Regulation of the Receptor Tyrosine Kinase AXL in Response to Therapy and Its Role in Therapy Resistance in Glioblastoma

Abstract

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM.

Keywords: R428; RTK-AXL; glioblastoma multiforme; post-translational receptor modification; radiation; temozolomide; tyrosine kinase inhibitor (TKI).

Figure 1

RTK-AXL undergoes post-translational modifications following temozolomide. (A,B) Fold gene expression changes in SF126; (A) und U118MG; (B) cell lines following 10 µm TMZ exposure at 24, 48 and 72 h (p = not significant); (C) Western blot with lysates of SF126 and U118MG cell lines treated with 10 µM TMZ for 48 h displaying full length RTK-AXL (FL-AXL) at 140 kDa and C-terminal RTK-AXL (CT-AXL) at 55 kDa; (D) Quantitative analysis of relative protein expression of FL-AXL and CT-AXL normalized to beta actin (p = not significant); (E) Effect of increasing doses of Temozolomide (TMZ) and repeated exposures (n = 5) on protein expression of FL-AXL and CT-AXL expression in U118MG (Western blot) and quantification of protein expression of FL-AXL and CT-AXL expression in U118MG. Ordinary one-way ANOVA, **** p < 0.0001 (F). (G) Activity of the TACE matrix metalloproteinase ADAM 10/17 following TMZ treatment in SF126 (ordinary one-way ANOVA, *** p < 0.005) und U118MG (ordinary one-way ANOVA, **** p < 0.0001) (H).

Figure 2

RTK-AXL undergoes post-translational modifications in a radio-resistant microenvironment. (A,B) Fold gene expression changes in SF126 (A) und U118MG (B) cell lines following 0, 2 and 6 Gy radiation. Ordinary one-way ANOVA, SF126: p = ns, U118MG: p = ns. (C) Western Blot with lysates of SF126 and U118MG cell lines treated with 0, 2 and 6 Gy radiation displaying full length RTK-AXL (FL-AXL) at 140 kDa and C-terminal RTK-AXL (CT-AXL) at 55 kDa. (D) Quantitative Western blot analysis of C (p = not significant). (E,F) Activity of the TACE matrix metalloproteinase ADAM 10/17 following radiation treatment in S126 (E) and U118MG (F). Ordinary one-way ANOVA, SF126: p = ns, U118MG: p = ns. (G) SF126 cultivated under hypoxia (1% O₂) for 3, 6, 12 and 24 h displaying HIF1 alpha, FL-AXL and CT-AXL compared to normoxia (21% O₂). (H) Quantification of protein expression of FL-AXL and CT-AXL in U118MG. Student’s t-test comparing CT-AXL 21% O₂ with CT-AXL 1% O₂, * p = 0.023.

Figure 3

AXL TKI R428 combined with temozolomide (TMZ) increases therapeutic effects: (A,B) colony formation assay (CFA) of SF126 and SF126-TR (A); and U118MG and U118-TR (B) cell lines under TMZ exposure. Paired t-test SF126 vs. SF126-TR: ** p = 0.006; paired t-test U118MG vs. U118MG-TR: ** p = 0.025; (C,D) representative images of the CFA of SF126 and SF126 temozolomide-resistant (TR); (C) and U118MG and U118MG-TR with and without TMZ treatment (D); (E) survival fraction of SF126 under TMZ and R428 exposure. Ordinary one-way ANOVA, SF126: p = ns; (F) survival fraction of U118MG under TMZ and R428 exposure. Ordinary one-way ANOVA U118MG ** p = 0.0069; (G) survival fraction of SF126-TR under TMZ and R428 exposure. Ordinary one-way ANOVA, SF126-TR: **** p < 0.0001. Student’s t-test comparing SF126 with SF126 + R428: ** p = 0.0015; (H) survival fraction of U118MG-TR under TMZ and R428 exposure. Ordinary one-way ANOVA U118MG *** p = 0.0002. Student’s t-test comparing U118MG with U188MG + R428: ** p = 0.0028.

Figure 4

AXL TKI R428 combined with radiation (RTX) increases therapeutic effects and endogenous RTK-AXL overexpression is related to RTX resistance: (A) Survival fraction of SF126 under radiation and R428 exposure. Ordinary one-way ANOVA, SF126: **** p < 0.0001; (B) Survival fraction of U118MG under radiation and R428 exposure. Ordinary one-way ANOVA, U118MG: p = ns; (C) survival fraction of SF126, SF126-DN and SF126-WT cell lines in colony formation assay under increasing dosage of temozolomide (TMZ). Ordinary one-way ANOVA, p = ns; (D) survival fraction of SF126, SF126-DN and SF126-WT cell lines in colony formation assay under increasing dosage of radiation (0, 2, 4, and 6 Gy). Ordinary one-way ANOVA, SF126WT at 2 and 6 Gy: **** p < 0.0001.