Bortezomib abrogates temozolomide-induced autophagic flux through an ATG5 dependent pathway

Mohummad Aminur Rahman; Agnete S T Engelsen; Shahin Sarowar; Christian Bindesbøll; Even Birkeland; Dorota Goplen; Maria L Lotsberg; Stian Knappskog; Anne Simonsen; Martha Chekenya

doi:10.3389/fcell.2022.1022191

Bortezomib abrogates temozolomide-induced autophagic flux through an ATG5 dependent pathway

Front Cell Dev Biol. 2022 Dec 22:10:1022191. doi: 10.3389/fcell.2022.1022191. eCollection 2022.

Authors

Mohummad Aminur Rahman^{1

2}, Agnete S T Engelsen^{1

3}, Shahin Sarowar¹, Christian Bindesbøll⁴, Even Birkeland¹, Dorota Goplen², Maria L Lotsberg^{1

3}, Stian Knappskog^{2

5}, Anne Simonsen^{4

6

7}, Martha Chekenya¹

Affiliations

¹ Department of Biomedicine, Faculty of Medicine, University of Bergen, Bergen, Norway.
² Department of Oncology, Haukeland University Hospital, Bergen, Norway.
³ Department of Clinical Medicine and Centre for Cancer Biomarkers, Faculty of Medicine, University of Bergen, Bergen, Norway.
⁴ Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway.
⁵ Department of Clinical Science, Faculty of Medicine, University of Bergen, Bergen, Norway.
⁶ Centre for Cancer Cell Reprogramming, Faculty of Medicine, University of Oslo, Oslo, Norway.
⁷ Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.

Abstract

Introduction: Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that pre-treatment of glioblastoma with bortezomib (BTZ) might sensitize glioblastoma to temozolomide by abolishing autophagy survival signals to augment DNA damage and apoptosis. Methods: P3 patient-derived glioblastoma cells, as well as the tumour cell lines U87, HF66, A172, and T98G were investigated for clonogenic survival after single or combined treatment with temozolomide and bortezomib in vitro. We investigated the requirement of functional autophagy machinery by utilizing pharmacological inhibitors or CRISPR-Cas9 knockout (KO) of autophagy-related genes -5 and -7 (ATG5 and ATG7) in glioblastoma cells and monitored changes in autophagic flux after temozolomide and/or bortezomib treatments. P3 wild-type and P3 ATG5-/- (ATG5 KO) cells were implanted orthotopically into NOD-SCID mice to assess the efficacy of bortezomib and temozolomide combination therapy with and without functional autophagy machinery. Results: The chemo-resistant glioblastoma cells increased autophagic flux during temozolomide treatment as indicated by increased degradation of long-lived proteins, diminished expression of autophagy markers LC3A/B-II and p62 (SQSTM1), increased co-localisation of LC3A/B-II with STX17, augmented and no induction of apoptosis. In contrast, bortezomib treatment abrogated autophagic flux indicated by the accumulation of LC3A/B-II and p62 (SQSTM1) positive autophagosomes that did not fuse with lysosomes and thus reduced the degradation of long-lived proteins. Bortezomib synergistically enhanced temozolomide efficacy by attenuating cell proliferation, increased DNA double-strand breaks, and apoptosis in an autophagy-dependent manner. Abolishing autophagy in ATG5 KOs reversed the bortezomib-induced toxicity, rescued glioblastoma cell death and reduced animal survival. Discussion: We conclude that bortezomib abrogates temozolomide induced autophagy flux through an ATG5 dependent pathway.

Keywords: apoptosis; autophagy flux; cell cycle kinetics; chemosensitization; glioblastoma (GBM); proteasome inhibitor bortezomib; secretome; temozolomide chemotherapy.