Defective M6P-dependent lysosomal sorting due to GlcNAc-1-phosphotransferase inactivation promotes breast cancer cell aggressiveness

Promoters

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

Prof. Michel Jadot, UNamur, Department of medicine, Molecular Physiology Research Unit (URPhyM), Laboratory of Intracellular Trafficking Biology (LBTI) - co-promoter

Jury

• Prof. Henri-François Renard, Université de Namur, URBC, NARILIS (Président)
• Prof. Marielle Boonen, Université de Namur, URPhyM, NARILIS (Promotrice et secrétaire)
• Prof. Michel Jadot, Université de Namur, URPhyM, NARILIS (Co-promoteur)
• Prof. Nicolas Gillet, Université de Namur, URVI, NARILIS
• Dr. Bassam Janji, Luxembourg Institute of Health, Department of Cancer Research
• Dr. Philippe Chavrier, Institut Curie, PSL University Paris

Summary

The N-acetylglucosamine-1-phosphotransferase (GlcNAc1PT) is a hexameric cis-Golgi resident enzyme composed of 2α, 2β and 2γ subunits. GNPTAB encodes the α and β subunits as a single polypeptide precursor, whose cleavage by site-1-protease is required for enzyme activation. This maturation step is supported by the recently discovered protein LYSET. Under its active form, GlcNAc1PT catalyzes the first step of Mannose-6-phosphate (M6P) residue synthesis on the oligosaccharide chain of acid hydrolase precursors. M6P residues are recognized by M6P receptors, which undertake the sorting of acid hydrolases to the endolysosomal system. GlcNAc1PT function is therefore essential for the biogenesis of lysosomes, which are hydrolytic organelles that mediate the degradation of endocytic and autophagic cargoes. Inactivation of GlcNAc1PT due to mutations in GNPTAB underlies the lysosomal storage disorder Mucolipidosis type II, which is characterized by hypersecretion of acid hydrolases lacking M6P residues and lysosomal accumulation of multiple substrates.

Interestingly, elevated frequency of mutations in lysosomal genes has been reported in several tumor types, including GNPTAB mutations in breast and endometrial tumors. As down-regulation of cation-independent M6P receptor, which likewise results in M6P pathway dysregulation, is known to promote aggressive traits in breast cancer, we wondered whether GlcNAc1PT inactivation would also affect cancer cell behavior.

Our work led to the discovery that BT549 breast cancer cells carry a homozygous mutation p.Y34* in LYSET and a heterozygous mutation p.Y916C in GNPTAB, the combination of which results in inactivation of GlcNAc1PT. Consequently, these cells fail to synthesize M6P moieties, leading to hypersecretion of acid hydrolases and subsequent lysosomal storage. By using a model whereby LYSET p.Y34* mutation was repaired using Crispr/Cas9, we uncovered that the inactivation of GlcNAc1PT modifies the surfaceome of BT549 cells, with important changes in cell-cell and cell-matrix adhesion molecules. Concomitantly, we found that these cells exhibit increased migration and invasion, the latter being supported by lysosomal cathepsin hypersecretion. We also found that GlcNAc1PT inactivation increases cell proliferation through increased IGF1R activation. M6P-bearing acid hydrolases are reported to induce the dimerization of CI-M6P/IGFIIR, which promotes the internalization of IGFII, another ligand of IGF1R. We propose that the absence of M6P synthesis in BT549 cells limits this clearance mechanism, resulting in increased IGFII extracellular levels and enhanced IGF1R activation. Moreover, we discovered that GlcNAc1PT inactivation causes increased reliance on extracellular nutrients, such as L-glutamine. Consistent with this observation, we detected higher levels of glutamine importer SCL38A2 at the plasma membrane of these cells. Lastly, we generated GNPTAB KO or LYSET KO Hs578t breast cancer cells, which both fail to synthesize M6P and present similar acid hydrolase secretion levels, supporting that a LYSET deficiency phenocopies GlcNAc1PT deficiency due to GNPTAB inactivation. Inactivation of GlcNAc1PT in Hs578t clones causes increased cell proliferation and marginally increased migration and invasion, with intriguing discrepancies between GNPTAB KO and LYSET KO clones.

In conclusion, through this work we show how GlcNAc1PT-inactivation and the resulting lysosomal defect as key determinants of cancer cell behavior, demonstrating that disruption of lysosomal homeostasis can promote multiple hallmarks of tumor aggressiveness, including enhanced proliferation, migration, and invasion. These findings provide mechanistic insight into the poorly understood association between lysosomal storage conditions and cancer. Our research also reveals potential opportunities for therapies. Moreover, the surfaceome modifications consequent to GlcNAc1PT inactivation provides new insight into the consequences of lysosomal storage on plasma membrane composition, and opens new research avenues into the mechanisms at the basis of these changes, while discrepancies between GNPTAB KO and LYSET KO Hs578t cells behavior suggest potentially unknown functions of LYSET, paving the way for future research on this recently discovered protein. Overall, this work sheds light on the relationship between lysosomal storage and cancer and deepens our understanding of the fundamental role of lysosomes in cancer biology.