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Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting - PubMed

  • ️Sun Jan 01 2017

Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by β1 integrin/JNK co-targeting

Anne Vehlow et al. Oncotarget. 2017.

Abstract

Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor β1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with β1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by β1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a β1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.

Keywords: GBM stem-like cells; JNK; orthotopic GBM mouse model; radiochemoresistance; β1 integrin.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have declared that no conflicts of interest exists.

Figures

Figure 1
Figure 1. Co-targeting of β1 integrin and JNK sensitizes GBM cells to radiotherapy

(A) Workflow of GBM sphere formation and clonogenic survival assay. (B) Relative sphere formation and basal surviving fraction of GBM stem-like cells (GS-8), patient-derived GBM cultures (DK32, DK41) and GBM cell lines (U343MG, DD-T4) upon treatment with AIIB2/SP600125 (EC10, EC50) or controls (IgG, DMSO). (C) Relative sphere formation and clonogenic survival upon treatment with AIIB2/SP600125 (EC10, EC50) and X-ray irradiation (2, 4, 6 Gy) (controls are IgG and DMSO). (B, C) Results are mean +/− SEM (n = 3–4, t-test). (D) Western blot analysis of β1 integrin, phospho-cJun (S63), cJun and β-actin of whole cell lysates from indicated GBM cells treated as indicated with AIIB2 (10 μg/ml), SP600125 (EC10), IgG control (10 μg/ml) and DMSO. Fold change is calculated by normalization to β-actin and IgG/DMSO controls according to representative blots.

Figure 2
Figure 2. Combined β1 integrin/JNK targeting blocks GBM cell invasion

(A) Work flow of 3D invasion assays. (B) Representative images of indicated GBM cells invading into 3D type I collagen 72 h after treatment with AIIB2/SP600125 (EC50) or control IgG/DMSO. Scale bar, 50 μm. (C) Invasion distance of indicated treated GBM cell populations. Results are mean +/− SEM (n = 3, t-test).

Figure 3
Figure 3. Dual inhibition of β1 integrin and JNK delays tumor growth and prolongs survival in combination with radiochemotherapy

(A) Treatment scheme of mice bearing tumors of GS-8_GFP/fLuc stem-like cells. (B) Left panel shows representative image of a mouse brain with GS-8_GFP/fLuc tumor. Scale bar, 2 mm. Region 1 and 2 corresponding to middle and right panel magnifications show invading tumor cells (IC) and blood vessel (BV) of GS-8_GFP/fLuc tumors, respectively. Scale bar, 100 μm. (C) Representative images from GS-8_GFP/fLuc tumors showing β1 integrin (green), phospho-JNK (T183/Y185) (red) or merge with nuclei (DAPI, blue). Scale bar, 100 μm. (D) Gadolinium-enhanced (Gd) T1-weighted magnetic resonance images (MRI) from mice bearing GS-8_GFP/fLuc tumors 24 days after transplantation and β1 integrin/JNK inhibition without or in combination with radiochemotherapy (RCT). Dashed lines delineate tumors. Lower images show luminescence analyses of representative GS-8_GFP/fLuc tumors 24 days after transplantation and indicated treatment. (E) GS-8_GFP/fLuc tumor growth time to 100 fold radiance after indicated treatment. Data are mean +/− SEM (6 - 8 mice per group, one-way ANOVA). (F) Survival of GS-8_GFP/fLuc mice treated as indicated. Kaplan Meier analysis includes 6 mice IgG/DMSO, 8 mice AIIB2/SP600125, 6 mice IgG/DMSO+RCT, 6 mice AIIB2/SP600125+RCT (two-sided log rank test, p < 0.009: IgG/DMSO+RCT vs IgG/DMSO, p < 0.02: AIIB2/SP600125+RCT vs IgG/DMSO+RCT).

Figure 4
Figure 4. β1 integrin/JNK co-targeting interferes with cell cycle regulatory networks

(A) Hierarchical clustering of altered phosphorylation sites (30% decrease, 50% increase, fold change) from phosphoproteome analysis of U343MG cells 1 h after indicated treatment with AIIB2, JNKi or AIIB2/JNKi (EC10) normalized to controls (percentage of phosphorylation site changes is shown on the right). (B) Percentage of phospho-sites showing 30% decreased and 50% increased phosphorylation upon indicated treatment among the 606 phosphorylations sited investigated in the phosphoproteome analysis. (C) Venn diagram analysis of altered phosphosites from (A). (D) Functional classification of altered proteins after treatment with AIIB2/SP600125 (EC10) in comparison to the IgG/DMSO controls. Enrichment of genes in signaling pathway: #p < 0.01 (Fisher's exact test). (E) Cytoscape-based molecular interaction network of β1 integrin and JNK isoforms (black) with altered (grey) and not altered (white) cell cycle associated proteins. (F) Fold change in phosphorylation of cell cycle associated proteins. Threshold of phosphosite changes (30% decrease and 50% increase) is marked in grey.

Figure 5
Figure 5. Block of β1 integrin and JNK signaling enhances radiation induced DSB by chromatin modification

(A) Western blot analysis of indicated proteins from whole cell lysates of U343MG and GS-8 cells treated with AIIB2/SP600125 (EC10) or control IgG/DMSO without and with X-ray irradiation (6 Gy). Fold changes are calculated by normalization to β-actin and IgG/DMSO controls according to representative blots. (B) Immunofluorescence analysis of nuclei with 53BP1-positive foci after indicated treatments. Scale bar, 10 μm. (C) Quantification of the number of DSB per cell at the indicated time points after treatment with AIIB2/SP600125 (EC10) or control IgG/DMSO without and with X-ray irradiation. (D) Basal surviving fraction of U343MG cells upon treatment with AIIB2/SP600125 (EC10), LBH589 (EC50) or a combination thereof compared to control treatment (IgG/DMSO). (E) Clonogenic radiation survival of U343MG cells treated as described in (H). (C–E) Results are mean +/− SEM (n = 3, t-test).

Figure 6
Figure 6. Inhibition of β1 integrin and JNK enhances G2/M cell cycle arrest

(A) Western blot analysis of indicated proteins and phosphorylation sites from whole cell lysates of U343MG cells treated with AIIB2/SP600125 (EC10) or control IgG/DMSO without and with X-ray irradiation (6 Gy). Representative blots are shown. (B) Quantification of ATM (S1981) and Cdc25A (S216) phosphorylation and p53 expression shown in (A). Fold changes are calculated by normalization to β-actin and IgG/DMSO controls. Results are mean +/− SEM (n = 3, t-test). (C) Flow cytometric cell cycle analysis (BrdU, propidium iodide) of U343MG cells 24 h after treatment with AIIB2/SP600125 (EC10) or control IgG/DMSO without and with X-ray irradiation (6 Gy). Representative dot blots are shown. (D) Quantification of flow cytometric analysis from (C) showing distribution of treated GBM cells into G1/G0, S and G2/M phase of the cell cycle. Results are mean +/− SEM (n = 3, t-test).

Figure 7
Figure 7. Scheme depicting the effect of the dual β1 integrin and JNK targeting approach on GBM radioresistance and invasion

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