pathway in neuroblastoma
Authors: Higashi Mayumi , Kin Kiyokazu , Tajiri Tatsuro 
Published: August 2018

Abstract
Background: Neuroblastoma is one of the most challenging malignant tumors in children. The 5-year survival rate among patients with progressive tumors is still 30-40%, even after the improvement of intensive therapies. In our study, we found that JNK and its downstream factor, STAT3, are novel targets for disrupting cancer stemness in neuroblastoma. JNK is one of the major MAPKs; it has been reported that JNK or STAT3 maintains the stemness of cancer cells. We analyzed the activity of the JNK-STAT3 cascade and the effects of inhibiting JNK or STAT3 in neuroblastomas. Methods: The CHP134, IMR32, IMR5, NLF, SK-N-AS, and SHEP neuroblastoma cell lines were used in this study. The hTERT-RPE1 (RPE1) cell line was analyzed as a normal cell control model. SP600125 and JNK-IN-8 (JNK inhibitors), and Niclosamide (a STAT3 inhibitor), were used in various concentrations. A cell viability assay was performed using the CellTiter-Blue (Promega) system. The gene expression was analyzed using a THUNDERBIRD SYBR-Green real-time PCR system (TOYOBO). An immunofluorescence analysis and immunoblotting were performed using antibodies for beta-3 tubulin (SIGMA-ALDRICH), JNK, p-JNK, STAT3, p-STAT3 s727, p-STAT3 y705 (all Abcam). Results: The IMR5, NLF, SK-N-AS neuroblastoma cell lines showed remarkably lower cell viability after treatment with SP600125 or JNK-IN-8, in comparison to nontreated cells. Treatment with JNK inhibitors led to growth retardation in RPE1 cells; however, this cell line showed differentiation rather than cell death. Cell death was more significant in cells with faster proliferation than in cells with slower proliferation, which showed differentiation after JNK inhibitor treatment. The phosphorylation of STAT3 serine 727 (s727), but not tyrosine 705 (y705), corresponded well to the JNK expression in neuroblastoma cells; cells with MYCN amplification or 1p deletion, both unfavorable prognostic markers, showed high levels of JNK expression and strong STAT3 s727 phosphorylation, while STAT3 y705 phosphorylation was not found in any cell lines. JNK inhibitor treatment resulted in a dose-dependent decrease in STAT3 s727 phosphorylation in IMR5 cells. The STAT3 inhibitor nicrosamide induced cell death at high concentrations and cell differentiation at low concentrations. The expression of CXCR4, a cancer stem cell marker, was downregulated by JNK or STAT3 inhibitors. Discussion: Targeting the JNK-STAT3 cascade induced both cell death and cell differentiation. Our results suggest that JNK-STAT3 maintains cancer stemness, and that the inhibition of this pathway disrupts the stemness in neuroblastomas. The JNK-STAT3 cascade is thus considered to be a novel target for neuroblastoma therapy.
Citation Format: Mayumi Higashi, Kiyokazu Kin, Tatsuro Tajiri. Disruption of cancer stemness by targeting the JNK-STAT3 pathway in neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B091.