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Gefitinib induces EGFR and α5β1 integrin co-endocytosis in glioblastoma cells

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Abstract

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.

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Acknowledgements

This research was funded by Ligue Contre le Cancer, Région Grand-Est, programme-inter region (No S17R417B). Elisabete Cruz Da Silva and Anne-Florence Blandin were PhD students funded by the University of Strasbourg. Marie-Cécile Mercier was a pharmacy intern funded by ARS Grand Est (regional health agency). We thank Romain Vauchelles and the PIQ platform (IBiSa Quest imaging facility) for their assistance in image quantification.

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  1. Anne-Florence Blandin, Elisabete Cruz Da Silva contributed equally to this work.

Authors and Affiliations

  1. Department of Oncologic Pathology, Dana Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA

    Anne-Florence Blandin

  2. UMR 7021, Laboratoire de Bioimagerie et Pathologies, Faculté de Pharmacie, CNRS, Université de Strasbourg, 67401, Illkirch, France

    Elisabete Cruz Da Silva, Marie-Cécile Mercier, Oleksandr Glushonkov, Pascal Didier, Nelly Etienne-Selloum, Monique Dontenwill, Laurence Choulier & Maxime Lehmann

  3. Département de Pharmacie, Centre de Lutte Contre le Cancer Paul Strauss, 67000, Strasbourg, France

    Nelly Etienne-Selloum

  4. UMR CNRS 7369, Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), Université de Reims Champagne Ardenne (URCA), Reims, France

    Stéphane Dedieu, Cristophe Schneider & Jessica Devy

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Contributions

Participated in research design: ML, MD, LC, PD. Conducted experiments: AFB, ECS, MCM, OG, NES, SD, CS, JD. Performed data analysis: AFB, ECS, MCM, SD, CS, JD. Wrote or contributed to the writing of the manuscript: AFB, ECS, SD, LC, ML. All of the authors reviewed the manuscript and approved the final version.

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Supplementary file1Supplemental Figure 1: Gefitinib provokes EGFR endocytosis in GBM cells. (A) Immunodetection of EGFR (red) and the endosomal marker EEA1 (green) after 4h treatment with DMSO (control) or gefinitib in LN443 and T98G GBM cells. Magnified images are from the inserts to the peri-nuclear area. Scale bar = 20 μm. (B) Quantification of EGFR/EEA1 colocalization following gefitinib treatment from 10–12 images (3 independent experiments). ***p < 0.001. (C-D) Endocytosis assays of EGF-Alexa488 was performed on LN443, T98G and LNZ308 cells during 1h in presence of gefitinib (20µM). The internalization was measured by integrating fluorescence density of 20-30 cells from 3 independent experiments. ****p < 0.0001. (E) Immunoblot showing α5 integrin and EGFR expression in the 4 cell lines used in this study. Supplemental Figure 2: Gefitinib provokes integrin re-localization in early endosomes. (A) Fluorescence microscopy images of U87 cells treated with gefitinib showing peri-nuclear co-localization of the β1 integrin (cyan) and the early-endosome marker Rab5 (red). (B) The Pearson correlation and Mender’s coefficient were used to quantify the degree of colocalization between the β1 integrin and Rab5. ***p < 0.001. Supplemental Figure 3: Second and third-generation TKIs also induce co-internalization of β1 integrin and EGFR during U87 GBM cell evasion. (A) Confocal images of U87 cells treated with vehicle (control) or TKIs gefitinib (20 µM), afatinib (5 µM), erlotinib (10 µM), dacomitinib (10 µM) or lapatinib (10 µM). Images are representative of 3 independent experiments. High-magnification images are from the inserts into the peri-nuclear area. Scale bar = 20 μm. (B) Quantification of the number of evading cells from U87 and U87α5- treated spheroids. Spheroids were incubated for 24 hours in the presence of DMSO or different TKIs (erlotinib, dacomitinib, lapatinib and afatinib) at the indicated concentrations. Nuclei were stained with DAPI and the number of evading cells was quantified using an ImageJ homemade plugin. Mean of 15 spheroids from 3 independent experiments. **p < 0.05, ***p < 0.001. Supplemental Figure 4: Gefitinib treatment provokes integrin/EGFR relocalization in endosomal compartments of GBM cell lines. Confocal images showing the intracellular distribution (perinuclear region enriched in endomembrane) of EGFR and β1 integrin in LN443, T98G and LNZ308 gefitinib-treated cells. Supplemental Figure 5: Gefitinib treatment does not affect the expression of total EGFR in U87 cells. Left panel: A) Protein expression of EGFR and α5 integrin in U87 GBM cells and U87α5- cells after 24h treatment with DMSO (-) or gefitinib 20µM (+). GADPH was used as loading control. Right panel: Histogram showing the quantification of GADPH-normalized EGFR level of 3 independent experiments. Data represented are the mean +/- s.e.m. B) Quantification of the ratio integrin/EGFR colocalized pixels in the perinuclear compartments of U87 or U87α5- cells that migrated at distance from spheroids after 24 hours of incubation in presence of 20µM gefitinib or DMSO (control). The degree of colocalization between the β1 integrin and EGFR was quantified using an home-made plugin with the ImageJ software. Data expressed as box and whiskers are from at least 30 cells from 10 different fields. Supplemental Figure 6: α5 expression does not affect U87 cell sensitivity to gefitinib in cell growth and cell survival experiments. (A) 2D growth curve of U87 and U87α5- cells in serum-containing medium. (B) Gefitinib dose-response curve on cells growth in 2D after 3 days of treatment. (C) Left panel: phase contrast images of spheroids after 8 days of treatment with indicated concentration of gefitinib. Right panel: dose-response curves of gefitinib on spheroid growth. D) Clonogenic assay in soft agar comparing U87 and U87α5- cell survival in presence of the indicated concentrations of gefitinib. (PPTX 20689 kb)

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Blandin, AF., Cruz Da Silva, E., Mercier, MC. et al. Gefitinib induces EGFR and α5β1 integrin co-endocytosis in glioblastoma cells. Cell. Mol. Life Sci. 78, 2949–2962 (2021). https://doi.org/10.1007/s00018-020-03686-6

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