Résumé
BACKGROUND – The N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-Ptase) catalyzes the first step of the mannose-6-phosphate (M6P) synthesis on lysosomal precursor proteins. GNPTAB codes for the α and ß subunits of the enzyme and is mutated in lysosomal storage disorders such as Mucolipidosis II, characterized by lysosomal enzyme hypersecretion. Intriguingly, the frequency of GNPTAB variants has been found increased in uterine and breast cancers. However, consequences of these mutations on GlcNAc-1-Ptase activity and on cancer cell behavior have yet to be investigated. Considering that acid hydrolase hypersecretion has been reported in many cancers and that it promotes cancer cell migration and invasion, it is worth considering that GNPTAB alterations could affect these processes.AIMS – Investigation of the consequences of GNPTAB mutations on GlcNAc-1-Ptase activity, expression, localization and on acid hydrolases sorting ; setting up of a siRNA-mediated knockdown model for GNPTAB in uterine cancer cell lines and investigation of the consequences on hydrolases sorting and cell properties (e.g. expression of EMT markers).
METHODS – Using western blottings, enzyme activity assays and immunofluorescence methods, we investigated the effect of mutations R334*, E632D, V872I, E1197K, R1237W and I1245K. Enzyme
assays and RT-qPCR were used to characterize the GNPTAB knockdown models in uterine cancer cell lines (HeLa, HEC-1-A and KLE cells).
RESULTS – We identified that R334* prevents GlcNAc-1-Ptase expression and fails to rescue acid hydrolase sorting in GNPTAB KO HeLa cells. The other mutants corrected acid hydrolases hypersecretion and most of them exhibited similar or slightly higher expression levels compared to the wild-type enzyme, except for E632D, which exhibited a 30% decrease of total expression level
(p=0,061). None of the mutations (except the R334*) impaired the maturation or localization of GlcNAc-1-Ptase in HeLa cells. We also identified conditions allowing for a >90% decrease of GNPTAB expression 48 and 72h after transfection of GNPTAB-targeting siRNAs in HeLa, HEC-1A or KLE cells. However, the knockdown of GNPTAB did not cause acid hydrolase hypersecretion, barely
affected the lysosomal population at a morphological level, and the expression of EMT markers remained unchanged.
CONCLUSION – Regarding GNPTAB knockdown using siRNAs, additional experiments will be needed to identify conditions that reproduce a lysosomal storage phenotype, and acid hydrolase hypersecretion, prior to conducting functional analyses to assess how these phenotypes affect cancer
cell behavior. Interestingly, we found that one of the mutations reported in uterine cancers (R334*) prevents the activity of GlcNAc-1-Ptase, and may therefore cause acid hydrolase hypersecretion. Introducing this mutation in the genome of uterine cancer cell lines using the CRISPR-Cas9 method could generate useful models to test if and ho the cell behavior (growth, migration, invasionproperties, expression of EMT markers,…) is affected by GlcNAc-1-Ptase inactivation.
| la date de réponse | 19 janv. 2022 |
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| langue originale | Anglais |
| L'institution diplômante |
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| Superviseur | Marielle Boonen (Promoteur) & Michel Jadot (Copromoteur) |