gms | German Medical Science

Objective: Developmentally conserved WNT-signaling pathway plays a crucial role in a physiological process of cell proliferation and migration. Moreover, it is associated with carcinogenesis and epithelial-to-mesenchymal-transition (EMT) in various tumors, including malignant gliomas. High WNT-activation was detected at the invasive margin in some cases of glioblastoma multiforme (GBM) that are confirmed to be highly invasive and highly resistant to current standard radio- and chemotherapies. A new compound, the porcupine inhibitor LGK 974, enables targeting the canonical WNT-pathway in tumor cells interfering with the WNT factor palmytoylation. In this study we tested LGK974 on two GBM cell lines, LN229 and JHH520, which show high intrinsic activation of WNT signaling pathway.

Method: Activity of the pathway was detected using a TCF/LEF reporter construct that enables sensible luciferase measurement proportional to intra-nuclear beta-catenin. The culture media was supplemented with 5uM of WNT inhibitor and changed every 48h hours. Cell viability after treatment with LGK974 was tested using Titer Blue Assay. The expression of AXIN2 and ZEB1 (WNT-pathway and EMT markers, respectively) were detected with RT qPCR. CD133 was used as a stem cell marker and CD133 positive cells were detected and sorted via FACS. In parallel, mRNA of 6 human GBMs was analyzed for WNT target gene expression and compared to investigated cell lines.

Results: In both cell lines 48h exposure to 5μM LGK974 resulted in significant inhibition of the WNT pathway and diminution of cell growth with consecutive apoptose induction, as schown by the presence of cleaved-capase. Under this condition more than 50% reduction of nuclear beta-catenin was observed. AXIN2 and ZEB1 were down-regulated over 70%. Cell growth was decreased by more than 40%. Additionally a decrease of CD133 positive cells following LGK947 treatment was abserved. Interestingly, FACS sorted CD133+ cells showed significantly higher intrinsic WNT-activity as compared to their CD133- counterparts.

Conclusions: WNT-inhibition in GBM cell lines with LGK974 significantly reduced cell proliferation and EMT process. The decrease of CD133+ cells following administration of WNT inhibitor indicates that this treatment might especially target tumor stem cells and is therefore contributing to the sustainable tumor growth arrest. Taken together, porcupine inhibition via LGK974 might play a role as a new, potent therapy against malignant gliomas.