Induction of Ca2+-dependent autophagy and concurrent lysosomal alkalinization underlies the cytotoxic effects of NNC-55–0396 on glioblastoma cells

Abstract

Diverse agents targeting (macro)autophagy, a critical metabolic stress response in cancer cells, have been proposed for cancer therapy. In previous studies, we showed that NNC-55–0396 (NNC) induces glioblastoma cell death by activating the Unfolded Protein Response (UPR) of ER stress and increasing cytosolic Ca²⁺ levels. Here, we report that NNC affects both ends of the autophagy process, causing extensive cytoplasmic vacuolation. Our results show that: (1) NNC induces autophagy downstream of UPR and Ca²⁺ signaling pathways, thus silencing IRE1α/JNK1 or inhibiting Ca²⁺/IP₃R signaling prevents NNC-induced vacuolation. (2) Silencing ATG5 delays cell death, indicating that autophagy induction plays a role in NNC's cytotoxic effects. (3) NNC and other Ca²⁺-mobilizing agents transcriptionally upregulate p62/SQSTM1, an autophagosome cargo receptor, highlighting a role for this protein in the response to NNC. (4) Studies using tandem fluorescent-tagged LC3 and electron microscopy, however, further reveal that NNC blocks late-stage autophagy that leads to enlarged degradative compartments accumulating ubiquitin-tagged cargoes. (5) Finally, NNC impedes pro-cathepsin-B processing, an effect that is reversed with a weak acid co-treatment, suggesting that lysosomal dysfunction due to increased intraluminal pH is the underlying cause of the autophagy blockade. Together, these findings underscore a multi-level dysregulation of autophagy that contributes to NNC's anti-tumoral effects.