Supplementary Material for: GSK3α/β: A Novel Therapeutic Target for Neuroendocrine Tumors?

<p><b><i>Introduction:</i></b> Glycogen synthase kinase 3α/β (GSK3α/β) is a serine/threonine kinase that plays a critical role in cancer. <b><i>Aims:</i></b> In this study, we evaluated the effects of the specific GSK3α/β inhibitor AR-A014418 in vitro to gain novel insights into GSK3α/β signaling in neuroendocrine tumors (NETs). <b><i>Materials and Methods:</i></b> Human NET cell lines (BON1, QGP1, H727, and GOT1) were treated with different concentrations of AR-A014418 alone and in combination with lovastatin, everolimus, 5-fluorouracil (5-FU), and γ-irradiation. <b><i>Results:</i></b> AR-A014418 significantly dose- and time-dependently decreased cell viability in all 4 NET cell lines through inhibition of epithelial growth factor receptor and mTORC1/p70S6K signaling, as well as cyclin D3 downregulation and induction of pChk1. In all cell lines tested, FACS analysis showed an AR-A014418-induced increase in the sub-G1 phase, reflecting cell death. Apoptosis induction was observed in H727, GOT1 and QGP1 cells, but not in BON1 cells. Furthermore, significant antimigratory effects upon GSK3α/β inhibition were found and were associated with β-catenin downregulation in all cell lines tested. Compensatory upregulation of pAkt and pERK in response to GSK3α/β inhibition was prevented by combining AR-A014418 with the ERK and Akt inhibitor lovastatin. Accordingly, the lovastatin/AR-A014418 combination was synergistic in BON1 and QGP1 cells. Moreover, AR-A014418 displayed promising chemosensitizing effects on 5-FU in QGP1 and slight radiosensitizing properties in BON1 and QGP1 cells. <b><i>Conclusion:</i></b> Our data provide new insights into the role of GSK3α/β in NETs and suggest that GSK3α/β inhibition could be a novel therapeutic option in NETs, especially in combination with lovastatin or 5-FU, depending on tumor entity.