Do HeLa cells knock-out for GNPTAB, which codes for α and β subunits of N-acetylglucosamine- 1-phosphotransferase, exhibit resistance to cytotoxic agents?

Abstract

Mutations in the GNPTAB gene cause Mucolipidosis II (MLII) and III, two human lysosomal storage disorders characterized by a defective form of N-acetylglucosamine-1-phosphotransferase (GlcNAc-1- PTase). This enzyme catalyzes the synthesis of Mannose-6-Phosphate (M6P) residues on newly synthesized lysosomal hydrolases, and these residues serve as targeting signals to lysosomes. Hence, in MLII or III, defective M6P synthesis results in acid hydrolase hypersecretion and accumulation of their substrates in lysosomes. Intriguingly, fibroblasts isolated from MLII patients exhibit more resistance to several cytotoxic agents. Since it has been reported that the frequency of GNPTAB variants is increased in breast and uterine cancers, we wondered whether GlcNAc-1-PTase inactivation in cancer cells could also confer them protection against induced cell death. To test this hypothesis, control and GNPTAB-KO HeLa cells were treated with different concentrations of chloroquine and doxorubicin. Taken together, the results of MTT assays and phase contrast microscopy analyses support that GNPTAB-KO HeLa cells are more resistant to the cytotoxic effect of those molecules. Moreover, caspase 3 activation upon treatment with doxorubicin was found lower in GNPTAB-KO cells compared to control cells, suggesting that the KO cells are more specifically resistant to caspase-dependent apoptosis. Lastly, we detected sustained levels of NF- κB in the KO cells after treatment, which might be among the mechanisms that confers them apoptosis resistance. Other mechanisms, including increased trapping of doxorubicin and chloroquine in their lysosomes, are also considered. Taken together, our findings raise the possibility that deregulation of M6P synthesis in cancer cells, and thus of acid hydrolases sorting to lysosomes, may confer resistance to some chemotherapeutic agents.

Date of Award	27 Mar 2023
Original language	English
Awarding Institution	University of Namur
Supervisor	Marielle Boonen (Supervisor)