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. 2023 Apr 6;25(4):648-661.
doi: 10.1093/neuonc/noac237.

Targeting integrin α2 as potential strategy for radiochemosensitization of glioblastoma

Irina Korovina  1   2 , Anne Vehlow  1   3 , Achim Temme  4   3   5 , Nils Cordes  1   2   4   6   3
Affiliations

Targeting integrin α2 as potential strategy for radiochemosensitization of glioblastoma

Irina Korovina et al. Neuro Oncol. .

Abstract

Background: Glioblastoma (GBM) is a fast-growing primary brain tumor characterized by high invasiveness and resistance. This results in poor patient survival. Resistance is caused by many factors, including cell-extracellular matrix (ECM) interactions. Here, we addressed the role of adhesion protein integrin α2, which we identified in a high-throughput screen for novel potential targets in GBM cells treated with standard therapy consisting of temozolomide (TMZ) and radiation.

Methods: In our study, we used a range of primary/stem-like and established GBM cell models in vitro and in vivo. To identify regulatory mechanisms, we employed high-throughput kinome profiling, Western blotting, immunofluorescence staining, reporter, and activity assays.

Results: Our data showed that integrin α2 is overexpressed in GBM compared to normal brain and, that its deletion causes radiochemosensitization. Similarly, invasion and adhesion were significantly reduced in TMZ-irradiated GBM cell models. Furthermore, we found that integrin α2-knockdown impairs the proliferation of GBM cells without affecting DNA damage repair. At the mechanistic level, we found that integrin α2 affects the activity of activating transcription factor 1 (ATF1) and modulates the expression of extracellular signal-regulated kinase 1 (ERK1) regulated by extracellular signals. Finally, we demonstrated that integrin α2-deficiency inhibits tumor growth and thereby prolongs the survival of mice with orthotopically growing GBM xenografts.

Conclusions: Taken together our data suggest that integrin α2 may be a promising target to overcome GBM resistance to radio- and chemotherapy. Thus, it would be worth evaluating how efficient and safe the adjuvant use of integrin α2 inhibitors is to standard radio(chemo)therapy in GBM.

Keywords: ATF1; ERK1; glioblastoma; integrin α2; radiochemoresistance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
High-throughput siRNA screening identifies new regulators of radiochemoresistance in human GBM cells. (A) U343MG cells were transfected with siRNAs targeting 53 focal adhesion proteins, receptor tyrosine kinases and chemokine receptors, and seeded for colony formation assay. Cells were treated with either 0.3 µM TMZ (DMSO as a control), 6 Gy X-rays (0 Gy as a control) alone, or in combination. (B), (C), (D), (E) Clonogenic survival of transfected U343MG cells upon indicated treatments was assessed. Data are presented as enhancement ratios (an average plating efficiency upon control siRNA treatment divided by an average plating efficiency upon target siRNA treatment). Sensitizing enhancement ratios of cells treated with control siRNA were set as 1 (black line), n = 3. Statistics of raw data were performed with unpaired two-tailed Student’s t-test.
Figure 2.
Figure 2.
Integrin α2-targeting effectively radiochemosensitizes GBM cells. (A) Comparative mRNA expression analysis of ITGA2 between GBM and normal brain using the GEPIA database (http://gepia.cancer-pku.cn). One-way ANOVA (P value cutoff .01). (B) ITGA2 mRNA expression in indicated anatomical structures from the Ivy GAP database. Each point corresponds to an individual laser-microdissected sample. (C) Graphs illustrating the correlation between overall survival and ITGA2 mRNA expression levels in GBM patients using Tumor Glioblastoma-TCGA-540-MAS5.0-u133a database (Log-rank test). (D) Western blot analysis for integrin α2 expression (β-actin as a loading control) from GBM cell lines and stem-like GS-8 cells. (E), (F) Analysis of the radiochemosensitizing potential of ITGA2 siRNA-mediated knockdown in 9 human GBM cell lines exposed to TMZ (DMSO as a control) and 6 Gy X-ray irradiation determined as clonogenic survival. Survival of control nonirradiated DMSO-treated cells was set as 100%, n = 3–6 (responders to TMZ and/or X-rays are highlighted in pink). (G) Growth curves of integrin α2-deficient and control A172 and U343MG cell cultures over 5 days, n = 3. (H) Percentages of BrdU+ and Ki67+ cells in integrin α2-deficient and control U343MG and A172 cells on day 1 after plating, n = 3. All data are presented as mean ± SEM with individual values. Statistical significance was analyzed with unpaired two-tailed Student’s t-test, *P < .05, **P < .01, ***P < .001.
Figure 3.
Figure 3.
Integrin α2-knockdown delays tumor growth and increases survival in vivo. (A) Graph illustrating the treatment scheme. GS-8 fLuc/GFP integrin α2-deficient or control cells were orthotopically transplanted into mice. Mice were treated with TMZ (22mg/kg)/4 Gy X-rays or 0.9% NaCl solution (as a control) on 3 consecutive days. (B) Representative Western blot showing integrin α2-knockdown efficiency in integrin α2-deficient and control GS-8 fLuc/GFP cells. (C) In vivo luminescence imaging of tumor-bearing mice on day 28 and day 56. (D), (E) Assessment of luminescence signal in indicated treatment groups on days 28 and 56 after transplantation, n = 7–20 mice per group. (F) Analysis of body weight changes in mice bearing integrin α2-deficient or control tumors on days 28 and 56 after transplantation upon indicated treatments, n = 7–20 mice per group. (G) Kaplan–Meier curves demonstrate the survival of mice with integrin α2-deficient or control tumors treated as indicated, n = 7 mice in untreated groups (NT), n = 19–20 mice in IR + TMZ treated groups. (H), (I) Graph and corresponding images show the number of GS-8 fLuc/GFP integrin α2-deficient or control cells invading the normal brain on day 21 after transplantation. Scale bar (H), 50 µm. All data are shown as mean ± SEM. Statistics were performed using an unpaired two-tailed Student’s t-test. For analysis of survival curves Gehan–Breslow–Wilcoxon test was applied, *P < .05, **P < .01, ***P < .001.
Figure 4.
Figure 4.
Integrin α2-knockdown modulates kinome profiles of GBM cells under radiochemotherapy. (A), (B) Diagrams illustrate predicted changes in activities of individual phosphotyrosine kinases (PTK) and serine/threonine kinases (STK) as well as overall alterations in STK- and PTK-profiles in integrin α2-deficient U343MG cells relative to controls upon irradiation (IR) + temozolomide (TMZ) or non-treated (NT) controls. Mean Kinase Statistic values of 3 independent experiments demonstrate changes in kinase activities. (C) Venn diagram of top 64 STK with the strongest activity reduction in integrin α2-deficient cells under IR + TMZ (Mean Kinase Statistic cutoff ≤ −0.5) in comparison to the untreated group (Δ Mean Kinase Statistic NT – (IR + TMZ) cutoff > 0.3). (D) KEGG pathway enrichment analysis including the top 64 deregulated STK identified in C. (E) Interaction analysis of 27 STK kinases associated with MAPK signaling pathway and deregulated in integrin α2-deficient cells upon IR + TMZ.
Figure 5.
Figure 5.
Integrin α2-deficiency regulates protein levels of ERK1 and ATF1 in GBM cells. Representative Western blots (A), (C) and densitometry analysis (B), (D) showing expression levels of indicated total proteins and their phosphorylated forms in integrin α2-deficient and control U343MG cells treated as indicated, n = 4–6. (E) Analysis of active ATF1 in U343MG cells upon integrin α2-knockdown and in controls with/without 6 Gy X-rays + TMZ. Western blots demonstrate ITGA2 siRNA knockdown efficiency, n = 3. Representative Western blots (F) and densitometry analysis (G) for ATF1-deficient and control U343MG cells treated as indicated demonstrating phospho-ERK1/2 (Thr202/Tyr204) and ERK1/2 expression levels, n = 3. (H) Analysis of ERK1 and ERK2 expression changes in integrin α2/ATF1-deficient cells 24 h after irradiation (IR) + temozolomide (TMZ), n = 5–6. All data are presented as mean ± SEM. Statistics were performed with unpaired two-tailed Student’s t-test. Multiple comparisons were performed using one-way ANOVA followed by Sidak test, *P < .05, **P < .01, ***P < .001, ****P < .0001.
Figure 6.
Figure 6.
Integrin α2-deficiency regulates the radiochemosensitivity of GBM cells via the ATF1/ERK1 axis. (A) Analysis of the clonogenic survival in U343MG cells transfected with control, ITGA2, ATF1, MAPK3 (targeting ERK1) or MAPK1 (targeting ERK2) siRNAs (and in combinations) upon indicated treatments, n = 4. (B) Growth rate of U343MG cells upon integrin α2 (ITGA2), ATF1, ERK1, or ERK2 knockdowns on day 5 after seeding, n = 3. (C) Analysis of radio(chemo)sensitizing effects of integrin α2 (E7820 and BTT3033) and ERK (SCH772984) inhibitors on U343MG cells, n = 4. (D) Differences in radiosensitizing efficiency in integrin α2-depleted U343MG cells transfected with either pTagGFP2-N-hATF1 or pTagGFP2-N-hERK1 vectors (empty pTagGFP2-N vector was used as a control) shown as Δ values of normalized plating efficiency (P.E.), n = 4. (E) Graph illustrating ITGA2, ATF1 and MAPK3 (=ERK1) mRNA levels in U343MG cells (upper panel) and mRNA-protein kinetics (lower panel) upon silencing of integrin α2 or ATF1, n = 3–6. (F) The indicated reporter constructs were transfected into integrin α2-deficient and control U343MG cells. Firefly in relation to Renilla luciferase levels were calculated. The empty vector pGL3-Basic was used as a reference, n = 3. N.D.— no signal detected. All data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Sidak (A, B) or Dunnett (C, E) post hoc tests, *P < .05, **P < .01, ***P < .001, ****P < .0001.
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