Abstract 4901: Inhibition of γ-secretase sensitizes drug-resistant neuroblastoma cells to doxorubicin
Authors: Solka Kathryn A. , Chu Fei , Qiu Yi-Yong , Clark Sandra H. , Madonna Mary Beth 
Published: August 2018

Introduction: Neuroblastoma is the most common extracranial solid tumor in childhood and accounts for ~15% of all pediatric cancer mortality. Approximately 650 new cases are reported annually in the USA, with 90% of cases diagnosed by 5 years of age. Novel therapies are needed as ~50% of cases are high-risk neuroblastoma with a poor (&lt;50%) five-year survival rate due to relapse and treatment resistance. Neuroblastoma cells undergo a mesenchymal change, including the activation of Notch pathway genes, in their transition to a drug-resistant state. The Notch pathway has both oncogenic and tumor-suppressive roles and has been implicated in epithelial-mesenchymal transition (EMT) and chemoresistance. This pathway can be blocked by γ-secretase inhibition, which prevents cleavage of the Notch receptor and therefore Notch signal transduction. Thus, targeting the Notch pathway has been considered a promising anticancer strategy. Underlying mechanisms of the Notch signaling pathway in drug-resistant cancers, including neuroblastoma, are not well understood.

Aim: Determine if inhibition of the Notch signaling pathway sensitizes chemoresistant neuroblastoma cells to doxorubicin.

Methods: Doxorubicin-resistant (DoxR) cells were generated by incubating doxorubicin-sensitive, wild-type (WT) patient-derived cell lines with incremental doxorubicin concentrations (1 x 10-9 to 1 x 10-6 M) over six months. Cells were considered chemoresistant after surviving five consecutive passages in 1 µM doxorubicin (MTT assay). Differentially expressed genes were identified by microarray (Illumina HumanHT-12 v4 Expression BeadChip) and validated by RT-qPCR. Cells treated with doxorubicin and/or DAPT (γ-secretase inhibitor) were evaluated for cell viability (MTT assay), proliferation (PCNA immunofluorescence and Western blot), senescence (SA-β-Gal immunocytochemistry and p21 Western blot) and proteomic profiling (2D-DIGE).

Results: Compared to WT cells, DoxR cells differentially expressed the following Notch pathway genes: downregulation of DLK1 and upregulation of HEY1 and SNAI2, as determined by microarray and RT-qPCR. Compared to WT cells, DoxR cells treated with both doxorubicin and DAPT exhibited significantly decreased cell viability, decreased proliferation and increased senescence. DAPT-treated DoxR cells differentially expressed nine proteins using 2D-DIGE: MCM3, MCM6, CAPN10, ACTN4, TUBA1B, HNRNPK, PDP1, NPM1 and BARD1.

Conclusion: The γ-secretase inhibitor DAPT sensitizes DoxR neuroblastoma cells to doxorubicin. Mechanisms underlying the synergistic effect of doxorubicin and DAPT in DoxR cells warrant further investigation.

Citation Format: Kathryn A. Solka, Fei Chu, Yi-Yong Qiu, Sandra H. Clark, Mary Beth Madonna. Inhibition of γ-secretase sensitizes drug-resistant neuroblastoma cells to doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4901.