Abstract LB-352: DUSP26 inhibition: a new therapeutic pathway in neuroblastoma.
Authors: Ma Irene T. , Fan Yihui , Patel Roma H. , Cheng Jin , Kim Eugene S. , Shohet Jason M. , Yang Jianhua , Vasudevan Sanjeev A. 
Published: August 2014

Introduction: Dual specificity phosphatase 26 (DUSP26) is overexpressed in high risk neuroblastoma (NB) and contributes to chemoresistance by inhibiting p53 function. In vitro, DUSP26 has also been shown to effectively inhibit p38 MAP kinase. We hypothesize that inhibiting DUSP26 will result in decreased NB cell growth in a p53 and/or p38 mediated manner.

Methods: We developed 2 small hairpin RNA's (shRNA) against DUSP26 and stably transduced them into NB cell lines. With these cell lines, we studied the effects of DUSP26 knockdown on anchorage independent growth, cell cycle, and in vivo tumor growth using an orthotopic NB mouse model. We then tested the effects of a known DUSP26 inhibitor, NSC-87877 (NSC), on NB cytotoxicity, in combination with doxorubicin and SB203580 (p38 inhibitor), and in vivo as a single agent.

Results: Both sh-RNA sequences achieved &gt;80% knockdown of DUSP26 expression. Colony formation in soft agar was significantly reduced (p&lt;0.05) in the sh-DUSP26 cell lines compared to control cell lines. Cell cycle analysis was also performed comparing the sh-Control and sh-DUSP26 cell lines that showed a G1 cell cycle arrest (46.8% vs. 80.6%, p&lt;0.03). In vivo, SH-SY5Y cells transduced with sh-DUSP26 had significantly lower tumor weights after 4 weeks of growth compared to the sh-Control transduced cells (1.8gm vs. 4.2gm, p&lt;0.01). Cytotoxicity assays testing NSC were performed on multiple NB cell lines. After 24 to 48 hours of exposure, NSC caused significantly decreased (p&lt;0.005) cell viability at 10µM in IMR32 (18% viable), NB-19 (40%), LAN-1 (41%), SH-SY5Y (53%), and SK-N-SH (38 %). In contrast, the SHEP cell line, known to have very little DUSP26 expression, actually grew in the setting of NSC treatment (200%, p&lt;0.005). In combination with doxorubicin (0.025 µM), NSC resulted in a significant additive decrease (p&lt;0.05) in cell viability for the MYCN amplified cell lines (IMR-32, 26% less; LAN-1, 24%; NB-19, 20%). The p53 pathway was evaluated by Western blot using both DUSP26 inhibition techniques, shRNA and NSC. Increased p53 stability and phosphorylation at Ser46 demonstrated a possible role for p38 MAP kinase. With NSC treatment, cells displayed increased p38 phosphorylation and PARP cleavage. This was further supported with the reversal of NSC-mediated cytotoxity (p&lt;0.01) by the p38 inhibitor SB203580 on IMR32 (54% vs. 124% viability) and NB-19 (69% vs. 102% viability). After 2 weeks of tumor growth in vivo, NSC was administered to mice once daily at 15mg/kg for a total of 21 days. Tumor weights were significantly decreased (p&lt;0.03) in the treated cohort of mice (0.1 gm, n=3) compared to placebo control (0.8 gm, n=2).

Conclusion: DUSP26 is an essential protein for NB cell growth in vitro and in vivo. By inhibiting DUSP26, pro-apoptotic pathways, such as p53 and/or p38, potentially become disinhibited. DUSP26 targeted therapy may prove to be a useful single agent or adjuvant to current chemotherapy for NB.

Citation Format: Irene T. Ma, Yihui Fan, Roma H. Patel, Jin Cheng, Eugene S. Kim, Jason M. Shohet, Jianhua Yang, Sanjeev A. Vasudevan. DUSP26 inhibition: a new therapeutic pathway in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-352. doi:10.1158/1538-7445.AM2013-LB-352