Deciphering the expression and function of the autophagy cysteine protease, ATG4B, in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) exhibits elevated levels of autophagy which promote tumor progression and treatment resistance. ATG4B is an autophagy-related cysteine protease under consideration as a potential therapeutic target, but is largely unexplored in PDAC. Here, I investigated the potential clinical and functional relevance of ATG4B expression in PDAC. Using two PDAC patient cohorts, I found that low ATG4B mRNA or protein expression was associated with worse patient survival outcomes, poorly differentiated PDAC tumors, and lack of survival benefit from adjuvant chemotherapy. In PDAC cell lines, ATG4B knockout (KO) reduced proliferation, abolished LC3B processing, reduced GABARAP and GABARAP-L1 levels and increased p62, GLUT1, PFK-P and ATG4A levels. In regards to metabolism, ATG4B KO lines exhibited an increase in glucose uptake, basal glycolysis, lactate efflux and reduction in mitochondrial function, which collectively reflect increased aerobic glycolysis activity and preference. Increased intracellular glutamine concentration, in combination with reduced relative abundance of -ketoglutarate and glutamine uptake in the ATG4B KO lines, suggest an increase in glutamine synthesis. Glutamine is known to play a role in cellular redox homeostasis and the increase in relative abundance of the glutathione synthesis intermediate, -glu-glu, suggests that glutamine might be used to produce the antioxidant glutathione in the ATG4B KO lines. ATG4B and ATG4A (ATG4BA) double KO lines displayed a further reduction in proliferation, characterized by delays in G1/S-phase transition and mitosis. ATG4BA KO cells exhibited reduced expression of CCND2, a G1/S phase protein, providing a potential explanation for the G1/S phase delay. The mitotic defect was associated with aberrant centrosomal accumulation of the unprocessed ATG4 substrate, pro-LC3B. Additionally, increased expression of the centrosomal proteins PCM1 and CEP131 was observed in the ATGT4BA KO lines. PCM1 and CEP131 are involved in doryphagy, the selective autophagic degradation of centriolar satellites, and thus defects in doryphagy could also contribute to the observed cell cycle delay. In aggregate, this work demonstrates cell cycle and metabolic consequences of ATG4B loss in PDAC cells that have important implications for therapeutic targeting of ATG4B in PDAC.