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

Introduction: Glycogen synthase kinase 3α/β (GSK3α/β) is a serine/threonine kinase that plays a critical role in cancer. Aims:
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). Materials and Methods:
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. Results:
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. Conclusion:
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.