M. Balamurugan1, S. Kunnimalaiyaan1, T. C. Gamblin1, M. Kunnimalaiyaan1 1Medical College Of Wisconsin,Division Of Surgical Oncology,Milwaukee, WI, USA
Introduction: Neuroblastoma (NB) is a highly malignant neuroendocrine tumor (NET) with high recurrence. NB is very rare in adults but usually present in early childhood. NETs express high levels of achaete-scute complex-like1 (ASCL1) protein and chromogranin A (CgA) peptide, two important NET markers. Despite recent advances, about 60% of patients with high-risk NB will have a recurrence and treatment options for these patients are limited. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target, as this pathway has been shown to be crucial in the management of other NETs. Recently, we have reported that treatment with GSK-3 inhibitor reduced growth of NB and it is associated with significant reduction in phosphorylation of GSK-3 alpha compared to GSK-3 beta. However, the role of GSK-3 isoforms on growth remains unclear. To understand the role of GSK-3 isoforms, we investigated the growth regulatory effects by depletion of each GSK-3 isoform individually in neuroblastoma.
Methods: SK-N-AS cells were transfected with shRNA of GSK-3 beta or alpha and selected on puromycin containing media to create GSK-3 alpha knockdown (GSK-3α- KO) or GSK- beta knockdown (GSK-3β-KO). As a control GSK-3-NC cells were created using shRNA against no-target, non-specific scrambled sequence. ShRNA mediated knockdown of GSK-3α or GSK-3β were confirmed at both mRNA and proteins levels by quantitative real time polymerase chain reaction (qPCR) and Western analysis respectively. Cellular proliferation of GSK-3α-KO or GSK-3β-KO cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, the effect of GSK-3 depletion in SK-N-AS cells on the levels of NET markers (ASCL1 and CgA), as well as cell cycle regulatory protein (cyclinD1) were determined by western blot.
Results: SK-N-AS cells transfected with shRNA against GSK-3 isoform (GSK-3α-KO or GSK-3β-KO) showed significant reduction in GSK-3 beta or alpha mRNA and protein. Cellular proliferation assay on GSK-3α-KO or GSK-3β-KO showed differential growth reduction compared to no-target, non-specific scrambled sequence (GSK-3-NC). The growth suppression effect by knockdown of GSK-3 is associated with decrease in cyclinD1. Importantly, knockdown of GSK-3 activity resulted in attenuation of NET markers ASCL1 and CgA.
Conclusion:Knockdown of GSK-3 beta in SK-N-AS cells partially inhibited the growth compared to GSK-3 alpha knockdown suggesting that GSK-3 alpha may play a role in cellular proliferation. Therefore, our findings warrants further investigation on the role of GSK-3 alpha in neuroblastoma.