Children with high-risk neuroblastoma face challenging treatment outcomes, including relapse with drug-resistant disease. Long non-coding RNAs (lncRNAs) are associated with cell differentiation, proliferation and survival by interacting with binding proteins. The objective of this study was to determine the role of the lncRNA NKAPP1 in neuroblastoma tumorigenesis and to identify small molecule compound inhibitors of NKAPP1.
Using human neuroblastoma tissue RNA sequencing data from 493 patients downloaded from the R2 platform [http://r2.amc.nl], the correlation between the expression of every transcript and event-free and overall survival in MYCN-amplified, MYCN-non-amplified, and the total cohort of neuroblastoma patients was examined using Kaplan-Meier curves. Multivariate Cox regression model was used to further evaluate the prognostic value of every transcript. Subsequently, NKAPP1 was knocked down by siRNA to examine neuroblastoma cell proliferation. Doxycycline (DOX)-inducible NKAPP1 shRNA stable Kelly and BE(2)-C cell lines were generated to investigate cell proliferation, colony formation, and cell survival/apoptosis.
NKAPP1 was identified as one of the transcripts, whose high expression correlated with poor event-free and overall survival in MYCN-amplified, MYCN-non-amplified, and the total cohort of neuroblastoma patients, stratified by the median level of expression. Multivariate Cox regression analysis showed that high levels of NKAPP1 expression in tumour tissues correlates with poor patient survival, independent of current prognostic markers. Knocking down NKAPP1 with NKAPP1 siRNAs, compared to control siRNAs, significantly reduced neuroblastoma cell proliferation. NKAPP1 knockdown using DOX-inducible NKAPP1 shRNA resulted in significantly less cell proliferation and colony formation, and induced neuroblastoma cell apoptosis.
Taken together, these data demonstrate that NKAPP1 promotes neuroblastoma cell proliferation, survival and colony formation, and that high levels of NKAPP1 in tumour tissues is an independent marker for poor prognosis in neuroblastoma patients. We are currently validating functional partner binding protein of NKAPP1 and will screen small molecule compounds for NKAPP1 inhibitors.