Pharmacologic Wnt Inhibition Reduces Proliferation, Survival, and Clonogenicity of Glioblastoma Cells

Abstract

Wingless (Wnt) signaling is an important pathway in gliomagenesis and in the growth of stem-like glioma cells. Using immunohistochemistry to assess the translocation of β-catenin protein, we identified intranuclear staining suggesting Wnt pathway activation in 8 of 43 surgical samples (19%) from adult patients with glioblastoma and in 9 of 30 surgical samples (30%) from pediatric patients with glioblastoma. Wnt activity, evidenced by nuclear β-catenin in our cohort and high expression of its target AXIN2 (axis inhibitor protein 2) in published glioma datasets, was associated with shorter patient survival, although this was not statistically significant. We determined the effects of the porcupine inhibitor LGK974 on 3 glioblastoma cell lines with elevated AXIN2 and found that it reduced Wnt pathway activity by 50% or more, as assessed by T-cell factor luciferase reporters. Wnt inhibition led to suppression of growth, proliferation in cultures, and modest induction of cell death. LGK974 reduced NANOG messenger RNA levels and the fraction of cells expressing the stem cell marker CD133 in neurosphere cultures, induced glial differentiation, and suppressed clonogenicity. These data indicate that LGK974 is a promising new agent that can inhibit the canonical Wnt pathway in vitro, slow tumor growth, and deplete stem-like clonogenic cells, thereby providing further support for targeting Wnt in patients with glioblastoma.

Figure 1

CTNNB1/β-catenin expression in surgical adult and pediatric glioblastoma specimens. (A) Glioblastoma in an adult with moderate cytoplasmic and weak nuclear β-catenin immunoreactivity in a subset of cells (inset, arrow). (B) A pediatric glioblastoma with weak β-catenin expression in cytoplasm and in scattered nuclei (inset, arrow). (C) A weak cytoplasmic staining pattern, but no nuclear protein, was detected in formalin-fixed pellets from the GBM1 neurosphere cell line. (D) Patients whose glioblastoma contain nuclear β-catenin have shorter overall survival than those without signs of WNT activity (median overall survival: 17 vs. 20 months, p = 0.8)

Figure 2

Quantification of the WNT target AXIN2 and clinical prognosis. (A) AXIN2 mRNA levels in snap frozen adult brain tumors (glioblastoma [GBM]: p349, p635, p636, p696, p770 and one LGG: p824), and brain tumor cell lines derived from adult GBMs (GBM1, GBM10, GBM14, JHH136, JHH520, AQH612, U87, U87NS and LN229) and pediatric brain tumors including diffuse intrinsic pontine glioma (SU-DIPG), anaplastic astrocytoma (AA: BT35), atypical teratoid malignant rhabdoid tumor (ATRT: BT40), low-grade glioma (LGG: Res186 and Res259), high-grade gliomas (pHGG: SF188 and KNS42) and medulloblastoma (MB: D283 and D425). (B) GBM patients with high AXIN2 have significantly shorter survival compared to patients with low AXIN2 (median overall survival: 208 days vs. 448 days) (z-score = +1.75, p = 0.06, TCGA dataset May 2015).

Figure 3

LGK974 suppresses canonical WNT activity. (A) WNT pathway activity assessed by T cell factor (TCF) luciferase reporters. (B) Introduction of mutant active CTNNB1 (S33Y) increased the TCF reporter signals in GBM1 and JHH520 cells. (C) LGK974 inhibits WNT activity in a dose-dependent manner. (D) Treatment with 5 μM LGK974 for 48 hours effectively inhibited WNT signaling in all glioma cell lines tested (p ≤ 0.05).

Figure 4

Decreased growth following WNT pathway blockade. (A-C) WNT inhibition significantly reduced cell growth as assessed with Titer Blue assay (A), cell proliferation assessed by fluorescence-based Ki67 quantification (B), and cell survival as assessed with Annexin V/Propidium iodide-based apoptosis and cell death quantification (C). *p ≤ 0.05 for all panels. 48h, 48 hours; 72h, 72 hours.

Figure 5

LGK974 promotes differentiation. (A) LGK974 treatment reduced the fraction of cells expressing the glioma stem cell marker CD133. (B) CD133-positive GBM1 cells have higher canonical WNT activity as compared to their CD133-negative counterparts. (C) Reduced expression of NANOG after LGK974 administration (p = 0.05). (D) Increased glial fibrillary acidic protein (GFAP) (all tested cell lines) and microtubule-associated protein 2 (MAP2) (LN229) 72 hours after administration of LGK974. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 6

LGK974 treatment significantly inhibited in vitro clonogenicity and reduced the average sphere diameter from (GBM1:147 μm to 87 μm, JHH520: 186 μm to 168 μm) compared to DMSO-treated control cells (p ≤ 0.001 for both parameters in both cell lines).