PhD thesis defense in biomedical and pharmaceutical sciences by Asena AYNACI

Consequences of GNPTAB-linked disruption of intracellular trafficking of lysosomal hydrolases on cancer cell behavior

Candidate

Asena AYNACI

Promoter

Prof. Marielle BOONEN, UNamur, Department of medicine, Molecular Physiology Research Unit (URPhyM), Laboratory of Intracellular Trafficking Biology (LBTI)

Jury

• Jean-Pierre GILLET, Président, Université de Namur
• Marielle BOONEN, Promotrice, Secrétaire, Université de Namur
• Michel JADOT, Co-promoteur, Université de Namur
• Isabelle MAYSTADT, Université de Namur et Institut de Pathologie & Génétique
• Christine GILLES, Université de Liège
• Stéphanie HERKENNE, Université de Liège

Summary

Mutations in GNPTAB, the gene encoding the catalytic α/β subunits of GlcNAc-1-phosphotransferase (GlcNAc-1-PTase), cause mucolipidoses II and III α/β, which are lysosomal storage disorders. Loss of GlcNAc-1-PTase function prevents the addition of the mannose-6-phosphate (Man 6-P) targeting signal to newly synthesized acid hydrolase precursors. Deprived of this signal, these enzymes are hypersecreted, leading to lysosomal depletion of active hydrolases and promoting intralysosomal accumulation of undegraded macromolecules.

GNPTAB mutation frequency also appears to be significantly increased in several cancers, particularly endometrial and breast cancers. However, the impact of Man-6-P pathway impairment on cancer cell behavior remains largely unknown. Defective targeting of acid hydrolases could remodel the tumor microenvironment, notably through pericellular proteolysis of the extracellular matrix. In parallel, loss of Man-6-P–dependent trafficking could alter clearance of the growth factor IGF-II, whose availability is regulated by endocytic mechanisms that depend, in part, on lysosomal cargo bearing multiple Man-6-P moieties. Finally, lysosomal storage may also affect the kinetics of activation and termination of growth-factor receptor signaling. On this basis, we hypothesized that GlcNAc-1-PTase deficiency may confer pro-tumorigenic phenotypes, particularly enhanced proliferation, survival, migration, and invasive potential.

To address this question, we first analyzed GNPTAB variants reported in endometrial tumors using the COSMIC and TCGA databases. This revealed a heterogeneous mutational landscape with respect to gene position and an enrichment in hypermutated subtypes (POLE/MSI). Six representative variants spanning distinct domains of GlcNAc-1-PTase were then selected and transiently expressed in HeLa GNPTAB^⁻/⁻ cells to assess their effects on enzyme expression and maturation, and on restoration of lysosomal targeting in these cells. This approach showed that the truncated variant p.R334* is not expressed and therefore cannot correct hydrolase hypersecretion, whereas the other mutants appear to retain at least partial activity.

We then performed a phenotypic and mechanistic analysis of the consequences of complete GlcNAc-1-PTase loss of function. Interestingly, we found that GlcNAc-1-PTase deficiency modifies the response to anticancer treatments. Specifically, GNPTAB inactivation confers resistance to multiple cytotoxic agents (doxorubicin, chloroquine, staurosporine, and paclitaxel), in association with reduced activation of an effector caspase. Our analyses highlight increased sequestration of weak bases within the expanded lysosomal compartment of GNPTAB^⁻/⁻ cells, potentially contributing to the reduced efficacy of doxorubicin and chloroquine, while leaving open the involvement of additional resistance mechanisms—particularly for staurosporine and paclitaxel, which are not weak bases. Mechanistically, we uncovered hyperactivation of IGF1R, consistent with increased extracellular availability of its ligand IGF-II, which would be less efficiently internalized by IGF2R (also known as CI-M6PR, reflecting its role in endocytosis of Man 6-P–tagged lysosomal hydrolases). Indeed, loss of Man 6-P signals on hydrolases is known to reduce IGF2R dimerization and internalization, thereby limiting lysosomal degradation of IGF-II. The resulting IGF1R hyperactivation is accompanied in our cells by activation of AKT, a kinase that participates in anti-apoptotic signaling cascades. Moreover, pharmacological inhibition of IGF1R restored apoptotic sensitivity of treated cells, regardless of the cytotoxic agent used. Together, these results identify the IGF-II/IGF1R/AKT axis as a central driver of the apoptosis resistance acquired by GlcNAc-1-PTase–deficient cancer cells, and reinforce the pivotal role of lysosomal biology in tumor aggressiveness.