Dual β1 integrin and JNK inhibition decreases radiochemoresistance and invasion of Glioblastoma stem-like cells


Dual β1 integrin and JNK inhibition decreases radiochemoresistance and invasion of Glioblastoma stem-like cells

Vehlow, A.; Klapproth, E.; Storch, K.; Temme, A.; Krause, M.; Cordes, N.

Abstract

Background: The poor prognosis associated with Glioblastoma multiforme (GBM) largely arises from the radiochemotherapy resistance of GBM cells and their aggressive infiltration and destruction of the healthy brain. β1 integrin and c-Jun N terminal kinase (JNK) are potential therapeutic targets in GBM owing to their regulation of radioprotective pro-survival and pro-invasive signaling hubs in Glioblastoma stem-like cells and the tumor bulk. Here, we evaluate the effect of simultaneous targeting of β1 integrin and JNK in combination with radiochemotherapy on clonogenic survival and invasion of patient-derived GBM stem-like cells and GBM cell lines in vitro and in an orthotopic GBM mouse model.
Methodology: The sphere forming capacity, clonogenic survival and invasion of patient-derived GBM stem-like cells (GS-8, DK41, DK42) and Glioblastoma cell lines (HT7606, T4, U343-MG) was analyzed upon irradiation (0-6 Gy X-ray), chemotherapy (Temozolomide, TMZ, IC10) or a combination of both. Simultaneously, β1 integrin was inhibited by the specific antibody AIIB2 and JNK activation was blocked by pharmacologic inhibition using SP600125 (IC10, IC50). In addition, tumor growth and survival of mice with orthotopic GBM were assessed upon dual β1 integrin/JNK inhibition combined with radiochemotherapy. Changes in signal transduction mechanisms were examined by means of Western blotting and broad-spectrum phosphoproteome analysis. Cell cycle changes were evaluated by FACS and alterations in DNA repair mechanisms were quantified by immunofluorescent staining of DNA double strand breaks.
Results: Co-inhibition of β1 integrin and JNK strikingly induces radiochemosensitization and blocks invasion of patient-derived GBM stem-like cells and GBM cell lines. In line, the combination of radiochemotherapy plus dual targeting significantly delays tumor growth resulting in prolonged median survival of mice bearing orthotopic GBM. Mechanistically, phosphoproteome and functional analysis reveals that the radiochemosensitizing potential of the β1 integrin/JNK co-inhibition is caused by a prolonged G2/M arrest upon irradiation due to a defective repair of DNA double strand breaks.
Conclusion: Here, we suggest co-targeting of β1 integrin and JNK as a promising approach to overcome radiochemoresistance of GBM in-vitro and in-vivo. Intriguingly, sphere forming capacity and invasion of GBM stem-like cell populations as well as clonogenicity and invasion of established GBM cell lines could be substantially reduced upon dual targeting simultaneously to conventional radiochemotherapy. Currently, ongoing studies are aimed at identifying possible bypass mechanisms.

  • Lecture (Conference)
    GBS-Jahrestagung, 26.-28.09.2016, Erlangen, Deutschland

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