Discoidin Domain Receptor 1 controls GBM radiochemosensitivity by modulating autophagy


Discoidin Domain Receptor 1 controls GBM radiochemosensitivity by modulating autophagy

Klapproth, E.; Vehlow, A.; Cordes, N.

Background: Glioblastoma multiforme (GBM) is characterized by genetic and epigenetic alterations in resistance-mediating genes and destructive infiltration of the surrounding brain. Cell adhesion molecules play an important role in therapy resistance. One of these cell adhesion molecules is the Discoidin Domain Receptor 1 (DDR1) facilitating binding to the extracellular matrix protein collagen type-1. Here, we evaluated the so far unknown role of DDR1 in GBM radiochemoresistance including analysis of the underlying molecular mechanisms.
Methods: DDR1 expression (tumor vs. normal) was investigated (Oncomine database). DDR1 targeting (DDR1-IN-1 inhibitor and siRNA) as single treatment and in combination with either irradiation or radiochemotherapy using Temozolomide (TMZ) was conducted to identify its radiochemosensitizing potential in GBM stem-like (GS-5, GS-8) and primary GBM cell populations (DK32, DK41, DK42) (Sphere Formation Assay, 0-6 Gy X-rays) as well as in an orthotopic GBM mouse model. Alterations of signal transduction upon DDR1 inhibition were examined by Western blotting and broad spectrum phosphoproteome analysis. A search for direct DDR1 binding partners was executed by sequential immunoprecipitation/mass spectrometry employing wildtype and truncated DDR1 variants and GST-pulldown.
Results: Database analysis revealed a 3-fold increased DDR1 expression in GBM compared with normal brain (Oncomine). GBM stem-like and patient-derived GBM cell cultures treated with DDR1-IN-1 showed significantly enhanced radiosensitivity in vitro. Intriguingly, a combined DDR1-IN-1/TMZ regimen plus irradiation significantly delayed tumor growth and prolonged survival of mice bearing orthotopic GBM. Mechanistically, a 14-3-3/Beclin-1 protein complex identified by MS/MS connects DDR1 to the pro-survival Akt-mTOR axis. Upon DDR1 inhibition, we observed dissociation of this protein complex followed by abrogated Akt-mTOR signaling, induction of LC3b expression and formation of LC3b-positive autophagosomes. Direct binding of 14-3-3 to DDR1 was confirmed by DDR1 deletion variants and GST-pulldown.
Conclusion: Our data demonstrate that DDR1 is a potential target in GBM and its pharmacological inhibition effectively mediates radiochemosensitization via induction of autophagy that is superior to the conventional therapy.

Keywords: GBM; DDR1; radiochemoresistance; autophagy

  • Contribution to proceedings
    Wolfsberg Meeting, 17.-19.06.2017, Ermatingen, Schweiz

Permalink: https://www.hzdr.de/publications/Publ-25184
Publ.-Id: 25184